in Uruguay. - prensamedica.com.uy

Transcripción

MARZO 2007; XXIX SUPL 1 : S01-S198
CONTENS
XXXI World Congress of the International Society of Hematology 2007
March 20-24, 2007 - Conrad Resort Casino - Punta del Este - Uruguay
Symposium
Education Session
Conference
Course
Oral Session
Poster Session
Prensa Médica Latinoamericana. Montevideo
ISSN 0250 - 3816
ÓRGANO OFICIAL DE LA SOCIEDAD DE MEDICINA
INTERNA DEL URUGUAY, SOCIEDAD URUGUAYA DE
NEFROLOGÍA, SOCIEDAD DE DIABETOLOGÍA Y
NUTRICIÓN DEL URUGUAY, SOCIEDAD URUGUAYA
DE ENDOCRINOLOGÍA, SOCIEDAD URUGUAYA DE
FARMACOLOGÍA Y TERAPÉUTICA, SOCIEDAD
URUGUAYA DE HEMATOLOGÍA, SOCIEDAD DE
TISIOLOGÍA Y ENFERMEDADES DEL TÓRAX DEL
URUGUAY, SOCIEDAD DE NEUROLOGÍA DEL
URUGUAY, SOCIEDAD DE GASTROENTEROLOGÍA
DEL URUGUAY, SOCIEDAD DE ONCOLOGÍA MÉDICA
Y PEDIÁTRICA DEL URUGUAY Y SOCIEDAD
URUGUAYA DE ATEREOSCLEROSIS.
•
DIRECTOR
Prof. Dr. Alfredo Álvarez Rocha
Profesor de Clínica Médica
Facultad de Medicina. Universidad de la República
Montevideo.
•
SECRETARIA CIENTIFICA
Dra. María Laura Llambí
Ex Profesora Adjunta de Clínica Médica
Facultad de Medicina. UdelaR. Montevideo
•
ASISTENTES DE DIRECCION
Prof. Dr. Gonzalo Aiello – Neumólogo
Prof. Dr. Álvaro Huarte – Internista
Prof. Dr. Pablo Muxi – Hematólogo
Prof. Dr. Raúl Pisabarro – Endocrinólogo
Prof. Dr. Leonardo Sosa – Internista
Prof. Dra. Verónica Torres Esteche – Internista
•
COMITÉ DE ARBITRAJE- Selección de trabajos
Prof. Dra. Adelina Braselli – Infectóloga
Prof. Dr. Daniel Bulla – Internista
Prof. Dr. Eladio García – Internista
Prof. Dra. Raquel Ponce De León – Internista
Prof. Dr. Carlos Romero – Cardiólogo
•
CO-SECRETARIAS CIENTIFICAS
Prof. Dra. Mariela Vacarezza
Profesora Adjunta de Cátedra de Enfermedades Infecciosas
Dra. Natalia Miranda
Asistente de Clínica Médica.
•
COMITÉ AD HONOREM
Prof. Dr. Nelson Mazzuchi – Nefrólogo
Prof. Dr. Mario Medici – Neurólogo
Prof. Dr. Ignacio Mussé – Oncólogo
Prof. Dr. Jorge Torres Calvete – Internista
•
CONSEJO EDITORIAL
Prof. Dra. Giséle Acosta – Anátomopatóloga
Prof. Dr. Juan Carlos Bagattini – Internista
Prof. Dra. María Cristina Belzarena – Endocrinóloga
Prof. Dr. Adriana Belloso – Internista
Prof. Dra. Mabel Buerger – Toxicóloga
Prof. Dr. Gaspar Catalá – Internista
Prof. Dr. Henry Cohen – Gastroenterólogo
Prof. Dra. Griselda de Anda – Dermatóloga
Prof. Dr. Francisco González – Nefrólogo
Prof. Dr. Ricardo Lluberas – Cardiólogo
Prof. Dra. Mirtha Moyano – Reumatóloga
Prof. Dra. Martha Nese – Hematóloga
Prof. Dra. Filomena Pignataro – Internista
Prof. Dr. Matías Pebet – Internista
Prof. Dr. Álvaro Pintos – Geriatra
Prof. Dr. Luis M. Piñeyro Gutiérrez – Neumólogo
Prof. Dr. Ricardo Roca – Patólogo
Prof. Dr. Carlos Salveraglio – Internista
Prof. Dr. Eduardo Savio – Infectólogo
Prof. Dr. Gustavo Tamosiunas – Farmacólogo
Prof. Dr. Miguel Torres – Oncólogo Radioterapeuta
•
SOCIEDADES CIENTIFICAS
Sociedad de Medicina Interna del Uruguay
Dra. Filomena Pignataro
Sociedad Uruguaya de Nefrología
Dra. Alicia Petraglia
Sociedad de Diabetología y Nutrición del Uruguay
Dra. Graciela Vitarella
Sociedad Uruguaya de Endocrinología
Dra. Anna Spitz
Sociedad Uruguaya de Farmacología y Terapéutica
Por Comis. Fiscal:
Dra. Carolina Seade
Sociedad Uruguaya de Hematología
Dr. Pablo Muxi
Sociedad de Tisiología y Enfermedades del Tórax del Uruguay
Dr. Enrique Invernizzi
Sociedad de Neurología del Uruguay
Dr. Hugo Tarigo
Sociedad de Gastroenterología del Uruguay
Dra. Elena Trucco
Sociedad de Oncología Médica y Pediátrica del Uruguay
Dra. Isabel Alonso
Sociedad Uruguaya de Aterosclerosis
Dra. Silvia Lissman
El Director Redactor Responsable, Secretarios Científicos, Asistentes de Dirección y el Comité de Arbitraje actúan con carácter de
Colaboradores Honorarios. Archivos de Medicina Interna está inscrita en el libro VI folio 289 del registro de Ley de imprenta.
ISSN 0250-3816. Arch.Med.Interna es publicado por Prensa Médica Latinoamericana D/L 341.462 / 07. Derechos de autor reservados:
Copyright by Prensa Médica Latinoamericana, Heber Saldivia.editor 2007. La reproducción total o parcial en forma idéntica o modificada escrita a maquina, por el sistema multigraph, mimeógrafo, impreso, fotocopia, scanner, medio electrónicos, etc., no autorizada
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ISSN 0250 - 3816
ÓRGANO OFICIAL DE LA SOCIEDAD DE MEDICINA
INTERNA DEL URUGUAY, SOCIEDAD URUGUAYA DE
NEFROLOGÍA, SOCIEDAD DE DIABETOLOGÍA Y
NUTRICIÓN DEL URUGUAY, SOCIEDAD URUGUAYA
DE ENDOCRINOLOGÍA, SOCIEDAD URUGUAYA DE
FARMACOLOGÍA Y TERAPÉUTICA, SOCIEDAD
URUGUAYA DE HEMATOLOGÍA, SOCIEDAD DE
TISIOLOGÍA Y ENFERMEDADES DEL TÓRAX DEL
URUGUAY, SOCIEDAD DE NEUROLOGÍA DEL
URUGUAY, SOCIEDAD DE GASTROENTEROLOGÍA
DEL URUGUAY, SOCIEDAD DE ONCOLOGÍA MÉDICA
Y PEDIÁTRICA DEL URUGUAY Y SOCIEDAD
URUGUAYA DE ATEREOSCLEROSIS.
CONTENIDO
XXXI Congreso Mundial de la Sociedad Internacional de Hematología 2007
20-24 Marzo 2007 - Conrad Resort Casino - Punta del Este - Uruguay
Simposio
Sesiones de Educación
Conferencias
Cursos
Sesiones Orales
Posters
Este suplemento de Archivos de Medicina Interna ha sido dirigido por la Prof. Dra. Martha Nese.
Los originales han sido aceptados y corregidos por el Director del Suplemento y no han estado sujetos al proceso de
revisión externa. Archivo de Medicina Interna y el Comité Organizador del XXXI Congreso Mundial de la Sociedad
Internacional de Hematología no aceptan ninguna responsabilidad respecto de los puntos de vista y afirmaciones mantenidas por los autores de sus trabajos publicados en este suplemento.
i
Arch Med Interna 2007; XXIX; Supl 1
© Prensa Médica Latinoamericana 2007 ISSN 0250-3816 - Printed in Uruguay - All rights reserved
XXXI World Congress of the International
Society of Hematology 2007
Dear colleague:
Welcome to the XXXI World Congress of the International Society of Hematology (ISH), to be held in Punta
del Este, Uruguay from 20 thru 24 March 2007.
It is really an honor for Uruguay to be the host of such an important international event. The Uruguayan
Society of Hematology (SHU) is working hard to make ISH 2007 a success to meet your expectations. An
impressive contingent of experts who are international referents in the proposed topics, are invited to cover the
great areas of Hematology.
Among the mainstays of our program we would like to emphasize the “Alfredo Pavlovsky Award”, to which Dr.
Mary Horowitz has been nominated for her contribution to research in blood and bone marrow transplantation.
The educational sessions will update the main hematological topics: leukemias, lymphomas, myelomas,
bone marrow transplantations, new stem cell therapies, thrombotic disorders, among others. Simultaneous
activities will be the IV International Congress of the GRCF (Flow Cytometry Group of the River Plate), the
courses in Molecular Biology, Laboratory, Pathology and transfusion Therapy, which are being organized together
with the respective Societies, will be instances of cooperation and exchange with related fields. The meeting of
the Latin American Cooperative Oncology Hematology Group (LACOGH) is another very relevant event among
the Congress activities. The presentations by the Young Hematologists will be a link to the future in the field of
research.
We hope that in your spare time you can enjoy the privileged natural setting of the region and its wonderful
beaches.
We are sure that ISH 2007 will have a high scientific level and will also be an excellent opportunity to meet
new and old friends to exchange experiences and the latest advances of our field.
The members of the Organizing Committee, the Uruguayan Society of Hematology, Punta del Este and
Uruguay are looking forward to welcoming you in Punta del Este in March 2007
Martha Nese, M.D.
ISH President
This number of Archivos de Medicina Interna was directed by Prof. Martha Nese, MD. The originals were accepted and corrected by the archive director, and
have not been revised externally. Archivos de Medicina Interna and the Organizing Committee of the XXXI World Congress of the International Society of
Hematology are in no way responsible for the contents of the papers, which are published as sent by the authors.
ii
A Farewell to Prof. Roberto De Bellis
Prof. Roberto De Bellis, MD, Honorary President of the XXXI Congress of the International Society of Hematology, died on 31 January 2007. He was our professor, colleague and friend. It is not easy to say goodbye to a
friend; strong emotions take hold, and one is unable to continue. I met Roberto when he was Associate Professor
of Internal Medicine at Prof. Manlio Ferrari’s Department, where I arrived as a resident.
He was passionate in his approach to the profession, and one of the great promoters of modern hematology
in this country; an excellent teacher, a brilliant speaker and a sharp and accurate critic.
He went through practically the whole teaching hierarchy of internal medicine, and also acted as Associate
Professor of Medical Pathology and advisor to the School of Medicine, simultaneously publishing a wide array of
papers of national and international relevance, for which he received many awards and honours.
However, in my view his greatest contribution to the profession was the creation of a hematological school.
He designed the project for the foundation of the Department of Hematology in 1980 and directed it until 2003.
At the School of Medicine, he trained an educational team that achieved recognition both in our country and
abroad. His role in the Sociedad de Hematología del Uruguay (Uruguayan Society of Hematology) was decisive
as a founding member and President. He also chaired the first congress of the Society in 1985.
In Uruguay he performed the first bone marrow transplantation, which made him one of the pioneers in the
region. For his outstanding work he received the highest national award in the field of medicine.
Recently he was involved in a new project, cellular therapy in neurodegenerative diseases, which he was
passionate about as was his wont. A few months ago he presented his preliminary results at the ASH congress,
which involved an extraordinary effort, as his health was seriously impaired by then.
With these few words we wish to pay a tribute to Roberto.
We are saying goodbye but we know that he is not leaving, that he will remain forever in the hearts of his
family, his friends and of those whose lives he saved or whose quality of life he improved.
His memory shall live forever in the history of Uruguayan medicine, and to paraphrase Manrique in his stanzas “On my father’s death”, Roberto lived his life in such a way that his life shall endure in death. Hasta siempre,
Roberto.
Martha Nese, MD
iii
Adiós al Prof. Roberto de Bellis
Despedimos al Dr. Roberto de Bellis “Presidente de Honor del XXXI Congreso de la Sociedad Internacional
de Hematología” que falleció el 31.1.07, quien fue nuestro Profesor, colega y amigo. Es difícil despedir a un
amigo sin que la emoción nos traicione y se nos haga un nudo en la garganta, conocí a Roberto cuando era grado
II de Clínica Médica en el Servicio del Prof. Manlio Ferrari, cuando llegue a su sala como practicante interna.
Fue un apasionado de su profesión y uno de los grandes impulsores de la Hematología moderna en el País,
excelente docente, brillante expositor, critico sagaz.
Recorrió prácticamente todo el escalafón docente en Clínica Medica, fue Prof. Agdo. de Patología Médica,
Consejero de la Facultad de Medicina, Fue autor de múltiples trabajos de relevancia Nacional e Internacional y
merecedor de numerosos premios y distinciones.
Pero creo que el aporte mayor en su vida profesional fue la creación de una Escuela Hematológica. Elaboró
el proyecto fundacional de la Cátedra de Hematología en 1980 y fue Director de la misma hasta el 2003.
Numerosos alumnos se formaron en esos años y hoy se han expandido por todo el Uruguay
Formo un equipo docente en la Facultad de Medicina reconocido en el País y en el extranjero.
Tuvo un papel protagónico en la Sociedad de Hematología del Uruguay de la que fue miembro fundador y
Presidente. Presidió también, el 1er Congreso de la Sociedad en 1985.
Inició los primeros trasplantes de Médula Ósea en el Uruguay en 1985 y fue uno de los pioneros en la región.
Por su trabajo recibió el gran premio Nacional de Medicina.
Fue Presidente de la Academia Nacional de Medicina.
Recientemente había comenzado el desarrollo de un nuevo proyecto, la terapia celular en afecciones neurodegenerativas, que abrazo cono siempre con gran pasión.
Hace apenas un mes presentó sus resultados preliminares en el Congreso Americano de Hematología,
haciendo un gran esfuerzo, porque su salud ya estaba seriamente quebrantada.
Queremos rendirle con estas pocas palabras un calido homenaje
Le estamos diciendo adiós, pero sabemos que no se va, que va a quedar para siempre en el corazón de su
familia, de sus amigos y el de todos aquellos a quienes les salvo o mejoro su calidad de vida.
Su recuerdo quedara indeleble en la Historia de la Medicina Nacional y como decía Manrique en las “Coplas a la muerte de su padre”, Roberto vivió la vida de tal suerte que viva quedará en su muerte. Hasta siempre
Roberto
Dra. Martha Nese
Supported by
Declaration of National Interest
Declaration of Tourist Interest
Declaration of Municipal Interest
Presidencia de la República
Ministerio de Salud Pública
Ministerio de Educación y Cultura
Ministerio de Turismo
Ministerio de Relaciones Exteriores
Intendencia Municipal de Maldonado
Academia Nacional de Medicina
Facultad de Medicina
Facultad de Enfermería
Escuela de Graduados
Sindicato Médico del Uruguay
Federación Médica del Interior
Colegio de Enfermería del Uruguay
Catedra de Hematologia
Sociedad de Anatomía Patológica
Sociedad de Hemoterapia e Inmuno-hematologia del Uruguay
Sociedad de Medicina Transfusional
Sociedad Oncológica Médica y Pediátrica del Uruguay (SOMPU)
Programa Nacional de Control de Cáncer
Comisión Honoraria de lucha contra el cáncer
Hospital Maciel
Grupo Latinoamericano de Hemaféresis (GHLEMA) (Latinoamerican Group of Hemapheresis)
World Apheresis Association (WAA)
(Asociación Mundial de Apheresis)
iv
Authorities
INTERNATIONAL SOCIETY OF HEMATOLOGY
LOCAL ORGANIZING COMMITTEE
ISH President
Martha Nese
Honorary President
Roberto De Bellis (†)
Chairman of Council – ISH
Guillermo J. Ruiz–Argüelles
President
Martha Nese
Secretary General – IAD
Norman Maldonado
Vice-president
Enrique Bódega
Secretary General – APD
Hidehiko Saito
Secretary
Alicia Magariños
Secretary General – EAD
Emin Kansu
Treasurer
Ernesto Novoa
National Councillor (Uruguay) ISH
Pablo Muxí
Members
Ada Caneiro
Agustín Dabezies
Adriana Cardeza
SOCIEDAD URUGUAYA DE HEMATOLOGÍA
SCIENTIFIC COMMITTEE
President
Pablo Muxí
Raúl Gabús
Pablo Muxí
Daniel Pieri
Ana María Otero
Secretary
Sebastián Galeano
NURSING COMMITTEE
NURSING COURSE SCIENTIFIC COMMITTEE
Nancy Seiler - President
Lilián Olivo - Vice-president
Alicia Reche - Secretary
Leda Berneche
Elvira Fernández
Clara Peña
Rosa Rigalli - Secretary
v
vi
International Faculty
Renán Acevedo (USA)
Eduardo Dibar (Argentina)
José María Aguado (Spain)
Guillermo Dighiero (Uruguay)
Elvira Álvarez Jara (Chile)
Juan Dupont (Argentina)
Mary Carmen Amigo (Mexico)
Thomas Elter (Germany)
Raúl Arce Levi (Paraguay)
Miguel Escobar (USA)
Alvaro Avezum (Brazil)
Silvina Estrada (Argentina)
Brady Beltrán (Peru)
Stefan Faderl (USA)
Raquel Bengió (Argentina)
Dorotea Beatriz Fantl (Argentina)
Pablo Bertín Cortés (Chile)
Julio Fernández Águila (Cuba)
Fernando Bezares (Argentina)
Mario Figueroa (Argentina)
Peter Borchmann (Germany)
Ricardo Forastiero (Argentina)
Silvia Brandalise (Brazil)
Robert Gallagher (USA)
Eduardo Bullorsky (Argentina)
Guy Garay (Argentina)
Richard Burt (USA)
Verónica García (Chile)
Roberto Cacchione (Argentina)
Bernadette Garvey (Canada)
Carmen Cao (Chile)
Maurice Genereux (Canada)
José Carnot (Cuba)
Isabel Giere (Argentina)
Antonio Carrasco (Peru)
Sergio Giralt (USA)
José Ceresetto (Argentina)
David Gómez-Almaguer (Mexico)
Juan Chalapud (Mexico)
Derlis González (Paraguay)
Gregory Cheng (Hong Kong)
Edward Gordon-Smith (UK)
Carlos Chiattone (Brazil)
Alexander Graham Turpie (Canada)
Rossana Clapsos (Argentina)
Paula Guggiari (Paraguay)
Guillermo Conte (Chile)
Gregory Hale (USA)
Marcela Contreras (UK)
Nelson Hamerschlak (Brasil)
Daniel Couriel (USA)
Héctor Hendler (Argentina)
Fabián Cusinato (Argentina)
Porfirio Hernández (Cuba)
Denise Helia de Lima (Brazil)
Mary Horowitz (USA)
Carmino de Souza (Brazil)
Gustavo Jarchum (Argentina)
vii
International Faculty
Emin Kansu (Turkey)
Nélida Noguera (Argentina)
Eva Kimby (Sweden)
Alberto Orfao (Spain)
Martin Korbling (USA)
Luis Palmer (Argentina)
Jorge Korin (Argentina)
Laura Pardo (Argentina)
Benjamín Koziner (Argentina)
Ricardo Pasquini (Brazil)
Gunnar Kvalheim (Norway)
Santiago Pavlovsky (Argentina)
Pierre Laneuville (Canada)
Armando Peña Hernández (Honduras)
Irene Larripa (Argentina)
Raúl Pérez Bianco (Argentina)
María Lazzari (Argentina)
Miguel Ángel Píris (Spain)
Howard Liebman (USA)
Carlos Ponzinibbio (Argentina)
Francesco Lo Coco (Italy)
Ramón Ramos (USA)
Oscar López (Argentina)
Edgar Gil Rizzati (Brazil)
Angelo Maiolino (Brazil)
Aníbal Robinson (Argentina)
Norman Maldonado (Puerto Rico)
Francesco Rodeghiero (Italy)
Marta Martinuzzo (Argentina)
Arlette Ruiz de Sáez (Venezuela)
Michael Mauro (USA)
Guillermo J. Ruiz-Argüelles (Mexico)
Lidia Medina (Chile)
Marcelo Russo (Argentina)
Ruben Mesa (USA)
Federico Sackmann (Argentina)
Mercedes Mijares (Venezuela)
Hidehiko Saito (Japan)
Jorge Milone (Argentina)
Jesús San Miguel (Spain)
Mariela Monreal (Argentina)
Julio Sánchez Ávalos (Argentina)
Emili Montserrat (Spain)
Miguel Sanz (Spain)
Ricardo Morilla (UK)
Adriana Sarto (Argentina)
Héctor Murro (Argentina)
Anne Tierens (Norway)
Arturo Mario Musso (Argentina)
Julie Vose ( USA)
Marina Narbaitz (Argentina)
Peter Wiernik (USA)
Bruno Nervi (Chile)
Brent Wood (USA)
Adrian Newland (UK)
viii
Local Faculty
Rosa Abel
Lilián Díaz
Sandra Monzón
Mariel Aguilera
Gustavo Dufort
Pablo Muxí
Esther Alonso
Diego Estol
Martha Nese
Gabriela Arana
Lina Foren
Ernesto Novoa
Graciela Areosa
Wilson França
Lilián Olivo
Laura Bello
Raúl Gabús
Ana María Otero
Beatriz Beñarán
Ana Galán
Juan Paganini
Leda Berneche
Sebastian Galeano
Carolina Pages
Enrique Bódega
Ana Garcia
Clara Peña
Rossana Bonomi
Carlos Ghiggino
Ana Perdomo
Luis Borche
Hugo Giordano
Susana Perdomo
Yanella Bornes
Marisa Gai
Daniel Pieri
Cristina Camejo
Elvira Gossio
Silvia Pierri
Ada Caneiro
Gabriela Gualco
Cristina Pintos
Cecilia Canessa
Cecilia Guillermo
Alfredo Prego
Adriana Cardeza
Olga Hernández
Alicia Reche
Luis A. Castillo
Martha Illa
Ismael Rodríguez
Cecilia Castro
Hugo Isaurralde
Ana Luz Rojo
Dardo Centurión
Patricia Kollar
Eduardo Savio
Alicia Ceres
Santa Leguiza
Nancy Seiler
Virginia Costa
Estela Lavalle
Blanca Stefano de Perdomo
Marta Da Cunha
Daniela Lens
Mario Stoll
Agustín Dabezies
Diego López
Adriana Tiscornia
Cynthia D’Almeida
Haydée López
Laura Topolansky
Gabriela de Gálvez
Milena López
Jimena Torterolo
Elena De Lisa
Alicia Magariños
Cristina Touriño
Jorge Decaro
Ana Mariño
Rosario Uriarte
Luz Deschenaux
Lem Martínez
Alicia Vaglio
Jorge Di Landro
Julio Medina
Ma. Lourdes Viano
Carina Di Matteo
Andrew Miller
Mercedes Zamora
Andrea Díaz
Índice
Aarvak, T., 164
Ardaiz, M., 156
Baubeta, A., 163
Acevedo, S., 171
Ardao, G., 128
Becerra-Artiles, A., 151
Acosta, G., 172, 191, 192, 193,
Arellano, G.J., 178
Bejar R, Y., 194
Aggio, M., 196
Argentieri, D., 185
Bellas, Carmen, 79
Agriello, E., 159, 161, 162
Armellini, A., 162, 183, 198
Belli, C., 171
Aguado, Jose M., 82, 83
Arones-V, A., 155
Bello, A.., 178
Aguilar, L., 191
Arriaga, F., 136, 144, 162
Bello, L., 175
Ahn, H.S., 139
Arrossagaray, G., 147, 171
Beltran Garate, B., 73, 127, 154, 155
Al Abri, Q., 142
Asano, Shigetaka , 100
Bencomo, A., 183
Al Haddabi, H., 142
Assis, A.M., 126
Bendek, G., 132
Al Kindi, S., 142
Astapenco, A., 172, 191, 193
Bene, L., 165, 174
Al Madhani, A., 142
Avila, E., 191
Benetti, F., 178
Al Tobi, F., 142
Avramidis Iakovos, I.G., 149
Bengiò, R, 4, 110, 124, 125, 171
Al Zadjali, S., 142
Azevedo, A.M., 167
Bengochea, M., 90, 163
Alberbide, J., 171
Azevedo, W., 167
Bentahar, A., 197
Beñaran, B., 126, 177, 187, 190, 191
Albuquerque, D.M., 126
Alejandre, M, 146
Bacovsky, J.M., 170
Bergmann, O., 169
Alfaro, E., 161
Baek, H.J., 139
Bernasconi, A., 161
Almeida Pereira, R.D., 167
Balanzategui, A., 198
Bertone, S., 161
Almeida, E. B., 170
Baltazar, S, 154
Bezares, F, 73, 127, 154, 156, 185
Almeida, Julia, , 55, 121
Bao-xia, D., 131
Bianchi, S., 186
Alonso, C., 161
Bar, D., 185
Bianchini, M, 125
Alonso, J, 147
Barbieri, B.E., 137
Bianco, S., 192
Altamirano-Ley, J., 87, 192
Barbón, M., 197
Bietti, J., 152, 195
Altman, Raúl, 188
Barcena, Paloma, 121
Bittencourt, H., 167
Alù, F, 125
Barrena, Susana, 121
Björkstrand, B., 169
Alvarez, A.I., 90
Barreto, W.G., 128
Bodega, E., 90, 186
Alvarez, I., 163
Barreyro, P., 147
Boggia, B., 188, 189
Amigo, Mary-Carmen, 14
Barrionuevo, C., 159, 160, 193
Boiron, J. M., 90
Andino, L., 196
Barros, J.C., 167
Bono, G., 110
Arana-Trejo, R.M., 165
Basso, G., 134
Bonomi, R., 125, 166, 168, 172
Aranha, F.J.P., 157, 167, 175
Batista, B., 191
Borbolla, J.R., 130, 133, 156, 186
Araos, Daniel, 35
Battaglia, L., 112
Borelli, G., 164, 186
Bortomioli, M., 185
Cardeza, A, 125
Ciudad, Juana, 120, 121
Bosch, I., 151
Cardoso, C., 144
Climent, C., 142
Boschi, S, 125
Cardoso, R.B., 157, 175
Collino, C.J.G., 159
Bouzas, L.F.S., 167
Carnot, J., 133, 141, 152, 156, 176, 179,
Colturato, V., 167
Bragós, Irma Margarita, 138
189, 192
Conte, Guillermo, 35
Brandalise, S.R., 92, 167
Carpio, N., 136
Corrado, C., 85, 110, 128, 137, 146, 147
Brandt, M., 162
Carrasco-Yalán, A., 127, 154, 155, 185
Corrales, M., 152, 195
Bravo L, A., 194
Carreño, R., 197
Costa, F.C., 126
Bridger, Gary, 89
Carretto, E., 90, 163
Costa, V, 125
Brignoni, S., 187, 191
Carvani, A., 148
Cottliar, A., 128
Briosso, J., 188, 190
Casagrande, G., 134
Cracco, E., 158
Brugnini, A., 137, 166
Casanova, L., 135, 159
Curi, L., 180
Brunsvig, A., 164
Castillo, H., 191
Cuttica, R., 152, 195
Brusa, B.G., 137
Castillo-Aguirre, J., 127, 154
Brusich, D., 178
Castro, N., 167
D’Antonio, C., 138
Brzosko, S., 143
Castro, R., 176
Daners, A., 162
Bussel, J., 130
Catovsky, Daniel, 122
Davalos, M., 179
Cauvi, F., 185
De Armas, R., 193
Caballero, R., 130, 133, 186
Caviglia, D., 185
† De Bellis, R., 126, 175, 177, 187, 191
Cabrera, A., 187
Cerruti, I., 127
De Brasi, C, 125
Cabrera, M., 183, 196
Cervellini, M., 160
de Cabo, E., 148, 197
Cabrera, S., 156
Cervetto, V., 152, 195
De Castro, R., 133, 141, 152, 156, 177,
Cacchione, R., 138, 156, 157, 179
Chabalgoity, J.A., 166
Calderón Garcidueñas, E. D., 164
Chacon, A., 166
De Galvez, G., 134, 147
Califani, S.M.V., 125
Chalapud Revelo, Juan Ramón, 87
de la Cueva, Paloma, 79
Calvo, H., 187
Chalapud, J., 192
De la Peña, P., 192
Campestri, R., 110, 138, 185
Chantada, G., 161
De La Rubia, J., 144, 163
Caneiro, A., 126, 175, 177
Chena, C., 147
De Rosa, C., 165, 174
Canessa, C., 186
Cheng, G., 130
De Souza, C., 176
Cantalapiedra, A., 197
Chiabrando, G.A., 159
de Souza, C.A., 126, 157, 167, 170, 175
Cantero, S., 144
Chillón, M.C., 198
De Souza, M.P., 167
Cantu-Rodriguez, O., 74, 133, 168
Chisesi, T., 158
Dearden, Claire, 122
Capetta, M., 125, 168
Choi, J.H., 144
deBosch, N., 151
Carballo, T., 152
Chuansumrit, A., 141
Decaro, J., 178, 182
Carbonati, V., 193
Chudý, P., 197
Dei Rossi, F., 158
Cismondi, V., 159, 161
Del Giudice, Ilaria, 122
179, 189, 192
Delamain, M.T., 126, 175, 176
Escamilla G, G., 194
Gabus, R., 71, 90, 185, 186
Delannoy, M., 160
Estigarribia, N., 160
Gaggero, M., 182
Demikhov, V.G., 181
Eugui, E., 192
Gale, R.P., 157
Demikhova, E.V., 181
Eui-Kyu, Noh, 153
Galeano, A., 159, 161
Dengra, C., 112
Eun Jung, Lee, 153
Galeano, S., 90
Galimberti, G., 195
Derakhshan, F., 189, 190, 197
Derakhshan, R., 189, 190, 197
Faderl, Stefan, 1
Galindo Delgado, Patricia, 184
Di Landro, J., 175
Fantl, D., 147, 171
Gallagher, Robert, 107
Di Matteo, C., 162, 168
Felice, M., 161
Gallego, M., 161
Di Paolo, D., 162
Fernández de Sevilla, Alberto, 79
Gallinger, M., 161
Di Tullio Budassi, L., 160
Fernandez Sasso, D., 112
Galzerano, Julia, 152
Dias, D.F., 170
Fernández Torres, J., 164
Gao-Sheng, H., 131
Diaz, A., 163, 175, 176
Fernández, Carlos, 120, 121
Garate, G., 138
Diaz, G., 161
Fernandez, G., 148
Garay, G., 138, 157, 179
Díaz, L., 134, 163, 172, 175, 176, 180,
Fernández, I, 85, 146, 128, 132, 166
Garbiero, S., 161, 162
Fernandez, J., 138, 157, 179, 183, 196
Garcés-Eisele, J., 136, 139
Díaz, Lilián, 152
Fernandez, V., 162
García Calvo, J., 198
Dighiero, Guillermo, 42
Ferreira, E., 167
García Marcos, M.A., 183, 197
Dilorenzi, N., 182
Ferreyros, G., 135, 159
Garcia Reinoso, F., 110, 124
DiPersio, John F., 89
Figueiredo, V.L.P., 128
García, H., 130, 133
Donato, Hugo C., 194
Figueroa, Gastón, 35
Garcia, J., 110, 124, 185
Doolittle, G.C., 194
Figueroa, M., 112
Garcia, M., 142
Drago, A., 196
Finizio, O., 165, 174
García-Cosío, Mónica, 79
Draper, R., 177, 187
Fishman, L., 160
García-Escobar, I., 148
Duarte, P., 179
Flores, G., 160, 171
García-Herrera, H., 186
Dueñas, M., 197
Flores, Juan, 120, 121
García-Laraña, José, 79
Dunlop, A.S., 171
Flores-Aguilar, Z.X., 140, 141
García-Sanz, R., 183, 198
Dupont, J., 138, 156, 157, 179, 191
Flores-Peredo, L., 165
Gargallo, P, 125
Duque, J., 191
Foncuberta, M., 156
Geffner, L., 178
Dyer, R., 135, 159, 160, 193
Foresto, P., 160
Georgescu, D., 124
Fradera, J., 166
Gianarelli, S., 179
Echeverría, O., 196
Fratazzi, C., 196
Giere, I., 166, 172
Eid, K.A., 167, 175
Fronzuti, A., 178
Giere, I.A., 110, 124
Elena, G., 152, 195
Fundia, A., 171
Giordano, H., 162, 166, 168
Elter, Thomas, 2
Funke, V.A., 126
Giralt, S., 140, 185
Encinas, C., 183
Furque, M., 162
Girtovitis Fotios, F.I., 149, 150, 195
191, 193
Godoy, W., 188, 190
Guy-Garay, E., 185
Jaime-Perez, J.C., 133, 140
Goldman, W., 152, 195
Guzman-Garcia, M.O., 142
Jait, C., 185
Jaldin-Fincati, J., 159
Goldstein, S., 171
Gologan, R., 124
Hale, Gregory A., 21
Jarque, I., 144
Gómez-Almaguer, D., 74, 97, 133, 139,
Halperin, N., 159
Johnsen, H., 169
Hamerschlack, N., 167
Jootar, S., 141
Gómez-Moreno, H., 155
Hamerschlak, Nelson, 188
Juan-hong, W., 131
Gonzalez de Castro, D., 171
Hamid, Maria., 188
Juni, M, 85, 128, 137, 146, 169
Gonzalez Pedroza, Lourdes, 184
Hassan, Fathelrahman., 188
González, D., 196
Hawk, K., 153
Kang, H.C., 144
Gonzalez, David, 122
Herena-Perez, Suzel., 168
Kang, H.J., 139
Gonzalez, F., 180
Hernández, C., 133, 141, 152, 156, 176,
Kapelushnik, Joseph, 132
140, 168,
Gonzalez, G., 148
177, 179, 182, 189, 192
Karpovitch, X., 133
González, M., 183, 198
Hernández, J.M., 183, 198
Kato, Shunichi , 100
González-Carrillo, M.L., 136, 168
Hernández-Campo, Pilar, 55
Kimura, E.Y.S., 128
Gonzalez-LLano, O., 133, 140, 168
Herrera-Garza, J.L., 133
Kitpoka, P., 141
Gonzalez-Pedroza, L, 184
Holt, Matthew , 89
Klausen, T., 169
Gordillo, F., 178
Hongeng, S., 141
Knudsen, L.., 169
Gordon-Smith, E.C., 101
Hoorfar, H., 189, 190, 197
Kook, H., 139
Graciani, I., 183
Huamani, J, 154
Körbling, Martin, 25
Grand, B., 138
Huamani-Z, J., 155
Korin, Jorge David, 5
Grandtnerová, B., 197
Hurtado de Mendoza, F., 127, 154, 155
Kostina, T.A., 181
Gregianin, M., 158
Hwang, T.J., 139
Kovaleva, L., 130
Kowalysin, R., 162
Gruber, Astrid., 169
Gualco, G., 128, 156
Ibarburu, S., 182
Koziner, B., 112
Guerra, T., 183, 196
Ichihara, E., 126
Koziner, Benjamín, 18
Guggiari, Paula Amante, 11
Ignacio-Ibarra, G., 165, 191
Krishnamoorthy, R., 142
Guiarte, M., 182
Infante, D., 172
Kubisz, P., 197
Guillermo, C., 163, 175, 176, 188
Intile, D., 137, 169
Kurchan, A., 162
Guirão, F.P., 128
Iommi, P., 159, 161, 162
Kvalheim, G., 164
Gunnar Kvalheim, 112, 117
Iriondo, N., 177, 187
Gutierrez, C., 140
Iris, A., 196
Labanca, L., 110
Gutierrez, Cesar Homero, 74
Isaurralde, H., 134, 163, 175, 176
Laca, L., 197
Gutiérrez, N., 183
Israel, Erena, 132
Lanari, E., 185
Landoni, A.I., 162, 186
Gutiérrez, O., 197
Gutierrez-Aguirre, C.H., 133, 168
Jae-Hoo, Park, 153
Larripa, I., 109, 125, 171
Larrosa, V., 182
Makri Lida, L.P., 150, 195
Mejía, M.D., 130, 133, 186
Lastiri, F., 127, 185
Makris Michael, M.P., 148, 150, 195
Mejía, O., 154
Lavagna, G., 163, 175, 176
Makris Pantelis, P.E., 148, 149, 150, 195
Melesi, S., 172, 191, 192, 193
Lavergne, M., 152, 195
Makris Sofia, S.P., 148
Mello, M.R.B., 167, 170
LeBourveau, P., 194
Málaga, J, 127, 154
Melo, F.C.B.C., 167
Lee, H., 182
Maldonado, Norman, 84
Melo, R.A.M., 167
Lee, J.H., 144
Malyszko, J., 143
Menárguez, Javier, 79
Lee, K.S., 144
Malyszko, J.S., 143
Mendez, M., 192, 193
Lee, S.H., 139
Mancini, M.M., 137
Mendoza, I., 196
Lenhoff, S., 169
Mandrile, L., 148
Merli, F., 158
Lens, D., 137, 166
Manera, G., 162
Mesa, Ruben A., 50, 60
Levi, Etai, 132
Manrique, G., 125, 166, 168, 172
Mettivier, V., 165, 174
Lima, C.S.P., 125
Mansilla, Mariela, 152
Metze, K., 125, 167, 176
Lo Coco, Francesco, 41
Manzano-Carlos, A., 168
Mezzano, R., 188, 189
Lodi, F.M., 167
Marín López, A., 164
Michael, J. Mauro, 44
Lombardi, M.V., 110, 124, 166
Mariño, A., 172, 191, 192, 193
Mijares, Mercedes, 14
López Berges, Maria Consuelo, 120
Marques Jr, J., 176
Milani, Angela Cristina, 138
López, Antonio, 120, 121, 151
Marsh, J.C., 101
Millán Rocha, M., 164
Lopez, O., 112
Martín Marcos, J.S., 183
Miller, A., 182
Lopez-Enriquez, A., 166
Martin, C., 136
Milone, G., 127, 156, 185
Lorand-Metze, I., 125, 126, 157, 167,
Martínez, C., 133, 141, 156, 176, 177,
Milone, J., 110, 124, 127, 156
170, 175, 176
179, 189, 192
Minarik, J., 170
Lordmendez J, D., 194
Martínez, C.A., 152
Minutti, M., 172
Loriya, S.S., 168
Martinez, L., 110, 124, 125, 166, 178,
Miranda, E., 126, 157, 175
LoRusso, P., 157
191
Miranda, N, 147
Lucero, G., 112
Martinez, N., 151
Moctezuma, A., 142
Lugo, Y., 191
Martínez-Murillo, C., 140, 141
Moidosky, M., 160
Luongo, A., 187, 191
Matiocevich, 161
Moiraghi, B., 110, 124
Lusis, M.K.P., 144
Matteo, C., 166
Mojtabavi Naini, M., 189, 190, 197
Matutes, Estella, 122
Monreal, M., 128
Machado, C.G.F., 167
Mayer, B., 130
Montalbán, Carlos, 79
Magalhães, K.G., 167
Mechoso, B., 173
Montante, A, 154
Magariños, A., 110, 124, 152
Medina M, M.L., 194
Montesinos, P., 163
Maiolino, A., 157, 167
Medina, Aurora, 24
Moraes, A.J.G., 157
Maiorano, M., 159, 161
Medina, J., 156
Morales, D., 127, 154
Makarian, F., 189, 190, 197
Medina, M.A., 178, 179
Morell, M., 152, 195
Moreno, G.S., 178
Novoa, E., 24, 177
Pathare, A.V., 142
Moreno, M.J., 179
Novoa, J.E., 126, 178, 179, 187, 188,
Paulino, G., 135
Moreno-Galván, M., 178
190, 191
Pavlov, A.D., 181
Morente, Manuel M., 79
Novoa, V., 160
Pavlove, M., 160
Morgan, Gareth, 122
Nucci, M., 157, 167
Pavlovsky, A, 85, 146
Morgan, L.R., 157
Nucifora, E., 171
Pavlovsky, C., 110, 124, 128
Morilla, Alison, 122
Nuñez, H., 178
Pavlovsky, M., 85, 110, 128, 137, 146,
Morilla, R., 122, 171
Nuñez, N., 160
169
Pavlovsky, S., 79, 85, 124, 128, 132,
Moro, M.J., 148, 197
137, 146, 166, 169, 172,
Morshchakova, E.F., 181
Ochoa Robledo, A., 164
Mountford, P., 85, 146, 169
Olazabal, E., 190
Pavlvovsky, M., 85, 146
Muciño-Hernández, G., 165
Oliveira, G.B., 167, 170, 175
Pavon, V., 183
Munro, 191
Olivera, P., 182
Pawlak, K., 143
Muñío, J., 133, 141, 152, 156, 176, 177,
Olivet, C., 178
Pebet, M., 187
Orfao, Alberto, 55, 120, 121
Pedrazzini, E., 128
Mur, N., 110, 162
Orihuela, S., 180
Perdomo, A., 163, 175, 176
Musso, Arturo Mario, 194
Ortega, F., 198
Perdomo, S., 163, 175, 176
Musto, M., 128, 156
Ortega, V., 128, 156
Pereira, A., 182
Muxi, P., 137, 175
Ortiz Calderón, P., 164
Pereira, F.G., 167, 170
Myslivecek, M., 170
Ortiz, G., 163
Peretz, F., 160
179, 189, 192
Pérez, D., 133, 141, 152, 156, 176, 177,
Mysliwiec, M., 143
Paciello, M.L., 136, 144
179, 189, 192
Pérez, G., 133, 141, 147, 152, 156, 176,
Nakaschian, P., 132
Paganini, Juan José, 22, 178
Narbaitz, M., 118, 128
Paganini, R., 178
Nasouhi Pur, S., 134
Pagnano, K.B.B., 126, 157
Pérez, José, 120
Navarro-Vázquez, M., 136
Pagnotta, P.E., 137
Pérez, L., 196
Negri Aranguren, P., 171
Pajuelo, J.C., 136
Pérez, V, 125
Negri, P., 110, 124, 156
Pakakasama, S., 141
Pezzullo, L., 165, 174
Nervi, Bruno , 89
Palmer, L., 76, 127
Piedra, J., 133
Nese, M., 28, 134, 147, 163, 172, 175,
Panero, J., 128
Piedra, P., 196
Panuncio, A., 192
Pier, D, 141
Nieto, V., 182
Papamichos Spyros, S.I., 195
Pierri, S., 137, 175
Nin, M., 180
Pardo, L., 128
Pilnik, N., 185
Noguera, Nélida Inés, 138
Paredes-Aguilera, Rogelio, 184
Pimentel, P, 154
Noriega, M.F., 128
Pasquini, R., 57, 167
Pintos, S., 110
Novis, Y., 167
Patel, A., 178
Pissano, S., 189
176, 180, 191, 192, 193
177, 179, 189, 192
Pithara Eleftheria, E.T., 150
Reis, A., 176
Ruiz-Argüelles, A., 136, 140, 142
Piwnica-Worms, David, 89
Remes, Kari., 169
Ruiz-Argüelles, G.J., 74, 97, 136, 139,
Pizarro, R., 135, 159, 160, 193
Rettig, Michael P., 89
Pizzolato, M, 85, 146
Reyes, G., 191
Ruiz-Reyes, G., 142
Pombo, P., 159, 161, 162
Reyes-Maldonado, E., 140
Ruíz-S, E., 165
Pons, E., 190
Reyes-Núñez, V., 136, 139
Ruiz-Sáez, Arlette, 104
Pontes, E.R., 167
Ribeiro, A.A.F., 167
Rumyantsev, A.G., 168
Portero, J.A., 198
Ribeiro, E., 125
Portillo, F., 179
Riera, L., 156, 157
Saad, S.T.O., 125
Portugal, K., 127
Rigacci, L., 158
Sabín, Pilar, 79
Poth, J., 182
Ríos, O., 192
Saccone, D., 178
Prates, V., 185
Riquelme, B., 160
Sackmann, F., 85, 132, 137, 146, 169
Pregno, P., 158
Ritchey, Julie K., 89
Saito, Hidehiko, 100
Prego, Alfredo, 13
Riva, L., 154, 155
Saleh, M., 130
Prior, Julie L., 89
Rivas, S, 154
San Miguel J., 120, 183, 198
Pritsch, O., 186
Rivas-Vera, S., 87, 192
Sancetta, R., 158
Provencio, Mariano, 79
Riveros, D., 138, 156, 157, 179
Sanchez Avalos, Julio C., 47
Rocco, S., 165, 174
Sánchez-Aguilera, Abel, 79
Qin-xian, B., 131
Rodriguez Grecco, I., 188, 189
Sánchez-Verde, Lydia, 79
Quadrelli, A., 173
Rodriguez, A., 127, 185, 191, 192, 193
Sandes, A.F., 128
Quevedo, E., 188, 190
Rodriguez, A.M., 172
San-Miguel, Jesús F., 40
Quijano, Sandra, 120, 121
Rodríguez, P., 191
Santana, J., 149, 151
Quinta, S., 193
Rodríguez, Y., 176, 177
Santarelli, R., 148
Quiñones, P., 127, 154, 155
Rojas O, E., 194
Santos, I.M.A.A., 144
Quiroz, A., 196
Rojas, A., 130, 186
Santucci, S.M.A., 137
Rojo, A.L., 126, 187, 191
Sanz Guilermo, G.F., 163
Raffo, C., 188
Romagosa, Vicens, 79
Sanz, G.F., 144
Ramirez, Pablo , 89, 178
Romero-Guzmán, Lina T., 184
Sanz, J., 163
Ramos, A., 196
Rong, L., 131
Sanz, M.A., 95, 136, 144, 163
Rams, L., 143
Roselli, M., 173
Sapia, S., 128
Raña, P., 162
Rosen, L., 157
Saralegui, P., 172, 191, 192, 193
Rasillo, Ana, 121
Rossi, J., 161
Sarti, E., 178
Rasmussen, A.M., 164
Rubio, P., 161
Sarto, Adriana, 7
Ravera, J., 179
Rudoy, S., 127
Savio, E., 156
Raviola, Mariana Paula, 138
Ruiz, M.A., 157, 167
Schiavo, L., 192
Redondo, C., 148
Ruiz, R., 130, 186
Scudla, V., 170
142, 168
Semochkin, S.V., 168
Taborda, M., 162
Venica, A., 171
Senent, M.L., 136
Tacchi, C., 171
Vera, A., 183
Sequeira, N., 178
Talavera, J., 191
Vera, L, 154
Shapiro, R., 182
Tarin-Arzaga, L., 74, 140
Vero, M. J., 193
Shin, H.J., 139
Te Kronnie, G., 134
Via, G., 110
Shubinaki, Giora, 132
Teper, S., 179
Vidal, J., 135, 159, 160, 193
Shütz, N., 132
Tiscornia, A., 163, 186
Vidriales, B., 120, 183
Sierra, J., 133
Toledo, R., 163
Vidurrizaga, M., 154
Silenzi, N., 162
Topolansky, L, 146, 163, 175, 176
Vigorito, A.C., 126, 157, 167, 175
Silva, M.C.A., 128
Torletti, F., 159
Villela, L., 130, 133, 186
Silveira, R.A., 126
Torres, H., 161
Vitolo, U., 158
Silveira, S., 182
Torres, W., 189
Vose, Julie, 17
Simões, B.P., 157
Touriño, C., 172
Simonet, S., 172
Trías, N., 137
Wen-qing, W., 131
Sirachainan, N., 141
Tripp, F., 191
Wiernik, Peter H., 91
Wilson, R, 148
Siufi, G.C., 128
Slavutsky, I., 128, 147
Sobrevilla-Calvo, P., 87, 192
Solimano, J., 179
Soo-Jin, Shin, 153
Ungkanont, A., 141
Uriarte, M.R., 125, 166, 168, 172
Xie-qun, C., 131
Uriarte, R., 110, 124
Uturubey, F., 186
Yamamoto, M., 128
Sosa, A., 179
Vaglio, A., 173
Yong-qing, Z., 131
Spaulding, A., 194
Valdés-Gómez, J.J., 127
Yoo, K.H., 139
Stanganelli, C., 147
Valdivieso, N., 135, 159
Young, Joo Min, 153
Stasko, J., 197
Valverde, J., 160
Steffano, B., 188
Varela, A., 182
Zarate, G., 148
Stemmelin, G., 185
Vassallo, R., 135
Zemanova, M., 170
Stevenazzi, M., 147, 163, 175, 176
Vazquez, I., 142
Zhe, W., 131
Stoll, M., 126
Vazquez, L., 196
Zimerman, J., 147
Stone, N., 130
Vazquez, M.L., 171
Zubillaga, M.N., 125, 166, 168, 172
Struck, R.F., 157
Veber, S., 152, 195
Zuffa, Z.E., 137
Sung, K.H., 139
Velez-Garcia, E., 166
Zulli, R., 167
Swebel, P., 193
Venchi, R., 162
Zunino, J., 163, 175, 176
S01
SCHERING SATELLITE SYMPOSIUM
Chronic Lymphoid Malignancies (CLM): The Next Chapter
Towards New Standards in the Treatment
of CLL
Stefan Faderl
The perception of CLL and the approach to therapy of patients
with CLL has undergone considerable change. Whereas CLL was
once considered a monotonous disease of predominantly older patients with a rather indolent disease course and without prospect for
cure, it is now increasingly appreciated that CLL can be quite the
opposite: a disease with enormous clinical diversity whose spectrum of clinical manifestations may range from indolent to rapidly
progressive. In addition, new therapies are emerging that are more
active than the traditional single agent chemotherapy approaches.
The combined impact of advances in the areas of pathobiology and
development of new therapies is leading to new concepts and strategies for patients with CLL.
The traditional approach to treatment of CLL based on single
agent chemotherapy (e.g. alkylators or nucleoside analogs) is
gradually being abandoned in favor of combination therapies. The
CLL4 trial of the German CLL study group randomized patients
younger than 65 years to fludarabine alone versus fludarabine plus
cyclophosphamide (FC).1 In 328 patients who were assessable
for response, FC produced significantly higher CR (24% versus
7%, p<.001) and overall response (OR) rates (95% versus 83%,
p=.001), longer median progression-free survival (PFS, 48 months
versus 20 months, p=.001), and more favorable median treatmentfree survival (37 months versus 20 months, p=.001). Although the
combination had a higher incidence of myelosuppression, there
was no increase in the number of infections. Similar results in favor
of FC over single agent chemotherapy were reported in randomized
studies from ECOG (E2997) and the UK Leukemia Research Fund
(LRF) CLL4 trials (Table 1).2,3
Table 1. FC versus F in Randomized Multicenter Trials
Study
German CLL Study
Group CLL41
FC
F
ECOG E29972
FC
F
UK LRF CLL43
FC
F
Median age
(yrs)
N
CR
(%)
OR
(%)
RD
58 (42-64)
59 (43-65)
180
182
24
7
95
83
48a
20
125
121
22
6
70
50
41a
18
176
176
38
15
90
77
62%b
31%
However, no improvement of overall survival has been demonstrated with FC.
Monoclonal antibodies (moabs) have revolutionized the therapeutic landscape for patients with lymphoproliferative disorders including CLL. The attraction of moabs is based on selective targeting
of tumor-relevant and more or less specific surface markers, and a
distinct mechanism of action involving elements of human effector
functions such as the complement system and ADCC (antibody-dependent cellular cytotoxicity). Rituximab (anti-CD20) has achieved
OR rates in 58% of untreated and symptomatic CLL patients (including a CR rate of 9%).4 In a recently conducted randomized
study of alemtuzumab versus chlorambucil as front-line therapy for
patients with progressive CLL, an OR and CR rate of 83% and 24%
has been reported for alemtuzumab compared to 55% and 2% for
chlorambucil, respectively (p<.0001).5 Alemtuzumab also had superior PFS, which was particularly pronounced in patients with 17p
abnormalities (10.7 months versus 2.2 months).
Building upon single agent experience of moabs, recent years
have seen an emergence of combinations of moabs with chemotherapy. In vitro data suggesting sensitization of CLL cells by rituximab to the cytotoxic and apoptotic effects of drugs such as fludarabine, and data showing downregulation of complement defense
proteins (CD46, CD55, CD59) by purine nucleoside analogs, led to
the design of chemoimmunotherapy regimens such as fludarabine,
cyclophosphamide, and rituximab (FCR), fludarabine plus rituximab
(FR), or pentostatin instead of fludarabine with cyclophosphamide
plus rituximab (PCR).6-8 The clinical experience of the three regimens is summarized in Table 2.
Table 2. Chemoimmunotherapy Experience in Untreated Patients
N
FR 6 *
FCR 7
PCR 8
Age (yrs)
51 63 (36-86)
224 58 (24-86)
64
NA
Rai ≥ 3 (%) β2M (mg/dL)
39
33
53
4.01
3.8
NA
CR OR
(%) (%)
47 90
70 95
41 91
* concurrent arm; NA, not available
62 (34-86)
NA*
RD, response duration;
a
median PFS in months; b 3-year PFS; * one third < 60 years, one
third > 70 years.
Comparisons to historical controls with fludarabine and fludarabine plus cyclophosphamide from the FR and FCR trial, respectively, have confirmed superior CR rates (70% for FCR compared to
35% for FC; p < .05), progression-free and overall survival in favor
of chemoimmunotherapy.7,9 Several combinations of chemotherapy
with alemtuzumab are now also in clinical trials. Fludarabine plus
alemtuzuamb in 36 patients with relapsed and refractory CLL patients has produced CR rates of 30% and OR rates of 83%.10 FCR
plus alemtuzumab (CFAR) has proved to be manageable and active
in relapsed patients and is currently investigated in symptomatic
untreated CLL patients with unfavorable β2M levels at diagnosis.11
S02
Another important observation that emerged from the chemoimmunotherapy experience has been the high number of molecular
responders as established by negative polymerase chain reaction
(PCR) testing or minimal residual disease (MRD) flow cytometry. In
the FCR trial, almost half of the clinic CR patients had a negative
PCR test and were therefore molecular responders. In a study by
the group of Hillmen et al., 91 patients with previously treated CLL
(about half of whom were refractory to purine analogs) received i.v.
alemtuzumab for a median of 9 weeks’ duration.12 The CR rate following alemtuzumab was 36% with an OR rate of 55%. Half of the
purine analog-refractory patients responded. Molecular responses
(negative for MRD flow cytometry) were achieved in 20% of the
patients. Interestingly, those patients achieving MRD-negative responses survived longer than did those without molecular responses. The therapeutic potential of moabs, especially alemtuzuamb,
is now extending into consolidation and maintenance therapy. In
the only randomized study to date, patients with CLL responding to
initial chemotherapy (either fludarabine or fludarabine plus cyclophosphamide) received either alemtuzumab or were observed.13 Of
11 patients on alemtuzumab, 2 converted to CR and 5 of 6 patients
achieved molecular remissions. On the other hand, 3 patients in the
observation arm progressed and no patient achieved a molecular
response. At a follow up of around 20 months, the alemtuzumab
group showed a significantly longer progression-free survival. However, the trial was fraught with a high toxicity rate in the alemtuzumab group necessitating modifications in the trial design.
Increasing use of moabs in CLL therapy led to the design of
effective treatment regimens with superior response rates and progression-free survival compared to more traditional CLL therapies.
Eradication of MRD and achievement of molecular responses has
become an important clinical trials endpoint and a major area of
current and future research in CLL therapy will center around further
refinement of consolidation and maintenance therapies. Important
questions remain with regard to: i) the validation of novel prognostic markers (cytogenetic-molecular markers, IgVH mutation status,
ZAP-70, CD38) and how to use those to identify patients whose
outcome can be improved by early initiation of therapy; ii) the further identification of molecular markers and how to develop biologydriven therapies as has been the case with alemtuzumab in patients
with 17p/p53 abnormalities; iii) the development of therapies specifically directed at older patients or those for whom chemoimmunotherapy is not considered beneficial; and iv) the ongoing search
for new and effective drugs. The combined efforts in these areas
will hopefully continue to provide hope for patients with a hitherto
considered incurable disease.
REFERENCES
1. Eichhorst BF, Busch R, Hopfinger G, et al. Fludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy
of younger patients with chronic lymphocytic leukemia. Blood
2006; 107: 885.
2. Flinn IW, Kumm E, Grever MR, et al. Fludarabine and cyclophosphamide produces a higher complete response rate and
more durable remissions than fludarabine in patients with previously untreated CLL: Intergroup Trial E2997. Blood 2004; 104:
139a.
3. Catovsky D, Richards S, Hillmen P. Early results from LRF
CLL4: A UK multicenter randomized trial. Blood 2005; 106:
212a.
4. Hainsworth JD, Litchy S, Barton JH, et al. Single-agent rituximab as first-line and maintenance treatment for patients with
chronic lymphocytic leukemia or small lymphocytic lymphoma:
a phase II trial of the Minnie Pearl Cancer Research Network. J
Clin Oncol 2003; 21: 1746.
5. Hillmen P, Skotnicki A, Robak T, et al. Alemtuzumab (CAMPATH®, MABCAMPATH®) has superior progression free survival (PFS) vs chlorambucil as front-line therapy for patients
with progressive B-cell chronic lymphocytic leukemia (BCLL).
Blood 2006; 108: 93a.
6. Byrd JC, Peterson BL, Morrison VA, et al. Randomized phase
2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with
Arch Med Interna 2007; XXIX; Supl 1: March 2007
7.
8.
9.
10.
11.
12.
13.
B-cell chronic lymphocytic leukemia: results from Cancer and
Leukemia Group B9712 (CALGB 9712). Blood 2003; 101: 6.
Keating MJ, O’Brien S, Albitar M, et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide,
and rituximab as initial therapy for chronic lymphocytic leukemia. J Clin Oncol 2005; 23: 4079.
Kay N, Geyer S, Call T, et al. Combination chemoimmunotherapy with pentostatin, cyclophosphamide and rituximab shows
significant clinical activity with low accompanying toxicity in previously untreated B-cell chronic lymphocytic leukemia. Blood
2006; 108: 15a.
Byrd JC, Rai K, Peterson BL, et al. Addition of rituximab to
fludarabine may prolong progression-free survival and overall
survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis
of CALGB 9712 and CALGB 9011. Blood 2005; 105: 49.
Elter T, Borchmann P, Schulz H, et al. Fludarabine in combination with alemtuzumab is effective and feasible in patients with
relapsed or refractory B-cell chronic lymphoycytic leukemia: results of a phase II trial. J Clin Oncol 2005; 23: 7024.
Wierda W, O’Brien S, Faderl S, et al. Combined cyclophosphamide, fludarabine, alemtuzumab, and rituximab (CFAR), an active regimen for heavily treated patients with CLL. Blood 2006;
108: 14a.
Moreton P, Kennedy B, Lucas G, et al. Eradication of minimal
residual disease in B-cell chronic lymphocytic leukemia after
alemtuzumab therapy is associated with prolonged survival. J
Clin Oncol 2005; 23: 2971.
Wendtner CM, Ritgen M, Schweighofer CD, et al. Consolidation
with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission – experience on safety and efficacy
within a randomized multicenter phase III trial of the German
CLL Study Group (GCLLSG). Leukemia 2004; 18: 1093.
Campath®/ Mab - Campath® :
combination therapy and minimal residual
disease management.
Thomas Elter
Assistant Professor
Department for Internal Medicine I, University of Cologne
Cologne, Germany
MRD-NEGATIVITY IMPROVES OVERALL SURVIVAL
The assessment of MRD is becoming increasingly important
in order to monitor the depth of response to therapy. In a study in
which 91 relapsed/refractory CLL patients received alemtuzumab
(MabCampath®), those achieving MRD-negativity (assessed using
4-colour flow cytometry) had a significantly better OS compared
with MRD-positive patients (median not reached at 6.5 years versus 60 months, respectively; P<0.001). Long-term clinical trial data
are eagerly awaited to confirm that MRD-negativity is a true marker
of improved survival. Agents such as alemtuzumab, that are able to
achieve MRD-negativity in a large proportion of patients, are likely
to become a standard part of the treatment algorithm in CLL. With
such agents we are now moving towards much longer remissions
(> 10yrs).
WHICH METHOD OF MRD ASSESSMENT?
A number of techniques are currently available for MRD assessment, with varying sensitivity. If MRD-negativity is to be used
as an endpoint in future clinical trials, the assessment techniques
needs to be standardized to allow meaningful comparisons between trials. Currently, 4-colour flow cytometry is considered to be
the most appropriate method in terms of sensitivity, applicability and
practicality.
S03
FIRST-LINE ALEMTUZUMAB CONSOLIDATION THERAPY
mode of action and is effective in poor-risk fludarabine-refractory
patients, providing a rationale for combining these agents to maximise response. Ongoing clinical studies are assessing other chemoimmunotherapy combinations eg, FC plus alemtuzumab and
CFAR (cyclophosphamide, fludarabine, alemtuzumab, rituximab).
The fludarabine and alemtuzumab (FluCam) combination was
evaluated in a phase II study enrolling 36 relapsed/refractory CLL
patients (mean number of prior chemotherapies 2.6). Patients received alemtuzumab 30 mg (after an initial dose escalation over
3 days) and fludarabine 30 mg/m2 on Days 1−3 for up to 6 cycles,
with restaging after cycles 2 and 4, and 1 month after the end of
treatment to confirm response and tolerability. Cumulative exposure
to both agents was reduced compared with the usual monotherapy
doses. The ORR was 83% (30% CR), and 52% achieved MRD-negative status. For patients achieving a complete remission (n=11),
the median OS has not yet been reached, and the median TTP
was 21.9 months. AEs were mild and occurred mainly in the first 2
cycles, suggesting a promising safety profile for FluCam (Elter T et
al. J Clin Oncol 2005;23:7024–31).
Consolidation therapy with alemtuzumab is a new concept in
the management of CLL, the rationale being to improve responses
to induction chemotherapy, with the aim of achieving MRD-negativity. In the German CLL Study Group CCL4B study, alemtuzumab
consolidation therapy (3 x 30 mg IV for 12 weeks) following fludarabine-based chemotherapy in 11 patients resulted in a significantly
longer progression-free survival (PFS; P=0.036) at a median followup of 24 months compared with no further treatment (Wendtner CM,
et al. Leukemia 2004;18:1093–101). Treatment was stopped early
in this trial owing to a high infection rate, which may have been related to the proximity of alemtuzumab treatment to prior chemotherapy
(median time interval between treatments 67 days), which resulted
into the recommendation for a longer treatment gap.
In a phase II study evaluating 34 patients (<65 years) with
CLL, alemtuzumab consolidation (10 mg subcutaneously three
times weekly for 6 weeks) improved the quality of responses to
fludarabine-based induction therapy. The complete response (CR)
rate improved from 35% after fludarabine induction to 79.4% after
alemtuzumab consolidation, and 19 patients (56%) achieved MRDnegativity. Subsequent peripheral blood stem-cell collection (PBSC)
was successful in 24 (92%) of 26 patients, and 18 patients underwent autologous PBSC transplantation (Montillo et al. J Clin Oncol
2006;15:2337–42)
In a further alemtuzumab consolidation study, patients responding to 4 cycles of fludarabine received alemtuzumab consolidation
(30 mg three times weekly for 6 weeks) either intravenously (iv) or
subcutaneously (sc). Both sc and iv alemtuzumab improved on the
response rates achieved following induction therapy (Table 1; Rai
K et al. Blood 2002;100:Abstract 772; Rai K et al. Blood 2003;102:
Abstract 2506).
Table 1. Intravenous or subcutaneous alemtuzumab consolidation therapy improved the quality of responses to fludarabinebased induction therapy in CLL. CR, complete response; ORR,
overall response rate
iv Campath1
sc Campath2
Induction
4 cycles fludarabine
25 mg/m2 days 1-5
(N=56)
CR: 4%
OR: 56%
(N=24)
CR: 4%
OR: 36%
Consolidation
6 wks Campath
30 mg TIW
(N=36)
CR: 42%
OR: 92%
(N=18)
CR: 22%
OR: 66%
CR: 27%
OR: 70%
CR: 18%
OR: 50%
Overall
CHEMO-IMMUNOTHERAPY IMPROVES RESPONSE IN RELAPSED/REFRACTORY CLL
Alemtuzumab and fludarabine are both effective as singleagents in CLL; in addition, alemtuzumab has a p53-independent
REDEFINING THE CLL GUIDELINES
Following the recent rapid advances in CLL treatment, it is
imperative that guidelines for assessing response to therapy also
evolve. The International Workshop on CLL (iwCLL) has recently
proposed revisions to the NCI-WG guidelines to reflect advances
such as prognostic factors, computed tomography scanning and
MRD assessment. Their key recommendations are shown in Table
2. We have to note that there should be a clear distinction between
recommendations for clinical practice and those for clinical trials.
Although MRD status is becoming a much more important part of
response assessment, it is possibly premature to recommend MRD
assessment in current routine clinical practice. However, MRD is
now considered an essential clinical trial end point to allow meaningful comparison of responses to highly effective new therapies
such as alemtuzumab.
Table 2. Recommended iwCLL inclusions to the updated NCIWG guidelines. CIRS, cumulative illness rating scale; CR, complete response; CT, computed tomography; MRD, minimal residual disease; PR, partial response
Recommendations for guideline revisions: ROUTINE PRACTICE
• Assess of ZAP-70 and CD38 status
• Determine cytogenetic abnormalities: 17p deletion, 11q deletion, 13q deletion
• Assess comorbidity i.e. CIRS, Charlson
• Revise definition of PR
Recommendations for guidelines revisions: CLINICAL TRIALS
• Include MRD-negative status as an end point
• Include bone marrow biopsy for safety assessment and CR
confirmation (not diagnosis)
• Assess IgVH mutation status
• Include HIV and hepatitis B/C virus infections
• Include CT scans
S04
NOVARTIS SYMPOSIUM
Chronic Myelogenous Leukemia (CML)
Imatinib Resistance in CML patients:
Study of Acquired Mutations in the BCR
ABL Kinase Domain
Raquel Bengió
Imatinib induces complete cytogenetic response (CCyR) in
82% of patients with CML in chronic phase. There is a subset of
patients who either failed to achieve or lose hematological/CCyR.
Kinase domain mutations have emerged as a potential cause for
treatment failure. Other mechanisms of resistance have been described.
We have performed a multicentric study in order to investigate
ABL kinase domain mutations, amplifications and quantification of
BCR-ABL transcripts by Q-PCR in Imatinib resistant patients.
Patients and Methods: A total of 96 patients (pts) with CML
treated with Imatinib were studied and 84 were evaluable. Forty
eight (57%) were in chronic phase (CP), twenty five (30%) were
in accelerated phase (AP), and eleven (13%) were in blast crisis
(BC).
RNA was extracted from peripheral blood samples and was
subjected to reverse transcriptase (RT-PCR) to obtain cDNA. One
fragment of the 1327 bp of BCR/ABL was amplified in the first cycle.
By a second cycle using specific primers a 579 bp region of ABL
was obtain. This region corresponds to exons 4-7 (ATP binding
pocket and activation loop of the kinase domain)
These PCR products were analyzed by conformation sensitive
gel electrophoresis (CSGE) screening and were sequenced using
automatic system.
The amplification of BCR-ABL rearrangement was studied
in interphase nuclei using Vysis extra-signaling probe. Real time
quantitative PCR (RQ-PCR) of BCR-ABL transcripts was performed
in a subset of 76 patients to assess molecular response, using Light
Cycler (Roche), Syber Green Method.
Results: Nineteen mutations from 84 evaluable patients were
detected (23%) Fourteen were in p- loop: G250E (2), Q252H (2),
E255K (2), L298V (1), V298F (1), E255V (2), L248V (1), T240A (1),
I253H (1), Y253H (1) and five in the Imatinib binding: T315I (1),
F359C (1), M351T (2) y E355G (1). One patient had BCR-ABL
amplifications with 4-6 signals in interphase nucleous. Five patients
had clonal evolution with double Ph chromosome. Most of mutations were found in patients in accelerated phase. The detection
occurred at a median of 59 months (range 2-154) after diagnosis.
Thirty three percent of cases studied with RQ-PCR, had null molecular response (<1Log Reduction). Conclusion: This is the first multicentric study in spanish-speaking South America. P loop mutations
were the most frequently founded. We found 23% of resistant cases
with point mutations, all of them located in the p-loop or imatinib
binding. This is an ongoing study and further recruitment is needed
to confirm these preliminary results. Early detection of mutations
can have prognostic implications and allow therapeutic intervention
such as dose escalation, combination therapy or second generation
tyrosine kinase inhibitors.
S05
GlaxoSmithKline SYMPOSIUM
A New Generation in Antithrombotic
Treatment: Fondaparinux
Established Role of Factor Xa inhibition in
Thrombosis Management
Jorge David Korin (Argentina).
Consultant in Hematology of the Sanatorio Los Arcos. Buenos
Aires.
Associate Profesor of the University of Buenos Aires.
Former President of the Argentine Society of Hematology
Former President of the Argentine Cooperative Group of Hemostasis and Thrombosis
INTRODUCTION:
Fondaparinux is a synthetic sulfated pentasaccharide (PS) of
1728 KD with selective inhibitory action against Factor Xa. The important amount of information generated with its clinical development during the last few years will be reviewed in this session. During my presentation I will concentrate on the breakthrough produced
by this agent to the existing paradigms in antithrombotic therapy as
well as the pharmacological properties of the product and the exciting advantages that this new drug provides for the clinicians.
PARADIGMS IN
FONDAPARINUX:
ANTITHROMBOSIS
QUESTIONED
BY
1) Because of its position at the merge of both pathways of clotting activation, Factor Xa has been seen as the main target for
slowing thrombin generation, since inhibiting 1 molecule of Factor Xa avoids 1000 molecules of thrombin. The clinical translation of this concept is that an exclusive Factor Xa inhibitor will
specially be useful in prophylaxis of venous and arterial thrombosis. Fondaparinux, however, has not only shown remarkable
potency in prevention of venous thromboembolism (VTE) and
arterial thrombosis during percutaneous interventions, but has
also been impressively effective in established DVT and PE as
well as in acute coronary syndromes (ACS).
2) One of the classical drawbacks of treating ACS with partial
thrombin inhibitors like unfractionated heparin (UFH) and lowmolecular weight heparins (LMWH) is the fact that they do not
penetrate in the nascent clot, thereby not completely inhibiting
thrombin bound to fibrin. Fondaparinux does not inhibit preformed thrombin at all but its results were superior to that obtained with UFH or LMWH in this setting. Only future head to
head comparisons with direct thrombin inhibitors will clarify this
issue.
3) Bleeding side effects of an antithrombotic agent were considered during the early 80`s probably related to the Anti-IIa activity. LMWH, with their Anti-Xa / Anti- IIa ratio of activity always
superior to 1, were theorized as potentially safer drugs than
UH –only shown in some meta-analysis-. Fondaparinux studies
have revealed that time window between surgery and post-operative injection is an important issue for clinical relevant bleeding in surgical patients.
4) A linear dose-response curve was considered desirable with
these agents. Fondaparinux exhibited this characteristic in
prophylaxis Phase II studies and not in therapeutics, perhaps
because the initial dose of 2.5 mg provided already a full antithrombotic action in clinical scenarios like ACS
5) Dosage of UFH or LMWH was related to the type of the thrombotic event: VTE prophylaxis < VTE treatment < Arterial and
heart thromboembolic disorders. Fondaparinux recommended
dose for DVT prophylaxis is the same that the one chosen for
treatment of ACS.
6) Nomograms for UFH and LMWH in VTE are tightly related to
aPTT or body weight, making their prescription rather complex.
Fondaparinux, on the contrary, exhibits a remarkable constant
effectiveness with only one dose between 50 and 100 kg
7) UH is the prototype of a drug with multiple properties besides
its role in the clotting cascade, affecting also several steps in
other processes, such as inflammation, fibrinolysis and angiogenesis. Fondaparinux has been shown as virtually devoid of
other actions than inhibiting thrombin generation. Less thrombin formation decreases TAFI activation, which can facilitate endogenous fibrinolysis. Perhaps these new drugs with only one
target will be preferred in the near future.
SUMMARY OF THE PHARMACOLOGICAL PROPERTIES OF
FONDAPARINUX:
Mechanism of action:
Fondaparinux acts as an indirect inhibitor of Factor Xa potentiating 500 times its inhibition by Antithrombin (AT), the main serpin
of the coagulation system. The drug binds with high affinity to the
binding site of AT for PS of natural GAGs, producing an irreversible
change of conformation of the serpin. As a result , an arginine group
is exposed that binds Factor Xa. The complex AT-Factor Xa then
losses its affinity for the drug, which is released to potentiate other
molecules of AT.
Pharmacokinetics:
Bioavailability 100%
Post Subcutaneous injection, the maximal peak occurs at 1.7 h
The distribution volume is 7-11 l.
C max and AUC increase 30% after several doses, and remain
stable after the 3rd day
• The drug has no liver metabolism, which implies no interactions
at the level of cytochromes or microsomes and the possibility of
its use in liver failure (taking into account the bleeding diathesis
of this condition)
• It has a renal clearance of 70% with a progressive prolongation
of the half life in case of aging or renal disease: The half life is
17 h in young adults; 21 h in the elderly; 29 h in moderate renal
failure and 72 h in severe renal failure
• There is no correlation between classic tests of coagulation like
Prothrombin Time, aPTT, Thrombin Time, Anti-Xa levels designed
for LMWH or UFH, and the clinical effects of Fondaparinux, making laboratory monitoring unnecessary
• No drug interactions have been found
• Recombinant Factor VIIa has been shown as an effective antidote
•
•
•
•
S06
REMARKS OF SOME
FONDAPARINUX:
RELEVANT
PROPERTIES
OF
a. It is synthetic, so full supply is guaranteed and contamination
with virus or prions, prevented.
b. The product is absolutely homogeneous, without batch to batch
differences.
c. Activity is expressed in mg (not in International Units) which is
easier to understand for patients and nurses.
d. Because of its long half life, only 1 subcutaneous injection per
day is required, eliminating the need for IV lines and repeated
punctures.
e. No cross-reaction with Heparin-PF4 antibodies and no platelet
aggregation induction have been demonstrated. Fondaparinux
has effectively been used in HIT patients in off-label reports.
Bauer KA., Eriksson BI., Lassen MR., Turpie AG Steering Committee of the Pentasaccharide in major knee surgery study. Fondaparinux compared with enoxaparin for
the prevention of venous thromboembolism after elective
major knee surgery. N Engl J Med 2001;345:1305-1310.
Turpie AG., Bauer KA., Eriksson BI., Lassen MR. Fondaparinux
versus enoxaparin for prevention of venous thromboembolism in
major orthopaedic surgery. A meta-analysis of 4 randomized, double blind studies. Arch Intern Med 2002;162:1833-1840.
Agnelli G. Bergqvist D, Cohen AT, Gallus AS, on behalf of the
PEGASUS investigators. A randomized double-blind study to compare the efficacy and safety of fondaparinux with dalteparin in the
prevention of venous thromboembolism after high-risk abdominal
surgery: the Pegasus Study. Br J Surgery 2005; 92: 1212-1220
REFERENCES:
Boneu B., Necciari J., Cariou R et al. Pharmacokinetics and tolerance of the natural pentasaccharide (SR90107/ORG31540)
with high affinity to antithrombin III in man. Thromb Haemost
1995;74:1468-1473.
Cohen AT, Davidson BL, Gallus AS, Lassen MR and ARTEMIS Investigators.
Efficacy and safety of fondaparinux for the prevention of venous
thromboembolism in older acute medical patients: randomised placebo controlled trial. BMJ 2006; 332 (7537): 325-329
Eriksson BI., Bauer KA., Lassen MR., Turpie AG. Steering Committee of the Pentasaccharide in hip fracture. Fondaparinux compared with enoxaparin for the prevention of venous thromboembolism after hip-fracture surgery. N Engl J Med 2001;345:1298-1304.
Buller HR., Davidson BL., Decousus H et al. Fondaparinux or
enoxaparin for the initial treatment of symptomatic deep venous
thrombosis: a randomized trial. Ann Intern Med 2004;140:867-873.
Lassen MR., Bauer KA., Eriksson BI., Turpie AG European Pentasaccharide elective surgery study (EPHESUS) Steering Committee. Postoperative fondaparinux versus preoperative enoxaparin for prevention of venous thromboembolism in elective hip-replacement surgery:
a randomised double-blind comparison. Lancet 2002;359:1715-1720.
Turpie AG., Bauer KA., Eriksson BI., Lassen MR PENTATHLON 2000 Steering Committee. Postoperative fondaparinux
versus postoperative enoxaparin for prevention of venous
thromboembolism after elective hip-replacement surgery: a
randomised double-blind trial. Lancet 2002;359:1721-1726.
The MATISSE Investigators. Subcutaneous fondaparinux versus
intravenous unfractionated heparin in the initial treatment of pulmonary embolism. N Engl J Med 2003;349:1695-1702.
The Fifth Organtization to Assess Strategies in Acute Ischemic Syndromes Investigators. Comparison of Fondaparinux and Enoxaparin
in Acute Coronary Syndromes. N Engl J Med; 354: 1464-1476
The OASIS-6 Trial Group. Effects of Fondaparinux on Mortality and
Reinfarction in patients with Acute ST-Segment Elevation Myocardial Infarction. The OASIS-6 Randomized Trial. JAMA 2006; 295:
1519-1530
Table 1 shows the medical-surgical scenarios where Fondaparinux has been investigated and briefly summarizes the results
obtained:
Study
Year
Scenario
Results (Risk Reduction)
Comparator
EPHESUS
PENTHATLON
PENTHIFRA
PENTAMAKS
PENTIHIFRA-PLUS
2001-2002
Prophylaxis of VTE in orthopedic
surgery
50% RR
Enoxaparin
96% RR
Placebo
PEGASUS
2005
Prophylaxis of VTE in high risk abdominal surgery
25% RR
Dalteparin
ARTEMIS
2006
Prophylaxis of VTE in high risk medical patients
50% RR
Placebo
MATISSE-DVT
2004
Treatment of DVT
Equivalence
Enoxaparin
MATISSE-PE
2003
Treatment of PE
Equivalence
UFH
OASIS-5
2006
Treatment of ACS
47% RR in major bleeding
17% RR in death
Enoxaparin
OASIS-6
2006
Treatment of STEMI
12% RR in death
19% RR in reinfarction
UFH
S07
SANOFI AVENTIS SYMPOSIUM
Venous Thromboembolic Disease(VTED)
Risk factors
Adriana Sarto (Argentina)
INTRODUCTION
Venous thromboembolic disease (VTD) continues to be a challenge for modern medicine due to its high mortality, and frequency
of sequels, and the high prevalence of predisposing risk factors in
both the general population as well as in patients seen for several
specific conditions1.
The VTD sequels are frequent, with a mortality estimated at
15% for each pulmonary thromboembolism (PTE) episode2. The
postthrombotic syndrome appears in 17–50% of patients who suffer
deep venous thrombosis (DVT), and chronic pulmonary hypertension occurs in 0.1–0.5% of PTE.
In 1884, Rudolph Virchow was the first one who proposed that
thrombosis was the result of at least 1 of 3 underlying ethiologic
factors: vascular endothelial damage, stasis of blood flow, and hypercoagulability of blood.
VTD, including deep vein thrombosis (DVT) and pulmonary
embolism (PE) is a complex disease which results from multiple
interactions between inherited and acquired risk factors. Those
generally defined as being persistent or transient factors and increased incidence of disease are considered to be causative. It is
important to recognize that the predictive values of these factors are
not equal. In assessing whether prophylaxis is indicated, physicians
should consider both the strength of individual risk factors and the
cumulative weight of all risk factors
In the last century, recognition that all DVT risk factors reflect
these underlying pathophysiologic processes and that VTE does not
usually develop in their absence. In a review of 1231 consecutive
patients treated for VTE, 96% had at least one recognized risk factor3. Furthermore, there is a convincing evidence that risk increases
in proportion to the number of predisposing factors4,5.
RISK FACTORS
1. Surgery:
Major General Surgery: The risk of VTE after major general
surgery has been extensively documented. Most investigators apply this term to patients who undergo abdominal or thoracic operations that require general anesthesia lasting >30 minutes6,7. In the
absence of prophylaxis, the risk of DVT is lower following spinal/epidural anesthesia than after general anesthesia8.
In patients undergoing general surgery without prophylaxis,
the rates of DVT and fatal PE range from 15% to 30% and from
0.2% to 0.9%, respectively9-12. Additional risk factors for thrombosis
in general surgery patients include cancer as the reason for surgery,
duration of procedure, previous VTE, advanced age, obesity, varicose veins, and estrogen use13.
Vascular surgery: Patients undergoing vascular surgery have
a high risk for VTE14. In a population-based study14 of 1.6 million patients, the incidence of symptomatic VTE within 3 months of major
vascular surgery was 1.7 to 2.8%. Potential thromboembolic risk
factors in vascular surgery include advanced age, limb ischemia,
long duration of surgery, and intraoperative local trauma, including
possible venous injury15. Preliminary evidence16,17 suggests that
atherosclerosis also may be an independent risk factor for VTE.
Gynecologic surgery: VTE is an important and potentially
preventable complication of major gynecologic surgery, with rates
of DVT, PE, and fatal PE comparable to those seen after general
surgical procedures18-20. Several factors appear to increase the risk
of VTE following gynecologic surgery, including malignancy, older
age, previous VTE, prior pelvic radiation therapy, and use of an abdominal surgical approach (in contrast to vaginal resection)14,21,22.
Gynecologic oncology patients are often elder, and they all have
cancer, with or without compression of the major pelvic veins by a
mass 21.
Urologic surgery: Venous thromboembolism is a common complication of major urologic surgery23 and VTE is considered to be the
most important nonsurgical complication following major urologic
procedures24-26. Between 1% and 5% of patients undergoing such
procedures experience clinically over VTE. Pulmonary embolism
remains being the most common cause of postoperative death in
these patients, and fatal PE has been estimated to occur in 1 of 500
patients27,28. Advanced age, malignancy, intraoperative lithotomy
position, and pelvic surgery with or without lymph node dissection
are established risk factors for VTE in patients undergoing urologic
surgery1.
Laparoscopic surgery: There is a considerable controversy
related to thromboembolic complications after these procedures29.
Bergqvist and Lowe concluded, in a recent review, that laparoscopic
cholecystectomy is a low-risk procedure such that routine VTE prophylaxis is probably not justified30.
Orthopedic Surgery: Patients undergoing major orthopedic
surgery, which includes elective hip and knee replacement and surgery for hip fracture, are particularly at high risk for VTE1.
- Elective hip arthroplasty: In patients undergoing elective total hip replacement in absence of any prophylaxis, the incidence
of venography detected DVT ranges from 40% to 60% and that of
clinically over VTE between 2% and 5%,1,31. Approximately 50% of
the venographically-detected DVT is proximal. Fatal PE occurs in 1
of 500 patients undergoing elective hip replacement 32, 33.
- Elective knee arthroplasty: Without prophylaxis, the rate of
venography-detected DVT in patients undergoing total knee replacement is 60%1. In these patients, 25% of venography-detected
DVT is proximal1.
- Knee arthroscopy: One early prospective study of 8,791 knee
arthroscopies, performed by 21 members of the Arthroscopy Association of North America56, reported symptomatic VTE in < 0.15%
of cases, with no fatal PE. In another series of 8,500 arthroscopic
procedures34, clinical DVT was reported in only four patients, with
no fatal PE. More recently, symptomatic, objectively confirmed DVT
was found in only 0.6% of 1,355 patients after diagnostic knee arthroscopy without the use of thromboprophylaxis, and only one patient developed proximal DVT35.
The prospective studies of knee arthroscopy, without thromboprophylaxis, but with routine screening for DVT, showed the rates of
DVT range from 2 to 18%36-44.
- Hip fracture surgery: Patients undergoing surgery for hip fracture have a very high risk of VTE. In the absence of any prophylaxis,
the rates of venography-assessed total and proximal DVT after hip
fracture are 50% and 27%, respectively1. In the 3 months after surgery, the rate of fatal PE ranges from 1.4% to 7.5%.
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Elective Spine Surgery: In patients undergoing elective spine
surgery, rates of clinically overt DVT (3.7%) and of PE (2.2%) have
been reported45. The incidence of venography-detected DVT has
been reported to be 18%46. Advanced age, cervical versus lumbar
surgery, anterior surgical approach, surgery for malignancy, prolonged procedure, and reduced preoperative and postoperative
mobility are risk factors for VTE in these patients1.
Neurosurgery: The rate of clinically overt VTE is 23% within 12
to 15 months after surgery for primary glioma1. Risk factors that increase the risk for VTE in these patients include intracranial surgery
in comparison to spinal surgery, surgery for malignancy, duration of
surgery, lower limb paralysis, and increased age47.
2. Fracture of the Pelvis, Hip, or Long Bones: Patients with
fractures of the pelvis or femur are also at high risk. The increased
risk after cast immobilization of tibial fractures is particularly well
documented, with overall VTE rate of 45%, but only one third of
those being symptomatic48.
3. Spinal Cord Injury: The overall incidence of DVT within
3 months of paralytic spinal cord injury is 38%; the corresponding
frequency of PE is 5%49. The risk appears higher during the first 2
weeks after injury, and fatal PE is rare >3 months after injury50,51.
4. Multiple Trauma: Geerts et al found DVT in 47% of trauma
patients, including proximal DVT in 12%. A low-risk group could not
be identified from risk factor profiles in these patients. Not only was
DVT found in 56% of patients with lower limb orthopedic or pelvic
injury, but 40% of patients in whom the primary site of injury was the
face, chest, or abdomen had DVT as well52.
5. Malignancy and chemotherapy: The frequency of VTE
increases 2- to 3-fold in patients undergoing surgery for malignant
disease compared with those undergoing surgery for nonmalignant
conditions. Because malignancy is commonly associated with other risk factors, the direct effect of malignancy on risk is uncertain
.Advanced cancers are associated with a high incidence of VTE,
especially cancer of breast, lung, brain, pelvis, rectum, pancreas,
and gastrointestinal tract53,54. Administration of chemotherapy and
thalidomide increases risk.55,56.
6. Myocardial Infarction (MI): The VTE risk of patients hospitalized with acute MI is comparable with that of moderate-risk general surgical patients (20% overall and 2% symptomatic) 57.
7. Congestive Heart or Respiratory Failure: Patients with
congestive heart or respiratory failure are also at risk of venous
thromboembolic complications. In MEDENOX trial, 15% of patients
with class III or IV heart failure treated with placebo had a confirmed episode of VTE58. Similarly, in PRINCE trial, 16% of patients
with class III or IV heart failure treated with low-dose subcutaneous
heparin developed VTE 59.
8. Prior VTE: Patients with a previous episode of VTE are at
greatly increased risk for recurrence, particularly when exposed to
high-risk conditions (eg, major surgery, prolonged immobility, or serious illness). In a case–control study, patients with a history of VTE
were 8 times more likely to develop a new episode during a subsequent high-risk period compared with patients without a history of
DVT or PE60.
9. Pregnancy and the Puerperium: A large population study
that retrospectively compared VTD risk of pregnant women (>28
weeks versus <28 weeks or non-pregnant), the authors observed a
greater incidence in the third quarter and in puerperium, with a relative VTD risk as high as 100 times during peripartum (2 days before
and one after delivery)61. The absolute risk (AR) of VTD is very low
in an average pregnancy: 1.23% women-years62. Some factors that
have been associated with an increased risk of VTD in pregnant
women are thrombophilia, bed rest for more than 3 days, previous
VTD, varicose veins in the lower limbs and age greater than 35 62-64.
The AR for pregnancy in thrombophilia (deficiency of antithrombin,
protein C, or protein S) was 4%63.
10. Oral contraceptives (OC): OC users have four to six
times more risk of VTD than women who do not use them, although
the absolute risk is low (between one and two cases per 10,000
women-year), 10% of these events being fatal (10 cases per million
women-year who use OC)65-67. Good quality studies have shown a
greater risk of VTD associated with third generation OCs in comparison to second generation OC, although these differences were
small (1.7 times greater risk)68. Obesity, smoking, and thrombophilia
have been demonstrated to increase VTD risk in OC users.
11. Hormone replacement therapy (HRT): Several studies
show the association of VTD and the use of HRT 102-105, . Overall the
absolute risk is low: 0.2–5.9% women-years. There is a greater risk
in the first 2 years, with disparities in the results for prolonged use,
while some studies found a decreasing risk over time until it probably equals baseline risk 69-72.
12. Selective modulators of estrogen receptors (raloxifene
and tamoxifen)
Tamoxifen users have an increased risk of VTD, although this
was non-significant in three of five studies available. One study
found a higher risk due to an increase of PTE cases in women over
50 years73. In the IBIS-I, the risk seemed to be related to a greater
incidence of secondary VTD events74. The AR varies between 3.6
and 12% women-year 73.
13. Thrombophilias
Antiphospholipid Antibody Síndrome: Thromboembolism rates
of 6% to 8% in otherwise healthy patients with lupus anticoagulant
have been reported74. In a case–control analysis involving participants in the Physicians Health Study, those with anticardiolipin antibody titers above the 95th percentile had a 5.3-fold increased risk
of developing DVT or PE over a 5-year period75. Prior thrombosis, a
lupus anticoagulant, and elevation of the IgG idiotype anticardiolipin
antibodies have all been suggested to increase the risk of thrombosis 76.
Antithrombin Deficiency: In unselected patients with VTE, the
frequency of AT deficiency is 1.1%77, compared with 2.4% (range
0.5–4.9%) in selected patients78,79. In general, patients with inherited AT deficiency are at greater risk for VTE than those with protein
C or protein S deficiency.
Protein C and Protein S Deficiencies: The prevalence of heterozygous protein C or protein S deficiency is low in the general
population, but 5% to 10% among selected patients with VTE80,81.
Defects in this natural anticoagulant system greatly increase the
risk of thrombosis: As many as 50% of heterozygotes up to 50 years
old suffer a thrombotic event80.
APC Resistance: Between 20% and 60% of patients with recurrent VTE display APC resistance on laboratory testing.81,82. In
the majority of cases, this is because of a mutation in the factor V
gene, labeled factor V Leiden. Approximately 4% to 6% of the general population are heterozygous for this trait (which is autosomal
dominant)81,83. Although APC resistance is still associated with a
3- to 7-fold increased risk of venous thrombosis81,83. In addition, the
factor V Leiden mutation can greatly enhance the thrombotic risk
from other factors. In a study of patients >70 years with at least 1
prior episode of venous thrombosis, 11% had the mutatio84. Up to
60% of women who experience VTE during oral contraceptive use
are APC resistant85. Finally, coinheritance of factor V Leiden with
other heritable thrombophilias has been shown to greatly increase
future thrombotic risk86.
Factor II (Prothrombin) G20210A: The prevalence of the prothrombin G20210A mutation is highest in white individuals of European descent, ranging from 1.7% to 3%87. The relative risk for
thrombosis associated with the 20210A allele was 2.8 (95% confidence interval, 1.4–5.6).
Coagulation Factors: Elevated levels of several coagulation
factors, including factors VIII, IX, and XI, have been linked with increased thrombotic risk88.
Hyperhomocysteinemia: Among 269 patients with first DVT
enrolled in the Leiden Thrombophilia Study, 10% had homocysteine levels above the 95th percentile (adjusted odds ratio for VTE
2.5, compared with healthy matched controls)89, and the same odds
ratio was found in a meta-analysis of 10 case–control studies90.
Patients with elevated homocysteine are also at increased risk for
recurrent VTE.
Aditional risks factors for DVT are: age, obesity, Immobility
and Varicose Veins91101
The risk factors for DVT could be classified in 3 categories: 1.
Strong risk factors (odds ratio >10), 2. Moderate risk factors (odds
ratio 2–9), Weak risk factors (odds ratio <2) (table 1)101.
In considering VTE prophylaxis, the physician should take into
account absolute and relative risks of VTE, potential benefits of
available prophylactic agents, possible complications (including the
risk of bleeding), and expense.
The American College of Chest Physicians published consensus guidelines for prevention of VTE. These guidelines, updated
every 2 to 3 years, have been widely adopted by physicians and
hospitals102.
Table 1. Risk factors for DVT (Anderson F and Spencer F)101.
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81. Pabinger I, Brucker S, Kyrle PA, et al. Hereditary deficiency of
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CHEST 2004; 126:338S–400S)
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Diagnosis of VTED
Paula Amante Guggiari
President of the Paraguayan Hematology and Hemotherapy
Society
Venous thromboembolic disease (VTED) is a major problem,
resulting in significant morbidity and mortality.Deep vein thrombosis (DVT) and pulmonary embolism(PE) are variants of the same
pathologic process.While one study found that almost 40% of patients with DVT did not present symptoms of PE, it is more common in patients with DVT than would be suggest by their clinical
presentation alone.
The diagnosis and treatment of pulmonary embolism demand
an interdisciplinary approach,combining medical,surgical and radiologic specialties.Despite substantial advances,mortality and recurrence rates remain high.
PE is the most common cause of preventable death in hospitalized patients.Of patient manifesting a VTE event (DVT and PE
with or without evidence of DVT), 25% die within the first 7 days
after onset. In addition , more than one third of the deaths from VTE
occur on the date of clinical symptom onset or following a silent
VTE event.
The natural history of DVT usually starts in the calves.The therapeutic implications of CVT are equivocal,but symptomatic calf vein
thrombosis (CVT) deserves the merit of anticoagulant treatment.
Nonextending CVT rarely causes adverse outcomes of clinical significant DVT or pulmonary embolism, wheareas popliteal , femoral ,
and ileofemoral DVT often does.
Venography is the “ gold standard” reference for the exclusion
and diagnosis of proximal DVT and CVT and has, by definition , a
sensitivity and specifity of 100% with a positive and negative predictive value of 100%. Because of its invasive nature and associated
side effects, contrast venography has not become a routine test for
venous thrombosis and has benn replaced by non-invasive test, of
which compression ultrasonography (CUS) is the best.
Patients with suspected deep vein thrombosis (DVT) are subjected to leg vein compression ultrasonography (CUS) wich confirms
DVT in only 20 to 30% of patients. A positive CUS is consistent with
DVT irrespective of clinical scores. The sequential use of a simple
clinical score assessment, a rapid sensitive enzyme-linked immunosorbent assay (ELISA) D-dimer test and CUS to safely exclude
DVT is promising. The clinical score is a validated clinical model of
complaints, signs, and symptoms, on the basis of which a pretest
clinical probability for DVT can be estimated as low, moderate, or
high. The safe exclusion of DVT by a rapid sensitive D-dimer test
in combination with clinical score or CUS necessitates a negative
predictive value of more than 99%. The negative predictive value
for DVT is determined by the sensitivity of the rapid ELISA D-dimer
test and the prevalence of DVT in subgroups of outpatients with
suspected DVT. The prevalence of DVT in outpatients with a low,
moderate, or high clinical score varies widely from 3 to 10%, 15 to
30% or more than 70%, respectively. A negative rapid ELISA D-dimer and a low clinical score (prevalence DVT 3 to 5%) will have a
very high negative predictive value of more than 99.5% to exclude
DVT without the need of CUS testing.In addition, a negative ELISA
D-dimer test and a first-negative CUS safely exclude DVT in patients with a moderate clinical score with a negative predictive value
of more than 99.5%, therefore obviating the need to repeat CUS.
The use of a rapid ELISA D-dimer testing in patients with a high
clinical score is not recommended. A negative CUS, a low clinical
score, and a positive ELISA D-dimer, even less than 1000 ng/ml
exclude DVT with a negative predictive value of more than 99%.
Patients with a negative CUS, but a positive ELISA D-dimer, and a
moderate or high clinical score have a probability of DVT of 3 to 5%
or 20 to 30%, respectively, and are thus candidates for repeated
CUS testing. The proposed sequential use of the clinical score assessment, a rapid ELISA D-dimer test, and CUS will be the most
cost-effective diagnostic strategy for DVT because of a significant
reduction of CUS examinations and time saved for the patient and
physician in charge.
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Clinical Model of Wells et Al30 for Predicting Pretest and Posttest Incidence of Proximal DVT in Outpatients with a First Suspicion
of DVT
The accurate detection of pulmonary embolism remains difficult , and the differential diagnosis is extensive
DIFFERENTIAL DIAGNOSIS OF PULMONARY EMBOLISM.
Pneumonia or bronchitis
Asthma
Exacerbation of chronic obstructive pulmonary disease
Myocardial infarction
Pulmonary edema
Anxiety
Dissection of the aorta
Pericardial tamponade
Lung cancer
Primary pulmonary hypertension
Rib fracture
Pneumothorax
Costochondritis
Pulmonary embolism can accompany as well as mimic other
cardiopulmonary illnesses.
Details should be regarding the patient`s history and a family history of venous thrombosis, as well as coexisting conditions,
environmetal risk factors, and hormonal influences should be taken
into consideration.
Dyspnea is the most frequent symptom of pulmonary embolism, and tachypnea is the most frequent sign. Whereas the presence of dyspnea, syncope, or cyanosis usually indicates a massive
pulmonary embolism,a finding of pleuritic pain, cough or hemoptysis often suggests a small embolism near the pleura.
On physical examination, findings of right ventricular disfunction include bulging neck vein waves, a left parasternal lift , an accentuated pulmonic component of the second heart sound, and a
systolic murmur at the left lower sternal border that increases in
intensity during inspiration.
Electrocardiography and chest radiography should usually be
incorporated into the diagnostic workup.
Specifically for the diagnosis of PE, wich includes all the
causes of negative CUS shows three imagery procedures:The spiral CT, the Ventilation-Perfusion Lung Scanning y pulmonary angiography .
For more than 30 years, ventilation-perfusion lung scanning
has been used as the imaging procedure for the evaluation of patients with suspected PE. A normal perfusion lung scan result excludes the diagnosis of PE. A high-probability lung scan has an 85
to 90% predictive value.
Pulmonary angiography is the” gold standard” test for the diagnosis of PE. This test may be used to identify thrombi in subsegmental pulmonary arterial vessels
The spiral CT emerged as a new non-invasive imaging modality for the research on patients with suspected PE. Spiral CT made
it possible to directly visualize segmental and some subsegmental
arteries using a single bolus of contrast, while passing a patient
through an x-ray beam.
D-dimer is a degradation product of cross-linked fibrin blood
clot. Levels of D-dimer are typically high in patients with acute venous thromboembolism. D-dimer levels may also be higher in a
variety of non-thrombotic disorders and circumstances, including
recent major surgery, hemorrhage, trauma, malignancy , or sepsis.
There , D-dimer assays are sensitive but non-specific markers for
venous thromboembolism. The most sensitive D- dimer test are the
ELISA.
In a direct comparison, Freyburger et al evaluated three conventional ELISA methods:the rapid ELISA VIDAS D-dimer assay
and various other rapid D-dimer test including Instant I.A., SimpliRed, and Nycocard, devoted to thrombosis exclusion against the
gold standard venography. The conventional ELISA methods and
the rapid ELISA had a sensitivity and a negative predictive value of
100% at specifity values between 34% and 52%.
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Direct Comparison of ELISA and Turbidimetric D-Dimer Tests Against Venography
REFERENCES
1. Moser KM,Fedullo PF,Litte John JK,Crawford R. Frequent
asymptomatic pulmonary embolism in patients with deep venous thrombosis.Jama 1994;271:223-5.
2. Goldhaber S.Pulmonary Embolism.Review article.N EngJ Med
1998;339:93-104.
3. Wells P.,et col.Seminars in thrombosis and Hemostasis
2000,26:643-656
4. Agnelli G.Unresolved Issues in the Prevention and treatment
of Venous Thromboembolism.Seminars in thrombosis and Hemostasis 2002,28:33-40.
5. Michiels J,Freyburger G,van der Graf F,Janssen M,Ooortwijn
W,Van Beek E.Strategies for the safe and effective exclusion
and diagnosis of deep vein thrombosis by the sequential use
of clinical score, d-dimer testing, and compression ultrasonography. Seminars in thrombosis and hemostasis 2000,26:657667.
6. Hirsh J,Hoak J.Management of deep vein thrombosis and pulmonary embolism:a statement for healthcare professionals.
Council on thrombosis,American Heart Association.Circulation
1996,93:2212-45
7. Geers WH,Heit JA,Clagett GP,et al.Prevention of venous thromboembolism.Chest 2001:119:132 S-175S.
8. Michota FA.Venous thromboembolism prophylaxis in the medically ill patient.Clin Chest Med 2003(24):93-101.
9. Cham MD,Yankelevitz DF,Shaham D,et al.Deep thrombosis:detection by using indirect DT venography.The Pulmonary Angiography-indirect CT Venography Cooperative Group.radiology
2000;216:744-751.
10. Hirsh J,Lee AY.How we diagnose and treat deep vein thrombosis.Blood 2002:99:3102-10..
Venous Thromboembolic Disease(VTED)
Endovascular procedures in VTED
Alfredo Prego
Chief Vascular and Endovascular Division HCFFA. Montevideo.
Uruguay.
ABSTRACT
Deep vein thrombosis(DVT) of the lower extremity is a significant clinical problem recognized due to the mortality and morbidity associated with its primarily complication the pulmonary
embolism(PE).
Less well recognized are the sequelar of DVT: the post thrombotic syndrome.
Regardless of the occurrence of PE, DVT can negatively impact patient outcomes and increased health care cost.
The goals of therapy are to prevent thrombosis extension and
embolizations restore venous patency and preserve vein value
function.
Anticoagulation is the accepted therapy for patient with VTED.
Endovascular interventions of acute DVT such as catheter directed thrombolysis, mechanical thrombectomy and inferior vena
cava(IVC) filter placement have received increased attention during
the last decade.
Thrombolytic therapy has the ability to produce a rapid and
more complete resolution of DVT reducing the risk of PE, but the
role prevent valve damage and posthrombotic syndrome remains
to be answer.
Because of the bleeding risk of thrombolysis, percutaneous
mechanical thrombectomy has emerged as a complementary tool
in the treatment of iliofemoral acute DVT.
Finally, at the present time we have a wide variety of IVC filters
designed for percutaneous insertion.
The accepted indications for placement was reported in the
Vena Cava Consensus Conference(2003).
With the advent of retrievable IVC filters the indications for PE
prophilaxis in high risk patients increased significantly.
A mayor advance is that retrievable IVC filter offer protections
against pulmonary embolism during the highest risk period, served
as an effective bridge to anticoagulation, and avoid the long term
sequelar of permanent filters.
S14
Venous thromboembolic disease in
systemic autoimmune conditions
Mary-Carmen Amigo
Rheumatologist
ABC Medical Center, Mexico City
Associate Professor, Universidad Nacional Autónoma de México
Clinical Investigador
Member of the Nacional Academy of Medicine, Mexico
Systemic autoimmune diseases share several properties that
sometimes make a specific diagnosis difficult. The diseases in
question are rheumatoid arthritis, systemic lupus erythematosus,
scleroderma, polymyositis, dermatomyositis and Sjögren´s syndrome. Several clinical and serologic features are shared to a variable extent by all of this conditions. In this occasion, we are going
to review venous thromboembolism in two diseases:
a) systemic lupus erythematosus as a classic example of a systemic autoimmune disease, and
b) rheumatoid arthritis as a systemic inflammatory disease with its
primary manifestation in the synovium
SYSTEMIC LUPUS ERYTHEMATOSUS
Systemic lupus erythematosus (SLE) is the prototypic systemic
autoimmune disease. It is a chronic inflammatory autoimmune disease affecting multiple organs. Thrombosis either arterial or venous has been reported in 7.2-12% of persons with SLE. Overall, a
high incidence rate of thrombotic events has been found in patients
with SLE from different populations. In a 10-year prospective cohort
study of European patients with SLE, the most frequent causes
of death were active SLE (26%), thrombosis (26%) and infection
(25%), with thrombosis dominating the second 5-year period of follow-up.
Venous thrombosis (VT) in SLE occurs through 2 major conditions: hypercoagulability and vasculitis. Hypercoagulability associated with the presence of antiphospholipid antibodies is one
of the major factors responsible for thrombosis in patients with
SLE. Antiphospholipid syndrome (APS) is an important predictor
of irreversible organ damage and mortality in patients with SLE.
Increased mortality linked to APS is due to thrombosis. Thus, the
subgroup of patients with definite APS has been identified as high
risk among those with SLE. In their meta-analysis of 29 published
series reporting over 1,000 SLE patients, Love and Santoro found
the prevalence of thromboembolic complication in SLE associated
with aPL to be 28%. However, in SLE, not all patients with aPL
develop VT and not all patients with VT have aPL. The incidence
of thrombophilic defects other than aPL in SLE patients appear to
be increased compared with the healthy population. Risk factors
other than aPL for the occurrence of VT in SLE were identified in a
multiethnic cohort (LUMINA). These factors were smoking, shorter
disease duration, older age, disease activity over time, and the average of glucocorticoids used. In addition, apart from aPL, acquired
or inherited thrombophilic defects have been demonstrated in these
patients. Alterations in protein C or protein S, polymorphisms of
plasma proteins such as factor V Leiden, prothrombin and methyltetrahydrofolate reductase have been reported in SLE patients. Low
levels of free protein S and high levels of C4 binding protein, a regulatory component of the classical pathway of complement, are common findings in SLE. Moreover, antibodies to protein S are frequent
in patients with SLE and their presence is associated with the clinical features of the APS. Hyperhomocysteinemia is a common and
potentially modifiable, independent risk factor for thrombotic events
in patients with SLE. Another risk factor for thrombosis in SLE patients is a defective fibrinolysis. Increased levels of plasminogen
activator inhibitor-1 as well as an increased fibrin polymerization
rate have been found in these patients.
Lupus-associated vasculitis may also increase the risk of
thombosis. Immune complex deposition releases inflammatory mediators that cause endothelial dysfunction, chronic vessel damage
and thrombosis. Anti-DNA antibodies are directly toxic to endothelial cells and, aPL and TNF-alpha might cooperate inducing endothelial perturbation. Other antibodies, such as anti-endothelial cell
antibodies seem as well to be related to disease activity in SLE and
can contribute to pathogenesis by activating endothelial cell-leukocyte interactions. In addition, patients with SLE have high levels
of circulating platelet-derived microparticles that correlate with an
increased potential to generate thrombin which is independent of
disease activity. Young female patients with SLE have high levels
of circulating apoptotic endothelial cells that correlate strongly with
pronounced abnormalities in endothelial function and elevated levels of circulating tissue factor, the major procoagulant in vivo.
It is now evident that there is a dynamic interaction between
coagulation and inflammation. Inflammation induces the expression
of tissue factor, reduces fibrinolytic activity through upregulation of
PAI and, impairs the anticoagulant effect of the protein C pathway
due to downregulation of thrombomodulin and a decrease of protein
S. Interestingly enough, heparin and warfarin have anti-inflammatory effect.
In summary, lupus patients are at significantly increased risk
for thrombosis, which is a multifactorial process. An approach to
thrombosis risk assessment (lupus disease activity/severity, traditional and lupus-related as well as acquired and genetic thrombotic
risk factors, and aPL profile) is important in the initial study of SLE
patients. However, at this moment, there are no evidence-based
recommendations for the primary thrombosis prevention.
RHEUMATOID ARTHRITIS
Rheumatoid arthritis is the quintessential chronic inflammatory
disease. Despite evidence supporting a link between inflammation
and thrombus formation, clinical practice and a thorough review of
the medical literature reveal few communications of RA-associated
venous thrombosis.
Rheumatoid arthritis (RA) is generally thought not to be associated with a predisposition to arterial or venous thrombosis. Raised
aPL antibodies are found in patients with RA with a mean prevalence
of 22%. However, it is the general belief that the majority of patients
have laboratory evidence of aPL without clinical consequences.
Some aspects deserve attention. It is well established that death
resulting from cardiovascular disorders contributes to the increased
mortality in RA. There is some vidence that there may be an association between aCL antibodies and ischaemic heart disease. Other
factors, particularly an altered lipid profile with increased Lp(a), increased homocysteine, or decreased protein S levels could represent partially modifiable risk factors in patients with RA.
On the other hand, some studies suggest that biologic agents
directed against tumor necrosis factor-α can induce anticardiolipin
antibodies (aCL). The absence of a strong relationship between
aCL antibodies and thrombosis in RA could be due to the finding
that aCL activity in RA appears to be independent of anti-B2GP1.
Finally, from a practical point of view, differentiation of a rupture of a Baker´s cyst (common in RA) from a true deep venous
thrombosis, especially in patients who are potentially susceptible
to thrombotic events, is extremely important. Both conditions are
similar enough that the rupture of the popliteal cyst is known as
pseudo-thrombophlebitic syndrome. It is not possible to distinguish
them by clinical examination. Ultrasonography is the test of choice,
avoiding erroneous diagnosis and treatment.
Treatment of Venous Thrombosis Disease
(VTED)
Mercedes Mijares, Venezuela
Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE)
are two clinical manifestations of VTE. DVT can lead to debilitating
postphlebitic syndrome in up to one-third of patients.
Anticoagulant treatment is essential to reduce morbidity and
mortality in patients with acute VTE. Rapid anticoagulation can only
be achieved with parenteral anticoagulants, such as heparin or Low
Molecular-Weight Heparin (LMWH).
The selective factor Xa inhibitor, Fondaparinux, has been recently introduced as an alternative to heparin or LMWH for initial
VTE treatment. Heparin, LMWH, or Fondaparinux should be administered for at least five to seven days. Vitamin K antagonists should
be initiated on the first day, or as soon as possible, in patients who
are candidates for an oral anticoagulant.
OBJECTIVES OF INITIAL TREATMENT OF VTE
The clinical objectives of the initial treatment of VTE are to
prevent fatal PE and recurrent VTE, thereby reducing the risk of
postphlebitic syndrome with an acceptable rate of bleeding complications.
HEPARIN AND VITAMIN K ANTAGONISTS
Heparin or LMWH, in association with vitamin K antagonists,
form the basis of currently recommended treatment for VTE. Provided there are no contraindications to anticoagulant therapy, heparin
or LMWH should be administered to patients with suspected VTE
while waiting for conclusive diagnosis tests. In patients with confirmed VTE, heparin or LMWH should be continued for at least five
days while waiting for the therapeutic effect of concomitant vitamin
K antagonists. Heparin or LMWH treatment should only be stopped
when the international normalized ratio (INR) is >2 for at least two
consecutive days. The therapeutic dose of warfarin varies from
patient to patient reflecting, differences in dietary vitamin K intake,
genetic polymorphisms in the enzymes involved in warfarin metabolism and administration of concomitant medications that suppress
or potentiate the anticoagulant effects of warfarin. Frequent coagulation monitoring is necessary to ensure that a therapeutic anticoagulant response is achieved with warfarin. Patients with unprovoked
VTE require anticoagulation therapy for at least 6 months.
Heparin is usually given as a continuous intravenous infusion,
but can be given subcutaneously. If it is given subcutaneously, higher doses of heparin are often needed to overcome its poor bioavailability after subcutaneous injection. There are studies which suggest that dose-adjusted heparin given subcutaneously (333 U/Kg
followed by twice-daily doses of 250 U/Kg) is as effective and safe
as continuous intravenous heparin.
Heparin produces an unpredictable anticoagulant response,
because non-specific binding to plasma protein happens. Therefore, anticoagulation monitoring is mandatory to ensure that a therapeutic response is achieved. The activated partial thromboplastin
time (aPTT) is the test most often used to monitor heparin to ensure
a therapeutic level of anticoagulation, although anti-factor Xa levels
also can be used.
It is important to achieve adequate anticoagulation early in the
course of VTE treatment, logistic regression analysis show that inadequate anticoagulation in the first 24 hours of treatment, as determined by a subtherapeutic aPTT, was associated with a high rate
of recurrent VTE.
An initial intravenous heparin bolus of 80 U/kg or 5,000 U
followed by a continuous intravenous infusion of 18 U/kg/hour or
30,000 U over 24 hours. The aPTT time should be measured 3 or 6
hours after the bolus and then 3 hours after each dose adjustment
or daily if no adjustment is necessary. Weight-adjusted nomograms
have advantages over fixed-dose nomograms.
The concept that subcutaneous heparin requires laboratory
monitoring has been challenged by a recent randomized open-label
study that showed that fixed weight-adjusted doses of subcutaneous heparin are effective for initial treatment of DVT.
Vitamin K antagonists should be initiated as soon as possible.
If warfarin is used, starting doses of 5 or 7.5 mg are preferred over
higher doses.
LMWH
The introduction of LMWHs simplified the treatment of VTE.
Weight-adjusted LMWH is the treatment of choice. LMWH has better bioavailability after subcutaneous injection than heparin. They
have a longer half-life than heparin and produce a more predictable anticoagulant response. These features allow once- or twicedaily subcutaneous dosing without coagulation monitoring. Consequently, the majority of patients with VTE can now be treated as
outpatients.
S15
Many studies have compared the efficacy and safety of LMWH
with heparin. While early clinical outcome-based trials reported
lower rates of recurrent VTE and bleeding with LMWH than with
heparin, more recent studies have shown comparable outcomes.
Recent meta-analyses of randomized trials comparing LMWH with
heparin for VTE treatment showed trend towards an advantage of
LMWH. Overall, LMWH and heparin appear to have similar efficacy
and safety. However, LMWH is more convenient to administer. An
intriguing finding is a reduction in mortality with LMWH that is confined to patients with cancer.
Several studies have showed the efficacy and safety of LMWH
in PE, with non-significant lower rates of recurrent VTE and mortality. Therefore, it is not clear whether the results can be applied
to patients with extensive PE or patients with massive PE. They
should not be administered LMWH. LMWH should be used in patients without signs of right ventricular overload.
Once- versus twice-daily LMWH for VTE treatment:
A systematic review of five studies showed no difference in
the rate of recurrent VTE in the two treatment regimens. Rates of
major bleeding and mortality were not statistically different between
the two treatment regimens. Assessment of thrombus regression
performed in two of these studies showed no difference between
the once- and twice-daily administrations. A subgroup analysis in
cancer patients receiving one specific LMWH suggested superior
efficacy of the twice-daily regimen.
FONDAPARINUX
Fondaparinux is a synthetic analogue of the pentasaccharide
sequence of heparin that, after binding to antithrombin, catalyzes
factor Xa inhibition. After binding with fondaparinux, antithrombin
undergoes a permanent conformational change that induces a 300fold increase in its affinity for factor Xa. Fondaparinux dissociates
from the complex antithrombin/factor Xa and is free to bind other
molecules of antithrombin while antithrombin remains permanently
bounded to factor Xa. Fondaparinux has 100% bioavailability after
subcutaneous injection. Its half-life, about 17 hours, allows a oncea-day administration for treatment of VTE. Fondaparinux has little or
no effect on routine tests of coagulation. It is excreted unchanged in
the urine, therefore is contraindicated in patients with renal failure.
Efficacy and safety of fondaparinux have been shown in several phase II and phase III clinical trials for the treatment of VTE.
Fondaparinux is approved in Europe and the United States for the
initial treatment of VTE. Fondaparinux was compared with LMWH
for treatment of DVT and with heparin for treatment of PE. Recurrent VTE occurred in 3.9% patients given fondaparinux compared
with 4.1% patients given enoxaparin. Rates of major bleeding were
similar in the two groups. Mortality rates were 3.8% and 3.0% in
patients given fondaparinux and LMWH, respectively.
In the second study recurrent VTE occurred in 3.8% patients
receiving fondaparinux as compared with 5.0% in patients receiving
unfractionated heparin. Major bleeding and mortality were similar in
the two groups. No statistically significant difference in efficacy or
safety was observed with fondaparinux as compared to standard
anticoagulant treatment. No difference was observed regarding
time to achieve a therapeutic INR in patients given fondaparinux
or standard anticoagulant treatment. Fondaparinux is more convenient to administer than heparin. Its only advantage over LMWH, is
the lower risk of heparin induced thrombocytopenia.
OUTPATIENT VTE TREATMENT
Out-of-hospital treatment of VTE is feasible for most patients.
This approach reduces health care costs and improves patient satisfaction. Several studies have compared out-of-hospital LMWH
with in hospital treatment with heparin. Rates of recurrent VTE with
heparin and LMWH were 8.6% and 6.9%, respectively (95% CI 3.6–
6.9), and bleedings were observed in 2.0 and 0.5% of the patients,
respectively. These findings suggest that home-treatment of DVT
with LMWH is feasible, effective and safe.
A number of factors can influence the choice for home or inhospital treatment as co-morbidity, bleeding risk, home care, concomitant symptomatic PE. Hospital admission and early discharge
may be an alternative strategy in medium or high risk patients.
S16
Home treatment seems a promising opportunity also in cancer
patients. There is no consensus regarding the home treatment of
patients with symptomatic PE. It is conceivable that this procedure
should be reserved to selected patients with PE and no signs of
right heart overload.
THROMBOLYTICS
The role of thrombolysis in patients with massive PE is clinically sound while its value in patients with normal blood pressure
and right ventricular dysfunction remain to be defined. Thrombolysis
has a reduced role, if any, in the treatment of DVT and should be
however reserved to individual cases.
NEW ANTICOAGULANT AGENTS
Several new anticoagulant agents for the treatment of VTE
are in different phases of clinical development. They can be distinguished in factor Xa inhibitors and thrombin inhibitors. They are
administered orally once or twice daily, except idraparinux that is
administered subcutaneously. All these new agents can be administered at fixed doses without laboratory monitoring.
IDRAPARINUX
Is a second generation synthetic pentasaccharide hypermethylated derivative of fondaparinux that binds antithrombin with such
high affinity that it assumes a prolonged half-life (130 hours) and
it can be administered once a week, without coagulation monitoring. Idraparinux exhibits complete bioavailability after subcutaneous
injection, binds only to antithrombin in plasma and produces a predictable anticoagulant response. idraparinux is excreted unchanged
via the kidneys. Therefore, the dose of idraparinux must be reduced
in patients with renal insufficiency and it is contraindicated in those
with renal failure. The safety of idraparinux in pregnancy is uncertain. Idraparinux was evaluated for the treatment of proximal DVT.
Bleeding complications were significantly lower in patients receiving idraparinux a dose of 2.5 mg in comparison to warfarin. Two
large clinical trials on the efficacy and safety of idraparinux 2.5 mg
administered subcutaneously once a week are currently ongoing in
patients with DVT and PE, respectively.
SSR126517E
It is a biotinylated form of idraparinux. Its advantage is that
it can be neutralized with intravenous recombinant avidin. Avidin
binds biotinylated fondaparinux with high affinity, and the complex
is then cleared quickly.
RIVAROXABAN
Is a potent and selective inhibitor of factor Xa. It is well absorbed from the gastrointestinal tract. The terminal half-life is about
9 hours at steady state so the drug is administered orally twice daily.
Food prolongs the time to peak plasma concentration and increases
drug exposure by 25 to 35%. Rivaroxaban exhibits a dual mechanism of excretion. About 65% is excreted via the kidneys, while the
remainder is excreted in the feces. Because of this dual excretion
mechanism, the drug may be less likely to accumulate in patients
with renal insufficiency. It is metabolized in the liver and shows some
interaction with potent inhibitors of CYP3A4, such as ketoconazole.
However, its potential for other drug-drug interactions is expected to
be low. Like other direct factor Xa inhibitors, rivaroxaban prolongs
INR and aPTT but its effects on these tests are relatively small at
therapeutic doses.
APIXABAN
A small molecule inhibitor that targets the active site of factor Xa, in a selective and reversible way and it inhibits factor Xa
bounded within the prothrombinase complex as well as the free enzyme. With repeated doses, the half-life is between 9 and 14 hours.
Therefore, once-daily administration may be possible. Apixaban is
oxidized to a phenol metabolite in the liver and CYP3A4 may be
involved in this metabolism. However, the potential for drug-drug
interactions with apixaban is expected to be low. Apixaban exhibits
a dual mechanism of excretion. About 25% is excreted via the kidneys, while the remainder appears in the feces. Apixaban prolongs
the INR and the aPTT in a concentration dependent fashion. However, its effect on these tests is minimal at concentrations that are
likely to be therapeutic.
XIMELAGATRAN
It is an oral direct thrombin inhibitor. The optimal dose of ximelagatran to be used for treatment of VTE was first assessed in a
phase II randomized compared with dalteparin followed by warfarin.
Similar rates of thrombus regression and clinically overt recurrence
were observed in the two treatment groups. Ximelagatran was compared with enoxaparin followed by warfarin, randomized, double
blind study in patients with DVT. The incidence of objectively confirmed recurrent VTE during 3 months following the index event was
similar in the two treatment groups. No difference was observed
concerning major bleedings and mortality.
The clinical development of ximelagatran has been recently interrupted due to a 6–10% incidence of increased levels of liver enzymes and in particular of alanine aminotransferase. The increase
occurred between three weeks and four months after starting treatment.
DABIGATRAN
The real advantage of this agent over warfarin is the potential
use at fixed daily doses without laboratory monitoring.
It is an oral thrombin inhibitor that is now under evaluation
for the treatment of DVT. Dabigatran etexilate is a double prodrug
that is converted by esterases into its active metabolite, dabigatran
(BIBR 953), once it is absorbed from the gastrointestinal tract. Once
in the liver, bioconversion of the prodrug is completed and about
20% is conjugated and excreted via the biliary system. The cytochrome P450 system plays no part in the metabolism of dabigatran
etexilate. Therefore, the risk of drug-drug interactions is low. The
bioavailability of dabigatran etexilate is only about 4%, so relatively
high doses of dabigatran etexilate must be given to ensure that adequate plasma concentrations are achieved. The half-life of dabigatran is approximately 14 to 17 hours after multiple doses have been
administered. With the long half-life, once-daily administration may
be possible for some indications.
Dabigatran is excreted unchanged via the kidneys. Consequently, plasma concentrations can increase in patients with renal
insufficiency.
FUCOSYLATED CHONDROITIN SULFATE (fucCS)
Is a new oral, potent, antithrombotic agent anticoagulant polysaccharide extracted from sea cucumber. It produces a dose-dependent increase in the plasma anticoagulant activity without modifying
the bleeding time. The anticoagulant activity of fucCS is related to
its capacity to increase thrombin inhibition by heparin cofactor II.
POTENTIAL ADVANTAGES OF NEW ANTICOAGULANTS
Fondaparinux and idraparinux have potential safety advantages over heparin and LMWH. They are likely to eliminate the risk
of heparin-induced thrombocytopenia. In addition, osteoporosis,
which can complicate long-term treatment with heparin, is unlikely
to occur with fondaparinux or idraparinux. They are less likely to
cause urticarial reactions at the site of subcutaneous injection than
heparin or LMWH.
Oral direct factor Xa or thrombin inhibitors have potential advantages over warfarin. These new agents have a rapid onset of
action, which could obviate the need for overlap with a parenteral
anticoagulant. With no food interactions, no genetic variations in
metabolism and minimal potential for drug-drug interactions, they
can be administered in fixed doses with little or no coagulation monitoring. These features render them more convenient to be administered instead warfarin.
S17
EDUCATION SESSION
Autologous Bone Marrow Transplantation (ABMT)
Transplantation for Follicular NonHodgkin’s Lymphoma
Julie Vose
Although most patients with follicular lymphoma have a relatively indolent course of their disease, it is not considered curable
with standard chemotherapy (1-4 ). Several studies have recently
analyzed the treatment changes over time and have identified possible improvements in the progression free survival and perhaps
overall survival when a monoclonal antibody is added to the therapy
(5,6). High dose chemotherapy and autologous stem cell transplantation is one of the options for patients with relapsed follicular
NHL. Several studies using autologous stem cell transplantation in
this patient population have demonstrated improved disease-free
survival but no consistent improvement in OS compared to a historically controlled population (7-9). The only randomized trial was conducted in Europe which randomized patients with relapsed follicular
lymphoma to standard chemotherapy vs. an unpurged autologous
stem cell transplant vs. a purged transplant (the CUP trial) (10). In
this trial, 140 patients with relapsed, chemosensitive follicular NHL
were randomized to one of the 3 arms. The OS at 4 years for the
chemotherapy arm was 46%, 71% for the unpurged transplant and
77% for the purged transplant. The 2-year PFS was 26% for the
chemotherapy arm, 58% for the unpurged and 55% for the purged
transplant. Significant reduction in the hazard rates for both PFS
and OS were present when comparing the chemotherapy arm to
the transplant arms. However, there was no difference between the
two transplant arms. Although the accrual goal was not met on this
trial, it did demonstrate an improvement in PFS and OS for those
patients in the transplant arms over standard chemotherapy.
The next issue to be addressed is which follicular lymphoma
patient population would benefit the most from the use of high-dose
chemotherapy and autologous stem cell transplantation. Various
studies have evaluated the prognostic indicators which predict for
better outcome with autologous transplant for follicular lymphoma
including chemotherapy sensitivity, bulk of disease, number of prior
chemotherapies received and IPI (7-9). Since it is relatively new,
the FLIPI index has been less utilized in transplant clinical trial evaluations. Our study has demonstrated a worse outcome for patients
with > 3 prior therapies, FL grade III disease, and a high FLIPI at
the time of transplant. Patients with all of these characteristics have
only a 5-year OS of 14% compared to an 82% 5-year survival with
none of these characteristics (Figure 1). This information should
assist us in considering autologous stem cell transplant for patients
before they have been heavily treated with chemotherapy or progressed to a higher grade of follicular lymphoma. Therefore, a standard autologous stem cell transplant should not be considered as a
last option for follicular lymphoma patients who have failed all other
options, but should be considered earlier in the course of disease
where the optimum benefit can be realized.
REFERENCES
1. McLaughlin P, Fuller LM, Velasquez WS, et al: Stage III follicular
lymphoma: durable remissions with a combined chemotherapyradiotherapy regimen J Clin Oncol 5: 867-874, 1987.
2. Flinn IW, Byrd JC, Morrison C, et al: Fludarabine and cyclophosphamide with filgrastim support in patients with previously
untreated indolent lymphoid malignancies. Blood 96: 71-75,
2000.
3. Czuczman MS, Weaver R, Alkuzeny B, et al: Prolonged clinical
and molecular remission in patients with low-grade or follicular non-Hodgkin’s lymphoma treated with rituximab and CHOP
chemotherapy: 9 year follow-up. J Clin Oncol 22: 4711-4716,
2004.
4. Marcus R, Imrie K, Belch A, et al: CVP chemotherapy plus rituximab compared with CVP as first-line treatment for advanced
follicular lymphoma. Blood 105: 1417-1423, 2005.
5. Fisher RI, LeBlanc M, Press OW, et al: New Treatment Options
Have Changed the Survival of Patients with Follicular Lymphoma. J Clin Oncol 23: 8447-8452, 2005.
6. Swenson WT, Wooldridge JE, Lynch CF, et al: Improved Survival of Follicular Lymphoma Patients in the United States. J
Clin Oncol 23: 5019-5026, 2005.
7. Bierman PJ, Vose JM, Anderson JR, et al: High-dose therapy
with autologous hematopoietic rescue for follicular low-grade
non-Hodgkin’s lymphoma. J Clin Oncol 15: 445-450, 1997.
8. Rohatiner AZ, Freedman A, Nadler L, et al: Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for follicular lymphoma. Ann Oncol 5: Suppl 2:
143-146, 1994.
9. Freedman AS, Neuberg D, Mauch P, et al: Long-term followup of autologous bone marrow transplantation in patients with
relapsed follicular lymphoma. Blood 94: 3325-3333, 1999.
10. Shouten HC, Qian W, Kvaloy S, et al: High-dose therapy improves progression-free survival and survival in relapsed follicular non-Hodgkin’s lymphoma: results from the randomized
European CUP trial. J Clin Oncol. 21: 3918-3927, 2003.
Figure 1. Outcome of FL Transplantation by Risk Factors
S18
Autologous stem cell transplantation for
aggressive non-Hodgkin’s lymphomas
Benjamín Koziner
HISTORICAL PERSPECTIVE – PARMA TRIAL AND CIMBTR DATABASE
Based on the rationale that high-dose chemotherapy (HDCT)
could overcome tumor-cell resistance, initial studies suggested that
HDCT, followed by autologous stem cell transplantation (ASCT),
was able to salvage patients with relapsed aggressive non-Hodgkin’s lymphomas (NHL) showing chemo-sensitivity to conventional
dose rescue regimens.
The Parma study included 215 patients with relapsed intermediate grade NHL that underwent salvage chemotherapy with
the DHAP regimen. The 109 patients with chemosensitive disease
were randomized to either autologous bone marrow transplantation (ABMT) or continuation of chemotherapy (1). The ABMT group
of patients showed a superior overall response rate (84% vs 44%,
median follow-up: 63 months), event-free survival (46% vs 12%, P
= 0.001) and overall survival (53% vs 32%, P = 0.038). Based on
these positive results, HDCT with ABMT became the accepted salvage modality for patients with relapsed chemosensitive NHL.
Using the International prognostic index (IPI) (2), to retrospectively analyze the Parma results, Blay et al. observed that ABMT did
not provide a survival advantage over conventional salvage chemotherapy for patients with an IPI of 0 (5-year OS: 51% vs 48%,
P = 0.59). In contrast, patients with an IPI > 0 had a significantly
superior survival if treated with ABMT (P < 0.05) (3).
The conclusions provided by the Parma trial in the present
context have to be reconsidered. Improvements in supportive care,
including the use of peripheral blood progenitor cells (PBPC), have
extended the use of SCT approaches to older patients. Furthermore, most centers will offer transplantation to patients who achieve
a partial as well as complete response to their initial chemotherapy,
and the use of PBPC has de-emphasized the requirement for an
uninvolved BM at the time of harvesting.
As part of the presentation, a review of the database on autotransplants for aggressive NHL of the CIBMTR will be presented,
kindly prepared by Manza Agovi, MPH from the Statistical Staff under the direction of Dr. Mary M. Horowitz.
Initially, ASCT relied on BM harvests. Subsequently, the advantage of growth-factor-mobilized PBSC over BM was shown
in retrospective, as well as several prospective randomized trials
(4)
. The mobilization of autologous hematopoietic progenitor cells
(CD34+) has been significantly enhanced not only by the administration of appropriate dosing of G–CSF but more recently by the
use of AMD3100 (inhibitor of SDF–1α/CXCR4 binding) alone or in
combination, offering an alternative for the subset of “poor mobilizers” that cannot qualify for autotransplantation (5).
Although autologous BM cells suspensions are frequently contaminated with malignant cells, the biologic relevance of graft contamination is unclear. A correlation between BM involvement and
disease recurrence has been demonstrated (6), but it is unclear if the
same conclusion applies to occult involvement of PBSC.
PREPARATIVE REGIMENS
Commonly used second-line regimens prior to ASCT for relapsed and aggressive NHL include at present not only DHAP, but
also ESHAP (etoposide, methylprednisone, cisplatin), mini-BEAM
(carmustine, etoposide, cytarabine, melphalan) and ICE (ifosfamide, carboplatin, etoposide). These regimens produce CR rates
of 25% to 35%. The addition of the anti-CD20 monoclonal antibody
Rituximab to ICE (R-ICE) increased the CR rate to 53% compared
with 27% for patients treated with ICE in a previous study (7). The
overall response rates did not differ between ICE and R-ICE and
OS was also the same for both groups. None of the patients in these
two series had received Rituximab as a component of initial therapy.
The effectiveness of adding Rituximab to second-line therapy for
patients previously treated with Rituximab remains unclear (8). There
are no current randomized trials that demonstrate a survival advantage for SCT in these diverse patient groups and it is unclear
whether HDT and ASCT will prove to be an effective salvage strategy for patients who relapse after these regimens.
No prospective randomized trials have compared the various
preparative regimens in use. TBI-containing conditioning programs
are often combined with cyclophosphamide, etoposide, or cytarabine.Non-TBI-containing regimens commonly include the nitrosourea
BCNU combined with other drugs (BEAM, BEAC, CBV). Carboplatin-based (ICE) or busulfan based regimens have been less commonly used. The influence of TBI on clinical outcome is unclear.
Other approaches being explored include the administration of escalated HDCT (Mega-CHOEP) followed by repetitive SCT (9), and
upfront double high dose chemotherapy (CHOP/DICEP) followed
by BEAM and SCT (10).
Khouri et al from the MD Anderson Cancer Center recently
reported on the concurrent administration of high dose Rituximab
before and after ASCT for relapsed aggressive B–cell NHL. The
overall survival rate at 2 years was 80% for the study group and
53% for the control group (P < 0.002) with no differences observed
in the median times of neutrophil recovery or incidence of infections
(11)
. However Hoerr et al observed a delay in platelet engraftment,
despite improvement in survival in patients that received Rituximab
on stem cell mobilization pre-autografting (12).
PROGNOSTIC FACTORS
It appears that relapsed NHL patients most likely to benefit
from ASCT are those with chemosensitive and limited disease, and
a prolonged initial remission. Those with very high-risk features
(high LDH, high IPI, short initial remission) do quite poorly, and
might derive benefit from more investigational approaches, such as
allogeneic transplantation, either the conventional myeloablative or
the most recently tried non-myeloablative modalities (13,14).
Gene expression profiling by micro-RNAs (mostly resorting to
Affymetrix V133A gene chips) is becoming a most useful and accurate tool to correctly identify diverse pathological subtypes of aggressive NHLs, mostly on the basis of levels of expression of c–myc
germinal center B–cell, MHC–class I and nuclear factor KB target
genes (15).
Another new diagnostic tool to be taken into account in the
evaluation of patients with aggressive NHL –candidates for ASCT–
is positron emission tomography (PET) using fluorine 18-fluorodeoxyglucose (18F–FDG–PET). Presence or absence of abnormal
uptake pretransplantation related to PFS and OS and has also
shown to be useful to evaluate response to ASCT and in the follow
up of patients (16).
TIMING OF ASCT
The role of ASCT as part of the initial management of aggressive lymphomas remains unclear. Certain subgroups, such as those
with intermediate and high-intermediate IPI scores, may derive benefit when transplanted in first CR. ASCT after shortened induction
regimens and for patients in PR seems not to be of benefit. In a
report dating back to almost 2 decades ago Gulati et al. at MSKCC
reported a DFS advantage (P < 0.002) for NHL patients with unfavourable prognostic features who underwent ABMT immediately
after induction of remission with 79% surviving at a median follow
up 49.2 + months compared with a median survival of 5.2 months
for those patients autotransplanted while in relapse and/or after
failing conventional treatment (17). However, a recent randomized
multi-institutional study by the EORTC has convincingly shown that
HDCT and ASCT does not confer benefit for patients with low or
low-intermediate IPI scores (18).
The prognosis for patients with relapsed chemoresistant disease and those with primary refractory disease is poor following
ASCT, with only 10–15% long-term survival. However, a retrospective analysis by Vose et al showed that patients achieving less than
a CR to initial therapy (primary refractory disease) benefit from subsequent immediate ASCT if they convert to CR following HDCT (19).
This patient subset had a significantly improved 3-year probability of
survival compared with those achieving a PR o less (68% vs 11%, P
< 0.001). Encouraging reports of durable remission with autologous
stem cell transplantation in high risk/relapsed HIV-associated lymphomas have been also published (20).
ROLE OF RADIOLABELLED MONOCLONAL ANTIBODIES
Using radiolabelled MAbs to deliver high doses of radiation to
a tumor while limiting the dose delivered to normal tissues and organs represents a most reasonable strategy. Both 131I tositumomab
(Bexxar) and 90Y ibritumomab tiuxetan (Zevalin) have been shown
to be active agents for patients with indolent and transformed CD20positive B-cell lymphomas, but only limited experience exists with
the use of these agents in diffuse large cell B lymphomas (DLBCL).
Most studies to date have included patients with various histologic
subtypes of NHL and have shown that both 131I tositumomab and
90
Y ibritumomab tiuxetan can be combined with standard high-dose
chemotherapy regimens without apparent additional toxicity or delay in engraftment. A recent report by Vose et al has explored the
use of 131I tositumomab plus BEAM in 23 patients with refractory
or multiply relapsed B-cell NHL, most of whom had DLBCL. The
investigators reported an overall response rate of 65%. At a median
follow-up of 38 months, the PFS and OS rates were 39% and 55%
respectively (21).
SPECIAL LYMPHOMA SUBTYPES
Mantle cell lymphoma (MCL)
High response rates and duration of responses have been
reported in newly diagnosed and relapsed MCL, especially when
ASCT was performed in first remission. Unfortunately, high recurrence rates are common, which are assumed to be related to the
presence of occult lymphoma cells in BM and PB of patients allegedly in clinical remission. Newer approaches incorporate the in vivo
purging with anti-CD20 MAb, Rituximab. Initial reports of stem-cell
collections after intensive chemotherapy and Rituximab administration, indicate effective purging and a high remission rate after
transplantation (22).
Recently, the HyperCVAD regimen has been recognized as an
effective upfront treatment of MCL. Khouri et al (23) evaluated the
role of ASCT following HyperCVAD–MTX/Ara–C. They reported that
at 3 years, the OS and EFS for previously untreated patients was
92% and 72%, respectively. For those patients who were previously
treated, the results were much worse, with the OS and EFS being
25% and 17%, respectively.
The European Mantle Cell Lymphoma Network reported on a
randomized trial comparing consolidation with myeloablative radiochemotherapy followed by autologous SCT to α–interferon (α–IFN)
maintenance in 1st remission after achievement of a CR or PR after
CHOP induction. Patients in the ASCT arm experienced a significantly longer PFS. The 3–year OS was 83% after ASCT vs 77% in
the α–IFN group (24).
High grade lymphomas (Burkitt’s and lymphoblastic)
Most published transplant series include only a small percentage of patients with Burkitt’s, Burkitt’s-like or lymphoblastic NHL. Although these diseases are highly curable in children, the long-term
prognosis in adults is generally poor. The European Bone Marrow
Transplant Registry has published the largest series of patients with
these diagnoses. In patients with Burkitt’s and Burkitt’s-like NHL the
3-year actuarial survival rates were 72%, 37% and 7% for patients
transplanted in first CR, chemosensitive relapse and chemoresistant relapse respectively (25).
In an international randomized study comparing ASCT versus
conventional dose consolidation and maintenance chemotherapy
that included 119 adult patients with lymphoblastic lymphoma, only
a trend for superior OS was observed with ASCT (56%) versus
conventional chemo (45%), P = 0.71 (26). Most recently, a common
report from the IBMTR and ABMTR concluded that alloSCT (76 pts)
for lymphoblastic lymphoma was associated with fewer relapses
than with ASCT (128 pts) but higher treatment related mortality.
However, survival did not differ significantly between the 2 groups
at 1 and 5 years (60% vs 49%, P = 0.09; 44% vs 39%, P = 0.47)
(27)
. The role of ASCT in the primary treatment of these disorders
remains uncertain.
S19
REFERENCES
1. Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in
relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma.
N Engl J Med. 1995;333:1540-1545.
2. A predictive model for aggressive non-Hodgkin’s lymphoma.
The international Non-Hodgkin’s Lymphoma Prognostic Factors
Project. N Engl J Med. 1993;329:987-994.
3. Blay J, Gomez F, Sebban C, et al. The International Prognostic Index correlates to survival in patients with aggressive
lymphoma in relapse : analysis of the PARMA trial. Blood.
1998;92:3562-3568.
4. Champlin RE. Peripheral blood progenitor cells: a replacement
for marrow transplantation? Semin Oncol. 1996;23:15-21.
5. Flomemberg N, Devine SM, DiPersio JF, et al. The use of
AMD3100 plus G-CSF for autologous hematopoietc progenitor cell mobilization is superior to G-CSF alone. Blood.
2005;5:1867-1874.
6. Vose JM, Sharp G, Chan WC, et al. Autologous transplantation
for aggressive non-Hodgkin’s lymphoma: Results of a randomized trial evaluating graft source and minimal residual disease.
J Clin Oncol. 2002;9:2344-2352.
7. Kewalramani T, Zelenetz AD, Nimer SD, et al. Rituximab and
ICE as second line therapy before autologous stem cell transplantation for relapsed or primary refractory diffuse large B-cell
lymphoma. Blood. 204;103:3684-3688.
8. Vose JM. Therapeutic uses of MAbs directed against CD20. Cytotherapy. 2000;6:455-462.
9. Glass B, Kloess M, Bentz M, et al. Dose-escalated CHOP plus
etoposide (MegaCHOEP) followed by repeated stem cell transplantation for primary treatment of aggressive high-risk nonHodgkin lymphoma. Blood. 2006;107:3058-3064.
10. Stewart DA, Bahlis N, Valentine K, et al. Upfront double highdose chemotherapy with DICEP followed by BEAM and autologous stem cell transplantation for poor-prognosis aggressive
non-Hodgkin lymphoma. Blood. 2006;107:4623-4627.
11. Khouri IF, Saliba RM, Hosing C, et al. Concurrent Administration
of High-Dose Rituximab Before and After Autologous Stem Cell
Transplantation for relapsed Aggressive B-Cell Non-Hodgkin’s
Lymphomas. J Clin Oncol. 2005;10:2240-2247.
12. Hoerr AL, Gao F, Hidalgo J, et al. Effects of pretransplantation treatment with Rituximab on outcomes of autologous stem
cell transplantation for non-Hodgkin’s lymphoma. J Clin Oncol.
2004;22:4561-4566.
13. Shipp MA, Ross KN, Tamayo P, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expresion profiling and supervised machine learning. Nat Med. 2002;8:68-74.
14. Hamlin PA, Zelenetz AD, Kewalramani T, et al. Age-adjusted
International prognostic Index predicts autologous stem cell
transplantation outcome for patients with relapsed or primary
refractory diffuse large B-cell lymphoma. Blood. 2003;6:19891996.
15. Shipp MA, Abeloff MD, Antman KH, et al. International consensus conference on high-dose therapy with hematopoietic stem
cell transplantation in aggressive non-Hodgkin’s lymphomas:
Report of the jury. J Clin Oncol. 1999;17:423-429.
16. Spaepen K, Stroobants S, Dupont P, et al. Prognostic value of
pretransplantation positron emission tomography using fluorine
18-fluorodeoxyglucose in patients with aggressive lymphoma
treated with high-dose chemotherapy and stem cell transplantation. Blood. 2003;102:53-59.
17. Gulati SC, Shank B, Black P et al. Autologous bone marrow
transplantation for patients with poor-prognosis lymphoma. J
Clin Oncol. 1988;8:1303-1313.
18. Kluin-Nelemans HC, Zagonel V, Anastasopoulou A, et al. Standard chemotherapy with or without high-dose chemotherapy for
aggressive non-Hodgkin’s lymphoma : randomized phase III
EORTC study. J Natl Cancer Inst. 2001;93:22-30.
19. Vose JM, Zhang M-J, Rowlings PA, et al. Autologous transplantation for diffuse aggressive non-Hodgkin’s lymphoma
in patients never achieving remission: A report from the Autologous Blood and Marrow Transplant Registry. J Clin Oncol.
2001;2:406-413.
S20
20. Krishnan A, Molina A, Zala J, et al. Durable remissions with autologous stem cell transplantation for high-risk HIV-associated
lymphoma. Blood. 2005;2:874-878.
21. Vose JM, Bierman PJ, Enke C, et al. Phase I trial of iodine131 tositumomab with high-dose chemotherapy and autologous
stem cell transplantation for relapsed non-Hodgkin lymphoma.
J Clin Oncol. 2005;23:461-467.
22. Magni M, Di Nicola M, Devizzi L, et al. Successful in vivo purging of CD34-containing peripheral blood harvests in mantle cell
and indolent lymphoma: evidence for a role of both chemotherapy and rituximab infusion. Blood. 200;96:865-869.
23. Khouri IF, Saliba RM, Okoraji GJ, et al. Long-term follow up
of autologous stem cell transplantation in first remission in patients with diffuse mantle cell lymphoma. Cancer. 2003;98:26302635.
24. Dreyling M, Lenz G, Hoster E, et al. Early consolidation by myeloablative readiochemotherapy followed by autologous stem
cell transplantation in first remission significantly prolongs progression-free survival in mantle-cell lymphoma: results of a pro-
spective randomized trial of the European MCL Network. Blood.
2005;7:2677-2684.
25. Sweetenham JW, Pearce R, Taghipour G, et al. Adult Burkitt’s
and Burkitt-like non-Hodgkin´s lymphoma– outcome for patients treated with high-dose therapy and autologous stem cell
transplantation in first remission or at relapse: results from the
European Group for Blood and Marrow Transplantation. J Clin
Oncol. 1996;14:2465-2472.
26. Sweetenham JW, Santini G, Oian W, et al. High-dose therapy
and autologous stem cell transplantation versus conventional
dose consolidation/maintenance therapy as post-remission
therapy for adult patients with lymphoblastic lymphoma: results
of a randomized trial of the European Group for Blood and Marrow Transplantation and the United Kingdom Lymphoma Group.
J Clin Oncol. 2001;19:2927-2936.
27. Levine JE, Harris RE, Loberiza FR, et al. A comparison of allogeneic and autologous bone marrow transplantation for lymphoblastic lymphoma. Blood. 2003;101:2476-2482
S21
EDUCATION SESSION
Cell Therapy
Cellular Therapy in Hematopoietic Cell
Transplantation
Gregory A. Hale
From Mismatched Family Member Donors
Associate Member and Clinical Director
Division of Bone Marrow Transplantation
St. Jude Children’s Research Hospital
Allogeneic hematopoietic stem cell transplantation (HSCT) is
curative for patients with high-risk or recurrent hematologic malignancies. However, only 25%-30% of patients have HLA-identical
sibling donors. For the remainder, only 40-50% of patients have an
appropriate unrelated donor identified, with a median time to HSCT
of 3-4 months. During this time, disease progression, infection, or
organ toxicity may make HSCT prohibitive or increase the risk of
transplant-related mortality. Highly motivated mismatched family
member (MMFM) donors are readily available for nearly all patients.
In this review, I will outline the current status of MMFM donor HSCT
summarizing outcomes, graft processing strategies, and the role of
cellular therapies.
Because of the high risk of severe GVHD, T-lymphocyte depletion of the graft is necessary, but at the risk of graft failure and delayed immune reconstitution. MMFM donor HSCT has historically
been reserved for patients who have failed all treatments, primarily
those with refractory or bulky disease or who are heavily pre-treated. Therefore, early studies reported survival rates after MMFM
HSCT of ≤ 25%. With improvements in T-cell depletion methodologies and supportive care and with the larger cell doses in peripheral
blood grafts, outcomes following MMFM donor HSCT have dramatically improved.1,2,3
One novel T-cell depletion strategies uses CD34+ selection of
grafts; a 5-log depletion of CD3+ cells can easily be achieved. This
methodology generates a graft containing megadoses of CD34+ hematopoietic cells to be generated, overcoming engraftment barriers.
These highly purified grafts have 98% purity, recover 75% of CD34+
cells, low CD3+ (0.5%) and CD19+ (0.04%) cells, making GVHD and
post-transplant lymphoproliferative disorder (PTLPD) rare.3 No
pharmacologic GVHD prophylaxis is necessary. However, graft
failure and delayed immune reconstitution remain problematic. In
early studies, overall survival increased to approximately 50% with
this T-cell depletion strategy.
Recently, less intensive T-cell depletion strategies using antiCD3 antibodies such as OKT3 have resulted in 3.5 log CD3+ depletion of the graft.4 This methodology results in a less purified graft
containing more CD3+ and CD19+ (5-15%) cells in addition to nonCD34+ cells (monocytes, hematopoietic precursors, and lymphocytes such as NK cells). These grafts have resulted in low rates
of graft failure with rapid immune reconstitution, and theoretically
low relapse rates due to additional NK cell content. However, the
risk of GVHD and PTLPD are increased compared to CD34+ selection. Post-HSCT pharmacologic prophylaxis is necessary. Overall
survival rates are shown to be 50% at 1 year post-HSCT in patients
with refractory disease.5
The immunosuppression and inflammation effects of conditioning are important. Recipients of myeloablative regimens are at
high-risk of regimen-related toxicities, particularly if they are heav-
ily pre-treated or have co-morbid conditions. In addition, thymic
epithelial damage from total body irradiation (TBI) can result in
impaired thymic function, leading to poor T-cell reconstitution. Recently, a report has shown that a reduced-intensity regimen consisting of fludarabine, melphalan, and thiotepa results in acceptable
engraftment and rapid CD3+ recovery compared to a TBI-containing
myeloablative regimen. Investigators have explored recombinant
human keratinocyte growth factor, a commercially available drug
used to prevent mucositis, to protect thymic epithelium. In animal
models, this agent protects the thymus, allowing more rapid CD3+
recovery following HSCT.6
After MMFM HSCT, patients must receive antiviral, antifungal,
and pneumocystis prophylaxis. Patients must be monitored closely
for evidence of viral reactivation. Routine surveillance of peripheral
blood for the presence of cytomegalovirus (CMV), adenoviral, and
Epstein Barr virus (EBV) DNA should be performed.7,8 Pre-emptive
antiviral therapy must be employed early: cidofovir for adenovirus,
ganciclovir for CMV, and rituximab for EBV. Viral reactivation can
further delay immune recovery making eradication of viruses difficult. In addition, the antiviral agents cause significant marrow and
renal toxicity. Investigators have infused non-alloreactive donor
hematopoietic cells containing significant numbers of CD3+ cells to
allow rapid immune recovery. Theoretically, this process would result in no GVHD while T-cell clones not recognizing host antigens
are generated. A recent study shows that this approach is safe and
feasible; however, some patients suffered significant GVHD.9
After MMFM HSCT, PTLPD occurs due to the imbalance between T and B cell content of the graft. PTLPD is exceedingly rare
in recipients of CD34+ selected grafts, due to the balanced depletion of T and B lymphocytes. OKT3-depleted grafts have a greater
B lymphocyte content, making PTLPD a more common problem.
Routine surveillance of peripheral blood for EBV is imperative, with
radiography for measurable disease in cases of rising EBV DNA
levels despite pre-emptive therapy with rituximab, which can also
be given prophylactically as a single dose on day of HSCT. Suspicious lesions should be biopsied with analysis for CD20 expression
and chimerism. While the majority of lesions following HSCT are
of donor origin, there is one case report of host-derived PTLPD.10
Pre-emptive or curative therapies include weekly rituximab, donor
lymphocyte infusions, and cytotoxic T-lymphocytes. Combined
CD3+ and CD19+ cell depletion of grafts is being studied for EBV
prevention.
Engraftment is typically the initial determinant of HSCT success. Delayed engraftment results in increased infection risk and
prolonged transfusion requirements. Graft rejection is more common in recipients of CD34+ selected grafts, due to the exceedingly
low CD3+ content. In heavily transfused immunocompetent patients, host-mediated anti-donor alloreactivity prior to HSCT must
be identified. Weekly monitoring of peripheral blood chimerism
is important. If rejection is a concern, T-cell chimerism should be
done.
GVHD has historically been a significant obstacle to successful
HSCT using MMFM donors, with up to 20%-50% of patients developing moderate to severe GVHD. With improved T-cell depletion
methodologies, hematopoietic grafts can be engineered to contain
specified quantities of specific cell populations. GVHD prophylaxis
is then determined by the CD3+ content of the graft and the potential
antileukemic benefit of GVHD in a certain patient population. Re-
S22
cent studies of CD3+ depletion strategies report < 10% of patients
develop severe GVHD. Higher rates of GVHD have been intentionally induced in patients with refractory disease in hopes of harnessing an antileukemic effect.
Post-HSCT donor-derived cellular therapies are lymphocytes
(DLI), cytotoxic T lymphocytes (CTL), NK cells, and CD34+ cell
boosts. DLIs are a well-established methodology to treat declining
donor chimerism, PTLPD, viral illnesses, and disease recurrence
but at the risk of GVHD. Therefore, the CD3+ content of the product
must be closely monitored. Published data indicates that CD3+ content of ≤ 2.5X104 CD3+ /kg in MMFM HSCT is associated with the
lowest risk of GVHD. CTLs have been successfully used for PTLPD
and viral illnesses. CTLs take weeks to generate, are not readily
available, and require significant laboratory support, yet have a low
GVHD risk. NK cell infusions are being studied to reduce relapse,
given published data showing decreased relapse rates in donorrecipient pairs who are KIR mismatched.11 In clinical trials, highly
purified CD56+ hematopoietic cells have been safely infused after
chemotherapy or alone.12,13 Investigators have demonstrated that
patients with delayed hematopoietic recovery or with poor immune
reconstitution may benefit from additional infusions of CD34+ hematopoietic precursors.
The ultimate determinant of HSCT success is the improvement
in overall survival, determined by disease recurrence and non-relapse mortality. For most patients requiring allogeneic HSCT but
who do not have an appropriately matched related or unrelated donor, HSCT is now a feasible alternative. Novel measures to prevent
non-relapse mortality and relapse are necessary to improve overall
survival. In the future, MMFM donor HSCT will likely be increasingly utilized for patient with malignant and non-malignant diseases.
Graft manipulation strategies will be studies to take advantage of
graft engineering measures to hasten immune reconstitution while
simultaneously decreasing disease recurrence rates and maintaining low GVHD incidence.
REFERENCES
1. Marks DI, Aversa F, Lazarus HM. Alternative donor transplants
for adult acute lymphoblastic leukaemia: a comparison of the
three major options. Bone Marrow Transplant 38(7):467-475,
2006.
2. Marks DI, Khattry N, Cummins M et al. Haploidentical stem cell
transplantation for children with acute leukaemia. Br J Haematol 134(2):196-201, 2006.
3. Lang P, greit J, Bader P et al. Long-term outcome after haploidentical stem cell transplantation in children. Blood Cells Mol
Dis 33(3):281-287, 2004.
4. Hale G, Chen X, Benaim E et al. Haploidentical transplantation
for children with refractory haematologic malignancies. European Group for Blood and Marrow Transplantation 2006. Bone
Marrow Transplant 37 Suppl 1:S250, 2006
5. Chen X, Hale GA, Barfield R et al. Rapid immune reconstitution after a reduced-intensity conditioning regimen and a CD3depleted haploidentical stem cell graft for pediatric refractory
hematological malignancies. Br J Haematol 134(4):524-532,
2006.
6. Min D, Taylor PA, Panoskaltsis-Mortari A et al. Protection from
thymic epithelial cell injury by keratinocyte growth factor: a new
approach to improve thymic and peripheral T-cell reconstitution
after bone marrow transplantation. Blood 99(12):4592-4600,
2002.
7. Yusuf U, Hale GA, Carr J et al. Cidofovir for the treatment of adenoviral infection in pediatric hematopoietic stem cell transplant
patients. Transplantation 81(10):1398-1404, 2006.
8. Kuehnle I, Huls MH, Liu Z et al. CD20 monoclonal antibody
(rituximab) for therapy of Epstein-Barr virus lymphoma after hemopoietic stem transplantation. Blood 95(4):1502-1505, 2000.
9. Amrolia PJ, Muccioli-Casadei G, Huls H et al. Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation. Blood
108(6):1797—1808, 2006.
10. Kasow KA, Leung W, Horwitz EM et al. EBV lymphoproliferative
disease of host orgin after haploidentical stem cell transplantation. Pediatr Blood Cancer (In press, 11/2006)
11. Leung W, Iyengar R, Triplett B et al. Comparison of killer Ig-like
receptor genotyping and phenotyping for selection of allogeneic
blood stem cell donors. J Immunol 174(10):6540-6545, 2005.
12. Iyengar R, Handgretinger R, Babarin-Dorner A et al. Purification
of human natural killer cells using a clinical-scale immunomagnetic method. Cytotherapy 5(6):479-484, 2003.
13. Triplett B, Handgretinger R, Pui CH et al. KIR-incompatible hematopoietic-cell transplantation for poor prognosis infant acute
lymphoblastic leukemia. Blood 107(3):1238-1239, 2006.
Stem Cells for Myocardiopathies
Juan José Paganini
Cardiac Surgeon, Asociación Española Montevideo Uruguay,
Hospital de Clinicas. [email protected]
INTRODUCTION
Congestive heart failure is one of the main causes for cardiologic morbility and mortality in the XXIst century (1,2). Patients in
advanced stages (NYHA functional classes III/IV) have an average
of 5 year survival rates below 50%, with an annual mortality of 40
– 50% (3), with high rates of re-hospitalization, morbility and high
related costs for health services. Etiology for dilated cardiomyopathy is 60% due to ischemic cardiomyopathy and 40% of idiopathic
– non ischemic origin.
This category of patients have been managed with medical
treatment (ACE inhibitors, diuretics, beta-blockers, spirolactone),
ventricular re-synchronization, ventricular assistance and heart
transplantation. For many years, heart transplantation has been the
surgical treatment of choice for patients with advanced heart failure. This procedure has been successful in many countries; however it presents many limitations, the most important ones being
the scarcity of donors and the contraindications of advanced age
and severe co-morbid situations (4). Moreover, there have been
frequent deaths during the prolonged periods in the waiting list for
organ reception.
The final stage of several heart diseases is congestive heart
failure determined by the quantitative deficiency of cardiomyocytes
and cardiac remodeling (5). Reversion of cardiac remodeling lies
in the possibility of myocyte regeneration and neo-vascularization
of affected areas. The goal of cellular therapy is the re-population
of the myocardium with cells capable of restoring contractility and
blood flow which will improve the systo-diastolic function of the
heart. The cells introduced must have the capacity for differentiation into cardiomyocytes or promote angiogenesis.
Several studies have shown that the adult bone marrow is
a rich reservoir of these pluri-potential, mesenchymal stem cells,
which contribute to functional neo-angiogenesis. Their beneficial
effects have been demonstrated in ischemic patients (6,7,8), and
more recently in non-ischemic patients (9). Our group conducted
two prospective, multicenter, studies of stem cell injection with positive results, in idiopathic and ischemic cardiomyopathy, in USA, Argentina and Uruguay.
METHODS
This studies were performed with the authorization of the Hospital authorities and ethics council; and the patients’ informed consent.
In ischemic patients we conducted a prospective randomized
study of autologous stem cell therapy in patients with heart faillure
requiring surgical revascularization.
The second group consisted in patients who presented dilated
idiopathic cardiomyopathy with a severe decrease in LVEF and
functional capacity. The goal of this study was to evaluate minimally
invasive surgical delivery of bone marrow cell therapy in patients
with non ischemic cardiomyopathy.
Ischemic patients, inclusion criteria was: Ischemic heart faillure
with EF<35% or less on two imaging studies. Two independent cardiologists evaluated EF. All patients had prior cardiac cathetheriza-
tion and optimal medical managment for heart faillure. All patients
were in NYHA class III/IV.They also had indication of myocardial surgical revascularization. Patients were exculded if: they were within
seven days of an acute coronary event, cancer present during the
last 5 years, presence of hematological diseases, leukocyte count
above 12000/cc or below 5000/cc, renal failure requiring hemodialysis, previous cardiac surgery, left ventricular aneurysm, valvular
heart disease requiring surgery, preoperative steroid therapy.
On the day of surgery, the patients were randomized to off
pump coronary artery by-pass grafting(OPCAB) or OPCAB plus
stem cell therapy. After this the patients were given a general anesthesia and monitoring lines were placed. If patients were in the OPCAB only group, then a standard sternotomy and off-pump coronary
artery bypass grafting was performed using both apical suction and
pressure stabilization of the heart. If patients were going to the stem
cell group after anesthesia, they were put prone and bone marrow
was harvested from the iliac bone.
Idiopathic patients inclusion criteria were: patients in NYHA
functional class III/IV, dilated idiopathic non-ischemic cardiomyopathy with LVEF<35%, optimal medical treatment including ACE
inhibitors, spironolactone, beta-blockers and diuretics at an average
85% of the maximum dose. Patients were excluded if congestive
heart failure decompensated in the last 6 days and all the other general criteria of ischemic patients. This group consisted of a prospective group of patients which were treated with minnimaly invasive
administration of stem cells.
STEM CELL EXTRACTION AND PROCESSING.
The patients were taken to the Operating Room monitored,
anesthetized and placed in the prone position, in both groups. Bone
marrow (BM) was harvested by hematologists in the team from the
iliac crests as is habitually done in the hematology and hemotherapy departments: Bilaterally between both posterior spinae. 400 to
500 were obtained and placed in a special container with 10000 U
of heparin and acetylsalicilate lysine to prevent coagulation. To minimize anesthesic time a special multi-channel harvest needle with
a 60 cc syringe. A sample is taken for a hematocrit, leukocyte and
mononuclear cell count: CD34+, CD34/CD45+. The BM is filtered,
the solution is mixed with 6% hydroethylstarch and the supernatant
is centrifuged. The pellet is washed with PBS and resuspended. By
differential weighing we can know exactly the weight of the BM harvest and we can collect 30 ml of the buffy coat plus 120 cc of plasma, obtaining a hematocrit of 20%. The buffy coat is then placed in
a rigid flask with 75 ml of ficoll-hypaque. After 30 minutes of centrifugation the upper layer is aspirated, leaving the mononuclear cell
layer at the interphase. The interphase cells are resuspended in
PBS and centrifugated. The supernatant is removed and the cell
pellet is again suspended in PBS. A new count of CD34+/CD45+
cells is performed. The resulting cell solution is resuspended in 30
cc of the patient own serum and 10 000 U of heparin sulfate. Cell
viability is established with a standard Trypan blue exclusion.
SURGICAL TECHNIQUE
Ischemic patients:
After OPCAB, the preselected sites of myocardial dyskinesis, akinesis and infarcted regions were injected with the stem cell
preparation, using a 22-25g apparatus. The placement of injections
was based on prior viewing of the echocardiogram as to prevent
direct introduction of cell into the ventricle based on wall thickness.
The cell preparation was injected in 1-2 cc aliquots as the needle
was withdrawn from the myocardium. The injections were separed
2 cm each other, and no direct coronary injections were performed.
Once this was completed, the chest was closed, drainage tubes
were placed, and the patient continued the normal postoperative
cardiac protocol. The remaining residual bone marrow were given
back intravenously.
Idiopathic patients
After harvesting of precursors from the BM the patient is placed
in a right lateral position with a 30° to 45° inclination from the horizontal plane of the right hemithorax.
Video-assisted surgery includes positioning three 10 mm trocars in the 3rd, 5th and 7th intercostal spaces: One for the camera
S23
and 2 for instruments. We use a double-lumen oro-tracheal tube
in this procedure, and the trocars are introduced once the left lung
has been isolated. The camera is inserted posteriorly in the 5th to
7th space (figure 1).
We then do a left minithoracotomy(5-7 cm), through where the
procedure is done(figure 2).
Exploration begins by opening the pericardium anteriorly to
the position of the frenic nerve. Injection in the selected areas is
performed, guided by the preoperative echocardiogram and avoiding intraventricular and intracoronary injection. We deliver aliquots
of 1cc separated by 2 to 3 cm and 3 to 5 mm in depth with a 23G
needle.
Once this has been done the trocars are withdrawn a thoracic
tube is put in place and the procedure is completed: The patient is
extubated and leaves the OR. Discharge occurs after 48 hours.
Follow-up in all groups is conducted by clinical examination,
EKG, X-ray films and echocardiogram at 6 months and 2 years, and
results were analyzed by independent cardiologists, not involved in
this study.
Statistical analysis applied the student test (SPSS program),
with p<0.05 were considered significant.
RESULTS
Ischemic group: 24 patients were enrolled in the randomized study. They had the following demographies, OPCAB vs
OPCAB+Stem Cells: Male/Female 9:3, mean age 63.6 vs 64.8
years and prior myocardial infarction 9:11. In OPCAB group, twelve
patients underwent succesful off-pump coronary artery bypass
grafting, by grafting the left internal thoracic artery to the left anterior descending artery and one patient also underwent a graft to
the posterior descending artery. In the OPCAB + stem group, 12
patients underwent successful grafting to the anterior descending
artery and one patient also underwent a saphenous vein graft to
circunflex artery. The median amount of bone marrow harvested
was 550cc , with a median processing time of two hours, and a median of 22 000 000 CD 34+/CD 45- cells in the final specimen. The
NYHA functional class at 6 months showed a drop in opcab from 3,4
to 2,7(p=0,001); however there was a larger drop in OPCAB + Stem
cells patients fron 3,5 to 0,7 (p=0,000000006). The mean ejection fractions for pre-operative, 1 month, 3 months and 6 months
were in OPCAB/ OPCAP + stem cells: 30,7%/29,4%(p=0,381);
36,4%/41,25%(p=0,002); 36,5%/44,66%(p=0,004), 37,2%/45,6%
(p=0,0007).
There was one hematoma from bone marrow harvest site.
There were no other complications(neurological,hematological,vas
cular, death or infection) neither did patients have arrhythmias.
Idiopathic group: 15 patients were enrolled for minimally invasive, toracoscopic video-assisted administration of stem cells. They
had the following demographics: Male/Female 12/3, mean age 71
vs 70. They all successfully recieved the cells. The mean amount of
bone marrow harvested was 250cc, and a a mean of 800 000 were
delivered. Cell viability was greater than 90% in all specimens. We
analized diferent variables preoperative and at 6 months : Ejection
fraction 26%/46%; end diastolic diameter 71/59mm; end sistolic diameter 50/42mm, NYHA class 3,4/1,3. There was one hematoma
in the punction site and no other complication.At two years not all of
the patients mantained the benefit in our patients.
DISCUSSION
The use of mesenchymal or stromal cells as precursors of
non-hemopoietic tissues was attempted for the first time by the
German pathologist Conheim in 1867 (10). It was later shown in
tissue cultures that they were capable of forming diverse tissues,
such as bone, cartilage, muscle, ligaments, tendons, etc (10,11),
and of intervening in tissue repair (10). An extremely interesting
study showed that stromal stem cells treated with 5 Azacytidine and
injected with the cardiomyocytes, transformed in cardiomyocytes
(12). The first reported case of BM cells applied to cardiomyoplasty
was by Weisel and Lee of Toronto University in 1999 (13).
This differentiation into myogenic lineage with development of
actin, myosin and tropomyosin was proved, as well as the presence of Conectin 43, a protein responsible for cellular inter-con-
S24
nection. This suggests the transformation into cardiomyocytes and
the electro-mechanical relationship of the cells (16). Improvement
of diastolic function, prevention of parietal thinning and decrease of
end-diastolic pressure were also shown.
Clinical application of this treatment was started in 2000, and
cases have increased throughout the world. Our studies with cases
of idiopathic and ischemic dilated cardiomyopathy show that there
are feasible procedures, that don´t increment nor surgical or postoperative morbility or mortality. They can be applied without using
complex technological devices, employing the equipment now at
our disposal in a third level hospital center, and costs are reasonable.
This technique has shown extremely good results in the prospective, randomized study in ischemic patients, taken to the OR for
myocardial revascularization: Marked improvement was observed
in ventricular ejection fraction and functional class in those receiving stem cells compared with those submitted to revascularization
alone. Clinical benefits were evident in improvement of functional
class, and no negative effects could be seen. These technique may
have its indication in patients with indication of CABG, CHF, low
ejection fraction and areas with vessels not suitable for revascularization.
Idiopathic patients receiving cell therapy obtained a significant
increase in LV ejection fraction and decrease of end-diastolic and
end-systolic ventricular diameters compared to values at baseline.
This increase in LVEF has allowed these patients to go from severely diminished values to a moderate decrease. Patients were
followed in the Heart Failure Unit. Evaluation at two years showed
that one of the patients had returned to pre-treatment values. The
other two showed the same functional and echocardiographic improvements.
Long-term results of this therapeutic modality are therefore still
unclear; however we consider it a valid option among other alternatives offered at present. The use of minimally invasive surgery with
thoracoscopy presents clear-cut advantages compared to other
methods used in delivering the cellular transplantation. It makes
thoracotomy or sternotomy unnecessary, thus decreasing surgical
morbility. The desired territories are injected under direct vision. It
is well tolerated and permits an early discharge. The percutaneous
technique is minimally invasive, but does not allow a precise identification of target areas.
CONCLUSIONS:
The procedure described is a feasible technique, with good
results in the short term, with low morbidity and mortality. Its place
in CHF, is still to be determined in bigger multicenter studies. Also
more controls, with PET, SPECT are required.
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Therapeutic Angiogenesis in Arterial
Ischaemic Limbs by Autologous Bone
Marrow Transplantation (ABMT).
The Conzi´s Effect in Human Diabetes
Mellitus.
Ernesto Novoa 1
Director of Clinical Hematology & Therapeutic Angiogenesis
Service
1
Police Hospital, Montevideo. Uruguay
Aurora Medina EP2
Scholarship “Angiogenesis”
Summary: therapeutic angiogenesis has recently been developed as a new method of treatment for several ischaemic diseases.
There is preliminary data suggesting that implantation of bone marrow-mononuclear cells into ischaemic limbs increases collateral
vessels formation. Aims:to evaluate viability of the therapeutic angiogenesis using hematopoyetic bone marrow progenitors mobilized
by G-CSF and safety of the procedure. Methods: 40 patients developing critical arterial limb ischaemia (candidates to amputation)
were included in this study. 23 men and 17 women. Median age 65
years old (44 – 86). Mobilized by filgrastim (Neupogen ®) 5 μg/kg
weight daily (5 days). Bone marrow harvest at 5th day. Local anaesthesia was employed in all the patients. Unmanipulated cells were
injected in the affected limb in 2 ml aliquots into the gastrocnemius
muscle. Each patient was evaluated regularly for rest pain, amount
of required analgesia , healing of the ulcers, peack walking time,
Doppler and angiographic findings. The mean number of injected
mononuclear cells was 1,9 x109/kg. All the patients received low
molecular weight heparin (nadroparin,Fraxiparine ®) 3800 – 5600
IU anti-Xa subcutaneously , aspirin 81 mg and pentoxifiline 400 mg
daily, as medical treatment after the procedure for at least 60 to 90
days. A control population of 39 vascular patients affected by critical
arterial limb isquemia was considered. They don’t received angiogenic treatment. Results: there were no deads secondary to the
procedure. 32 patients showed an improvement of all parameters.
On the control population, amputation was necessary in 87,2%. The
statistical differences betwen the two groups were highly significant
in favor of the angiogenic group. They were evaluated by the chi
square test and log rank test with a p value < 0,05. Conclusions:
autologous bone marrow transplantation can be performed safely
and appears to be a benefical therapy for selected patients with severe peripheral arterial disease. In adittion, some insulin dependent
diabetic patients, showed an important and maintained decrease
on blood sugar levels and insulin requirements after the cell therapy
procedure. Perhaps therapeutic angiogenesis will be in the future a
new way to treat diabetes mellitus and their vascular complications.
Going to the cure?.
References 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23
Key words: arterial limb ischaemia, autologous bone marrow
transplant, insulin dependent diabetes mellitus, angiogenesis.
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Shintani S, Morohara T, Ikeda H et al. Augmentation of postnatal Neovascularization with autologous bone marrow transplantation. Circulation 2001;103:897-95
Classification TASC. J of Vascular Surgery 2000;31:200.
Novoa JE. Diabetes, marcadores de trombosis y microangiopatia. Congreso Latinoamericano del Grupo Cooperativo Latinoamericano de Hemostasis y Trombosis(CLAHT) Rio de Janeiro,
Brasil, 2003.
Novoa JE. Angiogenesis terapeutica. Actualizaciones en Hemostasis y Trombosis. Coordinacion general: Ana Maria Otero.
Catedra de Hematologia. 17 de junio de 2004. Facultad de Medicina, Montevideo, Uruguay
Novoa JE. Angiogenesis terapéutica por trasplante de médula ósea en la arteriopatía de miembros inferiores. Simposio
CLAHT 2004. 20 al 22 de agosto de 2004. Lima, Perú.
Novoa JE, Medina MA & Gýanarellý S. Therapeutýc Angýogenesýs by stem cell transplant ýn the ýschemýc lýmb patýent:
a new therapeutýc aproach. XXXI Meetýng of the Slovakian /
Chek League Against Thrombosis. Martin,Slovakia. May 4-6,
2006.
Novoa JE & Medina MA. Angiogenesis Terapeutica en la Arteriopatia Periférica.Avances en Hematologia. Monterrey, Mexico
Junio 12-16 2006
Novoa JE, Medina MA & Gordillo F. The Conzi´s Effect in Human Diabetes Mellitus. From the Stem Cell to the Beta Cell.
XXXI World Congress of the International Society of Hematology (ISH). March 20-24 2007. Punta del Este, Uruguay (in
press).
Novoa JE, Medina MA & Gordillo F. Therapeutic Angiogenesis
in Arterial Ischaemic Limbs by Autologous Bone Marrow Transplantation (ABMT). XXXI World Congress of the International
Society of Hematology (ISH). March 20-24 2007. Punta del
Este, uruguay (in press).
Besalduch J, Lara R, Sampol A et al. Therapeutic Angiogenesis
in CriticalLimb Ischemia by Implantation of Autologous Hematopoietic Cells. Blood, Volume 106, issue 11, November 16, 2005.
S25
22 Statkute, L, Oyama, Y, Pearce, W, Yaung, K, Villa, M, Shook,
T, Clifton, R, Verda, L, Krosnjar, N, Burt, RK. Hematopoietic
AC133+ Stem Cell Therapy for Patients with Severe Peripheral
Vascular Disease. ASBMTR/IBMTR Tandem Meeting Honolulu,
Hawaii. February 2006.
23 Novoa JE, Medina MA, Gordillo F et al. Therapeutic Angiogenesis in arterial limb ischaemia by autologous bone marrow
transplant.Blood 2006;108:11(part 2) Abstract 5426.
Figure 1.
Therapeutic Angiogenesis. Ulcer leg in Charcot´s Syndrome
Insulin dependent Diabetes Mellitus. Before and after ABMT
Figure 2. The Conzi’s Effect in Human Diabetes Mellitus.
Stem Cell ‘Plasticity’: an Overview
Martin Körbling
Department of Stem Cell Transplantation and Cellular Therapy,
The University of Texas M.D. Anderson Cancer Center
Houston, Texas, USA
INTRODUCTION
Stem cells in any adult tissue are defined as being clonogenic,
having self-renewal capacity throughout lifetime and giving rise to
terminally differentiated cells of various cell lineages. In addition,
their differentiation pathway is unidirectional, passing through the
stage of lineage commitment and finally generating terminally differentiated cells, and adult stem cell differentiation is traditionally
S26
believed to be restricted to the tissue in which the stem cells reside
(hematopoietic stem cells generate blood cells, liver progenitor cells
[oval cells] produce hepatocytes and cholangiocytes, etc.). The latter two characteristics of adult stem cells are being questioned challenging a century old dogma of stem cell biology.
Lymphohematopoietic adult stem cells are the most thoroughly
characterized adult progenitor cells, mostly because of their easy
accessibility and more than 30 years of experience with their clinical use for transplantation to treat lymphohematopoietic disorders.
Using, among others, the Y-chromosome as a marker in a sex mismatched hematopoietic stem cell transplant model numerous reports over the past 5 years indicate that adult stem cells or their
progeny derived from hematopoietic tissue (bone marrow [BM], peripheral blood [PB], or umbilical cord blood [UCB]) not only generate
blood cells but also, at a much lower frequency, solid organ-specific
cells. Other reports, experimental and clinical, propagate cell fusion
and transfer of genetic material as the underlying event. Overall,
the mechanism(s) how these observations are explained are subject of intense and controversial debates throughout the literature
and scientific meetings.1,2
VARIOUS MODELS TO EXPLAIN HOW HEMATOPOIETIC TISSUE-DERIVED (STEM) CELLS EITHER GENERATE OR EXCHANGE GENETIC MATERIAL WITH SOLID ORGAN TISSUE
CELLS
There are essentially four models explaining how donor-derived cells originating from hematopoietic tissue are integrated into
host solid organ tissue:
1. Each tissue has its own circulating stem cell pool contributing
to lifelong tissue homeostasis. The best known circulating stem
cells are hematopoietic progenitor cells (CD34+38-)3 and mesenchymal stem cells (CD34-45-29+44+90+).4 More recently, circulating endothelial progenitor cells (EPC) (CD34+133+31+117bright
VEGFR-2+ vWF+ Tie-2+)5 have been identified, and, less well
characterized, circulating skeletal stem cells and smooth muscle stem cells. Those stem cells can be harvested in large numbers by continuous-flow apheresis6 to be injected locally at the
site of tissue damage.
2. Verfaillie’s group7,8 identified a very rare adult totipotent somatic
stem cell, also called multipotent adult progenitor cell (MAPC),
showing characteristics very similar to embryonic stem cells.
Those stem cells when transplanted into a host differentiate in
vivo into epithelium of liver, lung and gut besides generating
blood cells. It is therefore conceivable that a small reservoir of
primitive stem cells is available throughout lifetime to replenish
local stem cell pools in case of tissue damage or exhaustion.
3. Adult stem cells that differentiate inside their own tissue may
deviate from their preprogrammed pathway to generate under
conditions of stress (e.g., tissue injury) solid organ cells of a different tissue. This process is called “transdifferentiation”. Conclusive evidence for stem cell transdifferentiation comes from
experimental studies using either a mouse model with a genetic
defect (e.g.; tyrosinemia) that is repaired by injecting hematopoietic tissue-derived stem cells,9 or using a single hematopoietic stem cell differentiating in vivo into epithelial cells of liver,
lungs, GI-tract, skin, endothelial cells, glomerular mesangial
cells, skeletal muscle, and brain cells when transplanted into a
conditioned recipient.10,11,12 To underline the close relationship
between circulating stem cells and stationary solid organ tissue
stem cell pools, it is noteworthy that hepatic oval cells as part of
a solid organ tissue stem cell pool, share the same phenotype
with circulating stem cells being CD34+ CD90+ c-kit+ CXCR4+.13
4. Adult stem cells derived from hematopoietic tissue or their progeny may fuse with solid organ tissue cells to generate hybrid
cells. As first reported by two groups independently14,15 BM-derived cells fuse with hepatocytes to generate a polyploid mononuclear hybrid containing both donor and host genetic markers.
It is suggested that polyploid hybrid cells undergo a reduction
division with expulsion of chromosomes thus concealing their
fusion history. Fusion can even reprogram differentiated somatic cells and revert them back to a pluripotent state by activating Oct4, a gene essential for pluripotency.16 The physiological
purpose of adult cell fusion is speculative. As outlined by Helen
Blau17 fusion could be a means by which cells 1) deliver healthy
genetic material to dying cells (rescue function), 2) supply cells
with new genes (repair function), or 3) correct genetically defective cells such as in muscular dystrophy (gene replacement).
Fusion could even be considered a basic mechanism for keeping the adult cell systems intact throughout our lifespan.
Evidence in favor of both, stem cell (trans)differentiation and
cell fusion, may indicate that both mechanisms contribute to the
generation of solid organ-specific cells derived from hematopoietic
tissue. From a translational or clinical research point of view the
endproduct counts; it is crucial to find out about the function of those
cells and whether they are integrated into a functioning tissue.
ADULT HEMATOPOIETIC TISSUE-DERIVED STEM CELLS VERSUS EMBRYONIC STEM CELLS
The therapeutic use of human embryonic stem cells (ESC)
holds promise for repairing or generating solid organ tissue such
as slow-growing neuronal cells and pancreatic islet cells. ESCs differentiate into all three germ layers as do the rare MAPCs. The
long-term proliferative potential and self-renewing capacity of ESCs
is significantly higher than that of adult stem cells, two important requirements for successful tissue generation or repair. On the other
hand, when transplanted into severe combined immunodeficient
(SCID) mice ESCs form teratomas, part of the working definition of
an ESC. Such unwanted tumorigenicity sets them apart from adult
stem cells that have no known tumorigenic potential.
Whereas adult stem cells can be easily harvested from the
patient himself, ESCs and their tissue products have to cross the
HLA barrier when transplanted into an allogeneic recipient requiring
lifelong immunosuppression. Therapeutic cloning of human ESCs
by means of somatic cell nuclear transfer (SCNT) would bypass
HLA limitations. However, such an approach is being debated for
ethical reasons, and not considered practical for routine tissue repair purposes.
Most recently amniotic fluid stem (AFS) cells have been identified as a potential stem cell source that combines the phenotypes
and qualities of both, ECSs and adult stem cells, without being tumorigenic. Excess AFS are obtained from routine clinical amniocentesis specimens and considered a waste product.18
VARIOUS HEMATOPOIETIC TISSUE-DERIVED CELL SOURCES
FOR SOLID ORGAN TISSUE REPAIR
Among hematopoietic tissue, BM is not the only cell source
that can be used to generate solid organ-specific tissue. Since PB is
the only link between BM-derived cells and solid organ-specific tissue, it was a logical further step to investigate the possibility of PBderived cells generating non-lymphohematopoietic tissue. Orlic’s
group19 successfully demonstrated the repair of infarcted heart tissue in a mouse model by increasing the concentration of circulating
stem cells at the site of cardiac tissue damage. In a clinical setting,
we first reported the presence of XY-positive hepatocytes and epithelial cells of the skin and gastrointestinal tract in female recipients
of rhG-CSF-mobilized PB stem cell allografts from male donors.20
Donor-derived, non-lymphohematopoietic cells were identified at
frequencies ranging from 0% to 7% in the skin, gut, and liver. XYpositive cells were detected in liver tissue in these female recipients
as early as day 13 and as late as day 354 after transplantation.
Similar data for the generation of keratinocytes and GI-tract cells
have been reported by Hamatti et al.21 and Okamoto et al.22
EPCs are a circulating progenitor cell population that can easily be harvested in large quantities by continuous-flow apheresis
and locally injected for therapeutic vasculogenesis. The total number of EPCs available for treatment after CD133 selection is in the
range of forty millions.6
UCB is another PB stem cell source having the advantage of
being available in abundance. HLA restriction however limits its
clinical use the same way embryonic stem cells do.23
Multinucleated tissue cells such as muscle, liver and CNS tissue cells have been shown to incorporate genetic material from hematopoietic tissue cells preferentially by means of cell fusion.24,25
Circulating blood as a source of cells that deliver genetic material
to solid organ tissue cells is particularly appealing because of their
close proximity to solid organ tissue cells including intrinsic stem
cell pools.
POTENTIAL CLINICAL APPLICATIONS
It should be noted that, up to now, no clinical study has convincingly shown the repair of injured or the generation of new tissue
originating from adult hematopoietic cells including stem cells. This
is explained, among other reasons, by safety issues regarding stem
cell marking in an autologous transplant setting and by regulatory
issues. Nevertheless, experimental studies held promise of translating tissue repair data to a clinical level.
The potential clinical indications for tissue repair using cells
including stem cells derived from hematopoietic tissue are numerous including myocardial infarction, ischemic retinopathy, treatment
related pulmonary toxicity, skin and mucosa injury, spinal cord injuries and CNS diseases such as Parkinson and Alzheimer, and type
I diabetes. For a more detailed listing of the current literature and
potential clinical indications we would like to refer to our review article.26 The preferred clinical model for tissue repair is an autologous
transplant setting where the patient’s own hematopoietic tissue-derived stem cells are used. Cardiac tissue repair using autologous
hematopoietic tissue-derived stem cells is leading the field.27,28,29
BM- or PB-derived stem cells are applied via intravenous, intramyocardial, intracoronary, transendocardial, intramuscular, intravitreal,
or intraperitoneal administration.
Under steady-state conditions, cell repair originating from either tissue intrinsic stem cell pools or from PB, or both, is probably
an ongoing process throughout lifetime at a low frequency. As translational researchers we need to learn how to manipulate the system
to use it optimally for therapeutic purposes. Two approaches seem
to be promising:
1. manipulate the microenvironment at the site of tissue injury to
recruit circulating cells including stem cells (e.g.; SDF-1), and
2. increase the concentration of hematopoietic tissue-derived cells
including stem cells at the site of tissue injury by systemic cell
mobilization (cytokines).
SUMMARY
Regenerative Medicine as a new treatment modality by using
hematopoietic tissue-derived cells is at its very beginning. Whereas
there is convincing experimental evidence for both developmental
stem cell plasticity and cell fusion large animal and clinical data are
still scarce lacking the “sophistication” of small animal experimental
models. It remains to be shown whether promising treatment strategies can be designed to redirect hematopoietic tissue-derived cells
including stem cells to generate solid organ tissue in vivo.
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Krause DS. Lack of fusion requirement for development of bone
marrow-derived epithelia. Science 2004;305:90-93
2. Jang YY, Collector MI, Baylin SB, Diehl AM, Sharkis SJ. Hematopoietic stem cells convert into liver cells within days without fusion.
Nature Cell Biology 2004;6:532-539
3. Körbling M, Huh YO, Durett N, Mirza N, Miller P, Engel H, Anderlini P, van Besien K, Andreeff M, Przepiorka D, Deisseroth AB,
Champlin RE. Allogeneic blood stem cell transplantation: Peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+Thy-1dim) and lymphoid subsets, and possible
predictors of engraftment and graft-versus-host disease. Blood
1995;86:2842-2848.
4. Huss R, Lange C, Weissinger EM, Kolb HJ, Thalmeier K. Evidence
of peripheral blood-derived, plastic-adherent CD34-/low hematopoietic stem cell clones with mesenchymal stem cell characteristics.
Stem Cells 2000;18:252-260.
5. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M,
Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced
mobilization of bone marrow-derived endothelial progenitor cells
for neovascularization. Nature Medicine 1999;5:434-438.
6. Körbling M, Reuben JM, Gao H, et al. Recombinant human granulocyte-colony-stimulating factor-mobilized and apheresis-collected
endothelial progenitor cells: a novel blood cell component for therapeutic vasculogenesis. Transfusion 2006;46:1795-1802
S27
7. Jiang Y, Jahagirda BN, Reinhardt RL, Schwartz RE, Keene CD,
Ortiz-Gonzales XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du
J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM.
Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41-49.
8. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T,
Lenvik T, Johnson S, Hu W-S, Verfaillie CM. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291-1302.
9. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic
stem cells can differentiate into hepatocytes in vivo. Nature Medicine 2000;6:1229-1234
10. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S,
Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;
105; 369-377.
11. Grant MB, May WS, Caballero S, Brown GAJ, Guthrie SM, Mames
RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult
hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nature medicine 2002;8:607612.
12. Masuya M, Drake CJ, Fleming PA, Reilly CM, Zeng H, Hill WD,
Martin-Studdard A, Hess DC, Ogawa M. Hematopoietic origin of
glomerular mesangial cells. Blood 2003;101:2215-2218
13. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168-1170
14. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy
M, Lagasse E, Finegold M, Olson S, Grompe M. Cell fusion is
the principal source of bone-marrow-derived hepatocytes. Nature
2003; 422:897-901
15. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003; 422: 901-904.
16. Do JT, Scholer HR. Nuclei of embryonic stem cells reprogram somatic cells. Stem cells 2004;22:941-949
17. Blau HM. A twist of fate. Nature 2002;419:437
18. de Coppi P, Bartsch G, Siddiqui MM, et al. Isolation of amniotic
stem cell lines with potential for therapy. Nature Biotechnology
2007;25;100-106
19. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F,
Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone
marrow cells repair the infracted heart, improving function and survival. Proc. Natl. Acad. Sci. USA 2001; 98: 10344-10349.
20. Körbling M, Katz RL, Khanna A, Ruifrok A, Rondon G, Albitar M,
Champlin RE, Estrov Z. Hepatocytes and epithelial cells of donor
origin in recipients of peripheral-blood stem cells. The New England Journal of Medicine 2002; 346: 738-746.
21. Hematti P, Sloand EM, Carvallo CA, Albert MR, Yee CL, Fuehrer
MM, Blancato JK, Kearns WG, Barrett JA, Childs RW, Vogel JC,
Dunbar CE. Absence of donor-derived keratinocyte stem cells in
skin tissue cultured from patients after mobilized peripheral blood
hematopoietic stem cell transplantation. Experimental Hematology
2002;30:943-949
22. Okamoto R, Yajima T, Yamazaki M, et al. Damaged epithelia regenerated by bone marrow-derived cells in the human gastrointestinal
tract. Nature Medicine 2002; 8: 1011-1017.
23. Lu D, Sanberg PR, Mahmood A, Li Y, Wang L, Sanchez-Ramos
J, Chopp M. Intravenous administration of human umbilical cord
blood reduces neurological deficit in the rat after traumatic brain
injury. Cell Transplant 2002;11:275-281.
24. Camargo FD, Finegold M, Goodell MA. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners.
J Clin Invest 2004;113:1266-1270
25. Weimann JM, Charlton CA, Brazelton TR, Hackman RC, Blau HM.
Contribution of transplanted bone marrow cells to Purkinje neurons
in human adult brains. Proc Natl Acad Sci USA 2003;100:20882093
26. Körbling M, Estrov Z. Adult stem cells for tissue repair – a new
therapeutic concept? N Engl J Med 2003;349:570-582.
27. Lunde K, Solheim S, Aakhus S, et al. Intracoronary injection of
mononuclear bone marrow cells in acute myocardial infarction. N
Engl J Med 2006;355:1199-1209
28. Assmus B, Honold J, Schachinger V, et al. Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J
Med 2006;355:1222-1232
29. Schachinger V, Erbs S, Elsasser A, et al. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl
J Med 2006;355:1210-1221
S28
CONFERENCE
Hematopoietic Stem Cell Transplantation (HSCT) in
Uruguay.
Martha Nese
Clínica Hematológica. School of Medicine - Montevideo, Uruguay
One of the most dramatic contributions of the last fifty years
has been the demonstration that hematopoietic stem cells collected
from the bone marrow or peripheral blood can engraft in humans.
Since then, the use of allogeneic or autologous transplantation
(HSCT) has increased in patients with a variety of hematologic disorders.
The first allogeneic bone marrow transplantations in humans
were done in 1957 by Prof. E. Donnall Thomas, but the first successful HLA-identical sibling donor transplant was reported by
G. Mathé in 1968. In 1977, twenty years after his first trial E D.
Thomas reported the transplant outcomes of the first 100 patients
receiving high dose chemo-radiotherapy and allogeneic transplantations as treatment for refractory leukemia. In 1990 Prof. Thomas
received the Nobel Prize in Medicine.
The introduction of bone marrow transplantation in Uruguay to
improve the outcome of some hematologic malignancies was done
in 1985 by Prof. Roberto De Bellis. His team was one of the first in
South America.
Since then, the use of allogeneic or autologous bone marrow
transplantations (BMT) has increased with gradually improving results.
In 1987 Dr. De Bellis won the National Medicine Award for his
work in the field of Bone Marrow Transplantation.
Since 1995 The National Resource Fund (FNR), a non-governmental public body provides financial coverage for transplantation.
This is a solidarity system that enables a high-cost medical care for
the whole population, which was developed through the joint efforts
of both public and private sectors.
Four Highly Specialized Medical Institutes (IMAES), authorized
by the Ministry of Public Health, perform hematopoietic stem cell
transplantation (HSCT) in Uruguay. (Asociación Española, Hospital
Británico, IMPASA and Hospital Maciel)
This report evaluates annual numbers, indications, recipient
age, graft source, transplant regimens and the long-term results of
treatment and outcome of HSCT performed in Uruguay using data
derived from patients transplanted in 1996 - 2006 and the report
given by the transplant staff centers to FNR.
The annual numbers of blood and marrow transplantations
have decreased in the past few years both in Uruguay and worldwide (Figure 1, 2). The drop in autotransplants was due to a decrease in their use for solid tumors particularly for breast cancer.
The decline in allotransplant results from a decrease in their use
for chronic myelogenous leukemia as a result of the new therapy
with imatinib.
From 1996 to 2006, 1027 HSCT were performed in 969 patients, 849 (83% received autotransplants and 178 (17%) HLA identical sibling transplants (1) (Figure 3). Fifty eight were tandem
Because of the new approaches that have been developed in
the past several years, the average age of recipients for both autologous and allogeneic hematopoietic stem cells transplantation has
increased. Improvements in supportive care and innovations to decrease regimen-related morbidity and mortality may be responsible
for this trend. Forty-six percent of transplant recipients reported at
the FNR were older than 40 years of age, and 7% were older than
60. Trends in HSCT by recipient age and sex from 1996 to 2006 are
shown in Figure 4.
Figure 1. Trends in HSCT
FNR annual numbers 1995-2005
140
120
100
80
SCT
60
40
20
0
1995
97
99
1
3
5
Figure 2. Trends in HSCT FNR 2003- 2005a
120
100
80
Autologous
Allogeneic
Total
60
40
20
0
2003
2004
2005
Figure 3. Blood and Marrow Transplantation in Uruguay 1996
- 2006
Nº = 1027 SCT in 969 patients
178: 17 %
autologous
allogeneic
849: 83%
N= 58 2nd transplants
National Resource Fund (FNR)
S29
Figure 4. Trends in HSCT in Uruguay
Recipient Age FNR 1996- 2006
250
200
150
Total
Female
Male
100
50
0
<10
10a 19 20-29
30-39
40-49 50-59
>60
Figure 5. Indications for Hematopoietic Stem Cell Transplantation in Uruguay
350
300
250
200
allogeneic
autologous
150
100
Initially, transplantation was done with bone marrow grafts
(BM). From 1997, there was an increase in the use of peripheral
blood stem cell grafts (PBSC). Since 2001, this has been the predominant type of stem cells source used in allogeneic and autotransplants in Uruguay (Figure 6). These data show no significant
difference in the patient’s outcome between BM and PBSC.
The conditioning regimens more frequently used were: CVB,
BEAC, BEAM in Lymphoma; Bu/Cy in acute leukemia; Melfalan in
MM; Maxi-ICE in ST; and Cy/ATG in AA.
The disease status at transplantation among patients receiving transplants from 1996 to 2001(2) was; 49% in first complete remission (CR1), 22% in first partial remission (PR1), 11% in second
complete remission (CR2), 5% in second partial remission (PR2),
2% chronic phase (CF) and 3% refractory or relapse (R or REL)
Figure 7.
Hematological median time of recovery was: 10 and 12 days
for neutrophils (>0.5x109/l); 14 and 18 for platelets (>20x109/l) in
autologous and allogeneic hematopoietic stem cells transplantation
respectively. Hospitalization median time was 32 (+/-12), and 42
(+/- 17) days respectively.
Early mortality was lower after autotransplantation than after
allotransplantation, 100-day mortality from 1996 to 2001 after autotransplant was 7.5% and 30% in allogeneic (Figure 8). Death in
the first 100 days following allotransplantation was most commonly
due to graft-versus-host disease (GVHD), infections, or multi-organ
dysfunction. The stage of the disease at transplantation also had
an effect on 100-day mortality. Recurrence of primary disease accounted for the majority of deaths after autotransplantation.
Figure 7. Pretransplant disease status
FNR 1996- 2001
3%
50
2%
0
NHL
MM
HD
ALL
AML
AA
MDS
ST
CML
O
8%
5%
CLL
11%
49%
Figure 6. Stem Cell Source 95-06
22%
40
CR1
PR1
CR2
PR2
CF
R o Rel
Others
PB
Figure 8. 100-day Mortality
30
MO
FNR 96- 01
45
20
40
35
30
25
10
autologous
allogeneic
20
15
0
95
96
97
98
99
00
01
02
03
04
05
10
06
CITMO 2006
The most common indications for transplantation in Uruguay
have been allogeneic primarily for leukemia and autologous primarily for non Hodgkin’s lymphoma (NHL), myeloma (MM) and Hodgkin’s (HD) disease. In a total of 849 autotransplants, 55.5% were
lymphoma (NHL 291; HD 181), 17.4 % multiple myeloma (MM 147),
16% acute leukemia (AML 99; ALL 34), and 8 % solid tumors (ST
71). In 178 allotransplants the indications were 50% acute leukemia (AML 48; ALL 32), 20% chronic myelogenous leukemia (CML
35), and 13% aplastic anemia (AA 24), and 9.5% myelodysplasia
(SMD 17) (Figure 5).
5
0
NHL
AL
CML
Reduced-intensity conditioning regimens have recently been
introduced in Uruguay and we do not have any data yet.
Among patients receiving autotransplants the three-year probability of survival was: NHL 75%; HD 84%, MM 60% (Figure 9), AML
58% (Fig 10); ALL 57% (Fig 11). Some teams used maintenance
chemotherapy in ALL autotransplants in order to improve outcome
(abst. 127)
. In HLA identical sibling HSCT the three-year probability of
survival was: CML 46% (Figure 12); AML 37%; ALL 37%.
S30
Evaluations of transplant outcome with annual mandatory report could potentially improve our ability to demonstrate the effectiveness of HSCT.
In Uruguay the FNR guarantees and assesses the quality of
the care provided by transplant centers, the degree of efficiency
with which procedures are carried out and the results of the procedures that are performed.
Figure 9. Probability of Survival after Autotransplants
FNR Uruguay : 1996-2001
120
Nº = 290
100
80
106
60
136
48
40
HL
NHL
MM
84%
75%
60%
20
REFERENCE
1. The data in this report was submitted by the FNR register department, and this represents the information that the transplant
center staff gave to the FNR when the transplantations were
done, from 1996 to 2006.
0
0
90
365
730
2.- Patients follow up during the period 1996-2001 is reported
on the FNR web site by Correa F, Albornoz H, Cambogi R et
al ”2002 Programa de seguimiento de Trasplante de Médula
Osea”
1095
Figure 10. FNR : Probability of Survival after HSCT
for AML 1996-2001
120
ALPHABETICAL LISTING OF URUGUAYAN TRANSPLANT
CENTERS
Nº = 59
100
80
58%
60
37%
40
Autologous
Allogeneic
20
0
year
1
2
3
for ALL 1996-2001
120
Nº = 27
100
80
57%
autologous
allogeneic
60
33%
40
0
1
2
3
for CML 1996-2001
120
Nº = 24
100
80
60
CML 46%
40
20
0
day
90
365
730
1095
Hospital Británico
Director Técnico, Prof. Dr. Roberto De Bellis
Prof. Agdo. Pablo Muxi
Dr. Jorge Di Landro
Dra. Ada Caneiro
Dra. Laura Bello
Dra. Silvia Pierri
Dr. Andrew Miller
Dr. Diego Estol
IMPASA
Director Técnico Prof. Dra. Martha Nese
Prof. Adj. Cecilia Guillermo
Prof. Agda. Lilian Díaz
Prof. Adj. Hugo Isaurralde
Prof. Adj. Juan Zunino
Asistente. Laura Topolansky
Dra. Mariana Stevenazzi
Dra. Andrea Díaz
Dra Susana Perdomo
Dra. Ana Perdomo
Dra. Graciela Lavagna
20
year
Asociación Española (AEPSM)
Director Técnico adultos, Dr. Lem Martínez
Director Técnico pediatría, Dr. Luís Castillo
Dra. Adriana Cardeza
Dra. Lina Foren
Dra. Marcia Minutti
Dra Isabel Lopez
Dra. Ana Galán
Dr. Gustavo Dufort
Dr. Daniel Pieri
Dr. Agustín Dabezies
Dra. Mariela Catiglioni
Dra. Carolina Pages
Prof. Dr. Jorge Decaro
Dra. Mercedes Samora
Hospital Maciel
Director Técnico Dr. Enrique Bodega
Dr. Raúl Gabus
Dra. Alicia Magariños
Dra. Mercedes Zamora
Dra. Elena De Lisa
Dr. Wilson Franca
Dr. Juan Ferrari
Dra. Silvia Quiñones
S31
PUBLICATIONS LIST: URUGUAYAN TRANSPLANT CENTERS
1985-2006
1. INTRODUCCION ADAPTACION Y PERSPECTIVAS DEL
TRASPLANTE DE MEDULA OSEA EN EL URUGUAY DPTO.
HEMATOLOGÍA CLÍNICA DE BELLIS R, NESE M, MILLER A,
DI LANDRO J, CANEIRO A, ESTOL D, BERMÚDEZ J, BELLO
H, RUSSI J, QUADRELLI R, VIDAL J, TULLE S, STHANAN J,
VILA V, PEREZ CAMPOS H. 1ER PREMIO GRAN PREMIO
NACIONAL DE MEDICINA 10A.EDICIÓN.1986
2. PRIMEROS TRASPLANTES AUTOLOGOS DE MEDULA
OSEA EN EL URUGUAY DPTO. HEMATOLOGÍA CLÍNICA DE
BELLIS R; NESE M; MILLER A; DI LANDRO J; CANEIRO A;
ESTOL D 17 CONGRESO NACIONAL DE MEDICINA INTERNA. MONTEVIDEO 1986; 167-170
3. EFFECTIVITY OF A CONDITIONING REGIMEN FOR BONE
MARROW TRANSPLANTATION. DE BELLIS, R; NESE, M ;DI
LANDRO, J; CANEIRO, A. XXII CONGRESS OF THE ISH. MILAN. 1988.
4. PROFILAXIS DE LA ENFERMEDAD INJERTO CONTRA
HUESPED USANDO CICLOSPORINA Y ESTEROIDES CON
Y SIN TALIDOMIDA DPTO. HEMATOLOGÍA CLÍNICADE BELLIS R, NESE M, CANEIRO A, DI LANDRO J, MILLER A, SANTOS GW, VOGELSANG G B REVISTA DE LA SOCIEDAD DE
HEMATOLOGIA DEL URUGUAY 1990; 1(1): 26-29
5. PROPHYLACTIC ASSOCIATION OF THALIDOMIDE, CYCLOSPORINE AND STEROIDS FOR PREVENTION OF SEVERE
ACUTE GVHD IN BONE MARROW TRANSPLANTATION. DE
BELLIS, R ; NESE, M ; CANEIRO, A ET AL. 32ND. ANNUAL
MEETING OF ASH, XXIII CONGRESS OF THE ISH.BOSTON.1990.
6. GRAFT-VERSUS HOST DISEASE PROPHYLAXIS USING
CYCLOSPORINE AND STEROIDS WITH/WITHOUT THALIDOMIDE. DPTO. HEMATOLOGÍA CLÍNICA DE BELLIS R, NESE
M, CANEIRO A, DI LANDRO J, MILLER A, SANTOS GW, VOGELSANG BLOOD JOURNAL OF THE AMERICAN SOCIETY
OF HEMATOLOGY 1990; 76(10): ABST 2128
7. USO DE LOS FACTORES DE CRECIMIENTO EN EL
TRASPLANTE DE MEDULA OSEA.DPTO. HEMATOLOGÍA
CLÍNICA. DE BELLIS R, NESE M, DI LANDRO J- IV CONGRESO URUGUAYO DE HEMATOLOGIA. 1991: 103-105
8. SEGUIMIENTO CITOGENETICO Y MOLECULAR EN PACIENTES TRASPLANTADOS.DPTO. HEMATOLOGÍA CLÍNICA
URIARTE R, DE BELLIS R, NESE M, CARDOSO H IV CONGRESO URUGUAYO DE HEMATOLOGIA. 1991: 89-92
9. AUTOLOGOUS BONE MARROW TRASPLANTATION (ABMT)
USING EX-VIVO ETOPOSIDE (VP-16) WITH POOR RISK
LYMPHOMAS (LY) AND ACUTE LEUKEMIAS (LEUK) DPTO.
HEMATOLOGÍA CLÍNICA CIOBANU N, LAZARUS HM, DE
BELLIS R, ASCENSAO JA, SPARANO JA, GUCALP R, DUCTHER J, FOX RM, CREGER RJ, COOPER BW, GERSON LS,
NESE M, BELLO L, WIERNIK PH BLOOD JOURNAL OF THE
AMERICAN SOCIETY OF HEMATOLOGY 1993; 82 (10); ABST
2499
10. REMISSION OF PHILADELPHIA POSITIVE CHRONIC MYELOGENOUS LEUKEMIA ASSOCIATED WITH T(3;21) AFTER BONE MARROW TRANSPLANTATION. URIARTE MR,
MORI MA, DE BELLIS R, CARDOSO H. DIVISION CITOGENETICA, INSTITUTO DE INVESTIGACIONES BIOLOGICAS
CLEMENTE ESTABLE, MONTEVIDEO, URUGUAY. CANCER
GENET CYTOGENET. 1993 JUL 15; 68(2):122-5.
11. THALIDOMIDE PREVENTING GRAFT VERSUS HOST DISEASE. DPTO. HEMATOLOGÍA CLÍNICA DE BELLIS R, NESE
M, MUXI P, CANEIRO A, DI LANDRO J, MULLER A LA REVISTA. DE INVEST. CLINICA. XXV CONGRESS OF THE INTERNATIONAL SOCIETY OF HEMATOLOGY. MEXICO. APRIL:
1994; ABSTRACT 664, PAG. 353
12. TRASPLANTE DE MÉDULA ÓSEA EN LAS MUCOPOLISACARIDOSIS. MUXÍ P. VI CONGRESO
URUGUAYO DE
HEMATOLOGÍA 1995 MESA REDONDA: ACTUALIZACIÓN
EN TRASPLANTE DE MÉDULA ÓSEA
13. TRASPLANTE DE MEDULA OSEA AUTOLOGO CON PROGENITORES MEDULARES Y DE SANGRE PERIFERICA
DPTO. HEMATOLOGÍA CLÍNICA. NESE M, PERDOMO S,
PERDOMO A, GUILLERMO S, DIAZ L, ISAURRALDE H, GRIN-
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
BERG S, AGHAZARIAN M, VARANGOT M, MASI M, CAMEJO
REVISTA MEDICA DEL URUGUAY 1996; 12:106-111
TRASPLANTE DE MEDULA OSEA AUTOLOGO CON EXPANSION DE PROGENITORES Y TRATAMIENTO ANTIFACTOR
DE NECROSIS TUMORAL (TNF). DPTO. HEMATOLOGÍA
CLÍNICA NESE M, PERDOMO S, PERDOMO A, HISAURRALDE H, DIAZ L, GUILLERMO C, GRINBERG S, AGHAZARIAN
M, MASI M, CAMEJO
EN 25 CONGRESO NACIONAL
DE MEDICINA INTERNA, MONTEVIDEO: OFIC. DEL LIBRO.
AEM 1996; 203-205
COMPLICACIONES INFECCIOSAS EN EL TRASPLANTE
AUTOLOGO CON PROGENITORES MEDULARES (PM) Y DE
SANGRE PERIFERICA (PSP). DPTO. HEMATOLOGÍA CLÍNICA GUILLERMO C, NESE M, DIAZ L, ISAURRALDE H, GRINBERG S, PERDOMO S, PERDOMO A, MASI M, CAMEJO
E. EN 25 CONGRESO NACIONAL DE MEDICINA INTERNA,
MONTEVIDEO: OFIC. DEL LIBRO. AEM 1996; 212-214
TRASPLANTE DE MEDULA OSEA (TMO) AUTOLOGO CON
PROGENITORES MEDULARES Y DE SANGRE PERIFÉRICA (SCMO-SCSP) DPTO. HEMATOLOGÍA CLÍNICANESE
M, PERDOMO S, GUILLERMO C, DIAZ L, ISAURRALDE
H, GRINBERG S, AGHAZARIAN M, VARANGOT M, MASI
M, CAMEJO E. HEMO 96: 27-30 OCTUBRE 1996. PORTO
ALEGRE. BRASIL ABST. 189 P
TRASPLANTE DE MEDULA OSEA AUTOLOGO EN CANCER
DE MAMA DEP. DE HEMATOLOGÍA CLÍNICA CTMO DÍAZ L,
NESE M, GUILLERMO C; ISAURRALDE H; GRINBERG S;
PERDOMO S; PERDOMO A, AGHAZARIAN M, GARVINO C,
VARANGOT M, MASI M, CAMEJO E, CONGRESOS ONCOLOGICOS DEL URUGUAY. XII CONGRESOS INTEGRADOS
LATINOAMERICANOS DE CANCEROLOGIA..1996, ARCH.
DE MED. INT. PAG 45.
TRASPLANTE DE MEDULA OSEA AUTOLOGO CON EXPANSION DE PROGENITORES MEDULARES Y DE SANGRE
PERIFERICA DEP. DE HEMATOLOGÍA CLÍNICA CTMO NESE
M, PERDOMO S; PERDOMO A, GUILLERMO C, DÍAZ L, ISAURRALDE H; GRINBERG S; AGHAZARIAN M, MASI M,
CAMEJO E, CONGRESOS ONCOLOGICOS DEL URUGUAY.
XII CONGRESOS INTEGRADOS LATINOAMERICANOS DE
CANCEROLOGIA. 1996, ARCH. DE MED. INT. PAG 45.
TRASPLANTE DE MEDULA OSEA AUTOLOGO EN TUMORES GERMINALES DEP. DE HEMATOLOGÍA CLÍNICA
CTMO ISAURRALDE H; NESE M, DÍAZ L, GRINBERG S;
GUILLERMO C; PERDOMO S; PERDOMO A, AGHAZARIAN
M, VARANGOT M, GARVINO C,MASI M, CAMEJO E, CONGRESOS ONCOLOGICOS DEL URUGUAY. XII CONGRESOS
INTEGRADOS LATINOAMERICANOS DE CANCEROLOGIA..
1996, ARCH. DE MED. INT. PAG 46.
COMPLICACIONES
NO
HEMATOLOGICAS
EN
EL
TRASPLANTE DE MEDULA OSEA AUTOLOGO DEP. DE
HEMATOLOGÍA CLÍNICA CTMO GRINBERG S; NESE M,
GUILLERMO C; DÍAZ L,, ISAURRALDE H; PERDOMO S;
PERDOMO A, AGHAZARIAN M, MASI M, CAMEJO E, CONGRESOS ONCOLOGICOS DEL URUGUAY. XII CONGRESOS
INTEGRADOS LATINOAMERICANOS DE CANCEROLOGIA.
9-11. DIC. 1996, ARCH. DE MED. INT. PAG 45
TRASPLANTE AUTOLOGO DE MEDULA OSEA (TAMO).
ANALISIS DE LA MORBIMORTALIDAD EN LOS PRIMEROS
60 PACIENTES. DPTO. HEMATOLOGÍA CLÍNICA DIAZ L,
ISAURRALDE H, NESE M, GUILLERMO C, PERDOMO S,
PERDOMO A. XXVI CONGRESO NACIONAL DE MEDICINA
INTERNA. PUB. OFICIAL 1997:252-254
TRASPLANTE DE MÉDULA ÓSEA: MANEJO PREVIO POR
EL INTERNISTA. MUXÍ P. CONGRESO NACIONAL DE MEDICINA INTERNA 1998.
TRASPLANTE DE MEDULA OSEA AUTOLOGO (TMOA)
DPTO. HEMATOLOGÍA CLÍNICA NESE M. HEMASUR 98. 2931:1998 URUGUAY
ANTI-TUMOR NECROSIS FACTOR (TNF) TREATMENT IN
OUTCOME OF AUTOLOGOUS BONE MARROW TRANSPLANTATION. DPTO. HEMATOLOGÍA CLÍNICA NESE M;
GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG
S; PERDOMO S; PERDOMO A. IBMTR/ABMTR,1998 ANNUAL MEETING.KEYSTONE, COLORADO: 8-14/1/1998. ABST B
07
S32
25. LOW INCIDENCE OF COMPLICATIONS IN BONE MARROW
TRANSPLANTATION (BMT) WITH ANTI TNF TREATMENT,
CTMO IMPASA, DPTO. HEMATOLOGÍA CLÍNICA NESE M;
GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG S;
PERDOMO S; PERDOMO A; MASI M; CAMEJO E. BLOOD
VOL. 92, Nº10, SUPPL. 1 (PART 2 OF 2) 1998. ABST 4401.
26. MOBILIZED BONE MARROW (BM) AND LARGE VOLUMES
LEUKAPHERESIS DPTO. HEMATOLOGÍA CLÍNICA PERDOMO S; PERDOMO A; NESE M; GUILLERMO C; DÍAZ L; ISAURRALDE H; BLOOD VOL 92, Nº10, SUPPL. 1 (PART 2 OF
2) 1998. ABST 4311
27. COMPLICACIONES NO INFECCIOSAS EN EL TRASPLANTE
DE MEDULA OSEA AUTOLOGO (TAMO) CITMO GRINBERG
S; NESE M, DÍAZ L, GUILLERMO C; ISAURRALDE H; PERDOMO S; PERDOMO A, CAMEJO E, MASI M. HEMASUR 98.
29-31:1998 URUGUAY, PAG. 100
28. ANALISIS DE LOS EPISODIOS FEBRILES EN EL CURSO DEL TRASPLANTE DE MEDULA OSEA. CENTRO DE
TRASPLANTE DE MEDULA OSEA (CITMO) CITMO CECILIA GUILLERMO, MARTHA NESE, LILIÁN DÍAZ, HUGO ISAURRALDE, SUSANA GRIMBERG, SUSANA PERDOMO,
ANA PERDOMO, MIGUEL MASI, EDUARDO CAMEJO. HEMASUR 98. 29-31:1998 URUGUAY, PAG. 101
29. ACUTE EFFECTS OF AMINOFOSTINE USED DURING AUTOLOGOUS BONE MARROW TRANSPLANTATION FOR
SOLID TUMORS. (ABSTRACT). DE BELLIS R, MUXÍ P, DI
LANDRO J, CANEIRO A, PIERRI S, BELLO L. PROCEEDINGS ASCO. 1999
30. EFFECT OF CD34+ DOSE ON BONE MARROW RECOVERY IN STEM CELL TRANS-PLANTATION. R.GABUS,
A.MAGARIÑOS, M.ZAMORA, E.DELISA, AI.LANDONI,
H.GIORDANO, C.CANESSA, G.MARTINEZ Y E.BODEGA.
ABSTRACT. TANDEM MEETING OF IBMTR/ABMTR AND
AMERICAN SOCIETY OF BONE MARROW TRANSPLANTATION. KEYSTONE.COLORADO. EEUU. 28 FEBRERO-6
MARZO 1999.
31. TRASPLANTE AUTOLOGO DE MEDULA OSEA CUANTIFICACION DE CELULAS CD34 DEP. LABORATORIO CITMO GALVARINI E, CASTAGNO A, PERDOMO S, PERDOMO A, NESE
M. 2DO CONGRESO URUGUAYO DE BIOQUIMICA CLINICA
23-25 SET 1999
32. FEVER ANALYSIS DURIG BMT IN A SINGLE CENTER. DPTO.
HEMATOLOGÍA CLÍNICA CITMO NESE M; GUILLERMO C;
DIAZ L; ISAURRALDE H; GRINBERG S; PERDOMO S; PERDOMO A; MASI M; CAMEJO E.
IBMTR/ABMTR, ASBMT,
TANDEN BMT MEETINGS KEYSTONE RESORT, COLORADO.1999, ABST. B15, PAG. 35
33. EVALUATION OF HEMATOPOIETIC PROGENITORS IN HEMATOPOIETIC PROGENITOR CELL TRANSPLANT. CD34+
DOSE EFFECT IN MARROW RECOVERY. RETROSPECTIVE ANALYSIS IN 38 PATIENTS.” R.GABUS. A.MAGARIÑOS.
M.ZAMORA.
E.DELISA.
AI.LANDONI,
G.MARTINEZ,
C.CANESSA. H.GIORDANO Y E.BODEGA. HEMATOLOGY
AND CELL THERAPY. 41:171-177. 1999
34. TRASPLANTE DE MEDULA OSEA. EVALUACION DESDE
MAYO DE 1995 A JULIO DE 1998. DPTO. HEMATOLOGÍA
CLÍNICA CITMO NESE M; GUILLERMO C; DIAZ L; ISAURRALDE H; GRINBERG S; PERDOMO S; PERDOMO A;
REV. MED. URUGUAY 1999, 15:57-65
35. TRASPLANTE AUTOLOGO DE MEDULA OSEA (TAMO) EN
LNH. DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M; ISAURRALDE H; GUILLERMO C; DIAZ L; GRINBERG S; PERDOMO S; PERDOMO A; TOPOLANSKY L.. XIV CONGRESO
ARGENTINO DE HEMATOLOGIA. MAR DEL PLATA. 1999,
POST. 149, PAG.203
36. ENFERMEDAD DE HODGKIN EXPERIENCIA EN TRASPLANTE AUTOLOGO. DPTO. HEMATOLOGÍA CLÍNICA CITMO
NESE M; GUILLERMO C; ISAURRALDE H; GRINBERG S;
DÍAZ L; PERDOMO S; PERDOMO TOPOLANSKY L.
X I V
CONGRESO ARGENTINO DE HEMATOLOGÍA. MAR DEL
PLATA. 1999; POST. 150, PAG.203
37. TRASPLANTE AUTOLOGO DE STEM CELLS (TASC) EN
LINFOMAS DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE
M; GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
S; PERDOMO S; PERDOMO A; TOPOLANSKY L; MASI M;
CAMEJO E. ARCH. MED. INT. 1999; XXI; 3: 97-101
AUTOLOGOUS BONE MARROW TRANSPLANTATION
(ABMT) IN MALIGNANTS LYNPHOMAS WITH MOBILIZED
BONE MARROW AND PERIPHERAL BLOOD STEM CELLS
DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M; ISAURRALDE H; GUILLERMO C; DÍAZ L; GRINBERG S; PERDOMO S;
PERDOMO A; TOPOLANSKY L. BLOOD VOL. 94, Nº 10, SUPPL. 1 (PART 2 OF 2) 1999. ABST 5030, PAG. 403B
TAMDEM AUTOLOGOUS TRANSPLANT FOR MYELOMA
DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M; ISAURRALDE H; GUILLERMO C; DÍAZ L; GRINBERG S; PERDOMO S;
PERDOMO A; TOPOLANSKY L BLOOD VOL. 94, Nº 10, SUPPL. 1 (PART 2 OF 2) 1999. ABST 5031, PAG. 403B
TMO CON STEM CELLS PERIFERICAS MEDIANTE LEUCAFERESIS DE GRAN VOLUMEN CITMO NESE M, GRIMBERG S, PERDOMO S, PERDOMO A, ISAURRALDE H, DIAZ
L, GUILLERMO C, CADENAS G, LORENZO J.
X V I
CONGRESO DE LA SOCIEDAD LATINOAMERICANA DE ONCOLOGIA PEDIATRICA (SLAOP) 25-29. ABRIL. 1999, PORLAND- ESTADO DE NUEVA ESPAÑA-VENEZUELA
LINFOMA NO HODGKIN Y TRASPLANTE AUTÓLOGO DE
MÉDULA ÓSEA Y/O PROGENITORES PERIFÉRICOS, RESEÑA GENERAL Y ANÁLISIS DE RESULTADOS DE 13 AÑOS
DE LA UNIDAD DE TRASPLANTE DE MÉDULA ÓSEA DEL
HOSPITAL BRITÁNICO. MUXÍ P, DI LANDRO J, CANEIRO A,
PIERRI S, BELLO L, DE BELLIS R. ARCHIVOS DE MEDICINA
INTERNA . 2000.
EFFECT ON CD34+ DOSE IN BONE MARROW RECOVERY.
RETROSPECTIVE ANALYSIS IN 60 PATIENTS” R.GABUS.
A.MAGARIÑOS. M.ZAMORA, E.DELISA, AI.LANDONI,
F.UTURBEY, G.MARTINEZ, C.CANESSA, H.GIORDANO Y
E.BODEGA. TANDEM MEETING OF THE IBMTR7ABMTR
AND ASBMT. ANAHEIM. CALIFORNIA. EEUU. MARZO 26ABRIL 1. 2000.
EXPERIENCIA DEL CITMO EN TRASPLANTE AUTOLOGO
CON PROGENITORES DE MEDULA OSEA Y SANGRE PERIFERICA DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M;
GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG S; PERDOMO S; PERDOMO A; TOPOLANSKY L; MASI M; CAMEJO
E
4º ENCONTRO SOBRE TRANSPLANTE DE MEDULA
OSEA E HEMOPATIAS MALIGNAS. CURITIBA BRASIL. 2000,
ABST 189P
TRASPLANTE DE MÉDULA ÓSEA EN LAS ENFERMEDADES
AUTOINMUNES. MUXÍ P. V SEMINARIO INTERNACIONAL
INMUNOMODULACIÓN. 2000.
EMBARAZO Y TRASPLANTE DE MEDULA OSEA DPTO. HEMATOLOGÍA CLÍNICA CITMOISAURRALDE H, GUILLERMO
C, NESE M, DIAZ L, GRIMBER S, PERDOMO, S, TOPOLANSKY L, BUFANO G. XXIX CONGRESO NACIONAL DE MEDICINA INTERNA, PUB. OFICIAL, MONTEVIDO, URUGUAY.
2000, PAG. 302- 304
MEDULA OSEA MOVILIZADA Y LEUCAFERESIS DE GRAN
VOLUMEN CITMONESE M, PERDOMO S, PERDOMO A, LAVAGNA G, GUILLERMO C, DIAZ L, ISAURRALDE H, GRINBERG S, GALVARINI E, CASTAGNO G. VII CONGRESO
URUGUAYO DE HEMATOLOGIA 2000; PÁG. 25
TRASPLANTE AUTOLOGO CON PROGENITORES HEMATOPOYETICOS (AUTO-TPH). EVALUACION DE 1995 A 2001.
DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M; GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG S; PERDOMO S;
PERDOMO A; TOPOLANSKY L; LAVAGNA G, BUFANO G.
LIZARRALDE A, BAUBETA A. MASI M, CAMAJO E. ARCH.
MED. INT. 2001;XXIII;4: 187-193
INTENSIFICATION/ENHACEMENT TREATMENT WITH ABMT
IN LYMPHOMAS. INDICATION AND PROGNOSTIC FACTORS. EXPERIENCE IN ONE CENTRE: THE HOSPITAL MACIEL. A MAGARIÑOS, R.GABUS, M.ZAMORA, E.DELISA, AI
LANDONI, F.UTURBEY, C.CANESSA Y E.BODEGA.PRESENTACIÓN EN SESIÓN DE POSTERS. TANDEM MEETING OF
THE IBMTR/ABMTR AND ASBMT. KEYSTONE. COLORADO.
15-19 DE FEBRERO DE 2001.
SINGLE AND TANDEN AUTOLOGOUS HERMATOPOIETIC
SEM CELL TRANSPLANTATION (AHSCT) DPTO. HEMA-
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
TOLOGÍA CLÍNICA CITMO NESE M; GUILLERMO C; DÍAZ L;
ISAURRALDE H; GRINBERG S; PERDOMO S; PERDOMO A;
TOPOLANSKY L; BUFANO G, LIZARRALDE A; BAUBETA A;
MASI M; BLOOD; VOL 98, N°11:2001, ABST 5367
TRASPLANTE AUTOLOGO DE PROGENITORES HEMATOPOYETICOS (TAPH) EN LINFOMAS. DPTO. HEMATOLOGÍA
CLÍNICA CITMO NESE M; GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG S; PERDOMO S; PERDOMO A;
TOPOLANSKY L; BUFANO G. HEMASUR 2001, MAR DEL
PLATA ARGENTINA, ABST. 0 43, PAG. 106
DOBLE TRASPLANTE AUTOLOGO DE PROGENITORES HEMATOPOYETICOS (DTA). DPTO. HEMATOLOGÍA CLÍNICA
CITMO NESE M; DÍAZ L; GUILLERMO C; GRINBERG S; ISAURRALDE H; PERDOMO S; PERDOMO A; TOPOLANSKY L;
BUFANO G. HEMASUR 2001, MAR DEL PLATA ARGENTINA,
ABST. P 287, PAG. 169
TRASPLANTE DE PROGENITORES HEMOPOYETICOS
(TPH). DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M;
GUILLERMO C; DÍAZ L; ISAURRALDE H; GRINBERG
S; PERDOMO S; PERDOMO A; TOPOLANSKY L; LAVAGNA
G, BUFANO G. LIZARRALDE A, BAUBETA A. MASI M, XXX
CONGRESO NACIONAL DE MEDICINA INTERNA. MONTEVIDEO URUGUAY. 6-11, NOV:2001
TRASPLANTE AUTOLOGO DE PROGENITORES HEMOPOYETICOS EN LINFOMA DE HODGKIN DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M; GUILLERMO C; DÍAZ L;
ISAURRALDE H; TOPOLANSKY L; BUFANO G; GRINBERG S;
PERDOMO S; PERDOMO A; LAVAGNA G. XXXI CONGRESO
NACIONAL DE MEDICINA INTERNA. URUGUAY PUB. OFICIAL, ELECTRONICA. CD, 8/11/2002
TRASPLANTE DE PROGENITORES HEMATOPOYETICOS
EN PACIENTES CON MIELOMA MULTIPLE. DPTO. HEMATOLOGÍA CLÍNICA CITMO NESE M; DÍAZ L; GUILLERMO C;
ISAURRALDE H; TOPOLANSKY L; BUFANO G; GRINBERG
S; PERDOMO S; PERDOMO A, LAVAGNA G. XXXI CONGRESO NACIONAL DE MEDICINA INTERNA. MONTEVIDEO.
URUGUAY, PUB. OFICIAL, ELECTONICA, CD 8/11/2002
CASO CLÍNICO: ENFERMEDAD DE HODGKIN. TMO NO
MIELOABLATIVO. TERAPIA CELULAR. UTURBEY F, BORELLI G, GABÚS R, MAGARIÑOS A, DE LISA E, ZAMORA M, E
BODEGA. PRESENTACIÓN EN POSTER EN IX CONGRESO
URUGUAYO DE HEMATOLOGÍA. 10-12 DE OCTUBRE 2002.
AUTOLOGOUS STEM CELL TRANSPLANTATION (ASCT)
FOR POOR PROGNOSIS LYMPHOMAS DPTO. HEMATOLOGÍA CLÍNICA CITMO MARTHA NESE, CECILIA GUILLERMO, LILIÁN DÍAZ, HUGO IZAURRALDE, SUSANA GRINBERG, LAURA TOPOLANSKY, GLEDYS BUFFANO, SUSANA
PERDOMO, ANA PERDOMO, GRACIELA LAVAGNA BLOOD
VOL; 100;Nº:2002, ABSTRACT:5499, PAG. 479B
REPORTE DEL SERVICIO DE HEMATOLOGÍA DEL HOSPITAL MACIEL. NEWSLETTER ISCT (INTERNATIONAL SOCIETY OF CELL THERAPY) AUTOR: DR. RAÚL GABÚS. OCTUBRE 2003.
RESULTADO DE TRASPLANTE DE MÉDULA ÓSEA ALOGÉNICO (TMO) EN 7 PACIENTES CON APLASIA SEVERA
DE MÉDULA ÓSEA. MUXÍ PJ, PIERRI S, CANEIRO A, DI
LANDRO J, BELLO L, DE BELLIS R. MESA DE TRABAJOS
LIBRES. XXXII CONGRESO NACIONAL DE MEDICINA INTERNA. NOVIEMBRE 2003. 3ER PREMIO
LINFOMA NO HODGKIN. TRASPLANTE AUTOLOGO DE
PROGENITORES HEMATOPOYETICOS
(TAPH). DPTO.
HEMATOLOGÍA CLÍNICA CITMO MARTHA NESE; CECILIA
GUILLERMO; LILIAN DÍAZ; HUGO ISAURRALDE; LAURA
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2003; 1:09-14
MAINTENANCE TREATMENT AFTER AUTOLOGOUS BMT
IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) DEP. DE
HEMATOLOGÍA CLÍNICA CITMO MARTHA NESE, CECILIA
GUILLERMO, LILIÁN DÍAZ, HUGO ISAURRALDE, LAURA
TOPOLANSKY, GLEDYS BUFFANO, ALBERTO BAUBETA,
SUSANA PERDOMO, ANA PERDOMO, GRACIELA LAVAGNA
BLOOD VOL;102 ;Nº11: 2003, ABSTRACT: 5660, PAG. 483B
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61. TRASPLANTE DE MEDULA OSEA PANORAMA ACTUAL
DEP. DE HEMATOLOGÍA CLÍNICA CITMO NESE M “50 ANIVERSARIO DEL HOSPITAL DE CLINICAS “ CURSO DE ACTUALIZACION EN HOMENAJE AL PROF. DR. R. DE BELLIS.
23. SET. 2003 PUBLICACION ELECTRONICA
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PORTADORES DE LEUCEMIA MIELOIDE CRÓNICA. EXPERIENCIA DEL SERVICIO DE HEMATOLOGÍA Y TRASPLANTE
DE MÉDULA OSEA DEL HOSPITAL MACIEL, M.S.P. MAGARIÑOS A, BÓDEGA E, GABUS R, ZAMORA M, DE LISA E, BENGOCHEA M, SENATORE O, BONOMI R, URIARTE R, UTURBEY F, GALEANO S, BORELLI G*, POMOLI S, MARCHETTI N,
DE GIUDA R, SERVICIO DE HEMATOLOGÍA Y TRASPLANTE DE MÉDULA OSEA, HOSPITAL MACIEL, M.S.P., BANCO
NACIONAL DE ORGANOS Y TEJIDOS, LABORATORIO DE
CITOGENÉTICA Y BIOLOGÍA MOLECULAR, ASOC.ESP 1ª
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63. EL LABORATORIO EN EL TRASPLANTE DE MEDULA OSEA.
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CLÍNICA CITMO NESE M, GUILLERMO C; DÍAZ L, ISAURRALDE H; GRINBERG S; PERDOMO S; PERDOMO A,
TOPOLANSKY L, LAVAGNA G, LIZARRALDE A, BAUBETA
A, MASI M, CAMEJO E, LABORATORIO AL DIA Nº 10, OCT
2004, PÁG. 19
64. TRASPLANTE DE PROGENITORES HEMATOPOYETICOS
DEP. DE HEMATOLOGÍA CLÍNICA NESE M, PAGINA WEB
DEL DEPARTAMENTO DE MEDICINA. 2004
65. ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN MACIEL HOSPITAL, URUGUAY. SINGLE
CENTER STUDY. S.GALEANO, R.GABÚS, L.SERVENTE,
A.MAGARIÑOS, M.ZAMORA, E.DELISA, G.BORELLI,
R.DEGUIDA, N.MARCHETTI, S.POMOLI, E.BODEGA. ABSTRACT BOOK. 0939. EHA. ESTOCOLMO. SUECIA. HAEMATOGICA. JUNE.2005.
66. TREATMENT OF 22 PATIENTS WITH AMYOTHROPHYC
LATERAL SCLEROSIS, GRAFTING STEM CELLS THROUGH
NEUROENDOSCOPY. ROBERTO DE BELLIS, MD1, ALVARO
CORDOBA, MD2,*, PABLO J. MUXI, MD3,*, LAURA BELLO,
MD4,* AND ADA CANEIRO, MD5,* HEMATOLOGY, BRITISH
HOSPITAL, MONTEVIDEO, URUGUAY. BLOOD (ASH ANNUAL MEETING ABSTRACTS) 2006 108: ABST. 278.
67. HEMATOPOIETIC STEM CELL TRANSPLANTATION (SCT)
A SINGLE CENTER 10 YEARS EXPERIENCE GUILLERMO
C; DÍAZ L, ISAURRALDE H; TOPOLANSKY L; BAUBETA
A, LIZARRALDE A, TESTA G, PERDOMO S; PERDOMO A,
LAVAGNA G, NESE M. CENTRO IMPASA DE TRASPLANTE
DE MEDULA ÓSEA (CITMO), HEMATOLOGIC DEPARTMENT. MEDICINE DEPARTMENT, UNIVERSITY OF MEDICINE. MONTEVIDEO, URUGUAY. AMERICAN SOCIETY FOR
BLOOD AND MARROW TRANSPLANTATION MEETING 2006
(ABST. 282)
68. AUTOLOGOUS STEM CELL TRANSPLANTATION (ASCT)
FOR POOR PROGNOSIS NON HODGKIN LYMPHOMAS
(NHL). NESE M, GUILLERMO C; DÍAZ L, ISAURRALDE H;
TOPOLANSKY L ; ZUNINO J; PERDOMO S; PERDOMO A, ,
LAVAGNA G, DIAZ A; STEVENAZZI M;, BAUBETA A, CENTRO
IMPASA DE TRASPLANTE DE MEDULA ÓSEA (CITMO), HEMATOLOGIC DEPARTMENT. MEDICINE DEPARTMENT, UNIVERSITY OF MEDICINE. MONTEVIDEO, URUGUAY. BLOOD
VOL; 108; Nº11: 2006, ABST. 5430, PAG. 452B
69. LINEAGE SPECIFIC CHIMERISM ANALYSIS ALLOWS
EARLY DETECTION OF RELAPSES ALFTER ALLOGENEIC
STEM CELL TRANSPLANTATION. GALEANO S, GABÚS R,
BENGOCHEA M, BOIRON J-M, CARRETO E, BÓDEGA E, ALVAREZ I 11TH CONGRESS OF THE EUROPEAN HEMATOLOGY ASSOCIATION. ABSTRACT BOOK. 2006; 91(S1); 407.
ABSTRACT NO 1115. AMSTERDAN. HOLANDA. JUNE 2006
70. MAINTENANCE CHEMOTHERAPY POST AUTOLOGOUS
STEM CELL TRANSPLANTATION IN ADULT ACUTE LYMPHOCYTIC LEUKEMIA (ALL) TOPOLANSKY L, STEVENAZZI
M, ZUNINO J, DÍAZ. A, GUILLERMO C, DÍAZ L, ISAURRALDE
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H, PERDOMO S, PERDOMO A, LAVAGNA G, NESE M. INSTITUTION: CLÍNICA HEMATOLÓGICA. HOSPITAL DE CLÍNICAS, FACULTAD DE MEDICINA. CITMO.- IMPASA ISH 2007
PUNTA DEL ESTE URUGUAY (ABST127)
71. HEMATOPOIETIC STEM CELL TRANSPLANTATION (HCT)
FOR ACUTE MYELOID LEUKEMIA (AML). DIAZ A, TOPOLANSKY L, STEVENAZZI M, ZUNINO J, GUILLERMO C, DÍAZ L,
ISAURRALDE H, PERDOMO S, PERDOMO A, LAVAGNA G,
NESE M. INSTITUTION: CLÍNICA HEMATOLÓGICA. HOSPITAL DE CLÍNICAS, FACULTAD DE MEDICINA. CITMO.- IMPASA ISH 2007 PUNTA DEL ESTE URUGUAY(ABST128)
72. AUTOLOGOUS STEM CELL TRANSPLANTATION (ASCT)
IN MULTIPLE MYELOMA (MM). IMPACT OF SURVIVAL. ISAURRALDE H, DÍAZ L, GUILLERMO C, TOPOLANSKY L,
ZUNINO J, PERDOMO S, PERDOMO A, LAVAGNA G, STEVENAZZI M, DÍAZ A, NESE M. CENTRO IMPASA DE TRASPLANTE DE MÉDULA ÓSEA (CITMO), CLÍNICA HEMATOLÓGICA,
FACULTAD DE MEDICINA, UNIVERSIDAD DE LA REPÚBLICA, MONTEVIDEO, ISH 2007 PUNTA DEL ESTE URUGUAY
(ABST126)
73. HEMATOPOIETIC STEM CELL TRANSPLANTATION (SCT) A
SINGLE CENTER 11 YEARS EXPERIENCE. GUILLERMO C,
DÍAZ L, ISAURRALDE H, TOPOLANSKY L, ZUNINO J, STEVENAZI M, DIAZ A, PERDOMO S, PERDOMO A, LAVAGNA
G. BAUBETA A, NESE M, CITMO, CLÍNICA HEMATOLÓGICA.
FACULTAD DE MEDICINA MONTEVIDEO URUGUAY ISH
2007 PUNTA DEL ESTE URUGUAY (ABST 70)
S35
CONFERENCE
Prognostic Factors in Multiple Myeloma
Guillermo Conte, Daniel Araos, Gastón Figueroa
Hematology Department
Clinical Hospital University of Chile
INTRODUCTION
Multiple Myeloma (MM) is a heterogeneous illness at its biological and clinical levels, with a survival range between a few months
and more than 10 years. During the last decades significative advances have been achieved in the identification of prognostic clinical, biological, cytogenetics and molecular factors, which allow to
classify each patient in risk categories. In past years the increase
of the therapeutic strategies with new agents allowed to get a complete remission in a larger number of patients. With all this information, it will be easier to make a better therapeutic choice in the
different phases of MM.
EPIDEMIOLOGY
MM affects mainly elderly patients with a mean age of 65 years
at diagnosis. Only 1% of patients is under 40 years. It is more frequent in males (male-female ratio 1.4:1). The incidence increases
exponentially with age. The age-adjusted incidence in USA (SEER:
US Surveillance Epidemiology and End Results Programme) between 1992 and 1998 was 4.5 per 100,000 inhabitants per year
while in the European Union it was 5.72 per 100.000 inhabitants/
year in 1998. The MM incidence is higher in black population and
lower in the Asian population (1).
The survival rate of patients is variable. In the SEER Register,
survival at 5 years was 32% while in an English study the survival at
3 years was only 39% (2).
MONOCLONAL GAMMOPATHIES
Monoclonal gammopathies constitutes a spectrum of diverse
pathologies within which is MM. The most frequent is the monoclonal gammopathy of undetermined significance (MGUS), with a
prevalence of 3.2% in people older than 50 years, which increases
with age. The MGUS might be a potential precursor of MM, with a
risk of progression from MGUS to MM of 1% per year (3). The risk
of progression to MM is associated to 3 factors: serum M-protein
>15 gr/L, immunoglobulin no-IgG, abnormal free light chain ratio
(kappa/lambda <0.26 or >1.65). The risk of progression at 20 years
is 5% when no factor is found and 58% when the 3 factors are
present (4).
The diagnostic criteria of MGUS, asymptomatic MM (smoldering) and symptomatic MM are shown in Table 1 (5).
PROGNOSTIC FACTORS IN MULTIPLE MYELOMA
Prognostic factors can be classified into 3 groups:
a) Factors associated to the host
b) Factors reflecting the characteristics of malignant clone
c) Factors resulting from the interaction between the tumor and
the host (tumor burden, complications, response to treatment)
a) Factors related to the host
Age and the performance status of patients are important determinants of the prognostic of patients. Ages under 60-70 years are
associated to a prolonged survival versus elderly patients who are
associated to a poor prognostic independently of other risk factors.
The presence of a good performance status (ECOG 0-2) is also associated to a favorable prognostic (6).
Table 1. Diagnostic Criteria of MGUS, asymptomatic MM and
symptomatic MM
Diagnostic Criteria of the Monoclonal Gammopathy of Undetermined Significance
All 3 criteria are required:
1. Monoclonal paraprotein in serum <3gr/dL or in urine <1gr/24hr
2. Bone marrow plasma cells <10%
3. Normal calcium, hemoglobin and creatinine
No end organ damage related to plasma cell dyscrasia
(CRAB)
Asymptomatic Multiple Myeloma
1. Monoclonal paraprotein in serum >3gr/dL and/or plasma infiltration of the bone marrow >10%.
2. No end organ damage related to plasma cell dyscrasia
(CRAB)
Symptomatic Multiple Myeloma
1. Monoclonal plasma cells in bone marrow or plasmacytoma
2. Presence of monoclonal paraprotein in serum or urine
3. Presence of end organ damage felt related to the plasma cell
dyscrasia (1 or more criteria)
C} Increased calcium concentration >0.5 mmol/L (1mg/dL) over
the normal limit or >2.75 mmol/L (11mg/dL)
R} Renal failure: Creatininemia >173 mmol/L (1.96 mg/dL)
A} Anaemia: Hemoglobin <10 gr/dl or 2 gr/dl below the normal
range
B} Lithic lesions or osteoporosis with compression fractures
Others: Symptomatic hyperviscosity, amyloidosis, recurrent
bacterial infections (>2 episodes in 12 months)
b) Factors reflecting the characteristics of the malignant clone
This group of factors reflects morphological characteristics,
proliferative activity, immunophenotype, cytogenetics, and gene expression profile.
Morphology and proliferative activity:
An immature or plasmablastic morphology independently associated to a poor survival, even in patients treated with high doses
of chemotherapy (7). The proliferative activity of the plasma cell
measured by the Plasma Cell Labeling Index (PCLI), by bromodeoxyuridine administration or by flow cytometry with propidium iodide
has demonstrated that a higher proportion of cells in S-phase correlates with a worse prognostic (8, 9).
Immunophenotype:
Some studies have associated an immature phenotype (CD20,
CD45, sIg) with a poor prognostic (10,11). In a series of 587 patients, it was observed that the lack of CD56 and the overexpression
of CD19 and CD28 confer a poor prognostic while the CD117 acquisition was associated to a favorable outcome (12). Recently, the
CD200 expression, a transmembrane glycoprotein, which inhibits
S36
the immune response mediated by T cells, has been independently
correlated with a reduced event free survival (13).
Cytogenetics:
The cytogenetic feature constitutes one of the most significative prognostic factors in MM. In almost all MM cases it is possible
to identify cytogenetic abnormalities by FISH. On the other hand,
conventional cytogenetics has demonstrated abnormal karyotype
in one third of the cases, which is generally very complex, reaching
an average of 11 chromosomal abnormalities (14).
Ploidy: Using FISH it is possible to classify the karyotypes into
two groups: hyperdiploid and non-hyperdiploid (15). The hyperdiploid subtype is characterized by the presence of multiple trisomies
(more common chromosomes: 3, 5, 7, 9, 11, 15, 19, 21) and a low
frequency of translocations including IgH. Opposite to the previous
group, the non-hyperdiploid subtype is characterized by a high frequency of IgH translocations and lost of chromosomes, especially
chromosomes 13, 14, 16 and 18. The identification of hypodiploidy
is of clinical relevance since it is associated to a poor evolution
(16).
IgH translocations: The translocations involving the immunoglobulin heavy-chain locus (IgH) in 14q32, are a frequent finding
in MM patients (55-70%), even in those with MGUS (50%) when
they are studied by FISH. They are frequent in the non-hyperdiploid subtype of MM and their presence is of significative prognostic
importance (17).
The t(11;14)(q13;q32) is detected by karyotype metaphase in
5% and by FISH in 15-20% of MM patients. It is a frequent finding in
AL amyloidosis (50%) and in IgM myeloma (>90%). It produces the
overexpression of the cyclin D1 gene. Patients with this translocation present an oligosecretory disease or light-chain myeloma, expressing CD20 and lymphoplasmacytic morphology (18). Traditionally, the presence of t(11;14)(q13;q32) characterized a subgroup
of patients with a favorable prognostic; however, recent studies in
patients undergoing high-dose therapy do not support it (19,20) or
can even be an unfavorable factor in relapsed patients (21).
The t(4;14)(p16.3;q32) which is difficult to be detected by conventional cytogenetics, is detected by FISH in 15% of MM patients.
Genetically, its is characterized by the formation of a fusion gene IgHMMSET and overexpression of FGFR3. In patients presenting this
translocation, global gene expression analysis (GEP) has detected
expression changes of 127 genes (22). The t(4;14)(p16.3;q32) has
a marked unfavorable effect both in patients undergoing a standard
treatment (23) and in those treated with high-dose therapy (20,
24).
The t(14;16)(q32;q23), present in 5% of MM patients, is not detectable by conventional cytogenetics due to the telomeric position
of both loci. This translocation determines the overexpression of
c-maf which stimulate the progression in the cell cycle through the
overexpression of the cyclin D2 and promotes the interaction of the
myelomatous cell with the bone marrow microenvironment through
the overexpression of the β7 integrin. This last interaction increases
the secretion of VEGF. These phenomena are significative in the
survival of myelomatous cell (25) and its presence determines a
poor prognostic (23).
Chromosome 13q deletion: Using conventional cytogenetics,
it is possible to find out abnormalities of chromosome 13q in 10-20%
of the MM cases. With the FISH technique this increases to 30-55%
of the cases. Several studies have reported a strong association
between deletion of 13q identified by conventional cytogenetics and
a lower rate of responses, resistance to drugs and poor survival. On
the contrary, abnormalities detected by FISH have not been clearly
associated to a poor prognostic (26, 27).
One of the principal reasons in the difference between deletion
of 13q identified by conventional cytogenetics or FISH, is that the
first also measures proliferation and bone marrow plasmacytosis
(Rafael Fonseca MD, personal communication).
Other genetic alterations: Chromosome 17p13 deletion, the
locus of p53, detectable in 10% of patients is associated to the development of hypercalcemia and plasmacytomas. It is frequently
found in advanced stages of the illness and its detection confers a
poor prognostic (17).
The presence of activating mutations of K-ras has been detected in 35-50% of the MM patients. They are more frequent in
patients with t(11;14)(q13;q32) and correlate with advanced stages
and a shorter survival (17).
Recent studies suggest a strong prognostic association with
the overexpression of the CKS1B gene. The product of this gene is
part of a complex system that regulates the entrance into S-phase
of the cell cycle (28). The overexpression of the CKS1B gene correlates with an increase of the number of copies of the gene in the
chromosomal region 1q21 and it is associated to a lower survival
(29, 30).
Gene Expression Profiling (GEP):
Global gene expression analysis with microarrays techniques
has becoming a tool able to produce important changes in the prognostic of MM and classify them into different risk groups.
In MM this has allowed to identify the genes abnormally expressed in the five recurrent translocations of IgH: 11q13 (cyclin
D1), 6p21 (cyclin D3), 4p16 (MMSET and FGFR3), 16q23 (c-maf),
20q11 (mafB). With all this information, the Translocation Cyclin
(TC) System, which includes information of IgH translocations and
expression of cyclins D (Table 2) (31) was proposed.
The Arkansas group (32) has proposed a new classification of
the MM studying the GEP of plasma cells selected by its expression
of CD138 of 414 patients undergoing high-dose therapy. Analyzing
the expression of 1559 genes, they subclassified the cases into 7
subgroups so as to obtain a molecular classification of prognostic
value (Table 3). The overall survival (OS) data and event free survival (EFS) at 48 months (median follow-up of 36 months), points
out a clear difference between groups of high (PR, MS, MF) and low
risk (LB, HY, CD-1, CD-2) molecularly defined (EFS 68% vs 31%
y OS 79% vs. 51%). The multivariate analysis determined what
former survival independent predictors were: high-risk molecular
subgroups, abnormal cytogenetics, high levels of B2M and LDH.
Finally, this data has allowed the identification of the overexpression
of the cyclin D as a clue event in the pathogenic of MM (33).
Table 2. Molecular Classification TC
Group
TC1
TC2
TC3
TC4
TC5
Translocation
t(11;14)(q13;q32)
t(6;14)(p21;q32)
None
None
t(4;14)(p16.3;q32)
t(14;16)(q32;q23)
t(14;20)(q32;q11)
Gene
Cyclin D1
Cyclin D3
None
None
FGFR3/MMSET
c-maf
mafB
Cyclin D
D1
D3
D1
D2
D2
D2
D2
Ploidy
NH
NH
H
H-NH
NH>H
NH
NH
Frequency
15%
3%
37%
22%
16%
5%
2%
S37
Table 3. Molecular Classification of MM
Subgroup
Characteristics
3 years
EFS
44%
3 years
OS
55%
PR
Proliferation
LB
Low bone disease
MS
MMSET spike
HY
Hyperdiploid
CD-1
CCND1 spike
CD-2
CCND3 spike
MF
MAF or MAFB genes
Overexpression of numerous genes related to the cell cycle and proliferation, and cancertestis antigen genes. High incidence of abnormal metaphases
High expression of endothelin 1 (EDN1) and low expression of DKK1. Low frequency of
bone lesions
Expression of MMSET y FGFR3, associated to t(4;14)(p16.3;q32)
84%
87%
39%
69%
Overexpression of genes associated to bone metabolism, frequently hyperdiploid (90%)
72%
84%
Overexpression of cyclin D1 associated to t(11;14)(q13;q32)
82%
81%
Overexpression of cyclin D3 associated to t(6;14)(q21;q32)
86%
88%
Overexpression of MAF (t(14;16)(q32;q23) or MAFB (t(14;20)(q32;q11)
50%
71%
c) Factors resulting of the interaction between the tumor and
the host
Tumor burden:
The Durie & Salmon classification, which correlates with tumor
burden, has a prognostic value. The search of new factors identified B2M as a significative prognostic factor. Elevated levels of serum B2M are the result of the tumor growth and decrease in renal
function. This allowed establishing a new prognostic staging system
in MM (ISS International Staging System) (34). A record of 10,750
non-treated patients with symptomatic MM was used, achieving
the development of a classification system based on two variables:
B2M and albumin (Table 4).
Table 4. International Staging System (ISS)
Stage
population
%
I: B2M < 3.5 mg/L and albumin ≥ 3.5
g/dL.
28%
Survival
Median
(months)
62
II: B2M < 3,5 mg/L but albumin < 3.5
g/dL; or B2M 3.5 to < 5,5 mg/l
III: B2M ≥ 5.5 mg/L
33%
44
39%
29
Response to Treatment:
Using the criteria of Complete Remission (RC) of EBMT/IBMTR (negative immunofixation and <5% of plasma cells in bone
marrow) some groups have demonstrated that achieving RC is a
factor associated to a more favorable survival rate (41-43). In opposition to these results, a study of the SWOG group revealed that
time to first progression is a better predictor than the response to
the initial treatment (44).
PROGNOSTIC FACTORS IN PATIENTS SUBMITTED TO AUTOLOGOUS TRANSPLANT
Prognostic factors are valid in the two more used therapeutic
options: standard chemotherapy or autologous transplantation. In
the TT1 study of the Arkansas group, after a median follow-up of
12 years, factors associated to OS and EFS were the presence
of hypodiploidy and chromosome 13 deletion (45). A study of the
Mayo Clinic of 238 patients submitted to autologous transplant and
evaluated by FISH, revealed that the main independent predictors
(multivariate analysis) of survival were the pretransplant status (relapse versus plateau), PCLI, the presence of t(4;14)(p16.3;q32) and
chromosome 13 deletion (20).
NEW AGENTS
Disease complications:
Anemia, renal failure and thrombocytopenia have a significative impact in the prognostic of MM patients (35).
In the series of the Mayo Clinic (36) including 1,027 patients,
73% of the cases presented anaemia (Hb<12gr/dL), being severe in
7% (Hb<8gr/dL). The frequency of anaemia increases in advanced
stages of the illness and it is associated to a worse prognostic. It
can also affect significatively patients’ life quality (37). About 20%
of the MM patients present creatinine >2mg/dL at diagnosis and
a variable proportion of patients requires dialysis (2-5%). A study
of 756 patients revealed that the frequency of renal failure has not
diminished in the last 20 years and it is associated to a high mortality rate (38).
Bone lesions also affect the prognostic adversely. Traditionally,
bone lesions have been evaluated with X-Rays, but recently the
use of MRI or FDG-PET has allowed a more precise evaluation of
the bone and extramedular compromise, becoming a significative
prognostic technique (39, 40).
The new agents, especially thalidomide and bortezomib, have
modified the prognostic of the MM patient in relapse and refractory
setting, and possibly it is an alternative in the initial treatment.
A study of relapsed or refractory patients treated with thalidomide revealed that factors associated to a response based on a
paraprotein decrease were normal metaphases cytogenetics and
PCLI <0.5% (46).
In the SUMMIT study the predictive factors of response to bortezomib were: age <65 years and bone marrow plasmacytosis <50%
(47).
CONCLUSIONS
During the last decade significative advances have been
achieved in the knowledge of the MM biology. The progress in the
cytogenetics, and particularly Gene Expression Profiling (GEP) has
allowed the recognition of different subtypes of MM. If we add the
clinical and laboratory information, we can make a more precise
prognosis of each patient with therapeutic implications.
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2006. Abstract
13. Moreaux J, Hose D, Reme T, et al. CD200 is a new prognostic
factor in Multiple Myeloma. Blood. 2006 Aug 31; [Epub ahead of
print]
14. Tricot G, Sawyer JR, Jagannath S, et al. Unique role of cytogeneticss in the prognosis of patients with myeloma receiving highdose therapy and autotransplants. J Clin Oncol. 1997;15:265966
15. Fonseca R, Barlogie B, Bataille R, et al. Genetics and Cytogeneticss of Multiple Myeloma: A Workshop Report. Cancer
Res 2004;64:1546-1558
16. Smadja NV, Bastard C, Brigaudeau C, et al: Hypodiploidy is a major prognostic factor in multiple myeloma. Blood
2001;98:2229-2238
17. Stewart AK, Fonseca R. Prognostic and Therapeutic Significance of Myeloma Genetics and Gene Expression Profiling. J Clin Oncol 2005;23:6339-6344
18. Fonseca R, Blood EA, Oken MM, et al: Myeloma and the
t(11;14)(q13;q32): evidence for a biologically defined unique
subset of patients. Blood 2002;99:3735-3741
19. Avet-Loiseau H, Attal M, Moureau P, Carbonnel C, arban F, Harousseau JL et al. A Comprehensive Analysis of Cytogenetics
Abnormalities in Myeloma: Results of the FISH Analysis of 1000
Patients Enrolled in the IFM99 Trials. Blood 2005;106 (11) 622a
Abstract
20. Gertz Ma, Lacy MQ, Dispenzieri A, et al. Clinical implications
of t(11;14)(q13;q32), t(4;14)(p16.3;q32), and 17p13 in myeloma
patients treated with high-dose therapy. Blood 2005;106:28372840
21. Chng WJ, Mulligan G, Bryant B, Bergsagel PL. Survival of Genetic Subtypes of Relapsed Myeloma May Be Modulated by
Secondary Events. Blood 2006;108(11): Abstract 132
22. Dring AM, Davies FE, Fenton JA, et al: A global expressionbased analysis of the consequences of the t(4;14) translocation
in myeloma. Clin Cancer Res 2004; 10:5692-5701
23. Fonseca R, Blood E, Rue M et al. Clinical and biologic implications of recurrent genomic aberrations in myeloma. Blood 2003;
101:4569-4575
24. Gutierrez NC, Castellanos MV, Martin ML, et al. Prognostic and
biological implications of genetic abnormalities in multiple myeloma undergoing autologous stem cell transplantation: t(4;14)
is the most relevant adverse prognostic factor, whereas RB deletion as a unique abnormality is not associated with adverse
prognosis. Leukemia. 2006 Oct 5; [Epub ahead of print]
25. Hurt EM, Wiestner A, Rosenwald A, et al. Overexpression of
c-maf is a frequent oncogenic event in multiple myeloma that
promotes proliferation and pathological interactions with bone
marrow stroma. Cancer Cell 2004;5:191-9.
26. Chng WJ, Santana-Davila R, Van Wier SA, et al. Prognostic
factors for hyperdiploid-myeloma: effects of chromosome 13
deletions and IgH translocations. Leukemia 2006;20:807-13.
27. Chiecchio L, Protheroe RK, Ibrahim AH, et al. Deletion of chromosome 13 detected by conventional cytogenetics is a critical
prognostic factor in myeloma. Leukemia. 2006;20:1610-7
28. Reed S. Ratchets and clocks: the cell cycle, ubiquitylation and
protein turnover. Nat Rev Mol Cell Biol 2003;4: 855–864
29. Fonseca R, Van Wier SA, Chng WJ, et al. Prognostic value
of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma. Leukemia.
2006;20:2034-40.
30. Chang H, Qi X, Trieu Y, et al. Multiple myeloma patients with
CKS1B gene amplification have a shorter progression-free survival post-autologous stem cell transplantation. Br J Haematol.
2006;135:486-91
31. Bergsagel PL, Kuehl WM, Zhan F, et al: Cyclin D dysregulation:
An early and unifying pathogenic event in multiple myeloma.
Blood 2005;106:296-303
32. Zhan F, Huang Y, Colla S, et al. The molecular classification of
multiple myeloma. Blood. 2006;108:2020-8.
33. Bergsagel PL, Kuehl WM. Molecular pathogenesis and a
consequent classification of multiple myeloma. J Clin Oncol
2005;23:6333-8
34. Greipp PR, San Miguel J, Durie BG, et al. International staging system for multiple myeloma. J Clin Oncol 2005;23: 34123420
35. Blade J, Rosinol L. Renal, hematologic and infectious complications in multiple myeloma. Best Pract Res Clin Haematol
2005;18:635-52
36. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri
A, et al. Review of 1027 patients with newly diagnosed multiple
myeloma. Mayo Clin Proc 2003;78: 15-17
37. Birgegard G, Gascon P, Ludwig H. Evaluation of anaemia in patients with multiple myeloma and lymphoma: findings of the European Cancer Anaemia Survey. Eur J Haematol 2006;77:37886.
38. Eleftherakis-Papaiakovou V, Anagnostopoulos A, Bamias A, et
al. Renal Failure in Multiple Myeloma: Incidence, Correlations and Prognostic Significance. Blood 2006;108: Abstract
5000
39. Moulopoulos LA, Gika D, Anagnostopoulos A et al. Prognostic
significance of magnetic resonance imaging of bone marrow in
previously untreated patients with multiple myeloma. Ann Oncol
2005; 16:1824-1828.
40. Walker R, Haessler J, Tricot G, et al. Focal Lesion (FL) Identification by MRI and Metastatic Bone Survey (MBS) in Multiple Myeloma (MM): Laboratory Correlates and Prognostic
Implications for 611 Patients Receiving Total Therapy 2
(TT2). Blood 2006;108: Abstract 3496
41. Lahuerta JJ, Martinez-Lopez J, Serna JD et al. Remission status
defined by immunofixation vs. electrophoresis after autologous
transplantation has a major impact on the outcome of multiple
myeloma patients. Br J Haematol 2000; 109:438-446
42. Harousseau JL, Attal M, Moreau P, et al. The Prognostic Impact of Complete Remission (CR) Plus Very Good Partial
Remission (VGPR) in a Double-Transplantation Program
for Newly Diagnosed Multiple Myeloma (MM). Combined
Results of the IFM 99 Trials. Blood 2006;108: Abstract 3077
43. Alvares CL, Davies FE, Horton C, et al. Long-term outcomes of
previously untreated myeloma patients: responses to induction
chemotherapy and high-dose melphalan incorporated within a
risk stratification model can help to direct the use of novel treatments. Br J Haematol 2005;129:607-14.
44. Durie BG, Jacobson J, Barlogie B et al. Magnitude of response
with myeloma frontline therapy does not predict outcome: importance of time to progression in southwest oncology group
chemotherapy trials. J Clin Oncol 2004; 22:1857-1863.
45. Barlogie B, Tricot GJ, van Rhee F, et al. Long-term outcome
results of the first tandem autotransplant trial for multiple myeloma. Br J Haematol 2006;135:158-64
S39
46. Barlogie B, Desikan R, Eddlemon P, et al. Extended survival in
advanced and refractory multiple myeloma after single-agent
thalidomide: identification of prognostic factors in a phase 2
study of 169 patients. Blood. 2001;98:492-4.
47. Richardson PG, Barlogie B, Berenson J, et al. Clinical factors
predictive of outcome with bortezomib in patients with relapsed,
refractory multiple myeloma. Blood 2005;106:2977-81
S40
CONFERENCE
Novel therapies in Multiple Myeloma
Jesús F. San-Miguel.
Servicio de Hematología, Hospital Universitario de Salamanca
Centro de Investigación del Cáncer, Universidad de Salamanca (Spain)
The use of high dose chemotherapy followed by autologous
stem cell support (ASCT) has become the standard of care for young
myeloma patients. This has been based on the significant superiority of ASCT over standard chemotherapy in terms of response, time
to progression (TTP) and overall survival (OS), as demonstrated by
two randomized studies conducted by the IMF and MRC groups.
However, unfortunately all patients eventually relapse which suggests the need for alternative or complementary therapies in order
to maintain the responses. In addition, two other randomized trials
(Spanish/Pethema and USA intergroup) have failed to confirm such
a superiority of ASCT and this could be at least partially attributed to
the good results of the chemotherapy arm (VBMCP/VBAD). Moreover, the availability of new drugs such as Bortezomib and IMID´S (
thalidomide and Lenalidomide) with clear efficacy in MM makes it
necessary to re-evaluate the role of ASCT in the current and future
treatment of MM patients.
ASCT is currently being used in both newly diagnosed patients
with chemosensitivite and chemorefractory disease. For this latter
category ASCT is so far considered the treatment of choice and
performed directly without rescue treatment. However, novel drugs,
particularly in combination with Dexamethasone, with or without
other chemotherapeutic agents, such as Doxorubicin or Melphalan
are associated with high response rates (50%-75%, with 10%CR) .
Based on these positive results, it would be expected that all refractory MM patients will receive rescue treatment with novel agents
before ASCT. Accordingly, the efficacy of transplantation as rescue
treatment should be re-evaluated in prospective trials including the
novel agents. For patients entering into these trials, a clear distinction between those with progressive disease and those with stable
disease should be performed.
Regarding previously untreated MM patients, novel drugs
appear to be superior to conventional chemotherapy as debulky
pretransplant regimens. Using schemes with Bortezomib, Lenalidomide, or Thalidomide, the majority of patients respond (>80%) with
10%-30% CR rate. These schemes do not affect stem cell collection. Interestingly, in four pilot studies using Bortezomib regimens
it was observed that this CR rate was upgraded following ASCT,
which suggests that this novel treatment will not replace ASCT, but
will help to enhance its activity, although the EFS and OS of this approach is still unknown. By contrast, the Dutch/Hovon group have
shown that the initial advantage of the Thalidomide-based regimen
TAD versus VAD, was overcome following transplant.
As previously mentioned one important goal of novel treatments is to help to prolong the duration of the responses obtained
after transplant. The French group has recently shown that Thalidomide maintenance is clearly superior to no maintenance or Pamidronate alone in terms of EFS and OS. Similar results have been
reported by the Australian Group with the combination of Thalidomide and Prednisone. The Arkansas group has also observed that
the use of thalidomide as part of induction and maintenance phases
was associated with longer EFS however this doesn’t translate into
a prolonged OS. This raises an important concern about whether
novel drugs may induce more resistant relapses to salvage therapy
leading to shorter survival after relapse.
At present we have more questions than answers but our position is that ASCT represents an ideal treatment for reducing myeloma cell mass, with low toxicity, and therefore the novel drugs
should contribute to induce better responses before transplant and
to prolong EFS after it.
As far as elderly patients is concerned, the combination of Thalidomide with Melphalan- Prednisone (MP) is superior to MP alone,
as it has been demonstrated by the Italian and French Groups.
Moreover, the Spanish Group has also shown that the combination of Bortezomib with MP is also highly effective, even in patients
with poor cytogenetics. Similar results have been obtained in a pilot
study of Lenalidomide + MP. Accordingly, a new standard of care is
already available for elderly MM patients and this is based on the
combination of the new and old agents.
S41
TECNOFARMA ASOFARMA SYMPOSIUM
Advances in the biology and management of Acute
Promyelocytic Leukemia
Francesco Lo Coco.
Department of Hematology.
University Tor Vergata-Roma Italia.
OBJETIVES:
The objective of this conference will be to discuss most relevant issues concerning biology and management of APL with particular emphasis on those molecular and phenotypic features of the
disease which impact on targeted treatment. A number of practical aspects that may be frequently faced by hematologist in their
current practice will be addressed, including methodology for rapid
confirmation of diagnosis, supportive care, definition of response to
therapy etc.. Some relevant emerging concepts regarding therapy
will also be discussed. In addition to establish state of the art approach for front-line treatment, other open issues such as roile of
Ara-C, stem cell transplantation and place of new drugs will be analysed. A number of recent clinical studies strongly suggest that frontline treatment of this disease needs to be revisited. In particular,
the possibility of treating low-risk patients with a no-chemotherapy
approach warrants investigation by appropriately designed trials.
Hence, new directions for clinical investigation in this sense will be
provided.
S42
CONFERENCE
How should we assess prognosis in CLL?
Guillermo Dighiero
Executive Director of the Institut Pasteur of Montevideo
INTRODUCTION
Chronic lymphocytic leukaemia (CLL) is the commonest form
of leukaemia in Western countries and mainly affects elderly individuals. It follows an extremely variable course, with survival ranging
from months to decades. Available treatments often induce remissions, though nearly all patients relapse, and CLL remains an incurable disease. Recently, molecular and cellular markers have been
identified that may predict the tendency for disease progression. In
particular, the mutational profile of Ig genes (1) and some cytogenetic abnormalities (2) display strong prognostic value and raised
the question whether we should consider to move from the classical
Rai and Binet staging systems.
How should we assess prognosis in CLL?
In CLL, one-third of patients never require treatment and have
a long survival; in another third, an initial indolent phase is followed
by disease progression; the remaining third exhibit an aggressive
disease at the onset and need immediate treatment (3) The Rai and
Binet staging systems has allowed the division of patients with CLL
into three prognostic groups with good, intermediate and poor prognosis and provided a foundation that allowed clinicians to design
therapeutic strategies for the disease. However, neither the Rai nor
the Binet staging system is able to predict which patients among the
good prognosis group will develop progressive disease (4). Lymphocyte doubling time, serum levels of β2-microglobulin, thymidine
kinase and soluble CD23, as well as CD38 expression on malignant
cells can help predict disease activity . (5)
An initial description indicated that half of CLLs expressed
VH genes containing numerous somatic mutations(6). Two reports
demonstrated that the clinical behavior of CLL is related to the mutational status of immunoglobulin (Ig) genes (1) (7). CLLs with mutated Ig genes display a good prognosis and those with unmutated
Ig genes a poor prognosis. This observation has been extensively
confirmed (8-10) and it is well established that the mutational status
of Ig genes constitutes a strong prognostic indicator in CLL. The
mutational profile of Ig genes delineates prognostic groups within
all Binet’s stages (11).(9) . Interestingly, the rearrangement of a
specific VH gene, V3-21, has been associated with poor prognosis
whether mutated or not (12).
The presence in leukemic B cells of chromosomal deletions
at 11q23 or 17p13 (2) (10) (9) also constitute a strong prognostic
indicator in CLL. Döhner et al demonstrated in an extensive series
of CLL patients that chromosomal aberrations can be detected by
fluorescence in situ hybridization (FISH) in 82% of cases (2) The
presence of a 17p13 or 11q23 deletions is associated with poor
prognosis and predominates among advanced stages of the disease and among patients displaying unmutated V genes, whereas
isolated 13q14 deletions are associated with good prognosis, initial
stages of the disease and a mutated profile of Ig VH genes.
The VH mutational profile has the advantage that it remains
constant during clonal evolution, which contrasts with genomic
aberrations and serum markers. Since 11q23 or 17p13 deletions
are associated with poor outcome and an unmutated VH profile and
comprise about one-third of unmutated CLL, they might be considered as a subgroup of the unmutated group. Although CD38 expression is associated with poor prognosis, its relationship to Ig mutational status remains controversial. (7)(10) (13) can change during
disease evolution and there are concerns related to inter-laboratory
variations and the definition of the best cut-off value. (14) (5).
Since sequencing Ig V genes is costly, time consuming and
inaccessible for most medical facilities, the detection of appropriate,
reliable surrogate markers for IgVH mutational status has attracted
worldwide attention. Crespo et al developed a multiparameter flowcytometric test for ZAP-70 that showed 91% correlation with VH
gene mutational status (15). Subsequent studies have shown a less
good correlation (16, 17) . and a reliable and reproducible methodology for this test awaits development (18). Lipoprotein lipase that is
consistenly overexpressed among unmutated CLLs has also been
proposed as a surrogate marker (19). In contrast to ZAP-70, which
failed to segregate advanced forms of the disease, this marker comprise an independent prognostic factor for stage B and C patients
(19) (20).
Although, all these markers provide useful prognostic information, the mutational status of VH genes, FISH for del 17p and del
11q and CD38, are the most robust prognostic indicators. VH genes
and FISH have now been confirmed in prospective clinical trials and
influence outcome more than choice of treatment. Other markers
like ZAP-70, LPL, p53 functional tests, conventional karyotyping
and serum thymidine kinase, may provide useful prognostic information but require further evaluation. Markers like β -2 microglobulin, serum CD23, bone marrow biopsy, serum thrombopoietin and
serum IL-8, provide less useful information.
In a multivariate analysis both Binet staging and VH genes retain their independent prognostic significance in CLL and are complementary (9) (11). Thus, as shown in Table 1, the association of
the Binet staging, to the mutational profile of Ig genes and the 17p
deletion, that is the strongest independent prognostic markers, allows a new segregation of patients into 5 prognostic subgroups.
In conclusion, the recognition of novel biological variables has
had a major impact on our understanding of CLL. Some of them appear to be of considerable prognostic importance but as yet there
is no available evidence to suggest that changing therapeutic approaches on the basis of these results will lead to an improvement
in outcome. There is a pressing need for prospective clinical trials to
address the stratification of patients according to these factors.
Table 1. The combination of the Binet´s staging system with
the mutational status of IgVH genes and 17p deletion allows to
segregate CLL patients into 5 prognostic categories.
Prognostic groups
% of patients
Median survival
Mutated stage A *
43%
75% survival expectancy at 144 months
Unmutated stage A*
20%
97 months
Mutated stages B+C* 10%
120 months
Unmutated stages
B+C*
20%
78 months
17p deletion whatever the stage and
mutational status**
7%
36 months
* According to (11)
** According to (2)
1. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK.
Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia [see comments].
Blood. 1999;94(6):1848-54.
2. Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A,
Bullinger L, et al. Genomic aberrations and survival in chronic
lymphocytic leukemia. N Engl J Med. 2000;343(26):1910-6.
3. Dighiero G. Unsolved issues in CLL biology and management.
Leukemia. 2003 Dec;17(12):2385-91.
4. Dighiero G, Maloum K, Desablens B, Cazin B, Navarro M,
Leblay R, et al. Chlorambucil in indolent chronic lymphocytic
leukemia. French Cooperative Group on Chronic Lymphocytic
Leukemia. N Engl J Med. 1998;338(21):1506-14.
5. Montserrat E. Classical and new prognostic factors in chronic
lymphocytic leukemia: where to now? Hematol J. 2002;3(1):79.
6. Schroeder HW, Jr., Dighiero G. The pathogenesis of chronic
lymphocytic leukemia: analysis of the antibody repertoire [see
comments]. Immunology Today. 1994;15(6):288-94.
7. Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et
al. Ig V gene mutation status and CD38 expression as novel
prognostic indicators in chronic lymphocytic leukemia [see comments]. Blood. 1999;94(6):1840-7.
8. Maloum K, Davi F, Merle-Beral H, Pritsch O, Magnac C, Vuillier F, et al. Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia. Blood.
2000;96(1):377-9.
9. Oscier DG, Gardiner AC, Mould SJ, Glide S, Davis ZA, Ibbotson RE, et al. Multivariate analysis of prognostic factors in CLL:
clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood.
2002 Aug 15;100(4):1177-84.
10. Krober A, Seiler T, Benner A, Bullinger L, Bruckle E, Lichter P,
et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood.
2002 Aug 15;100(4):1410-6.
11. Vasconcelos Y, Davi F, Levy V, Oppezzo P, Magnac C, Michel
A, et al. Binet‘s staging system and VH genes are independent
but complementary prognostic indicators in chronic lymphocytic
leukemia. J Clin Oncol. 2003 Nov 1;21(21):3928-32.
S43
12. Tobin G, Thunberg U, Johnson A, Thorn I, Soderberg O, Hultdin
M, et al. Somatically mutated Ig V(H)3-21 genes characterize a
new subset of chronic lymphocytic leukemia. Blood. 2002 Mar
15;99(6):2262-4.
13. Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW,
Stevenson FK, et al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in
chronic lymphocytic leukemia, but CD38 expression may vary
during the course of the disease. Blood. 2002 Feb 1;99(3):10239.
14. Ghia P, Guida G, Stella S, Gottardi D, Geuna M, Strola G, et
al. The pattern of CD38 expression defines a distinct subset of
chronic lymphocytic leukemia (CLL) patients at risk of disease
progression. Blood. 2002 Oct 24;101((4)):1262-9.
15. Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic
leukemia. N Engl J Med. 2003 May 1;348(18):1764-75.
16. Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A,
Thomas PW, et al. ZAP-70 expression and prognosis in chronic
lymphocytic leukaemia. Lancet. 2004 Jan 10;363(9403):10511.
17. Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG,
et al. ZAP-70 compared with immunoglobulin heavy-chain gene
mutation status as a predictor of disease progression in chronic
lymphocytic leukemia. N Engl J Med. 2004 Aug 26;351(9):893901.
18. Marti G, Orfao A, Goolsby C. ZAP-70 in CLL: towards standardization of a biomarker for patient management: history of clinical cytometry special issue. Cytometry B Clin Cytom. 2006 Jul
15;70(4):197-200.
19. Oppezzo P, Vasconcelos Y, Settegrana C, Jeannel D, Vuillier F,
Legarff-Tavernier M, et al. The LPL/ADAM29 expression ratio
is a novel prognosis indicator in chronic lymphocytic leukemia.
Blood. 2005 Jul 15;106(2):650-7.
20. Heintel D, Kienle D, Shehata M, Krober A, Kroemer E, Schwarzinger I, et al. High expression of lipoprotein lipase in poor
risk B-cell chronic lymphocytic leukemia. Leukemia. 2005
Jul;19(7):1216-23.
S44
EDUCATION SESSION
Chronic Myeloproliferative Disorders
Chronic Myeloid Leukemia: Current
Management and Novel Therapies
Michael J. Mauro
Associate Professor, Center for Hematologic Malignancies, Oregon Cancer Institute, Oregon Health & Science University, 3181
SW Sam Jackson Park Road, UHN-73C, Portland, OR 97239
USA; email: [email protected]
INTRODUCTION
Advances in tyrosine kinase inhibitor therapy continue to morph
approaches to the treatment of chronic myelogenous leukemia
(CML). By 2007, we now have very highly active and durable primary therapy, imatinib mesylate; convincingly proven salvage therapy
options with dasatinib and nilotinib, the former agent FDA approved
in mid-2006; other salvage options ranging from additional multikinase (ABL, SRC, LYN, etc) inhibitors such as SKI606 (Wyeth) and
INNO-406 (Innovive) to the aurora kinase inhibitor MK0457 (Merck),
active against T315I mutant ABL kinase; and stem cell transplant,
continuing to be optimized with investigation into novel conditioning and GVHD prevention / management strategies. In the span of
less than a decade, CML therapy shifted dramatically away from the
primary immunotherapy based options of interferon and allografting
to an “era of kinase inhibitor therapy” which appears to be firmly
anchored in place for the foreseeable future.
DE NOVO CML TREATED WITH IMATINIB: IRIS TRIAL DATA
At 60 months, data from the IRIS trial1, a landmark trial comparing interferon based therapy and imatinib in newly diagnosed
chronic phase CML, now demonstrates the following cumulative
best response rates with imatinib: 98% complete hematologic remission (CHR), 92% major cytogenetic response (MCR) and 87%
complete cytogenetic response (CCR). A more pragmatic view of the
IRIS imatinib cohort must also include the event free survival rate
(83%) and the retention of patients (69%) after 60 months’ study. In
a breakdown of the status of patients from the IRIS trial randomized
to imatinib, there is a clear subset with imatinib “failure”- necessitating crossover to IFN or discontinuation for inadequate effect (14%)
and imatinib intolerance-- discontinuation for adverse events (4%).
An additional 3% of patients exited trial for perusal of SCT, implying
inadequate response to some degree; the remaining reasons for
exiting study are not therapy / response related and encompass an
additional 10%. The fact remains, however, that imatinib is highly
active primary therapy for the overwhelming majority of patients and
is a durable option for a similar proportion.
Of great interest is the kinetics of imatinib failure in the IRIS
cohort, now becoming apparent with longer follow-up. The rate of
all progression events, including cytogenetic and hematologic relapse within chronic phase and transformation to advanced phase,
is 18% after a median of 5 years. However, such events appear to
be most evident in the first 3 years of imatinib treatment, where progression to advanced phases of disease averaged 2% per year and
progression within chronic phase 5% per year. Year 4 then showed
diminution in these rates, and in year 5 both risks are less than 1%;
for those patients in CCR, the rate of progression to advanced CML
fell to zero at year 5. Such kinetics suggests an impending plateau
in progression-free survival, directly contradicting early pessimism
surrounding selective targeted therapy with imatinib. Indeed the
natural history of CML, particularly for patients in the chronic phase,
has been dramatically altered with current therapy and will likely
continue to a degree with additional advances forthcoming.
RESISTANCE TO ABL KINASE INHIBITORS
Imatinib does clearly have its limitations; during early development, clinical resistance, particularly in advanced phase, became
apparent and accelerated ongoing research into the “how and why”
of imatinib resistance, allowing for rapid development and validation of alternative therapies to circumvent mechanisms revealed.
Primary resistance to imatinib is defined as an inability to achieve
landmark response, whereas secondary resistance defines those
who achieve but subsequently lose relevant response. Persistence
or re-emergence of Ph+ hematopoiesis continues to firmly define
clinical resistance; when it occurs despite imatinib, or moreover ABL
kinase inhibitors as a class, a large proportion of cases (generally in
the rage of ~50%) have a consistent feature observed: acquisition
of a point mutation in the Abl kinase domain. Other mechanisms
are suspected in patients with wild-type ABL or those with Abl mutations predicted to respond to alternate kinase inhibitors who fail to
respond. BCR-ABL amplification at the genomic or transcript level2,3
has been implicated in imatinib failure, overexpression of other tyrosine kinases such as the Scr-related LYN kinase has been observed
in the case of BCR-ABL independent resistance, 4 and variability in
the amount and function of the drug influx protein OCT-1 has been
linked to relative insensitivity to kinase inhibition by imatinib5. It is
certainly worthwhile to add to this list the notion of CML “stem cell
resistance,” based in the ability of CML progenitors to exchange between a cycling and resting or “quiescent” (G0) state, the latter associated with minimal or no BCR-ABL expression and resulting lack
of effect of Abl kinase inhibitors6.
Regarding tyrosine kinase mutation, early investigation into
advanced phase CML cases of relapse first revealed critical single
amino acid substitutions (mutations)2 within BCR-ABL and reactivation of the kinase. Numerous reports followed, and the spectrum
of Abl kinase domain mutations observed in the setting of imatinib
spans the entire kinase domain with over 40 mutations identified7.
Abl kinase mutations generally cluster into four main categories and
are associated with particular numbered amino acid residues8: ATP
binding loop (p-loop), particularly Y253 and E255 mutants; T315
mutants; M351 mutants; and activation loop (a-loop), particularly
H396 mutants. Modeling of imatinib and other kinase inhibitors with
the crystal structure of the catalytic region of the Abl kinase suggests that mutations may interrupt critical drug contact points or
induce or favor a conformation of the Abl kinase in which drug binding is reduced or precluded. Now termed the “gatekeeper” position,
mutations at threonine 315 confer resistance both to imatinib and
“second generation” Abl kinase inhibitors nilotinib and dasatinib and
represent a new challenge already being engaged by newer inhibitors.
While it is accepted that expansion of a Ph(+) CML clone bearing an Abl kinase domain mutation may be associated with resistance to imatinib2,9-11 and also may herald progression to advanced-
phase disease12,13, the fact that mutations may be identified prior to
imatinib exposure and do not strictly correlate with clinical resistance
suggests a role for additional mechanisms to trigger outgrowth of
mutants, or that genesis of mutant clones reflects greater genetic
instability14. Cytogentic clonal evolution has been linked to mutation detection prior to imatinib and resistance to second generation
Abl kinase inhibitors where identified Abl mutations would predict
response. These observations support a continued role, past and
present, for clonal evolution in resistance and progressive disease.
It is important to note that while further cytogenetic abnormalities in
Ph(+) cells (Ph-positive clonal evolution) herald disease progression
and are a marker of accelerated phase disease, Ph(–) clonal cytogenetic abnormalities occur in the setting of CML response, may
become apparent only after significant reduction of the Ph(+) clone;
while associated with isolated reported cases of secondary hematopoietic disorders including MDS and Ph(–) acute leukemias15, this
phenomenon is usually benign in course and simply requires careful
observation in most cases.
Controversy surrounds several “early recognition” questions,
such as screening for mutations and defining change in minimal
residual disease relevant and linked to possible resistance. Beyond
the “signature” of a kinase domain mutation (the particular amino
acid substitution and kinase region being predictive), the “fitness”
of mutant clones—their ability to sustain proliferation with a relative
advantage over Ph(–) clones or wild type BCR-ABL—is most relevant to risk; screening for mutations prior to imatinib and for those
with stable minimal residual disease are thus scenarios that may
be misleading14,16. The area of minimal residual disease is “muddied” by variable precision in qPCR assays, with fluctuations in patient results common, and the fact that the threshold of “complete
molecular response”—where BCR-ABL transcripts are no longer
detectable—is as much a reflection of assay sensitivity as level of
patient response. Absence of detectable BCR-ABL transcripts in
laboratories with a high degree of sensitivity was previously felt to
not be a consistent finding even in the best responding patients17;
however, with time and continued improvement in depth of molecular response, a cohort of patients consistently without detectable
transcripts is emerging. For those patients with change in minimal
residual disease status, BCR-ABL transcript level increases as
small as a single observed18 or confirmed19 twofold (2 x) rise may
represent proliferating disease and have been associated with increase prevalence of kinase domain mutations and impending clinical resistance. However, a less subtle change such as a fivefold
(5 x) increase or a one-log increase (10 x) confirmed in a second
sample may be more readily identifiable, predictive with the majority
of current PCR labs’ techniques, and should warrant investigation
for molecular causes and closer follow-up.
MILESTONES IN THERAPY AND DETERMINING FAILURE /
SUBOPTIMAL RESPONSE
NCCN guidelines20 and a recent European LeukemiaNet consensus paper21 have summarized target responses at key time
points incorporating cytogenetic and molecular response and can
triage patients into categories of failure, suboptimal and optimal response, based on variance in risk of relapse or progression; a summary of generally accepted response targets for imatinib therapy is
S45
listed in Table 1. Achievement of CHR by the 3-month mark of therapy is deemed a minimum initial response and lack of hematologic
response by 3 months as failure. Failure to achieve any reduction
in Ph(+) cells by cytogenetic testing after 6 months of imatinib and
failure to achieve MCR after 12 months of imatinib therapy predicts
for less than 20% chance of subsequently achieving CCR. In contrast, much earlier (3 mo) cytogenetic response had been required
to optimized outcome in late chronic phase (post-IFN) patients22.
Response beyond these minimums, specifically CCR achieved by
12 months, certainly offers further risk reduction. Despite the variability and ongoing need for standardization of qPCR, it is clear
that one threshold level of BCR-ABL transcript reduction, agreed
upon to be a 3-log or greater reduction below standard baseline (a
major molecular response) occurring in the first 12–24 months of
imatinib therapy in the setting of CCR, confers maximal protection
from progression to advanced disease (projected transformationfree survival 100%) and the lowest rate of any disease progression
with 5 years’ follow-up1.
NEW THERAPY BEYOND IMATINIB
Less than ten years after imatinib first entered clinical trials,
at least two highly active second-line therapies to salvage imatinib
failure or intolerance have emerged. Such success stems from
extensive knowledge regarding the centrality of BCR-ABL in imatinib sensitive and resistant CML, the latter evidenced by selection
or genesis of clones in the case of resistant disease with restored
BCR-ABL kinase activity. Dasatinib (Sprycel; formerly BMS354825)
and nilotinib (formerly AMN107) address the gaps left by imatinib for
Ph+ leukemias and as well for potential use in other conditions with
relevant inhibitable kinases. Phase I trials in CML for both agents
have been reported23,24 simultaneously in paired articles with impressive activity demonstrated. Both compounds are noted in vitro
to be more potent inhibitors of Abl; nilotinib was developed from imatinib, modified to bind the Abl kinase with higher affinity and with less
stringent bonding requirements25, whereas dasatinib was developed
as an inhibitor of the Src kinase but found to inhibit BCR-ABL avidly
and in the active, as well as the inactive conformation26 required by
imatinib for binding. Both compounds inhibit Abl as well as all known
mutant Abl kinases in vitro except for one bearing threonine-to-isoleucine substitution at position 315 of Abl (T315I)27. Phase I studies
for both agents23,24 included patients with resistant chronic phase
disease (n = 40 for dasatinib, n = 17 for nilotinib), with slightly different entry criteria (mainly allowance for imatinib intolerant patients
[20% of the total] in the dasatinib study and patients with cytogenetic resistance only [i.e., still in CHR] in the nilotinib trial). The rate
of complete hematologic response was identical for both at 92%,
as was CCR at 35%, with an additional 10% of patients on dasatinib achieving partial cytogenetic response, bringing the totals for
MCR to 45% for dasatinib and 35% for nilotinib. No dose-limiting
toxicity was observed for dasatinib, with a range of 15–240 mg per
day administered; for nilotinib, dosing at 600 mg BID was limiting,
with associated liver (predominantly grade 3 indirect bilirubin and
transaminase) and pancreatic enzyme elevations (including grade 2
pancreatitis), as well as one grade 3 subdural hematoma. Extensive
monitoring for electrocardiographic changes from nilotinib revealed
a 5–15 msec increase in the corrected QT (QTcF). Pleural effusions
Table 1. Current Accepted Thresholds Defining Failure, Suboptimal response, and Optimal Response
3 Months
6 Months
12 Months
18 Months +
Failure
No hematologic
response
> 95% Ph+
> 35% Ph+
> 0% Ph+
Suboptimal Response
No complete hematologic response
35–95% Ph+
1–35% Ph+
0% Ph+, < 3 log reduction in
BCR-ABL transcripts
Optimal response
1–2 log reduction in
BCR-ABL transcripts
< 35% Ph+
0% Ph+, 3 log reduction in
BCR-ABL transcripts
0% Ph+, 3 log or greater
reduction in BCR-ABL transcripts
S46
deemed therapy related were observed in 15 of 84 dasatinib treated
patients overall in phase I (13% grade 3–4 in the myeloid blast crisis cohort) and were treated with diuretics and/or drainage. Other
higher-grade toxicity from dasatinib included edema, headache,
and elevated transaminase levels. Myelosuppression was observed
beyond the level seen with imatinib for both agents, and was more
pronounced with dasatinib; however, comparison may be difficult
due to the fact that patients with imatinib failure and intolerance may
be at greater risk due to longer disease duration or other factors. Activity was seen for advanced phases of CML and Ph+ ALL with both
agents in phase I. Of note, 70% of patients studied on dasatinib and
41% studied on nilotinib had Abl kinase mutations prior to therapy;
in both studies, presence of T315I mutant clones prior to therapy
precluded any response and at relapse, detection of T315I was a
common finding; patients with other mutations responded to both
agents, and patients without mutations responded as well.
Phase II studies for dasatinib28-31in all phases of CML and Ph+
ALL have been reported and supported rapid approval of the compound (named Sprycel) on 6/29/06 for both indications; the recommended dose is 70 mg BID. In the “Start-C” trial of dasatinib in CP
CML28, 60% of patients required dose reductions over time for toxicity and the median dose was closer to 100 mg per day; ongoing trials continue to explore dosing options for dasatinib, including varying total dose and QD versus BID dosing. Phase II data has been
presented for nilotinib, expanding experience with the 400 mg BID
dosing32-34, and both sets of data are summarized in Table 2. Results for both agents in chronic phase remain impressive, with the
majority of patients achieving sustained hematologic response and
approximately one-half MCR and one-third CCR. Advanced-phase
results show more limited salvage capability for both agents, particularly for the Ph+ acute leukemias, with early relapse common; in
accelerated-phase disease with both agents a subset of responders
remains fairly durable, albeit with limited follow-up.
High-dose imatinib early in disease continues to be studied in
comparison to standard dose, with randomized trials ongoing and
data forthcoming; further update of previously published single center experience35 now shows similar ultimate depth of response for
both 400 and 800 mg dosing, yet increased rapidity of response
and also potentially lower risk of progression for higher dose imatinib. A randomized trial of dasatinib (70 mg BID) versus imatinib
800 mg for patients with hematologic or cytogenetic resistance to
lower dose imatinib (400–600 mg36 reported early improvement in
CCR for dasatinib over high dose imatinib (21% vs. 8% at 3 months)
and prolonged time to treatment failure, prompting greater interest
in planned studies comparing dasatinib or nilotinib to dose esca-
lation of imatinib at earlier recognition of resistant or suboptimally
responding disease. Lastly, preliminary data from trials in chronic37
and advanced phase CML38 randomizing patients between once
daily versus divided (twice daily) dosing of dasatinib (and as well
100 mg versus 140 mg total dose for chronic phase) showed reduction in key dasatinib-related toxicities most linked to therapy interruptions and reductions, namely thrombocytopenia and pleural
effusions. Efficacy was identical for all arms studied; with further
follow-up, once daily dosing may become standard for dasatinib.
In addition to exploration earlier in the course of CML, the ubiquitous issue of “stem cell resistance” remains a challenge for new
Abl kinase inhibitors; with a goal of more definitive disease reduction
or potential elimination, dasatinib has been studied, and while able
to “reach” deeper into the earlier progenitor pool, the most primitive
CML cells remain resistant to both imatinib and dasatinib39.
With the ability to utilize Abl kinase and kinase mutant structure-function analysis, the aurora kinase inhibitor MK-0457 (formerly VX-680)40 and others are emerging with the expectation of
ability to overcome the T315I mutant kinase; MK-0457 is active in
patient cell samples in vitro bearing the T315I mutation and clinical
trial reports41 demonstrate activity in patients with advanced phase
CML / Ph+ ALL bearing the T315I mutation. Bosutinib (SKI606) is a
second Abl / Src inhibitor that in preliminary reports42 is highly active
with suggestion of less toxicity than other dual inhibitors; INNO-406
is a third dual inhibitor, against Abl and Lyn kinases and appears
promising in early trials43 as well as having potential for greater CNS
penetration.
CML: THE PAST (SCT) AND FUTURE
The use of combinations of agents to circumvent resistance
has strong rationale from in vitro studies, including combinations of
imatinib with both second-generation inhibitors44 and combinations
of nilotinib and dasatinib45, and clinical trials are planned in order to
explore development of a potential “cocktail” of kinase inhibitors to
obviate development of resistance. The role and timing of stem cell
transplant in the course of CML is a topic unto itself; however, allogeneic SCT remains an option offering long-term remission/”cure”
for CML. Although utilized differently in the current era of Abl kinase
inhibitors for CML, the potency of the graft-versus-leukemia effect
cannot be overlooked as a proper consolidation option after disease
salvage with second-generation inhibitors, or as an alternative to
unacceptably high relapse/progression risk most often associated
with disease unstable or unresponsive during nontransplant therapy. A potential algorithm for navigating current treatment options for
patients with imatinib-resistant disease is presented in Figure 1.
Table 2. Phase II Results for Nilotinib and Dasatinib in Imatinib Intolerant or Resistant Chronic and Advanced Phase CML
Nilotinib
(AMN107)
Sprycel (Dasatinib)
Disease State
N
Hematologic
Response (%)
CHR
(%)
MCR
(%)
CCR
(%)
Ref.
CP CML
279
NR
74
52
34
32
AP CML
64
59
23
36
22
33
MBC+ LBC CML
120
37*
NR
NR
6
34
Ph+ ALL
41
CP CML
387
NR
91
59
49
28
AP CML
174
64
45
39
32
29
MBC CML
109
34
27
33
26
30
LBC CML
48
36
29
52
46
31
Ph+ ALL
46
41
33
56
54
31
*Nilotinib response in Ph+ ALL was recorded as “complete response” (= hematologic recovery + < 5% marrow blasts);
Abbreviations: ALL, acute lymphoblastic leukemia; AP, accelerated phase; CCR, complete cytogenetic response; CP, chronic phase; LBC,
lymphoid blast crisis; MBC, myeloid blast crisis; MCR, major cytogenetic response; NR, not reported; Ph+, Philadelphia chromosome
positive;
S47
Figure 1. Potential Algorithm for Current Management of Imatinib Resistant CML in Chronic and Advanced Phase.a
Polycythemia Vera (PV)
Dr. Julio C. Sanchez Avalos
Polycythemia Vera is a clonal hematopoietic stem cell disease
characterized by a trilineage cell proliferation, primarily of erythroid
stem cells, with an increase in circulant erythrocytes, persistent
increase in Hb and GV, and less frequently leukocytosis, thrombocytosis, splenomegaly and hepathomegaly and other foci of extranodal erythropoyesis. Together with essential thrombocythemia
(ET) and idiopathic myelofibrosis (IMF), it is one of the chronic MPS
entities. (1)(2)
The evolution of the disease may be expressed in 2 stages:
polycythemic phase and post-polycythemic fibrosis. Different
evolving stages are currently identified according to clinical manifestations and laboratory tests, the most important being the “prepolycythemic phase” or “idiopathic polycythemia” or “early polycythemia”, which in some cases where erythrocytosis is the only
disorder in the course of a prolonged evolution would correspond to
“idiopathic erythrocytosis”. (3)
Differential diagnosis includes other secondary erythrocytosis
(SP) (e.g. apparent and relative erythrocytosis, idiopathic erythrocytosis, and secondary erythrocytosis, either congenital or acquired),
and is determined by clinical and laboratory parameters and special
studies used in different “diagnostic criteria”.
PV usually presents between the ages of 50-70, with slight predominance in males (58%). It shows a varying incidence, somewhat
related to ethnicity, ranging from 0.2/106 inhabitants/years in Asia
to 28/106 inhabitants/years in Sweden, with an overall mean of 210/106 inhabitants/years, a percentage below that for secondary
erythrocytosis. (1)(2)
CLINICAL MANIFESTATIONS
The most significant clinical manifestations, with implications
on patients’ morbidity and mortality, are the following: (1)(2)
1. Arterial
thrombotic
complications
(cardiac,
cerebral,
mesenteric,etc.), venous thrombosis (DVT, PTE, suprahepatic
(Budd Chiari), etc.12-39% at diagnosis and 10-25% during the
evolution). Physiopathology is related to hyperviscosity, leukocytosis and thrombocytosis, without excluding a functional im-
S48
2.
3.
4.
5.
6.
pairment of endothelial cells and the influence of general thrombotic risk factors.
Haemorrhage (2-20%), mainly in the oral and gastrointestinal
mucose.It is a consequence of thrombocytosis, frequently due
to acquired von Willebrand’s disease secondary to thrombocytosis
Hyperviscosity syndrome, causing facies pletorica, headache,
dizziness, eye abnormalities or ocular migraine, cognitive abnormalities, arterial claudication, erithromelalgia and distal
acrocyanosis of the hands and feet (sometimes associated to
platelet thrombosis in the microcirculation and inflammation of
the arteriolar wall), etc.
After some years of disease (10-15 years), a group of patients
may evolve into myelofibrosis with bone marrow failure (pancytopenia). Myelofibrosis is a consequence of stimulation with
cytokines delivered by the abnormal hematopoietic clone.
Evolution to MDS and AML (6-7% at 15 years), an incidence
that may be influenced by previous chemotherapy, radiation
therapy, and evolution to the myelofibrosis phase. It is a consequence of another clonal mutation, as leukemic cells are mutant
or non-mutant JAK-2. Other
manifestations include: aquagenic pruritus, fatigue, gout, renal
lithiasis, pulmonary hypertension, etc.
4. Leukocytosis: (60-70%). Neutrophilia with values >12.000/ul is
considered as a minor criterion. In smokers: >12.500/ul. Values
>25.000 can indicate a probable evolution to AML. Immature
leukocytes may be present, though with no cell atypia
5. Thrombocytosis: (50%) >400.000/ul is a minor criteria. Giant
and hypogranular platelets may be observed.
Erythrocytosis, leukocytosis and thrombocytosis are the expression
of clonal cell hyperreactivity to different stimulating haematopoietic factors: EPO, IGF-1, TPO, IL-3, G-CSF, SCF, etc.
6. Decreased ferritin level, and increased serum B12 level and
leukocyte alkaline phosphatase (LAP) score (>100)
NOVEL CRITERIA WITH DIAGNOSTIC USEFULNESS
1. Cytogenetic abnormalities(10): In 20% of patients with PV at
diagnosis and up to 80-90% patients after 10 year of evolution,
cytogenetic alterations detected by “chromosome banding” or
FISH have demonstrated:
REVIEW OF SEVERAL DIAGNOSTIC METHODS FOR PV
1. Erythrocyte Mass (RCM) is the measuring technique by means
of radioisotopes-labelled erythrocytes (CR51) and labelled albumin (I-125). To assess total volume and plasma volume, values
are expressed as related to body mass (Kg of body weight),
but for accuracy purposes, it is recommended to express them
as related to body surface area (sq. m of surface).
The value of this technique is currently being questioned, due to
its lack of standardization and its methodological complexity.(1)
In cases of PV clearly diagnosed by clinical picture, laboratory parameters and BM examination, it has shown suboptimal
sensitivity (76%) and low specificity (79%). It is useful to distinguish “true polycythemia” (1st and secondary), and according
to authors who still use it as diagnostic criterion, it is able to
differentiate PV from other MPS bearing similar abnormalities
to those of PV.(1)
The WHO criteria for polycythemia vera (2001) define the major diagnostic criterion as elevated RMC (>25% more than the
mean normal predicted value) or Hb >18.5 g/dL (male) or >16.5
g/dL (female) or greater than the 99th percentile of methodspecific reference range for age, sex, and altitude of residence
In the European Clinicopathological Criteria (ECP), the major
diagnostic criterion for classic PV is defined as Hb: >18.5 g/dl
(male) and 16.5 g/dl (female) and Htc:> 51% (male) and Htc:
48% (female), while RCM is an optional technique. In “early or
latent” PV : Htc:> 51% (male) and 43-48% (female)
True erythrocytosis is more useful in the absence of other secondary causes of erythrocytosis. The use of Hb and GV levels
as the only diagnostic criterion also has some limitations as
well, as the variables influencing these techniques do not allow
for a clear cut in the values that indicate a “true polyglobulia”.
2. Arterial oxygen saturation (SaO2): Diagnostic criterion: Saturation: <92%
3. Splenomegaly: Palpable or by ultrasound (>12 cm). Present in
70% of PV patients as a consequence of extramedulary hemopoiesis
Cr20p deletion (the alteration may be previous to the onset
of mutant JAK-2)
−
Cr8 and Cr9 trisomy. Chromosome 9p24, localizes the gene
of the JAK-2 kinase
−
DIAGNOSTIC CRITERIA
There is no clear clinical manifestation or laboratory finding to
diagnose PV; therefore, different criteria needed to be defined to allow for PV diagnosis. PVSG’s recommended criteria (1975) (4) were
the gold standard for many years, but the lack of inclusion of some
tests that have recently shown differential diagnostic usefulness
has lead to an update, according to the new criteria developed by
the WHO(5) and the European Clinicopathological Criteria (ECP).(6)
Other diagnostic criteria have been subsequently published: Polycythemia Rubra Vera Criteria (Pearson et al, 1996)(7), Guidelines
for the Diagnosis, Investigation and Management of Polycythaemia/ Erythrocytosis (Mc Mullin et al, 2005)(8), The Diagnosis of Polycythemia Vera: New Tests and Old Dictums (Tefferi, 2006) (9), etc.
−
2.
3.
4.
5.
6.
Abnormality in Cr13q, 5q, 7q, 1q, 5q, etc.(less specific, as
they are also present in other MPS o MDS). The presence
of Cr Ph+ excludes PV.
BM Biopsy (6) (11): Alterations typical of PV, which may differentiate the disease from other MPS and SP: Hypercellularity and
hyperplasia in all 3 hematopoietic series. Increase in immature
granulocytes and in megacaryocytes, with changes in size,
cluster formation and nuclear alterations. Increase in stromal
reticulin (grade 2-3) and absence of iron in macrophages. Absence of plasmatic cells, increase in hemosiderosis and apoptotic cells are frequent in SP. Investigation of mutant JAK-2
enhances BMB diagnostic value. Baseline BMB is useful to
assess the extent of fibrosis as well as to evaluate PV and assess therapeutic response.
Serum EPO levels (8) (9): A high level of EPO excludes a PV
diagnosis. Low levels are highly suggestive of PV (90-95% sensitivity and specificity). Normal levels of EPO do not rule out PV.
In the presence of elevated LAP, it is recommended to perform
BMB. A low level excludes SP. Mechanism is unknown, but it
may be associated to a decrease in the affinity of Hb for O2.
Other markers (10) which may possibly contribute to the development of the disease or otherwise constitute epiphenomena,
have been described lately and used as diagnostic criteria for
PV, though their use is limited by the complex methodology of
the test. These markers include: Polycythemia Rubra Vera-1
(PRV-1), Endogenous Erythroid Colony (EEC) formation “in
vitro” and Expression of Mpl (TPO receptor) and Serotonine
Level in Platelet-Rich Plasma (PRP).
Other alterations found in genomic and proteomic studies (microarray) and in molecular studies in patients with
PV (12): “Overexpression”(up-regulated) of 253 genes and low
expression (down-regulated) of 391 genes have been demonstrated. Sixty-four of these genes differentiate PV and SP.
Those best identified are: leucocystain, PIM-1, thrombomoduline, CEACAM-1, and NF-E2.
NF-E2 (Nuclear Factor Erythroid Derived-2) is a transcription
factor responsible for EEC and antiapoptosis in the absence of
EPO. It also stimulates thrombocytopoiesis.
Other significant findings: Overexpression of BCL-2 XL and
other antiapoptotic genes, constitutive phosphorilation of IGF-1
“like”- receptor, hypersensitivity of erythroid progenitors to hematopoietic growth factors (EPO, TPO, IGF-1, SCF, G-CSF),
IL-3, increase in telomerase activity (12) (13).
Deregulation in the expression of mi-RNA in PV erythroid progenitors cells (mi-RNA, small “non codified” RNA) that regulate
gene expression at post-transcriptional level(14). They regulate
several biological functions, including hemopoiesis. Findings
were made of different expressions of mi-RNA in PV, genes related to cell cycle regulation, JAK-2-STAT pathway, and genes
regulating “down-stream” JAK-2 molecules, which may play a
significant role in PV erythroid proliferation and activation. Their
quantitative expression by RT-PCR is currently being investigated miRNA: Let7, 16 and 26b and miRNA 27(14).
7. Mutation of the JAK-2 gene in MPS: The “loss of heterogeneity” (LOH) in chromosome 9 (p24), which included several
genes, among them JAK-2, was described in MPS in year 2000,
but it was only in 2005 that a mutation in gene JAK-2 was identified, with a valine substitution for phenylalanine in codon 617,
exon 14. The JAK-2 V617F mutation was described in a high
proportion of patients with PV, and to a lessen extent, in patients
with ET and IMF(15).
JAK-2 is a cytoplasmatic tyrosine kinase which triggers the transcriptional activity of several intracellular signaling pathways,
after activation of different growth factor membrane receptors
(type-1-citokines receptors) such as EPO, TPO, G-CSF, SCF,
etc. The main activated pathways are JAK-2 – STAT5 and
STAT3, PI3-K-AKT, RAS-MAPK, etc., regulating proliferation,
differentiation, cell cycle and apoptosis of hematopoietic cells.
Additionally, it stabilizes the EPO cell membrane receptor and
reduces MPL receptor (TPO) level. This finding strengthens the
concept that PV and other MPS are clonal hematopoietic stem
cell diseases, with an acquired mutation which is responsible
for their development and progression. Proposed role of JAK-2
V617F mutation, occurring at the JH-2 domain (pseudokinase):
interaction with the JAK—2 catalytic domain activation loop,
resulting in a loss of “self-inhibition” of its kinase activity and
leading to its “constitutive activation”.
The significance of the JAK-2 mutation has been demonstrated
in murine models, where hematopoietic stem cell transplantation with mutant JAK-2 induced alterations similar to those observed in PV (16).
Clinical findings in patients with MPS with mutant and non-mutant JAK-2 have shown some differences between both groups
and its influence on treatment. Study PT-1, comparing the effect of hidroxiurea and anagrelide in patients with ET at high
thrombotic risk, showed that patients with JAK-2 mutation had
higher hematocrite level, lower platelet count and lower level of
serum EPO, with a higher resemblance to PV than non-mutant
ET patients. Additionally, it has been shown that patients with
JAK-2 had higher incidence of thrombosis, and that the preventive superiority of thrombosis with hidroxiurea occurred in
patients with positive mutation. Furthermore, the comparison of
homozygous JAK-2 V617F mutation (20-30%) vs heterozygous
in patients with PV has shown that the homozygous phenotype
has a higher level of Hb, an increased rate of transformation
to myelofibrosis, and a higher incidence of pruritus(16). Recent
investigations in patients with PV have also shown that measuring the number of mutant alleles (mutant alleles / wild-type
alleles ratio) by PCR, and comparing them to different clinical
parameters, those with a higher number of mutant alleles had
higher levels of hematocrites and leukocytes, higher expression
of LAP, PVR-1, LDH level, splenomegaly, and a higher incidence
of thrombosis and treatment needs during the evolution(16) (17).
This would demonstrate that the number of “mutant alleles” is
more useful to differentiate between homo- and heterozygous
JAK-2 V617F phenotype, and that its measurement would be
important to stratify more severe and asymptomatic patients
with PV (17).
Incidence of the JAK-2V617F mutation in MPS depends on the
sensitivity of the test method, the most sensitive one being the
recognition of alleles by RT-PCR sequencing analysis. Using
this technique, the observed incidence was 93-97% for PV, 5057% for ET and 50% for IMF.
The presence of this mutation in patients with PV, and its absence in SP, would allow for a better and faster diagnosis of PV,
thus obviating more complex tests, e.g. erythrocyte mass and
other supplemental studies.
However, the absence of the JAK-2 mutation in a percentage
of patients with PV, and in a higher proportion in ET and IMF
patients, suggests that there may be additional mutations still
not identified.
Doubts remain, however, because a single mutation can contribute to the pathogeny of 3 different diseases and because
there are MPS wihtout mutation in JAK-2, which puts in doubt
the ethyologic relevance of this finding. A recent investigation
S49
conducted in 10 patients with erythrocytes with negative JAK2 V617F mutations showed that all of them had mutations of
JAK2 Exon 12, with deletion or mutation at different aminoacids level, 6 of which corresponded to PV and 4 to idiopathic
erythrocytosis. Clinical expression consisted in erythrocytosis,
thrombocytosis and increase in EEC, but they did not present
with leukocytosis(18). This would demonstrate that some idiopathic erythrocytosis would correspond to special or partial
forms of PV (early or latent PV). Stimulation of JAK-2 kinase
activity by these mutations (gain of function) results in a higher
level of erythrocytes than the JAK-2 V617F mutation. It was interpreted that a higher stimulation of JAK-2 is associated with
an increased erythropoietic activity, and that with a lower level
of stimulation, thrombopoiesis and leukopoiesis are stimulated
as well(18).
The identification of these mutations in the JAK-2 gene has
brought significant progress to the physiopathologic, diagnostic
and clinical interpretation of PV, ET and IMF, raising new questions for future investigations and potential new therapies with
inhibitors specific to the mutant JAK2 in this pathologies, as it
happened with CML.
As regards diagnosis, some authors currently propose diagnostic algorithms for PV in cases of significative erythrocytosis
(above the normal range), using only (low) serum EPO levels,
increased LAP score, bone marrow biopsy, with the characteristic histomorphological changes and mutation of positive JAK2(9), and some others just with abnormal erythrocytosis, if they
have the JAK-2 mutation associated (16).
Further experience is needed before definitive conclusions can
be drawn, including comparative evaluations of patients diagnosed based on classic criteria and patients diagnosed using
newly proposed criteria.
THE PROGNOSIS OF PV
The primary causes of mortality in PV patients are thrombotic
complications or haemorrhage, evolution to myelofibrois, with bone
marrow failure (pancitopenia), or transformation to AML.(1)(2)
Patients without treatment have a short survival time (<2 years).
Since more precise diagnostic criteria were defined and phlebotomy
treatment was initiated (thus maintaining normal Hb and hematocrit levels), survival has increased to >12 years. The association
of phlebotomy with some cytoreductive therapies, e.g. radioactive
phosphorus and clorambucil, reduces survival (9-10 years), as there
is an increase in mortality due to transformation to AML.
In the last years, the association of phlebotomy treatment and
cytoreductive agents with lower leukomogenic activity, as HU, IFN
and pipobroman, as well as the addition of aspirin as antithrombotic
agent, has enhanced survival, with an average survival time of >20
years.(1)(2)(9)
TREATMENT OF PV
Phlebotomy is the mainstay of therapy for PV, aiming at diminishing hematocrit and Hb levels to normal (< 45% male)(<42%
female), though some authors set adequate levels at <50%.
Phlebotomy should be normovolemic, and frequency should
be the necessary to maintain adequate levels of hematocrit and
Hb.(1)(2)(9)
The other treatment that has shown usefulness for the prevention of thrombosis (study ECLAP) is aspirin, taken at 100 mg/day
doses. It is recommended to use it in all patients(19). Patients with
active thrombosis should be treated with standard antithrombotic
therapy (Heparine- ACO) (20)
In cases of intolerance or aspirin-associated complications,
such as acquired vW ( FvW level, ristocetine cofactor <30%),
treatment should be interrupted or replaced by clopidogrel, until
the vW default improves by means of platelet cytoreduction (HU,
anagrelide, IFN) or plateletpheresis.(20)
The use of cytoreductive agents (HU, IFN, pipobroman) is recommended in patients with high thrombotic risk or in patients who
do not respond to phlebotomy and present with persistent elevated
erythrocytosis, leukocytosis, thrombocytosis, development of splenomegaly or evidence of increased fibrosis in the bone marrow.
S50
There are some useful treatment algorithms, such as therapeutic
guidance.
Overall, the most commonly used cytoreductive agent is HU,
but in resistant cases, IFN, pipobroman or busulfan can be used.
Some authors recommend IFN as the more efficacious agent (21),
though molecular response is minimal, as it happens with imatinib
(22).
However, high levels of molecular response have been reported
with the use of pegilated alpha-IFN (23). Treatment with “statins” has
been recently proposed, based on their multiple actions on PV(24)
TREATMENT OF PV AND PREGNANCY(25)
Cases of PV and concomitant pregnancy are uncommon (<50
reported cases), as the incidence of PV patients < 40 years old is
low (<15%)
Obstetrical complications in patients with ET and PV are higher
that in the general population, with a 50% fetal survival rate. Related complications are spontaneous abortion, late pregnancy loss,
delay in fetal development, and premature delivery.
Though levels of Hb, hematocrit and platelets tend to diminish
during pregnancy, it is however advisable to maintain platelet levels
<1.000.000/ul and hematocrit levels <40%, using phlebotomy and
cytoreductive agents with IFN.
It is advisable to perform ultrasound studies to assess fetal development, as well as uterine artery eccodopler, more frequently
than in normal pregnancy.
Recommended conduct during pregnancy is phlebotomy, aspirin and low molecular weight heparine (enoxaparine: 40 mg/day). In
cases of high risk pregnancy, it is advisable to add IFN (3.000.000
U/day) as cytoreductive agent. Aspirin and heparine should be interrupted before delivery or cesarean section, or to perform epidural
anesthesia procedures. They should be recommended post-partum and maintained at least 6 weeks post-partum.
FUTURE TREATMENTS IN MPS (PV, ET, IMF) WITH MUTANT
JAK-2
Molecules that have shown ability to inhibit mutant JAK-2 in in
vitro cell culture assays, animal models, and some Phase I studies
in patients:
- TK Inhibitors: AEE-788, CEP-701
- Histone Deacetylase Inhibitors: ITF 2357= Italfarmaco (Milan)
- Inhibitors specific to mutant JAK2: TG 101348, TG 101192, TG
101209, TG 101-345, Avicin-D, Tyrphostin (WP 1066), Atiprimod.
- Aurora kinases inhibitors = MK-0457
(Abstracts ASH, 2006)
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1. Tefferi, A., Spivak, J. “Polycythemia Vera: Scientifics advances
and current practice” Sem. Hematol., 42: 206-220, 2005
2. Cao, M., Olsen, R., Zu, Y. “Polycythemia Vera: new clinicopathologic perspectives” Arch. Pathol. Lab. Med, 130: 1126-1132,
2006
3. Michiels, J., Bernema, Z., van Bockstaele, D. et al. “Current diagnosis criteria for chronic myeloproliferative disorders, essential thrombocythemia (ET), polycythemia vera (PV) and chronic
idiopathic myelofibrosis (CIMF)”. Pathol. Biologie, 1-13, 2006
4. Berlin, N. “Diagnosis and classification of the polycythemias”.
Sem. Hematol., 12: 339-351, 1975
5. Jaffe, S., Harris, N., Stern, A. et al. “WHO classification of the
chronic myeloproliferative diseases (CMPD), polycythemia
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Classification of tumors of Haematopoietic and Lymphoid Tissues - Lyon, France. IARC: 31-42, 2001.
6. Michiels, J., Thiele, J. “Clinical and pathological criteria for the
diagnosis of essential thrombocythemia, polycythemia vera and
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profiling in polycythemia vera: overexpression of transcription
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14. Bruchova, H., Gaikwad, A., Mendell, J., Prchal, J. “Disregulated
expression of miRNAs in Polycythemia Vera erythroid progenitors”. Blood, 108: 1032a (Abstracts 3613), 2006.
15. Kralowics, R., Passamonti, F., Buser, A. Et al. “A gain of function
mutations JAK-2 in myeloproliferative disorders”. New Engl. J.
Med., 352: 1779-1790, 2005.
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Eng. J. Med., 355: 2452-2466, 2006.
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the JAK-2 V617F mutational load at diagnosis on mayor clinical aspects in patients with polycythemia vera”. Blood, 108: 6a
(Abstracts 5) 2006.
18. Scott, L., Tong, W, Ross, L. et al. “JAK-2 Exon 12 mutation in
polycythemia vera and idiopathic erythrocytosis”. New Eng. J.
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19. Landolfi, R., Marchioli, R., Kutti, J. et al. “Efficacy and safely of
low-dose aspirin in polycythemia vera”. New Engl. J. Med., 350:
114-124, 2004.
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32: 422-429, 2006.
Chronic Myeloproliferative Disorders
(Other than CML) on the subject State of
the art management of CMD: Implications
of Current Pathogenic Breakthroughs
Ruben A. Mesa
Associate Professor of Medicine - Division of Hematology
Mayo Clinic College of Medicine - Rochester, MN USA
[email protected]
The classic BCR-ABL negative myeloproliferative disorders
(MPDs) include polycythemia vera (PV), essential thrombocythemia
(ET), agnogenic myeloid metaplasia (AMM; includes post thrombocythemic (ET) and post polycythemic myeloid metaplasia (PV)
to form myelofibrosis with myeloid metaplasia (MMM)) 1. Clinically
these disorders share a variable spectrum of symptomatology arising from myeloproliferation (erythrocytosis, leukocytosis, or throm-
bocytosis) as well as target organ damage from the intramedullary
proliferative state (organomegaly2, vascular complications3.
The elucidation of the molecular defect in CML was a watershed event for that disease and eventually led to development of
effective targeted therapy (inhibition of the BCR/Abl product by
Imatinib Mesylate)4 for that disorder. In 2005 several independent
investigative groups, using a variety of methodologies, described an
activating point mutation in the pseudo-kinase domain of the tyrosine kinase JAK2 (JAK2 V617F) in the vast majority of patients with
PV and about half of those with ET and MMM 5-8. Therapeutic implications of the mutation, both prognostically and as a therapeutic
target remain uncertain but offer an exciting avenue of investigation
into MPD therapy. Additionally, novel therapies such as immunomodulatory agents (further lenalidomide analogs), hypomethylating
agents, proteosome inhibitors, and mTOR inhibitors are all being
evaluated in clinical trials to hopefully improve the efficacy of therapy for advanced MPD patients.
This manuscript will focus on limitations and opportunities with
current available therapeutic interventions, and hopes for future improvements. Currently there is no therapy has been shown to be
curative or prolong survival in MPD patients except allogeneic stem
cell transplantation. The concept of using stem cell transplantation
for the therapy of MMM is the most attractive given this MPD disorder is the most likely to decrease survival amongst those afflicted.
Initial reports with allogeneic transplantation in MMM have shown
that this therapy does have curative potential in these patients 9,10.
Recent reports describe a 58% 3 year survival in a group of 56
MMM patients (age 10-66), with a 32% non-relapse mortality rate
11
. The significant toxicity of full allogeneic transplant in MMM led to
non-myeloablative trials 12-14. The latter trials have ben encouraging
in terms of decreased non-relapse mortality, and increasing ages
of those successfully transplanted. However, allogeneic transplant
still carries a significant risk of graft versus host disease (at least
33%) and the exact role and benefit depends on the long term prognosis of the patient. There is currently no data on the use of stem
cell transplantation in stable phase ET or PV. Indeed, the significant
risks of any of the stem cell transplantation procedures make it difficult to justify this therapy for ET and PV given the overall modest
prognosis of these patients.
ESSENTIAL THROMBOCYTHEMIA AND
VERA: RISK MANAGEMENT APPROACH
POLYCYTHEMIA
Assessing short and long term risks: Patients with ET and PV
can potentially have life expectancies as long as age matched controls 15,16. However, such longevity amongst these patients is not
universal as both short and long term risks of both morbidity and
mortality exist.
Short term risks: ET and PV both share a risk of thrombosis
and hemorrhagic events. Muti-factorial analysis of risk factors have
shown that individuals with an age greater than 60 or those with
a prior history of thrombotic events have been found to be at high
risk of MPD associated thrombosis 17. Interestingly, the absolute
platelet count in and of itself has not been found to be an independent risk factor for thrombosis. Explanations for the lack of correlation between the platelet count and risk of thrombosis may at
least partially be due to the influence of activated neutrophils contributing to thrombotic diathesis 18. Intermediate risk of thrombotic
events has included those individuals felt to be at significant risk
of cardiovascular or cerebrovascular thrombotic events because of
hypertension, smoking, hyperlipidemia, etc. 19. Low risk individuals
have been defined by those lacking any of the previously stated
risk factors. Additional risk factors to be considered for hemorrhage
include extreme thrombocytosis, specifically a platelet count in excess of 1500 x 109/L as this can lead to imbalance between the
platelet value and existing coagulation factors 20. Additional risk factors for hemorrhagic events may be the acquisition of acquired von
Willebrand syndrome in patients with MPDs 21.
Long Terms Risks: ET and PV both have the risk of transformation to post thrombocythemic and post polycythemic myeloid
metaplasia respectively 15, with a 15 year cumulative incidence of
4 and 6% for ET and PV respectively. Additional risk of leukemic
transformation in these individuals (at 15 years) is 2% and 7% for
S51
ET and PV respectively (increased by radioactive phosphorus and
alkylator exposure 22). It should be noted that no medical therapy
has definitively helped to decrease the long term risks associated
with either of these disorders.
SHORT TERM GOALS
Prevention of Thrombosis and Hemorrhage: Management, and
prevention, of vascular events in MPD patients has relied upon a
risk stratified approach (according to the risks outlined above) 23.
This approach first uses low dose (40-100mg/day) of aspirin in most
ET and PV patients whom have no contraindication for aspirin use,
given the proven benefit in a large randomized placebo controlled
trial in PV patients which demonstrated a clear decrease in thrombosis 24. Second the use of phlebotomy for the control of erythrocytosis to a goal of a hematocrit <45% in males and <42% in females
for those with PV 25. Recent data regarding JAK2V617F mutant ET patients (whom have a PV like phenotype) 26 suggests these individuals may well also benefit from phlebotomy to control erythrocytosis
if present. Finally, cytoreductive therapy is employed to decrease
thrombotic or hemorrhagic risk in high risk ET/PV patients (and in
appropriate intermediate risk patients).
Cytoreductive Therapies: The choice of cytoreductive therapy,
and when to employ such therapy remains controversial in ET and
PV patients (see Table 1). All currently available agents have draw
backs in terms of their potential toxicities, expense, and questions
as to the efficacy achieving short and long term goals and will be
discussed. The main three agents currently include interferon alpha, hydroxyurea, and anagrelide. Additionally, other cytoreductive
agents are used sparingly because of their known propensity for
increasing the risk of leukemic transformation (radioactive phosphorus (P32) and alkylator therapy such as busulfan and melphalan).
These latter agents are best reserved for those individuals with limited life expectancy, or in whom the there is no other clinical choice
due to toxicity/intolerance to less toxic therapies.
PHARMACOLOGIC THERAPY OF MMM
The currently available therapeutic options for the therapy of
patients with myelofibrosis with myeloid metaplasia (MMM; therapeutically equivalent to those with chronic idiopathic myelofibrosis
(CIMF)) have been largely disappointing. Similarly, leukemic transformation (LT) of MMM or the MPDs occurs in approximately 1015% of patients27. Therapeutically these patient represent a very serious challenge (In a recent series of 91 consecutive MMM patients
that experienced LT 28), We found LT to be fatal in 98% of patients
after a median of 2.6 months (range 0-24.2). Survival was equally
poor regardless of whether patients received strictly supportive
care, low intensity therapy (i.e. low dose cytarabine) or induction.
The understanding of the pathogenetic mechanisms of CIMF/MMM
continue to grow as the molecular mechanisms potentially involved
with the pathogenesis of the disease such as the JAK2V617F5 and
the MPLW515L29 continue to be delineated. These molecular breakthroughs are added to previous findings of the mechanisms of myeloproliferation, and the aberrant stromal reaction in CIMF/MMM.
Pharmacologic therapies that are at various stages in the evaluation
process for treating CIMF/MMM will de discussed and are outlined
in (Table 2).
The Future
It is anticipated that therapeutic options for MPDs patients will
continue to improve as our understanding of the mechanisms of
the disease pathogenesis continue to improve. Many compounds
are currently in the earliest phases of testing to see if inhibition of
the wild-type JAK2 molecule will provide a therapeutic benefit. No
mature data exists in this arena, but is greatly anticipated. Additionally, as further information is gained as to disease mechanisms in
MMM patients whom do not have the JAK2 mutation (such as the
MPLW515L) additional therapeutic targets are anticipated.
S52
Table 1. Comparison of current cytoreductive agents in MPD patients
Agent
MPD Effect
Class
Route
Toxicities (partial list)
Ref
Myelosuppression
Oral ulcers
Leg Ulcers
Skin/ Nail changes
Alopecia
30 ,31
Main Line Cytoreductive Therapies in MPDs
Hydroxyurea
Decrease Leukocytosis
Decrease Thrombocytosis
Antimetabolite
Oral
Anagrelide
Platelet Reducing Agent Oral
Headache
Palpitations
Fluid Retention
Anemia
Arrythmia
Cardiomyopathy
Hemorrhage*
30,32
Interferon-Alpha
Decrease Leukocytosis
Biological
Confusion
Mood Deisorders
Fatigue
Fever
Thyroiditis
33-35
Busulfan
Myeloproliferation
Alkylator
Oral
Myelosuppression
Leukemogenic
36
P-32
Myeloproliferation
Radionuclide
IV
Myelosuppression
Leukemogenic
37
SQ
High Risk – Limited Use Cytoreductive Therapies in MPDs
SQ: Subcutaneous injections
*: In combination with aspirin 30
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S53
Table 2. Medical Therapies for Myelofibrosis with Myeloid Metaplasia (MMM)
Agent
Class
Route
Toxicities (partial list)
Ref
Available Agents
Erythropoietin
Growth Factor
SQ
Hypertension
Arthralgias
38
Danazol
Androgen
Oral
Hirsuitism
Edema
Thrombosis
Exacerbate prostate cancer
Liver adenoma
38
Thalidomide
Immunological
Oral
Neuropathy
Constipation
Thrombocytosis
39
Thalidomide w/steroids Immunological
Oral
Neuropathy
Constipation
Thrombocytosis
Hyperglycemia
40
Hydroxyurea
Antineoplastic
Oral
Myelosuppression
Leg Ulcers
41
Busulfan
Alkylator
Oral
Myelosuppression
Leukemogenic
36
Melphalan
Alkylator
Oral
Myelosuppression
Leukemogenic
42
P-32
Radionuclide
IV
Myelosuppression
Leukemogenic
37
2-CdA
Purine Nucleoside Analog
IV
Myelosuppression
43
Lenalidomide +/Prednisone
Immunomodulatory drug
Oral
Myelosuppression, rash, GI disturbances
44
Actimid (CC-4047)
Immunomodulatory drug
Oral
Myelosuppression, DVT
45
Sunitinib
Kinase inhibitor
Oral
Edema, fatigue, mucositis
46
Bortezomib
Proteasome inhibitor
IV
Peripheral neuropathy, hypotension, GI
disturbances
47
Dasatinib
Kinase inhibitor
Oral
Myelosuppression, fluid retention, fatigue
48
Bevacizumab
Anti-VEGF monoclonal antibody
IV
Hypertension, bleeding
49
GX15-170
Pan-Bcl-2-Inhibitor
IV
Somnolence, euphoria
50
Azacitidine
Hypomethylating agent
SC
Myelosuppression
51
GC-1008
Pan-specific human anti-TGF-β antibody IV
NA
52
Therapies for MMM in Development
Future Molecularly Targeted Approaches
JAK2 (?JAK-STAT)
Inhibition
Inhibitors of MPL mutants
SC: Subcutaneous
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by thalidomide-related immunomodulatory drugs. J Pharmacol
Exp Ther. 2003;305:1222-1232.
46. Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a
novel and potent inhibitor of vascular endothelial growth factor
receptor tyrosine kinases, impairs vascular endothelial growth
factor-induced responses and tumor growth after oral administration. Cancer Res. 2000;60:2178-2189.
47. Wagner-Ballon O, Gastinne, T, Tulliez, M, et al. Proteasome
inhibitor bortezomib can inhibit bone marrow fibrosis development in a murine model of myelofibrosis. Blood. 2005;106:(abstr 2582).
48. Lombardo LJ, Lee FY, Chen P, et al. Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS354825), a dual Src/Abl kinase inhibitor with potent antitumor
activity in preclinical assays. J Med Chem. 2004;47:66586661.
49. Cardones AR, Banez LL. VEGF inhibitors in cancer therapy.
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50. O’Brien S, Kipps, TJ, Faderl, S, et al. A phase I trial of the small
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51. Issa JP, Kantarjian H. Azacitidine. Nat Rev Drug Discov.
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52. Yingling JM, Blanchard KL, Sawyer JS. Development of TGFbeta signalling inhibitors for cancer therapy. Nat Rev Drug Discov. 2004;3:1011-1022.
S55
EDUCATION SESSION
Bone Marrow Failure
Contribution of Immunophenotyping to
the Diagnosis of Paroxysmal Nocturnal
Haemoglobinuria
Pilar Hernández-Campo, Julia Almeida Alberto Orfao
Centro de Investigación del Cáncer, Departamento de Medicina
and Servicio de Citometria; University of Salamanca, Salamanca,
Spain.
GPI-associated proteins are a relatively heterogeneous group of
proteins with differences in their pattern of expression, biochemical
structure and function (Table 1). Some GPI-asociated proteins (e.g.:
CD55 and CD59) are complement regulatory molecules, preventing
CD55+/CD59+ cells from complement mediated-lysis. While CD55
and CD59 as well as CD58, show a broad cellular distribution, being
present in virtually all haematopoietic cell types, other GPI-associated molecules show a much more restricted pattern of expression
(expression of CD66b is typically restricted to neutrophils and CD14
to monocytes)
INTRODUCTION:
FLOW CYTOMETRY IN THE DIAGNOSIS OF PNH:
Proxysmal nocturnal haemoglobinuria (PNH) is a clonal haematopoietic disorder (HPN) which involves an early haematopoietic
precursor with the ability to differentiate to red cells, platelets and
leucocytes. Typically in PNH patients, clonal cells coexist with normal haematopoietic cells, what may increase the difficulty of the
diagnosis of the disease.
From the clinical point of view PNH is characterized by the
presence of intravascular haemolysis in association with an increased susceptibility for infectious diseases and a higher risk of
thrombotic events. In the last decades, important advances have
been achieved as regards the understanding of the pathogenetic
mechanisms of the disease contributing to the decreased expression of membrane surface proteins in the altered cells. Accordingly,
at present it is well-known that PNH patients typically display a
somatic mutation of the glycosylphosphatydilinositol (GPI)-anchor
(PIG-A) gene localized in chromosome X, that encodes for an enzyme involved in the synthesis of GPI, a molecule which acts as an
anchor to the plasmatic membrane for a variety of cell surface proteins. Among these cell surface GPI-anchored proteins, molecules
involved in the regulation of complement capable of inhibiting formation and binding of activated complement proteins/complexes to
the surface of circulating autologous blood cells.
For many years, demonstration of the increased sensitivity of
red cells to undergo complement-mediated cell lysis was based
on the Ham’s and sccarose tests. Nevertheless, these techniques
are associated with important limitations. First, they have a limited
sensitivity and do not allow detection of small clones of pathological
cells represented at relatively low frequencies in blood, particularly
once patients have been transfused; Secondly, they just assess the
involvement of the erythorid lineage and do not provide information
about the status of leucocytes and platelets.
In the last decades, new advances in the understanding of
PNH have led to the development of new diagnostic approaches,
which include, among others: 1) the flow cytometric detection of
abnormally decreased expression of GPI-associated cell surface
proteins and, 2) detection of PIG-A gene mutations in PNH cells. In
this presentation we will first review the normal patterns of expression of PIG-associated proteins; in the second part we will review
the major contributions of flow cytometry immunophenotyping in the
diagnosis of PNH.
GPI-ASSOCIATED CELL SURFACE PROTEINS: MAJOR CHARACTERISTICS AND CELLULAR DISTRIBUTION.
Expression of surface proteins in haematopoietic cells uses different biochemical anchor mechanisms which include GPI bridges.
At present, the availability of monoclonal antibody reagents
directed against GPI-anchored proteins has allowed a better identification and characterization of clonal cells in the blood of PNH patients by flow cytometry. At present, general consensus exists about
the need to demonstrate the defect of at least two different proteins
in two different cell lineages in the diagnosis of PNH. Most frequently, assessment of both CD55 and CD59 expression is used for the
diagnosis of PNH. The advantage of using these two proteins for
the diagnosis of PNH relies on their broad cellular distribution and
the clinical relevance of their defective expression.However, important technical limitations may be associated with the assessment of
CD55 and CD59 in blood cells depending on the exaact sample preparatiuon procedure used and the great variability on total red cells,
platelets and leucocytes in identical volumes of blood from different
individuals. Accordingly, both markers typically display an optimal
pattern of staining on leucocytes if a direct immunofluorescence
stain-and-then-lyse procedure is used. Alternative micro techniques
have been developed which allow simultaneous assessment of expression of CD55, CD59 and other GPI-associated molecules in red
cells, platelets and different subsets of leucocytes (e.g.: neutrophils,
monocytes, lymphocytes)
Recent studies have provided in depth information about the
normal patterns of expression of a high number of GPI-associated
proteins in both the major and minor compartments of blood and
bone marrow cells from healthy controls. At the same time, these
type of studies have also allowed the identification of the most informative combinations of proteins and cell subsets to be studied
for the diagnosis of PNH. Of note, some of the GPI-associated proteins may also be anchored to the cell surface as a transmembrane
protein, which limits their diagnostic utility; as an example this is
the case of CD16 on NK-cells and of both CD58 and CD109. In
turn, despite the utility of CD55 and CD59, their combined use is
suboptimal for the assessment of specific subsets of leucocytes and
platelets since these antigens may be expressed at relatively low
levels in such cell subpopulations in normal individuals. In contrast,
other markers expressed at relatively high levels in different subsets
of normal haematopoietic cells show complete lack of expression in
clonal PNH cells, thus allowing for a clear discrimination between
normal and PNH cells. Accordingly, for specific cell populations
such as the neutrophils relatively large panels of proteins displaying
these features exist (CD16, CD24, CD55, CD59, CD66b, CD157),
while for other cell subsets the number of informative markers is
restricted (monocytes: CD14, CD55, CD157; B-cells: CD24, CD48,
CD52, CD55; CD4+/CD8- T-cells: CD48,CD52,CD55; eosinophils:
CD55, CD59; CD8+/CD4- T-cells: CD48, CD55) or even limited to a
single antigen (CD48 for CD56+ NK-cells, CD55 for BDCA3- nega-
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tive myeloid dendritic cells and CD56++ NK-cells and CD59 for red
cells).
In turn, flow cytometry not only allows the identification of altered PNH cells, but it also provides information about its representativeness in the sample and the degree of involvement of the
different subpopulations of haematopoietic cells. In this sense, it
should be noted that important differences exist in the proportion of
defective cells within the different compartments of blood cells from
individual PNH patients. Overall, the highest proportion of PNH cells
is typically detected for the neutrophils and monocytyes. In contrast, the proportion of PNH lymphocytes is typically lower or even
undetectable. Such differences have been associated with the longlife expectancy of lymphocytes in peripheral blood as compared to
most myeloid cell compartments.
3.
4.
5.
RELEVANT REFERENCES:
1. Hernández-Campo PM, Martín-Ayuso M, Almeida J, López A,
Orfao A. Comparative analysis of different flow cytometry-based
immnophenotypic methods for the analysis of CD59 and CD55
expression on major peripheral blood cell subsets. Cytometry,
2002; 50: 191-201.
2. Hernández-Campo PM, Almeida J, Sanchez ML, Malvezzi M,
Orfao A. Normal patterns of expression of glycosylphosphati-
6.
7.
dylinositol-anchored proteins on different subsets of peripheral
blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry B Clin Cytom. 2006
Mar;70(2):71-81.
Hernández-Campo PM, Almeida J, Matarraz S, de Santiago M,
Sánchez ML, Orfao A. Quantitative analysis of the expression of
glycosylphosphatidylinositol-anchored proteins during the maturation of different hematopoietic cell compartments of normal
bone marrow. Cytometry B Clin Cytom, 2007;72B:34-42.
Piedras J, López-Karpovitch X. Flow cytometric analysis of
glycosylphosphatidylinositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size. Cytometry, 2000;
42:234-238.
Olteanu H, Karandikar NJ, McKenna RW, Xu Y. Differential usefulness of various markers in the flow cytometric detection of
paroxysmal nocturnal hemoglobinuria in blood and bone marrow. Am J Clin Pathol, 2006;126:781-788.
Richards SJ, Rawstron AC, Hillmen P. Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry, 2000;42:223-233.
Hall SE, Rosse W. The use of monoclonal antibodies and flow
cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood, 1996; 87:5332-5340.
Table 1. Membrane proteins associated with GPI: functional characetristics and cellular distribution in peripheral blood.
PROTEIN
FUNCTION
DITRIBUTION
CD14
R-LPS
Monocytes
CD16
Fcγ receptor III
Neutrophils
NK-cells
CD24
Costimulatory molecule
Regulator of cell adhesion
Neutrphils
B-lymphocytes
CD48
Molecule involved in cell activation,
adhesion and T-cell costimulation
Monocytes,
Myeloid dendritic cells, T- B- and NK-cells
CD52
Adhesion molecule involved in T-cell
costimulation
Monocytes,
Myeloid dendritic cells, T- and B-cells
CD55
Inhibition of activation of C3
Red cells
Platelets
Leucocytes
CD58
Cellular adhesion (CD2 ligand)
Involved in signal transduction
Red cells
Platelets
Leucocytes
CD59
Binds to C5b-C8 (inhibits binding of
C8 to C9)
Red cells
Platelets
Leucocytes
CD66b
Adhesion molecule
Neutrophils
CD73
Ecto-5’-nucleotydase
T- and B-cells
CD87
Urokinase-PAR
Neutrophils
Monocytes
CD109
Unknown function
Neutrophils
Monocytes
Eosinophils
Myeloid dendritic cells
CD157
Ectoenzyme
Involved in signal transduction
Neutrophils
Monocytes
Fanconi Anemia
Ricardo Pasquini
Fanconi Anemia (FA) is a rare genetic disease, but it is the
most common inherited bone marrow failure syndrome, characterized by progressive marrow failure, congenital anomalies, and predisposition to develop leukemia and solid tumors. FA is included in
the group of chromosome instability syndromes, exhibiting a high
cellular hypersensitivity to interstrand DNA crosslinking agents,
such as cisplatin, mitomycin C (MMC), diepoxibutane (DEB) and
melphalan. The FA cells when exposed to crosslinking agents manifest chromosomal breakage and fusions with characteristic radial
forms consistent with an underlying genomic instability. FA is a recessive disorder with both autosomal and X linked patterns of inheritance. Recent identification of the responsible genes for FA has
changed the view of the molecular pathogenesis of the disease.
Genetically, FA is very heterogeneous disease since many genes
mutations may be responsible to similar clinical picture within the
expected spectrum of this disease. FA can be divided in at least
twelve complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, and
M) (table I), defined by cell fusion studies and 11 of the 12 genes
have been identified. Among 84% of patients fall within the subtypes
A, C, and G and the majority comprise the subtype A (table II). Many
of the FA genes encoded novel proteins of unknown function. FA
proteins A, B, C, F, G, L, and M associate in a nuclear core complex
and together with FA I protein are required for monoubiquitination
of the FA D2. Monoubiquitinated FA D2 has an important role in
DNA repair associated with other DNA repair proteins. Also, the FA
pathway interacts with additional DNA repair pathways involved in
tumor suppression (FANCD1/BRCA2, BRACA1, and NBS1) (figure
1). Defects of FA genes have been found in a wide variety of human cancers in the general population. Defects of DNA repair and
cell-cycle check-points such the defects of the FA pathway are possible mechanism of genomic instability in cancer and may also be
responsible for the hypersensitivity of cancer cells to certain types
of chemotherapeutical drugs and radiation.
Table I
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CLINICAL MANIFESTATIONS
Classical clinical features, such as growth retardation, small
head size, café-au-lait spots, upper limb abnormalities (thumbs,
hands, radii, ulnae), and renal structural abnormalities can be strong
diagnostic clues (table III), but it may occur in patient without congenital defects and can be diagnosed in adulthood. There is a correlation between the number of important congenital anomalies and
the early onset bone marrow failure as the first adverse outcome.
The relation between genotype and phenotype has not been clearly
established, but some specific mutation is associated to multiple
major physical anomalies and early onset hematological problems
(FANCC –IVS4+4A→T).
Table II. Fanconi anemia (FA) complementation groups.
FA
Subtype
Gene
Required for
FANC D2
Ubiquitination
Percentage
of FA
Patients
A
FANCA
yes
66%
B
FANCB
yes
rare
C
FANCC
yes
10%
D1
FANCD1/BRCA2
no
3%
D2
FANCD2
yes
3%
E
FANCE
yes
3%
F
FANCF
yes
2%
G
FANCG/XRCC9
yes
9%
I
?
yes
2%
J
FANCJ/BACH1/BRIP1
no
2%
L
FANCL/PHF9/POG
yes
rare
M
FANCM/Hef
yes
rare
*From Levitus M. Roolmans MA, Steltenpoo, J, et al. Blood.
2004; 1032498-2503
Figure 1. The Fanconi anemia pathway.
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PREDISPOSITION TO MALIGNANCY:
Table III
Anomaly
Skin pigment changes
Short stature
Upper limb abnormalities (thumbs,hands,
raddi, ulnae)
Hypogonadal and genitalia changes (mostly
male)
Other skeletal findings (head, face, neck,
spine)
Eyes/lids/ephicanthal fold anomalies
Renal malformations
Ear anomalies (external and internal), deafness
Abnormalities of hips, legs, feet, toes
Gastrointestinal and cardiopulmonary malformations
Approximate
Frequency (%)
65
60
50
Clonal hematological evolution to myelodysplastic syndrome
is found in some FA patients associate with deletions and translocations often involving chromosomes 1 and 7 with the propensity
to evolve to AML being this complication the most common malignancy found in FA patients. Liver malignancy and neck and head
squamous cell carcinoma are the most frequent solid tumors and
appear in FA patients older than 10 years and the average is 23
years (table IV).
40
30
25
25
10
Table IV – Observed cancers, ratio of observed to expected
cancers, and 95% Cls among North American respondents
with FA.
10
10
Young NS, Alter BP: Clinical features of Fanconi’s anemia. In
Young NS, Alter BP (eds): Aplastic Anemia, Acquired and Inherited. Philadelphia, WB Saunders, 1994, pp 275-309.
LABORATORY EVALUATION
The natural history of the hematological abnormalities is usually
a gradual onset of bone marrow failure with declining values in one
or more hematopoietic lineages. Thrombocytopenia usually is the
first to appear, following by granulocytopenia and anemia. Hematological abnormalities develop at median of seven years, ranging
from birth to 31 years. Severe aplasia develops in most cases, but
the full expression of the pancytopenia may take months to years to
appear. The red cells are often macrocytic and high concentration of
hemoglobin F is common. Mild dysplastic features is not rare at the
bone marrow cytology. The standard for the diagnosis of FA is the
MMC or DEB breakage test. The methods involve culturing replicative cells, usually PHA-stimulated peripheral blood T lymphocytes
or skin fibroblast in the presence of low doses of either MMC or
DEB, followed by examination of metaphase spreads for evidence
of chromosomal breakage and radial figures (figure 2). Each laboratory needs to standardize this method to establish ranges of normal
limits and those considered unequivocally diagnostic of FA. In few
FA patients, less than 10%, this test may be negative related to the
somatic mosaicism of the hematopoietic stem cell. In these cases,
the DEB test should be performed in cultured fibroblasts originated
from skin biopsy for the evidence of chromosomal breakage. Immunoblotting to detect ubiquitinated and non-ubiquitinated forms of
FANCD2 and retroviral complementation studies are useful methods for diagnosis and identification of the mutant gene.
Figure 2
CLINICAL MANAGEMENT
The median survival of patients with FA is approximately 30
years but prognosis is highly variable. The life-threatening early
event in the majority of the complementation groups is bone marrow
failure. The aim of treatment is to avoid the complications related to
cytopenias. Stem cell transplantation is the only option to establish a
normal hematopoiesis. Patients with significant pancytopenia, consisting of absolute neutrophils count <1000/μL hemoglobin <80g/dL
and platelets <40000/μL, who are otherwise healthy and have a
HLA-matched sibling donor are excellent candidates for hematopoietic stem cell transplantation (HSCT). FA patients are very sensitive
to chemotherapy, particularly to alkylating agents and radiation, and
the usual myeloablative preparative regimens result in very severe
or life threatening toxicity. Low doses of alkylating agents alone or
associated ATG are the present used conditioning regimens for FA
patients undergoing to HSCT utilizing an HLA-matched sibling donor. In this group of patients, the rejection rate is extremely low and
the overall survival is superior to 80%. The experience utilizing alternative donors, including mismatched related donors and matched
unrelated donors, is growing but the results have been significantly
inferior to those obtained with sibling donors. Approximately 60%
are long term survivors and the best results are reached when the
donor is HLA genotipically identical and the patient is free of infection, received few blood transfusions and no androgen was previously used. The ideal conditioning regimen has not yet been found,
but the addition of fludarabine and ATG has improved the outcome.
The rejection rate and the high incidence of severe GVHD are the
main barriers to improve the results in this context. Unrelated umbilical cord blood is an important source of hematopoietic stem cell and
more than one hundred transplants have been done in FA patients.
High number of nucleated cell infused and better matched degree
(5/6 and 6/6) are associated with better results. Over the last 20
years, the Bone Marrow Transplantation Center in Curitiba (Brazil)
transplanted more than 160 patients suffering from Fanconi Anemia
(table V). In figures 3 and 4 is illustrated this experience.
Not all patients are candidates for transplantation and certainly
they will need supportive measures. Red cell and platelets transfusions, infection control are common procedures. Androgens have
been used for a long time; particularly for patients requiring blood
transfusions. Oxymetholone is used more frequently and 50% of
the FA patients have blood counts improved and generally takes
1 to 2 months or more to reach the maximum response. Almost
all patients relapse when the androgen is stopped and liver toxicity should be monitorized, since enzymes elevations, cholestasis,
peliosis hepatis and liver tumors (adenoma) have been reported.
Gene therapy is appealing as the potential option to repopulate the bone marrow. Experience in humans is minimal; however
few research centers are already prepared to start enrolling eligible
patients.
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Figure 4.
Table V
SCHT IN FANCONI ANEMIA
Transplant Characteristics
(Bone Marrow Transplant Center – Curitiba/Brazil)
Number of Patients
RELATE
UNRELATE
111
57
Source of Stem Cell
bone marrow
110
20
cord blood
01
36
peripheral blood
00
01
NONE
103
20
YES 1mis/2mis
1/7*
20/17
# HLA Disparities
*Father; Mother; Sibling; Cousin; Grandparents
July/2006
Figure 3.
REFERENCES
Auerbach AD, Adler B, Chaganti RS: Prenatal and postnatal diagnosis andcarrier detection of Fanconi anemia by a cytogenetic
method. Pediatrics 67:128-135, 1981.
Bagby GC: Genetic basis of Fanconi anemia. Curr Opin Hematol
10: 68-76, 2003.
Bagby GC, Alter BP, Fanconi anemia. Seminars in Hematology,
43:147-156, 2006.
Boyer MW, Gross TG, Loechelt B, Leehuis T, Filipovich A, Harris RE:
Low risk of graft-versus-host disease with transplantation of CD34
selected peripheral blood progenitor cells from alternative donors
for Fanconi anemia. J Pediatr Hematol Oncol 25:890-895, 2003.
Futaki M, Yamashita T, Yagasaki H, Toda T, Yabe M, Kato S, et al:
The IVS4+4 A to T mutation of the Fanconi anemia gene FANCC
is not associated with a severe phenotype in Japanese patients.
Blood 95: 1493-1498, 2000.
Gluckman E: Radiosensitivity in Fanconi anemia: Application to
the conditioning for bone marrow transplantation. Radiother Oncol
18:88-93, 1990 (supp 1).
Guardiola P, Pasquini R, Dokal I, et al. Outcome of 69 allogeneic
stem cell transplantations for Fanconi anemia using HLA-matched
unrelated donors: a study on behalf of the European Group for
Blood and Marrow Transplantation. Blood 95:422-429, 2005.
Joenje H, Patel KJ: The emerging genetic and molecular basis of
Fanconi anemia. Nat Rev Genet 2:446-459, 2001.
Kennedy RD, D’Andrea AD. The Fanconi Anemia/BRCA pathway:
new faces in the crowd. Genes Dev. 19:2925-2940, 2005.
Kutler DI,Singh B, Satagopan J, Batish SD, Berwick M, Giampetro
PF, et al: A 20-year perspective on the International Fanconi Anemia
Registry (IFAR). Blood 101:1249-1256, 2003.
de Medeiros CR, Bitencourt MA, Zanis-Neto J, Maluf EC, Carvalho
DS, Bonfim CS, Funke VM, Setubal DC, Farah VM, Pasquini R.:
Allogeneic hematopoietic stem cell transplantation from na alternative stem cell source in Fanconi anemia patients: analisys of 47 patients from a single instituition. Braz J Med Biol Res 39 (10):1297304, 2006.
Rosensberg PS, Huang Y, Alter BP: Individualized risks of first adverse events in patients with Fanconi anemia. Blood104: 350-355,
2004.
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Rosenberg PS, Greene MH, Alter BP: Cancer incidence in persons
with Fanconi anemia. Blood 101: 822-826, 2003.
Rosenberg PS, Socie G. Alter BP, Gluckman E: Risk of head and
neck squamous cell cancer and death in patients with Fanconi
anemia who did and did not receive transplants. Blood 105:67-73,
2005.
Rubin CM, Arthur DC, Woods WG, Lange BJ, Nowell PC, Rowley
JD, et al: Therapy-related myelodysplastic syndrome and acute myeloid leukemia in children: Correlation between chromosomal adnormalities and prior therapy. Blood 78:2982-2988, 1991.
Shimamura A, Montes DO, Svenson JL, Haining N, Moreau LA, Nathan DG, et al: A novel diagnostic screen for defects in the Fanconi
anemia pathway, Blood 100: 4649-4654, 2002.
Toshiyasu Tanigushi and Ala D. D’Andrea: Molecular pathogenesis
of Fanconi anemia: recent progress. Blood 107:4223-4233, 2006.
Velazquez I, Alter BP: Androgens and liver tumors: Fanconi’s anemia and non-Fanconi’s conditions. Am J Hematol 77: 257-267,
2004.
Wagner JE, Eapen M, Macmillan ML, Harris RE, Pasquini R, Boulad
F, Zhang MJ, Auerbach AD: Unrelated donor bone marrow transplantation for the treatment of Fanconi anemia. Blood 109(5)225662, 2007.
Zanis-Neto, J et al. Low-dose cyclophosphamide conditioning for
haematopoietic cell transplantation from HLA-matched related donors in patients with Fanconi anemia. British Journal of Haematology 130:99-106, 2005.
MDS: Clinical Update 2007
Ruben A. Mesa, MD
Associate Professor of Medicine
Division of Hematology
Mayo Clinic
Rochester, MN, USA
[email protected]
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SYMPOSIUM
Latin American Cooperative Oncology Hematology GroupLacohg - clinical experiences
Mutational Status of Immunoglobulins Genes (VHIg) and CD38 Monoclonal Marker
in Chronic Lymphocytic Leukemia as
Prognostic Factors
Raúl Gabús
Servicio de Hematología. Hospital Maciel. Ministerio de Salud
Pública. MONTEVIDEO – URUGUAY
Project’s development. This study was the product of the CLL
cooperative working group between the Clinical Service of Hematology of Hospital Maciel (A.I. Landoni, E.Bodega MD), the Molecular Pathology Unit from Medicine and Science Public Universities
(O.Pristch, S.Bianchi MD) and the Flow Cytometry Martínez Prado
Laboratory (C.Canessa MD)
It was supported by Schering Laboratory.
INTRODUCTION.
Clinical rational.
Chronic Lymphocytic Leukemia (CLL) continues being the
most frequent leukemia in the western world and in most cases
affects patients over 60 years. Frequently, the diagnosis is precocious, finding a lymphocytosis in a routine screening blood test.
Usually the clinical course is indolent, over 50%, but its evolution can be unpredictable at the time of diagnostic. Nowadays the
diagnostic can be usually made near the sixties and in 20 a 30% on
patients beyond 55. That made a mayor life expectancy that justify
treatment research, that improve their clinical course.
The clinical course to an aggressive disease cannot be preestablished and there has been no biological prognostic factors for
many years that stratify this group of patients. The clinical classifications of Binet and Rai continue being the mayor value prognosis
parameters with significant statistical impact over survival but they
don’t respond to the clinical evolution questions, mainly in the indolent stages.
A third of the patients will never require treatment and will die
of non related cause disease.
Another third with indolent disease will evolve at some moment, will require treatment and will die frequently because of its
disease.
The remaining third group of patients will present aggressive
form of the disease at diagnoses, will require front treatment and
most of them will die of its disease.
Although the disease is considered up to now incurable, the
therapeutic attempts in order to obtain “complete remissions” in
youngest patients are higher.
The inclusion of new drugs to the treatment of Chronic Lymphocytic Leukemia, as the nucleoside analogues (Fludarabine) have
modified the clinical course of the disease in an important number
of patients. Although it is not clear if these have impact in a increase
of overall survival it must be accepted as an standard treatment of
frontline in CLL. The oral formulation of the Fludarabine is viable,
and improves the quality to bear the treatment in these patients with
the same results (Cazin et al)
The association of Fludarabine with other drugs as ciclophosphamide, the incorporation of monoclonal antibodies Anti CD 20
-Rituximab- (Schulz et al, Byrd et al CALGB) and antiCD52-Alem-
tuzumab Campath1H- (Lundin et al) and the transplant procedures
of hematopoietic progenitors, opened a promising door to achieve
complete remissions in this disease, even if the fact to obtain the
complete remission and increasing the overall survival will have to
be demonstrated.
The two challenging questions in the therapeutic strategy in
the CLL, are “when” and “how” to treat.
All the efforts aims to determine clearly the biological parameters that allow us to better define the groups of patients to treat in
the different therapeutic strategies.
Biological rational.
The CLL-B displays inmunophenotype characteristics:
Immunoglobuline of surface (sIg) of low intensity of fluorescence, co-expression CD5/CD20, CD20 of low intensity of fluorescence, CD23+, Cd22-/+, FMC7 -/-, CD10 -/-, CD79B -/-, CD11c-/+.
However aberrant markers can exist in the CLL-B, as the negative for CD5 or the positive for other antigens like CD22, FMC7,
CD11c, CD79b-/+; with homogenous or heterogeneous expression.
The prognostic value of inmunophenotype in Chronic Lymphocytic Leukemia as well as in others Chronic Lymphoproliferative disorders is not definitively established.
The study of different biological parameters: cytogenetic, immunophenotype and molecular markers, have been related with the
disease.
Many publications confer to the marker CD38 a value of worse
prognostic. (Damle RN et al., Ferrarini et al)
Döhner et al. have published a sub group of deletereous cytogenetic aberrations with prognostic value. The 17p (p53) deletion
and the 11q deletion are unfavorable prognostic factors and 13 q
deletion is considered by many authors as favorable prognostic factor.
The mutational profile of the variable genes of Immunoglobulins (VHIg) has been presented as a parameter of strong clinical predictive value. The studies of Hamblin et al. show clear differences
in the evolution of CLL according to the mutation or not of these
Immunoglobulins.
The technical complexity of this procedure makes its use difficult of routine. Different investigators tried to find markers that
surrogate this biological factor, and within them, Zap70 (Crespo et
al) seems to provide a good predictive values for IgVH mutational
status.
PRINCIPAL ENDPOINT
The main objective of this project was to evaluate and to compare the course of patients with a clear phenotype characterization
of CLL-B in relation with the genomic aberrations, the CD38 marker,
and the mutation status of the variable genes of the immunoglobulins (VHIg).
METHODOLOGY AND TECHNOLOGY
Immunophenotype by flow cytometry was made at time of
diagnosis, in flow cytometry of 3 colors of fluorescence (BD) by
triples markers (CD19,CD5,CD38) and tested the percentage of
CD19,CD5 cells who express CD38. The cut off level is defined in
30%.
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Mutational status of VH immunoglobulin genes:
Construction of a cDNA and genomic DNA bank originated
from Peripheral Blood Mononuclear Cells (PBMC) of patients with
B-CLL.
Identification of Ig VH, D and JH segments rearrangement present in the proliferative clone of B cells and their nucleotide sequence
analysis for each patient. Comparison between de malignant clone
gene sequence with the germinal configuration gene. (>98% of correlation: unmutated genes, and <98%: mutated genes)
Since the 12 Stage A mutated patients had no progression of
the disease and no need of treatment with a media follow up of 49.5
months, 5 of the 6 non mutated patients progressed and had NCI
criteria of treatment.
CLL - Progression free survival (PFS) and Binet stages
MATERIALS AND RESULTS
They were included 40 patients (55% females, 45% males)
with CLL diagnosed by inmunophenotype, included in a period of
three years (2003-2006) distributed in 20 patients Stage A, 10 patients Stage B and 10 patients Stage C (Binet classification), less
than 75 years old (average age of 61 years old 38-73) with a performance status <2, good cardiac and pulmonary function and non
pretreated patients.
The treatment schedule was watch and wait for Stage A and
Indolent Stage B patients.
Fludarabine iv or po for stage B aggressive patients. (25 mg/m2
iv x 5 days or 40 mg/m2 po x 5 days)
Fludarabine-ciclophosphamide for stage C patients (Fara 25
mg/m2 iv x 3 days or Fara 40 mg/m2 po x 3 days + Ciclophosphamide 300 mg/m2 iv /day x 3 days)
In 33 of 40 patients were performed the mutational status of
IgVH and all of them were studied with CD38 at time of diagnosis.
• 30 % of stage A patients were IgVH non mutated. This percentage is shown proportionally higher in advanced disease.
• 30% of Stage A and 20 % Stage B and C patients had a CD38+
marker. There were no significant differences between the 3
groups.
• From the 17 non mutated pts only 6 were CD38 + at diagnosis.
We remark that there were not done the CD38 at evolution and
progression, since this parameter may vary evolutionarily
CLL - stage A - Progression free survival (PFS) and IgVH mutational profile
Binet Stage Patients and Mutational Status of VHIg
12
10
8
mutated
Non mutated
6
4
2
0
A (n:18/20)
B (n:7/10)
3 of the 40 patients died: 1 from stage A () group and 2 from
stage C (Acute Pneumonia non related to fludarabine treatment)
C (n:8/10)
TREATMENT RESPONSE FOR 1ST. LINE THERAPY.
Binet Stage patients and CD 38 expression
% PATIENTS
80
70
60
50
NEGATIVE
POSITIVE
40
30
20
10
0
A (n:20)
B (n:10)
C (n:10)
BINET STAGE
In the 20 Stage A patients, there have been a significant difference in clinical evolution between the 12 mutated patients and the
6 non mutated ones.
21 patients received fludarabine regimen treatment.
20/21 evaluable patients underwent the 78.5 % of the preestablished protocol with the 1st. line regimen treatment.
17/21 evaluable patients obtained 88% of complete hematological response and 85% global mayor nodal response (66% CR
and 19% PR>50%).
The infectious toxicity was to consider since 3 patients underwent Acute Pneumoniae, one extend herpes virus, and one supurative cutaneous lesion.
There were no relevant severe III-IV hematological toxicity:
leucopenia 5/20 and trombopenia 1/20, without mortality evidence.
DISCUSSION
This study observed a direct relation between the non mutated
status of VHIg and the advanced stages of the CLL disease.
Stage A non mutated patients underwent a clear progression
profile and they required treatment (NCI criterias).
The CD38 + patient at time of diagnosis, didn’t show a significant correlation with the non mutated VHIg status.
It has prognostic value per se, and it can vary during evolution.
Even if it is not standard clinical practice to treat precociously
the stage A patients with poor biological prognostic factors, these
results promote the decision to include them into clinical trials in
order to evaluate the clinical impact and change the evolution of
the disease.
CLL an Overview
R Fernando Bezares.
Buenos Aires - Head Hematology Unit Hospital General de Agudos
Dr Teodoro Álvarez
Professor of Hematology.Sociedad Argentina de Hematología.
Universidad de Buenos Aires.
Member of Commitee trial in Lymphoma of GATLA GROUP
Chairman of CLL trials of GATLA & LACOHG (Latin American
Onco Hematology Cooperative Group)
The history of CLL begins in de 17th century when van Leewenhoeck invented the microscope. But it was Erlich, with the development of staining techniques in the last decades of the 17 Th century
(year 1800), who promoted the knowledge and understanding of
blood cells and their morphological differences.
Ray and Binet with their staging system introduced the first procedures that gave us some understanding of this disease.
The Spanish Hematological School contributed with two important prognostic factors, bone marrow infiltration patterns and Lymphocyte doubling time.
Further advances in immunology and flow cytometry resulted
in the establishment of monoclonal phenotypic profile enabling, safe
diagnosis.
However, CLL biology and treatment ran slowly until the end of
the last century when purine analogues and monoclonal antibodies
were introduced and cytogenetics together with molecular biology
offered new tools for a better knowledge of the cell involved.
Investigators from US, UK, Spain, and France were the first
groups to show interest in CLL. But in the last 20 years the addition
of Italian, German and others Groups has turned CLL into a glittering star in the Hematological Universe.
If we make a representative collage of CLL the picture would
probably be something like a snail climbing slowly up a hill until
the last 20 years when the immunophenotipe , FISH, Monoclonal
antibodies and molecular biology came into the CLL field working
as fast vehicles of progress for this uninteresting disease of the 70
and turning fascinating it into what today may be considered a preempting disease.
Recently in the last ASH meeting investigators from Mayo Clinic correlated Vimentin expression with mutational state of VH Ig and
Smudge cells(SC). They have being show a correlation between
TTT and OS according to the percentage of SC (more than 30%
good prognostic and low Vimentin expression). We are interested in
comparing SC with Vimentin expression by immuno histochemistry.
May be, as Nowakowski et all suggests, we may have obtained an
important and cheap prognostic factor with a microscope and MGG
staining when this paper is confirmed by others groups .
On finishing this overview, I would like to highlight a recent paper from Hoffbrand & Hamblin published in Leukemia Research.
These authors with a clinical criteria have came up again the concept of Benign Monoclonal Lymphocytosis for patients without adverse prognostic factors and stable disease that may have a life
expectancy similar to that of matched age and gender normal population.
Then the question remained open; CLL is only one disease?
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Fludarabine based regimens for indolent
NHL
MD Brady Beltran Garate
LACOGH gruop is conformed for several investigators from
Latinoamerica whose main objetive is to develop regional studies
in the branch of oncohematology.
Two studies has been developed into LACOGH for indolent
lymphomas . Both considered Fludarabine in association regimens
for naive and recurrent/refractory patients in the era pre-monoclonal antibodies.
A first study correponded to Milone et al. that reported Fludarabine, Mitoxantrone and Dexamethasone (FMD) for the first line
treatment of patients with Indolent Non-Hodgkin Lymphoma (NHL)
from de GATLA (Grupo Argentino para el Tratamiento de la leukemia aguda). Ninety-six patients were recruited. and sixty-nine
patients were valuable . FND treatment consisted of F 25 mg/m2 i.v.
(days 1–3), N 10 mg/m m2 i.v. (day 1) and D 20 mg (days 1–5) each
28 days for 6 cycles. Results: on this low grade NHL cohort showed
a response rate of 93% with 70% (48 pts) with complete response
(CR) and 23% (16 pts) with partial response; progressive disease
and non-response 7% (5 pts). The probability of event free survival
(EFS) and overall survival (OS) at 24 months was 60% and 90%
respectively.FND treatment demonstrated a high CR rate with low
toxicity and high probability of EFS and OS as previous experience
published in the literature. as first line of treatment in indolent Non
Hodgkin Lymphoma
A second study correponded to Baltazar et al that reported
Fludarabine and Mitoxantrone (FM) for the refractory/relapse treatment of B-cell low-grade non-Hodgkin lymphoma (NHL):. Fourtyeight patients were evaluated. Fourty-four pts. had follicular lymphoma and 4 small lymphocytic lymphoma. The median previous
treatment was 1 (range: 1-3). FM treatment consisted of F 25 mg/
m2 i.v. (day 1-3) and M 10 mg/m m2 i.v. (day 1) each 28 days for
6-8 cycles.Results showed a response rate (PR+CR) of 81% ,;progressive disease and non-response 19%. With a median follow up
of 17 months, OS at 24 months was 86% and disease Free Survival
(DFS) at 24 months 57.1% . Mortality rate was : 12,5% (6/48 patients), 5 of them because progressive disease. It study confirmed
that FM regimen is an effective and safe treatment for refractory/recurrent low grade NHL.
Both studies showed the high activity of Fludarabine regimens and low toxicity in the treatment of Latinoamerican patients
with Indolent non hodgkin lymphomas in the era pre-monoclonal
antibodies.
DFS eith FM in RR low grade NHL: LACOHG
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Non-myeloablative stem cell
transplantation. The Mexican approach.
Guillermo J. RUIZ-ARGÜELLES MD, FACP, FRCP (Glasg)
Centro de Hematología y Medicina Interna.
Clínica Ruiz de Puebla.
Puebla, MEXICO.
David GOMEZ-ALMAGUER MD
Hospital Universitario de Monterrey.
Universidad Autónoma de Nuevo León. Monterrey,
Monterrey, Nuevo León, MEXICO
Cesar Homero GUTIERREZ
Olga G. CANTU-RODRIGUEZ
Guillermo J. RUIZ-DELGADO MD
Luz. C. TARIN-ARZAGA MD
Nowadays allogeneic and autologous hematopoietic cell transplantation have become the treatment of choice for several malignant and non-malignant blood disorders. More than 50,000 transplants of hematopoietic cells from marrow, peripheral blood or cord
blood are being performed annually. The development of effective
control of graft versus host disease, anti-viral and antifungal drugs
and the shift to out-patient care have resulted in important reduction
of the cost of transplantation in the developed world. However, the
cost of the so called “conventional transplantation” is still unaffordable for the majority of the patients living in the developing world.
A question here is: How can we provide the best possible transplantation technique for the individual patient with scarce resources
without increasing the risk of failure?
In México we have made efforts to simplify the hematopoietic
cell transplantation methods, an experience that could be applicable
to other countries both in the developed and the developing world.
THE MEXICAN APPROACH
Non-myeloablative allogeneic stem cell transplantation (NST)
has been one of the most exciting developments in the treatment
of hematologic malignancies in the last years. Nine years ago, we
elected to employ in México a regimen to conduct NST, based in
those used in the developed world (1-3) , introducing some changes with the main goal of decreasing the cost of the procedure and in
turn, making it available to a larger number of patients. The salient
changes of our approach are: Use of cheapest and available drugs
(fludarabine, busulfan and cyclophosphamide), use of peripheral
blood stem cells and tailored number of apheresis sessions, elimination of prophylactic ganciclovir and intravenous IgG, outpatient
conduction, reduced number of blood products transfusions and reduced donor-lymphocyte infusions (4-5). The conditioning regimen
consist of oral busulphan, 4 mg / Kg on days - 6 and - 5; intravenous (i.v.) cyclophosphamide, 350 mg / m2 on days - 4, - 3 and - 2;
i.v. fludarabine, 30 mg / m2 on days -4, -3 and -2; oral cyclosporin A,
5 mg / Kg starting on day – 1 until day + 180 and i.v. methotrexate 5
mg / m2 delivered on days + 1, + 3, + 5 and + 11 (4-5).
a) Overall results: Using the “Mexican method” to conduct
NST, we have conducted over 300 allografts in patients with differ-
ent diseases: Chronic myelogenous leukemia, acute myelogenous
leukemia, acute lymphoblastic leukemia, myelodysplasia, thalassemia major, relapsed Hodgkin´s disease, Blackfan-Diamond syndrome, adrenoleukodystrophy, Hunter´s syndrome, aplastic anemia
and several solid tumors. In the whole group, the median granulocyte recovery time to 0.5 x 109/L was 13 days, whereas the median
platelet recovery time to 20 x 109/L was 12 days. Around one third
of the patients did not need red blood cell transfusions and also one
third did not need platelet transfusions. In more than 70% of cases
the procedure could be completed totally on an outpatient basis.
The follow up time of the patients ranges between 30 and 2000
days. In around 8% of individuals there was a graft failure and, since
the preparative regimen is non-myeloablative, all these patients
recovered endogenous hematopoiesis. Approximately 50% of the
allografted individuals developed acute graft versus host disease
(GVHD), and approximately 30% developed chronic GVHD. The
median post-allograft overall survival (SV) has not been reached
and the 2000 day overall SV is 54%. The 100-day mortality is 16%
and the transplant-related mortality is 20%. In the whole group of
patients, the median cost of each NST was 15 000 USD (6-14), a
figure which contrasts with that informed from the United States of
America, where a bone marrow transplantation using conventional
allografting has a cost of 200- 300 000 USD (15).
b) Chronic myelogenous leukemia (CML): We published initially a paper of 21 CML patients in different phases of the disease,
alografted in two institutions in México (Centro de Hematología y
Medicina Interna de Puebla and Hospital Universitario de Monterrey); in this study we found a 750 day overall SV of 60% (10). Later
on, in a group of 24 CML patients in first chronic phase, recruited in
a collaborative Group (Latin-American Cooperative Onco Hematology Group – LACOHG - ) with patients from six institutions located
in four Latin American countries (México, Venezuela, Brasil, and
Colombia), we obtained an 830-day disease free SV of 92% (16);
it was clear that the results were better allografting CML patients in
first chronic phase.
c) Acute myelogenous leukemia (AML): in a collaborative
study in three Mexican institutions: CHMI, HUM and Centro Médico
la Raza (CMR) of the Instituto Mexicano del Seguro Social, in a
group of 24 AML patients in different stages of the disease (first,
second and third remission) we found an 860-day SV of 66% (17).
These individuals were eligible for conventional allografting but
were given an NST using the “Mexican method” mainly for economic reasons. Later, on, analyzing separately the results in AML
according to the remission status, we found in another multicenter
study a 480-days SV of 50% for patients in first remission and 15%
for those in a second remission (18); accordingly, it seems to be
better to allograft patients with AML in first remission, but it is also
possible to rescue some AML patients when they have achieved a
second or further remission with this type of allografting.
d) Acute lymphoblastic leukemia (ALL): The results in this
disease have been dissapointing. In a group of 19 ALL patients
grafted in second remission or beyond in a single institution, we
found a 900 day overall SV of 22% with a median overall survival of
491 days; these data are similar to those obtained with allografting
after conventional conditioning and support the concept that malignant ALL cell “escape” from the graft versus leukemia effect which
is more clear and useful in other malignancies.
e) Aplastic anemia (AA): The “Mexican method” to conduct
non-ablative allografting has also been used to allograft patients
with severe AA. In a collaborative group of four Mexican institutions
(Centro de Hematología y Medicina Interna de Puebla, Hospital
Universitario de Monterrey, Centro Médico La Raza and Instituto
Nacional de Cancerología), 23 individuals with severe, refractory
aplastic anemia were allografted using peripheral blood stem cells
and a fludarabine based conditioning regimen ( without ATG) ; we
found a 1500 day SV of 91% (19), a figure which compares favorably
with those published using other types of conditioning regimens.
We have allografted also children and adolescents with the
“Mexican method” (20-21). Initially, it was considered that nonablative conditioning should be offered only to aged or debilitated
individuals, or with comorbidities; however it is clear that children
are the ones who suffer more the long-term consequences of the
aggressive conventional preparative regimens. Based in this idea,
we were the first to conduct non-ablative allografting in children with
malignant conditions (20), and we have found a very low prevalence of long-term complications with good results, mainly in nonmalignant hematological diseases. After our initial publication, other
groups have also engaged in allografting children using reducedintensity preparative regimens (22).
With the method that we have employed, we have also grafted
cord blood cells; the long-term survival of patients allografted with
cord blood cells obtained from both domestic and foreign cord blood
banks was in our experience of 40% at 6 years (23-24). We have
also been able to rescue individuals with relapsed Hodgkin´s disease and allografted small groups of patients with other diseases
such as multiple myeloma, myelodysplasia, chronic lymphocytic
leukemia, solid tumors, etc (4-5). Concerning the complications
of the allografting procedures and given the low hematologic and
extrahematologic toxicity of the “Mexican” conditioning regimen,
we have found that the proportion of individuals who develop a
nephrotic syndrome after the allograft is considerably lower than
that observed in patients given other types of non-ablative conditioning (25). By the same token, the transfusion requirements of
the patients allografted with this method are very low (26), and the
complications stemming from cytomegalovirus reactivation are exceptional (27). Along the same line, the 100-day mortality is 16%,
a figure which contrasts with that of conventional allografting than
can go up to 50%. We have also analyzed the significance of the
HLA disparity between donor and receptor, and we have found that
it is safe to conduct non-ablative allografting using our approach
in individuals who have either an HLA identical (6/6) or compatible
(5/6) sibling donor (28). Having increased the number of patients
allografted for acute leukemia and their follow up periods, we have
found leukemic relapses: They have occurred in 60% of patients
with ALL and in 50% of patients with AML. In a multicenter study
Universitario de Monterrey and Centro Médico La Raza) we have
found that extramedullary relapses are more frequent in patients
with AML than in those with lymphoid malignancies and that bone
marrow relapses are more frequent and aggressive in patients allografted for lymphoid malignancies (29). Interestingly, we have
also found that leukemic relapses in the hematopoietic cells of the
donor are not unfrequent (30) and that this complication should be
analyzed with more detail to further gain insight into the leukemogenesis events.
More than 95% of the patients who have been allografted in
México and other developing countries using the “Mexican approach” to conduct NST could not have afforded the cost of a
conventional or more expensive stem cell transplant. Prospective
studies will define if NST will eventually replace conventional stem
cell grafting; however, very frequently in developing countries, the
decision for a given patient is not between offering either a conventional bone marrow transplant or a NST; the decision has to
be made between NST or no other effective treatment. Because
of its cost, NST could be considered as an early treatment option
in countries where limited resources currently prevent the use of
conventional allogeneic bone marrow transplantation; role-definition and appropriate timing for this therapeutic approach in patients
are required. We are learning which malignancies are more susceptible to the graft versus tumor effect, one of the main effects of
NST in addition to the replacement of the bone marrow cells, and
as a consequence, we are also learning in which malignancies NST
is more useful. The “Mexican approach” to conduct NST has been
shown to be effective for allografting individuals with malignant and
non-malignant conditions. Despite the fact that ours and most studies with reduced intensity conditioning have a relatively short follow
up, there is information which indicates that the procedure is related
with lower toxicities and a lower prevalence and severity of GVHD,
with a similar efficacy as that of conventional allografting. Since this
method is more feasible and affordable for patients and physicians
in developing countries, the number of allografts in these places has
increased substantially, as well as the publications related to bone
marrow transplantation stemming from places where this therapeutic maneuver was considered as unaffordable previous to the development of this technology (31).
Allografting with reduced intensity conditioning may be related
with several disadvantages such as mixed chimerism and relapse
of the malignancy, however. NST has resulted not only in the prog-
S75
ress of knowledge, but also in the accessibility of many patients
to sophisticated therapeutic actions, in some cases, the only true
curative option for these individuals.
Figure 1. Overall survival of the patients given a non-myeloablative stem cell allotransplant using the “Mexican method”.
CML, CP = Chronic myelogenous leukemia in chronic phase;
AA = aplastic anemia; AML = acute myelogenous leukemia in
second or further remission; CML = chronic myelogenous leukemia in all phases; ALL = acute lymphoblastic leukemia.
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acondicionamiento no mieloablativo. Experiencia en una sola
institución. Bol Med Hosp Inf Mex 2005; 62: 88-95.
22. Spitzer TR: The expanding applications of non-myeloablative
stem cell transplantation. Pediatr Transplant 2003; 7:95-100
23. Ruiz-Argüelles GJ, Reyes-Núñez V, Garcés-Eisele J, Warwick
RM, McKenna L, Ruiz-Reyes G, Granados J, Mercado-Díaz
MA.: Acquired hemoglobin S trait in an adult patient with secondary acute myelogenous leukemia allografted with matched
unrelated umbilical cord blood cells using a non-ablative conditioning regimen. Haema 2005; 8: 492-496.
24. Mancías-Guerra C, Ruiz-Delgado GJ, Manzano C, DíazHernández MA, Tarín-Arzaga LC, González-Llano O, GómezAlmaguer D, Ruiz-Argüelles GJ.: Umbilical cord blood transplantation using non-myeloablative conditioning: The Mexican
experience. Hematology 2006, in the press
25. Ruiz-Argüelles GJ, Gómez-Almaguer D.: Nephrotic syndrome
after non-myeloablative stem cell transplantation. Brit J Haematol 2006, 132:801-802
27. Ruiz-Argüelles GJ, Gómez-Rangel JD, Ponce-de-León S,
disease. Am J Hematol 2004; 75;200-204
28. Ruiz-Argüelles GJ, López-Martínez B, Manzano C, GómezRangel JD, Lobato-Mendizábal E.: Significance of one human
leukocyte antigen mismatch on outcome of non-myeloablative
allogeneic stem cell transplantation from related donors using
the Mexican schedule. Bone Marrow Transpl 2005; 35:335339.
29. Ruiz-Argüelles GJ, Gómez-Almaguer D, Vela-Ojeda J, Morales-Toquero A, Gómez-Rangel JD, García-Ruiz-Esparza MA,
López-Martínez B, Cantú-Rodríguez OG, Gutiérrez-Aguirre
CH.: Extramedullary leukemic relapses following hematopoietic
stem cell transplantation with non-myeloablative conditioning.
Int J Hematol 2005, 82:262-265.
30. Ruiz-Argüelles GJ, Ruiz-Delgado GJ, Garcés-Eisele J, Ruiz-Argüelles A, Pérez-Romano B, Reyes-Núñez V.: Donor cell leukemia after non-myeloablative allogeneic stem cell transplantation: A single institution experience. Leukemia and Lymphoma
2006, in the press.
31. Gómez-Almaguer D. The simplification of the SCT procedures
in developing countries has resulted in cost-lowering and availability to more patients. Int J Hematol 2002; 76: 380-382 (supp
1)
ALEMTUZUMAB IN PERIPHERAL T-CELL
LYMPHOMAS
Luis Palmer
Peripheral T-cell lymphomas (PTLs) are uncommon, accounting for fewer than 10% of all non-Hodgkin lymphomas.1 The molecular pathogenesis of most PTLs is poorly understood and in the
WHO classification, clinical characteristics, in conjunction with morphological and immunophenotypic criteria, are relied on to define
most disease entities.2
Functionally, T cell Lymphomas are related to the two major
arms of the immune systems, the innate and the adaptive immune
system, PTCLs are derived from post-thymic cells and include a
number of distinct clinico-pathological entities such as angio-immunoblastic T-cell lymphoma, anaplastic large-cell lymphoma (ALCL),
enteropathy-type intestinal T-cell lymphoma, hepatosplenic Y/5TNHL, and subcutaneous panniculitis-like T-cell lymphoma.3 T h e
remainder cannot be categorized as any specific clinical or pathologicaí syndrome and are designated as PTCL unspecified. Success in therapy has lagged behind that of aggressive B-cell lymphomas with a poor prognosis 5-year survival. Apart from a subgroup of
ALCLs which are positive for the anaplastic lymphoma kinase (ALK)
protein, the outlook for this group of malignancies is very poor, with
3-year survival rates of around 20%. In a study by the Italian group
48.5% of the patients with PTCL (excluding ALCL and CTCL) died
within I year of diagnosis.4
Patients often present at older age (median 60 years) with
advanced-stage disease, and have unfavourable IPI scores (high
lactate dehydrogenase, bulky disease), B symptoms, and poor performance status.
A third of patients with PTCL and nearly all with NK-cell tumours
have extranodal disease. Progress has been slow due the rarity of
the disease, geographic variation, relative chemo resistance and
lack of randomized trials. Most peripheral T-cell lymphomas display
the phenotype: CD2+, CD3+, CD4+, CD5+, CD7~. Additionally,
CD30 and CD45 or CDI5 may be expressed, and the TCRy gene
is often rearranged. The t(2;5) translocation leads to over-expression of the gene for anaplastic lymphoma kinase (ALK) in patients
with anaplastic large-cell lymphoma of T-cell type and is associated
with a good prognosis. In PTCL unspecified, a number of different numerical and structural chromosomal abnormalities has been
detected. 5
The Working Formulation (WF) in 1980s failed to recognize the
B and T cell origins and grouped together disorders of different biologies. To day classification identifies disease entities based upon the
cell origin by immunophenotyping along with clinical, morphologic,
and genotypic data when available, despite it survival has remained
less than 30% for most types of peripheral T/NK neoplasm.
In the Western, nodal disease tends to predominate as the
common subtypes Anaplastic Large Cell Lymphoma, angioimmunoblastic T cell Lymphoma and peripheral T cell Lymphoma unspecified. In Asia the extranodal disease is more common particularly the
nasal type associated with Epstein Barr virus and endemic HTLV-1
has an important role in the incidence of Adult T cell Leukemia/Lymphoma.6
There is no consensus about the optimal therapy for PTLs, although recent reviews provide suggestions that are based on results
of anecdotal reports, small series, or phase II trials. In the other hand
the clinicopathologic dissociation where the pathology may appear
aggressive but the clinical course is indolent is another problem
for treatment principally in PTLs with cutaneous only involvement,
such primary cutaneous ALCL, the small or medium-sized PTCL
and subcutaneous panniculitis –like PTCL with a fulminant course
when associated with hemophagocytosis.7 These malignancies are
rarely curable, and for many patients survival is short (median of
6—8 months).9 With the exception of ALK+ anaplastic large-cell
lymphoma, PTCLs do not respond well to conventional combination
chemotherapy regimens; CR rates are usually <20% and of short
duration. Outcome is related to the PTCL subtype, stage at presentation, histology, age, and the presence of B symptoms.
The questions for PTCLs according therapy are.
Should CHOP be the standard?
The Intergroup trial in United State established CHOP as the
standard schema for Intermediate-grade lymphoma with PTCL included, but the complete remission range is variable but the 5 years
survival is no more than 30% except ALK-positive ALCL. Adding
others drugs or shortening the cycle is uncertain to had significant
improvement in survival. Others regimens of Europe or others entities of the United States have not shown benefits in PTCL, except
in elderly where the ACVBP shown better results but with a greater
treatment related mortality. 8
Some clinical or tumor factors predict prognosis?
Although the pathology has not consistently predicted prognosis some entities have the best as the ALK-positive ALCL, the
intermediate prognosis of PTCL/U and the worst in the systemic
extra nodal types. The IPI has been validated and a new model has
been proposed adding the bone marrow involvement to the others
factors. The 5 years survival varies from 65% with 0 factors to 18%
with 3-4 factors. Others factors as Ki-67, p53, chemokine receptors,
gene profiles and cytotoxic molecules are associated with worst
prognosis and short overall survival.
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What are the features of specific disease entities that warrant
disease-adapted strategies?
Anaplastic Large Cell Lymphoma (ALCL) subdivided into three
entities has different response where the ALK-positive ALCL has the
better prognosis (60-93% % years OS) compared with ALK-negative ALCL (11-46%) but it has poor prognosis with B symptoms, high
IPI, small cell variant and CD 56 +.9
Extranodal NK/T lymphomas have unique presentation depending upon the site of origin and have poor prognosis that depends upon the disease is small and confined to the nasopharinx
as opposed to disseminated.
Are there new agents that improve outcome?
The trials results in cutaneous T cell lymphomas as well aggressive T/NK leukaemia have introduced agents with activity in
PTCL that have included Gemcitabine with OR 60-70%, CR rate
11-20%;10 or the purine analogs pentostatin, fludarabine and clardibrine with activity in PTCL. The responses rate has been variable
with the different drugs OR for Pentostatin of 36-71% (CR 2-25%)
when combined with others drugs as cyclophosphamide, doxorubicin and alemtuzumab in a recent trial in PTCL in untreated patients
the CR rate was 78%.11
Immunotherapy
The humanized monoclonal antibody alemtuzumab binds to
the CD52 antigen, a glycoprotein which is widely expressed on normal and malignant B and T lymphocytes. Recently it has been demonstrated in a number of clinical trials that alemtuzumab has clinical
activity in mature T-cell diseases such as T-prolymphocytic leukaemia and cutaneous T-cell lymphoma, inducing responses in up to
two thirds of heavily pre-treated relapsed/refractory patients.12
Over the past 10 years unmodified monoclonal antibodies
have become established as an effective therapy for a number of
lymphoid malignancies. CD52 is a non-modulating antigen which
is expressed at high density on >95% of all normal and malignant
B and T lymphocytes, monocytes and macrophages1, but not on
haemopoietic stem cells. Campath-IH (alemtuzumab) was the first
fully humanized anti-CD52 monoclonal antibody to be used therapeutically in 1988 to treat a patient with a B-cell non-Hodgkins lymphoma
In vitro, alemtuzumab is active in complement-mediated lysis,
antibody-dependent cell cytotoxicity (ADCC), and apoptosis. It is
unclear which mechanism is most important for the therapeutic activity of this antibody ¡n vivo
Chemo-immunotherapy
With the success of chemo-immunotherapy combinations in
the B-NHLs, this approach is now being explored in PTCL. CHOP
plus alemtuzumab has demonstrated activity as first-line therapy in
PTCL patients. One dose of alemtuzumab (30 mg sub-cutaneously)
was administered with each of 8 courses of CHOP-21. Nine out of
12 patients completed the therapeutic regimen and were available
for evaluation. Five of the patients achieved CR, one PR and one
MR.13 After a median follow-up of 298 days, five out of nine treated
patients were alive. The Hovon group are also exploring the addition of alemtuzumab to CHOP for previously untreated patients, in
this case using a more intensive CHOP-14 schedule. The German
aggressive lymphoma study group are currently testing the value
of 4 weeks of therapy with alemtuzumab given as consolidation following induction therapy with 6 cycles of CHOEP-14. Results from
these trials are awaited.
An alternative combination using doxorubicin, cyclophosphamide, fludarabine and alemtuzumab as initial therapy or after first
or second relapse in 21 evaluable patients demonstrated an OR of
62%.14 Among newly diagnosed patients seven out of nine (78%)
achieved CR. Grade II—IV leukopenia developed in 81% of the
evaluable treatment cycles. The significantly higher response rates
in previously untreated patients receiving this regimen suggest that
the addition of alemtuzumab to induction chemotherapy may be valuable. Alemtuzumab has also been combined with platinum-based
regimens used as salvage therapy at relapse and prior to SCT. Wulf
et al have reported the success of a regimen incorporating standard
ICE (ifosfamide, carboplatin, etoposide) with alemtuzumab, given
S78
for 2 cycles prior to proceeding with reduced-intensity allograft. Of
ten patients, seven achieved CR with a survival of 4—13 months.66
Indeed, SCT — either autograft or allograft — should be considered for all patients in high-risk categories in first remission, and
for all patients in chemosensitive relapse.67 Alemtuzumab may be
of benefit used as part of a chemo-immunotherapy induction pretransplant, as an agent to purge residual disease prior to autograft,
or as part of the preparative regimen for allograft.
TOXICITY AND SIDE-EFFECTS OF ALEMTUZUMAB
The most significant side-effect is prolonged lymphopenia associated with reactivation of viruses (such as CMV and herpes) and
other opportunistic infections, particularly in heavily pre-treated patients. Use of antibacterial and antiviral prophylaxis is important and
should probably be continued for at least 3 months after completion
of therapy. In addition, monitoring for reactivation of CMV by PCR
and TBC in our countries is advisable during therapy, particularly in
the first 6 weeks.
There have been cases of bone-marrow aplasia which may
be related to viral reactivation, although this remains unproven. In
some cases the hypoplasia is due to haemophagocytosis. CD52
is not expressed on haemopoietic progenitors, and alemtuzumab
would therefore not be expected to cause direct toxicity to stem
cells.
CONCLUSION
Except for ALK+ anaplastic large-cell lymphoma, early-stage
MF and T-LGL leukaemia, T-cell malignancies have a poor prognosis with conventional therapy. Few patients achieve CR, relapse is
common and usually associated with chemo-resistance.
The role of alemtuzumab in Peripheral T-cell Lymphoma has
yet to be established, but preliminary results would suggest that the
antibody has clinical activity which may result into survival benefit
as more data become avail-able. However, alemtuzumab therapy
alone does not appear to be curative. Alternative strategies — such
as combinations with other agents either simultaneously or sequentially, consolidation of responses with SCT, and the use of maintenance therapy, need to be further explored if the natural history of
these aggressive malignancies is to be improved.
REFERENCES
1. Jaffe ES, Harris NL, Stein H, Vardiman J. J. Pathology and Genetics of Tumors of Hematopoietics and Lymphoid Tissues.
Lyon France: IARC Press: 2001.
2. Delves PJ, Roitt IM. The immune system. First of two parts. N
Engl. J Med. 2000; 343:37-49.
3. Savage KJ, Chhanabhai M, Gascoyne RD, Connors JM. Characterization of peripheral T.cell lymphomas I a single North American institution by the WHO classification. Ann Oncol. 2004; 15:
1467-1475.
4. Gallamini A, Stelitano C, Calvi R et al. Peripheral T-cell lymphoma unspecified (PTCL-U): a new
prognostic model from a retrospective multicentric clinical study.
B/ood 2004; 103(7): 2474—2479.
5. Zettl A, Rudiger T, Konrad MA et al. Genomic profiling of peripheral T-cell lymphoma, unspecified, and
anaplastic
large
T-cell
lymphoma
delinéales
novel recurrent chromosomal alterations. The American
Journal of Pathology 2004; 164: 1837-1848.
6. Arrowsmtih ER, Macon WR, Kinney MC,et al. Peripheral T-cell
lymphomas; Clinical features and prognostic factors of 92 cases defined by the revised European American Lymphoma Classification. Leuk. Lymph. 2003;44;241-249.
7. Ghobrial IM; Weenig RH; Pittlekow MR; et al. Clinical outcome
of patients with subcutaneous panniculitis-like t-cell lymphoma.
Leuk & Lymph; 2005; 46: 703-708.
8. Escalon MP; Liu NS; Yang Y, et al. Prognostic factors and treatment of patients with T-cell Non Hodgkin lymphoma. Cancer.
2005; 103:2091-2098
9. Susuki R; Kagami Y, Takeuchi K, et al. Prognostic significance
of CD 56 expression for ALK-positive and ALK-negative anaplastic large–cell lymphoma of T/Null cell phenotype. Blood.
2000;96:299-3000
10. Sallah S, Wan JY, Nguyen NP. Treatment of refractory T-cell malignancies using gemcitabine. Br J Haematol. 2001; 113:185187
11. Kurzrock R, avandi F. Purine analogues in advanced T-cell lymphoid malignancies. Elseivier. Semin. Hematol. 2006;43:s27s34.
12. Brady E.Beltran, Julia Humani Zavala, et al. Alemtuzumab in
Patients with Advanced Mycosis Fungoids: First Interim Report
ASH Annual Meeting Abstracts 2006 108: 4728
13. Enblad G, Hagberg H, Erlanson M et al. A pilot study of alemtuzumab (anti-CD52 monoclonal antibody) therapy for patients
with relapsed or chemotherapy-refractory peripheral T-cell lymphomas. 6/ood 2004; 103(8): 2920-2924.
14. Weidmann E, Hess G, Krause SW et al. Alemtuzumab, fludarabine,
cyclophosphamide, and doxorubicin; An effective first-line treatment
in peripheral T-cell lymphomas. Annals of Oncology 2005; 16
[Abstract 232].
S79
EDUCATION SESSION
Hodgkin Lymphoma
Molecular basis of the treatment failure in
Hodgkin Lymphoma
Tumor microenvironment and mitotic
checkpoint are key factors in the outcome
of classical Hodgkin Lymphoma
Abel Sánchez-Aguilera, Carlos Montalbán, Paloma de la Cueva, Lydia Sánchez-Verde, Manuel M. Morente, Mónica GarcíaCosío, José García-Laraña, Carmen Bellas, Mariano Provencio,
Vicens Romagosa, Alberto Fernández de Sevilla, Javier Menárguez, Pilar Sabín, María J. Mestre, Miguel Méndez, Manuel F.
Fresno, Concepción Nicolás, Juan F. García, Miguel A. Piris for
the Spanish Hodgkin Lymphoma Study Group.
From the Lymphoma Group (A.S.-A., P.C., M.A.P., and J.F.G.), the
Tumor Bank Network (M.M.) and the Histology and Immunohistochemistry Unit (L.S.-V.), Spanish National Cancer Centre (CNIO),
Madrid; the Department of Internal Medicine (C.M.), Pathology
(M.G.-C.), and Hematology (J.G.-L.), Hospital Ramón y Cajal,
Madrid; the Department of Pathology (C.B.) and Oncology (M.P.),
Hospital Puerta de Hierro, Madrid; the Department of Pathology
(V.R.) and Hematology (A. F.S.), Institut Catala d´Oncologia, Barcelona; the Department of Pathology (J.M.) and Oncology (P.S.),
Hospital General Universitario Gregorio Marañón, Madrid; the
Department of Pathology (M.J.M.) and Oncolology (M.M.), Hospital
de Móstoles, Madrid; and the Department of Pathology (M.F.F.)
and Hematology (C.N.), Hospital Central de Asturias, Oviedo; the
Department of Pathology, M.D. Anderson International, Madrid
(J.F.G.); Spain.
Around 20-30% of Hodgkin Lymphoma (HL) patients do not
benefit from standard therapies and finally succumb to their disease. The factors that influence the outcome of HL have not been
elucidated, underscoring the demand for the identification of biological risk factors and new therapeutic targets. We analyzed the geneexpression profiles of samples from 29 patients with advanced
classical HL treated with standard therapy, and compared the expression profiles of patients with favourable and unfavourable clinical outcome. Using supervised methods, we identified 145 genes
associated with outcome, which were grouped into four signatures
representing genes expressed by either the tumoral cells (genes involved in the regulation of mitosis and cell growth/apoptosis) or the
tumour microenvironment. A Gene Set Enrichment Analysis (GSEA)
allowed identifying the pathways whose expression is associated
with failure or progression in these cases, distinguishing tumoral
cells from the signals derived from the tumoral microenvironment.
Interestingly, the tumoral cells in cases showing an adverse outcome express a molecular signature reporting on G2/M transition
and chemo-resistance. The study of the genes expressed by the
Hodgkin environment identifies a molecular program derived from
multiple cell types and inflammatory background.
The relationship between the expression of eight representative genes and survival was successfully validated in an independent series of 235 patients by quantification of protein expression
levels on tissue-microarrays. Analysis of centrosomes and mitotic
checkpoint confirmed the existence of an abnormal transition
through mitosis in HL cells. Therefore, genes related with tumor microenvironment, cell growth/apoptosis, and regulation of mitosis are
associated with treatment response and outcome of HL patients.
The role of positron emission tomography
in the assesment of staging, monitoring
early response and restaging in Hodgkin
lymphoma. Implication to response
adapted-therapy.
S. Pavlovsky, MD. PhD.
Scientific Director
FUNDALEU, Hospitalisation and Clinical Research Center “Angélica Ocampo”
J. E. Uriburu 1450, Buenos Aires, Argentina.
CHEMORADIOTHERAPY CHANGES IN THE LAST TWO DECADES
The ABVD scheme has become the “gold standard” treatment
in Hodgkin lymphoma (HL). In several randomized studies it was
shown that it is superior to MOPP and similar to alternating and
hybrid MOPP-ABVD regimens with less toxicity (1,2). The combinedmodality of chemotherapy and radiotherapy showed better results
than both therapies alone mainly in patients who achieved partial
remission (PR) (3-8). In combination with chemotherapy, involvedfield radiotherapy (IFR) produced similar results to extended field
radiotherapy. Also, lower-doses were equivalent to higher-doses
(20 vs 30 vs 40 Gy) (9).
The purpose of the present strategy of treatment is to deliver
less cycles of chemotherapy and avoid radiotherapy in patients with
early complete response avoiding short and long term toxicity as
myelosupression, pulmonary fibrosis, cardiac failure, gonadal toxicity and second neoplasia specially AML/MDS (10). HL patients have
an excessive mortality directly related to these late treatment effects.
HISTORY OF POSITRON-EMISSION TOMOGRAPHY (PET)
Although PET has been used in cancer research for more than
two decades, its clinical application in oncology has only recently
found widespread use. The 18F-fluorodeoxyglucose (18 F-FDG) has
increased uptake in most types of cancer including HL as compared
with its uptake in most normal organs or tissues. However 18 F-FDG
uptake is usually also observed in infectious and inflammatory processes, thymic hyperplasia in younger patient, and bone marrow
hyperplasia after chemotherapy and/or use of haemopoietic growth
factors. The fundamental difference between PET versus computed
tomography (TC) and magnetic resonance (MNR) imaging is that
PET assesses metabolic characteristics of the tumor, whereas TC
and MNR asses the tumor´s anatomical or morphologic character-
S80
istics as density, size, and shape. The introduction of systems in
2001 that combined a PET scanner and CT scanner by computer
and more recently the design of a single instrument PET/TC with
improved resolution and sensitivity has increased its use in staging, monitoring early response to chemotherapy and restaging of
lymphomas.
PRETREATMENT STAGING
Several studies have shown that PET/TC is much sensitive in
detecting nodal and extranodal involvement of HL than TC alone.
PET/TC detects an additional number of sites not detected by CT
and bone marrow biopsy. PET/TC is able to detect focal or multifocal bone/bone marrow involvement with negative iliac crest bone
marrow biopsy. All these findings results in upstaging the disease in
about 15-20% of patients. A pretreatment PET/TC with iv contrast
provide an integrated functional/anatomical assessment of HL with
the added advantage of facilitating the interpretation of restaging
PET/TC (11-13). However due to cost and that PET/TC rarely will result in modification of the initial therapy especially if ABVD is used
in all stages, still its routinely use outside clinical trials is not common.
MIDTHERAPY PET/TC
Several studies have shown the prognostic value of PET/TC
after 2 to 3 cycles of chemotherapy especially using ABVD. All these
studies are retrospective without modification of the therapy according to the results. More than 90% of the patients PET/TC negative
are failure-free at 3 years while 0-39% of the patients PET/TC positive are failure-free at 3 years (P<0.0001) Table 1 (14-17). At present
several cooperative groups are designing studies using the PET/TC
after 2 to 3 cycles of chemotherapy to modify the therapy. The Argentine Group of Acute Leukemia (GATLA) has started a new trial
in August 2005 using in all the clinical stages three cycles of ABVD,
followed by a PET/TC three weeks later. Those patients PET/TC
negative considered in CR received no further therapy, those with
PR according to the new classification IHP received 3 more cycles
of ABVD followed by radiotherapy 25 GY to the PET/TC positive
sites. Up to November 2006, a total of 45 patients entered in the
study, 38 completed 3 cycles of ABVD and have been evaluated
with a PET/TC. Of them, 33 (87%) achieved CR, 4 (10%) achieved
a PR, completed six cycles of ABVD and radiotherapy achieving a
CR by PET/TC. One patient (3%) has progressive disease after 3
cycles of ABVD, received three cycles of ESHAP, achieved a PET/
TC negativity and has been consolidated with high-dose therapy
and an autograft. At present, with still a short follow-up, all the patients remain disease-free of their HL.
RESTAGING
The appropriate time point for restaging with PET at the conclusion of therapy for the validation of response varies with type
of administered therapy. To minimize the frequency of false-positive PET/TC an International Harmonization Project (IHP) recommends that PET/TC should be performed at least three weeks after
the completion of chemotherapy to avoid the increase intake in the
bone marrow due to recovery of myelosupression and or use of GCSF. Also is required 8-12 weeks after completion of radiotherapy
because acute inflammatory changes commonly seen in the first
few weeks after radiation can result in false positive (18). PET at restaging allows to distinguished between viable lymphoma cells and
necrosis or fibrosis in residual masses that can remain after treatment. More than two-thirds of residual masses by CT are seen as
negative by PET/TC with relapses occurring in less than 10% of
these patients, who can be safely observed. The other one–third
with positive PET/TC has a risk of progression or relapse in about
60-70% of patients. The revised recommendations for response criteria for lymphoma employed the PET/TC to confirm a complete remission PET negative. False positive findings at restaging with PET
include physiologic processes such as brown fat, infectious and inflammatory processes as pneumonia, histoplasmosis, and sarcoidosis and rebound thymic hyperplasia in children and young adult
patients. Most infectious or inflammatory processes are not very 18
F-FDG sensitive and do not usually cause a problem for an expert
PET reader. In cases of doubt a repetition after two or four months
or after antibiotic therapy will show a negativity uptake at the site.
The higher accuracy of PET/TC, as compared with CT could result
in cost saving with the avoidance of costly salvage treatments.
Due to the higher accuracy of PET/TC compared to TC the IHP
has revised the recommendation of response criteria for malignant
lymphoma of the CT-based International Workshop Criteria (IWC)
widely used since 1999 (Table 2) (19, 20). A new definition of CR,
PR, stable disease and progressive disease are based in PET/TC
negative or positive findings. The unconfirmed CR (CRu) will now
be eliminated. Those responses will be designed as CR if the PET/
TC is negative and PR if PET/TC is positive.
A recent comparison of the IWC with the new IHP using PET/
TC in the same group of patients with lymphoma show an increase
number of CR, no more CRu and decrease number of PR (Table
3) (21).
DETECTION OF RECURRENCE IN SYMPTOMATIC PATIENTS
The routine use of PET/TC after being confirmed a CR can
detect and localize recurrence among patients who have not symptoms, and start earlier a salvage therapy. This PET/TC application
is likely to become a routine practice in the near future replacing the
CT scan (22,23).
CONCLUSIONS
The wide availability of PET/TC, at reduced cost, will replace
TC, MNR, and Gallium Scan in the staging, early assessment of
therapy, staging after completion of therapy and long term follow up
in order to detect early relapse. At present, studies are designed to
evaluate early CR by PET/TC in order to avoid more cycles of chemotherapy and IFRT with the purpose of reducing long term toxicity,
especially mortality unrelated to Hodgkin lymphoma.
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of advanced Hodgkin’s disease with MOPP, ABVD, or MOPP
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7. Pavlovsky S, Corrado CS, Pavlovsky MA et al. Risk-Oriented
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11. Friedberg JW, Fishman A, Neuberg D, et al. FDG-PET is superior to gallium scintigraphy in staging and more sensitive in the
follow up of patients with de novo Hodgkin lymphoma: a blinded
comparison. Leuk Lymphoma 2004; 45: 85-92.
12. Weihrauch MR, Re D, Bischoff S, et al. Whole body positron
emission tomography using 18F-fluorodeoxyglucose for initial
staging of patients with Hodgkin´s disease. Ann Hematol 2002;
81: 20-25.
13. Hutchings M, Loft A, Hansen M et al. Positron emission tomography with or without computed tomography in the primary
staging of Hodgkin´s lymphoma. Haematologica. 2006;91; 482489.
14. Spaepen K, Stoobants S, Dupont P, et al. Can positron emission tomography with [(18)F]-fluorodeoxyglucose after fist-line
treatment distinguish Hodkin’s disease patients who need additional therapy from others in whom additional therapy would
mean avoidable toxicity. B J Haematol 2001;115:272-278.
15. Hutchings M, Mikhaeel NG, Fields PA, Ninan T, Timothy AR.
Prognostic value of interim FDG-PET after two or three cycles of
chemotherapy in Hodgkin Lymphoma. Ann Oncol 2005;16:11601168.
16. Hutchings M, Loft A, Hansen M, et al. FDG-PET after two cycles
of chemotherapy predicts treatment failure and progression free
survival in Hodgkin lymphoma. Blood 2006;107: 52-59.
17. Gallamini A, Hutchings M, Rigacci L et al. Advanced stage
Hodgkin lymphoma: The predictive value on treatment outcome
of early FDG-PET scan is independent of and superior to IPS
score. Blood 2006:108, Abstr 4592.
18. Juweid ME, Stroobants, Mottaghy et al. Recommendations of
the imaging committee of the International Harmonization Project (IHP) for FDG-PET (PET) use in patients with lymphoma J
Nucl Med 2006; 47:452 (Abstract #1681)
19. Cheson BD, Horning SJ, Coiffier B et al. Report of an International Workshop to standardize response criteria for nonHodgkin`s lymphoma. J Clin Oncol 1999;17:1244-1253.
20. Cheson BD, Pfistner B, Juweid ME, et al. Recommendations for
revised response criteria for malignant lymphomas. International
Harmonization Project (IHP). J Clin Oncol 2006;24,18S:423s.
21. Juweid ME, Wiseman G, Vose JM et al. Response assessment
of aggressive non Hodgkin lymphoma by integrated International Workshop Criteria and fluorine 18-fluorodeoxyclucose positron emission tomography. J Clin Oncol 2005; 23:4652-4661.
22. Jerusalem G, Beguin Y, Fassotte MF, et al. Early detection of
relapse by whole body emission tomography in the follow up of
patients with Hodgkin’s disease. Ann Oncol 2003; 14: 123-130.
23. Zijlstra JM, Linduer-vander Werf G, Hoekstra OS, Hooft L,
Riphagen II, Huijgens PC. 18F-fluoro-deoxyglucose positron
emission tomography for post-treatment evaluation of malignant
lymphoma: a systematic review. Haematologica 2006;91:522529.
S81
TABLE 1
Prognostic Value of early PET/TC after 2-3 cycles of ABVD to
predict failure-free survival (FFS) in Hodgkin lymphoma.
Therapy
ABVD
ABVD
ABVD
#
Pts
84
77
202
PET/TC
Negat.
Posit.
No % No %
71 85
13 15
61 79
16 21
164 81 38 19
% FFS 3 ys
Negat. Posit.
92
96
96
39
0
14
P<
Ref
0.0001
0.0001
0.0001
15
16
17
TABLE 2
Revised recomendations for response criteria for malignant
lymphoma (20)
PET avid histologies (PA): DLBCL, HL, FL, MCL.
• Complete remission: 1) No signs or symptoms of disease. 2)
PET (–) in PA lymphomas. 3) Normal bone marrow by morphology, or if indeterminate, negative by Immunohistochemistry, flow
cytometry and/or molecular genetic studies.
• CR unconfirmed (Cru) is no longer included.
• Partial remission: 1) >50% decrease in tumor size but PET(+)
at prior PA sites, or 2) >50% decrease in tumor size, but CT(+)
and PET(-) if PET negative prior to treatment. Bone marrow is
irrelevant if positive pre-treatment.
• Stable disease: is neither PR nor PD, PET(+) only at prior sites
of disease.
• Progressive/ relapsed disease: requires >50% increase in disease or new lesions that are PET(+) if PA lymphoma.
TABLE 3
Response and progression-free survival (PFS) at 3 years in 54
aggressive non-Hodgkin’s lymphoma according to the International Workshop Criteria (IWC) and IWC+PET. (21)
Imaging
Results
CR
CRu
PR
SD
PD
P value
#Pts
17
7
19
9
2
IWC
%
31
13
35
17
4
<0.021
% PFS
# Pts
74
86
62
33
50
35
0
12
6
1
IWC/PET
%
65
0
22
11
2
<0.0003
%
PFS
80
-42
17
0
CR: complete response; CRu: unconfirmed complete response;
PR: partial response; SD: stable disease; PD: progressive disease.
S82
EDUCATION SESSION
Infections In Immunosuppressed Patients
Early diagnosis of Fungal Infections and
Therapeutic Advances
Jose M. Aguado
University Hospital 12 de Octubre. Madrid, Spain.
Systemic fungal infections are significant causes of morbidity
and mortality in immunosuppressed patients. These infections are
difficult to definitively diagnose, and, until very recently, options for
therapy have been extremely limited. In order to effectively manage
patients at risk for these infections, clinicians must recognize the
factors associated with invasive mycoses and identify patients at
increased risk. The etiologic agents of these infections have also
continued to change. More infections are due to yeasts other than
Candida albicans, and unusual moulds have emerged as important
causes of infection in severely immunosuppressed patients.
Unfortunately, diagnostic tools have limited utility for many of
these infections. However, blood culture techniques for identifying
yeasts have improved, and advances have been made in non-culture-based diagnostics for Aspergillus. Of note, major advances
have been made with the introduction of new antifungals. A new
class of drugs that target the fungal cell wall, the echinocandins, is
now clinically available. In addition, the extended-spectrum azoles
offer coverage against moulds. New approaches, including antifungal prophylaxis in the highest risk patients, may further reduce complications of invasive mycoses.
Regardless of these advances, several issues continue to
make management of these infections challenging, including the
emergence of resistant organisms, the lack of reliable markers for
early invasive infection in high-risk patients, and limited evidence
of optimal strategies for utilizing the available antifungal armamentarium. This update reviews the risk factors for these infections, the
changing epidemiology of opportunistic mycoses, new diagnostic
tools, and current antifungal agents and strategies for their appropriate use in the therapy and prophylaxis of immunosuppressed patients at risk for invasive fungal infections.
It is hoped that improved recognition of high-risk patients combined with improved diagnostic techniques, and the availability of
new, less toxic, and potentially more effective antifungal agents will
improve outcomes in immunosuppressed patients with these infections.
For patients with candidemia, data shows that delays in institution of therapy are associated with poorer outcomes, so that early
institution of effective therapy in these patients is key. Echinocandins now available as broad-spectrum agents with minimal toxicity
(although expensive) could to improve outcomes; clinical trials are
needed to establish conditions and strategies for their optimal use.
In invasive aspergillosis, early, effective therapy has been
shown to be a critical feature leading to successful outcomes. Even
though the extended-spectrum azoles represent a major advance,
outcomes are still less than optimal in patients with disseminated
infection and in those with more severe immunosuppression. Nonculture-based diagnostics have improved for aspergillosis. Encouraging results by Maertens and colleagues showed that by employing
a combination of galactomannan, chest CT, and clinical presentation, invasive aspergillosis could be diagnosed earlier, which limited
empirical use of antifungal therapy and improved overall survival.
For other moulds, non-culture-based methods are unfortunately not
routinely available; therefore, the diagnosis is still based on culture
results and histopathology findings.
Combination therapy may improve outcomes, but clinical trials
are urgently needed to guide the choice of agents and the timing for
instituting these costly regimens.
Finally, prophylaxis of invasive fungal infection, particularly for
invasive moulds, in patient populations at highest risk may be an
important strategy for reducing morbidity and mortality of these often lethal infections.
S83
CONFERENCE
Advances in the treatment of cytomegalovirus infection
Jose M. Aguado
University Hospital 12 de Octubre. Madrid, Spain.
Cytomegalovirus (CMV) infection remains one of the most
important opportunistic infections in recipients of allogeneic bone
marrow transplant (BMT). CMV disease is still associated with significant mortality in recipients of an allogeneic stem-cell transplant.
Antiviral agents such as ganciclovir and foscarnet are highly effective against CMV and can improve the outcome of most patients
with CMV disease. Moreover, antiviral chemoprophylaxis with ganciclovir or aciclovir has been shown to reduce CMV infection and
CMV disease. However, antiviral drug therapy is often associated
with considerable toxicity and there is a certain risk of the development of antiviral resistance.
Pre-emptive antiviral therapy, administered after CMV infection
has been documented (by sensitive screening assays) but prior to
the development of clinical symptoms, have been shown to significantly reduce the incidence of CMV disease and has been associated with a reduced incidence of adverse effects.
Compared with antiviral prophylaxis, pre-emptive antiviral
therapy has the advantage that patients are stratified according to
individual risk factors (active CMV infection, viral load). This strat-
egy helps to reduce the number of patients treated and also the
duration of antiviral therapy, which might have important implications for adverse effects and the emergence of antiviral resistance.
However, sensitive screening is costly and must be performed on
at least a weekly basis. Therefore, antiviral prophylaxis remains an
attractive approach.
Valganciclovir has recently introduced clinical practice as a pivotal agent in antiviral prophylaxis that overcome the disadvantages
associated with ganciclovir, which include low oral bioavailability,
limited efficacy because of the development of viral resistance, and
the need for frequent administration, which can adversely affect patient adherence. Valganciclovir is rapidly converted to ganciclovir;
systemic exposure to the parent drug is low and short in duration.
The factors to be considered regarding the optimal method
for prevention include the cost of the preventive drug, the cost of
monitoring tests, and the potential for the emergence of resistance
to antiviral agents. In summary, universal prophylaxis or pre-emptive therapy are both valid methods for prevention, and there is still
debate in the literature regarding the optimal approach.
S84
SYMPOSIUM
Young Hematologists
Rituximab in Lymphoproliferative
Disorders and Immunologic Conditions
Norman Maldonado
The use of targeted therapy with monoclonal antibodies has
been the greatest advance in the treatment of many B cell lymphomas and immunologic conditions in the past decade. Rituximab is
a chimeric monoclonal immunoglobulin G kappa molecule whose
variable region (Fab fragment) is derived from mice and has specificity against CD 20 antigen which is expressed in B lymphocytes
and B cell lymphomas. The larger constant human region (Fc) which
fixes complement is composed of human sequences.
There are several mechanisms of action of this monoclonal antibody which includes; complement dependent cytotoxicity (CDC),
antibody dependent cellular cytotoxicity (ADCC) and apoptosis,
among others. A cellular immune mechanism has also been proposed. Cytokines have been used to potentiate the effect of rituximab. Interferon Alfa, growth factors such as GMCSF and interleukins have been used to potentiate the activity of rituximab with
promising results.
The first clinical studies of rituximab were done in Stanford University in patients with refractory follicular lymphomas. Out of 15
patients 3 responded. The next studies were phase 2 and showed a
50% response rate. The work by Coffier and collaborators with rituximab and the traditional CHOP regime in large cell lymphomas was
the most important study. It showed a definite benefit of R-CHOP
over CHOP in disease free survival, overall survival and complete
remissions. . There was no increase in toxicity. The study compared
196 patients given CHOP with 202 given R-CHOP between the
ages of 60 to 80 years old. The overall survival at two years was superior for the R-CHOP group. Disease free survival was better, 70%
versus 57%. Complete remission was 63% for CHOP and 76% for
R-CHOP. The GELA study published in 1999 showed the superiority
of R-CHOP in large cell lymphomas (DLCL).
Retrospective studies evaluate the effectiveness of rituximab
DLCL patients according to their Bcl-2 expression on the cell surface. In patients with Bcl-2 positive tumors which have a worse
prognosis, rituximab added to CHOP improves the response rate,
but this advantage was not seen in the Bcl-2 negative patients. This
suggests that patients with Bcl-2 negative may not warrant receiving rituximab, but this is controversial. Another study examined this
issue as well as the activity of Rituximab according to the Bcl-6
expression. Expression of the later molecule is associated with a favorable prognosis. The prospective study showed that patients who
are Bcl-6 –negative have a much better response to R-CHOP than
CHOP. The event free survival at two years was 76% versus 9%
and the overall survival (OS) was 79% versus 17%. There was no
benefit in the Bcl-6+ patients but the authors caution that this finding
needs independent confirmation. On the other hand, they couldn’t
confirm the previous retrospective studies which had identified that
Rituximab’s activity was limited to Bcl-2 positive subset in DLCLs.
The group at MD Anderson and Dr. Fernando Cabanillas
showed that in follicular lymphomas treated with fludarabine, novantrone and dexamethasone (FND) the addition of rituximab(RFND) improved the response rate and survival. There was no increase in toxicity
In the rescue regime of ICE (Ifosfamide, carboplatin and etoposide) the addition of rituximab improved the complete remissions
from 27% to 57%. In patients with Mantle cell lymphoma the addition of rituximab to hyperCVAD improves the response rate when
compared with its delayed application after chemotherapy was completed. There was no increase in toxicity except that it appears to
induce hypogammaglobulinemia in a significant fraction of cases.
In the salvage regime of ICE (Ifosfamide, carboplatin and etoposide) the addition of rituximab improved the remission rate from
27% to 57%. In patients with Mantle cell lymphoma the addition of
rituximab to hyperCVAD improves the response rate compared with
hyperCVAD alone.
A recent report by Dr. Thomas M. Habermann and collaborators from the Mayo Clinic evaluated the use of rituximab in older
patients with diffuse large cell lymphoma by giving it at the onset of
therapy R-CHOP or after CHOP in maintenance form (MR). The results confirmed previous studies the R-CHOP is superior the CHOP
with a three year failure free survival(FFS) rate of 53% and 46%
respectively. CHOP was compared with observation and with maintenance. Maintenance was superior 76% to 61% at two years but
only for patients who had not received rituximab during the induction phase. . There was no benefit of maintenance after R-CHOP in
patients who had received rituximab during induction.
However in patients with lymphoma and acquired immunodeficiency syndrome (AIDS) the addition of rituximab is associated
with increase toxicity and poor responses. It should not be used in
this setting although it remains unclear whether patients with AIDS
whose absolute CD4 count is not too low it might actually be of
benefit.
In patients with B cell chronic lymphatic leukemia (CLL) the
combination of fludarabine and or cyclophosphamide with rituximab
has been effective and has become a standard of care. In Waldenstrom’s macroglobulinemia rituximab has been effective although
the expression of CD 20 antigen in this disorder can be very variable.
The use of rituximab in autoimmune and imunologic diseases
has become very frequent. Recently the United States Food and
Drug (FDA) has approved the monoclonal antibody in rheumatoid
arthritis. Dr. Jonathan C.W. Edwards from London has reported the
findings in 161 patients divided in four groups. The control group
received methothrexate; another group received rituximab 1000 mg
on days 1 and 15; another received rituximab withy cyclophosphamide and another methothrexate and rituximab.The group receiving rituximab and methothrexate had a better response by 20%.
The next best group was rituximab with cyclophosphamide. The response criteria followed was according to the American College of
Rheumatology and the European League Against Rheumatism.
The use of rituximab has included many benign hematologic
conditions. Among the most frequently treated are idiopathic thromocytopenic purpuras (ITP), thrombotic thrombocytopenic purpura
(TTP), autoimmune hemolytic anemia (AIHA), cold agglutinin hemolytic anemia, post cardiac transplant pancytopenias and acquired
hemophilia, among others.
Among the most frequent immunologic conditions are systemic lupus erythematosus (SLE), Sjogrens syndrome, pemphigus,
vasculitis due to cryoglobulinemia, temporal arteritis, Wegener’s
granulomatosis, and post organ transplantation. It has also been
proven effective in graft versus host disease (GVH).
Rituximab is not free of side effects. The infusion, especially
the first one, can produce fever, chills, hypotension and bronchoconstriction. It has been associated, although rarely with death.
Hepatitis B and cytomegalovirus infections can be reactivated
and can be fatal. All patients should be tested for hepatitis B and if
positive treated for the hepatitis first. Tumor lysis syndrome should
be prevented especially in patients with bulky disease or chronic
lymphatic leukemia (CLL) with elevated blood counts. Prolonged
neutropenias has been observed especially in patients with CLL.
Dr. Fernando Cabanillas has documented a decrease in gamma
globulins.
Epratuzumab a humanized anti CD22 antibody has been developed and evaluated also. The mechanism of action of anti CD22 is
different and acts as an immunomodulatory agent in the sense that
it does not have direct cytotoxic effects. In other words, it doesn’t
posses CDC activity, but it does have ADCC activity. Epratuzumab
appears to potentiate the effects of rituximab and they can be used
together.
Pioneering but preliminary studies using rituximab in combination with IL-12 for patients with indolent and aggressive lymphomas
have shown a 69% response rate. The DNA immunostimulatory sequence (ISS) has also been combined with rituximab to potentiate
its effects in vitro, in animals as well as phase1 studies. It is capable
of generating antitumoral immunity. We do not have those results
yet.
Radioimmunotherapy using radionucleotides of monoclonal
antibodies tagged with Iodine131 an Yttrium 90 has added a new
dimension to the therapy of lymphomas. Some protocols also add
rituximab which improves the response rate.
Rituximab has become a multiple use monoclonal antibody
which prolongs life and increases the cure rate in some conditions.
Dr Antonio J. Grillo, a Puerto Rican physician, was the one who
developed rituximab for clinical use and to obtain FDA approval.
We are waiting for trials with other combinations including that with
other targeted therapies.
BIBLIOGRAPHY
1. Czuczman M, Grillo Lopez A, White C, Saleh M, Gordon L,
LoBuglioF, Jonas C, Klippenstein D, Dellaire B, Varns C. Treatment of patients with low grade lymphoma, the combination of
a chimeric anti-CD20 monoclonal antibody and CHOP Chemotherapy. J Clin Onco; 17(1):268-76, 1999.
2. Davis TA, Grillo Lopez AJ, White CA, McLaughlin P, Czuczman
MS. Link BK, Maloney DG, Weaver RL, Rosenberg J, Levy
R. Rituximab anti-CD20 monoclonal antibody therapy in non
Hodgkin’s lymphoma: Safety and efficacy of treatment J Clin
Onc 18 (17):3135-3143 September 2000.
3. Grillo Lopez AJ Radioimunotherapy with 90 yttrium Zevalin for
indolent and aggressive non Hodgkin’s Lymphoma. Haematol.
86: 65 -69, 2001.
4. Gordon L I, Solai-Celigny, P, Gascoyne R D, Freedman, AS,
Follicular lymphoma: Management options in the era of targeted
therapy.ASCO Educational Book pag 511-26 2005, Alexandria
VA.
5. Edwardsa JCW, Szczepanski L, Szechinski J, Filipowicz A,
Emery P, Close D R, Stevens R M, Shaw T, Efficacy of B-Cell
targeted therapy with rituximab in patients with arthritis, NEJM
350:2572-2581, 2004.
6. Coffier B, Pfreundschuh M, Stahel R, Vose J, Zinzani PL Aggressive lymphoma: Improving treatment outcomes with rituximab, Anticancer Drugs 2002 Nov 13 Suppl 2:S43-50.
7. Coffier B, Lepage E, Briere J, et al CHOP chemotherapy plus
rituximab compared with CHOP alone in elderly patients with
diffuse large cell lymphoma N. Engl J. Med 346(4), 235-242,
2002.
8. Winter JN, Weller DA, Horning SJ, et al, Prognostic significance
of Bcl-6 protein expression in DLBC treated with CHOP or RCHOP: a prospective correlative study, Blood 107:4207-4213,
2006.
S85
Prognostic Factors for Malignant
Transformation in Monoclonal
Gammopathy of Undetermined
Significance: Experience of one Institution
Sackmann F
Pavlovsky S
Pavlovsky M A
Corrado C
Pavlvovsky M
Fernandez I
Mountford P
Pavlovsky A
Pizzolato M, Alejandre M
Juni M.
FUNDALEU, Buenos Aires, Argentina.
BACKGROUND:
Monoclonal gammopathy of undetermined significance
(MGUS) is the most common plasma cell disorder and is a potential
precursor of multiple myeloma. It has a prevalence of 1 to 3% in persons younger than 50 years. It raises to 5,3% in persons between
50 and 70 years and up to 7,5% among those older than 85 years of
age. MGUS is defined by a monoclonal component (MC) in serum
amounting to 3 gr/dl or less, the absence of anaemia, lytic bone
lesions, renal insufficiency or hypercalcemia related to the monoclonal plasma cell proliferation and if performed, less than 10% of
plasma cells in bone marrow biopsy. It has an indolent evolution,
but some patients will develop a malignant neoplasm. The cumulative probability of progression is approximately 1% per year. Thus,
factors that identify patients who will progress are important. There
is a lot of data published in core medical journals, especially that
coming from Kyle, Cesana and Baldini, but we do not have any local information.
OBJECTIVES , METHODS AND STATISTICAL ANALYSIS:
The aim of this study was to evaluate whether any simple haematological parameter performed at diagnosis had prognostic value
for progression. Other end points were to determine the rate of progression, PFS and OS.
Medical records of the patients who were controlled in the
“Centro de Hematología Pavlovsky” with diagnosis of MGUS were
revised retrospectively from 1982 to 2006. Different variables at
diagnosis and evolution were analysed. MC was detected by protein electrophoresis on agars gel. The identification of MC was
performed by immunoelectrophoresis or immunofixation and was
quantified by radial immunodifussion.
The identification of prognostic factors was made using Cox
models. Rate of transformation, progression free survival (PFS)
and overall survival (OS) were calculated using the Kaplan Meier
method and the curves were compared with the log-rank test. The
PFS was defined as the time between diagnosis and progression
or death for the disease and OS, from diagnosis till death of any
cause or last visit.
The study was approved by FUNDALEU´s investigational committee and followed the recommendations of Good Clinical Practices and Helsinki Declaration and it’s following modifications. The
author has no conflicts of interest to disclose.
RESULTS:
Of the 281 patients with MGUS included, 171 (61%) were
women. The median age at diagnosis was 60 years. The median
MC value was 0.55 (range, 0.1 to 2.91 gr per decilitre) and 128
(46%) patients had less than 0.5 gr per decilitre. The type of immunoglobulin was evaluated in 270 patients and was IgG in 196 (70%),
IgA in 40 (14%), IgM in 33 (12%) and biclonal in 5 (1%). Light chains
were evaluated in 195 (69%) patients, and were kappa in 121 cases
S86
DISCUSSION AND CONCLUSIONS:
Figure 1: PFS of the patients with MGUS (86% at 10 years)
100%
86%
90%
80%
70%
23 / 254
60%
50%
40%
30%
20%
10%
0
24
48
72
96
120 144 168 192 216 240 264 288 312 336 360 384
months
Figure 2: OS of the patients with MGUS (90% at 10 years)
100%
90%
90%
13 / 254
80%
70%
60%
50%
40%
Progression Free Survival
30%
20%
10%
0%
0
24
48
72
96 120 144 168 192 216 240 264 288 312 336 360 384
months
Figure 3: PFS according to MC concentration
100%
89%
90%
80%
76%
< 1gr/dl: 9 / 222
> 1gr/dl: 14 / 58
70%
p = 0,0003
60%
50%
The rate of progression in our experience (9%, with a median
follow up of 66 months) is similar to the one published by Kyle et
al and slightly higher than the one described by Baldini et al (6.8%
at 70 months) and Cesana et al (5.8 at 65 months). The cumulative proportion of PFS (86% at 10 years) is a little lower than that
reported by Kyle, although the data is difficult to compare because
of the difference in follow up and number of patients.
According to previous publications, age, sex, haemoglobin levels, beta2 microglobulin, albumin, creatinine, MC immunoglobulin
light chains did not have prognostic value with respect to progression. Bence Jones was found to have a prognostic significance in
Baldini´s and Cesana´s experience but we could not find differences between those who had Bence Jones proteinuria and those
who had not, although 25% of our patients were not evaluated for
Bence Jones proteinuria. The variables that had influence in the
progression in our study were MC concentration and ESR. The patients with MC concentration higher than 1 gr/dl had 3.9 fold risk of
progression (CI95% 1.51 – 10.15). Patients with an abnormal ESR
had 2.17 fold risk of progression but as ESR HR CI95% is very near
1 (1.06 – 4.46) and it may be influenced by many external factors
(e.g. inflammation), the value of ESR as a prognostic factor may
doubtful.
Bone marrow plasma cell concentration has an established
prognostic significance. In our study, only 25% of the patients had
a bone marrow biopsy, as it is not a recommended procedure in
patients with a low MC. Analysing this subgroup alone, we found
that a concentration greater than 5% of bone marrow plasma cells
had 12.9 fold risk of progression than those with less than 5% (cox
analysis).
These data, MC concentration and bone marrow plasma cell
concentration as prognostic factors, agree with what it is published,
although other variables, as type of MC, Bence Jones or reduced
UI also described by some, did not have a prognostic value in our
experience.
Many years of observation generated the hypothesis that there
could be 2 types of MGUS: an evolving and a nonevolving type,
independent of the presence of initial prognostic factors. In this
way, evolving MGUS could be viewed as an early myeloma from
the beginning, whereas the nonevolving type would be a true stable
MGUS requiring input from a second trigger to initiate malignant
transformation. In the near future, microarray studies detecting genetic expression in these patients probably will help us to understand a little more about this disease.
Nowadays, based on our results and in what it is published,
one should be guided by MC concentration to identify those pa-
tients with a higher probability of progression and make a closer
follow up, in order to detect malignant transformation earlier. Until
this moment, no prognostic factor is able to replace periodic and
permanent follow up. Bone marrow biopsy should be performed in
those patients with high risk (MC greater than 1 gr/dl) or if any feature raises suspicion of the presence of myeloma or other malignant
disease.
(43%) and lambda in 74 (26%). Uninvolved immunoglobulins (UI)
were reduced in 56 patients (20%) and in 16 were not measured
(6%). Proteinuria was detected in 27 cases (10%). The bone marrow was examined in 70 patients (25%) and the median percentage
of plasma cells was 4 (range, 0 to 10). Only a few patients had
metaphase cytogenetic studies.
The median initial haemoglobin was 13.2 gr per decilitre (range,
8.3 to 16.8 gr per decilitre). Fifty-one patients had anaemia but, of
course, none related to the plasma cell disorder. The albumin was
measured in 273 patients and its value was normal in 242 patients
(86%). The median beta2 microglobulin was 2.5 ng per litre (range,
0.36 to 39 ng per litre) in the 184 patients evaluated and it was
above the normal level in 88 patients (31%). Finally, the ESR was
measured in 271 patients (96%) and it was normal, defined as lower
than 15 mm/hr, in 112 (40%) patients.
With a median follow up of 66 months (range 6.2 to 378), 23
patients (9%) evolved to a malignancy (18 to multiple myeloma, 4
to non Hodgkin lymphoma and 1 to amiloidosis. During the study,
13 patients died, 3 related to their disease progression, 8 died due
to other disorders (cardiovascular disease, other malignancy) and 2
of unknown cause. The cumulative probability of PFS and OS at 10
years was 86% and 90%, respectively.
MC concentration (p = 0.005, HR 3.9, CI95% 1.51 to 10.15)
and ESR (p = 0.003, HR 2.18, CI95% 1.06 to 4.46) were independent predictors of progression when analysed in a Cox proportional
model.
40%
30%
20%
0
24
48
72
96
120 144 168 192 216 240 264 288 312 336 360 384
months
S87
Figure 4: PFS according to ESR
Table 3: PFS of the patients with MGUS, multivariance analysis
(Cox model)
p
Beta
HR
IC 95%
1,36
Standard
Error
0,48
MC
concentration
0,005
3,9
1,51 – 10,15
Type of MC
Level of UI
Bence Jones
ESR
0,06
0,47
0,7
0,003
0,76
0,19
0,26
-0,09
0,41
0,16
0,26
0,36
2,13
1,12
0,9
2,18
1,05 – 4,82
0,82 – 1,55
0,55 – 1,17
1,06 – 4,46
Table 4: prognostic factors described by different authors
N
Follow-up (Md, months)
[CM]
Type MC
Bence Jones
Plasma cells in BM
Reduced UI
ESR
Rate of progression (%)
PFS (10 years, %)
OS (10 years, %)
# Median follow up, in years
Kyle
1395
15.4#
X
X
8
90
Cesana
1231
65
Baldini
335
70
X
Vuckovic
87
91
X
X
X
X
X
5,8
86
79
X
X
X
X
6,8
92##
Montoto
434
5.2#
X
X
CHP
281
66
X
X
83
80
85
9
86
90
## PFS at 60 months
Marked Improvement in Detecting the
Number of Involved Nodal Areas in
Lymphoma, Using 18 F- FDG – PET and CT
Scan.
OBJECTIVE
Juan Ramón Chalapud Revelo
Médico adscrito al servicio de hematología
Instituto Nacional de Cancerología México
Pedro Sobrevilla-Calvo
Silvia Rivas-Vera
Javier Altamirano-Ley.
Hematology, Instituto Nacional de Cancerologìa de Mèxico
Patient population
All FDG PET studies were done in patients with new diagnosis
or in recurrent disease of HL and NHL between April 2003 and April
2005, were valuated retrospectively. A total of 56 patients met the
following entry criteria: PET and CT before any therapy (chemo, immuno or radiotherapy), histopathology review at our institution.
The clinical stage was determined using the Ann Arbor classification (3) and NNA of disease as it is described in the FLIPI.
INTRODUCTION
PET
Positron emission tomography (PET) imaging with 18-fluoro2-deoxiglucose (FDG) is increasingly used for the initial evaluation
and staging of patients with Hodgkin’s lymphoma (HL) and nonHodgkin’s lymphoma (NHL) (1,2). However, the degree of concordance of PET and CT scanning for each site is not well defined. The
number of nodal areas involved (NNA) is a new prognostic factor in
follicular lymphomas as was demonstrated in the Follicular Lymphoma (FL) International prognostic index (FLIPI)(2). We hypothesized
that with the use of PET this evaluation would improve.
The objective of this study is to compare the number of nodal
areas involved with PET and TAC and to evaluate the cases of
agreement and disagreement for nodal areas, and clinical stage.
PATIENTS & METHODS
Patients were injected 370MBq of FDG intravenously. Images
were acquired from whole body. Plasma glucose measurements
were routinely obtained. Studies were performed using the ECAT
EXACT HR plus (Siemens /CTI, Knoxville, TN, USA) PETs. The HR
plus has a transaxial spatial resolution of 4.5 mm.
CT
Patients were injected intravenous contrast, and they were given oral contrast. Images were acquired from neck, thorax, abdomen
and pelvis with cross section of 3, 4, 10 and 10 mm respectively.
S88
Studies were performed using the Siemens, Semoton Volume helical and sequential of 4 multi detecting.
In this study, we examined the performance of CT versus FDGPET scanning, comparing; The Ann Arbor stage, NNA, and each
one of the nodal area (cervical, mediastinal, axillary, para aortic, inguinal) (figure 1). Bone marrow biopsy results were excluded from
this initial analysis
Table 1. Distribution of patients
STATISTICAL ANALYSIS
We used simple frequencies. Comparison of stage and NNA
were performed by the exact fisher test. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of
PET and CT for each nodal area were calculated.
RESULTS
The study population and histopathological diagnoses are
shown in Table 1. These consisted of 56 individuals; there were 32
females and 24 males (mean age 40 years, range, 15 to 74).
Among the 56 pts, 22 (39%) had discordant results between
PET scanning and CT scanning, that lead to a change in stage assignment. Among the discordant cases PET resulted in upstaging in
18/56 pts (32%), and down staging in 4/56 pts (7%) (table 2). Forty
four pts (79%) had discordant results in the number of nodal areas, among the discordant cases; PET detected more nodal areas
(36/56 pts, 64%) than CT in (8/56 pts,14%) (table 3).
The discordant cases for each nodal areas were distributed as
shown in table 4. Sensitivity, specificity, PNV, PPV is shown in table
5. PET was better than CT in all comparison.
DISCUSSION
PET had a higher detection rate for nodal areas, specifically
(axilar, mediastinal, and inguinal). PET provided accurate assessment of stage and NNA, Especially in the patients with low tumor
burden. PET and CT in combination detects more involved nodal
areas than each method by itself and both should be incluided in the
assessment of patients with lymphoma.
Table 2. Stage comparison between PET and CT
Figure 1.
Discordant findings at PET (upper row) and CT (lower rows) in
anaplastic large cell Lymphoma (ALCL) (A) The coronal images
obtained at PET show increased uptake of bilateral cervical, mediastinal, right cervical, para-aortic, right inguinal (stage IIII, NNA 6).
(B) The CT show a node right cervical (Stage I, NNA 1)
Table 3. Number of Nodal Areas comparison Between PET and
CT
S89
Table 4. Summary of PET/CT correlation with nodal areas
REFERENCES
1. Burton C, Ell P, Linch D. The role of PET imaging in lymphoma.
Brit J Haematol 2004:126;772-84.
2. Elstrom R, Guan L, Baker G, Nakhoda K, Vergilio J, Zhuang
H et al. Utility of FDG-PET scanning in lymphoma by WHO classification. Blood 2003; 101: 2875-6.
3. Solai-Céligny P, Roy P, Colombat P, White J, Armitage J, Arranz-Saez R, Wing Y, et al. Follicular Lymphoma International
Pronostic Index. Blood 2004; 14: 1258-65.
4. Rosemberg SA, Boiron M, DeVita VT Jr. Johnson RE, Lee BJ,
Ultman JE, Viamonte M Jr. Report of committee on Hodgkin’s
Disease Staging Procedures. Cancer Res 1971; 31:1862-13.
CXCR4/SDF-1is A Key Regulator for
Leukemia Migration and Homing to the
Bone Marrow And AMD3100 Increases In
Vivo Response to Chemotherapy
Bruno Nervi1, Matthew Holt2, Michael P Rettig2, Julie K Ritchey2,
Pablo Ramirez1, Julie L. Prior3, David Piwnica-Worms3, Gary
Bridger4, and John F DiPersio2
1
Universidad Catolica de Chile, Departamento de Hemato-Oncologia, Santiago, Chile, 2Division of Oncology, 3Department of Molecular Biology and Pharmacology Washington University School
of Medicine, Saint Louis, MO, United States, 63110 and 4AnorMed,
Inc., Langley, British Columbia, Canada.
Keywords: leukemia, AMD3100, stem cell mobilization
Hematopoietic stem cells (HSC) reside in the bone marrow
(BM) and interact with a highly organized microenvironment comprised of stoma cells and extracellular matrix. CXCR4/SDF-1 axis
regulates the trafficking of normal stem cells to and from the BM.
AMD3100 (AMD) is a competitive inhibitor of CXCR4 and a single
injection in mice and humans produces a rapid and transient mobilization of normal HSC from the BM into peripheral blood (PB).
We utilized a PML-RARα knocked-in mouse model of human acute
promyelocytic leukemia (APL) to study APL interaction with the
normal BM. Adoptive transfer of APL splenocytes into genetically
compatible mice (F1 B6 x 129) results in a rapidly fatal leukemia.
APL cells characterize by the CD34/GR1 co-expression. To more
efficiently track the leukemic cells, we transduced banked APL
tumors with a dual function reporter gene that encodes a fusion
protein comprised of luciferase, a bioluminescence imaging (BLI)
optical reporter gene, and EGFP for ex-vivo cell sorting. We hypothesize that we can overcome tumor resistance to chemotherapeutic
agents by interrupting the interaction that APL cells have with the
BM stroma. Upon intravenous (iv) injection of 106 APL cells into syngeneic unconditioned recipients, APL rapidly migrated to the BM
with increased BLI signal in the femurs, spine, ribs, and skull, at 4
days after injection, followed by spleen infiltration and by death due
to leukostasis by 14-16 days. Four mice were injected with 106 APL
cells iv on day 0. On day 12, mice received a single AMD injection
(5μg/kg/sq). We observed a rapid APL mobilization into PB. There
was a 3.5-fold increase in total WBC count and a 9-fold increases
in APL blast cells in PB compared to baseline. WBC and blast mobilization was transient and cell counts returned to baseline levels
within 12h. Unconditioned mice (n=28) were injected iv with 106 APL
cells. Engraftment of APL cells was evaluated weekly by PB flow
cytometry and BLI. By day 12 after APL injection all mice had ±5%
S90
APL cells in PB. 8 mice received AraC (500mg/kg/sq) on days 12
and 13, and another 8 mice received AraC + AMD (5mg/kg/sq) 1
hour before and 3 hours after each AraC injection. 6 mice received
only AMD and 6 control mice were observed. Total body BLI signal,
WBC, and blasts per μl of blood in days 19 and 23 were higher
in AraC versus AraC+AMD (p<0.004). Median survival for control,
AMD, AraC and AraC+AMD groups were 18, 19, 23 and 30 days
respectively (p<0.0006). Hemoglobin, platelet and granulocyte recovery post-chemotherapy was similar in both groups (p=NS). We
developed an in-vitro mouse stroma system to study engraftment,
ex-vivo mobilization and sensitivity to chemotherapy. In-vitro culture of APL cells with or without stroma showed no difference in
APL survival between AraC versus AraC+AMD by flow cytometry
or BLI (p=NS). Stroma offered a survival benefit versus no stroma
(p<0.0001). We injected 4 syngeneic mice with 106 APL cells iv. After 14 days mice were sacrificed and we collected and pulled blood,
spleen and BM; blast percentage was 47, 58 and 40% respectively.
We cultured ex-vivo cells from all three compartments with AraC
(25ng/ml). After 24 hours APL survival was 25, 80 and 60% respectively (p<0.006). We repeated the same experiment, but we did a
positive selection for CD34 to isolate APL cells from blood, spleen
and BM. Survival after ex-vivo AraC incubation was 32, 30, 34%
respectively (p=NS). In summary, we characterized a mouse leukemia model that “homes” preferentially to the BM microenvironment
similar to human AML. APL cells were mobilized from the BM into
PB after AMD3100 administration. Mobilized APL cells were found
to be significantly more sensitive to chemotherapy. These results
provide a foundation for future clinical trials with AMD3100 + chemotherapy in humans with AML.
Lineage Specific Chimerism Analysis
Allows Early Detection of Relapses After
Allogeneic Stem Cell Transplantation
Galeano, S.
PRO.IN.BIO, Montevideo , Uruguay.
Gabus, R.
Hospital Maciel, Montevideo, Uruguay
Bengochea, M.
Inst. Nacional de Donación y Trasplante, Montevideo, Uruguay
Boiron, JM.
Etablissement Francais Du Sang, Bordeaux, France
Carreto, E.
Bodega, E.
Hospital Maciel, Montevideo, Uruguay
Alvarez, A.I.
Background: Chimerism analysis is essential to verify the
origin of hematopoiesis after allogeneic stem cell transplantation
(SCT). Considering that after SCT, almost all relapses are recipient-derived, the reappearence of mixed chimerism or an increasing fraction of recipient-derived cells should prompt the suspicion of
relapse and be differentiated from graft failure or rejection. Furthermore, as reduced intensity conditioning SCT (RIC-SCT) emerge as
a frequent procedure, a correct interpretation of chimerism analysis
becomes imperative since transient mixed chimerism is frequently
observed after RIC-SCT and does not necessarily means an unwanted evolution. Aims: Evaluate the usefulness of our methodology of lineage-specific chimerism analysis to sensitively detect relapse early after conventional or RIC SCT. Methods: We performed
chimerism analysis in whole peripheral blood (PB) as well as in
the separated cells on days 14, at the time of neutrophil recovery
and monthly thereafter during the first year after SCT. Chimerism
was determined on PB by short tandem repeat (STR) analysis on
unfractionated PB or after cell separation (lineage-specific chimerism: positive selection of mononuclear cells using CD3, CD15 and
CD19 monoclonal antibodies conjugated with magnetic beads; Dynabeads®. DNA was obtained with the Miller method and samples
were used in a polymerase chain reaction to amplify 6 (D8S1130,
D21S1270, D6S1031, D3S2406, D9S938, IFNAR-ALU) or 10
(D16S2622, D1S1612, D2S1353, D22S685, D11S1392, D3S2398,
D5S2501, D15S657, D10S1237, IFNAR-ALU) STRs loci. Primers were marked with Cy5. Separation and detection of fragments
were done with ALF-Express® and infomative peaks were analyzed
with the AlleleLinks® software. Depending on the locus, sensitivity to detect mixed chimerism was evaluated in 1 to 5%. Results:
Fifteen patients were allografted at Maciel Hospital, Montevideo,
Uruguay, from january 2003 to december 2004 and those with at
least 1 chimerism analysis were included (n=13). Five patients relapsed during the first year after SCT. Three of them were detected
by chimerism analysis: in one case, mixed chimerism was observed
in the subpopulation compromised by the disease (CD19+ in B lineage ALL with CD19 positive blasts) while in the 2 other patients,
relapses were detected by an increasing recipient hematopoiesis
in unfractionated blood and CD3-CD19-CD15 subpopulations (AML
with CD15+, CD3- and CD19- blasts and ALL with CD19+, CD3and CD15- blasts). The other 2 patients had relapses of CML that
were detected by nested PCR for bcr/abl and cytogenetic analysis but did not show mixed chimerism. Conclusions: These results
suggest that, at least in some diseases, lineage specific chimerism
could be an alternative to other methods to increase sensitivity and
specificity of relapse detection.
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EDUCATION SESSION
Acute Leukemias
The Current and Future Management of
Acute Myeloid Leukemia in Adults
Peter H. Wiernik
Our Lady of Mercy Cancer Center, New York Medical College,
Bronx, New York, USA
Currently the best standard therapy for adults < 70 years of
age consists of induction therapy with three daily doses of idarubicin
and a seven-day continuous infusion of cytarabine. Some physicians still prefer daunorubicin or mitoxantrone instead of idarubicin,
but all relevant prospective, randomized trials demonstrate one or
more advantages of idarubicin over daunorubicin, and no studies
demonstrate an advantage for mitoxantrone over daunorubicin.
Furthermore, a meta-analysis of relevant raw data performed by
Wheatley et al1 confirmed the superiority of idarubicin over daunorubicin as an induction agent. Most studies in which daunorubicin was
used during induction employed three consecutive daily doses of 45
mg/M2. There is no evidence that 60 mg/M2 doses, as used by some
investigators2, lead to a better outcome than the lower doses. The
standard dose of cytarabine used in induction is 100 mg/M2 daily,
given as a continuous seven-day infusion. Doubling that dose3, or
even increasing it by a factor of 20 or 304 has resulted in little improvement, if any, in outcome of induction therapy. The addition of
etoposide to the standard anthracycline + cytarabine induction regimen has improved results in some 5but not all 6 studies.
There is general agreement that post-remission therapy is necessary to maximize disease-free and overall survival, but there is
no universally accepted post-remission therapy regimen. High-dose
cytarabine regimens have commonly been employed and seem to
be effective, especially in younger patients with favorable cytogenetics.7 There is little evidence that combining other drugs with highdose cytarabine post-remission improves results8. The optimum
dose of cytarabine as post-remission therapy has not been defined.
It seems clear from the original study by Mayer et al9 that a dose of
400 mg/M2 is inferior to 3 gm/M2 but doses in between those have
not been widely tested in an evaluable manner.
Despite the popularity of stem cell transplantation as a postremission therapy, outcome data are disappointing for both autologous 8,10 and allogeneic stem cell transplantation.10 In fact, Visani et
al 10 after an analysis of 344 papers concluded that there is no evidence that autologous stem cell transplantation is superior in terms
of overall survival to chemotherapy alone, and that no overall benefit of allografting on survival was demonstrated by any trial. Also of
note is the discovery that Hispanics allo- transplanted in the United
States had a significantly higher risk of treatment failure (death or
relapse) and overall mortality than Whites, for unknown reasons.11
G-CSF12 and GM-CSF13 have both been shown not to worsen
disease outcome when used as supportive care in patients with
AML. On the other hand, they may have the potential for inducing
secondary AML or myelodysplasia in certain solid tumor patients. A
doubling of the incidence of AML/MDS in 5,510 women treated with
adjuvant chemotherapy for breast cancer was observed in those
who received colony-stimulating factors compared with those who
did not.14
Patients with AML over age 65 years generally have a poorer
outcome with therapy than do younger patients, and controversy
exists as to whether older patients should be treated with regimens
used in younger patients, or with less intensive therapy such as lowdose cytarabine. Kantarjian et al15 analyzed the data for 998 patients
aged 65 years or more with AML or high-risk myelodysplasia treated
with intensive therapy in an effort to determine prognostic factors
for response and survival. The overall complete response rate was
45%. Poor prognostic factors for complete response and survival
were age >75 years, unfavorable karyotype, poor performance
status, longer duration of antecedent hematologic disorder and
abnormal organ function. Based on these prognostic factors, they
estimated that approximately 20% of the patients fell into a good
prognosis group with an expected complete response rate > 60%,
an induction mortality rate of 10% and a 1-year survival rate >50%.
Such patients would clearly be expected to benefit from standard intensive therapy. Appelbaum et al16 studied a similar group of almost
identical size. In addition to the prognostic factors noted above, they
found multidrug resistance protein in 33% of AML patients < age
56 compared with 57% of patients older than 75 years. Consistent
with the Kantarjian et al study15 they observed that 35% of patients
younger than age 56 had unfavorable cytogenetics, compared with
51% of patients older than 75 years. It seems advisable to treat
elderly AML patients with good prognostic factors as described in
these two studies with standard induction chemotherapy. It is not as
clear how to approach post-remission therapy. Standard high-dose
cytarabine is too toxic for most elderly patients. Doses of 1.0-1.5
gm/M2 have been well tolerated but not clearly effective.13
The best hope for improving therapy for adult AML is the development of new drugs with better activity against the disease. After a long draught, a number of recently introduced agents have
already demonstrated promise. Giles et al17 studied cloretazine in
patients age > 60 years with previously untreated AML. The drug
was given alone at a dose of 600 mg/M2 once, as induction therapy
to 104 patients with a median age of 72 years. No patient had a
favorable karyotype, and most had some significant organ dysfunction. The complete response rate was 28% and another 4% had a
complete response with incomplete recovery. The one-year survival
rate for the 32% of patients who were complete responders was
28%. There was minimal extramedullary toxicity in the study. The
drug causes DNA crosslinks. Its active metabolite has similarities
to that of carmustine (BCNU) but it yields more than twice the DNA
crosslinks, mole for mole, compared with carmustine.18 Burnett et
al19 administered clofarabine (a purine nucleoside analog) 30 mg/M2
daily for 5 days to 66 patients with a median age of 71 years. 62
had intermediate or poor risk cytogenetics. One course of drug was
given every 28-42 days and a maximum of 3 courses were given.
The CR + CRi rate was 29% and the one-year overall survival rate
for responders was 32% and 28% for non-responders. Interesting,
the one-year survival rate was identical for intermediate and poor
cytogenetics patients. Clofarabine appears to be more toxic than
cloretazine in the doses and schedules used. Serious renal toxicity
developed in about 18% of patients treated with the former, and
sepsis occurred in approximately 26% of those patients.
Several recent studies, if confirmed, will result in improved
treatment of patients with AML in the near future. Liu et al20 assessed response and survival in 60 patients with APL induced with
ATRA, 25 mg/M2 plus As2O3, 0.16 mg/kg and consolidated them
with 3 cycles of daunorubicin, cytarabine and homoharring-tonine,
and compared results with 56 historical controls induced with ATRA
S92
alone followed by postremission chemotherapy. The experimental
group also received 5 cycles of maintenance therapy with monthly
ATRA, followed by As2O3 daily for a month, which was followed by
weekly methotrexate for a month. There was no difference in CR
rate between the groups, which was low (56% v 51%). However,
at a median follow-up of 48 and 56 months, overall and event-free
survival were significantly longer in the study group (4-year overall survival 98.1% v 83.4%, and 4-year event-free survival 94.2%
v 45.6%).
The MRC21 studied the addition of gemtuzumab ozogamicin
(GO), 3 mg/M2 on day 1 of induction therapy with ADE, DA or FLAGIda in a randomized study of 113 patients <60 years old. CR rates
were not different (85%). At 3 years, disease-free survival was significantly different in favor of those who received GO (49% v 38%).
Toxicity was similar between the groups. Others22 have shown in vitro that cytotoxic activity of GO correlates with expression of protein
kinase Syk and that azacytidine upregulates Syk. In another in vitro
study Takahashi et al23 demonstrated a synergistic effect of As2O3
and FLT 3 inhibition on cells with FLT 3-ITD.
The best hope for real progress in the future is the identification
of specific genotypes that predict prognosis, or better yet, response
to a given agent. Probably with similar thoughts in mind, Schlenk
et al24 performed a retrospective analysis of 4 German AML Study
Group trials. The studies were of similar design and included 872
patients with a median age of 48 years. The results of gene analyses indicated that the 33% of patients found to be NPM1+ and FLT3
ITD – as well as those CEBPA+ had significantly higher response
rates than others (88% and 83% for the former and 66% for others). Furthermore, those favorable genotypes were associated with
significantly better relapse-free and overall survival. Others 25 have
confirmed in a larger study that if not associated with FLT3-ITD mutations, mutant NPM1 appears to identify patients with improved
response to treatment.
REFERENCES
1. Anon: A systematic collaborative overview of randomized trials comparing idarubicin with daunorubicin (or other anthracyclines) as induction therapy for acute myeloid leukaemia. AML
Collaborative Group. Br J Haematol 103:100-109, 1998.
2. Schiller G, Gajewski J, Territo M et al: Long-term outcome of
high-dose cytarabine-based consolidation chemotherapy for
adults with acute myelogenous leukemia. Blood 80:2977-2982,
1992.
3. Schiller G, Gajewski J, Nimer S et al: A randomized study of
intermediate versus conventional-dose cytarabine as intensive
induction for acute myelogenous leukaemia. Br J Haematol
81:170-177, 1992.
4. Kern W, Estey EH: High-dose cytosine arabinoside in the treatment of acute myeloid leukemia: Review of three randomized
trials. Cancer 107:116-124, 2006.
5. Bishop JF, Lowenthal RM, Joshua D et al: Etoposide in acute
nonlymphocytic leukemia, Australian Leukemia Study Group.
Blood 75:27-32, 1990.
6. Goldstone AH, Burnett AK, Wheatley K et al: Attempts to improve treatment outcomes in acute myeloid leukemia (AML) in
older patients: the results of the United Kingdom Medical Research council AML11 trial. Blood 98:1302-1311, 2001
7. Bloomfield CD, Lawrence D, Byrd JC et al: frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype.
Cancer Res 58:4173-4179, 1998.
8. Buchner T, Berdel WE, Schoch C et al: Double induction containing either two courses or one course of high-dose cytarabine plus mitoxantrone and postremission therapy by either autologous stem-cell transplantation or by prolonged maintenance
for acute myeloid leukemia. J Clin Oncol 24:2480-2489, 2006.
9. Mayer RJ, Davis RB, Schiffer CA et al: Intensive postremission
chemotherapy in adults with acute myeloid leukemia. Cancer
and Leukemia Group B. N Engl J Med 331:896-903, 1994.
10. Visani G, Olivieri A, Malagola M et al: Consolidation therapy for
adult acute myeloid leukemia: a systematic analysis according
to evidence based medicine. Leuk Lymphoma 47:1091-1102,
2006.
11. Baker KS, Loberiza FR Jr, Yu H et al: Outcome of ethnic minorities with acute or chronic leukemia treated with hematopoietic stem-cell transplantation in the United States. J Clin Oncol
23:7032-7042, 2005.
12. Heil G, Hoelzer D, Sanz MA et al: Long-term survival data from
a phase 3 study of filgrastim as an adjunct to chemotherapy in
adults with de novo acute myeloid leukemia
13. Rowe JM, Andersen JW, Mazza JJ et al: A randomized placebocontrolled phase III study of granulocyte-macrophage colonystimulating factor in adult patients (>55 to 70 years of age) with
acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood 86:457-462, 1995.
14. Hershman D, Neugut AI, Jacobson JS et al: Acute myeloid leukemia or myelodysplastic syndrome following use of granulocyte colony-stimulating factors during breast cancer adjuvant
chemotherapy. J Natl Cancer Inst 99:196-205, 2007.
15. Kantarjian H, O’Brien S, Cortes J et al: Results of intensive
chemotherapy in 998 patients age 65 years or older with acute
myeloid leukemia or high-risk myelodysplastic syndrome: predictive prognostic models for outcome. Cancer 106:1090-1098,
2006.
16. Appelbaum FR, Gundacker H, Head DR et al: Age and acute
myeloid leukemia. Blood 107:3481-3485, 2006.
17. Giles F, Rizzieri D, Karp J et al: Cloretazine (VNP40202M), a
novel sulfonylhydrazine alkylating agent, in patients age 60
years or older with previously untreated acute myeloid leukemia. J Clin Oncol 25:1-7, 2007.
18. Ishguro K, Seow HA, Penketh PG et al: Mode of action of the
chloro-ethylating moieties of the prodrug cloretazine. Mol Cancer Ther 5:969-976, 2006.
19. Burnett AK, Baccarani M, Johnson P et al: A phase II study of
clofarabine monotherapy first-line in patients aged 65 years or
older with acute myeloid leukemia for whom standard intensive
chemotherapy is not considered suitable. Am Soc Hematol
2006 abstract #425.
20. Liu YF, Zhu YM, Shi ZZ et al: Long-term follow-up confirms the
benefit of all-trans retinoic acid (ATRA) and arsenic trioxide
(As2O3) as front line therapy for newly diagnosed acute promyelocytic leukemia (APL). Am Soc Hematol 2006 Abstract #
565.
21. Burnett AK, Kell WJ, Goldstone AH et al: The addition of gemtuzumab ozogamicin to induction chemotherapy for AML improves
disease-free survival without extra toxicity: preliminary analysis
of 115 patients in the MRC AML15 trial. Am Soc Hematol 2006
Abstratc # 13.
22. Balain L, Ball ED: Cytotoxic activity of gemtuzumab ozogamicin
(Mylotarg) in acute myeloid leukemia correlates with the expression of protein kinas Syk. Leukemia 20:2093-2101, 2006.
23. Takahashi S, Harigae H, Yokoyama H et al: Synergistic effect
of arsenic trioxide and flt3 inhibition on cells with flt3 internal
tandem duplication. Int J Hematol 84:256-261, 2006.
24. Schlenk R, Corbacioglu A, Krauter J et al: Gene mutations as
predictive markers for postremission therapy in younger adults
with normal karyotype AML. Am Soc Hematol 2006 abstract
#4.
25. Thiede C, Koch S, Creutzig E et al: Prevalence and prognostic
impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 107:4011-4020, 2006.
Acute Lymphoblastic Leukemia In Children
Brandalise SR.
State University of Campinas, Pediatric, Hematology / Oncology.
Director Boldrini’s Pediatric Center.
e-mail: [email protected]
1. INTRODUCTION
Recent progress in the treatment of acute lymphoblastic leukemia (ALL) in children and adolescents is an example of great success in the context of modern medicine. The cure rates below 10%
in patients under 15 years of age observed in the 60’s, dramatically
jumped to 75% of event-free survival rates at the end of the 90’s
(24, 11). Besides treatment intensification based on combined chemotherapy and CNS directed therapy, better supportive measures
played an important role improving the survival. The prognostic
value of cytogenetic, immunophenotype, molecular biology and the
early response rate to treatment contributed to the patient’s stratification in different risk groups, allowing a definite application in clinical trials of the prognostic factors for risk-directed therapy. This resulted in a steady improvement in treatment outcome. Despite this
advances had been obtained in more developed countries, much
has to be gained in those countries with low economic income (9).
2. RECENT RESULTS FROM INTERNATIONAL PEDIATRIC COOPERATIVE GROUPS
In the last decades, ALL treatment results published by
prominent pediatric oncology institutions and cooperative groups,
were quite similar. In the 1990s, the 5-yr event-free survival rates for
childhood ALL generally ranged from 70 to 83 percent in developed
countries (21).The main common adopted strategy by those different clinical trials was based on therapy intensification during the first
six months of treatment, including remission-induction, followed by
consolidation (or intensification), reinduction and continuation treatment to eliminate residual leukemia. Therapy directed to the CNS
was given according to the patient’s risk of relapse.
Contemporary improved treatment has abolished the prognostic strength of many clinical and biologic variables that were previously related to outcome, as male sex (25,20) and
T-cell ALL or
mature B-cell ALL in children (12,17). Certain genetic abnormalities
presented on the leukemic cells have different prognostic value (1).
A poor prognosis is associated with t(4;11) and the MLL-AF4 fusion gene. However, patients with MLL-AF4 fusion that are over
one year of age, have a better outcome. Poor prognosis is also
related with the presence of t(9;22) with BCR-ABL fusion, which
increases in frequency with age. By the other side, in B-cell precursor ALL hyperdiploidy and t(12;21) with TEL-AML1 fusion gene,
confer a highly favorable prognosis. Childhood cases with trisomies
4, 10, and 17 may have a particularly favorable outcome (26). Considering the T-cell ALL phenotype, the presence of t(11;19) with
MLL-ENL fusion and over expression of the HOX11 gene confer a
good prognosis(1). Age and leukocyte count at diagnosis continue
to be strong prognostic indicators of outcome, mainly among patients with B-cell precursor ALL. Children with less than one year
of age represents the poorest risk group of patients, mainly those
with MLL translocations, with a dismal prognosis even with modern
treatments (22,19).
This knowledge is of great importance, mainly due the need
to identify a sub-group of ALL patients with very low risk of relapse
in whose treatment could be objectively reduced. The Children’s
Oncology Group recently has proposed in the Study AALL0331 a
four-category system that recognizes patients with a very low probability of relapse, with an estimated 5-yr event-free survival over
90%. In this COG’s Study the Standard Risk Group according to
Rome/NCI criteria is subdivided on three strata (SR-Low, SR-Average, SR-High) based on the presence of TEL-AML-1 fusion or triple
trisomy and low levels of MRD at end of induction for the group
with better prognosis. Increased therapy for the patients with resistant disease is today the best approach. Probably in the future,
new facts concerning the molecular pathogenesis of the disease, as
well as the knowledge of the patient’s pharmacogenetic factors, will
define new specific target therapies, with less toxicities and better
chances of cure.
3. CHILDHOOD ALL TREATMENT IN UNDERDEVELOPED
COUNTRIES
It is estimated that the number of children diagnosed with
cancer, in well developed countries versus those with limited economic resources, is approximately 22,000 to 33,000 versus 158,000
to 237,000 new cases / year (9). One third of these patients have
acute leukemia. In Latin America underdeveloped countries, when
given the patients equal access to effective treatment with modern
trials done at single institutions or in cooperative study groups, the
5-yr event-free survival rates are similar to those obtained in rich
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countries, despite the difficulties to introduce the routine cytogenetics and MRD analysis in most hospitals (5, 6, 7, 10, 14, 23).
Unfortunately, several countries in Latin America only provide
treatment for patients with medical or social securities (employed
parents), including a covering range from 40 to 70% of the population. Local and some International non-profit organizations help
these uninsurance children with cancer. Recent experiences in
Central America, as in El Salvador and Nicaragua, in a partnership
with St. Jude Children’s Research Hospital (Memphis, TNN) and
Monza (Italy) respectively, were highly effective with increasing better results , achieving a total survival rate for childhood cancer of
about 50% (15,4).The main point of these international experiences
was the establishment of twin programs with compromised institutions from developed countries, involved with medical and nurse
training, financial support to provide medical supplies and diagnostic resources, as well as adequate hospital and ambulatory facilities.
However, despite these focal efforts, treatment abandon and toxic
deaths due to neutropenia or immunossupression continue to be
important problems in countries with limited economic resources,
demanding a great challenge to reduce them (9).
With the objective to reduce relapses and the degree of immunossupression for low-risk ALL children, it was proposed by the
Brazilian Cooperative ALL-99 Protocol (GBTLI), the use of intermittent Methotrexate/6-Mercaptopurine in a randomized prospective
comparison with the traditional use of daily oral 6MP and weekly
MTX, given in maintenance therapy. This was based on a report
from the Japanese group study JCCLSG-5811 with ALL low risk
children, randomized to receive an intermittent regimen with MTX /
6MP or a continuous regimen with this pair of drugs during maintenance. The 4-yr DFS was 75.1% +-5.8% for the intermittent regimen
(cumulative dose of MTX 5,400 mg/m2) versus 49.7% +-7.3% for
the continuous regimen (cumulative MTX dosage of 2,240 mg/m2),
with a p value < 0.01 (13). This study, despite the small number of
registered patients (n=131), could demonstrate that infectious episodes related to the degree of immunossupression (Varicella/Herpes Zoster) were significantly higher in the group of patients with
the traditional use of 6MP/MTX, suggesting a worse function of their
immune system. Similar results were obtained by the Medical Research Council UKALL trials,1972-84 (18). In a previous published
study done by the MRC UKALL comparing three chemotherapy
regimens in ALL children, no Varicella/Herpes Zoster was seen in
the patients on intermittent maintenance (16) The pharmacological
rational for the sequential use of MTX and 6MP is that the previous
exposition to MTX diminishes the de novo purine synthesis, and increases the intracellular levels of 5 phosphoribosil-1-pirophosphate
(PRPP), resulting in higher capitation of the 6-MP nucleotide inside
the cell (3, 2,8). Another possible benefit with the intermittent use
of MTX / 6MP, is that decreasing the immunossupression degree,
it could be evoked the antileukemic activity of the immunological
system, thus controlling residual leukemic clones.
The eligibility criteria for the GBTLI ALL-99 low risk group was
based on NCI criteria with age between 1 to 9 years and WBC at diagnosis < 50,000/mm3. Immunophenotyping and cytogenetic findings were not used for risk classification. The study was designed
with a 2 x 2 factorial comparison to determine the event-free survival
(EFS), the disease – free survival (DFS) and toxicities according to
NCI criteria version 2.0 in patients receiving the maintenance therapy with MTX / 6MP given as intermittent regimen ( MTX 200 mg/m2
6h IV infusion each 21 days and oral 6MP 100 mg/m2 daily x 10
days, with 11 days rest ) or as continuous regimen (conventional
weekly IM MTX 25 mg/m2 and daily oral 6MP 50 mg/m2). The randomization of each of these two maintenance regimens was done at
the end of the late consolidation phase. All the chemotherapy could
be administered in an out-patient ambulatory clinic. No CNS radiation was given. Triple intrathecal therapy was administered each 8
weeks during all maintenance treatment.
The therapeutical approach of the Low Risk ALL patients was
based on the concept of double intensification (with MTX 2g/m2 x 4
and a delayed consolidation phase) of BFM components protocol.
Few patients from the Low Risk Group were not eligible for randomization. They were the slow responders ( WBC >5,000/mm3 with or
without peripheral blasts at Day 7 or those with M3 BM status at
Day 14 or BM status M2/M3 at Day 28 of the induction therapy ).
Day 7 WBC >5,000/mm3 were highly correlated to worse prognosis
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in the GBTLI ALL -93 protocol (16). Those patients move to the High
Risk Group, being analyzed as HR patients.
Till September 2006, 230 pts (44.9%) were randomized to the
Maintenance therapy for the Group 1 (continuous regimen) and 230
pts (44.9%) to Group 2 (intermittent regimen). Clinical and laboratorial data of the Low Risk ALL-99 GBTLI patients are summarized in
Table 1. The patients’ distribution according to known biological risk
features and the two randomized maintenance regimens is detailed
as follows. Group 1 (Continuous Regimen): T-ALL (6 pts), Calla
negative (8 pts), with t(9;22)(1 pt) and with t(4;11)(1pt). Group 2
(Intermittent Regimen): T-ALL (11 pts), Calla negative (12 pts), 3 pts
with t(9;22) and no one with t(4;11). Clinical evolution of all Low Risk
ALL-99 GBTLI patients is summarized in Table 2.
Table1. Clinical and Laboratorial data of the Low-Risk ALL-99
GBTLI patients
Ph+, 2 pts were T-ALL and one had CNS-3 at diagnosis. Additionally, 3 relapsed pts had t (12;21).
Deaths during the maintenance therapy occurred in 8 patients
out 460 (1.7%), being 7 pts from Group 1(continuous 6MP/MTX).
According to NCI Grade III and IV toxicities criteria, the continuous
use of 6MP/ MTX was associated with higher rates of significant
decrease of WBC / neutrophils counts and higher levels of hepatic
dysfunctions. Quite often, chemotherapy for Group 1 patients had
to be postponed. Grade III CNS toxicities occurred in 13/205 pts in
Group 1 and in 5/194 in Group 2 (intermittent MTX / 6MP).
In conclusion, with almost 2,5 yrs of mean follow-up the ALL
patients classified as Low Risk, treated with higher doses of MTX
during the maintenance therapy and with a sequential and intermittent use of 6MP, presented a better EFS (p=0.048), less hepatic
(p = 0.015) and CNS toxicities. Significant less infectious deaths in
maintenance therapy was registered in this group, probably related
to a better immune and/or bone marrow functional status. Besides
those mentioned measurable advantages, less visits to the hospital
and higher school attendance also contributed to a better quality of
life for those patients.
Finally, this maintenance therapy would be helpful not only
for children from underdeveloped countries, with enormous social
problems related with compliance and transportation facilities, but
to all the pediatric ALL low risk patients that deserves a better quality of life while on treatment. Further studies are necessary to understand the pharmacodynamic aspects and organ metabolism of
the drugs used in the maintenance phase, as well as, monitor the
financial impact and HRQOL parameters in a comparative study for
children with ALL.
4. REFERENCES
Table 2. Clinical Evolution of all Low Risk ALL-99 GBTLI patients
The estimated 5-yr Overall Survival of 512 consecutive Low
Risk patients treated according to the GBTLI ALL-99 Protocol is
88.1% ± 2.1%. The estimated 5-yr Event-Free Survival rate is 80%
± 2.9%. According to the maintenance regimen, the estimated 5-yr
EFS for Group 1 (continuous 6MP / MTX) is 80.2% ± 4.5% and for
Group 2 (intermittent MTX / 6MP) is 88.3% ± 3.7% (p = 0.048).
There were 26 relapses: 16 isolated BM (with equal number between the two regimens) and 10 extramedullary relapses, 8 of them
occurring in the Group 1. Among the 26 pts that relapsed, one was
1. Armstrong SA, Look AT. Molecular genetics of acute lymphoblastic leukemia. J Clin Oncol 2005; 23: 6306 -15.
2. Berkovitch M, Matsui D, Zipursky A, et al. Hepatotoxicity of
6-Mercaptopurine in childhood acute lymphoblastic leukemia:
Pharmacokinetic Characteristics. Med Ped Oncol 1996; 26: 85
- 89.
3. Bertino JR. Ode to Methotrexate. J. Clin Oncol 1993; 11: 5 -14.
4. Bonilla M, Moreno N, Marina N, et al. Acute lymphoblastic leukemia in a developing country: preliminary results of a nonrandomized clinical trial in El Salvador. J. Pediatr. Hematol. Oncol.
2000; 22: 495 - 501.
5. Brandalise SR, Odone V, Pereira WV, et al. Treatment results of
three consecutive Brazilian Cooperative Childhood ALL Protocol: GBTLI 80 – 82 and 85. Leukemia 1993; 7: 142 -145.
6. Brandalise SR. Prognostic Value of Day 8 peripheral blood response for children with acute lymphoblastic leukemia. NATO
ASI Series, 94. Gene Technology. Edited by Zander AR et al.
Springer-Verlag 1996, p. 421 - 28.
7. Brandalise SR, Viana, M, Pereira WV, et al. Dexametasone during induction, re-induction and maintenance pulses in low risk
ALL patients (Brazilian Cooperative ALL-93 Protocol - GBTLI
ALL-93. Journal Clinical Oncology, 41st ASCO Annual Meeting
Proceedings, Alexandria, v. 23, n. 16S, p. 810s. Jun. 2005. Orlando, FL. Suplemento. Ref. 8543.
8. Charlotte TC, Wollner N, Trippett T, et al. Pharmacologic
– Guided Trial of Sequential Methotrexate and Thioguanine in
children with advanced malignancies. J Clin Oncol 1994; 12:
1955 – 62.
9. Conter V, Rizari C, Sala A, et al. Leucemia linfoblástica aguda
infantil. In Tratado de oncologia Pediátrica, Edited by Sierrasesúmaga L et al. Pearson, 2006. Chapter 13, p. 268-273.
10. Felice MS, Zubizarreta PA, Alfaro EM, et al. Childhood acute
lymphoblastic leukemia: prognostic value of initial peripheral
blast count in good responders to prednisone. J Pediatric Hematol Oncol, 2001; 23 : 411 – 5.
11. Gaynon PS,Trigg ME, Heerema NA, et al. Children’s Cancer
Group trials in childhood acute lymphoblastic leukemia:19831995. Leukemia 2000; 14: 2223-33.
12. Goldberg JM, Silverman LB, Levy DE, et al: Childhood T-cell
acute lymphoblastic leukemia: The Dana-Farber Cancer Institute acute lymphoblastic leukemia consortium experience. J
Clin Oncol 2003; 21:3616-22.
13. Koizumi S, Fujimoto T, Takeda T, et al. Comparison of Intermittent or Continuous Methotrexate plus 6-Mercaptopurine in regimens for standard-risk acute lymphoblastic leukemia in childhood (JCCLSG – 5811). Cancer 1988; 61: 1292-1300.
14. Joannon P, Oviedo I, Campbell M, et al. High-dose methotrexate therapy of childhood acute lymphoblastic leukemia: lack of
relation between serum methotrexate concentration and creatine clearance. Pediatr Blood Cancer, 2004; 43: 17 – 22.
15. Masera G, Baez F, Marinoni M, et al. Pediatric HematologyOncology Centers in Low-Income and High-Income Countries:
Italy and Latin America. Am Soc Clin Oncol 2006; 6:543-47.
16. Papson NT, Cornbleet MA, Chessells JM, et al. Immunosupression and serious infections in children with acute lymphoblastic
leukemia: a comparison of three chemotherapy regimens. Br. J.
Haematol 1980; 45: 41-52.
17. Patte C, Ausperin A, Michon J, et al.The Societé Française
d’Oncologie Pediatrique LMB 89 protocol : highly effective
multiagent chemoterapy tailored to the tumor burden and initial
response in 561 unselected children with B-cell lymphomas and
L3 leukemia. Blood 2001; 97 : 3370-9.
18. Peto J, Eden OB, Lilleyman J and Richards S. Improvement in
treatment for children with acute lymphoblastic leukemia: The
Medical Research Council UKALL trials, 1972- 84, Lancet 1986,
1: 408-411.
19. Pui C-H, Chessells JM, Camitta B, et al. Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements. Leukemia 2003; 17: 700-6.
20. Pui CH, Sandlund JT, Pei D, et al. Improved outcome for children with acute lymphoblastic leukemia: results of Total Therapy Study XIII B at St Jude Children’s Research Hospital. Blood
2004; 104: 2690-6.
21. Pui CH and Evans WE. Treatment of Acute Lymphoblastic Leukemia. (Review article). N Engl J Med 2006; 354:166-78.
22. Rubnitz JE, Camitta BM, Mahmoud H, et al. Childhood acute
lymphoblastic leukemia with the MLL-ENL fusion and t(11,19)
(q23;p13.3) translocation.J Clin Oncol 1999; 1: 191-6.
23. Sackmann-Muriel F, Felice MS, Zubizarreta PA, et al. Treatment results in childhood acute lymphoblastic leukemia with a
modified ALL-BFM’90 protocol: lack of improvement in high-risk
group. Leukemia 1999; 23: 331 - 40.
24. Schrappe M, Reiter A, Riehm H , Zimmerman M, et al. Long
- term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Leukemia 2000; 14: 2205-22.
25. Silverman LB, Gelber RD, Dalton VK, et al. Improved outcome
for children with acute lymphoblastic leukemia: results of Dana
- Farber Consortium Protocol 91-01. Blood 2001; 97: 1211-8.
26. Sutcliffe MJ, Shuster JJ, Sather HN, et al. High concordance
from independent studies by the Children’s Cancer Group
(CCG) and Pediatric Oncology Group (POG) associating favorable prognosis with combined trisomies 4, 10, and 17 in children
with NCI standard-risk B-precursor acute lymphoblastic leukemia : a Children’s Oncology Group (COG) initiative. Leukemia
2005; 19: 734 - 40.
Front-line treatment of acute
promyelocytic leukemia (APL): The
PETHEMA protocol as a model for the ICAPL Project
Miguel A. Sanz
Hematology Service. University Hospital La Fe. Valencia. Spain.
Through the combination of all-trans retinoic acid (ATRA) and
chemotherapy, cure is now a reality for most patients with acute
promyelocytic leukemia (APL). In fact, several modern approaches
based on this combination have led to prolonged disease-free survival and potential cure for more than 80% of patients achieving
complete remission. The current consensus on the most appropriate induction therapy, once a diagnosis of APL has been confirmed
S95
at the genetic level, consists of the simultaneous administration of
ATRA and anthracycline-based chemotherapy.a,b,c The choice of anthracycline and whether it should be combined with other agents,
such as cytosine arabinoside, remain controversial. Exceptions
to the use of anthracycline-based induction regimens should be
considered only for individual patients in whom chemotherapy is
contraindicated. This is the case of patients with certain clinical
conditions such as severe organ failure, anticoagulant therapy, very
elderly patients, and others, in whom the toxicity of intensive chemotherapy is often unacceptable. For these cases, arsenic trioxide
(ATO) has recently emerged as a suitable alternative.d,e
Unlike induction therapy, there is not the same degree of consensus on the most appropriate consolidation therapy, except for
giving at least two or three cycles of anthracycline-based chemotherapy. Although the antileukemic benefit provided by the addition
of ATRA to consolidation therapy has not been demonstrated in randomized studies, historical comparisons of consecutive studies carried out separately by the GIMEMAf and PETHEMAg groups suggest
that the combination of ATRA and chemotherapy for consolidation
may also contribute to improving therapeutic results in APL. Another
interesting issue addressed in the aforementioned GIMEMA and
PETHEMA studies6,7 was the design of risk-adapted approaches to
administer distinct treatment intensities for consolidation based on
pre-defined risk of relapse.h According to these studies, this strategy seems a suitable approach to minimize therapy-related morbidity and mortality while maintaining the potential of cure for each
relapse-risk group. It is remarkable that both studies reported low
toxicity, high degree of compliance and high antileukemic efficacy
using ATRA combined with anthacycline monochemotherapy, especially in low- and intermediate-risk patients with APL.
Using monochemotherapy with anthracyclines for both induction and consolidation therapy, which led to a significant reduction in
treatment-related toxicity during the consolidation phase and a high
degree of compliance, the LPA96 study of the PETHEMA group
reported outcome results similar to those obtained in other major
studies using anthracycline-based chemotherapy combinations. In
November 1999, aiming to improve the antileukemic efficacy in patients with increased relapse risk, the PETHEMA started the trial
LPA99 based on a risk-adapted strategy. The results obtained in
the first 426 consecutive patients with newly diagnosed PML/RARα
positive APL who were enrolled in these two consecutive studies
(LPA96 and LPA99) were recently reported in Blood.7 This study,
which was recently updated including a significantly higher number
of patients and longer follow-up than in the first report,i shows that
combining ATRA with anthracycline monochemotherapy for induction and consolidation, followed by ATRA and low dose methotrexate
and mercaptopurine for maintenance therapy, results in extremely
high antileukemic efficacy, moderate toxicity and a high degree of
compliance in patients with APL. The novel addition of ATRA to consolidation therapy, combined with a moderate increase in the dose
of anthracycline for intermediate- and high-risk patients, resulted in
higher antileukemic activity with no additional severe toxicity.
Overall, 735 patients, ranging from 2 to 83 years of age, were
eligible for AIDA induction from November 1996 to June 2005. Remission induction rates were similar in both LPA96 and LPA99 trials,
89% and 91%, respectively. Induction failures were mainly due to
death during remission, confirming the virtual absence of drug resistance. It should be noted that the only 4 cases labeled as resistant
leukemia were evaluated too early for response, between the days
19 and 33 after completion chemotherapy. Today, it is well known
that a proportion of patients need up to 40 or 50 days to complete
terminal differentiation of blasts. The two major causes of failure
were bleeding and infection, accounting for around 5% and 3%,
respectively. No impact was observed in the mortality rate due to
hemorrhage according to the use of antifibrinolytic prophylaxis with
tranexamic acid nor in the morbidity and mortality rate associated
to the retinoic acid syndrome according to the use of prednisone
prophylaxis.
Regarding post-remission outcome of patients treated with the
currently ongoing risk-adapted protocol (LPA99), 2% of patients
died in remission. However, mortality rate in patients younger than
60 years was 0.6%, whereas it was significantly increased in elderly
patients (patients 60-70 years, 5.2%; patients older than 70 years,
19.2%). The 5-year disease-free (DFS) and relapse-free survival
S96
(RFS) is 89% and 91%, respectively, whereas cumulative incidence
of relapse (CIR) is 9%. These estimates still show significant differences according to WBC count. DFS and RFS at 5-years in patients
with less than 10 thousand leukocytes were 93% and 95%, respectively, with a CIR of 5%. In contrast, patients with more than 10
thousand leukocytes at presentation have a CIR at the same time
point of 22%. In conclusion, this updated analysis on a large series
of patients with APL confirms that a risk-adapted strategy combining
ATRA and anthracycline monochemotherapy provides a high antileukemic efficacy coupled with low toxicity and high degree of compliance. This improved antileukemic efficacy was certainly caused
by the modified consolidation therapy. Although it is unclear which
part of the reinforced consolidation therapy (ATRA or chemotherapy
or both) may have led to the impact observed in the outcome, it is
likely that the addition of ATRA has had a significant role. Based on
these results and those recently reported by the GIMEMA Group,6
we believe that the current consensus on the simultaneous administration of ATRA and chemotherapy for induction and maintenance
therapy of APL could be extended to the consolidation phase.
Once most of the objectives of the PETHEMA LPA99 study
were achieved, and based on the above outlined conclusions, a
new study (LPA2005) has been designed taking into account the
following considerations: (i) For induction therapy, no essentials
changes in the AIDA regimen have been made; (ii) For consolidation therapy, a risk-adapted strategy based on the combination of
ATRA and anthracycline monochemotherapy has been maintained
as backbone. Due to the low relapse rate observed in low- and intermediate-risk patients, only a slight reduction of mitoxantrone in
the second consolidation course and the addition of ATRA to the 3
consolidation courses have been proposed. However, the still unsatisfactory relapse rate observed in high-risk patients has induced
to reinforce consolidation chemotherapy with the addition of ara-C
to the idarubicin courses. This option was based on the results recently reported by the Italian GIMEMA group in high-risk patients
younger than 60 years;13 and (iii) Once demonstrated the benefit of
maintenance therapy in two randomized studies,5,9 no change has
been made for therapeutic phase.
An adapted version of the PETHEMA LPA2005 protocol, in
which idarubicin for induction and consolidation therapy has been
substituted by daunorubicin (equivalence, DNR 5 mg equal to IDA
1 mg), has been designed to be undertaken as a multinational mul-
ticenter non-randomized study in developing countries included
in the International Consortium of Acute Promyelocytic Leukemia
Project. The objectives, eligibility criteria, treatment and evaluation
criteria are unaltered, except for testing the efficacy and toxicity of
daunorubicin instead of idarubicin.
REFERENCES
1. Tallman MS, Nabhan Ch Feusner JH, Rowe JM. Acute promyelocytic leukaemia: evolving therapeutic strategies. Blood
2002;99;759-767.
2. Sanz MA, Martín G, Lo Coco F. Choice of chemotherapy in induction, consolidation and maintenance in acutepromyelocytic
leukemia. Baillieres Best Pract Res Clin Haematol 2003;16:43351.
3. Sanz MA. Treatment of acute promyelocytic leukemia. Hematology 2006; :147-155.
4. Sanz MA, Fenaux P, Lo-Coco on behalf of the European APL
Group of Experts. Arsenic trioxide in the treatment of acute promyelocytic leukemia. A review of current evidence. Haematologica 2005; 90:1231-1235.
5. Sanz MA, Lo-Coco F. Arsenic trioxide. Its use in the treatment of
acute promyelocytic leukemia. Am J Cancer 2006; 5:183-191.
6. Lo Coco F, Avvisati G, Vignetti M et al. Front-line treatment of
acute promyelocytic leukemia with AIDA induction followed by
risk-adapted consolidation: results of the AIDA-2000 trial of the
Italian GIMEMA group. Blood 2004;104:392[abstract].
7. Sanz MA, Martin G, Gonzalez M, et al. Risk-adapted treatment
of acute promyelocytic leukemia with all-trans retinoic acid and
anthracycline monochemotherapy: a multicenter study by the
PETHEMA Group. Blood. 2004; 104: 3490-3493.
8. Sanz MA, Lo Coco F, Martín G, et al. Definition of relapse risk
and role of non-anthracycline drugs for consolidation in patients
with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups. Blood 2000:96;12471253.
9. Risk-Adapted Treatment of Acute Promyelocytic Leukemia:
Updated Results of the Spanish PETHEMA LPA99 Trial Using ATRA and Anthracycline Monochemotherapy. J Clin Oncol
(Supp) 2005;23:563s[abstract].
S97
CONFERENCE
Bone Marrow Transplantation: A Simple Way to do it An Urgent Need in Developing Countries
Guillermo J. RUIZ-ARGÜELLES
Centro de Hematología y Medicina Interna. Clínica Ruiz de Puebla. Puebla, MEXICO.
David GOMEZ-ALMAGUER
Hospital Universitario de Monterrey. Universidad Autónoma de Nuevo León. Monterrey, Puebla, MEXICO
Guillermo J. RUIZ-DELGADO
Hospital Universitario de Monterrey. Universidad Autónoma de Nuevo León. Monterrey, Puebla, MEXICO
Non-myeloablative allogeneic stem cell transplantation (NST)
has been one of the most exciting developments in the treatment
of hematologic malignancies in the last years. In 1999, we elected
to employ in México a regimen to conduct NST, based in those
employed previously in Jerusalem (1), Houston (2) and Genoa (3),
introducing some changes with the main goal of decreasing the
cost of the procedure and in turn, making it available to a larger
number of patients. The salient changes of our approach are: Use
of cheapest and available drugs (fludarabine, busulfan and cyclophosphamide), tailored number of apheresis sessions, elimination
of prophylactic ganciclovir and intravenous IgG, outpatient conduction, reduced number of blood products transfusions and reduced
donor-lymphocyte infusions (4-5). The conditioning regimen consist of oral busulphan, 4 mg / Kg on days - 6 and - 5; intravenous
(i.v.) cyclophosphamide, 350 mg / m2 on days - 4, - 3 and - 2; i.v.
fludarabine, 30 mg / m2 on days -4, -3 and -2; oral cyclosporin A, 5
mg / Kg starting on day – 1 until day + 180 and i.v. methotrexate 5
mg / m2 delivered on days + 1, + 3, + 5 and + 11 (4-5).
a) Overall results: Using the “Mexican method” to conduct
NST, we have conducted over 300 allografts in patients with different diseases: Chronic myelogenous leukemia, acute myelogenous
leukemia, acute lymphoblastic leukemia, myelodysplasia, thalassemia major, relapsed Hodgkin´s disease, Blackfan-Diamond syndrome, adrenoleukodystrophy, Hunter´s syndrome, aplastic anemia
and several solid tumors. In the whole group, the median granulocyte recovery time to 0.5 x 109/L was 13 days, whereas the median
the preparative regimen is non-myeloablative, all these patents recovered endogenous hematopoiesis. Approximately 50% of the
allografted individuals developed acute graft versus host disease
(GVHD), and approiximately 30% developed chronic GVHD. The
median post-allograft overall survival (SV) has not been reached
and the 2000 day overall SV is 54%. The 100-day mortality is 16%
and the transplant-related mortality is 20%. In the whole group of
patients, the median cost of each NST was 18 000 USD (6-14), a
figure which contrasts with that informed from the United States of
America, where a bone marrow transplantation using conventional
allografting has a median cost of 300 000 USD (15).
b) Chronic myelogenous leukemia (CML): We published initially a paper of 21 CML patients in different phases of the disease,
alografted in two institutions in México (Centro de Hematología y
Medicina Interna de Puebla and Hospital Universitario de Monterrey); in this study we found a 750 day overall SV of 60% (10). Later
on, in a group of 24 CML patients in first chronic phase, recruited in
a collaborative Group (Latin-American Cooperative Onco Hematol-
ogy Group – LACOHG - ) with patients from six institutions located
in four Latin American countries (México, Venezuela, Brasil, and
Colombia), we obtained an 830-day disease free SV of 92% (16);
it was clear that the results were better allografting CML patients in
first chronic phase.
c) Acute myelogenous leukemia (AML): in a collaborative
study in three Mexican institutions: CHMI, HUM and Centro Médico
la Raza (CMR) of the Instituto Mexicano del Seguro Social, in a
group of 24 AML patients in different stages of the disease (first,
second and third remission) we found an 860-day SV of 66% (17).
These individuals were eligible for conventional allografting but
were given an NST using the “Mexican method” mainly for economic reasons. Later, on, analyzing separately the results in AML
according to the remission status, we found in another multicenter
study a 480-days SV of 50% for patients in first remission and 15%
for those in a second remission (18); accordingly, it seems to be
better to allograft patients with AML in first remission, but it is also
possible to rescue some AML patients when they have achieved a
second or further remission with this type of allografting.
d) Acute lymphoblastic leukemia (ALL): The results in this
disease have been dissapointing. In a group of 19 ALL patients
grafted in second remission or beyond in a single institution, we
found a 900 day overall SV of 22% with a median overall survival of
491 days; these data are similar to those obtained with allografting
after conventional conditioning and support the concept that malignant ALL cell “escape” from the graft versus leukemia effect which
is more clear and useful in other malignancies.
e) Aplastic anemia (AA): The “Mexican method” to conduct
non-ablative allografting has also been used to allograft patients
with severe AA. In a collaborative group of four Mexican institutions
Universitario de Monterrey, Centro Médico La Raza and Instituto
Nacional de Cancerología), 23 individuals with severe, refractory
aplastic anemia were allografted using peripheral blood stem cells
and our NST method; we found a 1500 day SV of 91% (19), a figure
which compares favorably with those published using other types of
conditioning regimens.
We have allografted also children and adolescents with the
“Mexican method” (20-21). Initially, it was considered that non-ablative conditioning should be offered only to aged or debilitated individuals, or with comorbidities; however it is clear that children are
the ones who suffer more the long-term consequences of the aggressive conventional preparative regimens. Based in this idea, we
were the first to conduct non-ablative allografting in children (20),
and we have found a very low prevalence of long-term complications with very adequate results, mainly in non-malignant hematological diseases). After our initial publication, other groups have also
engaged in allografting children using reduced-intensity preparative
regimens (22).
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With the method that we have employed, we have also grafted
cord blood cells; the long-term survival of patients allografted with
cord blood cells obtained from both domestic and foreign cord blood
banks was in our experience of 40% at 6 years (23-24). We have
also been able to rescue individuals with relapsed Hodgkin´s disease
and allografted small groups of patients with other diseases such as
multiple myeloma, myelodysplasia, chronic lymphocytic leukemia,
solid tumors, etc (4-5). Concerning the complications of the allografting procedures and given the low hematologic and extrahematologic
toxicity of the “Mexican” conditioning regimen, we have found that the
proportion of individuals who develop a nephrotic syndrome after the
allograft is considerably lower than that observed in patients given
other types of non-ablative conditioning (25). By the same token, the
transfusion requirements of the patients allografted with this method
are very low (26), and the complications stemming from cytomegalovirus reactivation are exceptional (27). Along the same line, the
100-day mortality is 16%, a figure which contrasts with that of conventional allografting than can go up to 50%. We have also analyzed
the significance of the HLA disparity between donor and receptor,
and we have found that it is safe to conduct non-ablative allografting
using our approach in individuals who have either an HLA identical
(6/6) or compatible (5/6) sibling donor (28). Having increased the
number of patients allografted for acute leukemia and their follow
up periods, we have found leukemic relapses: They have occurred
in 60% of patients with ALL and in 50% of patients with AML. In
a multicenter study (Centro de Hematología y Medicina Interna de
Puebla, Hospital Universitario de Monterrey and Centro Médico La
Raza) we have found that extramedullary relapses are more frequent in patients with AML than in those with lymphoid malignancies
and that bone marrow relapses are more frequent and aggressive
in patients allografted for lymphoid malignancies (29). Interestingly,
we have also found that leukemic relapses in the hematopoietic cells
of the donor are not unfrequent (30) and that this complication should
be analyzed with more detail to further gain insight into the leukemogenesis events.
More than 95% of the patients who have been allografted in
México and other developing countries using the “Mexican approach”
to conduct NST could not have afforded the cost of a conventional or
more expensive stem cell transplant. Prospective studies will define
if NST will eventually replace conventional stem cell grafting; however, very frequently in developing countries, the decision for a given
patient is not between offering either a conventional bone marrow
transplant or a NST; the decision has to be made between NST or no
other effective treatment. Because of its cost, NST could be considered as an early treatment option in countries where limited resources currently prevent usual allogeneic bone marrow transplantation;
role-definition and appropriate timing for this therapeutic approach in
patients are required. We are learning which malignancies are more
susceptible to the graft versus tumor effect, one of the main effects
of NST in addition to the replacement of the bone marrow cells, and
as a consequence, we are also learning in which malignancies NST
is more useful. The “Mexican approach” to conduct NST has been
shown to be effective for allografting individuals with malignant and
non-malignant conditions. Despite the fact that ours and most studies with reduced intensity conditioning have a relatively short follow
up, there is information which indicates that the procedure is related
with lower toxicities and a lower prevalence and severity of GVHD,
with a similar efficacy as that of conventional allografting. Since this
method is more feasible and affordable for patients and physicians
in developing countries, the number of allografts in these places has
increased substantially, as well as the publications related to bone
marrow transplantation stemming from places where this therapeutic maneuver was considered as unaffordable previous to the development of this technology (31).
Allografting with reduced intensity conditioning may be related
with several disadvantages such as mixed chimerism and relapse
of the malignancy, however. NST has resulted not only in the progress of knowledge, but also in the accessibility of many patients
to sophisticated therapeutic actions, in some cases, the only true
curative option for these individuals.
Figure 1.
Overall survival of the patients given a non-myeloablative stem
cell allotransplant using the “Mexican method”. CM, CP = Chronic
myelogenous leukemia in chronic phase; AA = aplastic anemia;
AML = acute myelogenous leukemia in second or further remission; CML = chronic myelogenous leukemia in all phases; ALL =
acute lymphoblastic leukemia.
REFERENCES:
1. Slavin S, Naparstek E, Nagler A, Ackerstein A, Kapelushnik J,
Or R.: Allogeneic cell therapy for relapsed leukemia after bone
marrow transplantation with donor peripheral blood lymphocytes. Exp Hematol. 1995;23:1553-62.
2. Giralt S, Estey E, Albitar M, van Besien K, Rondón G, Anderlini P, O´Brien S, Khouri I, Gajewski J, Mehra R, Claxton D,
Andersson B, Beran M, Przepiorka D, Koller C, Kornblau S,
Körbling M, Keating M, Kantarjian H, Champlin R.: Engraftment
of allogeneic hematopoietic progenitor cells with purine analogcontaining chemotherapy: Harnessing graft-versus-leukemia
without myeloablative therapy. Blood 1997; 89:4531-4536
3. Carella AM, Lerma E, Dejana A, Corsetti MT, Celesti L, Bruni R,
Benvenuto F, Figari O, Parodi C, Carlier P, Florio G, Lercari G,
Valbonesi M, Casarino L, De Stefano F, Geniram A, Venturino
M, Tedeschi L, Palmieri G, Piaggio G, Podesta M, Frassoni F,
Van Lint MT, Marmont AM, Bacigalupo A.: Engraftment of HLAmatched sibling hematopoietic stem cells after immunosuppressive conditioning regimen in patients with hematologic neoplasias. Haematologica 1998; 83:904-909
4. Ruiz-Argüelles GJ, Gómez-Almaguer D.: Breaking dogmata to
help patients: Non-myeloablative hematopoietic stem cell transplantation. Expert Opin Biol Ther 2004; 4: 1693-99.
5. Ruiz-Argüelles GJ.: The Mexican approach to conduct allogeneic stem cell transplantation: Braking dogmata and facing the
Matthew effect. Hematology 2005, 10 (Suppl 1):154-160.
6. Ruiz-Argüelles GJ, Ruiz-Argüelles A, Gómez-Almaguer D,
López-Martínez B, Abreu-Díaz G, Bravo G, Jaime-Pérez JC.:
Features of the engraftment of allogeneic hematopoietic stem
cells using reduced-intesity conditioning regimens. Leukemia
Lymph 2001, 42: 145-150.
7. Ruiz-Argüelles GJ, Gómez-Almaguer D, López-Martínez B,
Ponce-de-León S, Cantú-Rodríguez OG, Jaime-Pérez JC.: No
cytomegalovirus-related deaths after non-ablative stem cell allografts. Hematology 2002, 7:95-99.
Mexican schedule to conduct allogeneic stem cell transplanta-
9.
10.
11.
12.
13.
14.
15.
16)
17.
18.
19.
tion is related to a low risk of cytomegalovirus reactivation and
disease. Am J Hematol 2004; 75;200-204.
Ruiz-Argüelles GJ, López-Martínez B, Santellán-Olea MR,
Abreu-Díaz G, Reyes-Núñez V, Ruiz-Argüelles A, GarcésEisele J.: Follow up of hemopoietic chimerism in individuals
given allogeneic hemopoietic stem cell allografts using an immunosuppressive, non-myeloablative conditioning regimen:
A prospective study in a single instituition. Leukemia Lymph
2002, 43:1509-1511.
Ruiz-Argüelles GJ, Gómez-Almaguer D, López-Martínez B,
Cantú-Rodríguez OG, Jaime-Pérez JC, González-Llano O.:
Results of an allogeneic non-myeloablative stem cell transplantation program in patients with chronic myelogenous leukemia.
Haematologica 2002; 87: 894-896
Gómez-Almaguer D, Ruiz-Argüelles GJ, Tarín-Arzaga LC,
Ruiz-Argüelles GJ, López-Martínez B, Gómez-Rangel D, Estrada E, Marín-López A, Bravo-Hernández G, Hernández JM.:
Decreased transfusion requirements in patients given stem cell
allografts using a non-myeloablative conditioning regimen: A
single institution experience. Hematology 2003, 8: 151-154
Ruiz-Argüelles GJ, Morales-Toquero A, López-Martínez B,
Gómez-Almaguer D, Ruiz-Argüelles GJ, Ruiz-Argüelles A,
González-Llano O, Cantú OE, Hernández NE.: Hematopoietic stem cell allografts using a non-myeloablative conditioning
regimen can be safely performed on an outpatient basis. Bone
Marrow Transpl 2000; 25:131-133.
Thomas ED. Hematopoietic stem cell transplantation. Sci Am
1995; 272:38-47.
Ruiz-Argüelles GJ, Gómez-Almaguer D, Morales-Toquero A,
Gutiérrez-Aguirre CH, Vela-Ojeda J, García-Ruiz-Esparza MA,
Manzano C, Karduss A, Sumoza A, de-Souza C, Miranda E,
Giralt S; Latin American Cooperative Oncohematology Group.:
The early referral for reduced-intensity stem cell transplantation in patients with Ph1 (+) chronic myelogenous leukemia in
chronic phase in the imatinib era: Results of the Latin American
Cooperative Oncohematology Group (LACOHG) prospective,
multicenter study. Bone Marrow Transplant 2005;36:1043-7.
Ruiz-Argüelles GJ, Gómez-Almaguer D, Gómez Rangel JD,
Vela-Ojeda J, Cantú-Rodríguez OG, Jaime-Pérez JC, GonzálezLlano O, Herrera-Garza JL.: Allogeneic hematopoietic stem cell
transplantation with non-myeloablative conditioning in patients
with acute leukemia eligible for conventional allografting: A prospective study. Leukemia Lymphoma 2004; 45:1191-1195.
Gutiérrez-Aguirre CH, Cantú-Rodríguez OG, González-Llano
O, Salazar-Riojas R, Gonzalez-Maetinez O, Jaime-Pérez JC,
Morales-Toquero A, Tarín-Arzaga LC, Ruiz-Argüelles GJ, Gómez Almaguer D.: Non-myeloablative allogeneic hematopoietic
stem cell transplantation in patients with acute myelogenous
leukemia: The significance of the remission status. Biol Blood
Marrow Transpl 2005; 11 (Suppl 1):61-62.
Gómez-Almaguer D, Vela-Ojeda J, Jaime-Pérez JC, Guitiérrez-Aguirre CH, Cantú-Rodríguez OG, Sobrevilla-Calvo P, Rivas-Vera S, Gómez-Rangel JD, Ruiz-Argüelles GJ.: Allografting
in patients with severe aplastic anemia using peripheral blood
stem cells and a fludarabine-based conditoning regimen: The
Mexican Experience. Am J Hematol 2006, 81:157-161.
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21. Ruiz-Argüelles GJ, Morales-Toquero A, Gómez-Rangel JD,
López-Martínez B.: Trasplante de células hematopoyéticas
alogénicas en niños y adolescentes empleando esquema de
acondicionamiento no mieloablativo. Experiencia en una sola
institución. Bol Med Hosp Inf Mex 2005; 62: 88-95.
22. Spitzer TR: The expanding applications of non-myeloablative
stem cell transplantation. Pediatr Transplant 2003; 7:95-100
23. Ruiz-Argüelles GJ, Reyes-Núñez V, Garcés-Eisele J, Warwick
RM, McKenna L, Ruiz-Reyes G, Granados J, Mercado-Díaz
MA.: Acquired hemoglobin S trait in an adult patient with secondary acute myelogenous leukemia allografted with matched
unrelated umbilical cord blood cells using a non-ablative conditioning regimen. Haema 2005; 8: 492-496.
24. Mancías-Guerra C, Ruiz-Delgado GJ, Manzano C, DíazHernández MA, Tarín-Arzaga LC, González-Llano O, GómezAlmaguer D, Ruiz-Argüelles GJ.: Umbilical cord blood transplantation using non-myeloablative conditioning: The Mexican
experience. Hematology 2006, in the press
25. Ruiz-Argüelles GJ, Gómez-Almaguer D.: Nephrotic syndrome
after non-myeloablative stem cell transplantation. Brit J Haematol 2006, 132:801-802
disease. Am J Hematol 2004; 75;200-204
28. Ruiz-Argüelles GJ, López-Martínez B, Manzano C, GómezRangel JD, Lobato-Mendizábal E.: Significance of one human
leukocyte antigen mismatch on outcome of non-myeloablative
allogeneic stem cell transplantation from related donors using
the Mexican schedule. Bone Marrow Transpl 2005; 35:335339.
29. Ruiz-Argüelles GJ, Gómez-Almaguer D, Vela-Ojeda J, Morales-Toquero A, Gómez-Rangel JD, García-Ruiz-Esparza MA,
López-Martínez B, Cantú-Rodríguez OG, Gutiérrez-Aguirre
CH.: Extramedullary leukemic relapses following hematopoietic
stem cell transplantation with non-myeloablative conditioning.
Int J Hematol 2005, 82:262-265.
30. Ruiz-Argüelles GJ, Ruiz-Delgado GJ, Garcés-Eisele J, Ruiz-Argüelles A, Pérez-Romano B, Reyes-Núñez V.: Donor cell leukemia after non-myeloablative allogeneic stem cell transplantation: A single institution experience. Leukemia and Lymphoma
2006, in the press.
31. Ruiz-Argüelles GJ, Gómez-Almaguer D, Gómez-Morales E.:
Trasplante de células progenitoras hematopoyéticas. In Góngora-Biachi R. (editor) Hematología: Actualización 2004. Ediciones de la Agrupación Mexicana para el Estudio de la
Hematología A.C. Mérida, México. 2004. pp. 139-148.
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CONFERENCE
Cord blood transplantation in Japan: System, Finance and
Some Clinical Results.
Hidehiko Saito, Shunichi Kato, and Shigetaka Asano
Japan Cord Blood Bank Network
Cryopreserved umbilical cord blood from unrelated donors is
a useful source of hematopoietic stem cells. Unrelated cord blood
transplantation (uCBT) offers an alternative for patients who do not
have a donor in the family and bone marrow bank. uCBT has some
distinct advantages over bone marrow or peripheral blood stem cell
transplantation: no apparent risk for donor, rapid transplantation
without time-consuming donor coordination, and less-stringent HLA
requirements. The disadvantages of uCBT include increased infectious complications, delayed neutrophil and platelet recovery, and
high cost of collection and storage. We report here our experiences
on uCBT in Japan.
Japan Cord Blood Bank Network (JCBBN) was founded in
1999 on the initiative of the Ministry of Health, Labor and Welfare.
The network is now composed of 11 regional cord blood banks with
Japanese Red Cross central office serving as a headquarter. Using
uniform technical guidelines each bank collects, processes, HLAtypes, and stores cord bloods. All information regarding HLA types
and cell numbers is accumulated in a central inventory, where more
than 25,000 HLA-typed cryopreserved cord blood units containing
at least 6×108 cells are accessible to everyone on Internet. The online program is run in “first come-first serve” principle. The function
of JCBBN is coordination of safe, prompt, and impartial distribution
of cord blood units throughout Japan. We have estimated from HLA
diversity of Japanese population that a pool of 20,000 cord blood
units would be sufficient to serve at least one HLA-single locus mismached cord blood to 90% of population.
The Government annually allocates approximately 5 million
US$ to support JCBBN which then distributes the money to 11
regional banks according to the numbers of cord blood units preserved. Eleven regional banks are founded and managed by a variety of organizations; some are affiliated with Japanese Red Cross,
while others with University or NPO. The financial basis of regional
cord banks is not very stable, as Government grant is not sufficient
to support the activity. Thus, all banks are also dependent on donations and volunteer work.
As of August 31, 2006, 3218 uCBT have been conducted in
Japan. It is notable that this number accounts for more than half of
all uCBT performed in the world. Until 2001 the majority of uCBT
were performed in children. The number of uCBT in adults has
been rapidly increased since 2002 as a result of preservation of
cord blood units with increasing cell doses, and more than 80%
of uCBT were done in adults in 2005. Some results of uCBT procured by JCBBN are as follows. Our analysis of clinical outcomes
in 216 patients (median age: 6 years) with hematological malignancies showed that the overall survival rate at 3.5 years after transplantation was 32,6% (1). The Institute of Medical Science of the
University of Tokyo group compared the clinical outcomes of 45
adult patients who received uBMT and 68 patients who received
uCBT(2). Despite slow neutrophil and platelet recoveries, uCBT
group showed better treatment-related morbidity and disease-free
survival than uBMT group. A same group also reported an excellent
outcomes in 18 adult patients with acute myelogeneous leukemia
receiving uCBT; the 2-year probability of disease-free survival of
76% (3). More recent analysis by JCBBN revealed event-free survival of 64.0% and 45.7% for children with non-malignant (N=145)
and malignant disease (N=490), respectively.
The clinical outcomes in Japan as well as in other countries
showed that uCBT is a promising alternative for bone marrow or peripheral blood stem cell transplantation in both children and adults.
Our experience indicates the importance of an efficient national system for the collection, banking, distribution and use of cord blood for
patients with hematological malignancies.
REFERENCES
1. Nishihira H et al. The Japanese cord blood bank network experience with cord blood transplantation from unrelated donors for
hematological malignancies: an evaluation of graft-versus-host
disease prophylaxis. Brit J Haematol 120: 516-522, 2003.
2. Takahashi S, et al. Single institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood
104: 3813-3820, 2004.
3. Ooi J, et al. Unrelated cord blood transplantation after myeloablasive conditioning in patients over the age of 45 years. Br
J Haematol 126: 711-714, 2004.
S101
CONFERENCE
The pathogenesis of bone marrow failure:
Implications for treatment
E C Gordon-Smith and J C Marsh
St George’s, University of London
Bone marrow failure is defined by a primary deficiency of hemopoietic stem cell (HSC) function leading to loss of mature blood
cell production. The characteristic features of bone marrow failure,
best exemplified by acquired aplastic anemia (AA), are peripheral
blood pancytopenia with hypoplastic bone marrow, caused by absence of hemopoietic progenitor cells and their replacement by fat
cells.
PATHOGENESIS
The HSC in AA
The HSC population in the normal bone marrow produces
some 1011 cells daily with considerably greater proliferative capacity under stress. The HSC is defined by its ability to differentiate
into any lympho-hemopoietic cell lineage, by its high proliferative
capacity and its ability for self renewal. In clinical practice the HSC
is identified by the presence of the cell surface protein CD34 and
the absence of lineage specific markers. The population of cells so
identified is in fact highly heterogeneous. However, these markers
may be used to concentrate HSC from various sources and to purge
such sources of unwanted cells, either lymphocytes to avoid graft
versus host disease or the removal of malignant cells. Use CD34+
concentrates has been adopted in some centres for HSC transplantation, both allogeneic and autologous, though without really meaningful improvement in results. CD34+ lin- may be derived from the
bone marrow, the peripheral blood (usually after mobilisation with
G-CSF), umbilical cord blood (UCB) and fetal liver.
In AA the absolute number of CD34+ cells is reduced as well as
their proportion in remaining cells. Functionally they are abnormal
with a reduced proliferative capacity. The stroma in AA marrow is
only marginally disturbed and may be consequence of hemopoietic
loss rather than a cause. Recovery nevertheless may occur in AA,
particularly after immunosuppressive therapy (IS). A relatively small
number of surviving HSC may repopulate the marrow and produce
a normal peripheral blood count. In some cases it is possible to
show that the blood cells are derived from very few stem cells, oligoclonal hemopoiesis. It is also possible to demonstrate proliferative abnormalities in the recovered stem cell population. The clinical
consequence is the high risk of the emergence of abnormal clones,
which have a growth advantage over the AA stem cells (paroxysmal
nocturnal hemoglobinuria, PNH), and the risk of malignant mutations.
Etiology and epidemiology of AA
In Europe and the USA the incidence of AA is 1 –22 per million
per yearx. The incidence in Asia and probably Africa is 2 to 4 times
higherx. The reasons for the differences are not clear. Benzene is
a proven cause of AA but cases are rare since the introduction of
effective regulations on exposure. Pesticides have often been suggested but evidence is lacking. About 10% of AA follows an attack
of seronegative hepatitis though no infective agent has been identifiedx. A large number of therapeutic drugs have been implicated in
the cause of AA. The most widely quoted is chloramphenicol. The
incidence of AA following systemic exposure to chloramphenicol
was calculated to be about 1 in 64,000 an increased risk of some
15 fold. Even so the absolute risk is low and cases very uncommon
since the use of this very effective antibiotic has been limited. Gold
salts and non-steroidal anti- inflammatory drugs have an n increased
risk, as do antithyroid drugs and some drugs used in psychiatryx. No
mechanisms have been identified to account for the reasons these
drugs cause AA in a particular individual. There has been a delay of
about 2 – 4 months between exposure to the drug and the appearance of pancytopenia in those cases where exposure and onset
can be clearly defined, mostly patients exposed to gold salts. In
deciding whether a drug might be responsible for a particular case it
is important to confirm the drug was taken, that the time relationship
between exposure and onset makes the association likely and that
the drug has been implicated before. The importance lies in trying to
avoid re-exposure after recovery. The majority of AA is idiosyncratic
with no exposure history. When the severity of the AA is taken into
account there is no difference in natural history or response to treatment between idiosyncratic or other etiologies.
Immune processes in bone marrow failure
The success of treatment of AA with anti-lymphocyte globulin
(ALG) seems to confirm an autoimmune pathogenesis, first suggested by Georges Mathé. In vitro, removal of lymphocytes from
HSC culture may improve colony numbers in AA and addition reduce numbers in normal culture. Many other immune anomalies
have been reported in AA , reviewed by Youngx but none has been
a convincing pathogenetic marker and search for specific antigen
involvement unsuccessful. There is over representation of HLADR2 in patients with AA and in Japanese studies has been associated with a better response to cyclosporine x. In many patients who
respond to IS a small dose of cyclosporine is required to maintain
hemopoiesis. It is probable that the main cause of damage to HSC
in AA, or at least the perpetuation of damage, is an autoimmune
attack by cytotoxic lymphocytes, though the mechanism remains
unclear. Other autoimmune diseases are not more common in AA
patients or their families.
In hypoplastic myelodysplastic syndromes (MDS) similar immune anomalies to those of AA may be found and some patients
respond to ALG. There is obvious overlap between hypoplastic
MDS and AA which might indicate that immune mechanisms are
important in both syndromes.
Telomeres in Bone Marrow Failure
Telomeres are the region of highly repetitive DNA sequences
at the 3/ end of chromosomes which protect the chromosome from
loss of genetically functional DNA following replication. The telomere shortens with each replication. Cells which have many divisions over a lifetime, such as HSC, would run out of protective
telomere on the chromosomes if it were not for the action of telomerase, an enzyme which corrects telomere shorteneing. Telomerase is composed of two main parts, telomere reverse transcriptase
(TERT) which is the main active site and telomerase RNA component (TERC). Even with the action of telomerase the average length
of telomeres decreases with age.
In AA telomere length in blood cells is significantly less than
normal when age is taken into account. It may be that the abnormally short telomeres also contribute to the propensity to malignant
transformation. As the aplastic marrow recovers the telomeres
gradually return towards the age expected lenghth. In dyskeratosis congenita (DC), a congenital type of AA, telomere shortening
is more marked due to a constitutional failure of telomerase. The
defect arises from a number of different gene disorders which affect
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telomerase activity. The aplasia in DC usually develops in the 2nd or
3rd decade of life an is accompanied by other system abnormalities.
The most common discovered defect in the X linked DC is in the
gene encoding dyskerin (DKC). Dyskerin associatetes with TERC
and probably exerts its effect through telomerase deficient activity.
Other defects in autosomal inherited DC have involed TERT and
possibly TERC. The main importance of these observations for AA
is the absolute requirement to eliminate DC as a cause of aparrant
acquired disorder.
TREATMENT
The management of aplastic anemia (AA) has three main components. The first is to ensure that the diagnosis of acquired aplastic
anemia is correct and that there is no evidence for a genetic cause.
This phase includes the need to explain the protracted nature of the
disease and the possibility of complications. The second is to protect
and support patients from the consequences of pancytopenia and to
keep them alive so that the third stage, the introduction of treatment
designed to re-establish stem cell function, can be instituted. Support
is the key to successful management since recovery may occur even
after several years of apparently failed treatment.
The differential diagnosis includes myelodysplasia or acute
leukaemia with hypoplastic marrow, congenital aplastic anemias
including Fanconi anemia (FA) and DC. FA is excluded by determining chromosomal susceptibility of peripheral blood lymphocytes
to clastogenic agents (Di ethyl butane or mitomycin C). FA may
present in adult life without obvious somatic anomalies so the test
should be performed on all AA cases, at least up to the age of 40. FA
patients do not respond to IS and require modified conditioning for
HSC transplantation. DC may be X-linked or dominantly inherited
but evidence of the defect in parents or earlier generations may be
found only in systems other than haematopoietic, for example pulmonary fibrosis or osteoporosis. At present the diagnosis of DC can
only be made on clinical grounds though practical tests for telomere
length may become available in the future.
Once the diagnosis is established decisions on definitive treatment need to be taken promptly. First line treatment is determined
by the severity of the marrow failure, the age of the patient, the
availability of a suitable donor and the presence of co-morbidity.
Definitive treatment involves hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST).
HEMOPOIETIC STEM CELL TRANSPLANTATION
There is a strong relationship between age and outcome of
HSCT in AA. Children transplanted from HLA-identical siblings with
standard conditioning regimen have about 90% disease free survival, normal growth and development and normal fertility. Event free
survival falls to <50% in patients > 40years. Successful outcome
of transplant diminishes with matched unrelated donors and with
any degree of HLA mismatch. Many modifications of the standard
conditioning regimen with cyclophosphamide (CTX) have been
reported in attempts to improve outcome from alternative donors.
Published results are always encouraging but usually include small
numbers and the results of transplantation have improved more
or less steadily over the nearly 40years since the technique was
introduced into clinical practice by Donnell Thomas, Rainer Storb
and the Seattle groupx so that historical controls for small series are
unreliable. There are very few prospective studies.
HLA-matched Sibling Transplants
The current most widely used conditioning regimen for HLA
identical sibling BMT is cyclophosphamide (CY) 200mg/kg and ATG
with CSA and methotrexate as GVHD prophylaxis, although the
benefit of adding ATG has yet to be proven in a randomised study.
Long term overall survival is 80-90% although important differences
in survival exist according to patient age. Critical barriers to successful outcome remain, however, in particular chronic GVHD.
Graft rejection is still a problem for 5-15% of patients. Heavily
transfused patients are particularly at risk, emphasising the importance of early transplant before patients become sensitised from
multiple transfusions. G-CSF mobilised peripheral blood stem cells
(PBSC) have been used in many centres in an attempt to increase
the stem cell dose, to accelerate neutrophil and platelet recovery
and reduce graft rejection. However, a preliminary analysis of the
retrospective, combined CIBMTR/EBMT study comparing PBSC
with bone marrow as the source of stem cells for transplantation
in AA, showed no reduction in graft rejection using PBSC, more
chronic GVHD and worse outcome. Bone marrow remains the recommended source of stem cells for transplantation. Increased intensity of conditioning, with addition of fludarabine to CY/ATG, is
being evaluated as a new approach to overcoming graft rejection,
particularly in sensitised patients. The use of fludarabine-based
regimens warrants further evaluation in larger multi-centre studies,
and may also be more appropriate for older patients.
The incidence and severity of acute GVHD has decreased
with the introduction of cyclosporine and avoidance of irradiation.
However, chronic GVHD remains a problem, occurring in 25-40%
of patients and contributing to morbidity and mortality Risk factors
for chronic GVHD are acute GVHD, irradiation and increasing age.
The Seattle study also reported the unexpected finding that a marrow cell dose of > 3.4 x 108 nucleated cells/kg was also a risk factor
for chronic GVHD, but data on CD34 cell doses were not available.
In contrast, the use of anti-CD52 (Campath-1G and more recently
Campath-1H, Alemtuzumab) monoclonal antibodies results in a
low incidence of acute GVHD (14%) and virtually abolishes chronic
GVHD (4%). Although the overall graft rejection rate was 24%, using Campath pre-marrow infusion instead of both pre- and postinfusion, reduced rejection to 16% in a group of heavily sensitised
patients.
Matched Unrelated transplants
The recent CIBMTR retrospective study of severe AA patients
transplanted between 1988 and 1998 highlighted the poor outcome
(39% survival at 5 years) and high rates of graft rejection, GVHD
and infection, after MUD BMT. There was also no improvement in
outcome with time. In this study, HLA matching was determined
using only low resolution DNA typing for HLA-A, B, and DR loci.
In contrast, a Japanese study showed better survival (60%) for
HLA-A, B, DRB1 matched unrelated donor transplants, using high
resolution DNA typing techniques for matching. Attempts to reduce
graft rejection include the use of low dose TBI or a non-irradiation,
fludarabine-based regimen. In a more recent study from EBMT using fludarabine, low dose CY (1200mg/m2) and ATG, graft rejection was still a problem at 18%, particularly among older patients.
Although rates of acute and chronic GVHD were relatively low at
11 and 27%, respectively, and survival was 73% at 5 years. Other
approaches have been to use Alemtuzumab instead of ATG in an
otherwise similar regimen to EBMT , or G-CSF mobilised CD34+
selected PBSC .
Umbilical Cord Blood Transplants
A potential advantage of using umbilical cord blood (UCB) as
a source of stem cells for unrelated donor BMT in SAA is that HLA
mis-matching is better tolerated and so may be considered when
a fully matched marrow donor is not available. a good stem cell
dose is important to help maximise engraftment. The recent use of
sequential UCB transplants as a means of increasing the stem cell
dose for adults, has been successful in achieving a high engraftment rate in high risk MDS/AML. For individual patients, only one
of the two units engrafted long term. A higher CD3+ dose in the
engrafted unit led the authors to suggest that donor predominance
was immune mediated. In acquired AA, a recent study from China
demonstrated engraftment in 7 of 9 adults, with sustained mixed
chimerism. In four of the patients, two units of UCB were infused,
one of which engrafted each patient, as previously observed.
Immunosuppression
Immunosuppressive therapy for AA with anti-lymphocyte globulin (ALG) was introduced in 1977 following rabbit experiments by
Bruno Speck in Basle. ALG is prepared from a variety of human
lymphocyte cellular immunogens in different animal types, usually
horse or rabbit, by a number of companies. Anti-thymocyte globulin
(ATG) is the term used when the immunogen is mainly of thymocyte
cell or cell line origin. Preparations contain a “soup” of antibodies to
lymphocyte antigens and the dose varies between products. Addition of cyclosporin to ALG accelerates recovery of peripheral counts
and in some maintenance is cyclosporin dependent. About two
thirds of patients overall respond with partial or complete restitution
of blood counts following IST though continuing stem cell disorder
is identifiable even in the marrow of patients with long recovery of
normal counts. IST is effective at all ages though there is some decline in survival in older patients treatment may be successful even
in patients over 80.
Failure of IST requires a second line treatment. For patients
whose marrow function showed some response to IST, further
courses of ALG +/- additional IS agents are successful in some 5060% of cases, though response may be delayed. When there has
been no response at all to first line IST, further courses are unlikely
to produce improvement. For such patients, especially children and
young adults, HSCT with a degree of mismatch may be acceptable.
The observation that autologous reconstitution of marrow function
following a failed HSCT using high dose CTX led some groups to
advocate CTX, without stem cell transplant as rescue, for failed IST
or even first line. The disadvantage is the prolonged pancytopenia
and risk of infection that follows CTX.
RELAPSE AND COMPLICATIONS
Relapse, that is a return to transfusion dependence, occurs in
25-40% of patients treated with IST over the next 19 years. A return
to aplasia may follow a number of immunological stimuli such as
pregnancy or immunisation but is less common than the emergence
of abnormal cell clones. Paroxysmal nocturnal hemoglobinuria
(PNH) develops in about 20% of patients, though clinically irrelevant
populations of PNH cells may be found in most cases. AA and pancytopenia may develop in the course of PNH, a scenario which carries a poor prognosis. PNH clones may be transient or prolonged.
MDS with cytogenetic abnormalities develops in a further 10% or
so of patients and acute myeloid leukaemia in about 6%. The malignant transformation may take place in GPI- cells (a PNH clone)
or in the remaining GPI+ population. In AA with PNH clones, the
GPI- stem cells have a greater proliferative capacity than the GPI+
stem cells but less than that of GPI+ stem cells from normal donors,
indicating that the growth advantage which leads to the prevalence
of PNH in association with AA is a consequence of the damage to
the background hematopoiesis. Long term careful follow-up of AA
patients is a requirement for good management.
Conclusion
The outlook for patients with AA is now very good compared
with 40 years ago when HSCT was first used and 30 years ago
when IS therapy was introduced. However, there are still 1 in 3 or 4
patients who do not respond and much needs to be done. Insights
into pathogenesis are tantalising but so far have not informed treatment – rather the other way round!
S103
6
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8
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14
15
16
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•
•
•
REFERENCES
1
2
3
4
5
Heimpel H Epidemiology and etiology of aplastic anemia. In
Aplastic Anemia: Pathophysiology and treatment Ed Schrezenmeier H Bacigalupo A Cambridge University Press Cambridge
pp 97-116
Issaragrissil S Kaufman D Anderson T et al The epidemiology of
aplastic anemia in Thailand Blood 107: 1299-1307; 2006
Kaufman DW Kelly JP Levy M et al. Th drug etiology of agranulocytosis and aplastic anemia. Oxford University Press. New
York. 1991
Lu J Basu A Melenhorst et al Analysis of T-cell repertooire in
hepatitis associated aplastic anemia Blood 103: 4588-4593;
2004
Nakao S Takamatsu H Chuhjo T et al Identification of a specific
HLA class II haplotype strongly associated with susceptibility
to cyclosporine dependent aplastic anemia. Blood: 84: 42574261; (1994).
•
•
•
Young NS Hematopoietic cell destruction by immune mechanisms in acquired aplstic anemia. Semin Hematol 37: 3-14;
2000
Thomas ED, Storb R, Fefer A et al. Aplastic anaemia treated by
bone marrow transplantation. Lancet i: 284-289; 1972
Camitta BM, Thomas ED, Nathan DG et al Severe aplastic anemia: A prospective study of the effect of early marrow transplantation on acute mortality. Blood 48: 63-70; 1976
Speck B, Gluckman E, Haak HL and van Rood JJ Treatment of
aplastic anaemia by anti-lymphocyte globulin with and without
allogeneic bone marrow infusions. Lancet ii: 1145-1149; 1977
Lewis SM and Dacie JV The aplastic anaemia-paroxysmal nocturnal haemoglobinuria syndrome. British Journal of Haematology 13: 236-251; 1963
Marsh JC. Ball SE. Darbyshire P.et al British Committee for
Standards in Haematology. Guidelines for the diagnosis and
management of acquired aplastic anaemia. British Journal of
Haematology. 123:782-801, 2003
Marsh JC. Treatment of aplastic anaemia: first do no harm
Lancet. 356:1536-7, 2000
Marrone A. Walne A. Dokal I. Dyskeratosis congenita: telomerase, telomeres and anticipation Current Opinion in Genetics & Development. 15:249-57, 2005.
Dokal I. Dyskeratosis congenita in all its forms. British Journal of Haematology. 110:768-79, 2000.
Dokal I. Vulliamy T. Dyskeratosis congenita: its link to
telomerase and aplastic anaemia. Blood Reviews. 17:217-25,
2003.
Bacigalupo A, Brand R, Oneto R et al, Treatment of acquired
severe aplastic anaemia: bone marrow transplantation compared with immunosuppressive therapy – The European Group
for Blood and Marrow Transplantation experience. Seminars in
Hematology 37: 69-80; 2000.
Passweg JR. Perez WS. Eapen M.et al. Bone marrow transplants from mismatched related and unrelated donors
for severe aplastic anemia. Bone Marrow Transplantation.
37:641-9, 2006
Bacigalupo A. Locatelli F. Lanino E.et al. Fludarabine, cyclophosphamide and anti-thymocyte globulin for alternative
donor transplants in acquired severe aplastic anemia: a report from the EBMT-SAA Working Party. Bone Marrow Transplantation. 36:947-50, 2005.
Kahl C. Leisenring W. Deeg HJ. et al. Cyclophosphamide and
antithymocyte globulin as a conditioning regimen for allogeneic marrow transplantation in patients with aplastic
anaemia: a long-term follow-up. British Journal of Haematology. 130:747-51, 2005
Tichelli, A., Gratwohl, A., Nissen, C. & Speck, B. Late clonal
complications in severe aplastic anemia. Leukemia and Lymphoma, 12, 167-175, 1994
Socie G. Rosenfeld S. Frickhofen N. Gluckman E. Tichelli A.
Late clonal diseases of treated aplastic anemia. Seminars in
Hematology. 37:91-101, 2000
Tichelli A. Socie G. Marsh J. et al European Group for Blood
and Marrow Transplantation Severe Aplastic Anaemia Working
Party. Outcome of pregnancy and disease course among
women with aplastic anemia treated with immunosuppression. [Comment. Journal Article. Multicenter Study] Annals of
Internal Medicine. 137:164-72, 2002.
Gupta V. Gordon-Smith EC. Cook G. et al. A third course of
anti-thymocyte globulin in aplastic anaemia is only beneficial in previous responders. British Journal of Haematology.
129:110-7, 2005
S104
EDUCATION SESSION
Bleeding Disorders
Diagnosis of Congenital Coagulopathies in
Latin America
Arlette Ruiz-Sáez
Banco Municipal de Sangre, DC. Caracas, Venezuela
Von Willebrand disease (vWD) hemophilia A and B are the
most frequent inherited bleeding disorders, these include more
than 90% of all the congenital coagulopathies. The remaining group
of deficiencies considered as rare or recessively inherited coagulation disorders (RCD) include the hereditary defects of fibrinogen,
prothrombin, factors V, VII, X, XI, XIII and combined factor V and
VIII deficiencies and also these constitute an important group of
hemorrhagic coagulopathies.
The estimated prevalence for von Willebrand disease, based
on clinical symptoms, is 0.1%; for hemophilia 133 per million males
population while RCD overall population frequency is low. It is reported that homozygous deficiency of RCD varies from 1 in 300
000 for F VII to 1 in 5 millions for F XIII deficiency. The prevalence
of these disorders is influenced by the racial mix and frequency
of consanguineous marriages in the population, which could justify
its study in different regions. This is really important in Latin America (LA) where there is a very diverse population with many ethnic groups and different ancestries or races, the majority of which
are of Amerindian, African or European descent, or a mix of these.
Mestizos make up the majority of the population in several LatinAmerican countries.
The results of the 2004 WFH global survey show 16995 People
with Hemophilia (PWH) living in LA countries, but the proportion of
the estimated number of PWH identified in Latin-American countries varies from less than 10% up to almost 90%. In relation to other
coagulopathies, only 2358 vWD and 996 RCD patients have been
registered. Additionally, there are LA countries where diagnosis of
hemophilia and related disorders is based only on a clotting screening test.
On the other hand, recently there has been great interest in
establishing National and International registries of RCD. One of
these registries, the North American Registry reported a disproportionately large number of “Latinos” with factor II deficiencies (62%).
Data of the International Registry of Rare Bleeding Disorders available online at http://www.rbdd.org/join.html contain clinical and
therapeutic information of 277 individuals registered in 5 South and
1 Central America Centers. Patients were classified as with deficiency of FXIII 3.35%; FXI 31.77%; FX 17.69%; FVII 25.63%; FVVIII 6.5%; FV: 7.22%; FII 3.97% and as Afibrinogenemia 3.97%.
We could conclude that better registries are needed in order to
improve the knowledge of regional differences which may be important for the care and treatment of affected people and for designing
clinical trials for more effective and specific treatment strategies.
In relation to diagnosis, while most cases of hemophilia and
severe von Willebrand Disease (vWD) show a typical pattern of
clinical symptoms and mode of inheritance, some mild deficiencies and the RCD may present significant difficulties in diagnosis.
In this group, it has been reported 1.- A poor correlation between
phenotype and genotype. Some polymorphisms can influence the
phenotypic expression in F V, VII and XIII deficiencies 2.- Inter-indi-
vidual variation in bleeding phenotype amongst affected individuals.
Patients show varied clinical manifestations, different from the ones
observed in hemophilia, i.e. Epistaxis is frequent and thrombosis
has been reported in some cases. 3.- According to the laboratorial behavior, two phenotypes can be differentiated: Type I, characterized by concomitantly low levels of activity and antigen, as
the majority of factor V, XI and XIII deficient cases are, and Type
II characterized by a discrepancy between clotting activity and immunological assays antigen. In this last group, there are variants in
their response to different activators.
The quality of laboratory investigation is very important because patients can be diagnosed because of their clinical manifestations or through routine laboratory test or family studies. In this
regard, it is well known that the sensitivity of PT and APTT to the
presence of clotting factor deficiencies can vary between reagents
or assay systems, normal screening tests do not always exclude
the presence of mild deficiency states. Thus, laboratory diagnosis should include factor assays, vWF assays, inhibitor detection,
Internal Quality Control and External Quality schemes. Currently,
seven LA Centers have access to the IEQAS of the WFH.
Centers from Argentina, Mexico and Brazil have experience in diagnosis of gene defects in hemophilia and related disorders. Collaborative studies with other centers have improved the
understanding of genotype-phenotype relationship.
CLASSIFICATION OF CONGENITAL BLEEDING DISORDERS IN
VENEZUELA
We have analyzed the clinical history of patients referred to us
or cases evaluated during field visits programmed by our Center,
Centro Nacional de Hemofilia of the Banco Municipal de Sangre,
from Caracas, Capital city.
We have 2518 patients diagnosed. Distribution of inherited bleeding disorders is: Hemophilia: 1575 ( 62.6%); VWD 548
(21.7%) and 395 as RCD
(15.7%), out of these 128 (32.4%) were considered severe
deficiencies. The analysis of these cases is as follows:
Fibrinogen Deficiency:
In Afibrinogenemia it is well known that in spite of the infinitely
prolonged clotting assays the bleeding tendency is variable and it is
not more severe than in hemophilia. Also venous and arterial thrombosis has been reported, possibly related to an increase thrombin
generation. In the 5 cases of afibrinogenemia we have studied, we
confirmed the more common clinical features described such as
consanguinity in 84% of the cases, mucosal bleeding in 80%, umbilical cord bleeding in 60%, hemarthrosis 20% and one fatal intraabdominal bleeding. No thrombotic events have been observed so
far. In relation to the molecular defects, mutations tend to cluster in
the FGA gene. The most common mutation is a donor splice mutation in Intron 4(IVS4+1 G>T). In non-European patients the genetic
defects in FGG gene appear to be more frequent, but in two of our
mestizo cases it has been identified in the FGA gene..
We have identified 20 families with Dysfibrinogenemia, but only
7 patients from 4 families show a bleeding tendency, mostly mild. An
additional family showed a severe thrombotic phenotype. The variable clinical manifestations of these disorders could be observed
in our case of Hypo-Dysfibrinogenemia, which had a plasma fibrin-
ogen concentration of 45 mg/dl, but a severe bleeding tendency
characterized by very painful intra-osseous bleeding, splenic rupture and mucosal bleeding. Dysfibrinogenemic patients have been
studied for Grupo CLAHT investigators, cases have been reported
from Venezuela, Fibrinogen Caracas I to VI, Guarenas and La Victoria; Fibrinogen Lima and Fibrinogen Buenos Aires. Database of
fibrinogen mutations includes Fibrinogen Caracas I, II, V, VI, Guarenas, Fibrinogen Lima and Fibrinogen Puerto Rico (http://www.geht.
org/databaseang/fibrinogen).
Prothrombin Deficiency:.
Four cases are Type I or Hypoprothrombinemia, our 3 homozygous cases show severe bleeding manifestations: hemoperitoneum, hemarthroses and gastrointestinal bleeding in neonatal period. In hypoprothrombinemia the mutations described are inferred
to affect the folding or stability of the protein and have been identified in at least 16 cases. One missense mutation the Tyr44Cys
has been reported in a large Dutch family and recently it was also
identified in a Venezuelan one as Prothrombin Carora. In Type II
or Dysprothrombinemias the clinical picture is much more variable.
We identified an abnormal molecule in a small and isolated YukpaIrapa Amerindian tribe, We studied 146 individuals out of 6 hundred
(total population), we found that 7 individuals had factor II activity
~ 2%, for all methods employed, they showed a moderate bleeding tendency characterized by mucosal and post-trauma bleeding.
They were considered as homozygous. Additional 46 asymptomatic
cases with factor II between 15-53% were classified as heterozygous. In dysprothrombinemia 16 mutations have been described
that affect the catalytic function. A substitution of Gly548 to Ala has
been described as Prothrombin Perijá, and its homozygous expression in the Yukpa-Irapa sub-tribe population from Venezuela is very
high 4.9%.
In Puerto Rico prothrombin deficiency is the third most common coagulation factor deficiency. Four novel prothrombin mutations have been identified two of which were designated as Prothrombin Puerto Rico I Arg457Gln and Puerto Rico II Glu16Gln II.
Factor V Deficiency:
Five unrelated patients with F V plasma concentration < 2U/dl
showed a moderate to severe bleeding tendency. All have shown
hematomas, mucosal and oral bleeding during teething; a pleural
bleeding occurred in a newborn. A female patient presented recurrent hemarthrosis. Their bleeding time was normal. Two other
patients with factor V plasma level of 30% and 42% showed only
epistaxis and post-surgical bleeding respectively. Factor V deficiency have been reported in Perú, Mexico and in Puerto Rico
(Arg1002Stop mutation).
Combined FV and FVIII Deficiency:
In our center we have studied 20 cases from 14 families, with
plasma levels of factors V and VIII similarly diminished, that ranged
between 5% to 27%; around a fifth of them have had severe bleeding, the other 78% presented a rather moderate bleeding tendency.
One patient died due to HIV infection. Mutations have been identified in 4 families, 2 in the ER/Golgi protein LMNA1(ERGIC-53), (an
insG85-89 exon 1 in one family of Jewish ancestry, and delA270A735 exon 16, in a Venezuelan mestizo family). In the other two,
mutations in CFD2 gene on chromosome 2 have recently been
identified (20). Mutations have also been identified in an Argentinean patient.
Factor VII Deficiency (FVIID):
We have studied 71 affected individuals from 56 unrelated
families. As in other series, mucosal bleeding is the most frequent
symptom, observed in 89% of severe cases with FVII<3U/dl, hemarthroses and two episode of CNS bleeding were also observed in
this group. It is worth mentioning that one case with Factor VII less
than 3% was asymptomatic. All patients have a normal bleeding
time. Sixty four of our cases could be considered functional variants, with FVII antigen markedly higher than activity. In the international Registry of F VII Congenital Deficiency molecular defects
have been reported in 138 cases. Through a collaborative study
with Greifswald Group the molecular analysis of FVII gene in 23
Venezuelan families identified 16 different mutations in 2 homo-
S105
zygous and 12 compound heterozygous conditions. The mutation
Gly283Ser was found in four unrelated Type II patients and it was
associated to severe bleeding tendency as it has been reported in
other cases. Six cases carried the Arg304Gln mutation and showed
a mild phenotype. This mutation has been reported in other ethnic
groups and it is associated to different FVII levels, according to the
thromboplastin reagent used.
Factor VII deficiency was reported in 4.1% of 267 Brazilian patients registered in Campinas. Mutation Arg304Gln was the most
frequent genetic defect found..
Factor X Deficiency (FXD):
We have studied 45 patients from 36 unrelated families, 93%
of the cases were referred because of bleeding and 7% for an
abnormal coagulation test. The prevalence of this disorder in the
eastern part of the country is 8 times more frequent than in other
geographical areas. In 19 cases with FX levels ~2%, the 92% are
CRM neg or Type 1. We noted that 36% of our severe cases had
hemarthrosis and 100% referred mucosal bleeding, including menorrhagia, epistaxis and gum bleeding and hematuria. Two newborns had giant cephalhematoma and umbilical cord bleeding and 2
patients presented CNS hemorrhages. As in previous observations
some patients with FX levels between 20 to 41% have shown post
trauma or surgical related bleeding. All the relatives of the subjects
studied were asymptomatic (FX activity 41-80%).
Concerning the molecular defects, collaborative studies with
the Greifswald Factor X Deficiency Study Group has allowed the
analysis of 31 subjects from Venezuela and 20 from Costa Rica.
The high prevalence of the Gly(-20)Arg Mutation among the studied
Factor X families from Venezuela, as well as the Gly380Arg mutation in families from Costa Rica seems to be caused by a founder effect of the corresponding mutation in these regions. Mutation Gly(20)Arg have been previously reported as Factor X Santo Domingo
so future genetic studies in other LA countries could be useful. The
results also suggested that intracranial hemorrhage seems to be
associated with the mutation Gly380Arg while Gly (-20) Arg was
associated with hemarthrosis and menorrhagia.
Factor XI Deficiency:
This disorder has been reported mainly, but not exclusively, in
Jewish population. We have identified 133 individuals from 52 Venezuelan mestizo families. Nineteen patients with FXI levels <15U/dl
were classified as severe. The bleeding tendency is variable, depending on the type of mutation. We could confirm the described
characteristic clinical features: variable bleeding tendency poorly
correlated with the plasma factor level. Their clinical manifestations
ranged from a complete absence of symptoms to injury-related
bleeding that requires multiple transfusions. So, 52% of FXI severe
cases and 39% of the individuals with FXI between 18-53% were
bleeders. No spontaneous bleeding was observed. Blood products
were required in 41.2% of severe cases vs. 4.6 % of the mild ones.
Surgical procedures involving tissues with high content of plasminogen activators such as dental extractions, tonsillectomy, and urinary
and nasal surgery were associated with excessive bleeding. Association with low levels of vWF was observed in 2 cases and with
a mild hemophilia in one. We have not yet studied their molecular
structure, but 26 mutations that are found throughout the FXI gene,
have already been reported,.
Factor XIII Deficiency: Eight Venezuelan patients have been
classified as Type II or FXIII A Deficiency. Bleeding in CNS occurred
in 4 patients; one was fatal and one was recurrent, requiring prophylactic treatment every 3 weeks with FXIII concentrates. Umbilical
cord bleeding, wound healing impaired, delayed hemorrhages after
mild trauma and spontaneous bruising, have also been observed.
Consanguinity was present in 60%.
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S106
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Lefkowitz JB et al. J Thromb and Haemostasis, 2003, 1: 2381
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World Federation of Hemophilia Report on the Global Survey
2004, 2005
S107
COURSE
M Biology Symposium: Acute Myeloid Leukemia
THE MOLECULAR BIOLOGY OF APL: WHAT
CAN WE LEARN FROM A NEAR-CURED
DISEASE?
Robert Gallagher
An understanding of the molecular mechanisms involved in
APL has consistently been outpaced by advances at the clinical
level based on the empirical application of available therapeutics.
In 2007, 80% or more of newly-diagnosed APL cases can be cured
by state-of-the-art therapy, including combination therapy with anthracycline chemotherapy and all-trans retinoic acid (ATRA) without, and perhaps better, with arsenic trioxide (ATO). Despite this
molecular biology “lag”, APL has been an exceptional model for
advancing the understanding of molecular mechanisms involved in
various aspects of myeloid leukemia and, indeed, neoplasia more
generally. Important molecular discoveries continue to be made
related to the initiation and progression of the disease, response to
therapy, and basis of disease relapse.
The discoveries of the retinoic acid receptor-alpha (RARα)
gene in 1987 and the predominant fusion gene partner PML (for
ProMyelocytic Leukemia) in 1990, which together account for the
APL-specific 15;17 chromosome translocation, mark cardinal historical events for deciphering the molecular mechanisms involved
in APL (reviewed in Ref 1). In the interim, several additional fusion gene partners of RARα (generically, X-RARαs) have been
discovered with relatively minor variation of the APL phenotype, emphasizing that APL is fundamentally a disease of abnormal RARα.
Although each RARα fusion gene partner contributes some unique
features, all X-RARs function essentially as aberrant nuclear receptors that suppress the expression of genes modulated by normal
RARα at physiological ATRA concentrations (1-10 μM).
Important new information has recently emerged about the
detailed mechanisms by which PML-RARα subverts normal RARα
function on the non-rearranged allele---and beyond to further augment the transcriptional repressive state of nuclear chromatin. Normal RARα binds as a heterodimer with an RXR molecule to canonical sequence motifs in gene promoter regions called retinoic acid
response elements (RAREs), classically direct repeats of the hexamer AGGTCA separated by 2 or 5 nucleotides. In the absence of
ligand (ATRA), RARα recruits a complex of co-repressor proteins to
the RARE-containing gene promoter, which, most essentially, contain histone deacetylase (HDAC) activity that maintains the regional
chromatin in a reversibly repressed state. On ligand binding, there
is a configuration change in RARα that displaces the co-repressor
complex and recruits an alternative complex of co-activator proteins
that has histone acetylase (HAT) activity that restores the chromatin
to a transcriptionally open state. PML-RARα, in contrast to RARα,
binds to RAREs as homodimers, such that RXR, although present
in variable amounts, is not required for RARE interaction. It has
been recognized for several years that RARE-bound PML-RARα,
in fact, forms oligomeric complexes that recruit a much more dense
co-repressor complex than RARα. The assembly of these complexes is primarily dependent on the interaction of a domain in the
PML-region of PML-RARα, the coiled-coil region (analogous dimer-
ization interfaces are present in the other X-RARαs). This provides
a molecular explanation for the observation that the co-repressor
complex can be displaced and a co-activator complex recruited only
at higher, pharmacological ATRA concentrations (≥100 μM), which
can, then, overcome the differentiation block of APL cells. However, recent reports indicate that PML-RARα/co-repressor complex
uniquely interacts with other proteins that enhance methylation at
repressive chromatin sites in histone protein tails (histone H3 K9methyltransferases SUV39H1&2) and in gene promoter-region
DNA (DNA methyltransferases DNMT1&3a and methyl-CpG-binding protein MBD1)(reviewed in Ref 2). Additionally, PML-RARα has
been demonstrated to promiscuously bind to gene promoters that
contain only partial or aberrantly- spaced RAREs.3,4 This relaxed
DNA binding is likely related to the transcriptional activation of many
genes specifically by PML-RARα but not by RARα on exposure to
pharmacologic ATRA concentrations,4 possibly including a key APL
cell differentiation response gene CCAAT-enhancing binding protein-beta (C/EBPβ).5
The paradoxical flip-side to the requirement of PML-RARα for
the unusual sensitivity of APL cells to ATRA-induced differentiation
is documentation that PML-RARα is also an absolute requirement
for transformation of hematopoietic stem cells (HSCs), which has
been further localized to a sumoylation-dependent repression domain in the PML-region that recruits the repressor protein Daxx.3 In
murine model systems, it was recently demonstrated that the critical
function of this PML-region is the generation of RARα homodimers,
since it could be substituted by other appropriate recombinant constructs.3,6 In detail, these experiments supported the conclusion
that PML-RARα is not simply a double dominant-negative repressor but that it is a true gain-of-function mutation in which the altered
DNA binding specificity of PML-RARα is critical.3,6 In several mouse
models, a long latency period before the development of APL with
variable penetrance has been observed, indicating that PML-RARα
alone is insufficient and that secondary mutations are required. In
the early period, an increased self-renewal potential of HSCs but
virtually no other phenotypic alterations and very few gene expression differences from normal HSCs are detectable in PML-RARα
expressing HSCs,7 one of which may surprisingly but critically be
upregulation of the cyclin-dependent kinase inhibitor p21.(P.G.
Pelicci, 11th International Conference of Differentiation Therapy,
2006). In accord with the 2-hit hypothesis of leukemogenesis, the
transgenic introduction of an activated mutant receptor tyrosine kinase (RTK) effectively complemented PML-RARα to produce fullblown APL, suggesting that a single mutation in an RTK or a small
number of mutations in other growth-promoting genes may be sufficient for the genesis of APL.8 On the other hand, marked individual
heterogeneity in APL cell gene expression, as determined by microarray analysis, was observed in mice at the time of full-blown disease, suggesting diverse neoplastic progression,7 and in humans
at the time of disease presentation with selective downregulation
of DNA repair genes, additionally suggesting a predisposition to an
increased incidence of secondary mutations.4
Regardless of the genetic heterogeneity of APL, which requires further investigation, it is clear that PML-RARα expression
is required not only for the initiation but for the maintenance of APL.
This dependence of APL cells on PML-RARα makes it a premier example of a molecular pathway-addicted tumor cell that is highly vulnerable to targeted therapeutic attack.9 As indicated above, target-
S108
ed ATRA therapy attacks by activation of differentiation pathways,
initially by PML-RARα itself and, following its proteolytic destruction
after 12 to 24 hours, by liberated normal RARα. ATO attacks by
a complex, incompletely understood set of cellular reactions, but,
most essentially related to the unique hypersensitivity of APL cells
to ATO, this involves rapid degradation of PML-RARα via specific
targeting of the sumoylation-dependent Daxx-interactive repressor
domain of the PML-region.3 This results in apoptosis, mediated in
part through restoration of normal PML activity by p53-dependent
and –independent mechanisms, in cell growth inhibition, mediated
in part by restoration of the tumor growth factor-beta pathway, as
well as in differentiation, mediated in part through the liberation of
RARα and through mitogen-activated protein kinase-stimulated
phosphorylation of a nuclear co-repressor (see references in Ref
10).
While a source of vulnerability, PML-RARα is also a source
of resilience and adaptability in APL cells that manage to escape
therapeutic attack leading to disease relapse. Although it has
not been proven that APL cells continue to require the presence
of PML-RARα after relapse from ATRA and/or ATO therapy, this
is implied by the observations that it is always present at relapse
and that in 30% to 60% of cases it harbors mutations in the ligand
binding domain of the RARα-region.11 Additionally, the APL mutant-harboring subclone can emerge in the absence of selective
drug pressure, further implying that the mutant PML-RARα either
has acquired intrinsic properties that foster disease progression or
which has introduced a linked predisposition to acquire secondary
mutations with high autonomous selection capacity.12 These observations, together with studies performed with APL cells obtained
prior to treatment indicate that the PML-RARα fusion gene is the
molecular focus throughout the disease process and is likely the
most vulnerable point of therapeutic attack even after relapse and
the acquisition of targeted therapy resistance.
REFERENCES
1. Melnick A, Licht JD. Deconstructing a disease: RARα, its fusion
partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood. 1999;93:3167-3215.
2. Minucci S, Pelicci PG. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments of cancer. Nat Rev
Cancer. 2006;6:38-51.
3. Zhou J, Peres L, Honore N, Nasr R, Zhu J, de The H. Dimerization-induced corepressor binding and relaxed DNA-binding
specificity are critical for PML/RARA-induced immortalization.
Proc Natl Acad Sci USA. 2006;103:9238-9243.
4. Meani N, Minardi S, Sicciulli S, et al. Molecular signature of
retinoic acid treatment in acute promyelocytic leukemia. Oncogene. 2005;24:3358-3368.
5. Duprez EA, Koch H, Tenen DG. C/EBPbeta is an important target of PML/RARA during ATRA-induced differentiation of APL
cells. Blood. 2001;98:Abst 3462.
6. Sternsdorf T, Phan VT, Maunakea ML, et al. Forced retinoic acid
receptor α homodimers prime mice for APL-like leukemia. Cancer Cell. 2006;9:81-94.
7. Walter MJ, Park JS, Lau SKM, et al. Expression profiling
of murine acute promyelocytic leukemia cells reveals multiple model-dependent progression signatures. Mol Cel Biol.
2004;24:10882-10893.
8. Kelley LM, Kutok JL, Williams IR, et al. PML/RARα and FLT3ITD induce an APL-like disease in a mouse model. Proc Natl
Acad Sci USA. 2002;99:8283-8288.
9. Weinstein IB. Addiction to oncogenes--the Achilles heal of cancer. Science. 2002;297:63-64.
10. Joe YS, Jeong J-H, Yang S, et al. ATR, PML, and CHK2 play
a role in arsenic trioxide-induced apoptosis. J Biol Chem.
2006;281:28764-28771.
11. Zhou D-C, Kim S, Ding W, et al Frequent mutations in the ligand
binding domain of PML-RARα after multiple relapses of acute
promyelocytic leukemia: analysis from functional relationship to
response to all-trans retinoic acid and histone deacetylase inhibitors in vitro and in vivo. Blood 2002: 99:1356-1363.
12. Gallagher RE, Schachter-Tokarz EL, Zhou D-C, et al. Relapse
of acute promyelocytic leukemia with PML-RARα mutant subclones independent of proximate all-trans retinoic acid selection
pressure. Leukemia. 2006;20:556-562.
S109
COURSE
M Biology Symposium: Myeloproliferative syndromes
UNDERSTANDING THE MOLECULAR BASIS
OF MYELOPROLIFERATIVE DISORDERS:
GENOMIC APPROACH
Irene Larripa
The term myeloproliferative disorders (MPD) was first introduced by Dr. William Dameshek in 1951 (Dameshek, 1951) to include a spectrum of pathogenetically related disorders, such as:
chronic myeloid leukemia (CML), essential thrombocythemia (ET),
polycythemia vera (PV), myelofibrosis with myeloid metaplasia
(MMM) and diGuglielmo´s syndrome (now a day consider as an
erythroleukemia and excluded as a MPD). These entities are characterized by excessive production of blood cells by hematopoietic
precursors, where in addition to thrombotic and hemorrhagic complications leukemic transformation can occur.
Recently the World Health Organization (WHO) includes the
four classic MPD (CML, ET, PV, MMM) and in addition, chronic
neutrophilic leukemia (CNL), chronic eosinophilic leukemia (CEL),
hypereosinophilic syndrome (HES), systemic mast cell disease
(SMCD) and unclassified MPD (UMPD). Clonal hematopoyesis is
a key feature of these disorders. The lesion involved all myeloid
linages and, frequently, the B-cell linage are monoclonal, T cells,
however, are polyclonal (Spivak, 2004) (Liu, 2003). Hematopoietic
progenitor cells from the marrow or peripheral blood display altered
growth properties, proliferating in serum containing cultures in the
absence of exogenous hematopietic growth factors. Compared with
patients with CML, transformation to acute leukemia is far less common, especially in the absence of therapy with known mutagenic
agents (Kaushansky, 2005).
Currently MPD are entities molecularly characterized, this concept implies an invariable link to specific mutation that has been
shown to promote their growth-factor-independent cell proliferation
or cause the disease phenotype in animal models (Tefferi, 2006).
Within of MPD, CML was the first cancer to be invariably associated
with a cytogenetic alteration: the Philadelphia (Ph) chromosome
(Nowell, 1973). This marker represent a reciprocal chromosomal
translocation t(9;22) (q34;q11) (Rowley, 1973), this rearrangement
fuses the ABL gene from chromosome 9 with the BCR gene on
chromosome 22 (De Klein, 1982). The acquired somatic mutation
BCR/ABL is transcribed into a chimeric 8.5kb mRNA, that is translated into an oncoprotein BCR/ABL (p210) instead of the normal
ABL protein product (p145). BCR/ABL protein is a tyrosine kinase
that leads to both in vitro cell transformation and CML-like disease
in mice (Daley, 1990). The leukemogenic potential of p210 resides
in the fact that the normally regulated tyrosine kinase activity of ABL
protein is constitutively activated by the juxtaposition of BCR sequences, promoting dimerization of the oncoprotein. The two adjacent BCR/ABL molecules phosphorylate each other on tyrosine
residues in their kinase-activation loop (Mc Whirter, 1993).
Unlike CML, in which the t(9;22) characterizes almost all the
cases, chromosomal abnormalities are relatively rare in the MPD,
but a subset of CEL/HES show chromosomal translocations involving the PDGFRB gene at 5q33 or the FGFR1 gene at 8p11 or
PDGFRA gene at 4q12. Similar to the Ph chromosome these translocations result in the generation of in-frame fusion gene encoding activated forms of tyrosine kinases. The most common fusion
genes in these MPD cases are: ETV6/TEL-PDGFRB in the t(5;12)
(q33;p13), ZNF198-FGFR1 in the t(8;13)(p11;q12) and FIP1L1PDGFRA in del(4)(q12q12). The last one is a cryptic chromosomal
deletion of only 800kb, detected by FISH or RT-PCR. The list of
fusion partners of PDGFRB and FGFR1 is continuously expanding,
putting these genes among the oncogenes with the highest number
of fusion partners (De Keersmaecker, 2006), all of then produce
constitutively activated tyrosine kinases that promote proliferation
and survival pathways. In the remaining cases with MPD the presence of chromosomal abnormalities, are not a common event. Then
the search of candidate genes involved in the pathology is more difficult. However, studies based on proliferative defect and activation
pathways have demonstrated mutations in the c-KIT and JAK2 in
systemic mastocytosis and PV, ET, IM respectively
Systemic mastocytosis (SM) represent a clonal hematopoietic
stem cell disease with accumulation of mast cells in one or more
extracutaneous organs (intestine, spleen and/or bone marrow),
this disorder is molecularly heterogeneous. Cases with SM with
eosinophilia can present the fusion gene FIP1L1-PDGFRA, but in
the majority of cases has been linked to activating c-KIT mutations,
involving the catalytic kinase, yuxtamembrane or transmembrane
domains. KIT is a tyrosine kinase receptor codified by the protooncogene c-KIT, located in 4q12. Its ligand the stem cell factor is
essential for growth and survival of normal mast cells. Mutations
in human c-KIT at codons 560 (V560G) and 816 (D816V) of the
kinase domain, causes constitutive ligand-independent activation
and contribute to the abnormal proliferation and survival of the neoplastic cells (Longley, 1996) (Furitsu, 1993). The incidence of c-KIT
mutation present a high variation (30% -100%) depending of the
type of tissue and disease subtype. While kinase activity of wild
type KIT is inhibited by imatinib, this drug has no activity against the
D816V mutant (Akin, 2004), but second generation kinase inhibitor (PKC412 and AMN107) can overcome drug resistance (Gotlib,
2005) (von Bubnoff, 2005)
The biological behavior of PV, ET and IM (idiopathic myelofibrosis) is the presence of an erytroid progenitor growth independent
of exogenous erythropoietin (Epo). These endogenous erythroid
colonies have been used as an auxiliary diagnostic to distinguish
clonal vs secondary erythrocytosis. Epo is a primary growth factor in erythropoiesis, which prevents apoptosis and promotes cell
growth. Its receptor, Epo-R, is associated with JAK2 tyrosine kinase
(Witthuhn, 1993). Binding of Epo to Epo-R actives JAK2 and induce
activation of PI3K/Akt, STAT5 and ERKs necessary for growth and
expansion of erythroid progenitor cells. Four different laboratories
(Kralovics, 2005) (James, 2005) (Levine, 2005) (Baxter, 2005) have
recently described a single, clonal acquired point mutation on exon
14 of JAK2 kinase gene, located on chromosome 9p24. This point
mutation G>T at nucleotide 1849, leads to a valine to phenylalanine
substitution at codon 617 (V617F) in the JH2 (JAK2 homology-2)
or auto inhibitory, domain of JAK2. This change is observed in 65%
- 97% of PV patients, 23% - 57% of TE and 35% - 57% of IMF.
The variation in frequencies reported in the literature is most likely
due to technical differences and/or differences in the diagnostic
criteria (Bench, 2005). This mutation leads a constitutive tyrosine
phosphorylation activity that promotes cytokine hypersensitivity and
induces erythrocytosis in a mouse model (James, 2005), activating
STAT5, ERK/MAP kinase and PI3K kinase pathways (Pellaganti,
2003) with hypersensitivity to IL3, Epo and IGFI.
S110
Analysis of microarray permits to study the gene expression
profiling of different neoplasias, revealing the presence of molecularly distinct subgroups within a disease. Up to now there are few
papers using microarray en MPD. Pellagatti and Col have found
11 up regulated genes in PV that may represent a molecular signature for this disorder. Increase in the expression of protease
inhibitors, anti-apoptotic and survival factors. Studies performed
in CD34-derived megakaryocytic cells in ET vs healthy subjects
permitted to identify differentially expressed genes and disease
specific transcripts. The pro-apoptotic genes such as BAX, BNIP3
and BNIP3L were down-regulated; meanwhile IGI1-R, CFLAR and
SDF-1 were up regulated (Tenedini, 2004). Goerttler and col (Goettler, 2005) using cDNA arrays have defined a molecular signature
for PV composed of 64 genes, which correctly discriminated PV
from secondary erythropoiesis. In this paper the authors reported
over expression of the transcription factor NF-E2, which is over expressed 2 to 40 fold in megakaryocytic, erythroid and granulocytic
precursors of PV patients. The over expression of NF-E2 leads to
the development of erythropoietin-independent erythroid colonies.
The gene expression profiling can identify candidate genes involved
in the pathophysiology and generate a molecular signature to aid in
diagnosis of MPD.
Molecular and cytogenetic studies en MPD have demonstrated
that abnormalities in tyrosine kinase genes are a hallmark of this
group of pathologies. The knowledge of the genetic changes can
rapidly be translated into novel and more specific therapies. The
use of tyrosine kinase inhibitors has an important therapeutic consequence because they interact with specific etiologic targets
-
-
-
REFERENCES
-
-
-
Akin C., Fumo G., Yaruz A., et al. A novel form of mastocytosis
associated with a transmembrane c-kit mutation and response
to imatinib. Blood 103: 3222-3225, 2004.
Baxter E., Scott L., Campbell P., et al. Acquired mutation of
the tyrosine kinase Jack2 in human myeloproliferative disorders. Lancet 365: 1054 -1061, 2005.
Bench A., Pahl H. Chromosomal abnormalities and molecular
markers in myeloproliferative disorders. Semin Hematol 42: 196
– 205, 2005.
Daley G., Van Etten R., Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the
Philadelphia chromosome. Science 247: 824 – 830, 1990.
Dameshek W. Some especulations on the myeloproliferative
syndromes. Blood 6: 372 – 375, 1951.
De Keersmaecker K., Cools J. Chronic myeloproliferative disorders: a tyrosine kinase tale. Leukemia 20: 200- 205, 2006.
De Klein A., van Kessel A., Grosveld G., et al. A cellular oncogene is translocated to the Philadelphia chromosome in chronic
myelocytic leukemia . Nature 300: 765 – 767, 1982.
Furitsu T., Tsujimura T., Tono T., et al. Identification of mutations in the coding sequence of the protooncogene c-kit in a
human mast cell leukemia cell line causing ligand-independent
activation of c-kit product. J Clin Invest 92: 1736 – 1744, 1993.
Goettler P., Kreutz C., Donauer J., et al. Gene expression factor NF-E2. Br. J. Haematol 129: 138 – 150, 2005.
Gotlib J., Berube C., Growney J., et al. Activity of the tyrosine
kinase inhibitor PKC412 in a patient with mast cell leukemia
with the D816V KIT mutation. Blood 106: 2865 – 2870, 2005
James C., Ugo V., Le Couedic J., et al. A unique clonal Jak2
mutation leading to constitutive signaling causes polycythemia
vera. Nature 434: 1144-1148, 2005.
Kaushansky K. On the molecular origins of the chronic myeloproliferative disorders: it all makes sense. Blood 105: 4187
– 4190, 2005.
Kralovics R., Passamonti F., Buser A., et al. A gain of function
mutation of jak2 in myeloproliferative disorders. N Engl J Med
352: 1779 – 1790, 2005.
Levine R., Wadleigh M., Cools J., et al. Activating mutation in
the tyrosine kinase Jak2 in poltcythemia vera, essential thrombocythemia and myeloid metaplasia with myelofibrosis. Cancer
Cell 7: 387 – 397, 2005.
Liu E., Jelinek J., Pastore Y., et al. Discrimination of polycythemias and thrombocytoses by novel, simple, accurate clonal-
-
ity assays and comparison with PRV-1 expression and BFU-E
response to erythropoietin. Blood 101: 3294 – 3301, 2003.
Longley B., Tyrrell L., Lu S., et al. Somatic c-Kit activating
mutation in urticaria pigmentosa and aggressive mastocytosis:
establishment of clonality in a human mast cell neoplasm. Nat
Genet 12: 312 – 314, 1996.
McWhirter J., Galasso D., Wang J. A coiled-coil oligomerization domain of Bcr is essential for the transforming function of
Bcr/Abl oncoproteins. Mol Cell Biol 13: 7587 – 7595, 1993.
Nowell P & Hungerford D. A minute chromosome in human
chronic granulocytic leukemia . J Nat Cancer Inst 25: 85, 1960.
Pellaganti a., vetrie D., Langford C. Gene expression profiling
in polycithemia vera using cDNA microarray technology. Cancer
Res 63: 3940-3944, 2003.
Rowley J. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence
and Giemsa staining. Nature 243: 290 – 293, 1973.
Spivak J. The chronic myeloproliferative disorders: clonally and
clinical heterogeneity. Semin Hematol 41: 1-5, 2004.
Tefferi A., Gilliland G. Classification of chronic myeloid disorders: from Dameshek towards a semi-molecularm system.
Best Practice & Research Clinical Haematology 19: 365 – 385,
2006.
Tenedini E., Fagioli M., Vianelli N. Gene expression profiling
of normal and malignant CD34-derived megakaryocytic cells.
Blood 104: 3126 – 3135, 2004.
von Bubnoff N., Gorantla S., Kancha R., et al. The systemic
mastocytosis-specific activating cKit mutation D816V can be inhibited by the tyrosine kinase inhibitor AMN107. Leukemia 19:
1670 – 1671, 2005.
Witthuhn B., Quelle F., Silvennoinen O., et al. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
Cell 74: 227 – 236, 1993.
MONITORING RESPONSE TO IMATINIB
MESYLATE (IM) BY FLUORESCENCE IN
SITU HIBRIDIZATION (FISH) AND REALTIME QUANTITATIVE PCR (RQ-PCR) IN
CHRONIC MYELOID LEUKEMIA (CML)
PATIENTS (PTS) IN CHRONIC PHASE
(CP). EXPERIENCE OF ARGENTINA AND
URUGUAY.
Giere I A1,2, Pavlovsky C1 Lombardi M V1,2, Negri P1, Moiraghi B1,
Garcia J1, Pavlovsky M A1, Milone J1, Bengio R1, Campestri R1,
Labanca L1, Mur N1, Garcia Reinoso M F1,2, Via G1, Bono G1,2,
Pintos S1,2, Uriarte R3, Magariños A3, Martinez L3, Corrado C1,
Fernandez I1 and Pavlovsky S1.
1
CML Group, Argentina – 2 Dept. of Cytogenetic and Molecular
Biology, FUNDALEU, Buenos Aires, Argentina. 3CML Group,
Uruguay.
INTRODUCTION:
The degree of reduction of total leukemia cell mass by IM
treatment as measured by molecular response [MR] profile correlates with progression-free survival. Regular molecular monitoring
by RQ-PCR for bcr/abl transcript level detection is desirable and
should be considered into clinical practice.
PURPOSE:
MR assessment at 6 and 12 months after the first baseline
evaluation of residual Philadelphia(+), bcr/abl(+)by FISH and RQPCR methods in CP-CML IM Pts.
PATIENTS AND METHODS:
S111
A total of 177 1st CP-CML pts with CCyR imatinib treatment
(400mg/d) were studied since Nov. 2005. Pts were divided in two
groups according treatment: 85 cases (48%) as 1st line IM pts and
92 cases(52%) as 2nd line IM pts (previous IFN/Cytarabine treatment). The % pts according Sokal Index : Low risk, 72%; Intermediate risk, 19%; High risk, 9%. Complete cytogenetic response
(CCyR) was considered for 0%Ph(+) cells. For MR, a 4-log, 3-log
and 2-log reduction of bcr/abl transcript level from standardized
baseline value in untreated pts were defined as Complete (CMR),
Major (MaMR), and Minor (MiMR), respectively.
entered this study showed: 71% pts FISH (-) and 54% FISH(+) improved or maintained the molecular response. Of pts with CMR
or MaMR at baseline, 72% pts improved or maintained responses.
Similar rate was observed in the group with MiMR at baseline. Pts
FISH (+) and pts with no MR at baseline showed rising level of bcr/
abl transcripts in 46% and 66% cases, respectively. According Sokal Index, similar rates of better molecular outcomes were observed
in all risk levels. At this point, 42% of 1st line and 58% of 2nd line IM
pts achieved MaMR. Estimated % of all 1st line IM pts with CcyR
and MaMR are: 50 %, 32%, 50% and 25% by 1 yr, 2 yr, 3 yr or >4 yr
of IM treatment, respectively. For 2nd line IM pts the estimated rates
are: 75%, 45% , 40% and 49% by the same periods of treatment .
RESULTS:
CONCLUSION:
Baseline responses assessment in %Pts: 93% pts was
FISH(-) [0 % cells(+)]; 7% pts was FISH(+) [0,1 – 5% cells(+)],
CMR and MaMR: 40% ; MiMR: 49%, No MR:11%. CMR and
MaMR(>3LogRed) achievements were similar in pts with < 1yr and
> 2yrs of IM (42% Vs 47%). MR assessment at 6 moths once pts
This is an on going study. High rate (93%) of CcyR was observed in this study. Better molecular responses during minimal residual disease monitoring were observed in pts with CcyR. In order
to identify the late responders patients and the adverse profile to
disease progression further studies should be done.
S112
COURSE
Course on Hemapheresis
OBTAINING MONOCYTES FOR
PRODUCTION OF DENDRITIC CELLS BY
THE ELUTRA SYSTEM
Gunnar Kvalheim.
Department of Cellular Therapy, Rikshospitalet - Radiumhospialet
HF. University of Oslo, Norway.
In the present study we will report clinical experiences with
large-scale production of mRNA transfected monocyte-derived
DC used in phase I/II trials on patients with advanced malignant
melanoma and androgen resistant prostate cancer. 28 melanoma
patients and 23 prostate cancer patients have been included. The
apheresis product collected from Cobe Spectra contained a mean
value of 17.5% (5-34%) CD14+ cells. Further monocyte enrichment
was done by either immunomagnetic depletion (Isolex300i) or elutriation (Elutra). The immunomagnetic procedure which has until
recently been the standard method gave a mean purity of CD14+
cells of 51.7% (18-80%) with a yield of 67%. The elutriation procedure gave similar results both with regard to purity and yield. As
will be shown contamination of lymphocytes, NK-cells and granulocytes varied depending on enrichment techniques used. The
enriched monocytes were transferred to gas permeable VueLife
Teflon bags containing serum free medium (CellGro DC) supplemented with GM-CSF (2500U/ml) and IL-4 (1000U/ml) (Cellgenix,
Freiburg, Germany). Final cell concentration was 1x10E6 cells per
ml. After five days of incubation at 37˚C and 5% CO2, generated
immature DC’s were transfected with tumour-mRNA by electroporation. Following 2 days of culture in presence of IL-6 (1000U/ml),
TNF-α (10ng/ml), IL-1β (10ng/ml) and PgE2 (1ug/ml), cells show
DC morphology and characteristic high expression of CD40, CD80,
CD86 as well as HLA-DR with a variable expression of CD14 and
CD83. By introducing GM-CSF (2500U/ml) and IL-4 (1000U/ml) to
the maturation cocktail we found a dramatic change in CD14/CD83
ratio along with higher signals of CCR7, DC-SIGN and DC-LAMP.
The matured DC obtained represents a mean of 8.2% (1.9-19.6%)
of the monocytes cultured. Mean viability of DC was 95% (68-99%)
with a mean cell loss of 24% (20-34%). Mature DC’s were frozen
in aliquots at a cell concentration of 20x10E6, and stored in liquid
nitrogen until use. Freezing medium consisted of 10% DMSO and
50% HSA in CellGro DC medium. Thawing and washing of the frozen samples with PBS gave a mean viability of 86.6 % (56-98%). A
specific immune response against transfected DC’s was obtained in
22/41 evaluated patients (prostate cancer 12/19, melanoma 10/22).
In spite of that most patients included in the protocol had advanced
disease a clear correlation between immune response and survival
could be observed. We conclude that the Elutra System give high
yield and purity of monocytes and is the preferable method to use
for GMP production of mRNA transfected DCs. Recently we have
started a new project on adoptive T-cell therapy. Data will be presented showing that T-cell enrichment by the Elutra System follow
by ex vivo expansion of T-cells by CD3/CD28 beads and IL2 is also
very efficient method to obtain high numbers of activated T-cells for
clinical use.
LARGE VOLUME PERIPHERAL BLOOD
PROGENITOR CELL (PBPC) HARVESTS
FOR AUTOLOGOUS TRANSPLANTATION
IN PEDIATRIC PATIENTS WEIGHING LESS
THAN 15 Kg
Fernandez Sasso, D; Lopez, O; Dengra, C; Battaglia, L; Lucero,
G; Figueroa, M; Koziner, B.
Bone Marrow Transplant Unit, Instituto Argentino de Diagnóstico y
Tratamiento. Buenos Aires. Argentina.
1. ABSTRACT
PBPC are increasingly used in autologous transplantation,
mostly due to the achievement of a faster hematological recovery in
addition to a easier harvest. Processing of large blood volumes is
becoming common in pediatric patients in order to obtain adequate
numbers of CD34+ cells.
We carried out 48 procedures of large volume leukaphaeresis in 22 children with various malignancies weighing less than 15
kg. Adverse hemodynamic events were not observed in relation to
the harvest. However, a decrease in platelet count without clinical
repercussion was a common event. By obtaining an adequate number of progenitor cells, autologous transplantation could be carried
out without significant morbidity with satisfactory hematological recovery and duration of hospitalization.
2. INTRODUCTION
Autologous transplantation of PBPC is a potentially curative
procedure in the treatment of selected hematological and solid tumor malignancies [1] [2]. Until not long ago, hematopoietic progenitor cells were obtained by bone marrow aspirations. This procedure
was difficult to perform in pediatrics, since the patients underwent
multiple and bilateral bone punctures in the operating room, under
general anesthesia. Nowadays, the collection of PBPC by leukapheresis is replacing this practice [3][4].
At the Bone Marrow Transplant Unit (BMTU) of the Instituto Argentino de Diagnóstico y Tratamiento (IADT), collection of PBPC
in children is done through large volume leukapheresis (LVL). By
means of this technique, it is feasible and safe to obtain an adequate quantity of CD34+ cells by processing 3 or more volemias
from the patient. [5] [6]
This report details our experience with the use of this technique
of PBPC collection that permits the collection of a sufficient number
of PBPC without morbidity for the patient to be further used in autologous transplantation.
3. PATIENTS AND METHODS
Between May 2002 and May 2006, 48 collection procedures of
PBPC were carried out by means of the LVL technique in 22 pediatric patients, weighing less than 15 kg. (mean = 13 kg and range
= 9,2 to 15 kg). The clinical characteristics of the patients are sum-
marized in table 1. All the collections were carried out in order to be
used for autologous transplantation. The mean age was 2,2 years,
ranging from 0.7 to 4 years
The patients had the following diagnosis: neuroblastoma: 12,
medulloblastoma: 2, malignant germinal cancer: 1, astrocytoma: 1,
choroid plexus carcinoma: 2, anaplasic oligodendroglioma: 1, glioblastoma: 1; retinoblastoma: 2.
All patients that had bone marrow infiltration at some stage of
their clinical course had documented absence of residual disease
prior to the harvest of PBPC.
All patients were treated previously with chemotherapy according to the corresponding institutional protocol for the respective
diagnosis
a. Mobilization and placement of a vascular access
All patients were mobilized with G-CSF 10 μg/kg/day, which
was administrated subcutaneously throughout the 4 days preceding the collection. Twelve hours after the 4th administration of GCSF, the patients were hospitalized and underwent placement of
an Arrow 7 french double lumen central venous catheter (CVC) by
a vascular surgeon, in general in the subclavian veins. After the
patient came out from surgery, the procedure of aphaeresis began.
This central venous access, used for the collections, remained
through the course of transplantation for the administration of IV
medications, chemotherapy, transfusion of blood products and reinfusion of the PBPC .
b. Harvest of PBPC
Before LVL, the patient’s parents were asked to sign the informed consents authorizing the procedure, cryopreservation of the
material and further use for transplantation. Collections of PBPC
were carried out in the BMTU, supervised by a team of hemotherapists (Lopez, O and Figueroa, M; coauthors of this manuscript).
Parents stayed with the patients during the entire procedure. Only
one patient needed total sedation throughout the procedure due to
her neurological primary disease. The procedures were performed
on an ambulatory basis.
Collections were performed using a cellular separator of continuous flow COBE Spectra (Gambro BCT, Denver, CO, USA) version 6.1, CMN programmer (leukapheresis of mononuclear cells).
In all cases, the LVL was started at a speed of 10 ml/min which was
increased progressively to a maximum of 35 ml/min
c. Priming
The cellular separator was primed with 170 ml of red blood
cells, isogroup, Rh and phenotype matched white cell-depleted and
irradiated with 25 Gy, with the addition of human albumin 20% (50
ml) and normal saline solution in suitable quantities to carry the
same hematocrit from the separator to the patient.
d. Anticoagulant
ACD-A anticoagulant (citric acid, citrate of sodium, dextrose)was
used in 500 cc of solution with the addition of 3000 units of
heparin, which set the anticoagulant ratio to 26:1.
e. Determination of CD34+ cells
The determination of the percentage of CD34+ cells in the collected product was done in a sample taken out from the collection
bag after processing the second volemia. The counting of CD34+
cell by means of flow citometry was done following the guidelines of
the ISHAGE protocol [7].
f. Storage of CD34+ cells
During the period the patient received the regimen of high
dose conditioning chemotherapy, the collected product was stored
in liquid nitrogen at −196°C after progressive freezing using dimetilsulfoxide 10% in protein solution (albumin) as cryoprotector medium
g. Transplant
The patients received the previously criopreserved PBPC intravenously at least 48hs after completion of the conditioning chemotherapy regimen. The material infused had to be bacteriologically free of contamination. All patients received G-CSF at variable
S113
doses between 5-10 ug/kg/daily sc starting 4-7 days post transplant
until WBC recovery (neutrophils > 500/dl)
4. RESULTS
a. Assessment of leukaphaeresis parameters
All patients underwent LVL, defined as the process of collection of 3 or more volemias. The calculation of volemia was done by
entering the values of weight and height into the cellular separator
estimated at an average of 80 ml/kg.
The mean volume of processed blood was 4895 ml with a
range of 1910−8502 ml. The mean and range of volemia per patient was 1089 ml and 630−1464 ml, respectively. The mean and
range of processed volemias was 4.55 and 3−6.7, respectively. The
procedure lasted 190 min on the average, with a range of 124−261
min. The technical characteristics of the procedures are summarized in Table 2
The mean and range of apheresis sessions were 2 and 1−7
per patient, respectively. Sixteen patients (80%) required 1 or 2 procedures in order to reach a proper level of CD34+ cells.
Two patients (#16 and 22) were considered “poor mobilizers”
requiring discontinuation of the collection after 4 and 2 LVL, respectively. During the ensuing 3 weeks they received nutritional support
and supplements of hematinics. Prior to the repeat series of LVL,
they received G-CSF 16 ug/kg/sc/daily for 4 days which resulted
this time in the harvest of appropriate numbers of CD34+ cells.
b. Hematological parameters
All patients were controlled with hemograms before and after
each collection. The hemoglobin level increased after the collections, mostly due to the priming of the cellular separator with white
cell depleted red blood cells. At the start of the procedure,
the mean hemoglobin was 10.9 mg/dl and after it was 10,75 mg/
dl. Only one patient required RBC transfusion due to the development of a hemothorax after placement of a CVC. The platelet count
decreased after each collection. Patients started with a mean of
195,000 x mm3 platelets and ended with a mean of 74,000 x mm3
and a range of 32,000−219,000 platelets x mm3. Only 1 patient had
less than 35,000 x mm3 platelets at the end of the collection, with no
evidence of active bleeding.
c. CD34+
The mean number of CD34+ cells at the end of LVL was 4.72 x
106/kg, with a range of 2.33−7.85 x 106/kg.
d. Adverse events
In this series, only 1 patient complained of perioral paresthesias which reverted immediately and was not correlated with the
level of serum calcium. No patient showed vasovagal reactions.
One patient developed hemothorax following the placement of a
CVC, which did not require surgical measures and healed without
sequel .
e. Engraftment, transfusional needs and hospitalization
After receiving high dose chemotherapy, the patients underwent autologous transplantation of PBPC obtained by means of the
procedure of LVL. Patients received G-CSF at least 4 days after
infusion of PBPC. All of them achieved engraftment of neutrophils
≥ to 500/mm3 in peripheral blood) at a mean of 10,6 days postinfusion with a range of 9−13 days. The platelet count was ≥ to
20,000 x mm3 (with a variation of 48 hs from the last transfusion)
at a mean of 16,2 days post-transplantation of PBPC, with a range
of 8−37 days
All patients required transfusion of platelets from a single donor
by aphaeresis filtered and irradiated (mean = 4.5 units, range = 120 units).
Two patients did not need transfusion of red blood cells. Twenty out of 22 patients needed transfusions of a mean of 2.28 filtered
and irradiated units with a range of 1−8 units.
There was no mortality related to the transplantation procedure with mean and range of hospitalization days of 22.5 days and
18−42 , respectively.
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5. CONCLUSIONS
Since 1994 when Demeocq et al reported their experience in
low weight pediatric patients, [8] a large numbers of series argued
for the advantages of using PBPC instead of bone marrow cell suspension, including faster hematological recovery and less traumatic
and easy to perform procedures [9] [10] [11].
In pediatric patients, the procurement of PBPC has improved
considerably due to the use of LVL, a technique that requires the
processing of at least 3 volemias in a single session [12]. Different reports have claimed that this procedure courses with adverse
events, such as hypocalcaemia, vasovagal reactions and cytopenias requiring transfusion of blood products.
In the present series it was not required to check periodically
the level of calcium since symptomatic hypocalcemia due to citrate
was prevented by its combination with heparin.
Only one patient complained of perioral paresthesias but hypocalcemia was not confirmed in the laboratory. No vasovagal
episodes were observed, most likely due to the initial priming with
albumin and RBC.
Pain related to CVC placement was not significant, since catheters were implanted in the operation room under local anesthesia
and sedation. The only serious adverse event secondary to placement of CVC was hemothorax in one patient which resolved without
hemodynamic and/or ventilatory complications. This infrequently
reported event develops unrelated to type of catheter or apheresis
procedure used [13].
An event repeatedly mentioned in the medical literature is the
decrease in platelet count after collections. In our study, this decrease was less evident than commonly reported. We observed
a mean decrease of 63%, while in other studies, this value ranged
from a mean of 30 to 60%. Furthermore, this decrease in platelet
count was not clinically significant since it was not accompanied by
bleeding and did not require transfusion of blood products.
Unlike what has been described in other studies, our patients
completed their collections with similar hemoglobin values to the
starting level, mostly due to the priming of the cellular separator
with red blood cells , and except for the patient that developed hemothorax no other case required RBC transfusion.
In contrast with other reports that described mobilization protocols[14] [15] [16] using chemotherapy with G-CSF our patients only
received G-CSF, at 10ug/kg/qd x 4days.
The hematological recovery post transplantation assessed
by the time of achievement of neutrophil count over 500/mm3 and
platelets over 20,000/mm3 (without transfusion over 48hs) was similar to those described in other studies [5] [6] [7] [8] [9] [10]
In our pediatric population,, the use of LVL technique proved
being safe and effective. Patients did not develop adverse events
due to citrate, vasovagal effects nor apheresis-related complications.
REFERENCE
1. Urbano-Ispizua A, Schmitz N, de Witte T, Frassoni F, Rosti G,
Schrezenmeier H, Gluckman E, Friedrich W, European Group
for Blood and Marrow Transplantation. Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: definitions and current practice
in Europe. Bone Marrow Transplant. 2002 Apr;29(8):639-46
2. Hale GA; Autologous hematopoietic stem cell transplantation
for pediatric solid tumors. Expert Rev Anticancer Ther. 2005
Oct;5(5):835-46
3. William I. Bensinger, M.D., Paul J. Martin, M.D., Barry Storer,
Ph.D., Reginald Clift, F.I.M.L.S., Steven J. Forman, M.D., Robert Negrin, M.D., Ashwin
Kashyap, M.D., Mary E.D. Flowers, M.D., Kathy Lilleby, R.N.,
Thomas R. Chauncey, M.D., Rainer Storb, M.D., and Frederick
R. Appelbaum, M.D.
Transplantation of bone marrow as compared with peripheral blood
cells from HLA-identical relatives in patients with hematologic
cancers. NEJM 2001 January 18 Volume 344:175-1
4. Vicent MG, Madero L, Chamorro L, Madero R, Diaz MA Comparative cost analysis of autologous peripheral blood progenitor
cell and bone marrow transplantation in pediatric patients with
malignancies. Haematologica. 2001 Oct;86(10):1087-94
5. Sevilla J; Gonzalez-Vincent M; Large volume leukapheresis in
small children. Bone Marrow Transplant. 2003; 31,263-267
6. Kanold J, Halle P, Berger M, Rapatel C. Large-volume leukapheresis procedure for peripheral blood progenitor cell
collection in children weighing 15 kg or less: efficacy and
safety evaluation. Med Pediatr Oncol. 1999 Jan;32(1):7-10
7.
Serkes, S; Johnsen, HE. A European reference protocol
for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafos. Bone Marrow Transplant. 2001 Mar;27(5):463-70
8. Demeocq F, Kanold J,Chassagne J. Successful blood stem cell
collection and transplantation in children weighing less than 25
kg: a primer. Bone marrow transplan. 1994; 13: 43-50
9. Cecyn KZ, Seber A, Ginani VC, Goncalves AV, Caram EM,
Oguro T, Oliveira OM, Carvalho MM, Bordin JO. Large-volume
leukapheresis for peripheral blood progenitor cell collection in
low body weight pediatric patients: a single center experience.
Transfus Apher Sci. 2005 Jun;32(3):269-74.
10. Kanold J, Halle P, Berger M, Rapatel C, Palcoux JB, Rouzier
C, deLumley L, Vannier JP, Stephan JL, Demeocq F. Largevolume leukapheresis procedure for peripheral blood progenitor
cell collection in children weighing 15 kg or less: efficacy and
safety evaluation. Med Pediatr Oncol. 1999 Jan;32(1):7-10.
11. Diaz MA, Alegre A, Benito A, Villa M, Madero L. Peripheral
blood progenitor cell collection by large-volume leukapheresis
in low-weight children. J Hematother. 1998 Feb;7(1):63-8.
12. Gasova Z, Marinov I, Vodvarkova S, Bohmova M, Bhuyian-Ludvikova Z. PBPC collection techniques: standard versus large
volume leukapheresis (LVL) in donors and in patients. Transfus
Apher Sci. 2005 Apr;32(2):167-76.
13. Madero L, Ruano D, Villa M, Diaz MA. Non-tunneled catheters
in children undergoing bone marrow transplantation. Bone Marrow Transplant. 1996 Jan;17(1):87-9.
14. Watanabe H, Watanabe T, Suzuya H, Peripheral blood stem
cell mobilization by granulocyte colony-stimulating factor alone
and engraftment kinetics following autologous transplantation in
children and adolescents with solid tumor. Bone marrow Transplant. 2006 Apr; 37(7): 661-8
15. Diaz MA, Villa M, Alegre A, Lamana ML, de la Vega A, Granda
A, Madero L. Collection and transplantation of peripheral blood
progenitor cells mobilized by G-CSF alone in children with malignancies. Br J Haematol. 1996 Jul;94(1):148-54
16. Halle P, Kanold J, Rapatel C, Boiret N, Berger M, Stephan JL,
Albuisson E, Tournilhac O, Bonhomme J, Demeocq F. Granulocyte colony-stimulating factor alone at 20 micrograms/kg vs.
10 micrograms/kg for peripheral blood stem cell mobilization in
children. Pediatr Transplant. 2000 Nov;4(4):285-8
S115
TABLE 1 Clinical characteristics of the patients and myeloablative regimens
Pt
#
AGE(years)
WEIGHT (kg)
DIAGNOSIS
CONDITIONING REGIMEN
1
2
14
Neuroblastoma
Busulphan-Melphalan
2
3
13
Neuroblastoma
Carboplatinum-Etoposide-melphalan
3
2
13
Astrocytoma
Carboplatinum-Etoposide-Thiotepa
4
2
15
Choroid plexus carcinoma
5
2
13
Choroid plexus carcinoma
6
3
14
Medulloblastoma
7
3
14
Neuroblastoma
Busulphan-Melphalan
8
1
12
Neuroblastoma
Busulphan-Melphalan
9
2
14
Neuroblastoma
Busulphan-Melphalan
10
1
13
Oligodendroglioma
11
2
14
Germinal cancer
Carboplatinum-Etoposide-iphosphamide
12
1
9.8
Medulloblastoma
13
0.8
9.5
Glioblastoma
14
1
9.7
Neuroblastoma
Busulphan-Melphalan
15
3
15
Neuroblastoma
Busulphan-Melphalan
16
3
15
Neuroblastoma
Busulphan-Melphalan
17
0,9
10
Neuroblastoma
Busulphan-Melphalan
18
2
11,6
Retinoblastoma
19
3
14,8
Retinoblastoma
20
3
14
Neuroblastoma
Busulphan-Melphalan
21
2
11
Neuroblastoma
Busulphan-Melphalan
22
4
15
Neuroblastoma
Busulphan-Melphalan
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TABLE 2 Technical characteristics of the LVL procedures
Patient
#
APHAERESIS#
VOLEMIA(ml)
PROCESSED
VOLEMIAS
#
VOL. OF PROCESSED BLOOD
(ml)
FLOW
(ml/min)
4
950
3
1411
TIME
(min)
1
2
3,9
3667
22.5
154
3
4300
24
161
3
3
872
4
2
1100
4,1
3550
23.5
140
3,7
4100
18
261
5
2
910
5,5
5000
21
175
6
1
1330
5,2
6900
35
191
7
2
1410
6,0
8502
30
219
8
2
1199
4,9
5900
25
232
9
2
1330
5,0
6700
28
235
10
1
831
4,9
4100
30
140
11
2
950
5,8
5500
28
210
12
3
1057
4,5
4713
20
200
13
2
630
3,0
1910
12
153
14
1
1211
4,3
5200
24
216
15
1
993
4,6
4572
24
124
16
7
974
4,6
4513
16
207
17
1
1118
3,2
3550
18
165
18
1
839
4,7
3943
25
158
19
1
839
6,7
5600
25
182
20
2
1453
3,3
4825
19
226
21
1
1094
4,5
4950
24
205
22
4
1464
3,9
5708
23,5
240
TABLE 3 Parameters of hematological engrafment and hospitalization course
Pt
#
MNC yield
(x 108/kg)
CD34 x
106/kg
PLATELET
TRANSFUSION
#
RBCD TRANSFUSION
#
DAYS OF
HOSPITALIZATION
#
ENGRAFTMENT
> 500/mm3
(days)
RECOVERY OF
PLATELETS
>20,000/mm3
(days)
1
33,1
2,33
4
4
19
10
15
2
14,8
4,15
3
2
20
11
9
3
13,6
4,39
8
4
22
9
13
4
5,7
2,6
20
8
30
13
37
5
13,2
5,88
7
2
22
10
12
6
9,4
6,42
2
7
33
12
35
7
16,3
7,25
1
1
20
10
8
8
15,2
2,5
1
1
18
11
32
13
9
13
5,51
1
0
21
12
10
9,9
6,55
2
1
19
11
11
11
11,2
3,35
2
1
19
11
10
12
12,03
3,33
5
3
22
12
12
13
11,86
5,06
6
3
23
10
13
14
16,2
5,4
2
2
20
11
12
15
3,59
3,33
12
7
18
9
8
16
30,37
7,85
4
2
25
10
13
17
8,7
7,8
3
1
20
10
30
18
3,84
3,89
3
3
21
10
13
19
2,25
6,26
2
1
20
10
8
20
9,7
4,18
2
1
19
10
13
21
15,61
3,35
8
7
42
9
30
22
28,26
5,13
1
0
21
13
10
Stem cell collection in patients with
impaired mobilization of peripheral blood
progenitor cells.
Gunnar Kvalheim.
Department of Cellular Therapy, Rikshospitalet - Radiumhospialet
HF. University of Oslo, Norway.
High-dose chemotherapy (HDC) followed by autologous stem
cell transplantation (ASCT) is a standard treatment modality in
patients with lympoproliferative disorders, multiple myelomas and
some types of solid tumours. Mobilized peripheral blood progenitor
cells (PBPC) have become the main source for ASCT because they
give a faster haematological recovery and decrease both toxicity
and costs when compared to bone marrow (BM) stem cells. Mobilization of PBPC can be performed with G-CSF alone, or combined
with chemotherapy and G-CSF. One of the limitation of PBPC transplantation is that nearly 10-30% of patients are difficult to mobilize
or fail progenitor cell mobilization (Aurlien et al.1989 and 2001).
Factors influencing mobilization efficacy include disease characteristics and previous treatments (Bensinger, et al 1995, Haas, et
al 1994, Ketterer, et al 1998). There are no standard strategy for
patients who fail to mobilize an adequate quantity of PBPC after
G-CSF or G-CSF/chemotherapy (Stiff 1999), and the management
of these patients is critical because ASCT could cure the disease.
Different strategies have been tested in this type of patients. Some
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are suggesting a re-mobilization with high-dose G-CSF (Kobbe, et
al 1999). Others are proposing that the patients should have a rest
without any chemotherapy of 3-6 months before trying a new PBPC
mobilization (Watts, et al 2000). However, whatever being proposed
the general impression is that the majority of patients still fail to
obtain a sufficient numbers of PBPC mobilised. Some investigators
suggest that the use of G-CSF-primed BM may induce sustained
hematopoietic recovery in poor mobilisers (Lemoli, et al 2003),
while others have shown that the collection of large volume BM give
little or no advantage when compared with a standard bone marrow
harvest. (Watts, et al 1998). Recently a new agent, a CXCR4 antagonist, appears to enhance mobilization in poor mobilizers. If this
hold trough in a larger group of patients we are looking forward to
get access to this drug in the clinic (McGuirk, et al 2005).
It has previously been shown that ex vivo expansion of a small
volume of BM (80ml) could provide a sufficient dose of cells to assure haematopoietic reconstitution in patients who received high
dose therapy for breast cancer (Stiff, et al 2000). However, the haematological recovery was delayed in these patients compared to
what is observed after infusion of adequate doses of PBPC. When
small volume of ex vivo expanded BM were combined with suboptimal doses of PBPC a fast and substained engraftment was observed (Engelhardt, et al 2001, Pecora, et al 2001). Most of the
patients in this study were not defined as poor mobilisers. Recently
our centre has been involved in a multicentre study including only
poor mobilisers. In this study suboptimal doses of PBPC collected
over 3 days of leucapheresis and low volume ex vivo expanded BM
were used. The study is closed and our clinical experiences will be
presented.
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COURSE
Clinical Pathology (SUPAC)
MULTIPLE MYELOMA
Marina Narbaitz
Instituto de Investigaciones Hematológicas - Academia Nacional
de Medicina
Multiple myeloma (MM) is a neoplasm characterized by the accumulation of plasmablasts (PBs)/ plasma cell (PCs) in the bone
marrow and extraosseous tissues in a multifocal fashion. It represents 15% of all hematopoietic malignancies and accounts for
nearly 2% of deaths from cancer.
The diagnosis is based on a combination of clinical, pathologic and radiologic findings. The median age at diagnosis is 65-70
years.
Despite recent advances MM continues to be an incurable
plasma cell malignancy with a median survival of 3-4 years. Often it
is preceded by a premalignant tumor called monoclonal gammopathy of undetermined significance (MGUS).
The morphologic characteristics of plasma cells range from
mature-appearing cell to those resembling blasts. The pattern of
the plasma cell infiltrate in trephine biopsy specimens is interstitial,
focal, or diffuse.
Several studies have shown that a majority of MM tumors
have a translocation that non-randomly involves one of many potential chromosome partners. The prevalence of IgH translocations
is about 50%, whereas the prevalence of IgL translocations is no
more than 10-20%. About 40% of MM tumors have Ig translocations involving 5 recurrent chromosomal partners and oncogenes:
11p13 (cyclin D1)(15-20%); 4p16(FGFR3 y MMSET); 6p21 (cyclin
D3); 16q23(c-maf) y 20q11(mafB). Recurrent translocations appear
to be mediated mostly by errors in IgH switch recombination that
occur during the maturation of B cells in germinal center.
Chromosome content appears to identify 2 different, but perhaps overlapping, pathways of pathogenesis: a) nonhyperdiploid
tumors with a very high incidence of IgH translocations involving
the 5 recurrent parteners and a relatively high incidence of chromosome 13/13q14 loss; and b) hyperdiploid tumors, that include
about 50% of MM tumors, often have multiple trisomies involving
chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 and a substantially lower
prevalence of IgH translocations and monosomy of chromosome
13/13q14 compared with nonhyperdiploid tumors.
In addition to tumor mass and secondary features that represent a host response to MM, intrinsic properties of the tumor cell are
also informative in predicting prognosis and response to existing
therapies. It has been well documented that an unfavorable outcome is associated with each of the following: increased plasma cell
labeling index, tumor cells with abnormal karyopype, hypoploydia
compared with hyperdiploidia, monosomy of chromosome 13/13q,
monosomy of chromosome 17/deletion of p53 and activating mutations of K-Ras. More recently, it has become clear that specific
IgH translocations also have a profound prognostic significance. In
particular, patients with tumors that have a t (4;14) translocation
have a substantially shortened survival and patients with a t(14;16)
have a similarly poor if not worse prognosis. By contrast, patients
with tumors that have a t(11;14) translocation appear to have a better survival.
MIELODISPLADISC SYNDROME AND SMD/
SMP UNCLASIFAIED
Marina Narbaitz
Instituto de Investigaciones Hematológicas - Academia Nacional
de Medicina
MYELODISPLASTIC SYNDROME (MDS)
DEFINITION
The first and most commonly accepted definition of MDS is
ineffective hematopoiesis with sustained or prolonged cytopenias
with paradoxical hypercelullar bone marrow.
Myelodisplastic syndromes (MDS) are clonal stem cell disorders characterized by single or multilineage dysplasia, cytopenias,
in which bone marrow cannot produce blood cells effectively. The
blasts increase in variable number.
GENETICS
The genetic defects are unbalanced numeric abnormalities
that are associated with unmasking of oncogenes or inactivation/
deletion tumor suppressor genes, acquired defective DNA repair
mechanisms and intrinsic genetic instability and may also play a
role in oncogenesis. These studies have a major role in the evaluations and prognosis of MDS.
The most common abnormalities including chromosome 5q
and de novo 5q- syndrome are recognized as specific types of
MDS. There are other abnormalities related with chromosome 7,
17, 20 and 3.
EPIDEMIOLOGY
MDS can occur in patients of all ages, but more frequently in
elderly adults, and this is primarily (de novo) or secondarily occupational, environmental, and iatrogenic exposures.
CLASSIFICATION
The first and most utilized classification was the French-American-British (FAB) system which has been used since 1982, and is
useful in predicting rates of survival and transformation to AML. In
2000, the World Health Organization (WHO) published its classification of hematopoietic and lymphoid neoplasms, this included
the new concept to differentiate these disorders in clinicopathologis
entities. This included one special group to processes with overlapping MDS and MPD features
World Health Organization (WHO) Myelodysplastic Syndromes
RA
RARS
RCMD & RCMD-RS
RAEB-1 & RAEB-2
MDS Unclassified
MDS del (5q)
When the percentage of blast in BM is more than 20% is considered acute leukemia.
MORPHOLOGY
The blood changes are: single or multilineage cytopenias; leftward shift with myeloblast (< 20%) ; single or multilineage dyspoyesis; neutrophils with hipogranular cytoplasm and nuclear segmentation abnormalities; erytrhocyte dispoyesis with nucleated form;
platelet enlarged and hypogranulated abnormalities; immature
monocytosis; eosinophilia with dispoyesis.
The bone marrow abnormalities depend on the type of MDS. In
general there is hipercelularity, increased blast (< 20%),and erytroid
elements, increased dysplastic clustered megakariocyted, apoptotic
figures, ring sideroblasts and iron granules in erythroide cells.
The marrow biopsies have demonstrated that some MDS patients had clusters of blast cells in central marrow regions, rather
than being normally paratrabecular, referred to as abnormal localization of immature myeloid precursors (ALIP). Patients with these
morphologic findings had significantly shorter survival in all subtypes of MDS.
MYELODISPLASTIC/MYELOPROLIFERATIVE DISEASES
In clinical practice, it is recognized that some cases do not
distinguish between the two diseases, and these disorders exhibit
features intermediate between myelodisplastic and the more indolent CMPD. This is a difficult and the grey zone that represents a
clinicalpathological challenge.
In MDS, the stem cells do not mature into healthy with or red
blood cells or platelets. The immature blood cells, do not function
the way they should and either die in the bone marrow or shortly
after entering the blood, As a result, there are fewer healthy red
blood cells, white blood cells, and platelets.
In myeloproliferative diseases, a larger than normal level of
stem cells develop into one or more types of blood cells, resulting in
a gradual increase in the total number of blood cells.
When a MDS/MPS does not match any of these types, it is
called unclassifiable myelodysplastic/myeloproliferative disease.
These disorders are included in the WHO classification in the
category of MDS/MPS
and the definition is “clonal haematopoyetic neoplasm that
at the time of initial presentation have some clinical, laboratory, or
morphology findings that might support diagnosis with of a MDS
and other findings that are more consistent with chronic myeloproliferative disease (CMPD)”.
The BM is hypercelular due to proliferation of one or more myeloide lineages, and this proliferation may or may not be effective at
showing MDS or CMPD features. And the consequences are one
effective line and the all others infected with cytopenias.
PATHOGENESIS
In this disorder there could be abnormalities in the regulation of
the myieloide pathways for cellular proliferations, maturations, and
survival. And recurring chromosomal and molecular abnormalities,
like N-Ras mutations or RAS pa deregulations could be the pathway
of abnormal proliferation.
Classification
Myelodysplastic/Myeloproliferative Diseases
CMML
Atypical CML
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Juvenile CMML
MDS/MPD, unclassified
When the percentage of blast in BM is more than 20% is considered acute leukemia.
Chronic myelomonocytic leukemia (CMML): This is the most
frequent in this group. It is mainly a disease found in elderly populations. They usually have a very high white blood cell count in which
monocytosis is a defining feature.
The monocytosis is persistent in greater than 1x109 in the peripheral blood, chromosome Phi and BCR/ABL fusion genes are
negatives, blasts fewer than 20%, and displastic features involving
one or more myeloide lineages.
The 50% of people with this disease can have normal blood
counts with monocytosis , the cytopenias (neutropenia) and other
haematological disorders are similar to MDS
Bone marrow biopsy and blood smear present monocytosis
proliferations, disgranulocitopoyesis, and diserytropoyesis are present in more than 50% of the patients. There are megakariocytes
with abnormalities in more than 80% of the patients.
Because of the number of blast divided CMML in two groups
CMML- 1 with less than 55% in the blood, and less than 10% in BM,
and CMML- 2 blast 5-19% in blood or 10-19% in BM. The blast finding over 20% is considered acute myeloide leukemia.
Many patients have hepato-splenomegaly due to infiltration by
leukemia cells
Atypical chronic myeloid leukemia (aCML):
This is a rare disease that appears at the time of initial diagnosis with MDS and myeloproliferative syndrome features, without the
Phi chromosome or BCR/ABL fusion gene.
There is peripheral blood leukocytosis with increased number
of mature and immature neutrophils, dysgranulopoiesis, neutrophil
precursors equal to or greater than 10% of WBCs, with minimal or
no absolute basofilia and minimal or no absolute monocytosis (less
than 10% of WBCs).
The bone marrow biopsy presents granulopoyetic proliferation, with displastic features with or without dysplasia in the erytroid
and megakaryocytic lineages. The blasts are variable, but less than
20%.
Juvenile myelomonocytic leukemia (JMML): This type of leukemia is less than 3% of all leukemia found in children, but represents 20-30% of the MDS and CMPD. It tends to occur in very
young children, but the features are also similar in adults.
Myelodisplastic/ Myeloproliferative Diseases unclassified
This disorder has clinical, laboratory, or morphological findings
that support the diagnosis of MDS or MPD, but the criteria is not
absolute for one or the other without previous diagnosis of one of
them.
The bone marrow biopsy is hypercelular with hyperplasia of
any or all the myeloide lineages with dysplastic features.
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COURSE
FLOW CYTOMETRY
Minimal residual disease monitoring in
acute leukaemias: contribution of flor
cytometry immunophenotyping
Alberto Orfao, Antonio López, , Carlos Fernández, Sandra Quijano, Juan Flores, Belén Vidriales Maria Consuelo López Berges, José Pérez, Juana Ciudad, Jesús San Miguel
Servicio General de Citometria, Departamento de Medicina and
Centro de Investigación del Cancer, University of Salamanca and
Department of Haematology, University Hospital of Salamanca,
Salamanca, Spain
Introduction:
Minimal residual disease (MRD) in acute leukemias is used to
describe the persistence of relatively low levels of residual leukemia
cells, that persist after cytorreductive therapy at percentages below
the sensitivity limit of conventional morphological techniques(< 10
-2). In the last decade different methodological approaches have
been applied to the investigation of MRD in acute leukemia patients.
Based on these studies, consensus exist about the greater utility
of PCR-based molecular techniques together with multiparameter
flow cytometry , for the investigation of MRD in acute leukaemias.
Here we review the technical issues related to the flow cytometric
assessment of MRD in patients with acute leukemias and its major
clinical applications.
Immunophenotypic investigation of MRD in acute leukemia patients is typically based on the follow-up on aberrant phenotypes,
which are characteristic of leukemia cells while absent in their normal counterpart LAIP or leukemia-associated phenotypes). Such
phenotypic abnormalities are typically classified as: 1) aberrant expression of markers characteristic of a lineage different from that
of blast cells (cross-lineage antigen expression); 2) asynchronous
expression of antigens associated with specific maturation stages
within a given hematopoietic cell lineage; 3) antigen overexpression
(expression of an antigen at abnormally high levels); 4) altered patterns of forward (FSC) and sideward light scatter (SSC) of leukemia
cells, 5) absence of expression of a specific marker and; 6) ectopic
phenotypes.
MRD monitoring in acute myeloblastic leukemia (AML):
Despite the absence of general consensus about the exact frequency of LAIP in AML, most recent series indicate that they could
be present in the great majority of patients (>75%). In practice, such
variations are partially due to the use of different panels of reagents
for the characterization of AML blast cells both at diagnosis and
during follow-up. Of note, several reports have suggested that the
phenotype of AML blast cells is not always stable, the use of relativey large panels of 3- and more colour reagents being required
in such cases. Moreover, it has been claimed that in relapsed AML
there could be a lower number of LAIP, which would make MRD
follow-up more complex and difficult. In contrast, other groups suggest that LAIP are usually stable and the combined use of all LAIP
detected at diagnosis during follow-up would allow a reliable detection of leukemic cells in bone marrow samples in morphological
complete remission. Some of the LAIP suggested to be particularly
useful include asynchronous expression of the following antigens
(% of all AML cases): CD117+/CD33+/HLA-DR- (34%), CD34+/
CD117+/CD33+/HLA-DR- (21%), CD34-/CD15-/CD14-/CD33+
(19%), CD34+/CD33-/CD13+ (19%), CD117+/CD11b+ (13%); in addition, aberrant expression of CD2 (17%) together with other LAIP
(CD34+/CD56+, CD33-/CD15+, CD33-/CD13+, CD15+/CD117+,
CD15+(strong)/CD34+ and CD14+/CD34+) have also been found
at relatively high frequencies in AML. In addition, the CD15/C and/
or CD15/CD117 immunophenotypes have been described as useful for the follow-up of MRD among AML-M2 with t(8;21) and some
AML without differentiation.
The most commonly proposed strategies assess expression
of a large number of markers in 3 or more colours, at diagnosis, to
detected the maximum number of LAIP to be used during followup of the disease. From the clinical point of view different levels of
residual disease detected at specific time-points during follow-up
aretypically associated with different rates of disease-free survival.
An interesting alternative to the study of residual disease has
been the assessment of the relationship between CD34+CD33+
and CD34+CD19+ precursors in the patient bone marrow. A ratio
of ≥10 between the two parameters at the end of therapy has been
associated with shorter disease-free and overall survival rates.
Despite the applicability and prognostic utility of MRD studies
in AML, implementation of these flow cytometry approaches is still
complex and requires further efforts for increasing simplicity and
reproducibility.
MRD monitoring in acute lymphoblastic leukemia (ALL):
MRD immunophenotypic studies in ALL are also usually based
on the follow-up of LAIP detected at diagnosis. In contrast to AML,
LAIP are typically detected in all ALL patients (91% to 99%) independently of the age group and their B-cell precursor or T-cell origin.
In addition the sensitivity obtained for these phenotypes is typically
of ≤1x10-4. From the clinical point of view, detection of increasingly
high numbers of residual cells showing LAIP has been associated
with a poor prognosis independently of the time it is assessed (e.g.:
day 14, first BM in complete remission or end of therapy)
Different strategies have been proposed for the identification
of LAIP in ALL. Some studies have proposed the use of common 4
or more color combinations of reagents for the follow-up of MRD in
all B-cell precursor ALL patients. In line with this, it has been suggested that the CD10FITC, CD20PE, CD45PerCP, CD19APC and
the CD34FITC, CD9PE, CD45PerCP y CD19APC 4-colour combinations could allow detection of LAIP in virtually all (99%) B-cell
precursor ALL cases. In turn, others suggest that MRD investigation
should be focused on the LAIP detected at diagnosis; in this regard,
cross-lineage antigen expression (e.g. CD13, CD33 and CD66),
asynchronous antigen expression (e.g. CD10high/CD20high or
CD20+/CD45-/CD19+ blast cells), overexpression of CD10 and absence of expression of CD10 and CD34 among CD34+/CD19+ and
CD19+/CD10high B-cell precursors are frequently observed (30%
to 50%) in CD58B-cells are observed in up to ALL. Alternatively, it
has been proposed that the evaluation of the percentage of CD34+/
CD19+ or CD20-/CD19+ stage I hematogones/blasts could be easier, providing information with a similar clinical impact.
The BIOMED-I proposal of a standardized 3-color panel includes the following combinations of monoclonal LAIP): TdT/CD10/
CD19 (78%), CD10/CD20/CD19 (64%), CD34/CD38/CD19 (56%),
CD34/CD22/CD19 (46%) y CD19/CD34/CD45 (22%).
Regarding T-ALL a higher frequency of aberrant cases and
number of LAIP/case have been reported. Among other, aberrant
T-ALL phenotypes include cross-lineage expression of CD13, CD33
and CD19 (in up to two thirds of all T-ALL patients), asynchronous
expression of Tdt and sCD3 (around 40% of the cases), ectopic phenotypes (70%) and increased number of cells showing infrequent
phenotypes (88% of all T-ALL cases). Similarly to B-cell precursor
ALL, the BIOMED-I group also has proposed a consensus 3-colour
panel for ALL -TdT/CD7,citCD3, CD7/CD5/CD3, CD7/CD4/CD8,
CD7/CD2/CD3, and CD7/CD38/CD34 which could currently be updated to 6 or more colours with the new benchtop flow cytometers.
Identification and phenotypic
characterization of chronic
lymphoproliferative disorders
Alberto ORFAO, Paloma BARCENA, Antonio LOPEZ, Susana
BARRENA, Juan FLORES, Sandra QUIJANO, Carlos FERNANDEZ, Juana CIUDAD, Ana RASILLO, Julia ALMEIDA
Cytometry Service, Department of Medicine and Cancer Research
Centre, University of Salamanca, Salamanca, Spain
Flow cytometry has now been used for many years in the diagnosis and characterisation of haematological malignancies. Despite
the fact that flow cytometry can distinguish between normal and
leukemic cells based on the presence on the latter of aberrant phenotypic characteristics, in most occasions, flow cytometry immunophenotyping techniques are used only once diagnosis of an haematological disease has already been established by morphology.
Expansions of mature-appearing lymphocytes are frequently
detected in either peripheral blood or lymphoid tissues in either a
routine blood analysis or during physical examination. Additionally,
expansions of mature-appearing lymphoid cells can also be detected
in the cerebrospinal fluid, the skin and other tissues or body fluids. A
major goal of the study of those expansions of mature lymphocytes
in both peripheral and central lymphoid tissues is to establish (or
rule out) the clonal nature of the expanded cell population. Classically flow cytometry immunophenotyping techniques have proven to
be of great utility for the diagnostic screening of B-cell clonality while
they have failed to provide definitive results in most cases where
an expansion of either T or NK-cells are detected. Accordingly, the
screening (and frequently also the diagnosis) of B-cell clonality has
been based for many years in the existence of an excess of either Ig
k+ or Ig-lambda+ B-cells as detected by flow cytometry; in contrast
molecular techniques (e.g. Southern Blot) have been required as
the standard to detect clonality among T-cells.
Recent advances in the knowledge of the phenotypic differences existing between normal and leukemic mature lymphocytes,
together with the availability of multicolor flow cytometers and
large panels of high-quality fluorochrome-conjugated monoclonal antibodies directed against unique TCR-Vbeta families, have
change the way both B- and T-cell clonality can be performed; as
a consequence flow cytometry immunophenotyping has become a
primary diagnostic screening tool for B and T-cell clonality in this
area. Among others, such major advances include the possibility of
performing highly efficient (sensitive and specific) rapid and costeffective flow cytometry studies for the identification of the lineage
of the expanded lymphocytes. Such screening in peripheral blood
and lymphoid tissues can currently be done in a single tube combining five (CD3, CD4, CD8, CD56 and CD19) and seven (CD3,
CD4, CD8, CD56, CD19, sIgK and sIglambda) different antibodies,
respectively. These approaches typically provide a sensitivity and
specificity of > 90% as compared to conventionally used algorithms
for the diagnosis of clonality, results being obtained in a few minutes. Once B- or T-cell clonality are suspected, the identification of
the presence of cells carrying aberrant phenotypic features –which
are present in virtually all chronic lymphoproliferative disorders- that
show restricted usage of either an Ig light chain or a TCR-Vbeta
family, constitute unequivocal signs of B- and T-cell clonality. A similar situation occurs in those expansions of TCR-Vgamma/Vdelta.
S121
However due to the relatively restricted repertoire of TCR-Vgamma
and TCR-Vdelta, in these cases, further molecular confirmation of
clonality is frequently required.
In addition, flow cytometry immunophenotyping has also been
extensively used also for the phenotypic characterization of different subtypes of chronic lymphoproliferative disorders. Accordingly,
unique phenotypes have been associated with the most frequent Bcell disorders such as B-cell chronic lymphocytic leukaemia (B-CLL)
(sIgdim, CD20dim, CD22dim, CD79bdim, FMC7-, CD5+, CD23+),
hairy cell leukaemia (CD103+, CD11c+, CD25+), splenic marginal
zone lymphoma (CD25-, CD11c+), mantle cell lymphoma (CD5+,
CD23-, CD43+) follicular lymphoma (bcl2-high, CD10+, CD38+)
Burkitt lymphoma (bcl2-low, CD10+, CD38high), Waldenström
macroglobulinemia (CD25+, FMC7-/+, CD22dim, CD5-, CD23- in
the presence of clonal plasma cells), among others. Regarding,
T-cell neoplasias, flow cytometry immunophenotypes have also
been associated with specific disease groups such as T-LGL (perforin+/granzyme+), Sezary syndrome (CD7dim, CD3dim, CD2-dim,
CD28+, CD4+, CD45RO+) and T-prolymphocytic leukaemia (mainly
CD4+, CD7high, CD5+), among other conditions. As a result flow
cytometry immunophenotyping has become essential for the diagnostic classification of both B and T-cell chronic lymphoproliferative
disorders. At the same time, extensive characterization of neoplastic B- and T-cell phenotypes has shown the presence of aberrant
patterns of protein expression in virtually all cases, leading to the
possibility of using flow cytometry for monitoring residual disease
in these patients, after therapy. Although, few minimal residual disease studies have been reported in other disease conditions, in BCLL, they have proven the high sensitivity (between 10-4 and 10-5)
and specificity of the method and its clinical utility. At the same time,
the increased knowledge about the phenotypic aberrations present
in neoplastc cells from patients with chronic lymphoproliferative disorders has also facilitated the identification of an increasingly high
number of cases carrying two or more different, unrelated neoplastic cell clones, its frequency among B-chronic lymphoproliferative
disorders, being close to 5% of all cases.
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of reagents required to evaluate hematolymphoid neoplasias:
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Lochem EG, Hooijkaas H et al. Molecular and flow cytometric
analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations. Blood 2001; 98(1):165173.
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Balanzategui A et al. TCRalphabeta+/CD4+ large granular lymphocytosis: a new clonal T-cell lymphoproliferative disorder. Am
J Pathol 2003; 163(2):763-771.
- Lima M, Almeida J, Santos AH, Dos Anjos TM, Alguero MC,
Queiros ML et al. Immunophenotypic analysis of the TCR-Vbeta
repertoire in 98 persistent expansions of CD3(+)/TCR-alphabeta(+) large granular lymphocytes: utility in assessing clonality
and insights into the pathogenesis of the disease. Am J Pathol
2001; 159(5):1861-1868.
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DNA ploidy studies for diagnosis and characterization of blood
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disorders. Haemotologica 1998; 83(Sup):193-198.
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S122
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18(3):491-498.
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in a series of 467 patients with B chronic lymphoproliferative
disorders: basis for the design of specific four-color stainings to
be used for minimal residual disease investigation. Leukemia
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- Sandberg Y, Almeida J, Gonzalez M, Lima M, Barcena P, Szczepanski T et al. TCRgammadelta+ large granular lymphocyte
leukemias reflect the spectrum of normal antigen-selected
TCRgammadelta+ T-cells. Leukemia 2006; 20(3):505-513.
Stetler-Stevenson M, Braylan RC. Flow cytometric analysis of lymphomas and lymphoproliferative disorders. Semin Hematol
2001; 38(2):111-123.
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Combinations of ZAP-70,CD38 and IgVH
Mutational Status as Predictors of Time to
First Treatment in CLL
Alison Morilla, David Gonzalez, Ilaria Del Giudice, Ricardo Morilla, Estella Matutes, Claire Dearden, Daniel Catovsky,and Gareth Morgan
Section of Haemato-Oncology, Institute of Cancer Research,
United Kingdom.
Introduction
One of the most intriguing features of chronic lymphocytic leukaemia (CLL), is its clinical heterogeneity.
Although clinical staging systems still dictate management of
this disease, within two thirds of cases with early stage CLL, we remain unable to discriminate those patients who exhibit more stable
and indolent disease course from those who are likely to progress
and require treatment.
Recent advances in the biology of CLL have led to a greater
understanding of this condition and to the identification of new prognostic markers, which could provide more accurate prediction of
disease course in these CLL patients.
The mutational status of immunoglobulin heavy chain variable
region (IgVH) genes has been shown to be one of the most powerful predictors of overall survival (OS) and progression-free survival
(PFS) in CLL. Stage A CLL patients with unmutated IgVH genes,
have a significantly poorer survival than those with IgVH gene mutations (8 years versus 24 years) (Kuroyama et al. 2004; Tomlinson
et al. 2000)
Gene expression profile studies on CLL with mutated and unmutated IgVH have shown that these two groups exhibit rather similar genetic profiles, but differ in a small subset of genes. Amongst
these, ZAP-70 (Zeta-chain Associated Protein) RNA was shown to
be over-expressed in unmutated CLL. This finding has been confirmed by Chen et al (2002), who showed that ZAP-70 protein is
over-expressed in unmutated (100%) but rarely in mutated CLL
(10%). ZAP-70 expression has therefore been proposed as a possible surrogate marker for IgVH mutational status, since the latter is
technically challenging and not widely available in all laboratories.
ZAP-70 is a 70 kDa tyrosine kinase required for T cell receptor
signalling. It is expressed in normal T and NK cells but not in normal
resting B-lymphocytes. It has recently been shown to be involved in
B-cell signalling in CLL (Chen et al 2005).
Expression of ZAP-70 in lymphocytes can be detected by
different methods, including RT-PCR, western blot, immunohistochemistry and flow cytometry. While RT-PCR and western blot need
to be performed on purified CLL cells, multicolour flow cytometry
allows the simultaneous and selective analysis of ZAP-70 expression in specific blood lymphocyte populations. If standardised, this
assay could represent a practical alternative to the IgVH mutational
status analysis.
There have been a number of studies using flow cytometric
Zap-70 detection and these have shown an overall correlation between ZAP-70 expression and IgVH mutational status ranging from
72% to 90% concordance (Rassenti et al. 2004; Orchard et al. 2004
and Crespo et al.2003).
Recent reports have suggested that Zap-70 expression may
be more predictive of TFI, PFS and OS, than the IgVH status. (Rassenti et al. 2004)
However, it remains uncertain whether ZAP-70 expression has
independent prognostic value compared with other variables, such
as CD38 expression, Ig mutational status or cytogenetic abnormalities.
In 2005, reports by Schroers et al (2005) and Del Giudice et al
(2004) suggested that the combined analysis of ZAP-70 and CD38
could provide more refined information with respect to prognosis.
Both these studies demonstrated that CLL patients could be separated into three subgroups with good, intermediate and poor prognosis – with the discordant group showing intermediate prognosis
with respect to treatment free interval (TFI).
The aim of this study was to determine whether this model i.e.
the identification of three prognostic groups, remained valid when
IgVH mutational status was included in the equation and which
combinations of these three parameters provided the most useful
prognostic information, particularly with respect to TFI.
We studied 115 previously untreated patients. 99 of these were
defined as having progressive CLL requiring treatment and were
subsequently entered into the MRC CLL4 trial. The remainder were
from our own hospital and were defined as stage A.
Stage A 10%
Stage A progressive 30%
Stage B 37%
Stage C 23%
All patients were tested for all 3 parameters.
Mutational status was analysed by direct sequencing using
homology to germ line of 98% as cut-off. CD38 expression was determined by 3 colour flow cytometry and cut offs of 30% and 7%
were examined.
ZAP-70 levels were measured using the technique described
by Crespo et al using 4 colour flow cytometry an unconjugated ZAP
McAb clone 2F3.2 and using a direct comparison of ZAP expression
in T and B cells to determine ZAP positivity. A 20% ZAP-70 positivity in CD5+/CD19+ lymphocyte population was defined as ZAP 70
positive.
The impact of these parameters, singly and in combination, on
treatment free interval or TFI was analysed. With TFI being defined
as time between diagnosis and time of first treatment.
Results
Each prognostic parameter analysed independently showed
highly significant prediction of TFI in this group of patients.
We found that using a cut off of 7% for CD38 positivity as proposed by Thornton et al gave more significant prognostic information and so this cut off was used for all subsequent analyses.
The concordance between ZAP and mutational status was
68%, between CD38 and mutational status was 75% and ZAP and
CD38 were concordant in 67 %
Considering paired combinations of these three prognostic
markers, highly significant prognostic information is retained but
with a better separation of the good and poor prognostic groups with
discordant cases showing intermediate prognosis. (See Table 1)
The combination of CD38 and mutational status appeared to
provide the best discrimination between the good and poor prognostic groups with the least number of discordant cases (n=29).
Concordant cases of CD38 positive, ZAP positive were able to
positively predict unmutated status of the Ig gene in 94.1% of cases.79% of cases showed more than 2% of somatic mutations in the
concordant ZAP negative, CD38 negative patients.
The discordant ZAP/CD38 cases could be further stratified by
IgVH mutational status with mutated cases giving a median TFI of
42 months and the unmutated cases showing a median TFI of 19
months.
Combining all 3 prognostic parameters again defined three
prognostic groups with good, intermediate and poor prognosis, but
almost 50% of cases showed discordance for one or more parameter.
Focussing on these discordant cases, 40% were ZAP -/
CD38+/Unmutated with a median TFI of 25 months, comparable to
the worst prognostic group for all combinations.
The second largest group of discordant cases were ZAP -/
CD38+/Mutated
There was no preferential gene usage in either of these
groups.
In conclusion, the combination of all three biological parameters provides a more refined prediction of TFI in this group of patients.
The model proposed by Del Giudice and Schroers for defining
three prognostic groups when combining ZAP and CD38 is still valid
when IgVH mutational status is included in the analysis.
Combination of mutational status and CD38 expression gave
the best discrimination between good and poor prognostic groups
with the least number of discordant cases.
Analysis of ZAP and CD38, both detectable by flow cytometry,
when combined, continue to provide important prognostic information with respect to predicting time to first treatment without the
need for IgVH mutational status in concordant cases.
Discordant cases continue to raise questions regarding the biology of the CLL cells in these patients.
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References:
Kuroyama H, Ikeda T, Kasai M, Yamasaki S, Tatsumi M,
Utsuyama M, et al. Identification of a novel isoform of ZAP70, truncated ZAP kinase. Biochem Biophys Res Commun
2004;315:935 – 941.
Tomlinson MG, Lin J, Weiss A. Lymphocytes with a complex:
adapter proteins in antigen receptor signaling. Immunol
Today 2000;21:584 – 591.
Chen L, Apgar J, Huynh L, Dicker F, Giago-McGahan T,
Rassenti L, et al. ZAP-70 directly enhances IgM signaling
in chronic lymphocytic leukemia. Blood 2005;105:2036 –
2041.
Chen L, Widhopf G, Huynh L, Rassenti L, Rai KR, Weiss A,
Kipps TJ. Expression of ZAP-70 is associated with increased
B-cell receptor signaling in chronic lymphocytic leukemia.
Blood 2002;100:4609 – 4614.
Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ,
Gribben JG, et al. ZAP-70 compared with immunoglobulin
heavy-chain gene mutation status as a predictor of disease
progression in chronic lymphocytic leukemia. N Engl J Med
2004;351:893 – 901.
Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D,
Rozman M, et al. ZAP-70 expression as a surrogate for
immunoglobulin-variable-region mutations in chronic lymphocytic
leukemia. N Engl J Med 2003;348:1764 – 1775.
Del Giudice I, Osuji N, Matutes E, Morilla A, Morilla R,
Burford A, et al. ZAP-70 expression in CLL: correlation with
clinical and biological features. Blood 2004;104:529a.
Schroers R, Griesinger F, Trumper L, Haase D, Kulle B,
Klein-Hitpass L, et al. Combined analysis of ZAP-70 and
CD38 expression as a predictor of disease progression in Bcell
chronic lymphocytic leukemia. Leukemia 2005;19:750 –
758.
Treatment Free Interval (TFI )and Prognostic Factors
No.of
Cases
Mutational status
ZAP70≥20%
CD38≥7%
Mutation/ ZAP70
Mutation/CD38≥7%
ZAP70/CD38≥7%
Mutation/
ZAP70/CD38≥7%
Umutated
Mutated
Positive
Negative
Positive
Negative
ZAP70+/Unmutated
Discordants
ZAP70-/Mutated
CD38+/unmutated
Discordants
CD38-/Mutated
ZAP70+/CD38+
Discordants
ZAP70-/CD38ZAP70+/CD38+/Unmutated
Discordants
ZAP70-/CD38-/Mutated
68
47
37
78
79
36
35
36
44
59
29
27
34
48
33
32
57
26
Median
TFI
P Value
(months)
23
61
24
44
25
61
19
25
64
21
37
77
19
39
72
20
30
75
TFI=Time from diagnosis to date of first treatment.
0.00003
0.00055
0.0005
0.002
0.004
0.003
0.007
Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A,
Thomas PW, et al. ZAP-70 expression and prognosis in
chronic lymphocytic leukaemia. Lancet 2004;363:105 – 111.
Arch Med Interna 2007; Vol. XXIX; Supl 1
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ORAL SESSION
01 MYELOPROLIFERATIVE AND MYELODYSPLASTIC
SYNDROMES
002
025
EPIDEMIOLOGICAL DATA ON MYELODYSPLASTIC
SYNDROME PATIENTS FROM A ROMANIAN
SINGLE CENTER
MONITORING RESPONSE TO IMATINIB BY
FLUORESCENCE IN SITU HIBRIDIZATION (FISH)
AND REAL-TIME QUANTITATIVE POLYMERASE
CHAIN REACTION (RQ-PCR) IN CHRONIC
MYELOID LEUKEMIA (CML) PATIENTS (PTS)
IN CHRONIC PHASE (CP). EXPERIENCE OF
ARGENTINA AND URUGUAY.
R., Gologan1 *; D., Georgescu2; R., Gologan1; D., Georgescu2
*
Rumania - 1 Clinic of Hematology, Fundeni Clinical Institute; 2
Clinic of Hematology, Fundeni Clinical Institute
Introduction:
Since the World Health Organization (WHO) recognized MDS
as a disease entity only starting with 1997, epidemiological data on
MDS cannot be obtained from official statistics on morbidity and
mortality and have to be extracted from specialized registers.
Objective:
We present the first Romanian study on the incidence and
characteristics of MDS, based on the data existing in Fundeni Clinical Institute, Bucharest, the greatest hematological department in
Romania.
Materials and method:
The MDS files at diagnosis of the patients admitted during the
period 1982-2005, recorded in the registration forms provided by
the MDS Foundation (USA), represented the primary data-base.
The distribution by sex, age groups, subtypes (FAB) and the annual number of new cases were analysed comparatively with other
reference studies.
Results:
Four-hundred and twenty four cases of MDS were identified.
The distribution between sexes was relatively balanced with a slight
global preponderance of males ((M/F 1.26), except for refractory
anemia with excess of blasts (RAEB) 1.94. The median age was
62 years (16-91). Most of the patients (60.6 %) belonged to the
group of age 61-80, where all the subtypes of MDS had the highest
rates. A noticeable proportion (17%) had ages below 50 years, 25%
of which in the range 16-30. On the other hand, few cases (4%)
were above 81. Patients with refractory anemia (RA) and refractory
anemia with ringed sideroblasts (RARS) accounted for 44.5% of all
cases (RA 29%, RARS 15.5%), RAEB and RAEB in transformation 33%, chronic myelomonocytic leukemia 5.6% and unclassified
16.7%. The annual number of new cases was constantly low during
the period 1980-1989, but increased dramatically from 11 cases/
year in 1990 to a maximum of 48 cases/year in 1999, showing a
certain decrease afterwards. The subtypes with the most important
increase in time were RA and RARS.
Conclusion:
This study indicates an actual increase of the number of MDS
cases in Romania over the investigated period of time. Particularly,
a noticeable proportion of young patients and a low proportion of
patients ¡Ý 81years have been found, which make our findings closer to the Asian than to the Western MDS epidemiological results.
Pavlovsky, C1 *; Giere, IA1; Lombardi, MV1; Negri, P2; Moiraghi,
B3; Garcia, J4; Uriarte, R5; Magariños, A6; Martinez, L5; Garcia
Reinoso, F1; Milone, J7; Bengiò, R8; Pavlovsky, S1
*
Argentina - 1 FUNDALEU; 2 Inst.Privado de Hematología; 3 H. Ramos Mejía; 4 Hospital Privado de Cordoba; 5 Asociación Española
Primera de Socorros Mutuos; 6 H. Maciel; 7 ITMO; 8 Depto. De
Genetica - IIHEMA - Academia Nacional de Medicina
Introduction:
The degree of reduction of total leukemia cell mass by imatinib
as measured by FISH and RQ-PCR correlates with progressionfree survival. Dosis scalation is mandatory in pts with rising levels
of bcr/abl transcripts.
Objective:
To determine the potential of RQ-PCR according to duration of
complete cytogenetic remission (CCyR).
A total of 160 1st CP-CML pts with CCyR treated with 400 mg/d
imatinib were studied prospectively since november 2005. According to 1st line treatment pts were divided in two groups: IFN/Cytarabine, 83 pts (51%) and Imatinib, 77 pts (49%). At baseline all
pts were studied by FISH and RQ-PCR. Follow up: RQ-PCR was
performed at 6 and 18 months. Imatinib median duration was 26
months (range 6-64).
Results:
First FISH evaluation showed: 87% pts with 0% bcr/abl(+) cells,
13% pts with 0.1-5% bcr/abl(+) cells. Molecular responses (MR) in
160 pts were: Complete (CMR), >4 Log Red: 23%; Major (MaMR),
>3 Log Red: 17%; Minor (MiMR), >2 Log Red: 34%; Minor, < 2 Log
Red: 15%; Nule (NuMR),< 1 Log Red: 11%. No significant differences in MaMR and CMR were observed in pts with follow up < 12
months and > 24 months of imatinib (42% vs 47%). Follow up in 68
pts (6 months) showed: better MR in 13% pts, invariable in 68% pts
, worse in 19% pts. All pts are still in hematologic remission.
Conclusion:
In this on going study, 87% CML pts were FISH (-), 40%
achieved >3 Log Red MR (CMR or MaMR), 81% pts improved or
maintained the MR at 6 months of follow up.
045
THE PROGNOSTIC VALUE OF WPSS AS
COMPARED TO PHENOTYPIC FEATURES IN
MYELODYSPLASTIC SYNDROMES
Lorand-Metze, I.1 *; Califani, S.M.V.1; Ribeiro, E.1; Lima, C.S.P.1;
Saad, S.T.O.1; Metze, K.1
*
Brazil - 1 Faculty of Medicine, State University of Campinas, Brasi
Introduction:
Well established prognostic factors in myelodysplastic syndromes (MDS) are associated with the degree of peripheral blood
(PB) cytopenias, percentage of bone marrow (BM) blasts as well
as cytogenetic findings. Recently, a new prognostic score has been
described based on WHO classification, cytogentics and transfusion dependence. Some recent reports have stressed that maturation abnormalities, especially in the myelomonocytic series, detected by flow cytometric (FCM) studies show a correlation with IPSS
and WPSS.
Objective:
to examine the prognostic significance of FCM abnormalities
in MDS
we performed a quantitative FCM analysis of maturation of
erythroblastic, granulocytic and monocytic cell lines in newly diagnosed patients with confirmed MDS and examined the impact of
the abnormalities found on overall survival (OS) of the patients and
compared it with that of WPSS.
Results:
Among 31 patients that entered this prospective study, median
age was 60 years (18-93). According to the WHO classification, 11
were refractory anemia, 2 had sideroblastic anemia, 10 had refractory cytopenia with multilineage dysplasia, and 7 had refractory
anemia with excess of blasts. By WPSS, 6 had a very low, 9 had a
low, 9 intermediate and 6 a high risk. Median total number of FCM
abnormalities per patient was 3 (1-8). In the univariate Cox regression the following parameters had impact on OS: WPSS, number
of peripheral platelets and number of CD34+ cells besides SSC of
granulocytic precursors, MFI of CD13 of myelocytes - mature neutrophils and CD45 of mature neutrophils. The total number of abnormalities was also significantly associated with OS. In the multivariate analysis, only platelet number and CD13 of mature neutrophils
remained in the model.
Conclusion:
FCM parameters turned out as important prognostic parameters in MDS.
Supported by FAPESP and CNPq
041
OVEREXPRESSION OF BCR/ABL
REARRANGEMENT DETERMINED BY QRT-PCR
AND FISH RATIO
Bianchini, M1 *; Gargallo, P1; Alù, F1; De Brasi, C1; Bengiò, R1;
Larripa, IB1
*
Argentina - 1 Depto. De Genetica - IIHEMA - Academia Nacional
de Medicina
Introduction:
Resistance to Imatinib in the treatment of Chronic Myeloid
Leukemia (CML) is mainly associated to 3 mechanisms: acquired
mutations in the kinase domain of BCR/ABL protein, amplification
and overexpression of BCR/ABL rearrangement. Thus, the determination of molecular resistance is particularly important to improve
strategies to overcome resistance in CML patients. Amplification of
S125
BCR/ABL gene can be determined by interphase fluorescence in
situ hybridization (FISH) while BCR/ABL transcript expression can
be determined by quantitative real time PCR (qRT-PCR).
Objective:
With the aim to determine overexpression of BCR/ABL gene
we propose a method that correlates FISH and qRT-PCR, allowing to estimate the Expression Index (EI), calculated as [BCR-ABL/
ABL]/FISH.
The EI was determined for 70 CML patients in different disease
phases and clinical outcome, applying both methodologies (qRTPCR and FISH) to the same blood sample.
Results:
Expression Index values obtained from all patients were used
to calculate the median EI ratio (median 22.25% range 0.014 8.600). Statistical analysis was performed to stratify the patients in
different percentile groups while Mann-Whitney test was used to
evaluate different associations. Those cases included in percentile
85 showed an increment of EI above 1 Log respect to the median
value; this group was defined as patients with overexpression of
BCR/ABL. All of them were resistance to imatinib treatment; interestingly, any other cause of resistance such as, point mutations,
amplification and clonal evolution, could be described within this
group of Imatinib refractory patients.
Conclusion:
We speculate that, Philadelphia positive clone, overexpressing
BCR/ABL transcript, could be paradoxically maintained alive by the
presence of Imatinib which could partially inhibit oncogenic activity.
Thus, we conclude that screening patients for BCR-ABL overexpression could be cost-effective, since it would allow to optimize
treatment strategy.
181
RETROSPECTIVE ANALYSIS OF JAK2 V617F
MUTATION IN MYELOPROLIFERATIVE DISORDERS
(MPD) PATIENTS (PTS)
Manrique, G.1 *; Pérez, V1; Bonomi, R.1; Zubillaga, M.N.1; Capetta, M.1; Boschi, S1; Costa, V1; Cardeza, A1; Martínez, L.1; Uriarte,
M.R.1
*
Uruguay - 1 ASESP
Background: An acquired V617F mutation in the JH2 autoinhibitory domain of the JAK2 tyrosine kinase was recently demonstrated in the pathogenesis of polycythemia Vera (PV), essential
thrombocythemia (ET) and idiopathic myelofibrosis (IM). The reported frequencies of JAK2 mutation vary depending on diagnosis,
techniques and patients included.
Objectives: A retrospective analysis of JAK2 V617F status in
Ph(-)/BCR-ABL (-)MPD pts.
Material and Methods: DNA purified from granulocytes and/or
cytogenetic pellets from 58 pts (35 males, 23 females; aged: 5-83
ys) with clinical diagnosis of MPD Ph(-)/BCR-ABL (-) and 15 normal
controls were analyzed by two sensitive PCR based methods to
assess the JAK2 mutation: 1) allele specific JAK2V617F PCR mutation and 2) sequencing of PCR JAK2V617F mutation products.
Results: The JAK2-V617F mutation was demonstrated in 13
from 58 MPD pts: 8 pts had clinical diagnosis of PV; 3 pts of ET
and 2 pts IM: 2. Patients lacking JAK2-V617F mutation had clinical
laboratory features characteristic of MPD. Normal controls showed
absence of this mutation.
Conclusions: In our cohort of pts, JAK2-V617F mutation was
identifed in 14/58 MPD. In agreement with previous reports this mutation is present more frecuently in PV (8/13), than in others MPD.
This is an on-going study: larger pts series must be included to establish the incidence of this mutation in uruguayan MPD pts. The
identification of JAK2-V617F mutation provides a myeloid -specific
clonality assay and contributes to a more accurate diagnosis and
classification of the MPD.
S126
092
161
DETECTION OF BCR-ABL POINT MUTATIONS IN
PATIENTS WITH CHRONIC MYELOID LEUKEMIA
(CML) RESISTANT TO IMATINIB AND PROGNOSIS
JAK2 V617F MUTATION IN MYELOPROLIFERATIVE
DISORDERS
Silveira, RA1 *; Albuquerque, DM1; Assis, AM1; Ichihara, E1; de
Souza, CA1; Costa, FC1; Delamain, MT1; Vigorito, AC1; LorandMetze, I1; Miranda, E1; Funke, VA2; Pagnano, KBB1
*
Brazil - 1 UNICAMP; 2 UFPR
Introduction: Mutations in the kinase domain of BCR/ABL are
the most frequent mechanisms associated to Imatinib resistance.
Objective: Detect mutations in the kinase domain of BCR/ABL
in CML patients with primary or secondary resistance to Imatinib
and describe their clinical outcome after mutation detection.
Materials and method: We evaluated 20 CML patients with
primary or secondary resistance to Imatinib. After RNA extraction
from peripheral blood samples, amplification of the kinase domain
of ABL from BCR/ABL was performed, using a semi-nested RTPCR, to cover amino acids 244-486. PCR product was submitted to
direct automated sequencing and compared with normal sequences
of BCR-ABL gene (M14752, GenBank).
Results: We found seven mutations in 13 patients resistant
to imatinib: 5 in blast crisis (BC), 4 in accelerated phase (AP) and
4 in chronic phase (CP). Mutations identified: T315I (5), L248V (1),
G250E (1), F359V (2), M244V (1), E255K (2) and E279K (1). Seven
patients were treated with dasatinib. Patients with T315I mutation
(5) did not respond to treatment. Two are dead, two are currently
been treated with hydroxyurea in CP and AP, and one was submitted to bone marrow transplantation in AP. Patient with mutation M244V is still using dasatinib, presenting major cytogenetic
response. One patient with F359V mutation was submitted to a
non-myeloblative stem cell transplantation from an identical sibling
donor, in AP, but with no response. The F359V mutation was still
present after the SCT and the patient was included in a phase II
trial of dasatinib, which is on going and is in partial hematological
response. The other patients with P-loop mutations died in blast
crisis. In seven patients mutations were not found, but two patients
had clonal evolution.
Conclusion: Patient with P-loop mutations presented a worse
prognosis with rapid evolution to blast crisis and patients with T315I
mutation had no response to dasatinib, as recently described. We
identified two patients with non-P-loop mutations with good response to dasatinib. P-loop mutations have been associated with
poor prognosis, but some mutations may respond to new drugs.
Mutation detection is helpful in deciding strategies to overcome resistance to Imatinib.
Novoa, J.E.1 *; Stoll, M.1; Beñaran, B.1; Rojo, A.L.1; Caneiro, A.1;
† De Bellis, R.1
*
Uruguay - 1 Hospital Policial / CGM
Background: JAK2 V617F mutation in patients with myeloproliferative disorders (MPDs) represents a major advance in our
understanding of the pathogenesis of these diseases. JAK2 came
as a recognition to William Dameshek who demonstrated that classical MPDs shared phenotypical and a general pattern of clinical
evolution.Aim: to study JAK2 V617F mutation in a population with
different MPDs.To know how the identification of the JAK2 V617F
mutation could change our approach to patients. Methods: 28 patients with diagnosis of MPD were included on this study.15 men
and 13 women. Their ages ranged from 44 to 80 years old. All of
them were philadelphia chromosome negative and bcr/abl negative. Polycitemia vera 14/28, essential thrombocytemia 7/28 and idiopathic myelofibrosis 7/28. The elegibility criteria was JAK2V617F
genotype determination according to a polymerase chain reaction
(PCR-Restriction kit, ATGen Sistemas Moleculares) for the G1849T
variant. Results: the results are expressed in the table 1.
Table 1 - Detection of the mutation G1849T(V617F) in the
JAK2 gene__
Normal
PV
ET
IM
homozygote H e t e r o z y g o t e Homozygote (T/
(G/G)
(G/T)
T)
3/14
2/14
9/14
2/7
2/ 7
3/ 7
1/7
3/ 7
3/ 7
With respect to the homozygote patients, showed irrespective
to their
diagnosis, higher leukocyte count and hematocrit level. Platelets were unchanged. The frequency of splenomegaly were 43% in
heterozygotes and 73% in the group of homozygotes. Thrombosis
were 28,5% in heterozygotes and 53% in patients to the homozygotes group. Conclusions:the genotyping of JAK2 V617F may have
a role as prog nostic marker for the management of MPDs.
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ORAL SESSION
02 LYMPHOPROLIFERATIVE SYNDROMES
033
038
CUTANEOUS LYMPHOMAS: EXPERIENCE OF A
SINGLE INSTITUTION IN LIMA-PERÚ
FLUDARABINE, MITOXANTRONE AND
DEXAMETHASONE AS FIRST LINE TREATMENT
OF PATIENTS WITH INDOLENT NON-HODGKIN
LYMPHOMA (NHL): GATLA FIRST INTERIM
REPORT
Beltrán-Gárate, B; Málaga, J1; Portugal, K1; Morales, D1; Hurtado de Mendoza, F1; Castillo-Aguirre, J1; Quiñones, P1; ValdésGómez, JJ1; Carrasco-Yalán, A2
1
Hospital Edgardo Rebagliati, Lima, Perú.; 2 Hospital Edgardo
Rebagliati, Lima
Introduction:
The clinicopathologic characteristics of malignant lymphomas
may vary according to geography. We previously described Adult
T -cell leukaemia/lymphoma (ATLL) cases associated with human
T-cell lymphotropic virus type-I (HTLV-I) in their different clinical presentation: acute, lymphomatous, chronic and smoldering and the
recently primary cutaneous subtype in Perú (EHA 2001: abstract
129).
Objective:
To determine the relative frequency of cutaneous lymphomas
and evaluate the clinical relevance of the new WHO/EORTC classification in a General Hospital in Lima-Perú
We conducted a clinicopathologic retrospective study of primary cutaneous lymphomas diagnosed from 1997 to 2004 in our General Hospital. Clinical records, haematoxylin & eosin-stained slides
and immunohistochemical stains from 78 patients were reviewed.
HTLV-1 serology was made using ELISA and Western Blot method.
The statistical method was descriptive and survival was calculated
using the Kaplan-Meier method.
Results:
The mean age at time of presentation was 62 years and the
female/male ratio 1,5:1 . T-cell lymphomas were 88.6% and 11.4%
were B-cell lymphomas . Eight-six percent (67/78) were primary cutaneous lymphomas and fourteen percent (11/78) were secondary
cutaneous lymphomas. The most frequent primary cutaneous lymphomas was mycosis fungoides (MF): 44.7% (30/67); cutaneous /
smoldering ATLL sutypes included 13/67 (19.4%) patients; unspecified peripheral T-cell lymphoma 4/67 (6%), lymphomatoid papulosis
2/67 (3%), leg-type diffuse large B-cell lymphoma 2/67 (3%) , diffuse
large B-cell lymphoma 2/67 ( 3%) , subcutaneous panniculitis-like
T-cell lymphoma 2/67 ( 3%), one case of the following lymphomas:
anaplastic large cell, Sézary syndrome ,nasal type extranodal NK/
T-cell lymphoma, marginal zone B-cell lymphoma, follicle center
lymphoma and intravascular lymphoma; finally unclassifiable lymphomas 5/67 ( 7.4%). Most frequent secondary cutaneous lymphomas were acute and lymphomatous subtypes of ATLL with 72% of
the cases. Five-years overall survival for MF was 77%. The 5-years
overall survival for primary cutaneous ATLL lymphomas was 18%
and 0% for the secondary cutaneous ATLL group.
Conclusion:
In this retrospective analysis, both ATLL and MF are the most
frequent cutaneous lymphomas in our General Hospital. ATLL has
a poor overall survival.
Milone, G. *; Rodriguez, A1; Milone, J2; Bezares, F3; Rudoy, S4;
Palmer, L5; Cerruti, I6; Lastiri, F1
*
Argentina - 1 Instituto de Investigaciones Hematológicas, Buenos
Aires; 2 ITMO - Fundación Mainetti; 3 GATLA & LACOHG (Latin
American Cooperative Oncology Hematology Group); 4 Hospital
Santojanni; 5 Hospital Churruca; 6 IPROS, Rosario
Introduction:
Fludarabine (F) is licensed for the management of indolent
non- Hodgkin lymphoma in countries such as Canada and Switzerland. Clinical evidence suggests that fludarabine monotherapy
is as least as effective, than conventional therapies such as cyclophosphamide, vincristine, prednisone (CVP) for the first and second
line treatment of B-cell low grade NHL achieving objective response
rates. Better response rates can be achieved combining F with Mitoxantrone (N) and Dexamethasone (D) in indolent NHL patients
(pts). The GATLA (Grupo Argentino de Tratamiento de la Leucemia
Aguda) started a prospective multicenter national study to evaluate
the use FND as a first line treatment for low grade NHL.
Objective:
To assess the response rate, safety, disease free survival
(DFS) and overall survival (OS) of FND as first line treatment for
indolent NHL during (2002-2006).
Ninety-six patients in the period of January 2002 to April 2006
were recruited. Sixty-nine patients were valuable at the time of
analysis. Median age 54 years old (range: 21-79). Gender: male
51% and female 49%. Inclusion criteria for low grade NHL-LG was:
non-previous, age > 18 years old with symptomatic disease, ECOG
performance status 0-2 and written informed consent. Ann Arbor
staging: I: 5,8%, II: 14,5%, III: 24,6% and IV: 55%. FND treatment
consisted of F 25 mg/m2 i.v. (days 1-3), N 10 mg/m m2 i.v. (day
1) and D 20 mg (days 1-5) each 28 days for 6 cycles. All patients
received oral antibiotics for intestinal decontamination, antifungal
prophylaxis and Trimethoprim-Sulfamethoxazole as P. carinii prophylaxis for one year.
Results:
On this low grade NHL cohort the overall response rate (ORR)
was 93% (ORR) with 70% (48 pts) with complete response (CR)
and 23% (16 pts) with partial response; progressive disease and
non-response 7% (5 pts). The probability of event free survival
(EFS) and overall survival (OS) at 24 months was 60% and 90%
respectively. Two patients developed secondary malignancies after
treatment and one died. Only one patient died in CR
Conclusion:
In this population FND treatment demonstrate a high CR rate
with low toxicity and high probability of EFS and OS as previous
experience published in the literature.
S128
116
ANALYSIS OF 511 LYMPHOMA CASES IN
URUGUAY.
Gualco, G.1 *; Ortega, V.1; Musto, M.L.1; Ardao, G.1
*
Uruguay - 1 Hospital Militar // Laboratorio de Anatomía Patológica
Dr. G. Ardao
The study describes 511 cases of newly diagnosed lymphomas their frequency and distribution in a Uruguayan population. All
cases were classified following the WHO 2001, with appropriate immunophenotypification by the same pathologists. 204 cases from a
public hospital and 307 from a private laboratory.
There were 82.2% (422) Non Hodgkin Lymphomas (NHL) and
17.8% (89) Hodgkin Lymphomas (HL). 57.9% males.
NHL was 58.1% males. Mean age was 60 yo, (2 to 91). B cell
lymphomas (BNHL) were 92.7% and T cell lymphomas (TNHL) were
7.3%. BNHL subtypes distributed as follow: Diffuse large B cell was
the most frequent 37.9%, Follicular lymphoma 28.6%. Lymphocytic
lymphoma and Mantle cell lymphoma were 10 and 7.2% respectively; marginal zone lymphoma was 6.2% all the other subtypes were
less than 3% each. 27% with extra nodal presentation. The most
frequent TNHL were T Non otherwise specified 29%, Lymphoblast T
cell and Anaplastic large cell lymphomas were 22.6% each.
HL occurred in 57.3% (51) males. Mean age was 37.7 yo, significantly lower for males and showed bimodal distribution, (6 to
80). 95.5% were classic HL (CHL) and 4.5% were nodular lymphocyte predominance (NLP). CHL includes 68.2% nodular sclerosis,
21.3% mixed cellularity, 4.5% lymphocyte rich and 3.5% lymphocyte depletion. Male /female ratio was 1:1 for NS and 1.5 to 3:1 for
the other types. EBV was positive in 24% NS and 75% MC. NLP
represents 4.5%, all males, mean age 31.7.
The distribution of NHL B and T and also B subtypes is similar
to that observed in occidental world as male/female and age range.
The same is concluded to CHL with little less strong association
with EBV, more similar to studies in European population. This is
the first study in our country which takes an approximation to the
frequency and sex/age distribution in Hodgkin and Non Hodgkin
lymphomas.
130
MOLECULAR EVALUATION OF TELOMERE
LENGTH IN PATIENTS WITH MANTLE CELL
LYMPHOMA (MCL).
Cottliar, A.1 *; Panero, J.1; Pedrazzini, E.1; Noriega, M.F.1; Narbaitz, M.1; Slavutsky, I.1
*
Argentina - 1 Academia Nacional de Medicina
Background: Telomeres are repeated DNA sequences at
the ends of chromosomes, which play a key role in maintaining
chromosomal stability. Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma, genetically characterized by the
t(11;14)(q13;q32) and up-regulation of Cyclin D1 gene. Objective:
We assessed the molecular telomere length (TL) evaluation in patients with MCL and their correlation with genetic findings. Material
and Methods: Nineteen patients with MCL (14 males; mean age:
57.7 years; range 30-83 years) were studied. TL based on telomere
restriction fragment (TRF) assay on DNA samples from patients and
controls was evaluated. Cytogenetic studies, and fluorescence in
situ hybridization (FISH) and molecular analysis to detect Cyclin D1/
IgH rearrangement, were performed. Results: The TRF mean value
was significantly shorter in patients (4.33±0.72Kb) than in controls
(8.5±0.5Kb) (p<0.001). Ten patients (53%) had only one TRF peak
(4.37±0.68Kb). The remaining ones showed two TRF peaks, the
smaller representing the tumor component (4.12±0.78Kb) and the
other corresponded in size to TRF normal values (7.15±1.2Kb). Cytogenetic cultures were successful in 83% (15/18) of patients, and
abnormal karyotypes were observed in 47% of them. By molecular
approach, 58% of patients showed the Cyclin D1/IgH rearrangement. By FISH, all cases showed t(11,14) positive cells (mean:
39%; range: 4-97.2%). Only one TRF peak was observed in 77%
of patients with high frequencies of FISH positive cells (=20%)
compared to 33% of those with lower frequencies (p<0.01). Conclusions: Our results showed a significant TRF decrease in MCL
patients and suggest this parameter as a possible marker reflecting
tumor mass.
Key words: telomere length, TRF, mantle cell lymphoma, Cyclin D1.
173
THERAPY WITH FLUDARABINE,
CYCLOPHOSPHAMIDE AND RITUXIMAB (FCR)
FOR RELAPSED OR UNTREATED PROGRESSIVE
CHRONIC LYMPHOCYTIC LEUKEMIA (CLL): A
SINGLE CENTRE EXPERIENCE
Pavlovsky, M A1 *; Pavlovsky, C1; Pardo, L1; Sapia, S1; Monreal,
M3; Corrado, C1; Fernández, I1; Juni, M1; Pavlovsky, S1
*
Argentina - 1 FUNDALEU
Introduction: The combination of FCR has demonstrated superior rates of complete remission (CR), minimal residual disease
(MRD) negativity, disease-free survival (DFS) and overall survival
(OS) in previously treated and untreated patients with CLL, when
compared with historical control groups using fludarabine alone or
fludarabine and cyclophosphamide. Objectives: To evaluate the efficacy of FCR in improving CR, DFS and OS rates in patients previously treated with chlorambucil-prednisolone and untreated patients
with CLL. Patients and methods: A total of 45 CLL patients started
treatment with FCR. Forty-one patients completed treatment: 16
following previous relapse and 25 previously untreated with progressive disease, four patients are still receiving treatment. Median
patient age was 63 years (range 34-88 years). The majority of patients were Binet’s stage C; 8% stage A, 34% stage B, 58% stage C.
CD38 expression was positive (> 7% of cells) in 56% of patients and
negative in 44%. FCR consisted of: rituximab (375 mg/m2/day x 1),
fludarabine (25 mg/m2/day x 3) and cyclophosphamide (250 mg/
m2/day x 3), all given intravenously, every 4 weeks for 4-6 cycles.
CR was defined by CLL/NCI-WG criteria. Minimal residual disease
(MRD) negativity was < 1% of CD19 and CD5-positive cells in peripheral blood and bone marrow. Results: CR was observed in 74
% of patients, nodular partial remission in 16 % of patients, partial
remission in 8% of patients, stable disease in 2% and three patients
did not receive a bone marrow biopsy. Grade 3-4 neutropenia occurred in 33% of patients. Conclusions: FCR induces a high CR and
DFS rate and increases MRD negativity. Significantly higher DFS
rates were observed in patients who were CD38 negative.
194
PROGNOSTIC VALUE OF CD38 EXPRESSION
BY FLUORESCENCE INTENSITY HISTOGRAM IN
CHRONIC LYMPHOCYTIC LEUKEMIA
Barreto, W. G.1 *; Siufi, G. C.1; Silva, M. C. A.1; Sandes, A. F.1;
Kimura, E. Y. S.1; Figueiredo, V. L. P.2; Guirão, F. P.1; Yamamoto,
M1
*
Brazil - 1 Universidade Federal de São Paulo; 2 IAMSPE, São
Paulo, Brasil
Background-Patients with CLL can present a heterogeneous
clinical course. CD38 expression in B-CLL is an independent prognostic factor but its best cut-off level still has to be determined. Rassenti et al (2004) and Boonstra et al (2006) showed that CLL cells
express a heterogeneous pattern of CD38 intensity, suggesting that
its fluorescence intensity histogram (FIH) analysis may be useful
to evaluate the prognosis in this disease. Objectives-classify CLL
patients in groups according to the histogram of CD38 intensity expression and correlate these findings to prognosis. Methods-CD38
expression of 51 CLL patients was reanalyzed using FIH. Diagnosis of CLL was based on WHO criteria. The CD38+ histogram was
determined using a fixed threshold established from 10 patients
samples with a uniformly negative cell population for CD38. The
cursor was set at the right foot of this population, where <0.2% of
cells were classified as positive. Three types of FIH were unimodal:
negative (type I), weak expression (type II), strong expression (type
III) and one was bimodal (type IV). Different FIH types were evalu-
S129
ated according to the Binet´s staging, CD38 positive in percentages
and event-free survival (EFS) of patients. Results- According to FIH
23.5% of patients were type I, 39% type II, 14% type III and 23.5%
type IV. The mean percentage value of CD38 was: type I-2,1%, type
II-5,3%, type III-57,3% and type IV-33,2%. Type IV patients showed
an EFS lower than those with types I (p<0.05) and II (p<0.05). Types
III and IV had short EFS; 72% of patients with type IV and 75% with
type III FIH required treatment at time of diagnosis. Among Binet´s
A, patients with type IV showed EFS lower than those with other
histogram types (p<0.05). Conclusion-Expression of CD38 by FIH
is a very helpful tool in CLL patients especially in Binet´s stage A
with CD38 expression <30%.
S130
ORAL SESSION
03 MISCELLANEOUS TOPICS
026
065
ELTROMBOPAG INCREASES PLATELET
COUNTS DURING TREATMENT OF IDIOPATHIC
THROMBOCYTOPENIA PURPURA IN A
RANDOMIZED, DOUBLE=BLIND, PLACEBOCONTROLLED PHASE II STUDY
MEASUREMENT OF VASCULOENDOTHELIAL
GROWTH FACTOR (VEGF) IN A GROUP OF
PATIENTS WITH HEMATO-ONCOLOGICAL
DISEASES IN A SINGLE CENTER. PRELIMINARY
DESCRIPTIVE ANALYSIS
Cheng, G1 *; Saleh, M2; Kovaleva, L3; Mayer, B4; Stone, N4; Bussel, J5
*
Hong Kong - 1 Prince of Wales Hospital; 2 Geogia Cancer Specialists; 3 Hematology Research Center; 4 GlaxoSmithKline; 5 Cornell
University
Rojas, A.1 *; Villela, L.1; Caballero, R.1; Ruiz, R.1; Borbolla, J.R.1;
García, H.1; Mejía, D.1
*
Mexico - 1 Centro Médico ISSEMyM
Introduction:
Thrombocytopenia in idiopathic thrombocytopenia purpura
(ITP) is due to an imbalance between platelet clearance and inadequate platelet production. Eltrombopag olamine is a novel, small
molecule, that stimulates proliferation and differentiation of megakaryocytes and progenitor cells, ultimately increasing the number
of circulating platelets.
Objective:
Safety/efficacy of eltrombopag in patients with previously treated chronic ITP.
The safety and efficacy of eltrombopag were evaluated in a
multi-national, double-blind, placebo-controlled phase II trial in
adults with chronic, previously-treated ITP and platelets <30,000/
μL. Subjects were randomized (1:1:1:1) to placebo, 30mg, 50mg, or
75mg eltrombopag for up to 6 weeks and followed for 6 weeks after
discontinuation of study medication. The primary efficacy endpoint
was the proportion of patients with platelets > to 50,000/ìL after 42
days of dosing using last observation carried forward analysis. Randomization was stratified by splenectomy status, use of concomitant ITP therapy and platelet counts < to 15,000/ìL.
Results:
The intent-to-treat analysis included 117 subjects; 29 on placebo and 30, 30, and 28 on the 30mg, 50mg and 75mg eltrombopag
arms, respectively. The majority of patients were females (62%) and
Caucasian (68%). Median serum TPO levels at baseline and at Day
43 were : placebo (54ng/L; 56 ng/L), 30mg (46 ng/L;49 ng/L), 50 mg
(65 ng/L; 44 ng/L) and 75mg (53 ng/L;45 ng/L). During the study
32% patients received stable doses of concomitant ITP therapy. At
Day 43, a dose dependent increase in the proportion of responders
was observed: placebo (16%), 30mg (28%), 50mg (67%) and 75mg
(86%). The odds-ratio of treatment response to placebo was statistically significant in the 50 and 75mg arms (p<0.001). Median Day
43 platelet count was 16,000/ìL in the placebo group and 26,000/ìL,
128,000/ìL and 183,000/ìL in the 30mg, 50mg, and 75mg eltrombopag arms, respectively. The safety profile was similar across the
treatment groups.
Conclusion:
Eltrombopag at 50 and 75mg significantly increased platelet
counts during the 6 week treatment period compared to placebo.
The median serum TPO levels were within the range seen in adults
without ITP. No safety concerns were identified. Phase III trials of
eltrombopag in ITP are ongoing.
Introduction: Angiogenesis plays a very important role in the
development and progress of hemato-oncological diseases. There
have been reports on pro-angiogenic factors in anglo-saxons but no
report has been made in Latin Americans.
Objective: Assess VEGF levels in the serum of patients with
different hematological diseases previous to treatment.
Materials and method: Determination of VEGF was performed by ELISA with a commercial kit. Patients were divided in the
following groups: a) lymphoproliferative syndrome (LPS): NHL (B or
T), MM, ALL (B or T), HD, and b) Myelod syndromes (MS): MDS,
AML, CML, MPSC. We performed a descriptive analysis on each
patient and attempted to correlate these with the VEGF levels. The
VEGF normal levels used were 31 to 86 pg/dL.
Results: 59 patients (p) were included during September 2004
and April 2006, age mean 54 yo (range: 15-81), 59% male. 52 newly
diagnosed and 7 resistant to treatment. Mean follow up was 8.59
months (95%CI: 7.28 to 9.90). Mean VEFG was 191pg/dL (range
31-3064pg/dL). VEGF by disease group was as follows: a)SLP:
aggressive NHL (12p) mean 348.8pg/dL (95%CI: 222.5 to 475.1),
ALL (10p) mean 158.8pg/dL (95%CI: -17.8 to 335.38), MM (8p)
mean 496pg/dL (95%CI: -89.5 to 1081.28), indolent NHL (7p) mean
283.6 (95%CI: 63 to 504), HD (3p) mean 307.3 (95%CI: -119 to
734). b)MS: MDS-AML (7p) mean 247.5 (95%CI: -15.66 to 510.66),
MPSC (3p) mean 1237 (95%CI: 3855.2 to 6329).
Conclusion: VEGF is high in hemato-oncologic diseases like
LPSs and SMs. More patients need to be included to more accurately define VEGF levels. Our data reflect that some hemato-oncologic patients have an increase in VEGF and this might be used as
a therapeutic target.
Keywords: Angiogénesis, VEGF.
117
EFFECTS OF HUMAN BONE MARROW
STROMAL CELL LINE ON THE PROLIFERATION,
DIFFERENTIATION AND APOPTOSIS OF HL-60
CELLS
Rong, L.1 *; Gao-sheng, H.1; Zhe, W.1; Xie-qun, C.1; Qin-xian, B.1;
Yong-qing, Z.1; Bao-xia, D.1; Wen-qing, W.1; Juan-hong, W.1
*
China - 1 Xijing Hospital, Fourth Military Medical University
Introduction Rapid advances have been made in elucidating
the molecular mechanism of etiology and pathogenesis of leukemia,
while less attention has been directed toward examining the role of
the hematopoietic microenvironment(HM) in the initiation and progression of leukemia. As we know, HM can regulate hematopoiesis
through interactions with progenitor cells, hematopoietic cytokines
and the biosynthetic products of stromal and other cells. Acute myeloid leukemia(AML) initiates and progresses in the HM. Encounters between the HM and leukemic cells may affect the apoptosis,
differentiation and proliferation of leukemia cell.The bone marrow
microencironment is presumed to play an essentially regulatory role
in determining the fate of leukemic cells.Objective To investigate
the effects of human bone marrow fibroblastoid stromal cell line
(HFCL) on the proliferation, differentiation and chemosensitivity of
acute myeloid leukemia sensitive HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cell line in vitro co-culture. Methods By
setting up co-culture system of HL-60 or HL-60/VCR cells in direct
contact with HFCL cells, or with HFCL cells separated by transwell,
the cell growth curves were detected by cell counting, cell cycle by
flow cytometery(FCM). Cell differentiation was determined by morphologic observation ability of NBT cells and flow cytometric detection of expression of CD11b, CD14, CD13 and CD33.Exposing HL60 or HL60/VCR cells to different concentrations of topotecan(TPT),
morphologic evidence for apoptosis was determined by Wright-Giemsa and Acridine Orange/ethidium bromide(AO/EB) staining. Cell
S131
cycle, Sub-G1 and Annexin V FITC staining were detected by FCM.
To further study mechanism of HFCL cells on leukemic cells, we
compared the gene expression profiles of HL-60 cells without or
in direct contact with HFCL cells by Affymetrix GeneChip Human
Genome U133 setA. The expression of proliferation cell nucleus
antigen(PCNA), active caspase-3, bcl-2 and Pgp was detected by
Western blot. VEGF levels were evaluated by using commercial
ELISA Kits. Results Compared with leukemic cells alone, the proliferation of HL-60 and HL-60/VCR cells cocultured with HFCL cells
was inhibited. And NBT positive cells increased slightly. The percentage of G1 phase cells of HL-60 or HL-60/VCR cells cocultured
with HFCL cells was higher than that without HFCL cells, and that
of S phase cells was lower. The expression of CD11b and CD14
increased. The expression of PCNA was lower. HL-60 or HL60/VCR
cells treated by TPT were observed to have apoptosis characteristic morphological changes by Wright-Giemsa and AO/EB staining.
The percentage of Annexin V-positive cells and apoptotic cells decreased when they were cocultured with HFCL cells.The proportion of G0/G1 HL-60 or HL60/VCR cells treated with TPT increased
and the sub-G1 was 33.43% or 21.9%, but sub-G1 reduced after
in direct contact with HFCL cells. In the study of mechanism, after
direct contact with HFCL cells for 96h, the expression levels of 582
genes were up-regulated, and 1,323 genes were down-regulated
at least twofold. The expression change in some genes such as
HL14, VEGF was comfirmed by RT-PCR, Northern blot and ELISA,
respectively. Meanwhile, with treatment with TPT in vitro, the expression of activated caspase-3 was reduced and the expression of
bcl-2 increased in HL-60 or HL-60/VCR cells by co-culture of leukemic cells in direct contact with HFCL cells. However, the expression
of Pgp showed no change. Conclusion HFCL stromal cells could
inhibit the proliferation, induce the differentiation of HL-60 and HL60/VCR cells, and prevent TPT-induced apoptosis in HL-60 and HL60/VCR cells via modulation of Bcl-2 and active caspase-3. Many
genes might take part in the influence of HFCL cells on HL-60 cells,
which may give important insights into the interaction of bone marrow stromal cells and leukemic cells.
S132
POSTER SESSION
01.1 ACUTE LEUKEMIAS
001
ANALYSIS OF BONE MARROW LYMPHOPOIESIS
IN CHILDREN WITH ACUTE LYMPHOBLASTIC
LEUKEMIA DURING AND AFTER THE
CHEMOTHERAPY
Kapelushnik, Joseph1 *; Israel, Erena2; Levi, Etai2; Shubinaki,
Giora2
*
Israel - 1 Pediatric Hematology Soroka Medical and Ben -Gurion
U; 2 Hematology Soroka Medical and Ben -Gurion U
Introduction:
Study of lymphopoiesis during and after chemotherapy might
provide new data regarding the recovery of normal hematopoiesis.
Study of early lymphoid progenitors during chemotherapy might be
used for optimization of schedule for autologous BM transplantation. Significant progress had been recently achieved in antigenic
phenotyping of human B cells on different stages of maturation. It
rendered multiparametric flow cytometry as a powerful tool for analysis of BM lymphopoiesis in different hematological disorders
Objective:
The aim of this study was to characterize normal lymphopoiesis in patients with ALL in different phases of chemotherapy. We
used multiparametric flow cytometry to measure early B-, pro-B-,
pre-B, immature B and mature B cells in hematologically normal
childhood and adult BM and to establish reference ranges for normal BM lymphopoiesis.
Métodos y Materiales:
The samples of BM were collected in the end of 1st phase of
induction therapy, prior to re-induction, during or after re-induction,
twice during maintenance, after the end of treatment, and during
recovery.
Results:
Amounts
of
CD19+CD34+CD45dim
pro-B
cells,
CD10+CD19+CD20-CD45dim/+ pre-B cells and CD10+CD19
+CD20+CD45+ immature B cells were dramatically reduced after the
1st phase of induction and remained reduced until the end of therapy. Amount of CD10-CD19+ CD20+CD45++ mature B cells was not
changed after the 1st phase of induction, and declined significantly
after the end of induction therapy. Chemotherapy had variable and
inconsistent effect on the amount of CD19 -CD79a+TdT+CD45dim
early B-cell progenitors. Transient and moderate reduction of early
B-cell progenitors was found in all patients in the beginning of maintenance. Flow cytometric analysis of multidrug resistance (MDR)
by rhodamine 123 expel assay showed that CD10 +CD19+ immature B cells had lower MDR-activity than CD10-CD19+ mature
B cells. Analysis of lymphoid cells from patients with regenerating
BM revealed increased amount of CD19 -CD24+CD45dim B cells.
These cells expressed VpreB (CD179a) proteins hallmarked the
maturation of early B-cell progenitors into pre-B cells. Half of CD19CD24+CD45dim cells were TdT+. Few CD19-CD24+CD45dim B
cells might be also found in BM of patients during chemotherapy,
and most of them were VpreB-
Conclusion:
Our results showed that difference in MDR-activity between
immature and mature B cells might somewhat explain the different effect of chemotherapy on different B cells. Analysis of CD24
differentiation markers on B-cell progenitors showed that their expression seems to accompany the maturation of early B cells into
pro-B cells and occurs prior to the expression of CD19 pan-B cell
proteins .
047
THREE CASES OF LEUKEMIA AFTER IODINE - 131
EXPOSURE IN ONE INSTITUTION
Bendek, G1 *; Fernández, I1; Pavlovsky, S1; Shütz, N1; Nakaschian, P1; Sackmann, F1
*
Introduction: Second malignancies have been described after
treatment of thyroid carcinoma, especially in patients treated with
I131 and radiotherapy. The incidence of leukemia after I131 therapy
has been reported as 2%, the majority being acute leukemia.
Chronic myeloid leukemia (CML) is very rare with only 11 cases
reported until 2005. Most cases reported in the literature have
occurred after cumulative doses higher than 800 mCi.
Objective: review the cases of leukemia in patients with history
of papillary thyroid carcinoma who were treated in our institution.
Materials and method: we revised our records of patients with
both acute and chronic leukemia and selected those with a previous
history of thyroid carcinoma.
Results: case 1: a 39-year-old male with diagnoses of papillary thyroid carcinoma in 2001 had been treated with 300 mCi of
I131. In 2004, after 32 months he developed an acute promyelocytic
leukemia. He was treated with citarabina - idarrubicine - ATRA and
relapsed two years later. Case 2: a 49-year-old female with a history of papillary thyroid carcinoma was treated with 300 mCi of I131 in
1994. Eight years later, he was diagnosed with acute lymphoblastic
leukemia. She received FLAG-IDA and remained in complete remission for 4 years when she relapsed. She obtained a second complete remission with GATLA LLA-00 protocol and was transplanted
in august 2006. Case 3: a 37-year-old female with diagnoses of
papillary carcinoma in 2002 received 300mCi of I131. In March 2005
she developed chronic myeloid leukemia. She started treatment
with imatinib obtaining complete cytogenetic response.
Conclusion: acute or chronic leukemia can develop after I131
treatment and should be kept in mind in the follow up of patients
with thyroid carcinoma.
057
EVALUATION OF NUTRITIONAL STATUS,
ANTHROPOMETRICS, AND TOTAL BODY
COMPOSITION IN CHILDREN DIAGNOSED WITH
ACUTE LYMPHOBLASTIC LEUKEMIA.
Jaime-Perez, JC1 *; Gonzalez-LLano, O; Cantu-Rodriguez, O1;
Herrera-Garza, JL1; Gutierrez-Aguirre, CH2; Gómez-Almaguer,
D3
*
Mexico - 1 Hospital Universitario \”Dr. José E. Gonzalez\” de la
Facultad de Medicina de la UANL; 2 Servicio de Hematologia del
Hospital Universitario de la U.A.N.L.; 3 Hospital Universitario de
Nuevo León
Introduction: Acute lymphoblastic leukemia (ALL) treatment
adversely affects nutritional status through several mechanisms,
including emesis, diarrhea, anorexia, pain, and smell and olfaction
alterations. There are specific nutritional morbility effects due to administration of distinct drugs. Nutritional evaluation should be a fundamental component of the clinical history of any child diagnosed
with ALL.
Objective: The determination of nutritional status at diagnosis of ALL on 102 children of both sexes with a new diagnosis of
standard-risk ALL attending the hematology department of a public
university hospital in Northern Mexico during a period of five years.
Nutritional evaluation included nutritional history, serial anthropometrics, dual energy x-ray absorptiometry (DEXA) and detailed follow-up at each visit.
Materials and method: The nutritional status, with particular
attention to diet composition, of 102 children with ALL was assessed by a clinical nutritionist through a validated questionnaire;
serial anthropometrical determinations were performed. Dual energy X-ray absorptiometry (DEXA) for total body composition analysis
was carried out. Based on their BMI percentile, children were classified in four Body Mass Index (BMI) groups as underweight, normal
weight, at-risk for overweight, and overweight.
Results: Fifty three percent of our patients were boys (54) and
47 percent were (48) girls. Median values included age, 6.0 years;
weight, 23Kg, height, 118cm. Body mass index median value was
16.7. In seventy-eight patients in whom DEXA analysis of body
composition was possible, median body mass was 24,335g; 66.4%
of this amount was from lean tissue, and 23.5% from fat. Bone mineral content was Bone mineral content was 10.6%. The median
bone density was 0.754 g/cm2.
Conclusion: The majority of children with ALL attending our
center were well nourished at diagnosis; their body composition, as
evaluated by DEXA, was within the reference parameters of normality.
066
SAFETY OF INTRATHECAL RITUXIMAB AS
PROPHYLAXIS OR TREATMENT IN CD20+
ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) AND
AGGRESSIVE LYMPHOMA (AL): REPORT FROM A
MEXICAN PILOT STUDY.
Villela, L.1 *; Caballero, R.1; Sierra, J.2; Piedra, J.1; García, H.1;
Mejía, M.D.1; Karpovitch, X.2; Borbolla, JR.3
*
Mexico - 1 Centro Médico ISSEMyM; 2 Instituto Nacional de Ciencias Médicas y Nutrición; 3 School of Medicine, ITESM
Introduction: CNS involvement in ALL, as well as , AL carry a
very bad prognosis. The use rituximab has proved safe while given
to the CNS in some patients with lymphoproliferative disorders expressing CD20 respectively.
S133
Objective: To evaluate the feasibility of intrathecal rituximab in
patients with AL and ALL as prophylactic or treatment agent.
Materials and method: After approval from our ethics committee and obtaining signed informed consent, patients received 25
mg of intrathecal rituximab (IT-R), plus the standard systemic chemotherapy protocol in our department. Side effects were evaluated
according to the NIC toxicity scale.
Results: Nine patients have been enrolled since 2005. Eight
were male and 1 female, median age was 49 years (range 14-72).
A total of 60 IT-R were administered (median 7, range 3-15). Median follow-up was 9 months (range: 3-16 months). One patient had
initial CNS involvement with blastic type mantle cell lymphoma; this
patient received IT-R three times per week, plus two extra doses
after complete CNS remission. The patient achieved a complete
remission after 5 doses of IT-R which continues after autologous
stem cell transplantation. All other patients had risk factors that warranted prophylaxis. All side effects were temporary and no patient
has shown neither neurological nor other late toxicities. Side effects
included: headache 33.3%, temporally limb paresthesias 23.3%,
temporally limb pain 13.3% and nausea 11.6%; the majority (98.3%)
were grade I or II of NIC scale of toxicity.
Conclusion: The use of rituximab as IT-R is possible. We are
starting a phase II study using IT rituximab for prophylaxis and treatment, in a larger group of patients.
Key words: CNS infiltration, treatment, intrathecal, Rituximab.
098
RESULTS IN THE TREATMENT WITH
CHEMOTHERAPY IN ACUTE MYELOID
LEUKEMIAS. EXPERIENCE OF 20 YEARS.
Hernández, C.1 *; Pérez, D.1; Carnot, J.1; de Castro, R.1; Muñío,
J.1; Martínez, C.1; Pérez, G.1
*
Cuba - 1 Hospital “Hermanos Ameijeiras”
Background: Acute myeloid leukaemia (AML) represents 80%
of the total number of acute leukemias affecting human beings and
its prognosis remains very unfavourable. Objective: This paper was
aimed at showing the experience accrued by the hospital in the presentation and treatment of this disease with the use of chemotherapy. Material and Methods: A retrospective study was conducted
among 193 cases diagnosed with AML and treated at the Hematology Service for the last 20 years. Results: Average age of patients
was 42.7 years and the most frequent FAB subtype was M2. Non
M3 AML cases showed a complete remission index of 45.5% for
all the age groups and of 60.4% for under 60 year-olds. The global
survival rate was 8.9% after 5 years of follow-up for patients under
60 years, without survivors for the same length of time in the over 60
years-old group. Complication-free survival was 16.2% in general
at 5 years of follow-up. The patients with M3 AML had a complete
remission index of 75%. Global survival and complication-free survival at 5 years were 40.4% and 62.2% respectively. The 67.7% of
cases died from a disease resistant to treatment or from a relapse.
Conclusions: This study confirms others reports which show poor
outcome of patients with AML when chemotherapy is used as the
only treatment, with the exception of patients with M3 AML.
Key words: Acute myeloid leukemia, complete remission, global survival rate, complication-free survival rate
S134
073
siRNA-INHIBITION OF E2A-PBX1 IN PRE-B
LEUKEMIA CELLS
Casagrande, G1 *; te Kronnie, G1; Basso, G1
*
Italia - 1 University of Padova - Lab. Pediatric Onco-Hematology
Background and objectives. The t(1;19)(q23;p13) chromosomal translocation is detected in ~5-6% of childhood pre-B acute
lymphoblastic leukemias (ALLs) and usually results in E2A-PBX1
gene expression. Since the role of this chimeric gene during leukemogenesis is not yet fully understood, an approach to investigate its
function is to selectively deplete the E2A-PBX1 protein in pre-B leukemic cells and study the consequences of E2A-PBX1 inhibition.
Material and methods. First, we validated the delivery of siRNA
in the 697 pre-B ALL cell line (the cellular uptake was detected by
fluorescent confocal microscopy and FACS analysis). The ability of
the designed siRNAs to reduce E2A-PBX1 mRNA was measured
by real-time RT-PCR, and the E2A-PBX1 protein levels were analyzed 24 hr after transfection by Western Blotting. To better understand the role of E2A-PBX1 in human pre-B cells leukemogenesis
the study focused also on genes whose expression invariantly accompanies the t(1;19) translocation, and their transcription was detected by SYBR Green PCR.
Results. To a decreased E2A-PBX1 expression of 85-90%
corresponded a notable reduction in the protein level. In particular,
E2A-PBX1 silencing affected the EB-1 gene (which encodes for a
protein that could contribute to the transformed phenotype of pre-B
ALL), reducing its expression to 25%. Furthermore, the significant
decrease in the Wnt-16b mRNA levels (and not of the Wnt16a isoform of Wnt16 gene), observed consequently to the fusion gene depletion, confirms the hypothesis of Wnt-16b as target of E2A-PBX1.
The silencing of the fusion protein also induced apoptosis (2-fold
increase of apoptotic cell percent, compared to the mock control).
The effect on apoptosis was confirmed by silencing other two pre-B
ALL cell lines, with and without the t(1;19) translocation.
Conclusions. In summary, targeted-E2A-PBX1 inhibition leads
to an increase in apoptosis and to reduced expression of the EB-1
and Wnt16b genes, suggesting that their aberrant expression might
be a key-step in leukemogenesis in t(1;19)-positive pre-B leukemia.
Keywords: siRNA, leukaemia, fusion gene, E2A-PBX1, Wnt16,
EB-1.
191
DIAGNOSTIC AND THERAPEUTIC ADVANCES IN
ACUTE LEUKEMIAS
Nasouhi pur, S.
*
Iran - 1 University of Tabriz
1*
The acute leukemias account for approximatly 10% of all human cancers. They are a heterogeneous group of disorders in which
malignant clones arise from the lymphohematopoietic stem cell of
the bone marrow or its progenitors. Until recent years acute leukemias: Acute lymphoblastic leukemia(ALL), Acute myeloid leukemia
(AML), Acute biphenotypic leukemia, were almost always deadly.
Today improvements in diagnosis and treatment have dramatically increased survival rates for these disease .Dispit the increased incidance of acute leukemias and related diseases such
as nonhogkin,s lymphoma in some countries respectively due to
atomic bomb and explosions and also most likely due to AIDS, mortality rates for these diseases have remained relatively constant as
the result of improvements in therapy.
The laboratory plays a crucial role in diagnosis and therapy,
and new molecular technologies have greatly improved patient
care.
Currently, diagnosis of leukemias and lymphomas is based on
a combination of methods: clinical feature, microscopic examination of blood, bone marrow, cytochemical and immunohistochemical staning of specimences, immunophenotyping by flowcytometry,
cytogenetics, and molecular analysis , ultrastructural examination
fluorescent in situ hybridization(FISH). The combination of all of
these methods allows the identification and subclassification of
leukemias. Such subclassification is important because the specific
diagnosis guides clinicians in the selection of optimal therapy and
provides prognostic information.
Immunophenotyping by flow cytometry is now of major importance in the diagnosis of leukemia and lymphoma.Immunophenotyping is essential for confirmation of the diagnosis of ALL and acute
Biphenotypic leukemia, in the case of AML with no cytochemical
evidence of myeloid differentation(AML-M0) and in the acute megakaryoblastic leukemia(M7) that blasts have no cytological features
which permit their identification as megakaryoblast, it is essential.
Molecular diagnostic techniques include : Southern blot analysis and the polymerase chain reaction (PCR) for the analysis of
DNA and reverse transcriptasePCR(RT-PCR) for the investigation
of RNA. PCR can detect the presence of one abnormal cell among
a background of 1000 to 1000000 normal cells.
In addition diagnostic advances ,in recent years important advances have been achived in the treatment of patients with acute
myeloid leukemia. However, most of these advances have applied
to young AML patients , while elderly AML patients continue to face
a dismal outcome. Several studies have attempted to explain the
worse prognosis in elderly AML patients on biological grounds.
Complete treatment in children with ALL by current therapies is one
of the greatest achivements of modern oncology. Complete remission CR) rates in adults with ALL are 63%--86%.
In the future ,with continuing research, the molecular basis for
the malignant transformation of blood cells will be elucidated leading to leukemia-specific therapies.At the same time, the technologic
advances outlined in the preceding will allow the clinical laboratory
to keep pace with the scientific understanding of leukemias as well
as enhance diagnosis and monitoring of these diseases.
166
ALL-TRANS-RETINOIC ACID (ATRA) AND
PSEUDOTUMOR CEREBRI (PC) IN TWO YOUNG
ADULT WITH ACUTE PROMYELOCYTIC LEUKEMIA
(APL)
Díaz, L.1 *; Isaurralde, H.1; De Galvez, G1; Nese, M.1
*
Uruguay - 1 FACULTAD DE MEDICINA, UdelaR/ CITMO
PC is a neurological syndrome characterized by signs and symptoms of intracranial hypertension without evidence of infective or
space occupying lesions. ATRA is able to induce complete remission of APL in more than 80% of cases. PC associated with ATRA
have been described in pediatric patients. We report two case
observed in young adult age.
Case 1. 18-year-old female was found to have APL M3, 92%
promyelocytic in bone marrow; Case 2. 18-year-old female was
found to have APL M3, 60% promyelocitic in bone marrow. In both
cases Immunophenotyping using flow cytometry, karyotipe examination t(15;17) and molecular biology PML-RARa were compatible
with this diagnosis.
The administration of ATRA (45 mg/m2) and chemotherapy
(Cytarabine 100 mg/m2 x 7 days with Daunorubicin 45 mg/m2 x 3
days) was started at diagnosis. In case 1, after 8 days the patient
complaine headache and double vision and fundus oculi examination documented papilledema. Computed tomography and magnetic resonance imaging of the head showed no intracranial ab-
normalities, the cerebrospinal fluid was normal but with Increased
cerebrospinal fluid pressure (30 cm H2O). In case 2, after 90 days
the patient complaine headache, vomiting, and fundus oculi examination documented papilledema. Computed tomography and
magnetic resonance imaging of the head showed no intracranial
abnormalities. The cerebrospinal fluid was not examined. Evoked
potential tests were normal. The diagnosis in both cases were PC .
Symptom were improved by temporary ATRA discontinuation. The
patients completed chemotherapy protocol and achieved a complete remission.
190
ADULT T-CELL LEUKEMIA/LYMPHOMA
(ATLL) - DEMOGRAPHICAL, CLINICAL AND
INMUNOPHENOTIPICAL CHARACTERISTICS IN
PERÚ - 1996-2005
Vidal, J1 *; Dyer, R1; Valdivieso, N1; Pizarro, R1; Ferreyros, G1;
Casanova, L1
*
Peru - 1 Department of Pathology, Department of Medicine, Instituto Nacional de Enfermedades Neoplásicas “Eduardo Cáceres
Graziani”, Lima..
We compared the demographic differences of 92 cases of
adult
T-cell leukemia/lymphoma (ATLL), received by the National
Cancer Institute of Lima, Peru between 1996 and 2005. We found
that 52% of the patients came from the southern region of the Andes
where people of quechua origin predominates. The most common
clinical sub-type was acute type 61(66%), followed by lymphoma
29 (32%), chronic 1(1%) with 1(1%) smoldering cases. The female/male ratio was 1:4; the age range varied between 25 to 89
years, with an average of 51 years. The main clinical characteristics were: lymphadenopathy (89%), hepatomegaly (50%), skin lesions (42%) and splenomegaly (34%). Hypercalcemia was seen in
54%. Bone marrow involvement was noticed in 75% and in 67%
showed characteristic blood circulating neoplasic cells. Antibodies
to HTLV-1 virus were found in all cases. Bone marrow flow cytometer studies were contributory in 35 patients: 51% have the classical
inmunophenotype: CD3+, CD4+, CD5+, CD25+; Aberrant phenotype included: 4/35 (11.4%): sCD3- 1/35(2,8%): cyCD3+/sCD3- ;
3/35(8.6%): CD8+; 1/35(2,8%): CD8+/CD4+; 27/32(84,4%): CD25+;
22/31(70,9): TCR a/b and 2/20(10%) co-expressed CD56.
S135
197
TREATMENT RESULTS OF ACUTE
LYMPHOBLASTIC LEUKEMIA (ALL) ON CHILDREN
TREATED IN: ONCOLOGY INSTITUTE “DR.
HERIBERTO PIETER”
Vassallo, R; Paulino, G.1
1
Oncology Institute “Dr. Heriberto Pieter”
PURPOSE OF STUDY:
Determine the results obtained in patients with LLA treated with
BFM- 96 protocol, during the period May 2001- 2005 and compare
them with the of medical literature.
BRIEF DESCRIPTION OF THE PROJECT:
It’s a cohort, descriptive, retrospective study during May 20012005. Patients of both sexes, younger than 16 years old, with diagnosis of LLA were included and treated in the IOHP during this
period. The protocol to evaluate was the BFM-96
RESULTS 31 patients were diagnosed and treated, 70%
came from Santo Domingo, Santiago and Yamasá. 66% were male,
age average, 8 years old (rank 1-15 y.). The clinical manifestation shown more frequently was anemic syndrome (100%) followed
by fever (22%), bone pain (15%), Infectious Processes (11%) and
manifestations of bleeding (8%). The findings to the physical examination more frequently were Hepatomegaly (19%), splenomegaly
(17%), and Adenopathy (14%). 20 patients (60.6%) were L1; 11
ptes. (33.3%) was L2 and 2 ptes. (6%) L3. The inmunophenotype
was perform to 17 patients (51.5%), finding that 10/17 (58.8%) were
pre-B. (p<0.05 %). 5 patients were T type (29.4%) and 2 patient
was B type and pre-pre B type respective.
31 patients were treated with BFM-96 protocol, 30/31 achieves
the induction of remission (96%). 24/31 (77.4%) patients are in complete remission. 25 patients are alive at the time the study finished,
for a global survival of 80,6% and 50% 4 years survival. 6 patients
fell (19.35%), 3 in bone marrow and the rest 3 in CNS, mediastinum
and kidney . In 2 of them it was possible to obtain a second remission (33.3%) 5 of them was dead by failure to treatment.
Conclusion: BFM-96 protocol was effective in the induction of
remission in a 96% of the cases. Although the global survival is 80,
6% to 4 years is only 50%. Means of supp rt should be improved
and the protocol used to induce second remission should change.
S136
POSTER SESSION
02.1 MOLECULAR BIOLOGY
003
021
THE JANUS KINASE 2 (JAK2) V617F MUTATION
IN HEMATOLOGICAL MALIGNANCIES IN MÉXICO
SEROLOGIC DISCREPANCY BETWEEN THE
MOLECULAR BIOLOGY OF RHD NEGATIVE
Ruiz-Argüelles, GJ1 *; Garcés-Eisele, J2; Reyes-Núñez, V3; Navarro-Vázquez, M3; González-Carrillo, ML4
*
Mexico - 1 Centro de Hematologia y Medicina Interna de Puebla;
2
Laboratorios Clinicos de Puebla; 3 Laboratorios Clínicos de
Puebla; 4 Laboratorios Clnicos de Puebla
Arriaga, F1 *; Pajuelo, JC1; Pacielo, ML1; Senent, ML1; Martin, C1;
Carpio, N1; Sanz, MA1
*
España - 1 Hospital Universitario La Fe
Introduction:
In 1999 a bone marrow transplantaion program was started
in México using a non ablative conditining regimen which employs
fludarabine, cyclophosphamide and busulfan
Objective:
To asses the results of allografting individuals with different diseases using the ”Mexican method” to conduct allografting
Patients with different hematological diseases from Monterrey
(México), Puebla (México), México City (México), Valencia (Venezuela), Sao Paulo (Brasil) and Medellín (Colombia) were prospetively allografted using the \”Mexican method\”: oral busulphan, 4
mg / Kg on days - 6 and - 5; iv cyclophosphamide, 350 mg / m2 on
days - 4, - 3 and - 2; iv fludarabine, 30 mg / m2 on days -4, -3 and -2;
oral cyclosporin A, 5 mg / Kg starting on day - 1 until day + 180 and
iv methotrexate 5 mg / m 2 on days + 1, + 3, + 5 and + 11
Results:
We conducted over 300 allografts in patients with different
diseases: Chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplasia, thalassemia
major, relapsed Hodgkin´s disease, Blackfan-Diamond syndrome,
adrenoleukodystrophy, Hunter´s syndrome, aplastic anemia and
several solid tumors. In the whole group, the median granulocyte
recovery time to 0.5 x 109/L was 13 days, whereas the median
the preparative regimen is non-myeloablative, all these patents recovered endogenous hematopoiesis. Approximately 50% of the
allografted individuals developed acute GVHD, and approximately
30% developed chronic GVHD. The median post-allograft overall
survival (SV) has not been reached and the 2000 day overall SV is
54%. The 100-day mortality is 16% and the transplant-related mortality is 20%. In the whole group of patients, the median cost of each
NST was 18 000 USD . The best results were obtained in CML and
aplastic anemia, whereas the worse were obtained in lymphoblastic
leukemia, with intermediate results for myeloblastic leukemia
Conclusion:
The results using the ”Mexican method” obtained initially in
México have been reproduced in other Latin American countries.
The affordability of the procedure seems to be adequate, particularly for developing countries.
Introduction:
The RH locus consists of two genes: RHD and RHCE, both
of them have ten exons. They are 97% identical and are the result
of the gene duplication on chromosome 1p34-36. The antigen D is
determined by the presence of a normal RHD gene. About 15% of
Europeans don´t express an antigen D RHD caused by the RHD
gene deletion. About 1% of Europeans carry RHD alleles with aberrant structures encoding for disminished D inmunoreactivity (weak
D and hybrid antigens).
Objective:
Implications of antigen RHD with aberrant RHD alleles for disminished D immunoreacrivity.
We have studied 212 RHD negative patients with serologic
and molecular biology methods. Their phenotype was 108 dCcee,
53 dcE, 1 dCCe and 50 cde. Method: serologic test group ABO
RHD gel (Ortho and Diana-Gel). Adsortion-elution (Elu-Kit) and test
D partial (Diagast) and DVI (Diamed). Molecular biology: Extraction according to Kit QIAmp DNA Mini Kit (Quiagen). Multiplex RHD
PCR based on amplification of six RHD-specific exons in one reaction mixture. Six RHD-specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7 and 9 from 212 RHD negative blood
donors. Sequencing of 6 exons from genomic DNA: The nucleotide
sequencing was performed as described previously (Wagner et al).
Results:
28 of 212 cases were RHD negative with serologic methods
and RHD positive with PCR, 22 of 28 cases (78%) had the phenotype dCe, 5 (18%) had the phenotype dcE, and 1 case dce. The
exons 3, 4, 5, 6, 7 and 9 were present. Sequencing, 17 patients
showed Cde antigen, the most frequent allele was deletion of eleven nucleotides of exon 3 (5 patients) and 4 patients showed mutation 635G/T in exon 7. Five of 22 with phenotype Cde had antigen
Del (adsortion-elution) sequencing 3 of 5 was mutation of 885G/T in
axon 6, two patients with mutation 1227G/A exon 9.
Conclusion:
The frequency of RHD negative and gen RHD positive and secuencing were similar to other european population. Serologic discrepancy between the molecular biology of RHD is more frequent
in RH Cde (78%) and cdE(18%) genotype. Previous reports have
shown antigen Del in asian population but we have find 5 cases in
our population.
023
CLINICAL FEATURES AND PROGNOSTIC FACTORS
IN MYELOFIBROSIS WITH MYELOID METAPLASIA
(MMM): EXPERIENCE OF ONE INSTITUTION
Sackmann, F1 *; Pavlovsky, S1; Corrado, C1; Pavlovsky, M A1;
Juni, M1; Intile, D1
*
Introduction:
MMM is a heterogeneous disease. Prognostic factors (PF) that
identify patients (pts) at high risk who may benefit with aggressive
treatments are important.
Objective:
describe the pts with MMM and identify PF of survival. Other
end-points were progression free survival (PFS), leukaemia free
survival (LFS), overall survival (OS) and evaluate the role of LILLE
and Cervantes scores.
describe the pts with MMM and identify PF of survival. Other
end-points were progression free survival (PFS), leukaemia free
survival (LFS), overall survival (OS) and evaluate the role of LILLE
and Cervantes scores.
Results:
Nine pts had secondary MMM. Median age was 62 years (25
- 77). Splenomegaly at diagnosis was present in 29 pts. Median
haemoglobin was 10.8 gr/dl (6.3 - 17). Median white blood cells
was 9200 /ml (2800 - 25800). Median platelet count was 274000
(57000 - 4308000). Fifteen pts (41%) had an elevated lactic dehydrogenase (LDH) while median LDH of all the pts was 547 IU/L (177
- 3600). Most pts received more than one treatment. As first line,
7 received erythropoietin, 6 thalidomide, 6 hydroxyurea, 5 splenic
radiotherapy, 3 nandrolone. None of the variables evaluated had
prognostic value for PFS, LFS and OS. PFS, LFS and OS at 5 years
were 20%, 88% and 85% respectively, with a median follow up of
42 months (range 1 - 154). All the pts but 3 could be categorized
by LILLE and Cervantes scores. OS of low risk pts according to
LILLE was 92% and 62% for the intermediate risk (p=0.03). OS of
LR and high risk according to Cervantes score was 88% and 66%
respectively (p<0.05).
Conclusion:
MMM is an heterogeneous disease. LILLE and Cervantes
score are easy to use in clinical practice and are capable to identify
groups with different survival.
075
APOPTOTIC DEATH OF BCR-ABL-EXPRESSING
MYELOID PROGENITORS IN RESPONSE TO MTOR INHIBITOR RAD001 IS PROMOTED BY THE
NUCLEAR IMPORT OF C-ABL.
Mancini, MM1 *; Zuffa, ZE1; Brusa, BG1; Corrado, CP1; Pagnotta,
PE2; Barbieri, BE3; Santucci, SMA1
*
Italia - 1 Istituto di Ematologia e Oncologia medica Seràgnoli-Università di Bologna; 2 Dipartimento di Biochimica G.Moruzzi-Università di Bologna; 3 Istituto di Radioterapia L.Galvani-Università di
Bologna
Nuclear accumulation of c-Abl protein in response to genotoxic
damage addresses apoptotic cell death. The process is driven by
the disruption of its binding to 14-3-3 scaffolding proteins independent from c-Abl catalytic state and conditional upon 14-3-3 phosphorylation by the c-jun N-terminal kinase (JNK) (Yoshida et al Nat
S137
Cell Biol 7,278,2005). Our work moved from the observation that
p210 Bcr-Abl tyrosine kinase (TK) constitutive activation precludes
c-Abl nuclear import in response to gamma irradiation, advancing a
role for residual c-Abl protein “loss of function” in the apoptosis-resistant phenotype of Chronic Myeloid Leukemia (CML) progenitors.
In 32D cell clones transducing a temperature-conditional Bcr-Abl
mutant, active p210 TK is associated with the overexpression of 143-3sigma mostly driven by transcriptional events involving histone
H4 acetylation (but not methylation) status of gene promoter. P210
TK inhibition in response to 24 hour exposure IM is associated with
early downregulation of 14-3-3sigma and followed by c-Abl nuclear
import and commitment to apoptotic death. The two last events are
further enhanced by complementary inhibition of 14-3-3 binding site
by R18 peptide, supporting that the negative impact of p210 TK on
pro-apoptotic function of residual normal c-Abl arises from its effects on 14-3-3sigma. Previous studies proved that the activation
of p38 MAP kinase concurs to IM effects in CML (Parmar et al, J
Biol Chem 279,25345,2004). Interestingly, p38 MAP kinase is also
involved in tuberous sclerosis 2 gene protein (TSC2, also known
as tuberin) phosphorylation and enhanced binding to 14-3-3 possibly promoting the activation of IM-compensatory signals, including m-Tor (Li et al, J Biol Chem 278,13663,2003). Here we show
that m-Tor inhibitor RAD001 (kindly provided by Novartis) at 100 nM
concentration induces c-Abl nuclear import and apoptotic cell death
in 32D cell clones expressing active p210 TK. Both events are further and significantly enhanced by IM association. C-Abl nuclear
shuttling in response to RAD001 and IM combination proceedes
from Akt and p38 MAP kinase inactivating dephosphorylation and
14-3-3 phosphorylation at, a critical residue for client protein binding
including the apoptotic death effectors Bad, Bax and Ask1. In conclusion, our work supports the advantage of TK and m-Tor inhibitor
association for CML treatment.
174
DETERMINATION OF JAK2 V617F MUTATION IN
MYELOPROLIFERATIVE DISORDERS IN URUGUAY.
Lens, D1 *; Muxi, P1; Brugnini, A1; Trías, N1; Pierri, S1
*
Uruguay - 1 Depto Básico de Medicina, Clínica Hematológica.
Depto Clínico de Medicina. Hospital de Clínicas. Facultad de
Medicina.
Polycythaemia vera, essential thrombocytaemia and idiopathic
myelofobrosis are clonal myeloproliferative disorders (MPDs), characterized by excessive proliferation of one or more myeloid lineage
with overproduction of differentiated blood elements. Although there
are strict diagnostic criteria to define MPD subtypes, precise categorization remains a subject of debate and furthermore, it can be
difficult to differentiate some cases from reactive disorders.
During 2005, a single somatic activating somatic point mutation in the tyrosine kinase JAK2 gene was described. The mutation
is a G-C to T-A transversion at nucleotide 1849 of exon 14 resulting
in the substitution of valine to phenylalanine at codon 617: JAK2
V617F.
JAK2 V617F mutation is found in the great majority of patients
with-polycythaemia vera but also in other MPDs BCR/ ABL negatives. JAK2 v617F mutation represents a major breakthrough in understanding the molecular pathogenesis of classic MPD
In this work we will described the detection of this new mutation using a high sensitivity allele-specific PCR assay in 5 patients
with MPDs BCR/ABL negative: One patient with an erythrocytosis
and clinical suspicion for PV, 3 with the diagnosis of polycythemia
vera and one diagnosed as essential thrombocytaemia. The mutation was found in 4 patients. The importance of this mutation on
the diagnosis and treatment of the MPD BCR/ABL negative will be
discussed.
S138
077
089
HEREDITARY HEMORRHAGIC TELANGIECTASIA.
ENDOGLIN AND ALK MUTATIONS AND EFFECT OF
DANAZOL IN 20 PATIENTS WITH LOW FOLLOW
- UP
MOLECULAR BIOLOGY IN HETEROZYGOUS BETA
THALASSEMIA DIAGNOSIS
Fernandez, J(*); Riveros, D; Garay, G; Campestri, R; Garate, G;
Grand, B; Dupont, J; D’Antonio, C; Cacchione, R
(*)
CEMIC
Hereditary hemorrhagic telangiectasia (HHT) is a relatively
uncommon, autosomal dominant disorder characterized by telangiectases that develop in the skin, mucous membranes, and visceral
organs. Mucous localization may seldom bleed profusely, especially
epistaxis and upper gastrointestinal (GI) tract. Effective drug treatment is not well established, and multiple blood transfusions and
endoscopic or surgical procedures may be the ultimate solution to
the frequently bleeding HHT patient. Danazol (DZ) is a weak androgen that has been used in small series of HHT pts with ambiguous
results. Its toxicity profile in long standing administration is now well
known. Twenty pts had HHT with transfusion requirements (TR) and
were treated with DZ at 400-600 mg/daily for the initial three months
and 200-400 mg/daily thereafter as a maintenance treatment. At the
time of initiation of DZ therapy, median age was 54 yr-old (32-73),
10 were female and 10 male, and the median previous TR was 21
RBC units/yr. (2-66). All patients had epistaxis and oral cavity bleeding, with 4 additional upper gastrointestinal tract active bleeding that
were detected in 14 patients in which an upper GI endoscopy was
performed. One patient had a cerebral angioma, surgically treated.
One patient had pulmonary fistula. All patients had some kind of iron
treatment. DZ was the first drug treatment intended to reduce the
HHT bleeding in 12/20 pts. Nine subjects of 12 families were studied for endoglin and Alk mutations. Two had endoglin mutations, 6
had Alk mutations and in 1 pt, no mutation was detected. Median
follow-up was 6.2 years (0.8-14) and 2 pts were lost to follow-up at 4
and 11 years respectively. At three months of DZ therapy, 14/20 pts
(70%) showed a remarkable reduction of bleeding, and in 6 patients
that showed no response, DZ treatment was stopped. In 8 pts (40%)
TR dropped to none and in 6 pts median RT dropped from 22 RBC
units/yr to 10 units/yr. Two responders had a relapse with upper
GI tract bleeding and 1 pts with epistaxis within the first 2 years of
DZ treatment. Attempts to reduce the maintenance dose below 200
mg/daily were related to re-bleeding. None of the long standing DZ
therapy pts. had any significant toxicity. DZ treatment have shown
efficacy and safety in this cohort of HHT patients. Mecanism of action may involve the increase of synthesis or expression of ALK-1
dependent proteins and less likely of endoglin. We propose DZ as a
first line treatment for the transfusion dependent HHT patients.
Bragós Irma Margarita, Raviola Mariana Paula, Milani Angela
Cristina, Noguera Nélida Inés
Cátedra de Hematología. Facultad de Ciencias Bioquímicas y
Farmacéuticas. Universidad Nacional de Rosario. Argentina
High Hb A2 level is a criterion to identify heterozygous β-thalassemia. Nevetheless some carriers could be normal Hb A2 (β-silent) or have a carrier lower level, such as β+ I-6 carriers. We hereby
emphasize the importance of molecular biology to achieve reliable
diagnosis. In a previous work carried on in Rosario and influence
zone we studied six mutations in 124 β-thalassemia carriers (the
most frequent in Italy and Spain which are relevant given our ancestry). No significant differences were observed between MCV and
MHC since its either β0 or β+-thalassemia. Rosatelli in Italy studied
126 carriers and 7 mutations and found significant differences between MCV and MHC, with higher values in case of β+I-6 and –87
mutations. Stefanis did not find consistent correlations in 55 carriers
and the three most common mutations in Greece, i.e., β+I-6, β+I110 and CD39, with Hb, Htc and hematimetric indexes, and a light
MCV increase corresponding to β+I-6. In our study we were able
to identified three individuals bearing β+ I-6 mutation; their Hb A2
was 5.5, 5.6 and 4.9% that is similar to mean values in β+ mutation
carriers. Rosatelli reported HbA2 lower values in heterozygous for
β+ I-6 and β+ I-110 mutations in comparison with β039, β0 I-1 and
β+ II-745 phenotypes, however, these findings could not be related
with mutation severity, since a lower increase was observed in a
smooth (IVS I-6) or in a severe mutation (IVS I-110). Stefanis only
found statistical significant differences in Hb A2 in carriers lower β+
I-6 mutation values, in comparison with patients bearing IVS I-110
and CD39 mutations.
We hereby present a systemic lupus erythematosus patient
with 4.9 x 1012/l RBCs, 11.7 Hb g/dl, 36.8% Htc, 73.7 fL MCV, 23.4
pg MHC. RBC morphology: hypochromic microcytes. Normal iron.
Assuming that a patient could be a beta thalassemia carrier, Hb was
studied by electrophoresis which yielded the following values: HbA2
3.8%; Hb F 1%.
This study was considered not conclusive to support the diagnosis, and due to the bibliographical references analyzed, we
further performed the study of β+ 1-6 mutations by PCR-ARMS,
which provided a positive result and a conclusive diagnosis of beta
thalassemia carrier. Moreover, the presence of alfa 3.7 mutation
(GAP-PCR) that could have influenced the hematimetric indexes
was ruled out.
S139
POSTER SESSION
03.1 ALLOGENEIC TRANSPLANTATION
004
DONOR CELL LEUKEMIA AFTER NONMYELOABLATIVE ALLOGENEIC STEM CELL
TRANSPLANTATION: A SINGLE INSTITUTION
EXPERIENCE
Ruiz-Argüelles, GJ1 *; Garcés-Eisele, J2; Reyes-Núñez, V3; RuizDelgado, GJ4; Ruiz-Argüelles, A3
*
Mexico - 1 Centro de Hematologia y Medicina Interna de Puebla; 2
Laboratorios Clinicos de Puebla; 3 Laboratorios Clínicos de Puebla;
4
Hospital Universitario de Nuevo León
Introduction:
Leukemia relapse occurring in donor cells, so called donor cell
leukemia (DCL) after allogeneic hematopoietic stem cell
transplantation has been reported in the literature in less than
40 cases.
Objective:
To asses the prevalence of donor cell leukemia developing after non-myeloablative hematopoietic stem cell transplantation
In a group of 40 consecutive individuals with acute leukemia
allografted along an eight-year period in a single institution using a
non-myeloabative conditioning regimen developed in our country,
we assesed the prevalence of DCL
Results:
We identified two patients with DCL. Both of them were cases
of B-cell acute lymphoblastic leukemias and by means of both fluorescein activated cell sorting and molecular biology studies, the malignant cells were shown to be of donor origin.
Conclusion:
It is possible that the real prevalence of the donor cell leukemia
has been underestimate. The mechanisms of the malignant transformation of the donor cells are still largely unknown.
005
RESULTS OF THE ”MEXICAN METHOD” TO
CONDUCT ALLOGENEIC HEMATOPOIETIC STEM
CELL TRANSPLANTATION.
Ruiz-Argüelles, GJ1 *; Gómez-Almaguer, D2
*
Mexico - 1 Centro de Hematologia y Medicina Interna de Puebla; 2
Hospital Universitario de Nuevo León
Introduction:
In 1999 a bone marrow transplantaion program was started
in México using a non ablative conditining regimen which employs
fludarabine, cyclophosphamide and busulfan
Objective:
To asses the results of allografting individuals with different diseases using the ”Mexican method” to conduct allografting
Patients with different hematological diseases from Monterrey
(México), Puebla (México), México City (México), Valencia (Venezuela), Sao Paulo (Brasil) and Medellín (Colombia) were prospetively
allografted using the ”Mexican method”: oral busulphan, 4 mg / Kg
on days - 6 and - 5; iv cyclophosphamide, 350 mg / m2 on days - 4,
- 3 and - 2; iv fludarabine, 30 mg / m2 on days -4, -3 and -2; oral
cyclosporin A, 5 mg / Kg starting on day - 1 until day + 180 and iv
methotrexate 5 mg / m 2 on days + 1, + 3, + 5 and + 11
Results:
We conducted over 300 allografts in patients with different
diseases: Chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplasia, thalassemia
major, relapsed Hodgkin´s disease, Blackfan-Diamond syndrome,
adrenoleukodystrophy, Hunter´s syndrome, aplastic anemia and
several solid tumors. In the whole group, the median granulocyte recovery time to 0.5 x 109/L was 13 days, whereas the median platelet
recovery time to 20 x 109/L was 12 days. Around one third of the
patients did not need red blood cell transfusions and also one third
did not need platelet transfusions. In more than 70% of cases the
procedure could be completed totally on an outpatient basis. The
follow up time of the patients ranges between 30 and 2000 days. In
around 8% of individuals there was a graft failure and, since the preparative regimen is non-myeloablative, all these patents recovered
endogenous hematopoiesis. Approximately 50% of the allografted
individuals developed acute GVHD, and approximately 30% developed chronic GVHD. The median post-allograft overall survival (SV)
has not been reached and the 2000 day overall SV is 54%. The 100day mortality is 16% and the transplant-related mortality is 20%. In
the whole group of patients, the median cost of each NST was 18
000 USD . The best results were obtained in CML and aplastic anemia, whereas the worse were obtained in lymphoblastic leukemia,
with intermediate results for myeloblastic leukemia
Conclusion:
The results using the \”Mexican method\” obtained initially in
México have been reproduced in other Latin American countries.
The affordability of the procedure seems to be adequate, particularly
for developing countries.
072
ALLOGENEIC STEM CELL TRANSPLANT WITH TBI/
CY/CYTARABINE CONDITIONING FOR CHILDHOOD
PHILADELPHIA-POSITIVE ALL
Yoo, K.H.1 *; Kook, H.2; Lee, S.H.1; Sung, K.H.1; Koo, H.H.1; Baek,
H.J.2; Hwang, T.J.2; Kang, H.J.3; Shin, H.J.3; Ahn, H.S.3
*
Korea - 1 Samsung Medical Center; 2 Chonnam National University Hospital; 3 Seoul National University Children´s Hospital
Introduction: Allogeneic stem cell transplantation (SCT) is considered the best way to cure the Ph+ ALL. High-dose cytarabine
(HDC) has been used to treat patients with ALL who had relapsed
S140
or had been refractory to the standard induction chemotherapy.
Objective: We postulated that the addition of HDC to the standard conditioning regimen might decrease the relapse rate through
more effective eradication of residual leukemia before transplant.
So, we investigated the feasibility of HDC-containing conditioning
for the treatment of children with Ph+ ALL.
Materials and method: 13 consecutive patients with Ph+ ALL
aged 3.5-15.8 y (median 12.2 y) received allogeneic SCT at our
three cooperative institutions. Two patients (15.4%) were not in
CR1 at transplant. The sources of stem cells were as follows: unrelated BM (n=6), unrelated cord blood (n=5), matched sibling BM
(n=2). The conditioning regimen included HDC (3 g/m2/dose every
12 h x 4 doses), cyclophosphamide (60 mg/kg/d x 2 d), and total
body irradiation (TBI). TBI was delivered in three different manners
according to the each institutional guideline (1,000 cGy/3 Fr/3 d,
n=5; 1,320 cGy/11 Fr/4 d, n=5; 1,200 cGy/4 Fr/4 d, n=3).
Results: Grade 2-4 and grade 3-4 acute GVHD were developed in 6 (46.2%) and 3 (25.0%) patients, respectively. Four patients (33.3%) developed chronic GVHD (2 limited, 2 extensive).
Eleven patients are alive event-free with a median follow-up of 22
mo (range, 7-44 mo).
Conclusion: Our results suggest that adding HDC to the standard TBI/Cy conditioning is feasible in allogeneic HSCT for childhood Ph+ ALL.
Keywords: Philadelphia chromosome, Acute lymphoblastic
leukemia, Cytarabine, Children, Transplantation
031
ALEMTUZUMAB IN THE TREATMENT OF STEROID
REFRACTORY ACUTE GRAFT-VERSUS-HOST
DISEASE
Gómez-Almaguer, D1 *; Ruiz-Arguelles, G2; Gonzalez-LLano, O;
Gutierrez, C3; Jaime-Perez, JC3; Tarin-Arzaga, L3; Giralt, S4
*
Mexico - 1 Hospital Universitario de Nuevo León; 2 Centro de Hematología y Medicina Interna de Puebla; 3 Hospital
Universitario,UANL; 4 MD Anderson CC, USA
Introduction:
Corticosteroids therapy is the mainstay of treatment for GVHD,
however, it heavily impacts on post transplant morbidity and new
modalities are continually needed. Alemtuzumab a humanized
monoclonal antibody to CD52 has been used mainly as GVHD prophylaxis. Only a few patients have been treated with this antibody.
Objective:
Primary endpoints were response to treatment after 14 and 28
days. Secondary endpoints were side effects and incidence of infectious complications.
From December 2004 to May 2006, we recruited 13 steroid
refractory grade II-IV acute GvHD patients in a prospective trial
evaluating the efficacy of alemtuzumab (Campath 1H) after exclusion of other severe HST-related complications. Treatment consisted of Campath 1H 10mg given s.c. on days 1-5. Median age
was 33 years old (range:1-59) years, a fludarabine-based reduce
intensive conditioning (RIC) regimen was used and the hematopoietic cells were obtained from HLA-identical siblings in 12 cases and
one patient received stem cell from umbilical cord blood. All but one
received CSA and MTX for GvHD prophylaxis.
Results:
GvHD was classified as grade II in 5 patients, III in 6 and IV in
2; predominant organ affected was gut in 6 cases, skin in 7, liver in 4
and combination of gut and skin in 4 patients. In 6 of the 13 patients
the clinical manifestations of GVHD were noticed after the first 100
days of HSCT. Complete resolution of GvHD, partial response and
no response were seen respectively in 23%, 62% and 15%. Six
over the 13 patients were able to decrease steroid use. Five patients developed CMV (pp65) reactivation and 3 of them were successfully treated with valganciclovir. All patients maintained complete chimerism during and after alemtuzumab therapy, and after a
median follow-up of 5 months (range, 1- 18months), 8 remain alive,
3 without evidence of GVHD. Five patients died, 3 due to GvHD and
the others due to infectious complications.
Conclusion:
This preliminary study suggests that alemtuzumab is a welltolerated agent and has a beneficial effect in the treatment of refractory GvHD. It is only a pilot study and more studies are needed, but
we suggest that this modality could be used early in the management of these patients in order to improve quality of life and reduce
the long-term side effects of corticosteroids.
059
PROGESTRONE IN VITRO EFFECT ON
HAEMATOPOIETIC PROGENITOR CELLS FROM
UMBILICAL CORD BLOOD AND PERIPHERAL
MOBILIZED BLOOD
Flores-Aguilar, Z.X.1 *; Martínez-Murillo, C.1; Reyes-Maldonado,
E.1
*
Mexico - 1 Instituto Politécnico Nacional
Introduction: The haematopoietic progenitor cell (HPC) transplantation from umbilical cord blood has been in the latest times
another therapeutical option instead of bone marrow or peripheral
mobilized blood transplant. The main advantages of using umbilical
cord blood (UCB) as an alternate source of HPCs are the feasibility
to collect them, risk absences for donor, reduced risk to take infection and low incidence of graft versus host disease. Because of that
it has increased the interest to know the differentiation and maturation mechanisms that interact on HPCs from this source.
Objective: The objective of this work was to determine the effect of progesterone on the proliferation and differentiation of HPCs
obtained from UCB and peripheral mobilized blood (PMB) in clonogenic cultures.
Materials and method: It was obtained 9 alicuots from UCB
and 9 from PMB; the viability of the units was measured using trypan blue and 7-AAD, mononuclear cell count and CD34+/CD45+
determination with flow cytometry. 1 x 104 mononuclear cells from
UCB and 5 x104 mononuclear cells from PMB were inoculated for
each milliliter of culture medium in culture boxes (3 cm diameter),
we used specific culture medium for stem cells (Methocult H3444)
each sample was cultured with different concentration of progesterone:21.5 ng/mL, 39.75 ng/mL, 121.5 ng/mL, 400 ng/mL and without progesterone (control) The cultures were incubated on a CO2
chamber at 37ºC. with different concentration of progesterone.
Results: All the units cultured were on the range between 8599% of viability , the amount of CD34+/CD45+ cells was between
0.020x106 - 6x106/mL. The summatory of the different colony types,
CFU-M 177 CFU-E, 10 BFU-E, 28 CFU-G, 19 CFU-GMM and CFUM of UCB and 456 CFU-E, 10 BFU-E, 90 CFU-G, 370 CFU-GMM
and 80 CFU-M of SPA was the highest on controls and we observed
a decrement between 30-50% with progesterone cultures´ treatment not until development with the 400 ng/mL concentration. We
found significative differences for BFU-E from both sources.
Conclusion: We concluded that progesterone has an inhibitory effect on HPCs from both sources and this can explain why the
cells from PMB are at quiescent phase at the moment of collection
and the mieloprotective effect of high levels of progesterone in patients under chemotherapy.
060
UMBILICAL CORD BLOOD BANKS, AN OPTIMAL
THERAPEUTIC ALTERNATIVE FOR THE
AUTOLOGOUS TRANSPLANT. ANALYSIS OF ONE
THOUSAND PROCESSED BLOOD UNITS.
Flores-Aguilar, Z.X.1 *; Martínez-Murillo, C.1; Pier, D1
*
Mexico - 1 Banco de Cordón Umbilical S.A. de C.V.
Introduction: In the last years the source of hematopoietic
progenitors has been growing. The frequency of hematopoietic
stem cells in cord blood varies between 0.2-1%. The first transplant of cord blood was performed in 1988 between HLA identical
siblings for the treatment of Fanconi´s anemia, achieving a complete reconstitution of the lymph and hematopeietic systems. Until
then, the knowledge of the biological characteristics of the umbilical
cord blood (UCB) has been growing and the advantages for its use
in transplants are increasing. Among the principal advantages of
using UCB as a source of hematopoietic progenitors we can find:
the ease of collection, lack of risk for the donor, reduction in the
transmission of infection, easy accessibility of the cryopreserved
samples and less incidence of graft vs. host disease, because of
the immaturity of the newborn immune system.
Objective: To describe the collection and storage process that
is done in the Umbilical Cord Bank, Banco de Cordón Umbilical in
Mexico City.
Materials and method: CRYOPRESERVATION. Processing
of the UCB was done in a biosafe class III system, the cryopreservation was done in a freezing chamber (Cryomed) following a decrease in temperature of -1°C per minute and the storing of the
units was done in Nitrogen tanks in vapor phase at a temperature
of - 195°C . A count of mononucleated cells was done after cellular
recuperation as a measure of quality control as well as a count of
CD34+/CD45+ cells by flow cythometer (FacsCalibur).
PARALEL BANK. Serological tests where performed on all
units from the mother´s serum for the markers of: HIV, VHB; VHC
, Brucella, VDRL and Tripanosoma cruzi. Sterility tests where performed pre and post freezing.
Results: Up to know we have processed one thousand two
hundred units of UCB for autologous transplantation, the average
volume collected was 68 ml, none was positive for the markers
mentioned above, the average number of mononucleated cells was
2.5 X108 and of CD34+ cells was 1.3X106 .
Conclusion: The collection and storage of UCB units in blood
banks that have a national accreditation (NOM) with the right infrastructure, that follow international standards (NETCORD), are
a useful alternative for the Mexican population which has become
interested in using this options to guarantee the possibility to have
UCB stored to use in transplants.
150
HEMATOPOIETIC STEM CELL TRANSPLANTATION.
(HSCT) IN ADULT ACUTE LEUKEMIAS (AL)
De Castro, R.1 *; Carnot, J.1; Muñío, J.1; Pérez, G.1; Martínez, C.1;
Hernández, C.1; Pérez, D.1
*
Cuba - 1 Hospital ”Hermanos Ameijeiras”
Background: HSCT is an important procedure in treatment of AL in
adults. Allogenic HSCT yields best results than autologous, but the
latter could be done if no suitable donor is available. Objectives:
To show experience in our hospital in transplanting 48 adults with
AL. Material and Methods: 48 adults treated in our hospital for AL
who obtained remissions underwent either allogenic or autologous
HSCT, the latter were performed without criopreservation, keeping the bone marrow (BM) or peripheral blood (PB) at 4 degrees
for not more than 56 hours. AML patients 37 and ALL 11. 35 patients in 1CR, 11 in 2CR,1 inPR and 1 in relapse. BMT 42 and PB
S141
SCT 6 .Autologous 33 ,allogenic 14, singenic 1. AML: Age 16-52
years,median 37,2. FAB classification M1 5, M2 8, M3 8, M4 11,
M5 2, M7 1, not classified 2. Time from remission to transplant 1-16
months, median 8,9. Autologous 24, Allogenic 12. BM 9, PB 3 (1
related, 1 not related, 1 not myeloablative) 1 singenic.. Conditioning
regimens are shown. ALL: Age 16-39 years, median 28,2. FAB L1
3, L2 8, Phenotype B 3, T 2.Time from remission to SCHT 0,7-19
months, median 8.Autologous 9 (BM 7, PB 2) Allogenic 2, both
BMT. Conditioning regimens showed. Results (AML): Hematopoietic recovery within normal limits. GvHD present in 7 out of 12 cases (
58,3% allogenic). 2 deaths from GvHD (16,7%). Transplant Related
Mortality (TRM) 7 (18,9%).Relapses 19 / 37 (51,3%.) Deaths 28 /
37 (75,6%). Follow up period between 1 and 18 years In allogenic
transplants Disease Free Survival (DFS) = 61% at 4 years and
Overall Survival (OS)=55% at 4 years. In autologous, DFS=24%
at 4 years and OS=34% at 4 years. (ALL):Hematopoietic recovery
within normal range. GvHD was present in both allogenic transplants in grade I-II No mortality . Relapses 4 / 11 (36,3%) Autol. 3 /
9 Follow up period 1 to 18 years. Deaths 6 / 11 (54,5%) TRM 1
(9,09%) DFS Autologous = 61% at 4 years OS Autologous= 60% at
4 years Allogenic only 2 cases. Conclusions: In AML, results of allogenic HSCT acceptable but much better than in autologous. TRM
high. In ALL very few cases but good results in autologous HSCT.
Key Words: Hematopoietic Stem Cell Transplantation (Autologous and Allogenic) - Acute Leukemia (Myeloid and Lymphoblastic)
- Disease Free and Overall Survival.
120
OUTCOMES OF TRANSPLANTATION WITH
RELATED DONOR (RD) AND UNRELATED DONOR
(URD) STEM CELLS IN CHILDREN WITH SEVERE
THALASSEMIA
Hongeng, S.1 *; Pakakasama, S.1; Chuansumrit, A.1; Sirachainan, N.1; Kitpoka, P.1; Ungkanont, A.1; Jootar, S.1
*
Tailandia - 1 Ramathibodi Hospital, Mahidol University
Introduction: Allogeneic stem cell transplant (SCT) from an HLA
identical family donor is an accepted option for severe thalassemia.
However, the availability of matched RD is only 30%. Therefore,
unrelated donor is another option.
Objective: The aim of this study is to explore the outcomes of
allogeneic SCT with RD and URD in our institution.
Materials and method: We studied 59 consecutive patients
(pts) who received RD (n = 36) or URD (n = 23) stem cells (SCs)
for severe thalassemia between September 1992 and September
2006.
Results: The characteristics of both groups were followed.
The conditioning regimen consisted with busulfan 16 mg/kg,
cyclophosphamide 200 mg/kg for RD and additional ALG (Fresineus) 40 mg/kg for matched unrelated donor (URD) and CBT and
busulfan 8-12 mg/kg, fludarabine 175 mg/m2, ALG, + thiotepa, and
+ total lymphoid irradiation for NST. GVHD prophylaxis was cyclosporin (CSA) and MTX for RD, FK 506 and MTX or MMF for URD,
cyclosporin, and prednisolone for CBT, and FK 506 or CSA and
MMF for NST.
Result: The 2 yr thalassemia free survival rate for RD group
(gr.) is 81% and URD gr. 70% (p = 0.6). The 2 yr overall survival
rate for RD gr is 95% and URD gr. 82 % (p = 0.5). Two (5%) pts of
RD gr. and 3 (13%) of URD gr. had severe acute GVHD grade 34. Three pts of RD gr. had chronic GVHD (2; limited, 1;extensive).
Three pts of URD gr. had limited chronic GVHD. Rejection rate of
RD gr. is 13% (n=5). Two of 5 RD pts who had graft failure after first
transplant were successful for second transplant. Rejection rate of
URD gr. is 13% (n=3).
Conclusion: The outcomes of transplantation with RD and
URD stem cells in children with severe thalassemia are both equally
favorable. Further study should be investigated in a larger group of
patients to confirm our findings.
S142
POSTER SESSION
04.1 ANEMIAS
006
NOSOGRAPHIC PERFORMANCE OF THE RED
CELL DISTRIBUTION WIDTH (RDW) FOR THE
DIAGNOSIS OF THALASSEMIA
RUIZ-REYES, G1 *; RUIZ-ARGUELLES, GJ2; GUZMAN-GARCIA,
MO1; RUIZ-ARGUELLES, A1
*
Mexico - 1 Laboratorios Clinicos de Puebla; 2 Centro de Hematología y Medicina Interna de Puebla
Introduction:
The definite diagnosis of thalassemia is based upon relatively
complex laboratory tests, hence, these syndromes might be underestimated in the routine clinical setting.
Objective:
To analyze the nosographic performance of the Red Cell Distribution Width for the presumptive diagnosis of thalassemia.
500 consecutive individuals identified in Laboratorios Clínicos de Puebla with red blood cells showing either hypochromia
(MCH<24 pg) and/or microcytosis (MCV <75 fl in women or <80 fl
in man), with or without anemia, were prospectively accrued in this
study, along a 16 month-period. Iron deficiency, b and a-thalassemia were searched by definite methods.
Results:
Out of the 500 consecutive cases with red blood cell hypochromia or microcytosis, 394 ( 78.8%) were found to have iron deficiency, 37 cases had b-thalassemia, 11 cases had a-thalassemia, while
in 58 cases (11.6%) a definite diagnosis could not be established.
Red cell distribution width (RDW) was significantly lower in the thalassemic patients than in the iron deficient group, and it proved to
bear high nosographic sensitivity and specificity for the diagnosis of
either a or b thalassemia.
Conclusion:
The thalassemic syndromes should be suspected in individuals with red blood cell microcytosis and / or hypochromia, with or
without anemia, showing very low RDW values. These individuals
should be further tested for thalassemia.
010
ANTI-U IN A SICKLE CELL TRAIT PREGNANT
PATIENT : CASE REPORT
Garcia, M1 *; Climent, C2; Moctezuma, A3; Vazquez, I4
*
Puerto Rico - 1 Department of Pathology and Laboratory Medicine, UPR Medical Sciences Campus; 2 University of Puerto Rico;
3
Medical Sciences Campus; 4 Department of Pathology and
Laboratory Medicine
Introduction:
A 32-year old black female with sickle cell trait, G2P1A0, is admitted to the hospital for labor. She had a 39 week gestational age,
and no prenatal care. There no history of systemic illness, intra-
venous drug abuse or previous transfusions. Her hemoglobin was
13.8 g/dL. As part of her pre surgical protocol, a type and screen
was ordered. The patient was group B, Rh negative with a positive indirect antibody test. The antibody reacted with all the reagent
cells of the panel except autologous cells, indicating the presence
of an alloantibody against a high frequency RBC antigen. The phenotype of the patient was significant for S-s-. At this time anti-U
was suspected and the sample was sent to a reference laboratory
that confirmed anti-U specificity. The baby RBC\’s phenotype was
identical to the mother and the direct antiglobulin test was negative,
excluding the possibility of hemolytic disease of newborn. Anti-U
is a rare red blood cell antibody that has been found exclusively in
blacks. The U antigen belongs to the MNS complex system of over
40 antigens carried on two glycophorin molecules. The M, N, S, s
and U antigens are the most important antigens of this system. The
antibodies to S, s and U antigens are capable of causing hemolytic
transfusion reactions and hemolytic disease of the newborn. Red
cells that lack S and s may be negative for a high incidence antigen
U, and persons who lack U make anti-U after sensitization. The
frequency of S-s-U- phenotype among black population is less than
1% and has not been described in Caucasians. Whenever an antibody against a high frequency RBC antigen is identified on black
pregnant women, anti-U must be rule-out.
016
IMPLEMENTING NEONATAL CORD BLOOD
SCREENING WITH HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY IN THE DIAGNOSIS OF
HAEMOGLOBINOPATHIES: INTERIM ANALYSIS
Al Zadjali, S1 *; Al Kindi, S1; Al Tobi, F1; Al Haddabi, H2; Al Abri,
Q1; Al Madhani, A3; Krishnamoorthy, R4; Pathare, AV1
*
Omán - 1 Sultan Qaboos University Hospital; 2 Sultan Qaboos
University; 3 New Sohar Hospital; 4 Inserm U763,Hopital Robert
Debre
Introduction:
High performance liquid chromatography [HPLC] is a powerful
tool to screen newborns for haemoglobinopathies.
Objective:
The aim of the study was to ascertain the feasibility of cord
blood screening in the Sultanate of Oman in an effort to determine
the prevalence of haemoglobinopathies by a cost-effective method.
Neonatal screening includes cord blood samples collection,
screening and follow up of all newborns with abnormal results. A
total of 5176 consecutive cord blood samples were screened for
presence of possible haemoglobinopathies by HPLC using Biorad
Variant ÉÉ program between April 2005 & August 2006. Complete
blood counts [CBC] were also obtained on Cell Dyn 4000 automat-
S143
ed blood cell counter. All samples were then processed to isolate
and store mononuclear leukocytes for subsequent molecular diagnostics.
Results:
The findings indicated a 37.33% incidence of á-thalassaemia,
based on significant amounts of Hb Barts on HPLC and low mean
cell volume [MCV] & mean cell haemoglobin [MCH] on the CBC.
Furthermore, the overall incidence of other haemoglobinopathies
was 10.28%, with 5.64% incidence of sickle haemoglobin. On
HPLC, D-window, E-window and C-window were present in 1.02%,
0.77% and 0.09% of the samples respectively. Since HPLC cannot diagnose beta thalassemia major at birth, in samples with HbA
below 10%, the beta globin gene was directly sequenced including the promoter, all exons and introns in the abnormal samples.
[n=143] Additionally, direct sequencing of abnormal samples with
HbS,[n=292] HbD,[n=53], HbE[n=40] and HbC[n=5] was also performed on ABI Prism 3100 analyzer to assign the genotype status to
these subjects and was used to validate the HPLC results.
dialyzed patients. In hemodialyzed patients hepcidin, correlated
significantly with triglycerides, albumin, creatinine, urea, residual
renal function and hsCRP. Multiple regression analysis in hemodialyzed patients showed that hepcidin was independently related
to creatinine, triglycerides and residual renal function. In patients
with chronic renal failure hepcidin, correlated significantly with total
protein, albumin, creatinine, and eGRF. In the healthy volunteers
hepcidin was related to triglycerides and ferritin.
Conclusion:
Oman with a population of varied ethnicity, high rates of consanguinity and inter-cousin marriages, has an increased prevalence
of haemoglobinopathies. Although the prevalence of a-thalassaemia is high, it is not a clinically significant problem with only occasional cases of HbH. Between group differences were significant for
RBC count, MCV, MCH, MCHC and the red cell distribution width
(RDW), which along with Hb Barts, and HPLC results can successfully predict the correct underlying diagnosis. Key Words: Neonatal,
Cord Blood, Screening, HPLC, Haemoglobinopathy
036
035
Introduction:
In kidney transplant recipients endothelial dysfunction and atherosclerosis are almost universal, as well as cardiovascular complications. Inflammatory markers have been shown to play a role
in the pathogenesis and progression of atherosclerosis, regarded
as a chronic inflammatory condition. Iron metabolism is disturbed
in chronic inflammatory diseases i.e, atherosclerosis. Hepcidin
(liver-expressed antimicrobial peptide, LEAP-1) is an acute phase
reactant protein produced in the liver, with intrinsic antimicrobial activity.
HEPCIDIN: A LINK BETWEEN ANEMIA AND
INFLAMMATION IN PATIENS WITH CHRONIC
RENAL FAILURE?
Malyszko, J1 *; Malyszko, JS2; Pawlak, K1; Brzosko, S2; Rams,
L1; Mysliwiec, M2
*
Polonia - 1 Medical University; 2 Department of Nephrology and
Transplantology
Introduction:
Hepcidin is a small defensin-like peptide whose production by
hepatocytes is modulated in response to anemia, hypoxia or inflammation. Both anemia of renal disease and anemia of chronic inflammation are commom in renal failure.
Objective:
Hepcidin correlations with markers of iron status, erythropoietin therapy and markers of inflammation in patients with chronic
renal failure on conservative treatment, maintained on peritoneal
dialyses, on hemodialyses and in the healthy volunteers.
Iron status (serum iron, total iron binding capacity-TIBC, ferritin, total saturation of transferin-TSAT), complete blood count,
creatinine, albumin, serum lipids were assessed using standard
laboratory methods. Hepcidin, soluble receptor of transferin-sTFR
and high sensitivity CRP were measured using commercially available kits.
Results:
Serum iron, TIBC, TSAT, erythrocyte count, Hb, Ht, platelet
count, albumin were lower in peritoneally dialyzed patients when
compared with the control group. Ferritin and hepcidin were significantly higher in chronic renal failure, peritoneally dialyzed and
hemodialyzed patients relative to healthy volunteers. Hepcidin correlated positively with albumin, ferritin, iron, transferin saturation,
hsCRP and sTFR and negatively with erythrocyte count, MCV in
peritoneally dialyzed patients. In multiple regression analysis albumin, ferritin and hsCRP were predictors of hepcidin in peritoneally
Conclusion:
Elevated hepcidin levels in peritoneally dialyzed patients may
be due to. Elevated hepcidin in all groups of patients studied may
be due to functional iron deficiency and anemia as well as low grade
inflammation, frequently encountered in this population and mainly
to impaired renal function. Further studies are needed to evaluate
the hypothesis of hepcidin being a molecular link between anemia,
and inflammation in patients with kidney diseases.
HEPCIDIN, AN ACUTE PHASE PROTEIN AND
A MARKER OF INFLAMMATION IN KIDNEY
TRANSPLANT RECIPIENTS WITH AND WITHOUT
CORONARY ARTERY DISEASE
Malyszko, J1 *; Malyszko, JS2; Pawlak, K1; Mysliwiec, M2
*
Polonia - 1 Medical University; 2 Department of Nephrology and
Transplantology
Objective:
Cross-sectional study was performed to assess possible relations between hepcidin and inflammatory markers in kidney transplant recipients with and without coronary artery disease.
Iron status, complete blood count, creatinine, albumin, lipids
were assessed using standard laboratory methods. GFR was estimated using MDRD formula. Hepcidin, high sensitivity CRP, IL-6
TNFá, soluble receptor of transferin-were measured using commercially available kits.
Results:
Kidney transplant recipients with CAD were older with higher
hepcidin, hsCRP, IL-6 and TNF alpha, sTFR, ferritin, and lower cholesterol than patients without CAD. In kidney transplant recipients
hepcidin, correlated significantly, in univariate analysis, with total
protein, ferritin, time after transplantation, creatinine, eGFR (simplified MDRD), cholesterol, neutrophil count, hsCRP, IL-6 and tended
to correlate with TNF á. Multiple regression analysis showed that
hepcidin was independently related to GFR, ferritin, cholesterol and
hsCRP.
Conclusion:
Elevated hepcidin in kidney allograft recipients may be due not
only to impaired renal function, but also to low-grade inflammatory
state (as reflected by hepcidin correlations with hsCRP, IL-6 and
ferritin).
S144
063
APLASTIC CRISIS OF HEREDITARY
SPHEROCYTOSIS DUE TO PARVOVIRUS B19
INFECTION
Lee, K.S.1 *; Lee, J.H.1; Choi, J.H.1
*
Korea - 1 Kyungpook National University School of Medicine
Introduction: Hereditary spherocytosis (HS) is the commonest cause of inherited hemolytic anemia in Korea. In HS patients,
parvovirus B19 infection causes transient severe anemia, so called
aplastic crisis.
Objective: This study designed to characterize the clinical
features and laboratory findings of HS patients with and without
aplastic crisis, and define the serologic responses of parvovirus B19
infection in HS patients who presented with aplastic crisis.
Materials and method: We reviewed the medical records of
the patients with HS visited at Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea from June 1995 to
Feburary 2006. HS was diagnosed as anemia with reticulocytosis,
negative Coombs’ test, indirect hyper-bilirubinemia, spherocytosis,
positive osmotic fragility test and splenomegaly. Aplastic crisis was
defined as fever, abrupt onset of severe anemia and reticulocytopenia. And human parvovirus (HPV) B19 infection was proven by the
presence of Ig M antibody to HPV B19 and/or the detection of virus
DNA using the PCR technique.
Results: Thir-teen cases were diagnosed as HS. 6 were
boys and 7 girls, the mean age at diagnosis was 3.9 years (range:
0.2~8.3 years), and family history was positive in 10 cases (76.9
%). One case was showed aplastic crisis as initial presentation and
was confirmed as HS in recovery phase. Another one case was
diagnosed as HS at previous hospital. The mean hemoglobin (Hb)
was 9.5 g/dL (range: 7.9~11.1 g/dL), the mean reticulocyte count
was 11.9 % (range: 6.0~25.4 %), the mean reticulocyte index (RI)
was 4.3 (range: 2.7~8.5) and the mean indirect bilirubin was 2.6
mg/dL (range: 1.0~6.6 mg/dL) at initial diagnosis as HS in 11 cases. All cases had spleno-megaly (mean size: 2.0±0.4 cm) and 3
cases (27.3%) also had hepatomegaly (mean size: 0.7±0.2 cm).
Osmotic fragility test was performed at 7 cases (53.8%) and all of
them showed increased osmotic fragility compared with control (the
mean NaCl concentration(%) was 0.60±0.03% at begin of hemolysis and 0.45±0.02% at complete of hemolysis). Aplastic crisis was
seen in 9 patients (69.2 %) at the mean age of 7.0 years (range:
5.3~10.8 years) and there was no second episode at same patient.
In aplastic crisis patients, the mean Hb was 4.3 g/dL (range: 2.8~6.8
g/dL), the mean reticulocyte count was 1.6 % (range: 0.5~4.6 %),
the mean RI was 0.2 (range: <0.1~0.8) and the mean indirect bilirubin was 2.1 mg/dL (range: 0.6~4.9 mg/dL). And liver (mean: 2.9±0.7
cm) and spleen size (mean: 5.0±0.8 cm) were increased. HPV B19
IgM and/or HPV PCR were positive in all aplastic crisis patients.
Splenectomy was performed in 7 cases (53.8 %) at the mean age
of 7.7 years (range: 6.3~10.2 years).
Conclusion: There were 13 cases of hereditary spherocytosis
and 9 cases of aplastic crisis due to parvovirus B19 infection. We
have to inform the patients and their family about the importance of
aplastic crisis with ubrupt onset of severe anemia.
Keywords ; hereditary spherocytosis, aplastic crisis, parvovirus
B19 infection
019
RITUXIMAB MONOTHERAPY FOR COLD
AGGLUTININ DISEASE. REPORT ON 18 PATIENT
FROM A SINGLE INSTITUTION
Arriaga Chapper, F1 *; Jarque, I1; Paciello, ML1; Cantero, S1; De
La Rubia, J1; Sanz, GF1; Sanz, MA1
*
España - 1 Hospital La Fé. Servicio de Hematología
Background: Cold agglutinin disease (CAD) is an acquired
autoimmune hemolytic anemia mediated by cold-reactive autoantibodies carbohydrate antigens, causing hemagglutination, complement-mediated hemolysis and C3d positive direct antiglobulina test
(DAT). Clinically is characterized by chronic anemia, episodes of
acute hemolysis occur after cold exposure. Conventional therapias
for CAD are largely ineffective, but remissions after treatment with
the anti-CD20 monoclonal antibody rituximab are increasingly being reported. Patients and Methods: A total 18 CAD (12 women, 6
men) with a median age of 48 years (20-86 years) were treated in
our center between may 2002 and may 2006, CAD was idiopatic
in 8 cases and associated with other conditions 10 (systemic lupus erythematous in 5, chronic lymphoproliferative disorder in 3,
and unrealated donor cord blood transplant in 2). Seven patients
have received previous therapy with corticoids and others therapy,
without response. Hemoglobin concentration before treatment
ranged from 4,9-10g/dL. Median IgM anti-I titers was 1/512 (range
1/128-100.000). Rituximab was given as single agent in doses of
375m/m2 on days 1, 8, 15, 22. Complete remision (CR) was defined as at 1,5g/dl increase of hemoglobin concentration together
with <10 g/dL with a 30% reduction of titer anti-I and reticulocytes.
Results: The overall response rate was 61% (11 patients), with 9
patients achieving complete remission (50%) and PR in two cases.
Of the 7 non-responders (38%). Median duration of response was
24 months (5-48). Of the 7 non responders, 5 died from disease
progression and 1 died for hemolysis and 1 remains alive with transfusion dependence. No serious infusion-related adverse events occurred with rituximab.Conclusion: Rituximab is a safe and effective
therapeutic option and should be considered as first-line for patients
with CAD.
058
HEMOGLOBINOPATHIES PREVALENCE IN
PATIENTS WITH HEMATOLOGICAL NEOPLASIA
UNDER CHEMOTHERAPY IS THE SAME AS
GENERAL POPULATION IN NITEROI, RIO DE
JANEIRO
Kang, HC1 *; Cardoso, C1; Santos, IMAA1; Lusis, MKP1
*
Brazil - 1 Universidade Federal Fluminense
Introduction: Sickle cell anemia, arised from a point mutation in b globin gene, is well studied and important progresses were
done, increasing the survival from16 years in the beginning of 20th
century to over 60 in 21th century. The evolutionary advantage for
survival in the endemic areas of malaria, sickle trait, has interested
to many researchers, because was associated with sudden death,
in study carried through american recruits. Ocasionally appear reports about association of hemoglobinopathies with other disorders
and still has controversy in literature about sickle trait be or not be
an health problem. Interesting case reports of patients with association of hemoglobinopathies and hematological neoplasias rised
a question about the co-morbidity in our population, in Niterói, Rio
De Janeiro, in wich, previous study show prevalence in 5,1 % of S
hemoglobin, including homozigotes and in Rio de Janeiro state the
prevalence is 3,9%.
Objective: Evaluate the frequency of hemoglobinopathies in
200 patients with hematolgical neoplasias under chemotherapy.
Materials and method: The study, approved for the ethics committee of HUAP-UFF was carried through in 200 patients
of Clinical Hematology Service. The design study focused on a
selected population and used haemoglobin electrophoresis on cellulose acetate in alkaline buffer. Briefly, blood samples were colected with EDTA, centrifugued, plasma dischaged and erythrocytes
washed two times with saline solution. The erythorcytes was hemolized with 1% saponin, applied on cellulose acetate strip, previously
humidified with TRIS buffer, pH 9,1. After 45 minutes, 245 volts, the
strip was diped in Ponceau S, discoloured with Acetic Acid 5%, fixed
with methanol and transparentizased. The electrophoresis system
and densitometer was purchased from Biosystems®.
S145
Results: Our results show same prevalence of S haemoglobinophaties as in general population, 3,5% and was found one (0,5%)
C hemoglobin trait and one with SC (0.5%) heaemoglobinopathy.
However, their distribution inside of the groups was not homogeneous as espected, when lymphocytic neoplasias was 67% of our
population, and in literature we find more association with lymphocytic neoplasias. Our data show higher frequency of associations
with chronic myeloprolyferative disorders. As additional data we
observed 15.5% of thalassemia beta. .
Conclusion: The hemoglobinopathy do not change the frequency of neoplasia. More investigation need to be performed about
the implications of the presence of sickle trait in genesis, diagnosis
and prognosis of patients with hematological neoplasias.
S146
POSTER SESSION
05.1 MONOCLONAL GAMMOPATHIES AND RELATED
DISORDERS
022
125
PRONOSTIC FACTORS FOR MALIGNANT
TRANSFORMATION IN MONOCLONAL
GAMMOPATHY OF UNDETERMINED
SIGNIFICANCE (MGUS): EXPERIENCE OF ONE
INSTITUTION
POEMS SYNDROME: CLINICAL CASE.
Sackmann, F1 *; Pavlovsky, S1; Pavlvovsky, M A1; Corrado, C1;
Pavlovsky, M2; Fernández, I1; Mountford, P1; Pavlovsky, A1; Pizzolato, M2; Alejandre, M2; Juni, M1
*
Argentina - 1 FUNDALEU; 2 Centro de Hematología Pavlovsky
Introduction:
MGUS has a prevalence of 1 to 3%. It has an indolent evolution, but some patients (pts) will develop a malignant neoplasm.
Thus, factors that identify pts who will progress are important.
Objective:
asses the value for malignant transformation of simple haematological or biochemical parameters detected at diagnosis. Rate of
transformation, progression free survival (PFS) and overall survival
(OS) will also be calculated.
This is a retrospective analysis of 236 pts with MGUS. PFS
and OS were calculated using the Kaplan Meier method and the
curves were compared with the log-rank test. The identification of
prognostic factors was made using Cox models.
Results:
Median monoclonal component (MC) was 0.6 gr/dl. In 74% of
the cases it was IgG, 17% IgA, 9% IgM and 0.5% biclonal. The light
chain was Kappa in 61% of the pts. Bence Jones was detected
in only 12%. Uninvolved immunoglobulins (UI) were reduced in
20% of the pts. Bone marrow analysis were performed in 64 pts.
Median bone marrow plasma cells was 4%. Median haemoglobin
was 13.3 gr/dl. Lactic deshidrogenase, albumin, B2microglobulin
and erythrocyte sedimentation rate were normal in 97%, 87%, 53%
and 42% respectively. With a median follow up of 58 months (6.2
- 375 months), 17 pts (7%) evolved to a malignancy (15 multiple
myeloma, 1 NHL, and 1 amyloidosis). The cumulative probability of
transformation to malignant neoplasm was 12% at 10 years. PFS
and OS at 10 years 88% and 95%, respectively. MC concentration
(p = 0.04, HR 4.73, CI 1.06 - 21) and reduction of UI (p = 0.01, HR
5.8, CI 1.48 - 22.7) had prognostic value for progression.
Conclusion:
MC concentration and UI level at diagnosis identify a subgroup
of pts with higher risk of transformation
TOPOLANSKY, L1 *
*
Uruguay - 1 CAMDEL
Objective: to report an unusual disease, and update this pathology.
Case: 48 year old male with diagnosis of chronic inflammatory
demyelinating poliradiculopathy (CIDP) in 2000. Add strength loss,
lower extremities oedema, darker skin, erectile dysfunction in 2005.
Physical exam: hiperpigmentation, palpebral oedema, gynecomastia, splenomegaly G1, lower extremities oedema, distal extremities
weakness with areflexia and sensibility loss. Laboratory: WBC 9,4 x
109/l; Hb: 16g/dl, Plts: 537x109/l. Ca: 9.8 mg%; Creatinine: 1mg/dl;
serum protein electrophoresis and immunofixation: PT 6.8 g%,
Alb: 3.1 g%; and ?: 1.4g%; small amount of serum M protein Ig
G lambda, in beta globulins region. Crioglobulinemia: negative.
Testosterone: 139 ng/dl. Uroproteinelectrophoresis and uroimmunofixation : small amount of Ig G lambda in beta globulins region.
Rx: sclerotic lytic lesion in 10th left rib and sclerotic lesion in 11th left
rib. BMO: normal. ß 2 microglobulin 2190 ug/l. POEMS is caracterized by neuropathy, serum M protein, organomegaly, endocrin
abnormalities, skin changes, papiledema, ascites, thrombocytosis and bone osteosclerotic lesions. Differential diagnosis: MGUS
associated with neuropathy, CIDP, systemic amyloidosis, cryoglobulinemia, and MM. Diagnosis: major criteria, polineuropathy,
monoclonal component; minor criteria: bone osteosclerotic lesions,
Castleman´s disease, organomegaly, edema, endocrin abnormalities, hiperpigmentation, multiple hemangiomata, and white nails.
Etiology is unknown. Herpes virus 8 (HV8), has been associated
in 78% of cases. There are elevated cytokines like VEGF, IL 1ß,
TNF-a and IL-6. There are 99 patients reported in the Mayo Clinic
(1975 - 1998). The general survival is 12 - 33 months. With hematopoietic stem cells transplantation, median survival is 13.8 years.
When osteosclerotic lesions are localized radiation therapy is the
treatment of choice.
S147
129
172
ABERRANT METHYLATION OF TUMOR
SUPPRESSOR GENES (TSG) IN MYELOMA
MULTIPLE (MM) AND CHRONIC LYMPHOCYTIC
LEUKEMIA (CLL).
PRIMARY AMYLOIDOSIS WITH PREDOMINANT
CARDIAC AFFECTION. CASE REPORT
Chena, C.1 *; Stanganelli, C.1; Barreyro, P.1; Arrossagaray, G.1;
Fantl, D.2; Zimerman, J.2; Corrado, C.1; Slavutsky, I.1
*
Argentina - 1 Academia Nacional de Medicina; 2 Hospital Italiano
Background: Aberrant gene promoter methylation is an epigenetic mechanism whereby gene expression is abrogated. It is therefore potentially involved in silencing of tumour suppressor genes
during carcinogenesis. Objective: To evaluate the methylation status of different TSG involved in regulation of the cell cycle: p15INK4b,
p16INK4a, p14ARF, SOCS-1, p27KIP1, RASSF1A and TP73, in MM and
CLL patients. Material and Methods: We have studied 30 MM (14
males; mean age 67.6 years; Durie-Salmon clinical stages: I: 24%,
II: 12%, III: 64%) and 35 CLL patients (19 males; mean age 66.2
years; Rai clinical stages: 0: 32%, I-II: 38%, III-IV: 30%). Moreover,
8 monoclonal gammopathies of undetermined significance (MGUS)
(3 males; mean age 68.6 years) were also included. Methylation
status from DNA samples was performed using Methylation Specific PCR (MSP) technique. Results: In MM, methylation of SOCS-1,
TP73, p14ARF, p15INK4b, p16INK4a and RASSF1A genes was detected
in 61%, 36%, 21%, 18%, 14% and 4% of cases, respectively. Methylation of at least one of these TSG was observed in 82% of cases.
MGUS showed methylation of p15INK4b and SOCS-1 in 13% of cases
and of p14ARF and TP73 in 25% of patients. In CLL, methylation of
TP73, p15INK4b and p16INK4a genes (90%, 9% and 4%, respectively)
was observed. All patients showed unmethylated p27KIP1 gene. Conclusions: A different methylation pattern was observed in MM and
CLL. The genes most frequently affected by aberrant methylation
were SOCS-1 in MM and TP73 in CLL. The high incidence of these
two latter alterations suggests that they would be potential targets
for therapeutic intervention.
Key words: methylation, MM, CLL.
CC and SC have contributed equally to this work.
De Galvez, M. G.1 *; Stevenazzi, M1; Perez, G1; Miranda, N1;
Alonso, J1; Nese, M1
*
Uruguay - 1 Clinical Department of Medicine. Clinical Hematology.
Faculty of Medicine. Montevideo Uruguay..
Primary amyloidosis is a disease characterized by tissue deposits of immunoglobulin light or heavy chains associated with
monoclonal gammopathy.
Infiltrative cardiomyopathy is present in 37.4% of cases in
which the clinical presentation may include: congestive heart failure, arrhythmia, syncope and sudden death. Cardiac involvement
presenting as the dominant manifestation of the disease, is the
most important adverse prognostic factor.
Case report: a 52- year- old man, with history of severe aortic
stenosis who has undergone a mechanical valve replacement, in
treatment with ACEI, oral anticoagulants, and furosemide; presented with refractary congestive heart failure, effort syncope; at the
physical examination: macroglossia and bipalpebral ecchymosis.
The electrocardiogram showed low voltage and the echocardiogram with doppler, and transesophageal echocardiogram, exhibed
increase in septal thickness and decrease in left ventricule size, severe diastolic dysfunction , mechanical aortic valve in good position
with good function according with infiltrative cardiomiopathy diagnosis. Laboratory evaluation: serum protein electrophoresis : serum M
protein in gamma globulins region of 1.8 gr/dl; immunofixation: IgG
lambda; Nephelometry: IgG 2.35gr/dl; bone marrow aspirate found
10% of plasma cells . Amyloidosis was confirmed histologically by
subcutaneous biopsy.
Evolution: the patient was refractary to treatment and suffered
sudden death.
S148
POSTER SESSION
06.1 HEMOSTASIS AND THROMBOSIS
049
048
ACQUIRED AMEGACARYOCYTIC
THROMBOCYTOPENIA PURPURA (AATP)
SUCCESSFULLY TREATED WITH ANTITHYMOCYTE GLOBULIN (ATG)
TEN YEARS FOLLOW UP OF HEREDITARY
HAEMORRHAGIC PARIENTS IN DENTAL PRACTICE
Fernandez, G.1 *; Mandrile, L.2; Gonzalez, G.1; Carvani, A.2; Santarelli, R.1; Wilson, R2; Zarate, G.1
*
Argentina - 1 Htal Pirovano Buenos Aires; 2 Htal Paroissien Buenos Aires
Introduction: The provision of dental care in hereditary hemorrhagic patients was always a challenge. Pain and bleeding kept
patients away from the dentist. The last 10 years we are trying to
make dental procedures more comfortable and less complicated to
this group of patients.
Objective: In order to accomplice that, we thought that prevention must be the key word regular preventive checkout examination
- teeth cleaning - instructions early care of dental problems fillings,
endodontic treatments, teeth scalings to avoid teeth loss
Materials and method: In 65 patients, 213 teeth extractions
were realized and 15 minor surgical cases. Concerning haemophiliacs we administered 40 unit BW of recombinant factors. All
the previous treatment protocols were accompanied by strenuous
measures of local haemostasis (we replaced surgical with collagen
fleece, splint with fibrin glue and then with pressing tampon due to
problems that occurred. Of course important was the use of 10%
tranexamic acid solution as local anti-fibrinolytic treatment.
Results: In these ten years that we follow up this group of patients a great number of dental actions were performed (250 tooth
cleanings and scaling, 353 tooth fillings, 57 endodontic treatments,
213 teeth extractions, 15 minor surgical interventions and 85 deciduous teeth extractions).
Conclusion: By avoiding haemorrhagic complications and
providing painless conditions of treatment we restored the confidence in the dentist’s face and reassured the acceptance of preventive dental measurres
Introduction: Acquired amegacaryocytic thrombocytopenia is
a rare disorder of unclear etiology characterized by severe thrombocytopenia, preservation of erythroid and myeloid cell lines, and
absence of megacaryocytes in the bone marrow, pathogenic mechanisms unclear and consequently several therapies are used.
Objective: We reported two cases with AATP, an uncommon
and severe disease, which had been successfully treated with
GAT.
Materials and method: Clinical evaluation of two patients, a
19-year-old woman, and 31-year-old man with severe trombocytopenia and AATP diagnosis. We show the unsuccessful use of corticoid and favorable response with GAT.
Results: Case 1: A man with serious bleeding syndrome
was hospitalized in 2000.The platelet count was 5000/mm3.
The bone marrow aspirate and biopsy showed the absence of
megacaryocytes,and normal granulocyte and eritroyd precursors.
Cytogenetic was normal. No definable etiology has been found.
He was treated with prednisone, without response. Then, GAT was
started and resulted in favorable response. He has had normal
platelet recount for 6 years
Case 2: In 2005, a woman consulted for pallior and menorrhagia.The blood test showed anemia and platelet count was 3000/
mm3.The bone marrow aspirate and biopsy showed absence of
megacaryocytes, without others abnormalities. Flow citometry and
cytogenetic was normal.No definable etiology has been found. She
received two pulses of metilprednisolone and prednisone, without
response.After that, she was treated with GAT, and the platelet count
increased to 90.000/mm3.The bone marrow showed some megacaryocites. At the same time the patient presented an ostemeylites
which was treated with ATB, mero-imiperen, and the platelets count
decreased to 5000/mm3. She also developed an avascular necrosis
secondary to corticoid. Then,she received IgG IV without response.
When the infection was controlled, in August 2006, she was retreated with GAT, and she had a successful response.The platelet count
in October 2006 was 150.000/mm3.
Conclusion: The AATP is an uncommon disease in which several empirical therapies are used. In these patients the GAT has
shown to be the most beneficial treatment.
Makris Sofia, SP; Makris Michael, MP1; Makris Pantelis, PE1
1
Aristotle University of Thessaloniki
015
HEPARIN-INDUCED THROMBOCYTOPENIA: TWO
CASE REPORTS
de Cabo, E.1 *; Moro, M.J.1; García-Escobar, I.1; Redondo, C.1
*
España - 1 Hospital de León
Introduction:
Thrombocitopenia is a relatively frequent and usual clinical
complication of heparin therapy. Some patients receiving heparin
and heparin -based products have an immune-mediated reaction
due to the development of heparin -induced antibodies. This reaction le ads to a highly specific and paradoxical form of thrombocitopenia (HIT). Unlike other types of drug -induced thrombocitopenia,
HIT promotes thrombosis rather than bleeding, so HIT should be
suspected in patients who experience thrombotic events despite ad
equate anticoagulant therapy.
Objective:
Early identification and treatment of HIT can preventmore serious complications associated with this disorder (e.g., exarcebation
of venous thormbosis, limb gangrene and skin necrosis). Both, arterial and venous thrombosis, c an arise from a single episode of HIT.
Routine assessment of platelet counts is necessary with heparin
therapy. Treatment with a direct thrombin inhibitor, such as lepirudin, is an effective strategy in reversing the thrombocitopenia associated with HIT an d reducing its complications.
To illustrate this issue we present two cases diagnosed at our
institution last year.
Results:
Case 1: 66 years old woman who received Sodic Heparin for
a pulmonary thromboembolism. She suffers a thrombocitopenia of
34000 p latelets/mm 3 in the 12th day of heparin -therapy (basal
platelets count of 243000/mm 3) with a exacerbation of venous
thrombosis in form of massive pulmonary thromboembolism. The
heparin therapy was took out and lepirudin therapy was started.
The PF4 IgG antibodies we re detected. The platelets grew up to
175000/mm 3 in four days and the thromboembolis evolutioned
properly. Case 2: 58 years old woman who received Sodic Heparin
for a pulmonary thromboemboli sm. In this case, the thrombocitopenia was of 39000 platelets/m m 3 in the 7th day of heparin-therapy
(basal platelets count of 214000/mm 3) with a exacerbation of venous thrombosis in form of massive pulmonar thromboembolism.
For this reason the patient had to be assessed by intensive care unit
for 3 days. Close to the other case, lepirudin therapy was started.
and the PF4 IgG antibodies were detected after Sodic Heparin was
stopped. The platelets grew up to 147000/mm 3 in four days and the
thromboembolis evolutioned properly.
Conclusion:
Conclusions: induced by heparin thrombocitopenia is a commun and serious problem during Heparin therapy, and should be
take in account in orther to avoid complications of embolism therapy.
017
RELAPSE THROMBOTIC THROMBOCYTOPENIC
PURPURA IN A 32 Y/O DIABETIC MALE WITH
DEFICIENCY OF ADAMTS 13 PROTEASE, TREATED
SUCCESFULLY WITH PLASMA INFUSIONS.
Santana, J.1 *
*
Puerto Rico - 1 UPR Medical School
Introduction:
Thrombotic Thrombocytopenic Purpura (TTP) is diagnosed by
identifying a microangiopathic hemolytic anemia and thrombocytopenia in a very sick patient usually with fever, renal failure and
neurologic deficits. The deficiency of a protease, ADAMTS 13, that
cleaves a specific peptide bond in the Von Willebrand Factor(VWF)
units causes accumulation of this VWF proteins, platelet clumping
and microthrombosis. By providing this protease in Fresh Frozen
Plasma the platelet clumping and microangiopathy stops. Autoantibodies to this protease has been described and measured, both
conditions giving the same clinical presentation.
Objective:
The ADAMTS 13 protease was measured in this patient by
Elisa technique at Dr.Tsai Laboratory.
The ADAMTS 13 protease was measured in this patient by
Elisa technique after recovering from the second episode of TTP,
on two different occasions, 0.22u/ml and 0.23u/ml (normal 1.0 +
0.2u/ml. in active TTP <0.1u/ml) Plasma mixing studies detected
the presence of ADAMTS 13 inhibitors on both occasions.
S149
Results:
This 32 y/o diabetic had his first episode of TTP in January 2004
and was treated successfully with Plasmapheresis and steroids
requiring four weeks of treatments. He relapsed eighteen months
later with microangiopathy, severe thrombocytopenia (2,000/ul) and
mucosal bleeding. Plasma infusions (1-1.5 liters per day) with high
dose steroids where given daily for two weeks with platelets transfusions in the first few days. He recovered counts by the second
week and was followed in the clinic until complete platelet recovery
by the fourth week. Since this initial report he has relapsed twice
in a six month period, the first after uneventfull cholecystectomy
and the second after an episode of Bronchitis. Both episodes where
treated succesfully with Plasma Infusions and Steroids, recovering
in 4-5 days.
Conclusion:
TTP is a fatal disease if not diagnosed and treated promptly.
Although plasma exchange by Plasmapheresis has been recommended mainly for volume management, plasma infusions and steroids should not be delayed. Platelet transfusions may be given to
bleeding patients without adverse consequences.
051
HAEMARTHROSIS AS COMPLICATION OF
SUPERWARFARIN POISONING
GIRTOVITIS Fotios, FI1 *; AVRAMIDIS Iakovos, IG1; Makris Pantelis, PE1
*
Grecia - 1 Aristotle University of Thessaloniki
Introduction: Poisoning with long-acting anticoagulants is known
to lead to disturbance of haemostasis. Such haemorrhagic complications, like the ones after poisoning with rodenticide, are often
difficult to identify
Objective: To present, for the first time in literature, a case
of haemarthrosis as a haemorrhagic complication of superwarfarin
poisoning.
Materials and method: CASE REPORT
A 67-year-old man was admitted to our clinic with melaena, epistaxis and haemarthrosis in his left knee. He received portion of rodenticide substance 15 days ago. 2 days after the reception he was
hospitalized for 6 days with melaena and epistaxis. Screening tests
of haemostasis revealed undetermined PT (INR) and aPTT. Initially,
the patient was treated with 4 units of fresh frozen plasma (FFP)
and 3X10mg of Vitamin K iv per day, for 6 days. For further investigation he was admitted in our unit, where, during the first 7 days
of hospitalization, had also melaena. We administrated supporting
treatment (PPIs and 6 units of red cells) and we successfully treated
epistaxis with topical haemostasis. Haemarthrosis was treated with
FFP (4 unitsX2 per day, for two months). Initial screening; 1st day:
INR=7.15, PT=60.6”. aPTT=79.9”, Hct=28%. 2nd day: INR=15.2,
PT=107.8”, aPTT = 95”, Hct= 23.5%. After the third day, values of
INR varied from 2.04 to 4.78. Immunological and biochemical tests,
levels of electrolytes, complete study of haemostasis, microscopic
examination of excrements for fat and undigested fibers, tests for
viruses and complete study of liver function (for latent hepatic insufficiency) were performed.
Results: We found very low levels of vitamin K dependant factors (II, VII, IX, X, protein C and S), which were normalized after the
administration of FFP and Vitamin K. Levels of the rest of the factors
were normal. Erosions in antrum, bulb, and 2nd section of duodenum were revealed endoscopically, while the psychiatric estimation
revealed a disturbed personality.
Conclusion: Acquired disturbances of haemostasis after
poisoning with superwarfarin (rodenticides) substances were described in several cases and have often led to death. This is why a
long duration treatment and follow-up are required. Haemarthrosis
as a complication of superwarfarin poisoning is presented for the
first time in literature
S150
054
ACUTE DIETARY EFFECT ON PLATELETS’
AGGREGATION
Makri Lida, LP; Makris Michael, MP1; GIRTOVITIS Fotios, FI1;
PITHARA Eleftheria, ET1; Makris Pantelis, PE1
1
Introduction: Dietary effect on cardiovascular disease is well
known. Elevated levels of coagulation factor VII have been associated to increased risk of coronary heart disease and seem to be
affected by a diet rich in saturated fatty acids and proteins. On the
contrary, a diet rich in ?3-unsaturated fatty acids seem to reduce
levels of coagulation factor VII and have a similar effect on platelets’ function. Generally, a long-term diet rich in ?3-unsaturated fatty
acids seems to affect haemostasis, as it has been proved by the
Mediterranean diet.
Objective: The aim of the study is to control the acute effect of
a diet rich in ?3-unsaturated lipids on platelets’ aggregation.
Materials and method: 109 students (23-25 years old, 61
male and 48 female) followed a dietary program designed by our dietician, for three days. It consisted of fruit, vegetables, olive oil and
fish rich in ?3-unsaturated lipids (salmon-5.04%, sardine-8.88% and
tuna-4.8%). Blood sample was taken just before and after the diet.
Female subjects gave blood near the 20th day of their menstrual
cycle. We used P.I.C.A-aggregometer with the stimulators ristosetin, collagen and ADP.
Results:
Χ*
SD**
Risto
Before
%
81.37
9.72
After
%
82.29
8.64
ADP
Before
%
78.21
13.72
After
%
79.81
11.03
Colla
Before
%
80.26
9.41
After
%
80.56
8.43
(X: mean value, SD: Standard deviation)
Conclusion: Paired-t test revealed no significant difference
(p<0.1) in platelets’ aggregation with any of the stimulators. Thus,
a diet rich in ?3-unsaturated lipids doesn’t seem to have an acute
effect on aggregation
055
DIETARY EFFECT ON VITAMIN K-DEPENDED
COAGULATION FACTOR
Makri Lida, LP; Makris Michael, MP1; GIRTOVITIS Fotios, FI1;
PITHARA Eleftheria, ET1; Makris Pantelis, PE1
1
Introduction: Dietary effect on cardiovascular disease is well
known. Elevated levels of coagulation factor VII have been associated to increased risk of coronary heart disease and seem to be
affected by a diet rich in saturated fatty acids and proteins. On the
contrary, a diet rich in ¦Ø3-unsaturated fatty acids seem to reduce
levels of coagulation factor VII and have a similar effect on platelets¡¯ function. Generally, a long-term diet rich in ¦Ø3-unsaturated
fatty acids seems to affect haemostasis, as it has been proved by
the Mediterranean diet.
Objective: The aim of the study is to control the acute effect
of a diet rich in ¦Ø3-unsaturated lipids on the levels of vitamin Kdepended factors.
Materials and method: 56 students (23-25 years old, 33 male
and 23 female) followed a dietary program designed by our dietician, for three days. It consisted of fruit, vegetables, olive oil and fish
rich in ¦Ø3-unsaturated lipids (salmon-5.04%, sardine-8.88% and
tuna-4.8%). Blood sample was taken just before and after the diet.
Female subjects gave blood near the 20th day of their menstrual
cycle. We used DADE-Behring BCS and confirmed the results with
STA-compact of STAGO
Results:
Factors
Pr C a
PrC b
PrS free a
PrS free b
II a
II b
VII a
VII b
IX a
IXb
Xa
Xb
Mean V
SD
93,5
15,2
92,2
15,3
106,5
22,7
96,0
18,0
107,9
13,0
91,6
13,8
89,2
12,8
88,9
12,8
87,1
12,1
108,7
23,1
104,1
20,6
106,1
22,5
Conclusion: There was no acute effect of a diet rich in ¦Ø3unsaturated fatty acids on the levels of vitamin K-depended factors,
not even on the ones with a short half life.
S151
POSTER SESSION
07.1 INFECTIOUS DISEASES
061
018
CHARACTERIZATION OF MICROVESICLES FROM
IMMUNE CELLS INFECTED WITH DENGUE VIRUS
PREVALENCE OF NOSOCOMIAL BLOODSTREAM
INFECTIONS IN THE ONCOLOGY UNIT AT THE
UNIVERSITY TERTIARY HOSPITAL: 9 YEARS
EXPERIENCE
Bosch, I1 *; Becerra-Artiles, A1; deBosch, N2
*
USA - 1 Umass Medical School; 2 Banco Metropolitano de Sangre
de Caracas
Introduction: The infection with dengue virus takes place in
immune cells, like Monocytes and B cells. Microparticles/microvesicles are shedded particles of 200 nm- 700 nm diameter collected
in plasma or supernatant of cell in culture derived from a variety of
immune cells. They have Annexin V positive staining and they are
the product of apoptosis and the normal cell life. They have one lipid
by-layer and proteins and nucleic acid internally. The composition
of the membrane resembles that of the cell membrane that give
their origin. Their presence in circulation have been associated with
endothelium activation, including pro-coagulation function. The microvesicle may therefore, be implicated in the pathology observed
during dengue virus infection
Objective: Caracterize the protein composition of microvesicles produced by human immune cells B and Monocytes and their
role in the activation of endothelial cells in vitro. Compare microvesicles from un-infected and infected conditions to draw hypothesis of
virus-induced cell activation through microvesicle production.
Materials and method: Ultracentrifugation, Flowcytometry,
PAGE, Mass Spectrometry, immunofluorescence were among the
techniques that were used to characterized B and Monocyte derived
microvesicles. Cell lines used were LG2 cells (human B cells overexpressing MHC Class II) and THP-1-DCSIGN (human monocytic
cell line over-expressing DC-SIGN, putative receptor for dengue virus). PMBCs were obtained from healthy donors and dengue virus
serotype 2 New Guinea C was utilized as prototype virus.
Results: All microvesicles derived from PBMCs, B cells and
monocytes were positive for CD45 by Flowcytometry. Proteomics
using MS quantitative analysis (iTraq) determined the proteins that
were differentially present in microvesicles of human B cells infected
with dengue virus. Membrane (MHC class I and II), cytoplasmic (actin) and nuclear proteins (histones) were identified. Of those, plasma membrane proteins were confirmed by Flowcytometry. Moreover, microvesicles fused to human endothelial cells (HUVEC) in a
time-dependent manner and the gene expression of HUVECs were
studied. Implications of microvesicle formation in dengue infection
are discussed.
Conclusion:
The number of microvesicles derived form infected cells increased. The fusion capacity of microvesicles derived from infected
cells was greater than that from un-infected cells. The proteomics
analysis showed differential expression of proteins between the infected and uninfected. The Global gene expression of endothelial
cells in the presence of microvesicles will be presented. From the
present data, we concluded that clinical studies should include microvesicle isolation from acute dengue infections. It is important to
study the microvesicles in vivo and their possible role in dengue
pathogenesis.
Santana, J.1 *; Lopez, A.1; Martinez, N.1
*
Puerto Rico - 1 UPR Medical School
Introduction:
Nosocomial bloodstream infections represent an increased
risk factor for morbidity and mortality among immunecompromised
patients hospitalized in Oncology units. We evaluated retrospective surveillance reports from cultures isolated at our Oncology Unit
from 1995 to 2004. We treat Acute and Chronic Leukemias, Lymphomas, high dose chemotherapy with marrow rescue and ventilator support as needed.
Objective:
To review all bloodstream infections in a nine year period at
our Oncology Unit.
Surveillance and captured monthly reports of cultured isolated
organisms were evaluated and quantified in total absolute numbers
as well as percentages from bloodstream infections observed during the period. To minimize error variable and maintain accuracy,
only one person was allowed to tabulate data.
Results:
Total percent of isolated organisms increased from 19.6% to
37.5% Gram (+) organism increased from 8.5% to 18.7% Gram () organisms increased from 6.2% to 16.8%. Fungal infections increased from 9.1% to 18.2% Besides coagulase negative Staphylococci, S. aureus and Enterococci prevalence increased from 0.85%
to 2.9% and from 0 to 5.1% respectively. Among Gram (-) P. Aeruginosa and Acinetobacter species increased from 1.2% to 2.1% and
from 0.2% to 2.5% respectively. Non albicans Candida, C. tropicalius, C. parapsilosis and Trichosporium have steadily increased
during this period.
Conclusion:
Nosocomial bloodstream infections are a serious complication
associated with morbidity and mortality in inmunecompromised patients. In our study, data review, Gram (+) organisms S. aureus and
Enterococci as well as Gram (-) P.Aeruginosa and Acinetobacter
species had the greatest increase in prevalence, however fungal
infections are increasing. No Aspergillus species has been isolated
in a twenty year period in this Unit.
S152
122
THROMBOCYTOPENIA IN HIV INFECTED
CHILDREN IN AN ARGENTINE HOSPITAL
Elena, G.1 *; Lavergne, M.1; Veber, S.1; Goldman, W.1; Corrales,
M.1; Bietti, J.1; Morell, M.1; Cuttica, R.1; Cervetto, V.1
*
Argentina - 1 Reumatology and Hematology-Oncology Deparment
Introduction: Thrombocytopenia can occur in 20-30% of pediatric patient with HIV at sometime during the course of their disease
or be the initial clinical manifestation. HIV directly causes thrombocytopenia in most of the patients. The two components of pathophysiology of HIV associated thrombocytopenia are: 1) Immune mediated destruction of platelet 2)A deffect in bone marrow production
as result of the interaction between HIV and megacariocitos through
several pathways. With progression of the underlying disease other
causes appear such as: opportunistic infections, medication side
effects and infiltrative diseases.
Objective: Describe the population of HIV-infected children
with Immune Thrombocytopenic Purpura (ITP) in our institution for a
period of 10 years (May/1996-May 2006) treatment and evolution.
Materials and method: We studied retrospectively 12 children
diagnosed as ITP in association with HIV, treated in Hematology and
Immunology department. The data colleted from clinical records.
Results: We diagnosed 12 patients as ITP, 9 female and 3
male patients, age of presentation: X: 76.7 m (3 - 144m). ITP was
the initial clinical manifestation in 3 patients (25%) and the others
patients occurred X: 29 m (1-87 m) after the diagnosis of HIV. At the
time of the ITP they were classified in the following categories: N1:
1; A1: 1; A2: 1; A3: 1; B3: 3; C2: 2; C3: 4. Six patients were treated
with HAART. The initial count of platelet was X: 36000/mm3 (800064000/mm3). Only 7 patients received treatment for ITP, ?-globuline: 2/7; steroid: 2/7; and both 3/7. Nine patients got remission, 2
no remission and 1 was lost in follow up. From the 3 patient who had
ITP as initial presentation only 2 received HAART, and one of them
had remission.Thrombocytopenia and microangiopathic anemia associated with HIV was excluded.
Conclusion: * We observed that 7% of the children with ITP in
our hospital had HIV and their initial clinical manifestation was ITP
(25%). * ITP occurred in patients with worse status of HIV disease,
but the mortality associated with thrombocytopenia was 0%.
Key words: HIV, Thrombocytopenia.
141
INFECTIOUS COMPLICATIONS IN RECIPIENTS OF
HEMATOPOIETIC STEM CELLS TRASPLANTS.
Muñío, J.E.1 *; Carballo, T.1; Carnot, J.1; de Castro, R.1; Pérez,
G.1; Martínez, C.A.1; Hernández, C.1; Pérez, D.1
*
Background. The infectious complications in the first 30 days
after
hematopoietic stem cells trasplants ( HSCT ) are frequently
causes of morbidity and potential mortality in patients who underwent this procedure.Objetives. To know the characteristics of the
infectious complications in the first 30 days after HSCT and it influense in the outcome. Material and Methods. We characterize the
infectious complications diagnosed in 130 HSCT carried out to 129
patients older than 15 years old in our series. Results. A total of 226
infectious events were diagnosed in 128 of the 129 patients who
underwent HSCT ( 99.22 % of morbidity ). Major frecuency ocurred
among days + 4 - +6 after HSCT. Bacteriemia was the most frecuenty clinical form of debut when Absolute Neutrophil Count
( ANC ) < 100 x mm3. Coagulase - negative Staphylococci
was the most frecuently isolated organism. ANC > 500 x mm3 was
reached among days +15 - +16, but in 46 patients receiving Granulociyte Colony Stimulating Factor ( G - CSF ) was among days +12
- +13 after HSCT. The infectious complications were direct causes
of death in 6 out of 129 patients ( 4.65 % of mortality ) in this period.
Conclusions. The infectious complications in recipients of HSCT
were important causes of morbidity and mortality in our series.
Keywords. Hematologic Stem Cell Trasplant ( HSCT ) recipients, infectious complications.
152
MONONUCLEOSID SYNDROME (MNS), IMMUNE
THROMBOCYTOPENIA RELATED TO HIV (ITPHIV), SOLITARY PLASMOCYTOMA (SP) AND
NO HODGKIN LYMPHOMA(NHL) IN THE HUMAN
IMMUNEDEFICIENCY VIRUS (HIV) INFECTION.
Mansilla, Mariela1 *; Díaz, Lilián1; Galzerano, Julia1; Magariños,
Alicia1
*
Uruguay - 1 CASMU)
Background: the hematologic alterations are noticed during all
the course of HIV infection. The presence of MNS or ITP addresses
the suspicion of primary HIV infection and its etiopatogenia may
be entailed to HIV.. The neoplasia’s apparition like than SP or NHL,
remains the chronic infection, put the evidence that the immune
system is altered.
Objective: of the present paper is to analyze the epidemiology
and the prognostic of MNS,ITP,SP and NHL in our patients with
HIV infection, whose were detected and followed at our hospital.
Material and methods: we have reviewed the clinical charts of
644 patients with diagnostic of HIV infection between 10/1988 to
6/2006. We have selected 28(4.3%) with diagnostic of MNS, ITP,
SP and NHL. We didn’t include the following disgnostics: anemias,
leucopenias, or thrombotic complications. We have analyzed the
following variables: age, sex, hematologic and infectious chronology diagnostic ( synchronous or metachronous) and established
treatments.
Results: 28/244(4.3%) of hematologic complications were distibuted as follow: 20 were men (76%) and 8(24%) women; age average was:33 y.o.; the frequency of the investigated pathologies were:
MNS, 11/28(39%); ITP,7/28(2.5%); SP, 2/28(7%); NHL8/28(28%).
Synchronous in all MNS(11/11); in 3 ITP(3/7) and 2 of NHL(2/8).
The SP were metachronous 8 (both patients were under ARVT
more than 2 years ago). 3/11 MNS were HIV primary infection and
they were diagnosed in the last 18 months, and were under ARVT.
The 7 ITP, 3 needed palliative splenectomy and 4/7 received ARVT(
zidovudine included). All patients with NHL diagnosed after 1997 received ARVT and PQT; 2 have dead at the first month on oncologic
treatment (B high grade) and 3 are alive.
Conclusions: The MNS’s apparition drives the diagnostic suspicion of HIV primary infection and it gets the profitable ARVT. With
diagnostic of ITP-VIH the terapeutic tools availables are variable
and aren’t different to the ITP without HIV infection. This variety
hosts in the lack of definitive and longer results. The NHL are benefit
the early diagnostic and the combinig treatments, ARVT plus PQT.
175
ENHANCED BACTERIOCIDAL FUNCTION BY
WKYMVM IN PATIENTS WITH ACUTE LEUKEMIA
Hawk, K.1 *; Young, Joo Min1; Soo-Jin, Shin1; Eui-Kyu, Noh1;
Eun Jung, Lee1; Jae-Hoo, Park1
*
Korea - 1 Division of Hematology-Oncology, Ulsan University
Hospital, University of Ulsan College of Medicine
Acute leukemia (AL) is a disease of challenge in that many
patients die not only from eventual relapse but also from infection
during treatment. In this regard efforts for decreasing infectious
mortality will help increasing survival. Our previous study revealed
that leukocyte bacteriocidal functions in chemotherapy-treated
cancer patients were decreased and were stimulated by a novel
hexapeptide, WKYMVm. We evaluated the leukocyte bacteriocidal
function in patients with AL and searched whether WKYMVm can
enhance the bacteriocidal function in patients with AL. On induction chemotherapy, blood sampling was performed at diagnosis and
repeated weekly from start day of chemotherapy until patient died
or complete remission was achieved. Tests were done weekly until
white blood cell (WBC) count reached up to 1000/mm3 and platelet
counts were stable without transfusion after consolidation chemotherapy. Fifteen AL patients and 2 healthy controls were enrolled.
Diseases were acute myeloid leukemia (14 patients) and acute lymphoblastic leukemia (1 patient). Eight (53.5%) were male and me-
S153
dian age was 55 years. Median WBC, absolute blast count (ABC)
and absolute neutrophil count (ANC) at diagnosis were 5280/mm3
(range 840-315100), 162.9/mm3 (range 0-286741) and 796.4/mm3
(range 126-7336), respectively. Mean values of bacreriocidal activity
at diagnosis were increased by concentrations of WKYMVm (13.4
at 0 nM; 24.9 at 1 nM; 33.3 at 10 nM; 38.5 at 100 nM; p<0.001),
which were also increased in normal samples (20.6 at 0 nM; 41.6 at
1 nM; 51.6 at 10 nM; 66.4 at 100 nM; p<0.001). At each concentrations of WKYMVm, the bacteriocidal activities were inferior to those
of normal control (p=0.029, at 0 nM; p=0.015, at 1 nM; p=0.015,
at 10 nM; p=0.015, at 100 nM). The bacteriocidal activities were
increased (p=0.008, at 0 nM; p=0.015, at 1 nM; p=0.011, at 10 nM;
p=0.021, at 100 nM) compared with the corresponding values when
patients achieved complete remission (CR). However, bactericidal
activities by stimulation of WKYMVm were inferior to normal control
even in CR (p=0.036, at 1 nM; p=0.036, at 10 nM; p=0.036, at 100
nM) although base line value were similar (p=1.0, at 0 nM). After
hematological recovery of consolidation chemotherapy, the bacteriocidal activities of patients were similar to those of normal control
(p=0.533, at 0 nM; p=0.133, at 1 nM; p=0.133, at 10 nM; p=0.133,
at 100 nM).
In conclusion, the bacteriocidal activities in AL patients were
severely decreased at diagnosis and could be enhanced by WKYMVm. Near normal bacteriocidal activities can be achieved when 1
nM or more concentration of WKYMVm is applied in patients with
AL. At the end of consolidation, bacteriocidal activities and stimulation by WKYMVm in patients were almost recovered.
S154
POSTER SESSION
08.1 LYMPHOMAS
029
030
FLUDARABINE AND MITOXANTRONE FOR THE
REFRACTORY RELAPSE TREATMENT OF B-CELL
LOW-GRADE NON-HODGKIN LYMPHOMA (NHL):
FIRST INTERIM REPORT LACOHG
FOXP3 EXPRESSION IN DIVERSE T-CELL
LYMPHOMAS
Baltazar, S1 *; Pimentel, P2; Vera, L2; Bezares, F3; Málaga, J4;
Huamani, J2; Montante, A5; Rivas, S6; Carrasco-Y, A2; BeltránGárate, B
*
Mexico - 1 Centro Médico Noreste Monterrey México; 2 Hospital
E. Rebagliati-Lima-Perú; 3 GATLA & LACOHG (Latin American
Cooperative Oncology Hematology Group); 4 Hospital Nacional
del Sur de Arequipa; 5 Hospital General de México OD; 6 IN de
Cancerología, México DF
Introduction:
Clinical evidence suggests that fludarabine (F) monotherapy
is as least as effective, than conventional therapies such as cyclophosphamide, vincristine, prednisone (CVP) for the first and second
line treatment of B-cell low grade NHL achieving objective response
rates. Better response rates can be achieved combining F with Mitoxantrone (M) in low grade NHL even in refractory relapsed (RR)
patients (pts). The Latin American Cooperative Oncology Hematology Group (LACOHG) proposed a multicenter study in Latin American countries in 2002 to use FM in RR B-cell low grade NHL.
Objective:
Assess the response rate, safety, disease free survival (DFS)
and overall survival (OS) of FM in RR B-cell low grade NHL
Fourty-eight patients in the period of January 2003 to February
2006 were evaluated. Fourty-four pts. had follicular lymphoma and
4 small lymphocytic lymphoma. Median age 63.5 years old (range:
24-83). Gender: female 56% and male 44%. Inclusion criteria for
low grade NHL-LG was: any previous treatment excluding autologous transplantation, Ann Arbor stage II to IV , age > 18 years
old, ECOG performance status 0-2 and written informed consent.
ECOG performance status 0: 2%, 1: 71% and 2: 27%. Ann Arbor
staging: II: 2%, III: 29% and IV: 69%. International Prognostic Index
(IPI): 0-1: 19%, 2-3: 71% and 4-5: 10%. Median previous treatment
was 1 (range: 1-3). FM treatment consisted of F 25 mg/m2 i.v. (day
1-3) and M 10 mg/m m2 i.v. (day 1) each 28 days for 6-8 cycles.
Results:
Overall response rate (PR+CR) was 81% (ORR); progressive disease and non-response 19%. With a median follow up of
17 months, OS at 24 months was 86% (DE 5.2%) and DFS at 24
months 57.1% (DE 11.3%) . LDH in serum was not an adverse
prognostic factor for DFS and OS. Safety: on the 286 cycles in 48
pts, the toxicity was: 18 episodes of grade 3-4 neutropenias, 15
episodes of grade 3-4 thrombocytopenia, 7 episodes of grade 1-2
nausea/vomiting, grade 1-2 diarrhea in 4 pts, 8 pts were admitted to
the clinic, 11 fever episodes, 2 allopecia, 4 pts developed grade 1-2
peripheral neuropathy and infections 7%: one case herpes zoster.
Mortality rate: 12,5% (6/48 patients), 5 of them because progressive
disease. No cardiac toxicity was reported.
Conclusion:
FM is an effective and safe treatment for RR low grade NHL.
Beltrán-Gárate, B; Quiñones, P1; Morales, D1; Huamani, J1;
Mejía, O2; Malaga, J3; Castillo-Aguirre, J1; Riva, L1; Hurtado de
Mendoza, F1; Vidurrizaga, M4; Carrasco-Yalán, A5
1
Hospital E. Regagliati Martins - Lima - Perú; 2 Hospital del Cuzco
- Peru; 3 Hospital de Arequipa; 4 Schering Peruana; 5 Hospital
Edgardo Rebagliati, Lima
Introduction:
Foxp3 is a key regulatory gene required for the development
and function of: (1) regulatory CD4+CD25high T cells (Treg) specialized in maintaining the balance between immunity and tolerance
and (2) activated conventional CD4+CD25low T cells without suppressive activity. Until now it is not yet possible to study human
FOXP3+ Treg irrespective of their CD25 expression. Previous studies had reported FOXP3+ T cells in Adult T-cell Leukemia/Lymphoma cells (ATLL) related to HTLV-1 .
Objective:
To determine the specifity and prognostic value of the FOXP3
expression in T-cell lymphomas.
A retrospective study was performed on 46 samples collected
from diverse T-cell lymphomas in our institution. A highly sensitive
immunohistochemical method was used to demonstrate FOXP3
protein expression with a mouse monoclonal antibody (clone 236A/
E7ABCAM) in most formalin-fixed paraffin-embedded tissue sections from lymph nodes, skin, bone marrow and extranodal sites
samples as cavum and stomach. We did not co-stained with CD25
and considered a FOXP3+ tissue when positivity was > 80% of toumor cells.
Results:
Among the 46 evaluable T-cell lymphomas collected, 33 were
ATLL, 7 mycosis fungoides (MF) and 6 unspecified peripheral Tcell lymphomas (U-PTCL). Among the 33 ATLL: lymphomatous=17,
acute=11, smoldering=1, chronic=1, cutaneous=1 and undefined=2.
FOXP3 expression in tumour cells was detected in 24% (8/33) of
ATLL cases, was negative in MF tumour cells and detected in 33%
(2/6) of U-PTCL. Interestingly FOXP3 expression between 30-40%
was expressed in 3 MF cases. Among the ATLL cases FOXP3 positivity were obtained in 35% (6/17) of lymphomatous type; 18% (2/11)
of acute ones and none in others ATLL types studied. We failed to
demonstrated any correlation between FOXP3 status and survival.
Conclusion:
FOXP3 is expressed in ATLL and T peripheral lymphoma. Treg
presence in the tumour environment plays an important immune
system response role and its identification should be fundamental.
S155
032
034
ALEMTUZUMAB IN PATIENTS WITH ADVANCED
MYCOSIS FUNGOIDES: FIRST INTERIM REPORT,
LACOHG-PERÚ (LATIN AMERICAN COOPERATIVE
ONCOLOGY HEMATOLOGY GROUP)
AGGRESSIVE PRIMARY CUTANEOUS CD8POSITIVE EPIDERMOTROPIC CYTOTOXIC T-CELL
LYMPHOMA TREATED WITH ALEMTUZUMAB.
Beltrán-Gárate, B; Huamani-Z, J ; Arones-V, A ; Hurtado de
Mendoza, F1; Carrasco-Yalán, A1; Gómez-Moreno, H3
1
Hospital Edgardo Rebagliati, Lima; 2 Hospital Militar Central,
Lima; 3 INEN, Lima, Perú.
1
2
Introduction:
Alemtuzumab (Campath®/Mabcampath®, anti-CD52 humanized monoclonal antibody) has recently been shown to be effectiv
e in the treatment of diverse hematological malignancies. Mycosis
fungoides (MF) is low grade cutaneous T-cell lymphoma with indolent course and good prognosis while response to chemotherapy
is achieved.
Objective:
We started a prospective phae II study in refractory relapse
MF cases with Alemtuzumab (ClinicalTrial.gov Identifier: NCT
00157274)
From July 2005 to April 2006 a total of eight patients were
recruited from 2 centers in Lima-Perú with hystophatological diagnosed of advanced refractory relapse MF. Inclusion criteria include:
above 18 years old, ECOG status 0-2, no active infections, no
more than 3 previous chemo±radiotherapy, HTLV-1 negative, HIV
negative, abnormal renal or hepatic function and written informed
consent. Median age 64 years old (range: 36-72). Five were male.
Median number of previous therapies 2 (range: 2-3). Original treatment scheduled was planed as Alemtuzumab 30 mg i.v. tiw per 12
weeks with a gradually escalated doses during the first week (3, 10,
30 mg). Trimethoprim/sulphamethoxazole and aciclovir prophylaxis
was given. Median Alemtuzumab total dose was 283 mg (range:
123-706) over a median of 5 weeks of treatment (range: 3-15). The
first four pts. received the programmed schedule dose and because
toxicity the subsecuent 2 pts. received Alemtuzumab 30 mg i.v. tiw
for 4 weeks and then 30 mg i.v. weekly and the last 2 recruited pts.
received Alemtuzumab 10 mg iv. tiw for 4 weeks them after 10 mg
i.v. biw and finally 10 mg i.v. weekly. CMV monitoring with pp65 was
performed in the first five pts. and qualitative PCR in the last 3 pts.
Results:
Seven patients were evaluable for response, overall response
rate (ORR) was 57% (4/7), with two patients achieving complete
remission (CR) and two patients with partial response (PR) and
three patients with progressive disease (PD) during treatment. Response duration was brief with duration less than 3 months in all
cases (table 1). Median Pruritus Analogue Scale was reduced from
4 to 1. Grade 1 neutropenia in one pt. and grade 1 thrombocytopenia in one pt. One patient developed urosepsis caused by E. Coli.
No cardiac toxicity was reported. Kaposi sarcoma progression was
discovered in one patient.
Conclusion:
Alemtuzumab shows promising clinical activity in patients with
advanced MF previously treated. Alemtuzumab dose reduction in
combination should be explore in advance MF.
Beltrán-Gárate, B; Quiñones, P1; Carrasco-Yalán, A2; Riva, L1;
Hurtado de Mendoza, F1
1
Hospital Edgardo Rebagliati, Lima, Perú.; 2 Hospital Edgardo
Rebagliati, Lima
Introduction:
Aggressive primary cutaneous epidermotropic cytotoxic
CD8(+) T-cell lymphoma is an entity included in the new WHOEORTC classification as cutaneous T-cell Lymphoma. It is characterized by epidermotropic CD8(+) cytotoxic T cells proliferation
and an aggressive clinical behavior. Clinical pattern are localized or
disseminated eruptive papules, nodules and tumors. Alemtuzumab
(Campath®/Mabcampath®, anti-CD52 humanized monoclonal antibody) has recently been shown to be effective in the treatment
of diverse hematological malignancies, including B-cell chronic
lymphocytic leukemia and T-cell prolymphocytic leukemia, further
Alemtuzumab is being explored in T-cell lymphomas.
Objective:
We report a case of a patient with primary cutaneous aggressive epidermotropic cytotoxic CD8(+) T-cell lymphoma treated successfully treated with Alemtuzumab which showed previously refractoriness to other cytotoxic and retinoid drugs.
A 34 years-old women presented tumors with pruriginous
plaques and descamative lesions in arms , thorax and left leg. The
skin biopsy confirmed primary cutaneous CD8-positive epidermotropic cytotoxic T-cell lymphoma, with positive immunohistochemistry to CD3, CD8 and CD45, and negative to CD 56. Patient was
HTLV-1 & HIV negative
Results:
She received á-Interferon 9 millions three times per week with
poor response, with progressive disease showing multiple tumors
lesions. After this she received radiotherapy achieving partial response with a short control disease period. After progression she
was scheduled to receive CHOP with poor response. The patient
was scheduled to receive i.v. Alemtuzumab10mg three times a
week for five weeks, total Alemtuzumab dose was 123 mg achieving
partial response with tumor mass and nodular lesions reduction and
the analogue visual score of pruritus decreased from 7 to 2. Quantitative CMV PCR was positive at five weeks and treatment was
discontinued and started anti-CMV therapy. There were not other
side effects. The time disease control with Alemtuzumab was four
months. After relapsed, further therapy with bexarotene 150 mg per
day plus á-Interferon 3 millions three times a week and gemcitabine
plus vinorelbine were unsuccessful to achieve any control disease.
Patient died 26 months from the diagnosis.
Conclusion:
To our knowledge this is the first reported case with shows
Alemtuzumab effectiveness in the treatment of aggressive primary
cutaneous CD8-positive epidermotropic cytotoxic T-cell lymphoma.
This treatment should be explored using subcutaneous administration whether as a maintenance or combined therapy with other
agents.
S156
119
040
A PHASE 2 STUDY OF Y-ZEVALIN IN RELAPSED
REFRACTORY NON-HODGKIN’S LYMPHOMA:
PRELIMINARY REPORT OF THE ARGENTINEAN
COOPERATIVE GROUP
90
Cacchione, R1 *; Milone, J2; Bordone, J2; Dupont, J1; Milone, G3;
Riveros, D1; Negri, P4; Ardaiz, M5; Riera, L1; Foncuberta, M6;
Bezares, F7
*
Argentina - 1 CEMIC; 2 ITMO; 3 FUNDALEU; 4 H. Paraná; 5 H.
Ramos; 6 Fleming; 7 GATLA & LACOHG (Latin American Cooperative Oncology Hematology Group)
Introduction:
90Y-Zevalin (90Y-ibritumomab tiuxetan), a radiolabeled antibody to CD20, has shown promising activity in this patient population.
Objective:
We present the initial outcome of a phase 2 trial conducted
using 90Y-Zevalin, for relapsed refractory FL and transformed lymphomas
Between September 2005 and November 2005, we recruited
10 patients (6 male/4 female; median age = 56 yrs [range: 45-71
yrs]) with platelets >100,000/mm3 and bone marrow involvement
<25% for this trial. Nine pts had FL and 1 pt had mantle cell lymphoma. Four pts had bulky disease (largest diameter >5 cm). Three pts
were Ann Arbor stage I or II and 7 pts were stage IV. Three pts had
1-2 previous cycles of therapy and 7 pts had 3-5 previous cycles.
Three pts underwent previous autologous transplant. All 10 pts had
previously received chemoimmunotherapy with rituximab. Pts received rituximab 250 mg/m2 IV on Days 0 and 7. After the second
dose of rituximab, pts received 11 MBq (0.3 mCi) 90Y-Zevalin per
kg or 15 MBq (0.4 mCi) 90Y-Zevalin per kg based on platelet counts
with a maximum dose of 32 mCi. Blood counts were monitored
weekly until week 10 post-treatment and monthly thereafter. Patient
tumor response was reevaluated 3 and 6 months after treatment
according to standard criteria.
Results:
Five pts received a complete dose of 0.4 mCi 90Y-Zevalin
per kg and 5 received a reduced dose of 0.3 mCi 90Y-Zevalin per
kg. The overall response rate was 60% (CR=5, PR=1). The 5 pts
with a CR remained disease-free 8 months later. The pt with a PR
progressed with adenopathies and visceromegaly 7 months after
treatment. Toxicities were mainly hematologic: 5 pts required GCSF because of grade 3 or 4 neutropenia and 3 of them developed
neutropenic fever. Four pts required platelet transfusions and 2 pts
required red blood cell transfusions. Two pts were admitted briefly
because of hematopoietic toxicity. All 3 pts with previous autologous
transplant required G-CSF and transfusion support, and 2 of them
were admitted to the clinic because of hematologic toxicity, with recovery by week 9 post-treatment
Conclusion:
The use of 90Y-Zevalin in relapsed/refractory NHL setting resulted in better response rates and longer DFS. Our finding with
50% CR in a heavily pretreated cohort, including 42.8% CR in stage
IV pts and 33% after autologous transplantation is encouraging
HODGKIN’S LYMPHOMA AND HIV. CASES
REPORT
Savio, E1 *; Cabrera, S1; Ortega, V2; Medina, J1; Pérez, G1; Gualco, G2; Musto, M2
*
Uruguay - 1 Cátedra de Enfermedades Infecciosas; 2 Hospital
Militar // Laboratorio de Anatomía Patológica Dr. G. Ardao
Epidemiological studies have demostrated an increased risk
of some non AIDS-defining malignancies. Hodgkin’s lymphoma
(HL) represents the most common of non AIDS-defining tumors;
8-17 fold incresed risk than expected. Clinicopathologic features
are differents from that HL in the general populations: extranodal
disease, widespread extent and systemic B symptoms at presentation, higher incidence of unfavorable histologic subtypes and linked
pathogenically to Epstein-Barr virus (EBV). An improved survival
has been observed since the introduction of HAART in response to
chemotherapy and autologous stem cell transplantation. We report
2 cases of HIV-HL. Both patients were suspicious of disseminated
tuberculosis at diagnosis. Clinicopathologic features and survival:
case 1- men, 47 yo, 80 CD4 (cell/mm3), viral load (copies/mm3)
<50, HAART +, extranodal involvement (bone marrow and liver),
histological type: mixed celullarity, EBV +, survival: 2 months; case
2: men, 35 yo, 200 CD4 (cell/mm3), viral load (copies/mm3) <50,
HAART +, extranodal involvement (bone marrow and lung); histological type: lymphocyte depletion, EVB +, Survival: 24 months.
Case 1 died without specific treatment. Case 2 was treated
with 8 cycles of doxorubicin, blemoycin, vinblastine and dacarbazine and is in complete remission. HAART may not prevent excess
risk of some non AIDS-defining malignancies. The peculiar clinicopathologic characteristics of the HIV-HL and the concomitant opportunistic infections make it difficult to diagnose this malignancy. HL
were 9% of all the lymphoma diagnosis in our HIV + patients.
097
CLINICAL AND EPIDEMIOLOGICAL STUDY OF 644
PATIENTS WITH NON-HODGKIN’S LYMPHOMA.
Hernández, C.1 *; Muñío, J.1; Pérez, D.1; Carnot, J.1; de Castro,
R.1; Martínez, C.1; Pérez, G.1
*
Background: Non-Hodgkin’s lymphomas include a variety of
diseases with different clinical course, pathology, prognostic and
treatment. Material and Methods: A retrospective study was conducted in 644 patients with diagnosis of non-Hodgkin’s lymphoma
that received attention at the service of Haematology of “Hermanos Ameijeiras” Hospital for the last 20 years. Objective: Determine
the clinical and epidemiological characteristics of this disease on
its onset. Results: The males:females ratio was 1.02:1. 68.7 %
were white and the age group 55-74 was the most affected. The
ganglionar involvement prevailed over the extraganglionar, with a
higher incidence in the cervical region. The bone marrow was the
most common extranodal site affected. The primary extranodal affectation was present in 15 % of the cases. The general symptoms
were detected in 27.9 % of these patients. Aggressive histology predominated among the indolents and the most observed histological
type was the diffuse of large cells. On diagnosis, 66.1 % were in
advanced stages. Statistical significance was found on relating the
aggressiveness degree to the presence of general symptoms and
stages. Conclusions: Only little differences were found in this study
in relation with the reviewed literature.
Key words: Non-hodgkin’s lymphomas, epidemiology, neoplasm staging
080
FRONT-LINE THERAPY WITH EARLY
INTENSIFICATION AND AUTOLOGOUS STEM CELL
TRANSPLANTATION VERSUS CONVENTIONAL
CHEMOTHERAPY IN UNSELECTED HIGH RISK,
AGGRESSIVE NON-HODGKIN´S LYMPHOMA
PATIENTS. A PROSPECTIVE RANDOMIZED
GEMOH REPORT
Cardoso, RB1 *; Nucci, M2; Vigorito, AC3; Maiolino, A2; Simões,
BP4; Lorand-Metze, I5; Aranha, FJP5; Miranda, ECM5; Pagnano,
KBB5; Ruiz, MA6; Moraes, AJG7; De Souza, CA5
*
Brazil - 1 Marilia Medical School; 2 Federal University of Rio de
Janeiro; 3 State University of Campinas e Vera Cruz Hospital; 4
University of São Paulo; 5 State University of Campinas; 6 São
José do Rio Preto Medical School; 7 Campinas Oncology Center,
Campinas
Introduction:
this trial compares conventional chemotherapy (VACOP-B)
with early intensification with high-dose sequential chemotherapy
(HDS) and autologous stem cell transplantation (ASCT)
Objective:
as front-line therapy in patients with high risk non-Hodgkin\’s
lymphoma (NHL).
This prospective multicenter randomized trial was conducted
between September 1998 and July 2003. At diagnosis, patients
with aggressive high risk [intermediate-high (HI) and high-risk (HR)]
NHL (intermediate/high-grade and high-risk) according to the International Prognostic Index (IPI) were randomized between conventional chemotherapy VACOP-B 12 weeks (arm A) with an abbreviated of VACOP-B (6 weeks) regimen followed by HDS and ASCT
(arm B). Twenty-seven patients were randomly assigned to arm A,
and twenty-nine patients were randomly assigned to arm B.
Results:
The complete remission rate was 52% in arm A and 55% in
arm B. Eleven patients (22%) died early due to lymphoma progression. Treatment-related death occurred in three patients in arm A
and four in arm B (during ASCT). In arm B, 38% of patients did not
undergo HDS and ASCT. According to the intention-to-treat basis at
a median follow-up of 23 months, 5-year overall survival probability
in arms A and B was 47% and 40% ( P=NS); progression-free survival was 47% and 30% (P =NS) and disease-free survival was 97%
and 47% (P =0.02), respectively.
Conclusion:
Abbreviated chemotherapy followed by intensification with
HDS-ASCT seems not to be superior to conventional chemotherapy
in patients with HI/HR aggressive NHL. Considering the low DFS in
arm B, we can not consider HDS and ASCT as primary therapy for
this category of patients.
095
ZIO-201 A NEW ALKYLATOR FOR LYMPHOMA
ACTIVE IN IFOSFAMIDE (IFOS) RESIDENT
CANCERS
Gale, R.P.1; Morgan, L.R.1; Struck, R.F.; Rosen, L.1; LoRusso, P.1
1
Southern Research InstituteBackground
High-dose IFOS is a pro-drug metabolized to isophosphoramide
mustard (IPM) and used to treat advanced lymphoma. There are
several important problems. IPM is a bi-functional alkylator that irreparably cross-links DNA via G:C base sequences resulting in cell
death. Because IPM is directly active several problems associated
with IFOS are solved: (1) toxicity: IFOS metabolites causing hemorrhagic cystitis and confusion are not produced; (2) inter-subject vari-
S157
ability: reduced because metabolic activation is not needed; and (3)
resistance: because IPM is not deactivated by ALDH it is active in
IFOS- and CPA-resistant cancers where resistance is caused by increased ALDH. We recently stabilized IPM with lysine (IPM-L; ZIO201) and tested it in preclinical models and in a phase-1 trial.
Methods
Preclinical studies, phase-1 study and pharmacokinetics.
Results
ZIO-201 was 10-30-fold more active than IFOS or CPA in most
leukemia/lymphoma studies in in vitro models and in cancer-bearing mice. ZIO-201 also killed CPA- and IFOS-resistant leukemia/
lymphoma in cancer-bearing mice. In a phase-1 trial, ZIO-201 was
given daily for 3 consecutive d every 3 w without mesna to 20 subjects with advanced cancers. MTD was 445 mg/me2/d. The DLT
was proximal renal tubular acidosis; there was no hemorrhagic cystitis or CNS-toxicity. Bone marrow toxicity was modest. 1 subject
with mesothelioma had stable disease >13 mo; another with sarcoma responded clinically. Pharmacokinetic studies at MTD showed
a tmax=13 min (SD±14 min), Cmax=24.6 μg/mL (SD±13.2 μg/mL),
t1/2=37 min (SD±9 min) and AUC0-8=0.81 mg•min/ml (SD±0.45
mg•min/ml).
Conclusion
These data suggest a role for ZIO-201 in lymphoma including settings with CPA- and IFOS-resistance caused by increased
ALDH. There was no hemorrhagic cystitis or CNS-toxicity; bone
marrow-toxicity was modest. Doses achieved with ZIO-201 at MTD
are comparable to IFOS doses of 15-30 g/me2, substantially more
than can given safely in ICE or MIME. Because of modest bone
marrow toxicity, ZIO-201 can be given to persons with bone marrow
failure. Plasma levels at MTD exceed the IC50 of lymphoma cells
in experimental models. A phase-1/-2 study in lymphoma will begin
soon; data will be presented.
079
90Y-IBRITUMOMAB TREATMENT FOR RELAPSED
AND/OR REFRACTORY B CELL TYPE NONHODGKIN`S LYMPHOMA
Cacchione, R1 *; Dupont, J1; Riera, L1; Fernandez, J1; Garay, G1;
Riveros, D1
*
Argentina - 1 CEMIC
The radionucleid conjugate with monoclonal antibodies
(antiCD20/90Y-ibritumomab tiuxetan) have been aproved for the
treatment of relapsed, refractory and transformed (high grade) follicular lymphomas. Between September and November 2005, ten
patients with refractory/relapsed lymphoma were enrolled. Median
age was 56 yrs old (45-71). Four were women and 6 were men.
Nine were follicular and 1 was mantle cell lymphoma. Four patients
had bulky disease, 4 had bone marrow involvement and 7 had
stage VI disease. Time from diagnosis was 0-3 years in 2 pts, 3-6
in 3 pts and over 6 years in 5 pts.. Three pts had received 1-2 previous treatments, and 7 pts had received 3-5 previous treatments
including autologous bone marrow transplantation. All had received
anti-CD20 monoclonal antibody therapy. No pts recived previous
radiotherapy. 90Y-Ibritumomab (Zevamab NR Schering Argentina)
was administered at 0,3 or 0,4 mCi, based on initial platelet count.
Seven days before, and the same day of the inmunoconjugate administration, pts received rituximab 250 mg/m2. Six pts responded,
(5 CR, 1 PR major) and 4 pts continued in remission at 11 months
of follow-up. Five pts required filgrastim administration for neutropenia, 4 pts required platelet transfusions, 3 pts had neutropenia
plus fever, 2 pts required red blod cells transfusion, and only 2 pts
had to be admitted for complicated pancytopenia. Three pts with
previous bone marrow transplantation, required filgrastim, transfusions and 2/3 had febrile neutropenia; their cytopenias were not
persistent. Our experience shows 50% CR. Even heavily treated
pts, that had previous bone marrow transplant were able to receive
radioimmuno conjugate, although they required extra support. Our
experience favours the use of 90Y-Ibritumomab tiuxetan in relapsed
and refractory lymphomas even they had received previous bone
marrow transplantation.
S158
196
WHAT’S THE SIGNIFICANCE OF FDG-PET/
CT SCAN AT DIAGNOSIS OF NON HODGKIN
LYMPHOMAS?
Sancetta, R.1 *; Gregianin, M.1; Dei Rossi, F.1; Cracco, E.1; Pregno, P.2; Vitolo, U.2; Rigacci, L.3; Merli, F.4; Chisesi, T.1
*
Italia - 1 Ospedale Civile \”Umberto I\”, Venezia-Mestre; 2 Az.
Ospedaliera \”S. Giovanni Battista\”, Molinette-Torino; 3 DAC - Università di Firenze, Firenze; 4 Arcispedale S. Maria Nuova, Reggio
Emilia
Background: Correct staging is important for the appropriate
treatment in lymphoma patients. Most cancers, including lymphomas, metabolize glucose at abnormally high rate and so FDG-PET/
CT is an important tool in the evaluation of patients with lymphoma.
Many authors in these last years have shown the importance of
FDG-PET/CT analysis at diagnosis of lymphomas and the differences according to histologic subtypes. Aims: The IIL (Italian Lymphoma Intergroup) evaluated:1) the role of FDG-PET/CT versus CT
scanning in the staging of Non-Hodgkin´s lymphoma, 2) the significance of FDG-PET/CT according to histologic subtypes, 3) the ability of FDG-PET/CT in showing extranodal localizations. Methods:
We have retrospectively analysed at diagnosis 108 patients (pts)
(54 male, 54 female) with both FDG-PET/CT and conventional CT
scanning. The histologic subtypes were: diffuse, large B-cell lym-
phoma (LBCL) 50 pts (46%), follicular lymphoma (FL) 37 pts (34%),
marginal zone lymphoma (MZL) 7 pts (6%), mantle cell lymphoma
(MCL) 4 pts (4%), Burkitt and Burkitt-like lymphoma (BL) 4 pts (4%),
primitive mediastinal B-cell lymphoma 2 pts (2%), other lymphomas
(small lymphocytic, peripheral T-cell, extranodal, lymphomatoid
granulomatosis) 4 pts (4%). Results: We have evaluated nodal (18)
and extranodal (12) stations. Considering all cases, the agreement
between FDG-PET/CT and CT scanning was 89% in nodal stations
and 95% in extranodal ones, while discordance was 9% (7% toward PET/CT and 2% toward CT), and 5% (4% toward PET/CT and
1% toward CT) respectively. The percentage was similar in all the
different histologic subtypes. The extranodal localizations in which
there were more discordances were spleen (7 pts), liver (6 pts), and
bones (17 pts). FDG-PET/CT upstaged 27/108 pts (25%) and for
16% of pts the upstaging modified therapy (0 → III-IV in 4 pts (4%),
I→ III-IV in 3 pts (3%), II → III-IV in 10 pts (9%). The FDG-PET/CT
downstaged only 9/108 pts (8%): II→ I in 1 pts (1%), III-IV → II
in 5 pts (4%), I → 0 3 pts (3%). Conclusions: FDG-PET/CT and
CT scanning are concordant, for nodal and extranodal localizations,
in staging of Non-Hodgkin lymphomas. FDG-PET/CT shows more
nodal localizations (7%) and extranodal localizations (4%) than CT
scanning. There isn´t s substantial difference in concordance between FDG-PET/CT and CT scanning according to the various histologic subtypes. It is important to have FDG-PET/CT baseline for
early and late evaluation during and after therapy. FDG-PET/CT is
essential for staging lymphomas also as exclusive method.
S159
POSTER SESSION
09.1 Flow Cytometry
FC_01
STATISTICAL CRITERIA TO ESTABLISH OPTIMAL
ANTIBODY DILUTION IN FLOW CYTOMETRY
ANALYSIS.
Collino, CJG1 *; Jaldin-Fincati, J1; Chiabrando, GA2
*
Argentina - 1 CEQUIMAP y CIBICI-CONICET; 2 CIBICI-CONICET
Background: In direct techniques of flow cytometry, the optimal
antibody dilution or titer point is established from the plateau area
of the antibody titration curve. However, the plateau area is defined
without any statistical criteria, which may lead to an incorrect selection of antibody dilution. Objective: We apply statistical criteria to
establish the optimal antibody dilution for CD14, CD8, CD4, and
CD3 analysis by flow cytometry in peripheral whole blood. Methods:
The unpaired t-test (two-tail P value) was used as statistical criteria
to analyse the titration curve of the following monoclonal antibody
panels: CD14-FITC, CD8-FITC, CD4-RD1, and CD3-PC5. Results:
Using the unpaired t-test (two-tail P value) the plateau area from
the antibody titration curve was fitted when two consecutive antibody volumes showed mean peak of channel fluorescence (MPCF)
values not significantly different. When the antibody was used at
volume corresponding to that of the antibody titration point, the flow
cytometry analysis of whole blood samples with different density of
cell antigens can be correctly discriminated. Conclusion: This statistical criteria allows the fitting of the plateau area of MPCF versus
antibody volume and consequently, to define the optimal antibody
dilution. Key words: Statistical criteria; Unpaired t-test; Antibody titration; Flow cytometry; Mean peak of channel fluorescence (MPCF).
FC_02
COEXISTANCE OF TWO ABNORMAL POPULATION
IDENTIFIED BY FLOW CYTOMETRY
Cismondi, V1 *; Maiorano, M1; Galeano, A1; Iommi, P2; Pombo,
P2; Halperin, N3; Torletti, F4; Agriello, E2
*
Argentina - 1 Centro de Diagnóstico Molecular S.A., Bs.As.; 2
Servicio de Hematología, H.I.G. Dr. José Penna, Bahía Blanca; 3
Servicio de Inmunogenetica, Hospital de Clinicas, UBA, Bs.As; 4
CAPGRI S.A., Posadas. República Argentina
Background: Chronic Lymphoproliferative disorders (CLPDs)
are a heterogeneous group of diseases that result from the proliferation and accumulation of mature-appearing aberrant lymphocytes
arrested at a given stage of differentiation. Multiparameter flow cytometry (FC) allows the characterization of a particular cell within
a large cell population. Objectives: to report the presence of two
different populations of neoplastic lymphocytes in the same patient.
Material and methods: peripheral blood, bone marrow and body flu-
ids collected from patients diagnosed with CLPD were evaluated by
FC. When monoclonality is detected in a screening panel, samples
are stained with additional markers for further characterization of
cells. Results: the presence of two abnormal populations of lymphocytes was observed in 8 cases. All samples include chronic lymphocytic leukemia associated phenotype (AP)(CLL) population and
one of the following: hairy cell leukemia AP(n:1), B-cell lymphoma
CD5(-)CD23(-) (n:4), Follicular lymphoma AP (n:1), T-CLPD (n:1)
and a CLL AP with a distinct surface immunoglobulin light chain
(n:1) Conclusions: Flow cytometry is a high-sensitivity technique
that allows the finding of more than one abnormal population in
a single sample. The correct identification of the abnormal populations at diagnosis allows the adequate evaluation of them in the
follow up.
Keywords: flow cytometry, cronic lymphoproliferative
disorders,co-existing populations.
FC_03
ADULT T-CELL LEUKEMIA/LYMPHOMA
(ATLL) - DEMOGRAPHICAL, CLINICAL AND
INMUNOPHENOTIPICAL CHARACTERISTICS IN
PERU - 1996-2005
Vidal, J.1 *; Dyer, R.1; Valdivieso, N.1; Pizarro, R.1; Ferreyros, G.1;
Barrionuevo, C.1; Casanova, L.1
*
Perú - 1 Department of Pathology, Department of Medicine, Instituto Nacional de Enfermedades Neoplásicas “Eduardo Cáceres
Graziani”
We compared the demographic differences of 92 cases of
adult
T-cell leukemia/lymphoma (ATLL), received by the National
Cancer Institute of Lima, Peru between 1996 and 2005. We found
that 52% of the patients came from the southern region of the Andes
where people of quechua origin predominates. The most common
clinical sub-type was acute type 61(66%), followed by lymphoma
29 (32%), chronic 1(1%) with 1(1%) smoldering cases. The female/male ratio was 1:4; the age range varied between 25 to 89
years, with an average of 51 years. The main clinical characteristics were: lymphadenopathy (89%), hepatomegaly (50%), skin lesions (42%) and splenomegaly (34%). Hypercalcemia was seen in
54%. Bone marrow involvement was noticed in 75% and in 67%
showed characteristic blood circulating neoplasic cells. Antibodies
to HTLV-1 virus were found in all cases. Bone marrow flow cytometer studies were contributory in 35 patients: 51% have the classical
inmunophenotype: CD3+, CD4+, CD5+, CD25+; Aberrant phenotype included: 4/35 (11.4%): sCD3- 1/35(2,8%): cyCD3+/sCD3- ;
3/35(8.6%):
CD8+; 1/35(2,8%): CD8+/CD4+; 27/32(84,4%):
CD25+; 22/31(70,9): TCR a/b and 2/20(10%) co-expressed CD56.
Keywords: Adult T-cell leukemia/lymphoma, immunophenotype
S160
FC_04
HYDROA-LIKE CUTANEOUS T-CELL LYMPHOMA
WITH BONE MARROW INFILTRATION DETECTED
BY FLOW- CASE REPORT
Vidal, J.1 *; Dyer, R.1; Barrionuevo, C.1; Pizarro, R.1
*
Perú - 1 Department of Pathology, Department of Medicine, Instituto Nacional de Enfermedades Neoplásicas “Eduardo Cáceres
Graziani”
There are reports of patients from Asia and Latin America with
a hydroa vacciniforme (HV)-like eruption, a cutaneous lymphoma
that affects children, called angiocentric Tcell lymphoma of childhood. The lymphoma cells displayed T-cell cytotoxic phenotype. No
bone marrow infiltration has been reported. We describe a 13-years
old girl, YRI, HC 396345 with an 18 months history of weight loss,
fever, cutaneous edema, blisters and scars on the face. Physical
examination showed: jaundice, hepatomegaly, edema and lymphadenopathy. Laboratory tests: Hb: ?114gr/L WBC: 3.7 Plts: 206,
HTLV 1 (Neg). Myelogram showed 18% lymphocytes, with irregular shape, intermediate chromatin, some fusiforms cells, and few
hystiocites with hemophagocytosis. Cytogenetic studies showed
hypodiploidy and the immunophenotype was CD45/ CD5 (Bright),
CD3/CD8, TCR AB, CD7/CD2.
Key words: T cell cutaneous lymphoma; angiocentric T ell lymphoma of childhood, inmunophenotype bone marrow infiltration
at room temperature. During this incubation time, aliquots of treated
RBCs were re-suspended in PBS 300 mM pH 7.4 solution at six different times: 1, 5, 15, 30, 45 and 60 minutes to be analyzed.
The flow cytometry measurements were performed using a
standard instrument (Coulter Epics XL-MCL), which measures the
forward light scattering intensity (FSC) and the 90º side light scattering intensity (SSC) at logarithmic amplification. 100,000 events
were acquired in list mode at every sample run. The list mode files
were processed by using a computer software Win MDI version
2.8.
Results: We present the FS and SS dot plot and histogram in
which can observe that during RBCs storage the SS and FS values
decrease according to the discocyte-echinocyte transformation. All
over this storage time, the SS value tends to recover the initial value
with fresh frozen plasma treatment in function of the incubation time
but the same behavior was not observed in the FS value, where all
values tend to declined.
Conclusions: Data obtained with flow cytometry turned out to
be useful to evaluate the RBCs characteristics and their reversibility properties as storage time increases, because it is possible to
count a great number of cells in a few minutes and evaluate them
in a multi-parametric way. This method is a non-conventional tool to
evaluate accurate RBCs behavior during storage under blood bank
conditions, but unlike conventional microscopy, flow cytometry is
not affected by the operator’s subjectivity. Therefore, flow cytometry could also be applied to quality control protocols in transfusion
medicine.
FC_06
FC_05
FLOW CYTOMETRY METHODOLOGY APPLIED TO
THE STUDY OF RBC CHARACTERISTICS AND ITS
RE-ESTABLISHMENT AFTER STORING
Di Tullio Budassi, L.1 *; Foresto, P.1; Delannoy, M.1; Valverde, J.2;
Riquelme, B2
*
Argentina - 1 Fac.Cs.Bioquímicas y Farmacéuticas; 2 Fac.
Cs.Bioquímicas y Farmacéuticas / Instituto de Física Rosario
Introduction: It is well known that red blood cells (RBCs) show
characteristic shape changes, especially discocyte-echinocyte
transformation when they are stored. The normal discocyte represents an equilibrium state between two opposing shape changes:
the echinocytic and the stomatocytic transformation. Transformation discocyte-echinocyte is influenced by many factors such as
ATP depletion, intracellular calcium increase, pH changes and alterations in the composition of the cell membrane. During storage,
lyso-phosphatidylcholine is produced from phosphatidylcholine,
which accumulates in cell membranes and is a potent echinocytogenic stimulant. Even though the RBC shape transformations have
a potential reversibility, these alterations may alter transfusion effectiveness.
Objectives: The aims of this study were to analyses the discocyte-echinocyte transformation and evaluate the re-establishment
of the stored erythrocyte characteristic after incubation in autologous plasma by means of flow cytometry.
Materials and Methods: Concentrated RBCs prepared from a
healthy donor’s whole blood and collected with CPDA-1 solution
were stored under blood bank conditions during 45 days at 4°C. A
sample of these concentrated RBCs was taken every week after
donation in order to be analyzed. Then, 500μl of RBCs and 500μl
of autologous plasma were mixed and incubated during 60 minutes
CD38 AS A PROGNOSTIC MARKER IN B CHRONIC
LYMPHICYTIC LEUKEMIA AT DIAGNOSIS:
RETROSPECTIVE STUDY
Novoa, V.1 *; Nuñez, N.1; Pavlove, M.1; Fishman, L.1; Moidosky,
M.1; Peretz, F1; Estigarribia, N.1; Flores, G.1; Cervellini, M.1
*
Argentina - 1 Unidad Inmunología. Servicio de Hematología. Hospital ”Carlos G. Durand”
Resumen: Chronic lymphocytic leukemia is a disease among
the so-called SLPC, its clinical stage is based on the Rai-Binet System. However this criteria doesn´t predict the patients evolution. Expression of CD38 molecule in clonal cells has been considered as
a prognostic marker. Our aim was to analyze the disease evolution
in 16 patients, the CD38 expression at diagnostic stage and the
need at treatment. Materials and method.: 16 patients ( 12 males
and 4 females ) were studied during 5 years ( 2000-2005). CD38
expression was evaluated by flow cytometry ( FACSort BD) with 3
colour staining at diagnostic and the Binet System was applied for
clinical classification . Results: from 9 patients CD38 + , 8 (88%) required treatment. From 7 patients CD38 -, only 2 (35%) were treated. According to Binet System : 10 patients belonged to Group
A : those with CD38 +(4 patients) required treatment all of them,
and in the remaining 6 patients group that were not treated only
one was CD38+. All patients in Group B (2 patients ) were CD38
(+) and required treatment and the 4 patients belonging to Group
C (2 CD38(+), 2 CD38(-)) disease progressed and died. Discussion: Our results suggest that the CD38 (+) shows a tendency to
progress to advanced clinical stages which required treatment. But
the low number of patients and the brief period of follow-up doesn´t
allowed us to obtain statistic conclusions. This is important in Group
A of Binet System at diagnostic stage because we could recognize
subgroups with an aggressive evolution.
FC_07
TRANSIENT MYELOPROLIFERATIVE
DISORDER (TMD) IN DOWN SYNDROME
(DS): IMMUNOPHENOTYPIC ANALYSIS USING
MULTIPARAMETER FLOW CYTOMETRY (FC)
Agriello, E.1 *; Iommi, P.1; Pombo, P.1; Garbiero, S.1; Cismondi,
V.2; Maiorano, M.2; Galeano, A.2; Diaz, G3; Matiocevich, 3; Torres, H4
*
Argentina - 1 Servicio de Hematología, H.I.G. Dr. José Penna,
Bahía Blanca; 2 Centro de Diagnóstico Molecular, Bs.As; 3 GHS; 4
SAP
Background: Children with DS are at a higher risk of developing
Acute Leukemias (AL) compared with the general pediatric population. Also, 10% of neonates with DS may develop a TMD, which is
an abnormal proliferation of myeloid blasts in blood that resolves
generally without therapeutic intervention. Objective: to evaluate
inmnunophenotypic patterns in TMD cells by FC and its contribution
for diagnosis. Material and Methods: Peripheral blood and bone
marrow from four patients with DS were studied by FC. The age
range was from birth to 2 months. Results: In all cases elevated
percentages of myeloid cells with abnormal immunophenotype were
found. However, without megakariocytic antigen expression (CD61,
CD42a, CD41). All blast cells expresed CD34, CD117, HLA DR,
CD13, CD33 and were negative for CD15 and CD16. There was
also abnormal expression of CD56, CD4 and CD7 in some cases.
Conclusions: TMD is frequently found in newborn patients with DS.
This pathology must be suspected and differentiated from AL since
the spontaneous remission may occur in 70% of the cases. Taking
into account that blast cells from TMD and AML are very similar immunophenotyphically, both, the physician and the cytometrist must
be careful in evaluating and interpreting the results.
Keywords: transient myeloproliferative disorder, acute leukemia, flow cytometry
FC_08
IMMUNOPHENOTYPE IN ACUTE LYMPHOBLASTIC
LEUKEMIA (ALL) PATIENTS YOUNGER THAN 1
YEAR OLD AT DIAGNOSIS WITH ALTERATIONS IN
THE 11Q23/ MLL GENE
Gallinger, M *; Felice, M1; Alonso, C1; Gallego, M1; Bernasconi,
A1; Alfaro, E1; Rubio, P1; Rossi, J1
*
Argentina - 1 Hemato/oncology,Immunology and Cytogenetic
Departments
Acute leukemia (AL) in patients (pts) younger than 1 year old
presents with unique clinical and laboratory features, treatment response and outcome. Around 80% of them have cytogenetic alterations involving band 23 of the long arm of chromosome 11, where
the MLL (mixed lineage leukemia) gene is localized. Given the particular gene profiling and biological characteristics of this group of
S161
pts [most commonly immature immunophenotype, CD10 negative
B cell precursor (Bcp), and the poor treatment response], the MLL+
AL has been proposed to be considered as a different entity.
Materials and methods: Sixty two pts <1 year old were admitted to our Hospital between January 1990 and November 2006, 54
of them were evaluable for this presentation. All cases were analyzed by flow cytometry and MLL gene involvement was assessed
by conventional cytogenetic, SPLIT-FISH and/or RT-PCR.
Results: 40 out of 54 cases were acute lymphoblastic leukemia (ALL) with 11q23/MLL rearrangements and were treated with
ALL chemotherapy protocols, 39 of them presented Bcp ALL and
1 disclosed an ambiguous phenotype (T/B/myeloid). The analysis
of the maturational stage of the 39 Bcp cases revealed: 22 Pro-B
(56.5%), 15 Pre-B (38.5%), 1 Common (2.5%) and 1 Mature (2.5%)
cases. CD10 was expressed in 6 cases (5 Pre-B and 1 Common).
Of note, CD45 expression was more intense and homogeneous in
the 11q23/MLL Bcp ALL group compared to the blasts of Bcp ALL
cases, without this alteration. During their evolution, 4 pts developed lineage switch from ALL to AML, keeping the same cytogenetic/molecular findings.
Conclusions: We found a higher incidence of CD10+ cases
(15%) than that described in the literature, in our setting. The immature phenotype prevalence, the detection of blasts with ambiguous
lineage and the occurrence of lineage switches support the notion
of a common lymphoid-myeloid precursor as target of the malignant
transformation in this differential subset of leukemia.
FC_09
INFREQUENT IMMUNOPHENOTYPIC FINDINGS
OF B-LINEAGE NEOPLASMS IN CHILDHOOD:
EXPERIENCE IN A SINGLE INSTITUTION.
Bertone, S *; Rossi, J1; Bernasconi, A1; Gallego, M1; Alonso, C1;
Chantada, G1; Alfaro, E1; Felice, M1
*
Argentina - 1 Hemato/oncology,Immunology and Cytogenetic
Departments
A clear correlation exists between morphologic, immunophenotypic and cytogenetic/molecular findings in B-cell malignancies.
Mature B (Bm) phenotype [surface Immunoglobulin (sIg)+] is associated to L3 morphology and t(8;14)(q24;q32) or its variants, while B
cell precursor (Bcp) acute lymphoblastic leukemia (ALL) phenotype
corresponds to L1/L2 morphology and several different cytogenetic
alterations. The correct characterization and interpretation of results
in these malignancies, both at the moment of initial diagnosis and
relapse, are essential for the selection of the most appropriate treatment, taking into account that it should be different for Bcp ALL or
Bm leukemias with t(8,14).
From August 1988 to December 2006, 928 patients (pts) with
diagnosis of B-lineage ALL were admitted to our Hospital, 27 presented Bm and 901 Bcp phenotypes.
The aim of this presentation is to describe 4 pts that did not
show the usual correlation of the different parameters at diagnosis,
and another one (pt 5) who presented an intra-lineage switch at
relapse, which determined a change of treatment, in spite of the
initial diagnosis findings.
S162
Pt
Age/ sex Clinical/ Laboratory findings WBC/ mm3 Platelets/ mm3 Hb /dl Morphology Immunophenotype
12y/F
uric acid LDH
15900
48
14.1
L3
Common
7y/M
uric acid LDH
15800
21
5.1
L3
Common
Skin and subcutaneous nod3
9mo/M
11000
104
7.2
L1
Mature B (sIg+) l
ules
Lymphoid
4
7mo/F
Skin nodules
12500
424
10.0
Mature B (sIg+) l
(histology)
5 Diagnosis 12y/M
Hepato- splenomegaly
8300
17
8.9
L1
Pre B
5 Relapse
L3
Mature B (sIg+) l
1
2
In spite of a clear L1 morphology, monoclonality for sIg was
assessed in pt 3, while in other 2 with conspicuous L3 morphology,
immunophenotyping revealed Bcp (sIg-) ALL. Another unexpected
finding was the change both in morphology and cytogenetics in pt
5 from the initial diagnosis to relapse. Pts 1, 2 and the relapse of
pt 5 were treated with protocols for Bm ALL, while pts 3, 5 (initial
diagnosis) and 4 received schemes for Bcp ALL. All pts achieved
complete remission.
Conclusions: These results emphasize the importance of determining sIg in all cases, despite morphological findings. The availability of all these diagnostic tools (morphology, immunophenotype,
cytogenetics and molecular studies) in proper timing was essential
for the administration of the adequate specific treatment schedule
in this unusual group of pts.
FC_10
PHENOTYPICAL PATTERNS ASSOCIATED TO
SPECIFIC GENOTYPES IN ACUTE LEUKEMIAS
(AL).
Agriello, E1 *; Pombo, P2; Iommi, P2; Fernandez, V1; Garbiero,
S1; Brandt, M1; Di Paolo, D1; Silenzi, N3; Furque, M3; Manera, G3;
Taborda, M3; Mur, N3; Venchi, R3; Kurchan, A3; Raña, P3; Kowalysin, R4
*
Argentina - 1 Hematology Department, HIGA Dr. Penna, Bahía
Blanca / Hematological Group of the South; 2 Hematology Department, HIGA Dr. Penna, Bahía Blanca; 3 Hematological Group of
the South; 4 Hospital Zatti
Background: the expression of certain protein patterns is associated to specific genotypical groups. Objective: to learn to associate phenotypical patterns as predictors of specific genetic aberrations. Material and Methods: only AL diagnosed cases assessed
by means of multiparameter flow cytometry (MFC) with specific
combinations for this objective in which later the chromosomic abnormality was confirmed by such confirmation techniques as cytogenetic studies using G banding, FISH or Molecular Biology were
considered. Results: Regarding Acute Lymphoblastic Leukemias
B (ALL): in 2 ALL Pro B cases with CD15+ and CD65+ expression (associated to myeloid line) the presence of the abnormality in
11q23 t(4;11) was assumed, in 4 adult ALL B cases with characteristic pattern CD34/CD38 and CD13heterogeneous the presence of
t(9;22) was predicted. Regarding Acute Myeloid Leukemias (AML)
in 19 M3(4M3v) cases t(15;17) was predicted, in 4 AML cases with
CD19 expression t(8;21) was predicted. Conclusion: the importance
of the MFC result lies on the fact that it is fast. The milestone is
to know the combinations determined so as to reach and predict
these genetic aberrations. This is done with an exhaustive analysis
of leukemic patterns regarding normal patterns and evaluation of
Citogenetic / Molecular findings
t(8;14)
t(8;14)
SPLIT signal for MLL+/MLLENL+
Normal
add(5)(p?)
t(8;14)
aberrant expression combinations. The objective of this association
is just to be a guide for the chromosomic study and to know the
prognosis of the AL type fast. The main usefulness lies in the fact
that it cooperates in later direction of the confirmation study type
depending on the type of leukemia so as to optimize resources.
Keywords: acute leukemia, flow cytometry
FC_11
CONTRIBUTION OF FLOW CYTOMETRY IN
SOLID FRESH TISSUES EVALUATION OF
HEMATOLOGICAL MALIGNANCIES. STUDY OVER
A 6 YEARS PERIOD IN THE AEPSM.
Di Matteo, C.1 *; Landoni, A.I.1; Daners, A.1; Arocena, A.1; Giordano, H.1
*
Uruguay - 1 Laboratorio de Biología Molecular, ASESP
Immunophenotyping of hematological malignancies has become one of the most relevant clinical applications of flow cytometry. Its great utility in blood and bone marrow, where cells are naturally suspended has already been established. Among other flow
cytometric approaches, immunophenotypic analysis of solid fresh
tissues has become particularly important for diagnosis and characterization of lymphoid malignancies. The advantages of flow cytometry are based on its sensitivity, specificity, simplicity and speed,
and it provides a better way for the simultaneous quantitative assessment of multiple antigens in large number of cells, even in small
samples. In addition, it offers objective criteria for interpretation of
results. Recent studies show that flow cytometry analysis in tissuebased lymphoproliferative disorders is an excellent complement to
microscope-based traditional diagnostic methods and improve diagnostic accuracy and precision over other diagnostic techniques.
A number of protocols are available for disagreggating fresh tissue samples into suitable single-cell suspensions. These protocols typically involve either enzymatic digestion (e.g. collagenous)
or mechanical chopping and filtering. In all situations, the fresh
tissues samples should processes within 24h of collection to ensure that there is a good viability, e.g. at least 80% viable cells.
We present our experience over a period from 2001 to 2006 in
the analysis of lymphoid fresh tissues. Most samples were lymph
nodes. We detected 55 lymphoproliferative disorders. The diagnosis was based in three colors combination panels with the antibodies CD19, CD20, FMC7, CD5, CD23, CD22, CD10, CD38,
CD43, CD79a, CD4, CD8, CD3, CD56, CD7, CD2, lambda, kappa.
We conclude that flow cytometry immunophenotyping is an excellent complement to microscopy in the analysis of lymphoid fresh
tissues.
S163
ORAL SESSION
04 STEM CELL TRANSPLANTATION – CLINICAL AND
EXPERIMENTAL
020
070
AUTOIMMUNE HEMOLYTIC ANEMIA FOLLOWING
ALLOGENEIC HEMATOPOIETIC STEM CELL
TRANSPLANTATION
HEMATOPOIETIC STEM CELL TRANSPLANTATION
(SCT) A SINGLE CENTER 11 YEARS EXPERIENCE.
Arriaga, F ; Sanz, J ; Montesinos, P ; Ortiz, G ; Sanz Guilermo,
GF1; Sanz Miguel Angel, MA1; De la Rubia Javier, J1
*
España - 1 Hospital Universitario La Fe
1*
1
1
1
Introduction:
Hemolytic anemia (AIHA) after allogeneic HSCT has been less
frequently described and it is characterized by the presence of autoantibodies from donor origin targeting donor derived red blood cell.
The aim of the study was to analyze the incidence and risk factors
for the development of AIHA, as well as its prognosis and response
to treatment in a series of patients undergoing allogeneic HSCT at
a single institution.
Objective:
The incidence of hemolytic anaemia in the progenitor hematopoyetic cell transplantation.
Between January 1996 and June 2004, 272 adult (18 year or
older) patients underwent allogeneic HSCT for the treatment of a
variety of malignant hematopoietic disorders at ours hospital. Direct
antiglobulin test (DAT) was performed in routine pretransfusional
compatibility testing or after clinical suspicion of immune hemolysis.
AIHA after HSCT was diagnosed:1) positive DAT, 2) positive indirect
antiglobulin test with broad reactivity to RBC in serum and eluate, 3)
clinical and laboratory evidence of hemolysis (increase of LDH and
bilirrubin levels, decrease of hemoglobin and haptoglobin levels or
increase in transfusion requirements), 4) other causes of immune
hemolytic anemia were excluded.
Results:
Twelve patients developed AIHA after HSCT at a median time
of 147 days (range, 41-170), giving a cumulative incidence at 3
years of 4.44%. Eight cold antibodies (IgM) and 4 warm antibodies (IgG) were detected. In the multivariate analysis, HSCT from
unrelated donors (Odds ratio 1.47, P=0.014) and the development
of chronic extensive GVHD in patients that survived after day 100
(Odds Ratio 6.74, P=0.006) were the only independent risk factors
associated to AIHA. Only two patient are still alive. The remaining
patients died mainly due to infection or GVHD between 3 and 28
months after HSCT.
Conclusion:
AIHA was never the primary cause of death but added morbidity in patients with other concomitant complications. AIHA after
HSCT is a relatively common and clinically significant complication
that often contributes to the morbidity and mortality of these critically
ill patients. Patients undergoing HSCT from unrelated donors and
those who develop chronic GVHD are specially predisposed for this
complication, for which we need to be specially cautious.
Guillermo, C.1 *; Díaz, L.1; Isaurralde, H.1; Topolansky, L.1;
Zunino, J.1; Stevenazi, M.1; Diaz, A.1; Perdomo, S.1; Perdomo,
A.1; Lavagna, G.1; Baubeta, A.1; Nese, M.1
*
Uruguay - 1 CITMO/ Facultad de Medicina, UdelaR
We evaluated the results of SCT between 1995-2006. We performed 346 SCT; 301 autologous (ASCT): NHL 101, HL 77, MM 55,
AML 32, ALL 13, ST 23; 45 allogeneic (ALSCT): AL 11, CML 12, MDS
9, AA 6, others 7. Twenty ASCT and 10 ALSCT were tandem. Median age was 43 years (3-65), 171 male and 130 female for ASCT;
39 years (6-58), 29 male and 16 female for ALSCT. The conditioning regimens were: CVB, BEAC, BEAM in Lymphoma (Lym); BuCy
in AL; Melfalan in MM; Maxi-ICE in ST; Cy-ATG in AA. We used a
maintenance treatment in ALL with Mtx and 6 MP. Stem cells mobilized with G-CSF were obtained from BM in 16, BM+PB in 127 and
PB in 203. The median MNC and CD34 infused were 9x108/kg and
8x106/kg in ASCT; 6x108/kg and 7x106/kg in ALSCT. Hematological
recovery median time was: 10 and 12 days for neutrophils; 14 and
18 for platelets in ASCT and ALSCT respectively. 100 days mortality (TRM) was 3% in ASCT, 5% in tandem SCT and 33% in ALSCT,
hospitalization median time was 22, 25 and 46 days respectively.
Overall survival (OS) in ASCT was: NHL 59%; HL 79%; AML 39%;
ALL 75%; MM 38%; germinal tumor (GT) 62%. ASCT tandem was:
MM 62%, NHL 31%, HL 60%, in ALSCT was: AA 33%, CML 40%;
AML 33%; MDS 25%. These data show: no significantly difference
in the patients outcome between BM and PBSC; ASCT has a low
TRM. The higher relapse rate in ALL and GT was in the first year,
and AML and Lym in the first 4 years, and then the survival curve
reaches a plateau. The tandem SCT in MM lets see a significantly
better OS at 8 years. Lymphomas that relapse after the first ASCT
can benefit from a second transplant. We think, higher OS in ALL,
than the literature reports, is in relationship with the maintenance
treatment. We emphasize the ALSCT higher mortality, without relapse after first year except in MDS.
118
KIR GENES IN UNRELATED AND HAPODENTICAL
HEMATOPOIETIC CELL TRANSPLANTATION (HCT)
Bengochea, M.1 *; Carretto, E.1; Tiscornia, A.1; Toledo, R.1; Alvarez, I.1
*
Uruguay - 1 INDT
Introduction: Killer Ig-like receptor (KIR) are found on the surface of human NK cells and some T-cell subsets existing in both
inhibitory and activating isoforms. The KIR gene family consists
of 15 genes and 2 pseudogene located on chromosome 19q13.4.
There is high polymorfism in the number and type of the genesand
a variability in the protein expression level. HLA-C and HLA-B are
ligands for KIRs. HLA-C are grouped acoording to amino acid at
S164
position 80 asparagine or lysine in C1 or C2 groups respectively.
The influence of KIR gene matching/mismatching on transplantation outcome is being investigated.
Objective:To initiate KIR genes typing for patients and donors
undergoing matched unrelated and haploidentical transplant and
to analyze retrospectively HLA-B, HLA-C and KIR-ligand matching.
Materials and Methods: Were selected 16 individuals from 8
hematopoietic cell transplantation: 5 unrelated and 3 haploidentical.
Typing for KIR genes were carried out using the KIR GENOTYPING SSP- PCR (Pel Freez) from genomic DNA. 20 primer
pairs were used to identify the 14 known KIR genes and 2 pseudogenes.
Results and Conclusions: There were no barriers to obtain the
KIR typing data in our laboratory. We showed the Kir genes frequency in this sample and analized KIR and HLA genotypes and
group A or B KIR haplotypes and the HLA-C or Bw (KIR ligands)
genotypes between recipient and donor. KIR A and B haplotype
were present 16/16 and 10/16 samples, and C1 and C2 HLA group
was present 8/15 and 14/15 respectively. 2/5 unrelated HCT were
performed with HLA-C incompatibility and one of them was KIRligand missmatched.
These preliminary information impulse us to propose a prospective study that correlate clinical data, to optimize the donors
selection.
Toxoplasma and syphilis) in a serum bank sample (umbilical cord
serum). Results. Whit this kind of studies we guarante that the unit
that was sent to the transplant center it’s in the better condition to
transplant: BLOOD GROUP. No difference was found between the
baseline cord blood samples and the segment removed from the unit
post-thawing.HUMAN LEUKOCYTE ANTIGEN (HLA). The results
of these pre-transplant quality control tests are not 100% comparable. A mismatch was found in a sample between the initial umbilical cord blood HLA result and the segment results. The conclusion
was that the external lab that processed the sample made a band
interpretation error in the initial sample. FLOW CYTOMETRY. In all
the flow cytometry tests, a decrease of up to 50% was observed
in the post-thawing segment cell viability, this dramatic drop in cell
viability (50%) that occurs with flow cytometry tests is not observed
when viability is assessed with the trypan blue technique upon
performing clonogenic cultures.CLONOGENIC CULTURES. The
results showed mean E-clone values (total CFUs/CD34+ ratio) of
22.07%, which are above the recommended ranges (>6), the mean
UFC-T x 106 value is 0.96. SEROLOGIC MARKER DETERMINATION. The serologic tests performed in a serum bank sample taken
from the units undergoing pre-transplant quality control reported a
100% correlation with the baseline tests. Conclusions Implementing a pre-transplant quality control system within a Umbilical Cord
Blood Bank is essential to the quality assurance of a hematopoietic
progenitor cell unit to be infused, and should be systematically performed in all the HPC units
180
171
PRE-TRANSPLANT QUALITY CONTROL
OF UMBILICAL CORD CRYOPRESERVED
HEMATOPOIETIC PROGENITOR CELL UNITS: A
MANDATORY ACTION TO ASSURE ENGRAFTMENT
IN VITRO CD4+ T CELLS EXPANSION:
INDUCTION OF REGULATORY T CELLS WITH
SIROLIMUS AND CD3/CD28 DYNABEADS
Calderón Garcidueñas, E. D.1 *; Ochoa Robledo, A1; Fernández
Torres, J1; Millán Rocha, M1; Ortiz Calderón, P1; Marín López, A1
*
Mexico - 1 Centro Nacional de la Transfusión Sanguínea
Introduction. The transplant of umbilical cord hematopoietic
progenitor cells has remarkably increased, because have certain
advantages when compared with other sources of hematopoietic
progenitor cells (HPC) like: a sufficient number of HPC for transplantation, great proliferation capability, decreased alloreactivity
and a great cellular plasticity. Maybe their major advantage is immediate availability, because are frozen at -196ºC The frozen and
defrozen procedures of the cells to transplant can produce damage
to them that’s why it is essential to establish a quality control process that is applied to the umbilical cord blood unit after cryopreservation and before transplantation, so that the transplant center can
guarantee a better chance of umbilical cord blood unit engraftment
in the patient. Material and methods. This study was conducted at
the Umbilical Cord Blood Bank (CordMX), CNTS Mexico City. The
methodology used to collect, process and cryopreserve the hematopoietic progenitor cells (HPC) from the umbilical cord blood (UCB)
was based on the international NETCORD-FAHCT standards. The
UCB was obtained in a sterile-bag closed system, an automated
cell separation and concentration system was used to process the
UCB (SEPAX-Biosafe). Cryopreservation of HPC was performed
with a controlled freezing system and the final storage was made
in liquid nitrogen at -196º C (Bioarchive System TG3626. Pre-transplant quality control was performed in 25 umbilical cord blood (UCB)
in a sample of the unit without danger to the complete unit, the studies that include the pre-trasplant quality control are: Transfutional
security with Blood group determination and Rho tests (Diana Gel
Grifols) as well as medium- and high-resolution HLA typing (PCR
SSP Dynal Biotech) and hematopoietic security with flow cytometry
(FACScalibur, BD) viability with blue tripan, and clonogenic cultures
(Stem Cell Technologies). To complete the control Infectious serology was once again performed (HIV, HBsAg, HCV, Chagas, CMV,
Borelli, G1 *; Aarvak, T2; Brunsvig, A1; Rasmussen, AM3; Kvalheim, G1
*
Norway - 1 Department of Cellular Therapy, Rikshospitalet-Radiumhospitalet HF.; 2 Dynal-Invitrogen, Oslo, Norway.; 3 Department
of Immunology, Rikshospitalet-Radiumhospitalet HF.
Background.- T regulatory cells (Treg) are a subset of T lymphocytes defined by CD4+CD25+ markers and high FOXP3 expression. They play a key role in self-reactivity and alloreactivity control.
In allogenic hematopoietic stem cell transplantation (HSCT), these
cells could be potential useful in reducing graft versus host disease
without impairing graft versus tumor effect.
Objective.- To optimize the in vitro T- cell expansion conditions
for clinical grade production of Treg cells.
Material and methods.- CD4+ cells were obtained by positive and negative immunomagnetic selection from peripheral blood
derived lymphocytes. CD4+ cells were stimulated with anti CD3/
CD28-coated Dynabeads at a bead/cell ratio of 3:1. The cells were
cultured with X-VIVO20 media, autologous plasma, IL2, IL4 and with
and without Sirolimus. At day 12 cells were washed and depleted
of CD3/CD28 Dynabeads. After 24 h re-incubation with CD3/CD28
Dynabeads, cytokine secretion was analyzed in the supernatant by
BioPlex. Cell phenotype and FOXP3 expression was evaluated by
flow cytometry and suppressive capacitive was measured using
standard proliferation assay.
Results.- 80% of CD4+ cells cultured with Sirolimus expressed
CD25 and secreted low levels of both Th1 and Th2 cytokines. In
contrast, only 20% of CD4+ cells cultured without Sirolimus expressed CD25 and secreted high level of Th1 cytokines. Culture
conditions either with or without Sirolimus resulted in the same number of FOXP3+ cells. However, cells cultured with Sirolimus showed
strong suppressive capacity and could suppress CD4+CD25- T cell
proliferation by 80% even at 1:32 ratio of CD4+CD25-:CD4+CD25+
cells.
Conclusions.- Sirolimus induces generation of Treg cells with a
strong inhibitory power over CD4+ CD25- cells proliferation.
S165
ORAL SESSION
05 ACUTE MYELOBLASTIC LEUKEMIA
008
042
ACUTE MYELOID LEUKEMIA IN THE ELDERLY,
INTENSIVE OR MAINTENANCE THERAPY? OUR
EXPERIENCE IN PATIENTS OVER 65 YEARS.
FLT3 GENE INTERNAL TANDEM DUPLICATION
(ITD) MUTATIONS IN PATIENTS WITH ACUTE
MYELOID LEUKEMIA (AML).
Mettivier, V1 *; Pezzullo, L1; Finizio, o1; Rocco, S1; Bene, L1; De
Rosa, C1
*
Italia - 1 Haematology Division A.Cardarelli Hospital
Arana-Trejo, RM1 *; Muciño-Hernández, G2; Ruíz-S, E2; FloresPeredo, L3; Ignacio-Ibarra, G4
*
Mexico - 1 Genética, Hospital General de México and Laboratorio de OncoHematologia, SC.; 2 Genética, Hospital General de
México; 3 Laboratorio de Análisis de OncoHematologia, SC.; 4
Laboratorio de Análisis de OncoHematología, SC.
Introduction:
The treatment of acute myeloid leukaemia in elderly with age >
65 years is still debated. In literature numerous studies have valued
the feasibility of intensive chemotherapy in these patients.
Objective:
The aim of the study is to value the difference in EFS and OS
among 2 groups of AML elderly patients treated with intensive chemotherapy (IC) or maintenance (M).
From June 2001 to May 2006 we have treated in our Division
54 AML patients, 30 male and 24 female with median age of 73
years (66-90 years). 27 patients (16 M and 11 F with median age
of 71 years) have received intensive chemotherapy ( I.C. Flag and
MICE) and 27 (14 M and 13 F with median age of 78.5 years) have
received maintenance (low dose cytarabine and/or support).
Results:
In IC group 12 patients (45%) have obtained to complete remission (CR) with to EFS and OS media of 4, 47 and 7, 15 months
respectively, the rate of TRM has been of 25%. In the M group the
CR has been documented in 8 patients (30%) with to EFS and OS
media of 4,22 and 4,94 months respectively (graph 1-2).
This results have shown a best rate of CR in the IC group but
the OS and EFS difference is not statistically significant in the two
groups (p: 0.7).
Conclusion:
In conclusion the Intensive chemotherapy has not improved
the survival in AML elderly patients. New therapeutics strategy is
necessary for to improve the EFS and OS in these patients.
Interesting is the use of specific monoclonal antibodies (anti
CD33) in this poor disease especially in maintenance after a CR
obtainable with an intensive or low dose chemotherapy.
Introduction:
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase,
it`s mainly expressed by early myeloid and lymphoid progenitor
cells. FLT3 mutations are the most frequent genetic lesion seen in
acute myeloid leukaemia (60%) (AML). Two distinct forms of FLT3
gene aberrations have been identified, internal tandem duplication
(ITD) in the juxtamembrane domain and point mutation within the
activation loop of the tyrosine kinase domain (D835), both have
been associated with poor clinical outcome.
Objective:
In this study we have examined the ITD FLT3 gene mutations
in patients with AML and we discuss the relationships between the
mutations and patient prognosis.
A total of 20 patients with diagnosis of AML de novo were
studied for the presence of FLT3 mutations by SSCP assay. DNA
was obtained using DNAzol (Invitrogen); its quality was evaluated
through electrophoresis and spectrophotometry. ITD was examined by amplifying the JM domain from exons 11 and 12. The PCR
products were denatured and loaded into 7.5% polyacrylamide gel
and after electrophoresis, the gels were stained with a silver stain.
ITD DNA fragments were detected as two bands of higher molecular weight than the wild-type FLT3 fragment (at 240 and 330 bp).
These higher molecular weight fragments were then isolated and
sequenced.
Results:
ITD mutations were detected in three of the 20 cases (15%).
Sequence analysis of ITD mutation samples showed tandem duplications, confirming and characterizing the ITD in these samples.
The karyotype were normal in two cases and t(6;11) was present
in one patient con ITD mutation. The treatment was the same in all
cases and the patients with normal karyotype and FLT3/ITD+ had
complete remission, but they had relapsed after 8 months.
Conclusion:
In AML with normal karyotype the FLT3/ITD+ can be the most
important factor predicting for relapse and we have confirmed in a
large cohort of Mexican patients.
S166
064
ACUTE PROMIELOCYTIC LEUKEMIA, TWELVE
YEARS EXPERIENCE AT THE UNIVERSITY
HOSPITAL,SAN JUAN,PUERTO RICO
Lopez-Enriquez, A.1 *; Fradera, J.1; Velez-Garcia, E.1
*
Puerto Rico - 1 University of Puerto Rico
Introduction: Acute Promielocytic Leukemias (APL) are a
unique example in carcinogenesis, of maturation arrest at the promielocyte stage, associated with a chromosomal reciprocal translocation of a portion of chromosome 15 and 17 with the formation of fusion proteins between the PML gene and the alpha retinoic receptor
site. The discovery that the Trans-retinoic Acid compound induced
maturation of the promielocyte, has contributed to increased the
curability of this disease.
Objective: To decrease the tumor burden with chemotherapy
followed by induction of differentiation and maturation of the promyelocyte with ATRA
Materials and method: Since 1994 when Transretinoic Acid
(ATRA) became available to us, we developed a protocol incorporating this drug to the standard regime of induction chemotherapy
for Acute Leukemias used in our Institution of seven days of continuous infusion of Cytosine-Arabinoside (Ara-C) and three days of
Daunorubicine (7+3), starting the ATRA on day 14 at 45mg/m2 and
continued for 120 days. Two to three more courses of consolidation
with high dose Ara-C, Ara-C with Daunorubicine or Daunorubicine
alone where given.
Results: We have treated 65 patients with APL since 1994 up
to February 2006. Sixty three (63) patients received 7+3+ATRA,
one received ATRA with Arsenic, one patient received Arsenic only.
Fourteen of sixtyfour (14/64) died early in the first two weeks of Induction of bleeding and sepsis for a 21% early death rate. Fortynine
out of Fifty patients (49/50) went into Complete Remission for a
98% rate. Three patients developed Atra Syndrome, they were mistakenly given Atra in the first few days of Induction, two responded
to steroids and went into remission but the other one died with the
Atra Syndrome in respiratory failure. Thirty three has remained in
complete remission with a range of two to twelve years for a rate of
67%. Ten patients (20%) relapsed within the first two years. One of
them was an HIV patient, another relapsed three more times even
after autologous transplant and died six years later.
Conclusion: Acute Promielocytic Leukemias are nowdays a
potentially curable disease. The initial high early mortality needs to
be addressed with a more aggressive support system. A 98% complete remission rate for Induction Chemotherapy is extraordinary,
no ATRA Syndrome when the Atra is given on the 14th day of treatment reduces further morbidity and mortality in this group of patients. Four of the ten patients that relapsed received daunorubicin
as single agent in consolidation.
132
ACUTE PROMYELOCYTIC LEUKEMIA (APL):
GENETIC CHARACTERIZATION OF 78
ARGENTINIAN AND URUGUAYAN PATIENTS.
Uriarte, M.R.1 *; Giere, I.2; Zubillaga, M.N.1; Chacon, A.2; Bonomi,
R.1; Lombardi, V.2; Giordano, H.1; Fernandez, I.2; Manrique, G.1;
Matteo, C.1; Pavlovsky, S.2; Martínez, L.1
*
Uruguay - 1 ASESP; 2 Fundaleu
Background: APL requires accurate and rapid diagnosis of
PML-RAR? transcript to implement specific therapy, prognostic assessment and MRD monitoring. FLT3 gene mutations have been
detected in 30% of APL pts in association with aggressive disease.
Objectives: Genetic characterization and prognostic value of
Flt3 mutations in 78 APL pts from Argentina and Uruguay.
Material and Methods: Flow-cytometry, cytogenetic and nested-PCR studies were done at presentation and during follow-up.
Results: 74/78 pts showed PML-RARa/t(15;17) (+) with isoforms: L (59%); S (36%); V (5%). Twelve pts died early and MRD
monitoring was performed in 54 pts: showing, after induction, CCR
93% and 7% persistence of MRD. After consolidation 53 pts (98%)
remained in molecular remission (2% died during consolidation) Actually, 45 pts (84%) are alive with no evidence of MRD. The remaining 8 cases died by hematologic relapse preceded by PCR (+). FLT3
gene status was established in 42 APL pts at presentation. Twelve
(28.6 %) pts showed FLT3 mutations: 8 (19%) were ITD (+) and 4
(9,5%) showed D835 mutation. 10/12 FLT3 (+) pts (83%) showed
S-isoform and 2 pts (17%) were L-isoform.
Conclusions: 1) S-isoform is most frecuently among children;
2) FLT3 mutations were associated with S-isoform (p<0,00032);
3) RT-PCR (+) after consolidation predicts relapse, remarking the
prognostic value of RT-PCR. Larger number of pts is required to
fully address the association of FLT3 mutation with poor clinical outcome.
Keywords: APL, MRD, FLT3, PML-RARa
176
SALMONELLA AS A VECTOR FOR NEW
IMMUNOTHERAPIES IN AML
Lens, D1 *; Brugnini, A1; Chabalgoity, J. A.1; Lens, D1
*
Uruguay - 1 Depto. Básico de Medicina, Depto. Desarrollo Biotecnológico, Facultad de Medicina.
Immunotherapies may play a major role in eradicating minimal
residual disease in leukemia. We have assessed an immunotherapy
protocol based on the use of live attenuated Salmonella as a vector
for cytokine genes in a leukemia model. Using the WEHI3B myelomonocytic leukemia cell line and Balb-C mice, we successfully
developed a reproducible AML model, where animals died between
day 20 and 35 after inoculation. Leukemia progression can be followed by flow cytometry by quantifying the CD45low/SSClow population on peripheral blood.
For assessing Salmonella based immunotherapies, three
groups of leukemic mice received at day 10, S. Typhimurium harboring a plasmid encoding murine IL-4 gene (SL-IL4), the empty
plasmid (SL) or no treatment (C). Tumor kinetics, survival time and
immune response were all evaluated.
SL-IL4 exhibited delayed tumor growth: at day 19, 71% of this
group remained leukemia-free in comparison with 43% and 38% for
the SL and C groups respectively (p<0.05). Disease progression is
paralleled by a marked reduction in the number of B cells in bone
marrow. Interestingly, SL-IL4 showed an increased B cells repopulation with numbers similar to those of naïve animals. In spleen,
SL-IL4 showed a significant increase of T cells as compared with
untreated animals, mainly due to an increment of CD4+ population
(27.3% vs 14.8%, p<0.05).
Antibody responses against WEHI3B antigens were evaluated
by ELISA. Only SL-IL4 developed detectable level of anti- WEHI3B
antibodies.
Overall these results showed that cytokine-gene therapy using
Salmonella as a vector can be the basis of new effective immunotherapies for leukemia.
S167
ORAL SESSION
06 ACUTE LYMPHOBLASTIC LEUKEMIA
050
093
LOOKING FOR NEW PROGNOSTIC FACTORS IN
ACUTE LYMPHOBLASTIC LEUKEMIA
CLINICAL OUTCOME OF ALLOGENEIC
PERIPHERAL BLOOD STEM CELL TRANSPLANTS
(PBSCT) IN ACUTE LYMPHOBLASTIC LEUKEMIA
(ALL).
Mello, M.R.B.1 *; Pereira, F.G.1; Magalhães, M.G.1; Melo, F.C.B.C.1;
Melo, R.A.M.1; Machado, C.G.F.1; Metze, K1; Lorand-Metze, I.1
*
Brazil - 1 Department of Internal Medicine and Pathology, Hemocentro, State University of Campinas, and HEMOPE, Recife
Introduction: Treatment of acute lymphoblastic leukemia
(ALL) is stratified according to several well-known risk factors such
as immunophenotype (T or B), peripheral leukocyte number at diagnosis, and cytogenetics.
Objective: to study the prognostic value of parameters of nuclear texture analysis in ALL.
Materials and method: in newly diagnosed patients we obtained clinical and laboratory data, as well as those of quantitative
flow cytometric analysis, DNA ploidy and proliferation rate. BCR/
ABL, AF4/MLL, E2A/PBX1 and TEL/AML1 were examined. Blasts
from the diagnostic bone marrow aspirate were digitalized, segmented and morphometric and texture parameters of nuclei were
examined.
Results: we studied 49 cases: 18 with age <18 years and 31
>18 years. T-ALL: 11 cases. Median age: 19 years, median PB leukocyte count: 81.5x109 /l. B-ALL: median age: 29 years; median
PB leukocyte count: 32.7x109 /l. Gene rearrangements were only
found in B-ALL: BCR/ABL in 4 cases and AF4/MLL and E2A/PBX1
2 cases each. T- and B-ALL differed also by MFI of CD45 and morphometric features such as entropy and 4 parameters of the co-occurrence matrix. In the survival analysis (Cox) several models containing age, PB leukocyte count, integrated optical density, minimal
grey value and parameters of the co-ocurrence matrix were found.
Gene rearrangements were excluded from these models.
Conclusion: morphometric parameters were able to separate
T and B ALL and had an independent prognostic value. As in the
present study the frequency of detected gene rearrangements was
low and the digitalization of cytologic slides is getting more used,
especially for registration of cases in multicentre studies, it is worthwhile to validate these results in a larger cohort of patients.
Vigorito, AC1 *; Aranha, FJP1; Oliveira, GB1; Eid, KAB1; Zulli, R1;
Colturato, V2; Azevedo, W3; De Souza, MP2; Lodi, FM3; Bittencourt, H3; Castro, N4; Barros, JC4; Pontes, ER5; Brandalise, SR5;
Almeida Pereira, RD6; Ribeiro, AAF6; Novis, Y6; Hamerschlack,
N6; Ferreira, E6; Azevedo, AM7; Magalhães, KG7; Bouzas, LFS7;
Ruiz, MA8; Maiolino, A9; Nucci, M9; Pasquini, R10; De Souza,
CA1
*
Brazil - 1 UNICAMP; 2 Jaú Cancer Hospital; 3 UFMG; 4 Santa
Casa SP Medical School; 5 Boldrini Children Hospital; 6 Albert Einstein Hospital; 7 INCA; 8 SJRP Medical School; 9 UFRJ; 10 UFPR
Introduction: HSCT is a valid alternative as post-remission
therapy in ALL.
Objective: Our aim was analyzed outcome of 97 ALL patients
with HLA identical sibling donors who underwent an allo PBSCT.
Materials and method: Median age was 24 ys (2-45), advanced disease was present in 74%, conditioning without irradiation was 56%; GVHD prophylaxis with MTX/CsA was 91%; CD34+
median was 4.6X106/kg (1.2-24)
Results: the median follow-up for surviving pts was 22 months
(1.6-93). Median day for neutrophils and platelets engraftment
was 15 and 13, respectively; no TBI conditioning, no MTX/CsA,
and aGVHD were associated significantly with faster neutrophils
engraftment; no MTX/CsA and aGVHD with platelets. Cumulative incidence (CI) for ³ 2 aGVHD was 45%, extensive cGVHD
50%; aGVHD in patients who received TBI conditioning was 34%
(P=0.04). The estimates of OS and DFS at 92 months was 21%
and 31%, respectively; OS for patients >36ys was 16% (P=0.04),
1ª RC vs others for patients with aGVHD 11% (P=0.03); there
was a trend towards better OS and DFS in patients with cGVHD
(54%, 63%; P=0.07,P=0.06). CI for relapses was 60%; relapses for
cGVHD pts were 36% (P= 0.05), and there was a trend towards
higher relapses in advanced disease (66%,P=0.06). TRM was
64%; in those patients with aGVHD, 73% (P=0.008). In multivariate analyses no MTX/CsA and aGVHD correlated with the speed
of platelets engraftment (P=0.001, P=0.006); TBI conditioning was
associated with less aGVHD and TRM (P=0.05, P=0.001); aGVHD
had a negative impact on OS with higher TRM (P=0.02, P=0.02).
Conclusion: Although not confirmed in the multivariate analyses, fewer relapses, and a trend towards better OS, and DFS was
found in patients with extensive cGVHD. Further follow up will be
necessary to confirm these results.
S168
027
PROGNOSTIC VALUE OF DIFFERENT PROFILES
OF THE EARLY RESPONSE TO THERAPY IN
ADOLESCENTS AND YOUNG ADULTS WITH ACUTE
LYMPHOBLASTIC LEUKEMIA
Semochkin, S.V.1 *; Loriya, S.S.1; Rumyantsev, A.G.1
*
Rusia - 1 Federal scientific clinical center for pediatric hematology,
oncology and immunology
Introduction:
The early response evaluated during induction therapy is essential for patient risk-stratification in childhood acute lymphoblastic
leukemia (ALL). However, there are few data available looking in
this feature in particular at adolescents and young adults with ALL
treated under pediatric protocols.
Objective:
The aim of the study was to assess the predictive value of the
early response to therapy in adolescents and young adults with
acute lymphoblastic leukemia.
During 1991-2003 in this study 124 (m-77, f-47) patients (pts)
from 10 till 29 years of age with a de novo ALL have been enrolled.
49/39.5% pts have been treated under protocol ALL-MB-91 and
75/60.5% pts - ALL-BFM-90m. The early response has been esteemed on number of circulating blasts on 8 day and status of a
bone marrow on 15 and 33/36 days of treatment. The status of a
bone marrow anticipated on the standard scale: M1 (<5%) and M2
(5-25%) and M3 (>25% blasts).
Results:
6-years event free survival (EFS) has made 61.3% and an overall survival (OS) has made 65.3%. The survival rate of patients with
the poor response to steroids on 8 day (> 1000 blasts / μl) in 2 times
has lower in comparison with patients with the good response: EFS
33.3 vs. 69.4% (p=0.006). The status M3 on 15 day of treatment had
adverse prognostic value: EFS 33.3% (M3: 21/102/20.6% pts) vs.
73.7% (M2: 19/102/18.6% pts) and 71.0% (M1: 62/102/60.8% pts)
(p<0.001). The status M2 on 15 day had no poor prognostic value
(p>0.05). EFS of patients with the poor response on 8 day but with
the favorable status of M1/M2 on 15 day has better in comparison
with patients with the good response on 8 day but with adverse status M3 on 15 day: 57.1 vs. 46.2% (p=0.375). Status M2/M3 on 33
/ 36 day has the strongest adverse prognostic factor: EFS 16.7%
(M3: 6/109/5.5% pts) and 28.6% (M2: 7/109/6.4% pts) vs. 72.9%
(M1: 96/109/88.1%) (p<0.001). Early response to therapy did not
depend on age and a sex of patients and the therapeutic protocols
(p > 0.05).
Conclusion:
The early response to therapy is the important prognostic factor
in treatment of adolescents and young adults with ALL.
052
NON-MYELOABLATIVE STEM CELL
TRANSPLANTATION IN PATIENTS WITH
RELAPSED ACUTE LYMPHOBLASTIC LEUKEMIA
(ALL).
Gómez-Almaguer, D1 *; Gutierrez-Aguirre, CH2; Cantu-Rodriguez, OG2; Gonzalez-LLano, O; Herena-Perez, Suzel3; Manzano-Carlos, A3; González-Carrillo, ML4; Ruiz-Argüelles, GJ3
*
Mexico - 1 Hospital Universitario de Nuevo León; 2 Servicio de
Hematologia del Hospital Universitario de la U.A.N.L.; 3 Centro de
Hematologia y Medicina Interna de Puebla; 4 Laboratorios Clnicos
de Puebla
Introduction: Despite the optimal use of the antileukemic
agents, reported cure rates no exceed 40% in high-risk ALL adult
patients. The use of hematopoietic stem cell transplantation is other
option in these patients and non-myeloablative conditioning is a
friendly alternative to the conventional and more toxic myeloablative
radio-chemtotherapy scheme, but there is very limited information
using this kind of transplantation in ALL.
Objective: We prospectively evaluated the therapeutic value
of non-myeloablative conditioning HSCT in 43 high risk ALL patients
in second remission
Materials and method: Using a HSCT schedule, 43 ALL highrisk patients were prospectively allografted in México, using HLAidentical siblings as donors. All patients received oral busulphan 4
mg / Kg/2 days, i.v. cyclophosphamide 350 mg /m2/3 days and i.v.
fludarabine 30 mg /m2/3 days; oral cyclosporin A 4 mg / Kg was
started on day - 1 and i.v. methotrexate 5 mg / m2 was delivered
on days + 1, + 3, + 5 and + 11. Median age of the patients was 19
years; there were 19 females. Patients received a median of 5.0 x
106/ Kg CD34 cells.
Results: Median time to achieve above 0.5 x 109/L granulocytes was 14 days, whereas median time to achieve above 20 x
109/L platelets was 15 days. Thirteen patients (30%) are alive 491
days (median) after the HSCT. The 861-day probability of survival
is 22%, whereas median survival is 200 days. Ten patients (23%)
developed acute graft versus-host disease (GVHD), and 8 patients
(18.6%) developed chronic GVHD. Twenty eight (65%) patients
showed relapse, in 9 cases despite the GVHD. Thirty patients died
between day 47 and 1050 after the HSCT, most of them (70%) of an
ALL relapse. The 100-day mortality was 25.5 %.
Conclusion: Relapse remains the first cause of death in highrisk ALL patients. Non-myeloablative HCST seems to have limited
therapeutic effect in ALL patients with advanced disease. New
ideas and emerging strategies should be employed in order to improve the outcome of these patients, like enhancement of graft-versus leukemia effects and the use HSCT in first complete remission.
124
ACUTE LYMPHOBLASTIC LEUKEMIA (ALL):
DETECTION OF PROGNOSTICALLY SIGNIFICANCE
FUSION-GENES BY MULTIPLEX-POLYMERASE
CHAIN REACTION (M-PCR)
Manrique, G.1 *; Capetta, M.1; Zubillaga, M.N.1; Bonomi, R.1; Di
Matteo, C.1; Giordano, H.1; Uriarte, M.R.1
*
Uruguay - 1 ASESP
Background: The identification of specific chimeric genes related with ALL is relevant for clinical assessment: allowing therapeutic implementation, monitoring minimal residual disease (MRD) and
risk-stratification patients (pts) assignment.
Objectives: To standardize a rapid, specific and sensitive molecular method to detect simultaneously the most prognostic relevant chromosomal translocations in ALL.
Materials and Methods: Bone marrow and peripherical blood
samples from 82 ALL pts (61 pediatric and 21 adults) previously
analyzed by flow cytometry and cytogenetic techniques were studied by RT-M-PCR (reverse transcriptase M-PCR) for the TEL/AML1,
BCR/ABL; MLL/AF4 and E2A/PBX1 fusion genes.
Results: B clonality was demonstrated by flow cytometry in all
patients; 26 from 82 samples (32%) were PCR (+) at diagnosis for
one of the following specific fusion gene: BCR/ABL (6 adults/3 children); MLL/AF4 (8 children) E2A/PBX1 (1 children). All of these pts
showed the corresponding cytogentic translocation. The TEL/AML
fusion gene, undetectable by cytogentic methods was identified in
samples from 8 children. During follow-up to evaluate treatment effectiveness MRD was established in 8 pts predicting hematological
relapse.
Conclusions: The M-PCR is a very useful tool for a fast and
sensitive identification of prognostically significance fusion genes
and early diagnosis of relapse risk in leukemia. This assay shows a
good correlation with the cytogenetic findings, allowing the detection
of t(12,21) undetectable by conventional cytogenetics.
Keywords: ALL, fusion genes, multiplex-PCR
S169
ORAL SESSION
07 MULTIPLE MYELOMA
043
024
DOUBLE VERSUS SINGLE AUTOLOGOUS STEMCELL TRANSPLANTATION FOR MULTIPLE
MYELOMA: A REGION BASED STUDY IN 485
PATIENTS FROM THE NORDIC AREA
PROGNOSTIC FACTORS IN MYELODYSPLASTIC
SYNDROMES (MDS): EXPERIENCE OF ONE
INSTITUTION
Björkstrand, Bo1 *; Klausen, TW2; Remes, Kari3; Gruber, Astrid4;
Knudsen, LM2; Bergmann, OJ5; Lenhoff, S5; Johnsen, HE6
*
Suecia - 1 Huddinge University Hospital; 2 Herlev University
Hospital; 3 University Central Hospital; 4 Karolinska Hospital; 5 Lund
University Hospital; 6 Aalborg Hospital, Aarhus University
Introduction:
Autologous stem cell transplantation is now considered the
standard of care in young patients with multiple myeloma (MM).
Objective:
The available results of randomized studies are in favor of tandem autologous transplantation; however the effect on long term
survival is unclear. During 1994-2000 we have conducted sequential registration trials in the Nordic area, including a regional phase II
registration study of double autologous stem-cell transplantations.
We have registered a total of 485 previously untreated patients
under 60 years at diagnosis who were treated with single (Trial
NMSG #5/94 and #7/98 (N=384)) or double (Trial HKTH (N=101))
high dose melphalan (200 mg/m2) therapy + autologous stem cell
transplantation.
Results:
A complete or a very good partial response was achieved by 40
percent of patients in the single-transplant group and 60 percent of
patients in the double-transplant group (P=0.0006). The probability
of surviving event-free for 5 years after the diagnosis was 25 (1832) percent in the single-transplant group and 44 (33-55) percent in
the double-transplant group (P=0.0014). The estimated overall 5year survival rate was 50 percent in the single-transplant group and
50 percent in the double-transplant group (P=0.9). In a multivariate
analysis of variables including single versus double transplantation,
beta-2 microglobulin level, age, sex and disease stage only beta2 microglobulin came out significantly (p<0.0001) and (p=0.001)
for overall and event free survival respectively. In accordance with
these results a 1:1 case-control matched comparison between double and single transplantation did not identify significant differences
in overall and event free survival.
Conclusion:
As compared with single autologous stem-cell transplantation
up front double transplantation did not seem to improve the final
outcome among patients with multiple myeloma in the Nordic area.
Sackmann, F1 *; Pavlovsky, S1; Pavlovsky, M A1; Mountford, P1;
Juni, M1; Intile, D1
*
Introduction:
the natural history of MDS complicates therapeutic modalities.
The International Prognostic Scoring System (IPSS) has become
the gold standard for risk assessment in patients (pts) with MDS.
The WHO classification has also a prognostic value.
Objective:
evaluate the prognostic value of the WHO classification and
the IPSS with respect to leukaemia free survival (LFS) and overall
survival (OS) in MDS pts.
125 pts with MDS were evaluated retrospectively. Clinical and
haematological features at diagnoses were recorded. LFS and OS
were calculated using Kaplan Meier method and the curves were
compared with the log rank test.
Results:
27% had refractory anaemia (RA), 2% RA with ringed sideroblastos (RARS), 44% refractory citopenia with multilineage dysplasia (RCMD), 10% RA with excess blasts, type 1 (RAEB-1), 9%
RAEB-2, 1% 5q- syndrome and 8% MDS unclassified (MDS-U). Cytogenetic analysis were performed in 84% of the pts; 14% could not
be analysed, 24% were normal and 62% had abnormalities. According to the IPSS, 24% had low risk, 51%, inter-1, 19%, inter-2 and
6%, high risk. Forty-one pts died, 55% with acute leukaemia (18%
overall). Considering the WHO classification, LFS for pts with RA/
RARS, RCMD/RCMDRS, RAEB-1 and RAEB-2 was 100%, 80%,
45% and 27% respectively (p = 0.00001) and OS for these groups
was 88%, 68%, 25% and 18% respectively (p = 0.00001), with a
median follow-up of 35 months (2 - 229 months). According to the
IPSS, LFS at 4 years for the low, inter-1, inter-2/high risk pts was
92%, 82% and 44% respectively (p = 0.00004) and OS for these
groups was 72%, 61% and 29%, respectively (p = 0.00004).
Conclusion:
WHO classification and IPSS identify groups with different LFS
and OS in our study. Thus, treatment strategies must be based on
theses classifications.
S170
044
111
EXPRESSION OF CD56 AS A PROGNOSTIC
FACTOR IN MULTIPLE MYELOMA
CORRELATION BETWEEN THE UPTAKE OF TC99M-SESTAMIBI AND PROGNOSTIC FACTORS IN
PATIENTS WITH MULTIPLE MYELOMA
Almeida, E. B.1 *; Pereira, F. G.1; Oliveira, G.B.1; Dias, D.F.1; Mello, M.R.B.1; De Souza, C.A.1; Lorand-Metze, I.1
*
Brazil - 1 Department of Internal Medicine and Hematology/Hemotherapy Center, State University of Campinas - UNICAMP
Introduction:
Aberrant expression of CD56 is the most common phenotypic
abnormality in multiple myeloma (MM). However, its absence seems
to be associated with a more aggressive form of the disease.
Objective:
to compare the prognostic significance of CD56 expression
with other known risk factors.
we performed a quantitative analysis of the expression of CD56
(MFI) by flow cytometry on bone marrow myeloma cells in newly
diagnosed patients, and compared its impact on overall survival with
International Prognostic Score (IPI) and laboratory data as well as
mean fluorescence intensity of SSC, CD45 and CD38 and presence
of deletion of chromosome 13. The patients were treated according
to the Brazilian Cooperative Myeloma Study.
Results:
Until now, 31 patients entered the analysis: 13 males and 18
females. Median age: 60 years (46-74). By the IPI staging, 10 patients were stage I, 8 were stage II and 13 were stage III. Expression
of CD56 was found in 68% of the cases. D13 was found in 23% of
the patients. MFI of CD56 had a positive correlation with the gamma
peak (r=0.57) and was inversely correlated to proteinuria (r -0.45)
and IPI. No correlation between this value and the MFI of the other
flow values was found. Concerning survival, in the univariate analysis, only hemoglobin value, IPI and MFI of CD56 had a significant
influence. All three variables remained in the model in the multivariate analysis (Cox model): IPI and hemoglobin as unfavourable and
MFI CD56 as favourable factor. Presence of D13 had no impact on
overall survival.
Conclusion:
among our patients, the quantitative value of the expression of
CD56 was a significant favourable parameter, more important than
levels of beta2-microglobulin and cytogenetics.
Bacovsky, J.M.1 *; Myslivecek, M.1; Scudla, V.1; Minarik, J.1; Zemanova, M.1
*
República Checa - 1 University of Olomouc
Backround: Multiple myeloma is a malignant disease characterised by clonal proliferation and accumulation of neoplasticly
transformed B-line elements, producing monoclonal immunoglobulin (MIG) demostrable in serum and/or urine.
Technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) has
been shown to be useful in identifying several types of tumors, such
as breast, brain, thyroid gland, malignant lymphomas and multiple
myeloma.
Methods: In this study, 102 patients with multiple myeloma
(MM) and 32 patients with monoclonal gammopathy of undetermined significance (MGUS) had been evaluated for correlation
between 99mTc-MIBI and biochemical and hematological markers of
activity of the disease.
Results: Significant statistical correlation was found between
summary score (SS) of 99mTc-MIBI scintigrams and beta2-microglobulin (p < 0.001), monoclonal immunoglobulin level MIG (p<
0.001), serum thymidinekinase - sTK (p < 0.001), CRP (p < 0.05)
and cross- linked carboxyterminal telopeptide of type I collagen ICTP (p< 0.05) bone marrow plasmocytosis -Pb (p < 0.001) and
hemoglobin Hb ( p < 0.001).
All 32 patients with MGUS had physiological activity of 99mTcMIBI scintigrams.
Technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) is a
useful indicator of activity of MM and helps in differentiating between
multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS).
Founded by grant IGA CR MHCR NC 7503-3/2003 and MSM
6198959205
S171
POSTER SESSION
02.2 CYTOGENETICS AND FLOW CITOMETRY
112
CYTOGENETIC CHARACTERIZATION AND
PROGNOSIS OF MYELODYSPLASTIC SYNDROME
ARGENTINE POPULATION
Belli, C. *; Acevedo, S.1; Arrossagaray, G.1; Bengió, R.1; Flores,
G.2; Goldstein, S.2; Negri Aranguren, P.3; Larripa, I.1
*
Argentina - 1 IIHEMA, Academia Nacional de Medicina; 2 HIGA “Dr
C Durand”, Bs As; 3 Hosp.”San Martín”, Paraná
Myelodysplastic Syndromes (MDS) comprise a group of heterogeneous hematological disorders with risk of leukemic evolution
(LE). The French-American-British (FAB) cooperative group classifies them into five morphological entities and the International
Prognostic Scoring System (IPSS) proposes four groups of risk on
the basis of clinical and cytogenetics variables. Objectives: to characterize the cytogenetic profile, to test its prognostic value and to
apply the IPSS in our MDS population.
Materials and methods: bone marrow samples from 275 MDS
patients classified according to FAB (128 RA, 27 RARS, 69 RAEB,
23 RAEBt and 27 CMML) were short-term cultured. Results: karyotypes were obtained from 228 patients and 89 (39%) showed cytogenetic aberrations. Chromosomes 5, 7 and 8 were the mostly
involved, either as a sole abnormality or in complex karyotypes. Although no particular aberration was associated to any FAB subtype,
their frequencies were increased according to the subgroup risk:
33% RA, 44% RARS, 45% RAEB, 76% RAEBt and 39% for LMMC.
The karyotypes were also subdivided according to IPSS cytogenetic risk into 147 good, 47 intermediate and 34 poor. In addition,
the IPSS was fully applied and the patients were stratified into 64
Low, 92 Intermediate (Int)-1, 43 Int-2 and 29 High. Also the median
Survival (SV) and the time to LE (median follow-up: 22 months)
were determined for FAB classification, cytogenetic groups of risk
and for the IPSS, the three of them showed statistical significances
for predicting outcome. Conclusions: Our results showed that cytogenetic, FAB and IPSS are important tools for accessing prognosis
in MDS patients.
Keys words: MDS, cytogenetic, leukemic evolution
131
DETECTION OF MICROSATELLITE INSTABILITY
AND LOSS OF HETEROZYGOSITY IN
MYELODYSPLASTIC SYNDROMES
Vazquez, M.L.1 *; Belli, C.1; Tacchi, C.1; Fantl, D.1; Venica, A.1;
Alberbide, J.1; Nucifora, E.1; Larripa, I.1; Fundia, A.1
*
Argentina - 1 IIHEMA, Academia Nacional de Medicina / Hospital
Italiano
The onset of myelodysplastic syndrome (MDS) has been associated to genomic instability, which is frequently due to deficiencies
in mismatch repair (MMR) or tumor-suppressor (TSG) genes. To
verify whether both mechanisms might be involved in MDS genomic
instability, microsatellite instability (MSI) and loss of heterozygosity
(LOH) were analyzed. Bone marrow samples from 21 untreated patients (12 females/ 9 males) with a mean age of 70.7 years (range
38-93) were studied. Myelodysplastic DNA was obtained from nonadherent mononuclear cells, while constitutional DNA was extracted
from polymorphonuclear cells. Nine STR chosen from the MSI colon cancer reference panel (BAT 25, BAT 26, D2S123 and D18S5)
or located at points involved in MDS and acute leukemia (D5S209,
CRTL, CSF1RT, D7S525 and TP53), were analyzed by PCR and
electrophoresis on native polyacrylamide gels silver stained. FLT3
mutations were also studied. The colon panel revealed MSI only
in one patient (4.8%) at D18S58. Employing hematological STR, 5
cases (23.8%) presented alterations at 1 or 2 STR: CSF1R-T, CRTL
and TP53, being classified as MSI-low according international criteria. Two cases presented LOH at TP53, D5S209 and D7S525. The
mean frequency of mutated STR/individual showed a significant
difference between hematological markers (0.08± 0.03) respect to
colon STR (0.01±0.03) (Student test, p=0.007), suggesting that colon markers are insensitive for MDS. Only 1/10 cases showed one
D835 FLT3 mutation in addition to MSI and LOH. The low frequency
of LOH suggests that putative TSG at the loci studied are not involved in genomic instability. These findings allows to identify a subset of patients with MSI-low, not due to MMR mutations but probably
related to other genetic alterations, critical in MDS development.
147
DETECTION OF T CELL CLONALITY BY FLOW
CYTOMETRY AND PCR
Dunlop, A.S.1 *; Gonzalez de Castro, D.1; Morilla, R.1
*
UK - 1 ICR
T-Cell receptor (TCR) clonality has traditionally been established using molecular methods, a commercial kit is now available,
the IOß Mark kit, to detect clonality by flow cytometry (FC) This
allows the rapid identification and quantification of 24 specificities
of the Vß family and covers around 70% of the TCR Vß repertoire.
The aim of this study was to determine whether the Vß kit is comparable to polymerase chain reaction (PCR) in detecting clonality
in mature T cell disorders. 51 patients with T cells lymphocytosis
were submitted for Immunophenotyping followed by morphological
examination and analysis of clonality by PCR using primers specific
for the Vß-Jß families. Patients were studied with the Vß kit using
a BD Facscalibur and cellquest pro software. CD3 was used as a
gating strategy to isolate the T cells in each of 8 tubes containing
antibodies targeted against 3 of the Vß families. Final diagnosis for
the 51 patients were; 16 cases of T cell lymphocytosis, 9 of T-PLL,
4 of ATLL, 9 of LGL expansions, 7 of LGL leukaemia and 6 of peripheral T-NHL. Of the cases studied 40 were found to be clonal by
PCR. By flow cytometry 37 cases were defined as clonal by applying the kit reference range, and 31 by applying a 1.6 fold increase
above the reference range. All cases of T-PLL and LGL leukaemia
were identified to be clonal using both PCR and FC. Clonality was
not detected in 1 case of ATLL using both techniques. 1 case of pe-
S172
ripheral T-NHL was found to be clonal by PCR and not FC irrespective of the normal range used. In the group with the diagnosis of T
lymphocytosis clonality was found in 56% of the cases by PCR. It
was in this group that the normal range applied for FC assessment
was most relevant to determine clonality. Likewise in cases of LGL
expansion although all but 1 case was found to be clonal by both
techniques, the application of relevant cutoff values was essential
in identifying the restricted Vß family. We further studied 17 cases
clonal by both methods and we were able to confirm the specificity
of the V? family involved.
069
A COMPLEX KARYOTIPE WITH AN EXTRA
COPY OF CHROMOSOME 1 INVOLVED IN
THREE DIFFERENTS TRANSLOCATIONS IN A
MYELODISPLASTIC SYNDROME (MDS) PATIENT
Minutti, M1 *; Simonet, S1; Infante, D1; Bonomi, R.2
*
Uruguay - 1 Asociación Española Primera de Socorros Mutuos; 2
ASESP
Introduction: Myelodisplastic syndromes (MDS) are heterogeneous family diseases. Chromosomal findings are one of the
most important parameters for diagnostic and predicting evolution
to acute myeloid leukemia (AML) and are included in International
Prognostic Scoring System.
Objectives: Report a patient with a MDS, in which banding
techniques allowed to determinate a complex karyotype constituted
by an extra copy of chromosomes 1 involved in three translocations
with different partners.
Material and methods: a 75 years old male was referred to our
laboratory catalogued as a refractory anemia. After 1month of cobalamin treatment no response was observed. Cytogenetic analysis
was performed by standard procedures from bone marrow (BM) aspiration after 48h of culture. Fifteen metaphases were examined by
GTG banding and karyotiped according to the ISCN. Two months
after cytogenetic study and being treated with grow factor and erythropoietin, the transformation in AML was observed.
Results: a complex karyotipe with the following structural abnorma-lities, add(2)(q37); add(11)(p15), add(11)(q25) was established. The analysis of this three segment allowed to establish the
presence of an extra copy of chromosome 1, that break in 1(p13)
and 1(p32) and translocated to the partner chromosomes before
indicated.
Conclusions: cytogenetic analysis is an important parameter
in MDS evaluation. The identification of new breakpoints and rearrangement allow deeper understanding in ethiopathogenic mechanism.
165
CHRONIC MYELOYD LEUKEMIA (CML):
CYTOGENETIC AND MOLECULAR THERAPEUTIC
MONITORING
Bonomi, R.1 *; Simonet, S2; Infante, D2; Manrique, G.1; Zubillaga,
M.N.1; Giere, I.3; Pavlovsky, S.3; Uriarte, M.R.1
*
Uruguay - 1 ASESP; 2 Asociación Española Primera de Socorros
Mutuos; 3 Fundaleu
Introduction: Different therapeutic approaches like bone marrow transplantation (BMT), interferon (IFN) and Imatinib (IB) are
been established in CML to achieve an eradication or reduction of
Ph-chromosome/BCR-ABL fusion gene.
Objectives: Molecular monitoring of BMT, IFN and IB treatments in 45 CML pts that achieve complete cytogenetic response
(CCR).
Material and Methods: A total of 591 pts with a suspected diagnosis of myeloproliferative disease (MPD) were referred to our laboratory from different health institutions between 1994-2006 to establish karyotype and BCR-ABL status. 247 pts were Ph/BCR-ABL
(+) confirming CML diagnosis. Treatment response was evaluated
during follow-up in 117 pts Ph (+) using GTG banding and nested
-polymerase chain reaction (PCR). Quantitative PCR (Q-PCR) was
available in 13 pts in CCR with IB therapy for at least six months.
Results: CCR was achieved in 45 from 117 pts who received
different treatments: BMT (n=10); IFN (n=5); IFN/IB: (IB with previous IFN) (n=17) and IB (n=13). Two of them showed trisomy 8 in
Ph (-) clones. The nested PCR performed in 41 from 45 CCR pts
showed different BCR-ABL status: a) molecular remission (n=14): 5
TMO; 5 IFN; 3 IB; 1 IFN/IB, b) transient relapse (n=4) 3TMO/ILD,
1IFN/IB, c) persistence (n= 23), 12 IFN/IB, 11IB. In 13 from 23 CCR
pts with persistence of BCR-ABL expression, Q-PCR demonstrated: complete molecular response (CMR) (n=5); mayor molecular
response (MMR) (n=3); and minor molecular response (MmR) (n=
5)
Conclusions: Qualitative and quantitative PCR detection of
BCR-ABL mRNA expression is a reliable tool to monitor the tumour
burden of CCR CML pts during follow-up.
168
MIELODISPLASTIC SYNDROMES.
VALUE OF THE MORPHOMETRIC AND
IMMUNOHISTOQUEMISTRY WITH P53 AND CD34
AS PROGNOSTIC FACTORS.
Mariño, A1 *; Melesi, S1; Touriño, C1; Rodriguez, A. M.1; Astapenco, A1; Saralegui, P1; Diaz, L1; Nese, M1; Acosta, G1
*
Uruguay - 1 Hospital de Clinicas. School of Medicine. Montevideo.
Uruguay
Introduction The mielodisplastic syndromes are an heterogeneous group of clonal hematopoyetic disorders characterized by
displasia at the level of one or more of the mieloid cell lines. Since
the year 1982 the Franco American British Classification (FAB) has
allowed to carry out a morphological, clinical, biological and genetic
correlation of these entities. At the level of BM the blastic counting
represents an element of prognostic values together with the study
of peripheric blood and the analysis of the citogenetic alterations
recognized by the International Prognostic Score System ( IPSS1997) .Materials and methods. Seventy five patients diagnosed as
SMD are analyzed and were studied by biopsy of bone marrow between 1990-2003. Results: The Mielodisplasic Síndrome, predominated in patients older than 65 years old, with male prevalence,
the clinical presentation was with one or several citopenias, with a
larger relationship between the number of citopenias and a shorter
possibility to outlive. In the bone marrow an abnormal localization
of immature precursors was identified, in 29 cases of aggressive
subtypes of syndromes mielodisplasic. The intense immunoreactivity for the CD34 and p53 in these agregates, such as the blastic
counting superior to 15 % , would both be risk markers towards
the leucaemia transformation. Dismegacariopoyesis with marked
fibrosis en 36 cases, in 10 cases megakaryocytes discariotic, hypolobulated with macrocitosis and aberrant expression for CD34.
The p53 was immunoreactive in all the cases with excessive blastic
counting. Conclusions. These findings are coincidental with the referred ones in the literature, as well as the association of cromosomic anomalies in the 5q.
S173
193
CYTOGENETIC EVOLUTION OF A PATIENT AFTER
MULTIPLE CHEMOTHERAPEUTIC AGENTS
TREATMENT: A CASE REPORT WITH CHRONIC
MYELOID LEUKEMIA
Mechoso, B1 *; Vaglio, A1; Roselli, M1; Quadrelli, A1; Quadrelli, R1
*
Uruguay - 1 Instituto de Genética Médica. Hospital Italiano.
Chronic myeloid leukemia is genetically characterized by the
presence of the reciprocal translocation t(9;22)(q34;q11), resulting in a BCR/ABL gene fusion on the derivative chromosome 22
called the Philadelphia chromosome. In most instances the t(9;22),
or a variant thereof, is the sole chromosomal anomaly during the
chronic phase of the disease, whereas additional genetic changes
are demonstrable in 60-80% of cases in blast crisis. Chromosome
segments often involved in structural rearrangements include 1q,
3q21, 3q26, 7p, 9p, 11q23, 12p13, 13q11-14, 17p11, 17q10, 21q22,
and 22q10. The cytogenetic evolution patterns vary significantly in
relation to treatment.
The aim of this presentation is to communicate the cytogenetic
evolution of a case after therapy with interferon-alpha, hydroxyurea
and in the last two years only with imatinib because of the resistance at the previous therapy.
Since the diagnosis, sixteen years before, the only aberration
was the t(9;22). In the last year it was also founded a new clone with
a secondary change, a 7p11 deletion:
(46,XY, t(9;22) (q34;q11)[50%]/46,XY, del(7)(p11), t(9;22)
(q34;q11) [50%]).
It is remarkable that associated with imatinib treatment the primary clone presented whole remission, while the last one has kept
during the course disease to the patient death.
Considering that the clinical impact of additional cytogenetic
and molecular genetic aberrations is most likely modified by the
treatment modalities used, the follow-up of these patients may offer
guidelines to the accurate clinical management in these hematologic disorders.
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POSTER SESSION
03.2 AUTOLOGOUS TRANSPLANTATION
007
009
MAINTENANCE WITH VERY LOW DOSE
THALIDOMIDE AFTER AUTO-SCT IN MULTIPLE
MYELOMA: LOW TOXICITY AND IMPROVED
OUTCOME
PURGING IN VIVO AND AUTOLOGOUS STEMCELLS TRANSPLANTATION IN POOR PROGNOSYS
PATIENTS WITH LYMPHOPROLIFERATIVE
DISEASE
Rosa, C1
*
Rosa, C1
*
Introduction:
High dose therapy with single or double transplantation (autoSCT) has improved prognosis of multiple myeloma (MM). New
drugs are promising in upfront therapy while the role of maintenance
is still debated. Thalidomide (thal) is an active drug in the treatment
of myeloma, and is been investigated as first line therapy. It could be
useful in the control of minimal residual disease.
Objective:
We used thal as maintenance after autologous transplantation
(single or double) and compare the outcome with other maintenance
or none.
From January 2001 to May 2006 28 patients (14 males and
14 females) with MM have been treated in our institution. Median
age was 59 years (range 40-72). 13 were IgG, 8 IgA, 1 IgD, 4 light
chains and 2 plasma-cell leukaemia. Treatment was 4 cycles of VAD
regimen followed by auto-SCT. 9/28 performed double auto-SCT.
3 months after SCT, 14 patients (10 single and 4 double SCT) began thal 50 mg/die as maintenance therapy. 14 patients (9 single
and 5 double SCT) received IFN-g (4/13), dexa (3/13) or no therapy
(7/13). The 2 groups were regarding the type of myeloma: 7 IgG, 3
IgA, 3 light chains and 1 plasma-cell leukaemia in the thal group; 6
IgG, 5 IgA, 1 IgD, 1 light chains and 1 plasmacell leukaemia in the
other. Response to SCT: 4 CR, 9 PR and 1 NR in the thal group; 7
CR, 6 PR and 1 NR in the other.
Results:
Relapsed in the thal or no thal groups 5/14 (35%) and 8/14
(57%) patients respectively. Median follow up from the diagnosis
was 33 months (range 7-151) for every group. After auto-SCT, in the
thal group the median PFS were 44 months and OS were 71% projected at 50 months, in no thal group the median PFS and OS were
10 and 21 months respectively.The difference between the 2 groups
is statistically significant for PFS (p: 0,001), and not significant for
OS from diagnosis (p: 0,057) even if difference (70% vs. 15% projected a 100 months) appears clear.
Thal was administered for a median period of 12 months, being neurological toxicity the main reason of suspension in 3/14 patients (21%). Neurological toxicity grade I-III was present in 65%
of patients, while haematological toxicity grade I occur in 55% of
patients.
Conclusion:
In conclusion, in a small number of patients low dose thal as
maintenance after auto-SCT resulted in an improved PFS and OS
when compared with other or none maintenance, with acceptable
toxicity.
Introduction:
Autologous stem cell transplantation is a efficacy therapy for
limphoproliferative disease. However a concern with the procedure
is the potential of malignant cells to reinfuse with stem-cell graft. In
the past five year, investigators have used rituximab to purge malignant cells in vivo without any manipulation in vitro.
Objective:
From April 2003 to May 2006 we have treated with Autologous
stem cell transplantation, purged in vivo with monoclonal antibodies, 13 patients (2 F; M 11 median age: 56 years) with limphoproliferative diseases to poor prognosis (2 Burkitt lymphoma; 3 mantle
cells; 3 CLL; 1 NHL- peripheral T cells; 2 follicular and 2 large cells)
and we have evaluated the results and the feasibility.
In all patients, the purged in vivo, has been effected administering a dose of monoclonal antibodies (anti CD20 in B-NHL and anti
CD52 in CLL and T-NHL) before the harvest and after the infusion
of the stem-cells. To the transplantation 3 patients were in CR (2
Burkitt lymphoma and 1 mantle cells) 7 in PR (1 CLL; 2 mantle cells;
2 follicular and 2 large cells lymphoma) and 3 in resistant disease (2
CLL and 1 NHL peripheral T cells) All patients have harvest (median
CD34:4 x106/Kg) and median minimal residual disease in the harvest has been < to 2%. All the patients have been conditioned with
BEAM and the graft are documented in 12/13 patients (1 patient is
dead to the day +4 for gastric haemorrhage) with neutrophils> 1000
in media to day + 14 (range 10-19 days)
Results:
After transplantation 12/13 patients were in CR, a day +60 the
MMR in bone marrow was <0, 5% (range 0-0, 3%). With a median follow-up of 8 months after transplantation (range 2-36) 11/12
patients are in CR (one patient with burkitt lymphoma is relapsed
extra-nodular at months +3 and died for disease a months + 5 after
transplantation). One patient (CLL) is died at months + 7 for interstitial pneumonia. The DFS and EFS projected at 36 months are of
the 85% and 75% respectively.
Conclusion:
In conclusion the purging in alive with antibodies monoclonal,
effected during the harvest that immediately after the infusion of the
stem-cells, allows to get besides a graft with least residual disease
in this cohort (patients with poor prognosis) and the preliminary results they seem excellent.
The principal problem in these patients have been primarily the
infectious and gastro-intestinal complications, these has been correlated to patients much treated and in disease. These data suggest
treating in first line, with transplantation of stem-cells purged in vivo
with monoclonal antibodies to eradicate the MRD, patients to poor
prognosis or with chronic limphoproliferative disease.
153
“HIGH DOSE SEQUENTIAL (HDS) FOLLOWED
BY AUTOLOGOUS BONE MARROW
TRANSPLANTATION (ABMT) AS SALVAGE
TREATMENT IN ADVANCED HODGKIN’S DISEASE
(HD)”.
Delamain, M.T.1 *; Cardoso, RB1; Oliveira, GB1; Lorand-Metze, I1;
Vigorito, AC1; Aranha, FJP1; Eid, KA1; Miranda, E1; De Souza,
CA1
*
Brazil - 1 Hematology and Blood Transfusion Center, State University of Campinas - UNICAMP / BRAZIL
Introduction: ABMT has been proposed as a salvage treatment of resistant / refractory HD. HDS using Cy 7g/m2 for debulking
and PBPC mobilization followed by MTX 8g/m2 and then VP-16 2g/
m2 before ABMT, described by Milan group, seems to be effective
in these patients.
Objective: We report the results obtained using this procedure
in 31 patients with HD who failed conventional therapy or relapsed
within 24 months of conventional chemo +/- radiotherapy.
Materials and method: Patients were treated with HDS followed by ABMT receiving BEAM (BCNU, Etoposide, Ara-C and Melphalan) as conditioning regimen.
Results: The median age was 25 years (12-61), 20 male
(64,5%) and 11 female (35,5%). The histology at diagnosis showed:
19 (61,3%) nodular sclerosis; 9 (29%) mixed cellularity; 2 (6,5%)
lymphocyte-depleted and 1 (3,2%) lymphocyte-rich HD. Bulky disease was presented in 15/31 (49%) patients and 6/31 (20%) had
bone marrow infiltration. The Overall survival (OS) and disease free
survival (DFS) were 49% (n=31) and 60% (n=15), respectively, in
1825 days. The OS was 62% for patients with 0-1 prognostic factors
at diagnosis and a shorter OS was observed in patients with more
than 2 prognostic factors (P=0.004). Bulky disease , histology type
and bone marrow involvement did not correlate with poor outcome.
Fifteen patients died, 8/15 due to progressive disease (53%), 5/15
due to toxicity after the HDS (33%) and 2/15 due to toxicity after
ABMT (13%). Status presens for alive patients after a median time
of 783 days (range 50-1929) from transplant is: CR 11 ( 35,5%),
partial response 2 (6,5%) and 4 in progressive disease (12,9%).
Conclusion: We conclude that HDS followed by ABMT is an
effective salvage regimen for patients with resistant/refractory Hodgkin’s disease and probably induces a long and stable CR mainly in
chemosensitive patients. Patients presenting >2 prognostic factors
at diagnosis presented worst outcome.
S175
or following remissions, and they had more than one schedule of
chemotherapy. At the time of transplantation, 18/46 (39%%) were in
first CR and 28/46 (61%) in partial remission (PR). One (2%) out of
these had a progressive disease. The average time from diagnosis
to ABMT, was 25 months (6-120). Conditioning regimen was BEAC
in 37/46 (80%) and BEAM in 9/46 (20%).
Results: Recovery time of white blood cells was 16 days (724) and for platelets 11 days (6-35). The median follow-up was 73
months (2-117). 33/46 patients (75%) are in CR up to date. 2/46 in
PR still alive. In three cases, the follow-up was lost. Two patients
died by causes related to the procedure (one of a sepsis, one of a
veno-occlusive disease.
Conclusion: ABMT in a long period of follow up has shown its
value in the treatment of this malignant lymphoma.
126
AUTOLOGOUS STEM CELL TRANSPLANTATION
(ASCT) IN MULTIPLE MYELOMA (MM). IMPACT
OF SURVIVAL
Isaurralde, H1 *; Díaz, L1; Guillermo, C1; Topolansky, L1; Zunino,
J1; Perdomo, S1; Perdomo, A1; Lavagna, G1; Stevenazzi, M1;
Díaz, A1; Nese, M1
*
Uruguay - 1 CITMO/ FACULTAD DE MEDICINA,UdelaR
Between 1995 and 2005, we performed 329 hematopoietic
stem cell transplantation (HSCT), 286 autoulogous and 43 allogeneic.
Long term results of treatment and outcome of HSCT in 46
patients with MM, 54 ASCT, 1 RIC allo (9 in tandem, 8 auto - auto, 1
auto - allo) were analyzed. Median age was 54 years (range 33-65),
21 male, 25 female. At diagnosis 4 patients were IA; 20 IIA; 15 IIIA;
1 IIB; 6 IIIB. The monoclonal Ig was: IgG 24; IgA 12; light chains
10. Disease status at HSCT was: CR1: 22 patients, PR1:26; PR2:
1, PR3: 1, relapse: 5. Conditional regimen was Melphalan in ASCT
and Melphalan -Fludarabine in the RIC allo. Stem cell source was
bone marrow (BM) in 1, BM and peripheral blood (PBSC) in 10 and
PBSC in 44.
The median of CD34 infused was 9x 106 x Kg. The median of
bone marrow recovery (NCC > 500 x 109/L) was 11 days (7-17), and
platelet (>20000 x 109/L) 15 days (11-100). The median of hospitalization was 21 days in ASCT and 38 in RIC allo. The median followup was 4,5 years (0-9) in tandem ASCT and 2 years (0-7) in single
ASCT. The median overall survival in single ASCT was 4 years.
The median overall survival in tandem HSCT was not reached with
an overall survival of 62% at 11 years. We concluded high dose
therapy and ASCT is an effective therapy for patients with MM with
better results in long follow up for tandem HSCT.
074
AUTOLOGOUS BONE MARROW
TRANSPLANTATION IN HODGKIN´S DISEASE
Muxi, P J1 *; Pierri, S1; Bello, L1; Caneiro, A1; Di Landro, J1;
† De Bellis, R
1
*
Uruguay - 1 British Hospital
Introduction: From 1984 to 2006, 43 patients with Hodgkin
Lymphoma received high dose therapy and rescued with autologous bone marrow (ABMT) or peripheral stem cells, in the British
Hospital
Objective: Evaluate this procedure to improve Hodgkin´s disease treatment
Materials and method: A population of 24 females and 19
males, with a median age of 31 years (17-55y) was studied. 3 patients received two ABMT. 18 patients(39%) were in an early stage
of their disease, and 25 (61%) were in advanced condition. Of this
population, 18/43 were in first complete remission (CR), after one
schedule of chemotherapy. The remaining 28/43 were in second
127
ROLE OF MAINTENANCE CHEMOTHERAPY AFTER
AUTOLOGOUS STEM CELL TRANSPLANTATION IN
ADULT ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)
Topolansky, L.1 *; Stevenazzi, M.1; Zunino, J.1; Díaz, A.1; Guillermo, C.1; Díaz, L.1; Isaurralde, H.1; Perdomo, S.1; Perdomo, A.1;
Lavagna, G.1; Nese, M.1
*
Limited experience is available on the feasibility and efficacy
of maintenance treatment after autologous stem cell transplantation
(ASCT) in acute lymphoblastic leukemia (ALL). Long term results of
treatment and outcome of maintenance chemotherapy after ASCT
in ALL were analyzed. From 1995 to 2006, 13 adult ALL patients
>16 years old received ASCT at the Bone Marrow Transplantation
Unit of IMPASA (CITMO). There were 10 male and 3 female patients with a median age of 34 years (range 16-57 years). Diagnosis: ALL T-cell 1, early Pre B 1, pre B 4, B 2, Phi+ 1 and un-
S176
specified 4. The conditioning regimen was Bu/Cy. Disease status at
ASCT was first complete remission (CR) in all cases. 12/13 patients
received maintenance chemotherapy with methotrexate (MTX) and
6-mercaptopurine (6MP) after ASCT for 2 years, in attempt to decrease relapse rate. Median follow-up was 7 years. The medians
to reach neutrophils >0.5x109/l and platelets >20x109/l were 10 and
14 days, respectively. The early treatment-related mortality (TRM)
(<100 days) was 0%. With a median follow-up of 7 years, 10 patients (76%) are alive, 8 in CR, 1 in relapse, 1 has lost follow-up
and 3 died (23%). Overall survival (OS), at 10 years was 75%. Conclusions: Those patients receiving two drugs maintenance chemotherapy had lower relapse rates and higher OS than most reported
using the conventional approach of autographs without post transplantation chemotherapy. Our data suggest that maintenance chemotherapy after ASCT improve the disease outcome of adult ALL.
A large number of cases and randomized trial are needed to take
definitive conclusion.
128
HEMATOPOIETIC STEM CELL TRANSPLANTATION
(HCT) FOR ACUTE MYELOID LEUKEMIA (AML)
Diaz, A.1 *; Topolansky, L.1; Stevenazzi, M.1; Zunino, J.1; Guillermo, C.1; Díaz, L.1; Isaurralde, H.1; Perdomo, S.1; Perdomo, A.1;
Lavagna, G.1; Nese, M.1
*
The aim of this prospective analysis was to evaluate the 100
days mortality (TRM) and overall survival (OS) of HCT in a population of adults patients with AML, by giving auto or allogeneic HCT.
We performed 38 HCT, 32 autologous and 6 allogeneic from 1995
to 2006, 23 males and 14 females, with a median age of 37.5 years
(15-61) and a median follow-up of 2 years (0-11). Diagnosis (FAB):
M0 1; M1 5; M2 11; M3 8; M4 7; M5 1; M6 2; M7 1 and biphenotypic 1. At transplant three patients (8%) had refractory disease to
standard induction; 28(74%), were at first complete remission (CR),
3 (8%) at second CR; 1(2.7%) in relapse, and 2 (5%) were in third
relapse or more. The preparative regimen was Busulfan Cyclosphosphamide (BuCy) in 92% (35 pts); the median of mononuclear cells
infused was 9x108/kg (1.54-23) and CD34: 7x106/kg (2.64- 25). The
bone marrow recovery (NBC>0.5x109/L) was 11 days (8-24 days),
and platelet recovery (>20x109/L) 19 days (10-86). Red blood cells
transfusion was required with a median of 2 units (0-26) and 2 (0-17)
single donor platelet concentrates. There were 38 febrile episodes,
16 bacterial infection (42%), 2 mycotic infection, and 17 suspects infections (44%). The median hospitalization days for allogeneic HCT
was 41 (22-59) and for autologous 27 (20-67). TRM was 33% and
3% respectively. Overall survival at 11 years was 39% for autologous HCT, with a median survival of 1543 days, and 33.35% OS for
allogeneic with a median of 231 days. Autologous HCT in this trial
of adult’s patients had successfully outcome with low toxicity, TRM
and a good OS. The number of allogeneic HCT is very low, so we
cannot consider definitive conclusions.
139
EFFECTIVENESS OF HIGH DOSE
CHEMORADIOTHERAPY AND AUTOLOGOUS
HEMATOPOIETIC CELL TRANSPLANTATION
IN ADVANCED HODGKIN’S DISEASE: SINGLE
CENTER EXPERIENCE
Carnot, J.1 *; Rodríguez, Y.1; Castro, R.1; Muñio, J.1; Pérez, G.1;
Martínez, C.1; Hernández, C.1; Pérez, D.1
*
Background: In advanced Hodgkin’s Disease (HD), high-dose
therapy and autologous hematopoietic cell transplantation (AHCT)
is the best option in patients who fail to enter complete remission
with initial therapy or relapses after a complete remission. The purpose of this study was to evaluate the results of AHCT in 17 patients with HD. Patients and Methods: We analyzed the outcome of
17 patients with relapsed or refractory HD , who underwent AHCT
between November 1991 and December 2005. The median age
was 34 years (range, 21 to 51 years); 5 patients (30%) were transplanted in second complete remission (2nd CR), 6 (35%) in relapsed (4 chemosensitive and 2 chemoresistant) and 6 (35%) who
fail to enter complete remission with initial therapy. We performed
autotransplants using non-cryopreserved unmanipulated bone
marrow (12 patients ) or peripheral progenitor cells (5 patients) and
using a 2 - 3 days conditioning regimen: CVB in 6, (35%) patients,
cyclophosphamide± etoposide/ total body irradiation in 5 (30%),
and other regimens in 6 (35%).
Results: At the end of the treatment program, 8 patients (47
%) are alive ( 5 CR, 2 CR uncertain and 1 relapse). After a median
follow-up 40 months (range 1 - 144), the probabilities of 5-year
progression-free survival (PFS) and Overall Survival (OS) rates
were 52% and 38% respectively. There were 9 decease and the
major cause of death was progression/relapsed (6 cases) There
were 2/17 (12 %) deaths due to treatment-related toxicity within the
first 100 days after transplantation. Conclusion: AHCT can lead to
durable remissions in patients with relapsed or refractory HD
Key words: autologous transplant, Hodgkin’s Disease , highdose therapy,
083
OPTIMIZATION OF CD34+ COLLECTION FOR
AUTOLOGOUS TRANSPLANTATION USING
EVOLUTION OF PERIPHERAL BLOOD CELL
COUNTS AFTER MOBILIZATION CHEMOTHERAPY
Delamain, M.T.1 *; Lorand Metze, I1; Marques Jr, JFC1; Reis,
ACR1; De Souza, CA1; Metze, K1
*
Brazil - 1 Hematology and Blood Transfusion Center, State University of Campinas - UNICAMP / BRAZIL
Introduction:
Intensive chemotherapy with peripheral blood progenitor cells
(PBPC) rescue is an important therapeutic procedure in hematological malignancies.
Objective:
To establish the parameters that best predict the day to start
harvesting of PBPC .
Analysis of the data of patients with hematological malignancies, who underwent mobilization of PBPC . Mobilization: cyclophosphamide (4 or 7g/m 2) and G-CSF. Influence of age, sex, diagnosis,
number of previous chemotherapy lines (nr CHT), peripheral blood
counts at day D0, day of neutrophils <1.0x109/l and day of nadir
(including the interval between these two days - delta) on harvesting
was investigated. Multivariate linear correlation models were built in
order to predict the day of best harvesting. The quality of the models
was evaluated using the Kolmogorof Smirnov test.
Results:
134 patients were analysed: 36 Hodgkin\’s lymphoma (HL), 65
B-large cell lymphoma (NHL) and 33 multiple myeloma (MM). Median age: 28, 40 and 48 years respectively. Day of apheresis presented a correlation with number of previous chemotherapy lines,
hemoglobin value of day 0, day of granulocytes < 1.0x109 /l, day of
nadir and dosis of mobilization therapy. However, for each disease
a different model could be established.
HL: day of apheresis = delta x 1.5 + 9.1; NHL: delta x 0.6 + 10.8
; MM: delta x 1.6 + 8.8
Conclusion:
each disease has its own pattern of mobilization dynamics. The
most important parameter, common to all patients, was the velocity
of decline of the neutrophil count after mobilization chemotherapy.
140
HIGH DOSE CHEMOTHERAPY WITH
AUTOLOGOUS STEM CELL TRANSPLANTATION IN
AGGRESSIVE NON-HODGKIN’S LYMPHOMA - A
SINGLE CENTER EXPERIENCE
Rodríguez, Y.1 *; De Castro, R.1; Muñío, J.1; Pérez, G.1; Martínez,
C.1; Hernández, C.1; Pérez, D.1
*
Background: Combination chemotherapy can cure patients
with agresivo non-Hodgkin\’s lymphoma (NHL), but those who suffer treatment failure or relapse still have a poor prognosis. Highdose therapy with autologous hematopoietic cell transplantation
(AHCT) can improve the outcome of these patients. The purpose
of this study was to analyse the results and prognostic factors influencing overall survival (OS) and progression-free survival (PFS) in
34 patients diagnosed with aggressive NHL treated with high-dose
therapy and AHCT. Patients and Methods: We analyzed the outcome of 34 patients aged < 60 years with aggressive NHL (Intermediate and High Grade lymphomas, Working Formulation classification ), who underwent AHCT between february 1985 and December
2005. At transplantation, median age was 36 years (range 17-58),
17 patients (50%) were transplanted in first complete remission
(1st CR), 6 (18%) in second CR (2nd CR), 4 (12%) in relapsed
chemosensitive and 7 (21%) in first partial remission after fail to
enter complete remission with initial therapy. We performed autotransplants using non-cryopreserved unmanipulated bone marrow
(24 patients ) or peripheral progenitor cells (10 patients) and using
a 2 - 3 days conditioning regimen: , cyclophosphamide / etoposide/
total body irradiation in 17 patients (50%), cyclophosphamide /total
body irradiation in 14 (41%), and other regimens in 3 (9%). Results:
At the end of the treatment program, 23 patients (68 %) are alive (
19 CR, 3 CR uncertain and 1 relapse). After a median follow-up 59
months (range 5 - 184), the probabilities of 5-year progression-free
S177
survival (PFS) and Overall Survival (OS) rates were 79% and 67%
respectively. There were11 decease and the major cause of death
was progression/relapsed (5 cases) There were 2/34 (6 %) deaths
due to treatment-related toxicity within the first 100 days after transplantation. Two variables influenced PFS and OS : disease status
at transplant and bulky disease. The probabilities of 5-year PFS
and OS were better in patients transplanted in 1st CR and without
bulky disease. Age-adjusted IPI at transplantation didn\’t influence
in PFS or OS. Conclusions: Our results confirm that the use of highdose therapy with AHCT in a group of patient with is able to alter
the course of the illness and to produce a SG and SLE prolonged
NHL is able to alter the course of the illness and to produce a OS
and PFS prolonged
159
ALPHA INTERFERON AFTER AUTOLOGOUS
BONE MARROW. TRANSPLANTATION IN
LYMPHOPROLIFERATIVE DISORDERS
Novoa, E.1 *; Beñaran, B.1; Caneiro, A.1; Iriondo, N.1; Draper, R.1;
† De Bellis, R.
1
*
Uruguay - 1 FEMI/ Hospital Británico
Background:It has been known since the early 1930s that cells
infected with viruses are capable of protecting other cells from viral infection. Minimal residual disease (MRD) is the main cause of
relapse and death, after autologous bone marrow transplantation.
Objectives:to study the efficacy and safety of IFN-á as maintenance
therapy after ABMT in patients with chronic lymphoproliferative disorders. Methods: 80 patients treated by ABMT were evaluated; 32
non Hodgkin lymphoma, 30 Hodgkin’s disease & 18 multiple myeloma patients. Gender: 39 men and 41 women. Age from 18 to
60 years old (media 45). On the day 90 after ABMT, they started
receiving IFN-á(Roferon-A ®) 3 MU, subcutaneously, three times
a week, during 12 to 18 months (media 14 months). The historical control population (CP) was selected from the international
literature.(Horning SJ. Cancer 1985;56:1305-1310: Hodgkin’s disease - Rohatiner AZS. Br J Cancer 1987; 55:225-226 - Osteborg
A. Eur J Haematol 1990;45:153-157). Results: no patient’s dead as
a consequence of IFN-á treatment. 80% of side effects in patients
receiving interferon were acute. The majority of them are constitutional.The hematologic effects of IFN therapy include leuko penia,
anemia and thrombocytopenia.The most frequent neurologic side
effects were depression, confusion and mental slowing. Overall survival (OS) and disease - free interval survival (DFI-S) was evaluated
in each group of patients. Hodgkin’s disease 89% (75% CP), DFI-S
84% (64% CP); non Hodgkin’s lymphomas 87% (55% CP), DFI-S
56% (39% CP); multiple myeloma 92% (52% CP), DFI-S 66% (33%
CP). Log rank test was highly significant in favor of the population
treated with IFN-? (p<0,001). Conclusions: IFN-? has clearly demonstrated its usefulness inducing prolongation of disease free and
overall survival in lymphopro liferative disorders after ABMT.
S178
POSTER SESSION
03.3 HEMOPOIETIC STEM CELL TRANSPLANTATION
071
107
VIDEO-ASSISTED THORACOSCOPY INJECTION
OF AUTOLOGOUS STEM CELLS IN IDIOPATHIC
CARDIOMYOPATHY; FOLLOW UP AT TWO YEARS
KIR ANALYSIS IN PEDIATRIC BONE MARROW
TRASPLANTATION.
Paganini, J.J. ; Brusich, D. ; Fronzuti, A. ; Paganini, R. ; Saccone, D.1; Martínez, L.3; Decaro, J.4; Geffner, L.5; Patel, A.6; Benetti, F.5
*
Uruguay - 1 Department of Cardiac Surgery; 2 Department of
Cardiac Surgery - Heart Failure Unit; 3 Hematology Department; 4
Hemotherapy Department; 5 Benetti Foundation for Cardiovascular
Surgery; 6 Department of Cardiothoracic Surgery, University of
Pittsburgh - Department of Cardiothoracic Surgery, Baylor University Medical Center, Dallas
1*
1
1
2
Introduction: Dilated idiopathic cardiomyopathy in advanced
stages presents an ominous outcome in a short period of time. Early
results of cellular therapy for this condition have been most promising.
Objective:
Materials and method: 3 patients with dilated cardiomyopathy
were submitted to minimally invasive video-assisted cellular therapy,
using autologous CD34+ stem cells. These patients were receiving
maximum medical treatment (85% of maximal doses), in functional class III/IV and with a LVEF of <35%. The stem cells are harvested at the onset of surgery, and after processing are delivered
into the myocardium. The pericardium is opened by three trocars
inserted at the 2nd, 4th and 7th intercostal spaces for thoracoscopy
and a small thoracotomy. By this means the whole of the left ventricle can be controlled and the injections performed at the desired
myocardial areas.
Results: The treatment was successful in the three patients
treated with cellular therapy. The mean amount of bone marrow harvesting was 250cc, with an average of 800,000 cells/kg. (CD34+).
Neither deaths nor complications occurred. There was an improvement in their clinical functional class (NYHA) from 3.4 to 1,3(p<0.05)
at 6 months. Echocardiograms before and after six months showed
a marked increase of LVEF and decrease of end - diastolic and systolic diameters of the left ventricle. After two years one patient did
not maintain this LVEF improvement.
Conclusion: The cellular therapy procedure shows clear benefits on cardiac function in this selected population. Larger, double-blind controlled trials are necessary in order to define long and
short-term results of this therapeutic modality.
Moreno, G.S.1 *; Arellano, G.J.1; Ramírez, P.A.1; Bello, A.1; Sarti,
E.2; Moreno-Galván, M.1
*
Mexico - 1 Hospital Infantil de México, “Federico Gómez”; 2 CENAVECE SSA
Natural Killer (NK) cells may be involved both in allogenic bone
marrow transplantation (BMT) rejection and graft-versus-host disease (GVDH). The physiologic functions of NK cells appear to be
regulated by inhibitory an non-inbitory receptors including the killer
cell immunoglobulin-like receptors (KIR). The role of genes KIR in
transplantation alloreactivity has been studied but it´s need to be
futher investigated. The aim of thiss study was to describe and
evaluated the KIR polymorphisms, HLA-typing and quimerisms, on
BMT evolution in five Mexican pediatric patients. A descriptive serial cases study was done. KIR genotype was examinted by PCR
with sequence-specific primers for 2DL1, 2DL4, 3DL1,3DL2, 2DS3,
2DS4, 3DS1and KIRE.4. Chimerism was performed by microsatellites analysis. HLA typing was done by low resolution techniques.
Since October, 2001 to September, 2005 we have five patients submited to BMT (two ALL, one CML and two aplasic anemia). Our
results show that 6 of 8 KIR genes were always identical in donors and recipients (2DL1, 2DL4, 3DL1,3DL2, 2DS3 and 2DS4).
Three patients showed GVHD, one of them had a difererence in
KIR3DS1, and the others had “included” at 100% the KIR genotype
(recipent-donor). Two patiens were suitables, one had 100% KIR
“included”, full quimerism and three HLA diferrences, the second
had differences in KIR 3DS1 and KIRE.4. We could not calculate
statistical analysis because the number of cases. Our data will be
help to created a “genotyping combination” (KIR, quimerisms and
HLA) to predict which patients could be develop GVHD or rejection.
Killer cell immunoglobulin-like receptors, bone marrow transplantation, genotyping
154
THERAPEUTIC ANGIOGENESIS IN ARTERIAL LIMB
ISCHAEMIA BY AUTOLOGOUS BONE MARROW
TRANSPLANTATION
Novoa, J.E.1 *; Medina, M.A.1; Gordillo, F.1; Sequeira, N.1; Nuñez,
H.1; Olivet, C.1
*
Uruguay - 1 Hospital de San Carlos, MSP-ASSE
Background:therapeutic angiogenesis has recently been developed as a new method of treatment for several ischaemic diseases.
There is preliminary data suggesting that implantation of bone marrow-mononuclear cells into ischaemic limbs increases collateral
vessels formation.Aims:to evaluate viability of the therapeutic angiogenesis using hematopoietic bone marrow progenitors mobilized
by G-CSF and safety of the procedure.Methods: 40 patients de-
veloping critical arterial limb ischaemia (candidates to amputation)
were included in this study. 23 men and 17 women. Median age 65
years old (44 - 86). Mobilized by filgrastim (Neupogen ®) 5 μg/kg
weight daily (5 days). Bone marrow harvest at 5th day. Local anaesthesia was employed in all the patients. Unmanipulated cells were
injected in the affected limb in 2 ml aliquots into the gastrocnemius
muscle. Each patient was evaluated regularly for rest pain, amount
of required analgesia , healing of the ulcers, peak walking time,
Doppler and angiographic findings. The mean number of injected
mononuclear cells was 1,9 x109/kg. All the patients received low
molecular weight heparin (nadroparin,Fraxiparine ®) 3800 - 5600
IU anti-Xa subcutaneously , aspirin 81 mg and pentoxifiline 400 mg
daily, as medical treatment after the procedure for at least 60 to 90
days. A control population of 39 vascular patients affected by critical arterial limb isquemia was considered. They don’t received angiogenic treatment.Results:there were no deaths secondary to the
procedure. 32 patients showed an improvement of all parameters.
On the control population, amputation was necessary in 87,2%. The
statistical differences betwen the two groups were highly significant
in favor of the angiogenic group. They were evaluated by the chi
square test and log rank test with a p value < 0,05. Conclusions:autologous bone marrow transplantation can be performed safely and
appears to be a benefical therapy for selected patients with severe
peripheral arterial disease.
155
THE CONZI’S EFFECT IN HUMAN DIABETES
MELLITUS: FROM BONE MARROW PROGENITOR
CELLS TO BETA CELLS
Novoa, J.E.1 *; Medina, M.A.1; Portillo, F.1; Ravera, J.1; Sosa, A.1;
Gianarelli, S.1
*
Uruguay - 1 Hospital Policial
Background:cell therapy represents a potential cure for diabetes me llitus.Stem cells have the potentiality to proliferate and differentiate into any type of cell. Bernat Soria showed how to guide stem
cells in animals to differentiate in specific cell lineages.Therapeutic
angio genesis has recently been developed as a new method of
treatment for several ischaemic diseases by stem cell transplantation. Objectives: to present the reduction of hyperglycemia and
insulin requirements, after autologous bone marrow trasnsplant as
treatment of arterial limb ischaemia.Methods:a 75 years old male,
diabetic, requiring insulin two times daily from the last 7 months.
Mobilized by filgrastim (Neupogen ®) 5 μg/kg weight daily (5 days).
Bone marrow harvested at 5th day. Local anaesthesia. Unmanipulated cells were injected in the affected limb ml He has Charcot´s
syndrome in the right leg.Very intensive leg pain, requiring frequent
analgesia. Doppler and angiographic findings showed severe bilateral arterial ischaemic disease.The number of injected BM mononuclear cells were 1,9 x109/kg in the first transplant and 1,6 x109/kg in
the second one.Results: an important and maintained decrease on
blood sugar levels and insulin requirement was observed after bone
marrow-derived stem cells transplant. Simultaneously, leg pain, local skin temperature and ulcers of the legs improve until normalization.Conclusions:our preliminary data suggest that autologous
bone marrow transplantation for therapeutic angiogenesis, can induce the recipient´s own cells to prolipferate, to produce insulin and
control blood sugar. Therapeutic angiogenesis by autologous bone
marrow trasnplantation can be performed safely and appears to be
a benefical therapy for selected patients with severe peripheral arterial disease; perhaps it can induce insulin-secreting cells in diabetic
patients. Going to the cure?.
S179
090
RENAL INVOLVEMENT IN HEMATOPOIETIC
PROGENITOR CELL TRANSPLANTATION.
Pérez, D.1 *; Carnot, J.1; Hernández, C.1; de Castro, R.1; Muñío,
J.1; Martínez, C.1; Pérez, G.1
*
Background: Hematopoietic progenitor cell transplantation
(HPCT) is a common treatment for many patients with hematologic
and non hematologic diseases, and renal dysfunction is a relative
common complication after this procedure. Objective: Evaluate renal
involvement in patients who underwent HPCT. Material and Methods: A retrospective and descriptive study was conducted among 97
patients that underwent HPCT between 1985 and 2004. Results: An
incidence of this complication of 27.5 % was observed in the analysed sample. The most frequent renal alterations were acute renal
dysfunction and failure. (51.6 and 22.5 %, respectively). During the
first 120 days of the transplantation, there was a predominance of
acute renal dysfunction, whereas in the period of more than 120
days there was a prevalence of radiogenic nephritis. The prevailing
causes were the multifactorial (54.2 %) and nephrotoxicity due to
cyclosporin A (17.1 %): up to 30 days, the multifactorial (72.7 %);
between 31 and 120 days, the nephrotoxicity due to cyclosporin A
(71.4 %); and in the period over 120 days, the radiations (50 %).
More alterations were observed in the allogenous transplant recipients (61.5 %) than in the autologous transplant recipients (15.4 %).
Among those who underwent chemotherapy + radiotherapy as a
conditioning regime (30.8 %) there were also more alterations than
among those who received only chemotherapy (11.7 %). Conclusions: The knowledge of renal complications is very important in
order to avoid them or treat them promptly.
Key words: Hematopoietic progenitor cell transplantation, renal
involvement, nephropathy .
078
POST TRANSPLANT MALIGNANCIES AFTER
RENAL TRANSPLANTATION IN A SINGLE
INSTITUTION OVER A 20 YEARS PERIOD
Cacchione, R1 *; Dupont, J1; Teper, S1; Garay, G1; Fernandez, J1;
Duarte, P1; Solimano, J1; Moreno, M. J.1; Davalos, M1; Riveros,
D1
*
Argentina - 1 CEMIC
Cancer incidence is increased after renal allograft due to immunosupressive treatment regularly given to prevent organ rejection. Mortality due tosecondary malignancies may account for
30% of the deaths causes of renal transplant (RT) recipients. Most
prevalent neoplasms (Neo) are cutaneous, lymphoproliferative
diseases and Kaposi’s sarcoma (KS). We review the incidence of
secondary tunors in 513 renal transplants performed at our institution during a 20 year. Fifty one per cent of RT were of cadaveric
origin. All patients received cyclosporine A (Cya), Azathioprine and
steroids as immunosupressive regimen, and 20% received either
antithymoglobulin, rapamycin or tacrolimus. We detected 43 Neo’s
(8.38%). Hematological Neo’s accounted for 13 pts. (2.53%), cutaneous (including 1 melanoma of donor origin): 12 (2.33%), hepatic: 5 (1%), KS: 4 (0.8%), gastrointestinal (GH): 4 (0.8%), lung: 2
(0.4%), breast 2 (0.4%) and coriocarcinoma 1 (0.2%). Out of 13 hematological Neo’s, 9 (69%) were non Hodgkin’s lymphoma (NHL), 2
(15%) Hodgkin’s disease (HD), 1 (8%) multiple myeloma (MM) and
1 (8%) acute myeloblastic leukemia (AML). Median time from RT to
secondary Neo was 5 years (1-20), without difference between non
hematologic and hematologic Neo’s. 5/9 pts with NHL were alive
and free of disease after +0.1, +1.2, +2, +3 and +7 years after NHL
was diagnosed. Four pts died at 0.2, 0.4, 0.6 and 7 years after NHL
diagnosis. The cause of death in latter was NHL with acceptable re-
S180
nal function. Two NHL pts had evidence of EBV infection by PCR in
the DNA obtained from tumor cells. One out of 2 HD pts is alive +8
years and the other had died 0.5 yrs after HD. One patient with MM
is alive in complete remission at 1 yr. One pt with AML died within
3 months after diagnosis. In all cases the first therapeutic attempt
was the reduction of thr immunosupressive drug dosage. Seven pts
required further treatment: chemotherapy 4 pts, radiotherapy 1 pt,
rituximab 1 pt and surgical tumor resection 1 pt, because absence
of response to ini tial therapy. In our review, secondary Neo’s to RT,
represented 8.3%. Hematological Neo`s were as frequent as cutaneous Neo`s and KS. Near 50% of secondary lymphomas remained
alive inthis cohort. Their outcome may not be worse than the “de
novo” lymphomas. Viral infections (CMV and EBV), immuinosupression and environmental factor have been considered risk factors or causative of secondary Neo`s. It is now acceptable that new
immunosupressive agents may provide less secondary neoplasms
in the setting of solid organ transplantation
162
POST TRANSPLANTATION
LYMPHOPROLIFERATIVE DISORDERS (PTLD) IN
RENAL TRANSPLANT RECIPIENT.
Diaz, L1 *; Nin, M2; Orihuela, S3; Curi, L4; Gonzalez, F3; Nese, M1
*
Uruguay - 1 Clínica Hematológica. Hospital de Clínicas, Facultad
de Medicina, Universidad de la República, Montevideo Uruguay.; 2
Nefrología. Instituto de Nefrología y Urología. Hospital de Clínicas,
Facultad de Medicina, Universidad de la República, Montevideo
Uruguay.; 3 Nefrología. Instituto de Nefrología y Urología. Dpto
Clínico de Medicina. Hospital de Clínicas, Facultad de Medicina,
Universidad de la República, Montevideo Uruguay.; 4 Instituto de
Nefrología y Urología. Hospital de Clínicas, Facultad de Medicina,
Universidad de la República, Montevideo Uruguay.
The aime of this review is to report the cases, clinical presentation and treatment of PTLD in 1000 renal transplant recipients
performed between 1981-2006.
PTLD CHARACTERISTICS AND OUTCOMES
Case 1 Case 2 Case 3 Case 4 Case5
Age/sex 48/male 46/male 34/male 50/male 43/male
Donor Alive Cadaveric Cadaveric Cadaveric Cadaveric
IS CsA +ster CsA+Aza+Ster CsA+Aza+Ster CsA+Aza+Ster
Aza+Ster
Induction IS No No No No No
Acute Rejection No No Yes Yes Yes
Rejection Treatment No No MP MP + ALG MP
Years to diagnosis 10 11 5 14 6
Localization Bowel Nodal Nodal Gastric Bowel
Histology LNH B LNH B LNH B LNH B Malt HP+ LNH B
Graft involve No No No No No
Treatment Reduc IS+PQT Reduction IS+PQT No Reduction
IS+HP treatment Reduc IS+PQT
Survival Yes at month24 Yes at month 36 Died at diagnosis Yes
at month 36 Died at month 60
Relapse treatment No Yes/PQT+ anti-CD20 No No No
Renal failure No No No No No
The incidence of PTLD was 0,5%. All were LNH-B late developing. Extranodal disease was the more frecuent presentation. The
EBV serostatus were unknow at transplantation. Reduction of immunosuppression in 4 patients was the first intervention without lost
graft. The optimal management of this disorders include maintaning
a high index of suspicion and identification of risk factors.
S181
POSTER SESSION
04.2 ANEMIAS
144
BLUNTED ERYTHROPOIESIS AND RECOMBINANT
HUMAN ERYTHROPOIETIN THERAPY IN ANEMIC
PREGNANT WOMENBLUNTED ERYTHROPOIESIS
AND RECOMBINANT HUMAN ERYTHROPOIETIN
THERAPY IN ANEMIC PREGNANT WOMEN
Demikhov, V.G.1 *; Morshchakova, E.F.1; Pavlov, A.D.1; Demikhova, E.V.1
*
Rusia - 1 Federal Research Center for Hematology, Oncology and
Immunology, Ryazan Branch
There is evidence that anemia in pregnancy is mainly due to
iron deficiency (ID). That is why, iron therapy is the basic treatment
of anemic pregnant women all over the world. However, there often found resistance to the treatment of anemia with iron. It is very
likely that anemia in pregnancy has a more complex pathogenesis,
than ineffective erythropoiesis, caused by iron or folate deficiency.
In the present study, we investigated the adequacy of the erythropoietin (EPO) production for the degree of anemia in pregnant
women and evaluated efficacy of rHu-EPO combined with oral iron
in the treatment of anemic pregnant women. We suppose that the
inadequately low production of EPO for the degree of anemia is a
possible mechanism of anemia development in pregnancy. The use
of rHu-EPO combined with iron is effective method in the therapy
of anemic pregnant women, who had been ineffectively treated with
iron alone.
Key words: anemia, pregnancy, erythropoiesis, rHuEPO
143
IS THE LINK BETWEEN PROINFLAMMATORY
CYTOKINES, HEPCIDIN AND ANEMIA IN
PREGNANCY?
Demikhov, V.G.1 *; Morshchakova, E.F.1; Pavlov, A.D.1; Kostina,
T.A.1
*
Rusia - 1 Federal Research Center for Hematology, Oncology and
Immunology, Ryazan Branch
Anemia in pregnancy has a more complex pathogenesis, than
ineffective erythropoiesis, caused by iron or folate deficiency. According to last our investigations the main mechanism of anemia in
pregnancy pathogenesis is inadequately low production of EPO for
the degree of the anemia. Nevertheless causes of blunt erythropoiesis in anemic pregnant women are uncleare. AIM: An influence of
some cytokines and hepcidin in anemia of pregnancy pathogenesis
to determine. MATERIALS AND METHODS: A total 62 pregnant
women were tested. All pregnant women were divided into 3 groups
on the basis of iron status and Hb level: group1 - iron deficiency
anemia (IDA) – 18, group 2 - anemia with normal iron status and
inadequately low production of EPO for the degree of the anemia
– 20 and group 3 - pregnant women with normal Hb levels - 24.
Control group consisted of 11 non-pregnant healthy women. We
determined serum levels of IL-6, IL-8, INF-γ and TNF-α by using
commercial ELISA kits. Serum pro-hepcidin concentrations were
measured immunoenzymometrically by using Hepcidin Prohormone ELISA (DRG, Germany) kits. RESULTS: Concentrations of
TNF-α in all pregnant’s sera samples and control were zero. The
significant elevated serum levels of IL-8 and INF-γ observed at all
pregnant women groups versus control (Table). Serum INF-γ concentration in IDA pregnant women (group1) was significant higher
than in group 2: 414.7±131.6 ng/L and 95.9±30.0 ng/L respectively
(p< .05). Increased IL-6 serum level was in group 3 only: 78.4±35.1
ng/L vs 3.13±3.13 ng/L in control (p< .05). Group 3 pregnants have
had significant decreased serum level of hepcidin: 9.6±2.34 μg/L vs
30.2±8.62 μg/L in control (p< .05). A considerable inverse correlations (r) between EPO and hepcidin, EPO and IL-8, EPO and INF-γ
serum levels were found in IDA pregnant women. CONCLUSIONS:
Apparently during pregnancy there is an alteration in maternal immunity within the uterus where innate, proinflammatory immune responses are tightly regulated to prevent immunological rejection of
the fetal allograft. Disruption of the delicate balance of cytokines by
bacteria or other factors increases production of proinflammatory
cytokines at the maternal-fetal interface and leads to blunt erythropoiesis and anemia. Significant increased production of proinflammatory cytokines in IDA is relation to stimulated placental production of inflammatory cytokines by redused O2 apparently. Detected
considerable inverse correlation between EPO and hepcidin serum
levels indicates that hepcidin may be one of inhibiting erythropoiesis
factors during pregnancy.
Table. Serum levels of cytokines and hepcidin
groups of pregnants
Groups
IL-6
IL-8
INF-γ
ng/L
ng/L
ng/L
Iron deficiency
35,6±
38,5±
414,7±
anemia
22,2
7,95**
131,6**
(group 1) n=18
Anemia with normal
52,7±
73±
95,9±
iron status
32,5
29,6*
30**
(group 2) n=20
Pregnants with
78,4±
27,4±
39,4±
normal Hb level
35,1*
5,99**
9,43**
(group 3) n=24
Healthy non-preg3,1±
10,2±
6,9±
nants (control) n=11 3,13
0,97
2,87
* - p< .05
** - p < .01
in different
Hepcidin
μg/L
32±
12,31
11,0±
3,74
9,6±
2,34*
30,2±
8,62
S182
146
TREATMENT OF AUTOIMMUNE HEMOLYTIC
ANEMIA WITH INTRAVENOUS IMMUNOGLOBULIN
IN A PATIENT WITH COLD AGGLUTININ
DISEASE ASSOCIATED WITH WALDENSTROM’S
MACROGLOBULINEMIA
Lee, H.1 *; Shapiro, R.1; Poth, J.1
*
USA - 1 Dominican Hospital
Background: Intravenous immunoglobulin (IVIG) is considered
to be a treatment option for autoimmune hemolytic anemia. However, little information exists about utilizing IVIG to manage autoimmune hemolytic anemia due to cold agglutinin disease.
Summary: We report a case of Waldenstrom’s macroglobulinemia with autoimmune hemolytic anemia due to cold agglutinin
disease who has remained in remission for nine months since treatment with IVIG. The patient is an elderly man who has suffered
several episodes of hemolytic anemia requiring transfusions for a
hemoglobin as low as 5 g/dL. His haptoglobin was decreased at
less than 10 mg/dL, and lactate dehydrogenase was increased to
780, consistent with hemolysis. Cold agglutinin titers were positive
at 1:32,768 dilution. He received a course of rituximab, but experienced another hemolytic episode associated with pneumonia.
He was hypogammaglobulinemic with an immunoglobuin G of 490
mg/dL, and has a detectable immunoglobulin M (IgM) type kappa
monoclonal protein with normal IgM level. In order to prevent infections which may be an exacerbating cause of his hemolytic anemia,
the patient was given IVIG. Thereafter, his hemoglobin normalized,
and he has remained stable.
Conclusion: Cold agglutinin disease is seen with certain infections such as mycoplasma pneumonia, although this is associated
with polyclonal IgM production. Whether or not the response observed here is due to prevention of potentially precipitating infections, or due to an immune mediated response from IVIG therapy
is unclear. Further study of the use of IVIG in such individuals is
warranted.
Keywords: Waldenstrom’s macroglobulinemia, cold agglutinin
disease, autoimmune hemolytic anemia, intravenous immunoglobulin
151
IRON METABOLISM, ANEMIA AND PREGNANCY
Ibarburu, S.1 *; Nieto, V.1
*
Uruguay - 1 Banco de Previsión Social
OBJECTIVES: To study changes in maternal Iron Metabolism during normal pregnancies and their effects on fetuses in a
not supplemented population.To establish diagnosis and treatment
guidelines for our population.
MATERIAL AND METHODS: 725 pregnant women were studied. 3 blood samples were taking from each patient before week
20, between weeks 20 and 29, and week 30 until the end of pregnancy. Blood samples from the cord during delivery were taken..
Hemograms and ferritin analysis were performed on all samples.
International WHO limits and the Indian Council of Medical Research Categories of Anemia were used for anemia diagnosis and
classification.
RESULTS AND DISCUSSION: The data analysis shows that
25.4% of our population has mild anemia and 7.7% has moderate anemia, without iron supplements intake, without any clinical
effects on the mother or the fetus during pregnancy. There were no
cases with severe or very severe anemia On newborns no effects
were detected.The significant finding is that ferritin values drop dramatically, especially after week 25, even with normal hemoglobin
values, in 66.9% of the cases, and with mild anemia in 25.4% of
them. In these cases there were no clinical effects on mothers or
fetuses.This finding is a characteristical trait of our population, For
such purposes, we suggest as diagnosis and treatment guidelines:
1)nutritional assessment in order to improve and maximize the diet
iron intake. 2)Maternal ferritin dosification and hemogram twice at
the first control visit and between weeks 24 and 28. 3)Ferrous salts
administration only to those patients whose ferritin at the beginning
of controls is lower than 80 μg/l, and to those whose values are
lower than 14 μg/l in the second control.
186
ERITHROCYTE ANTIBODIES IN PREGNANT
WOMEN
Pereira, A.1 *; Silveira, S.2; Hernandez, C.2; Varela, A.2; Gaggero,
M.2; Olivera, P.2; Dilorenzi, N.1; Guiarte, M.1; Larrosa, V.1; Miller,
A.1; Decaro, J.2
*
Uruguay - 1 National Blood Transfusion Service; 2 Transfusion
Medicine Department of the Social Security
Objective. Investigate the prevalence of erithrocyte antobodies
(EA) in pregnant women, the specificity and the possibility of these
causing Haemolytic Disease of the Newborn (HDN).
Materials and methods. 14,860 pregnant women attending the Social Security maternity during the period: 01/01/2001 to
31/12/2005 were screened for EA using the immunoprecipitation gel
test (DIAMED). The ID-Cards Type and screen A-B-D (VI-)3 AHG
cards were used to determine the blood group and Indirect Coombs
Test (ICT) was performed on AHG DIAMED cards and the three vial
(I-II-II) phenotyped erythrocytes (DIAMED) to determine the presence of EA. Antibody specificity was determined at the Servicio Nacional de Sangre Immunohematology Reference Laboratory using
LISS-Coombs and enzyme cards and the DIAMED 11 cell panel.
Results. EA were found in 157 (1.05%) of the samples studied,
Table 1. 153 of these are alloantibodies of which 84 (55%) were
found in Rh-Positive women and 69 (45%) were Rh-Negative. AntiD was found in 55 (80%) of Rh-Negative women, Table 2. In RhPositive pregnant women 36 (42.8) had antibodies with a specificity which has been reported as capable of causing HDN, whilst in
Rh-Negative women 66 (95.6%) had antibodies with a specificity
reported to cause HDN. In all patients studied 104 (0.7%) had antibodies capable of causing EA, of these 98 (94%) belonged to the
Rh and Kell blood group systems. Autoantibodies were found in 4
patients, Direct and Indirect Coombs Test positive, these were identified as antiphospholipid antibodies (APL).
Conclusions. Erythrocyte antibodies were found more frequently in Rh-Positive than in Rh-Negative pregnant women. In 102
of the cases the specificity of the antibody present indicates that it
can cause HDN, this is more frequent in Rh-Negative 95.6% than
in Rh-Positive 42.8% of the patients. Less than 1% of all patients
studied have EA capable of causing HDN. Despite Rh-immunoprofilaxis, Anti-D is still the EA most frequently detected, whereby
greater emphasis is needed to ensure that Rh-immunoprophilaxis is
administered according to current standard regulations. Alloinmmunization to other erythrocyte antibodies is increasing, efforts should
be made to transfuse only fully phenotyped Rh-Hr and Kell blood
to women of child-bearing age. These results reinforce the need to
test all pregnant women for erythrocyte antibodies, irrespective of
there Rh type.
Key words: pregnancy, Rh-Negative, Erythrocyte antibodies,
Haemolytic Disease of the Newborn.
184
AUTOIMMUNE HEMOLYTIC ANAEMIA
ASSOCIATED TO TREATMENT WITH IMATINIB
MESYLATE. A CASE REPORT
Fernández, J1 *; Guerra, T1; Cabrera, M1; Vera, A1; Bencomo, A2;
Pavon, V2
*
Cuba - 1 University Hospital Dr. Gustavo Aldereguía Lima; 2 Institute of Hematology and Immunology
Imatinib mesylate (Glivec ®, Novartis) is an inhibitor of protein
tyrosine kinase BCR- ABL which stop selectively the proliferation
and induce the apoptosis in leukemic cells Philadelphia +. Gastrointestinal, and skeletal muscle disorders as well as headache are
the adverse events communicated with higher frecuency in the
clinical trials with this drug. Neutropenia, thrombocytopenia, and
anemia due to medullar depression are among the hematic events.
A woman is presented with a diagnosis of chronic myelocitic leukemia (CML) in accelerated phase who after starting a treatment with
Glivec ® had a reversible medullar aplasia and hemolytic anemia
with a positive direct antiglobulin test mediated by IgG and C3.
The presence of anti-erythrocytary antibodies in the eluate of the
hematies, and in the alloantibodies anti Rh E, and the anti Kell as
reagent in the indirect antiglobulin probably stimulated through
previous transfusion of concentrate of hematies were shown. The
patient was treated with prednisolone and azathioprine together
with imatinib achieving leukemia hematologic remission and complete cytogenetic response. The few cases reported with hemolitic
anemia by Glivec ® are due to the mixture of the medication to the
proteins of the membrane of the erythrocite with formation of antibodies which recognize antigen determinants of the hapten-type
drug, and not the production of antibodies vs specific proteins of the
membrane of the hematie as in our case.
195
VALUE OF THE RETICULOCYTE HAEMOGLOBIN
IN PATIENTS WITH SECONDARY ANAEMIA TO
MALIGNANT HEMOPATHIES IN TREATMENT WITH
ERYTHROPOIESIS-STIMULATING AGENTS (ESAS)
Armellini, A.1 *; San Miguel, J.1; García Marcos, M. A.1; Martín Marcos, J. S.1; González, M1; Vidriales, B1; Gutiérrez, N1;
Hernández, J. M.1; Encinas, C1; Graciani, I1; García-Sanz, R.1
*
España - 1 Hospital Universitario de Salamanca
Introduction
The Erythropoiesis-stimulating agents (ESAs) are indicated in
the supportive treatment of the anaemia in malignant hemopathies
(HM). However, the response rate is very variable due to different
factors, for example: the functional iron deficiency (FID) which is
responsible for the important number of the therapeutic failure. This
problem can be solved and an early detection will allow to optimize
the use of the ESAs in the clinical practice. The level of the reticulocyte haemoglobin which is useful for the diagnostic of the FID, at the
present can be easily obtained with the new Autoanalyzers.
S183
AIMS
To evaluate which patients treated with ESAs, present FID and
improve the terapeutic indication.
To determine if the level of the reticulocyte haemoglobin predicts the response in patients with HM treated with ESAs.
Patients and Methods
Reticulocyte haemoglobin level was analysed by two automatic
counters Sysmex XE-2000, Roche and ADVIA® 2120 Hematology
System, Bayer Diagnostic, following the manufacturer’s instructions
and the required standard qualities of the Spanish Hematology and
Hemotherapy Asociation (AEHH). The FID was defined using the
Thomas Plot algorithm. This uses the following parametres: C - reactive protein, ferritin, serum soluble transferrin receptor and reticulocyte haemoglobin. The evaluation was performed at the beginning
of the treatment and in the first, third, sixth and twelfth week of the
treatment once it was iniciated. The response was defined as the
increase of at least 0.5 g/dl regarding to the initial value of the haemoglobin. The response was defined as very early response (third
week), early response (sixth week) and global response (twelfth
week). We included 26 patients (13 men and 13 women) age was
64±15 years and their diagnosed was: Multiple Myeloma (n= 13),
Non Hodgkin Lymphoma (n=7), Hodgkin Lymphoma (n=2), Chronic
lymphocytic Leukemia (n=3) and one plasma cell Leukemia. Twelve
of them were treated with Epoetin beta and three with darbepoetin
alfa at standard doses.
Results
The levels of analyzed parameters in patients with and without
FID on the third and the sixth week are as follows.
Parameters Initial N = 25 Patients without FID Patients with
FID
3ªW, n=17 6ªW, n=14 3ªW, n=5 6ªW, n=4
Hb (g/dl) 9,6±0,9 9,7±2 11±1,2 10,1±0,8 10,2±1,2
Reticulocyte haemoglobin. (pg) 36,3±4,4 34,4±4,4 35,9±4,1
28,4±3 28,9±6,6
Fe (μg/dl) 101,3±66,2 88±48 72,8±28 48,8±24 43,9± 12
Ferritin (ng/dl) 1284,8 (46-15917) 631(32-3530) 1093(42-8686)
518(37-1542) 260(19-468)
Soluble Transferrin receptor (mg/dl) 3,6±2 5,4±1,5 5,5±2,3
6,7±2,7 7,7±2,4
Index Saturation (%) 42,3±29,6 37,6±25 40±26 23±18 21±10
Regarding the response on the sixth week, patients with favorable response showed the initial reticulocyte haemoglobin higher
than patients who did not respond (67% vs 33%) (p=0.056).Thus,
81% of the responding patients had a reticulocyte haemoglobin
higher than 36 pg, while this percentage was only 36% for the non
responders.
The 53% of patients without FID in the sixth week of the treatment reached early response. However, only 20% of the patients
with FID reached early response, despite in all of them iron replacement therapy was indicated.The response of treatment with ESAs
was 26%, 45.5 % and 58% on the third, sixth and twelfth week of the
treatment, without clear correlation with FID.
Conclusions
The high level of reticulocyte haemoglobin at the initial treatment with ESAs seems a favorable of response predictor. This fact
with its easy determination justifies its use in the baseline evaluation
of the patients with HM who are planned to receive treatment with
ESAs.
S184
POSTER SESSION
05.2 LYMPHOPROLIFERATIVE SYNDROMES
012
013
LABORATORY AND BONE MARROW FEATURES IN
HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS
IMMUNOCHEMOTHERAPY IN HEMOPHAGOCYTIC
LYMPHOHISTIOCYTOSIS
Lina T. Romero-Guzmán, LTRG1 *; Rogelio Paredes- Aguilera,
RAPA1; Patricia Galindo Delgado, PGD1; Lourdes Gonzalez Pedroza, LGP1
*
Mexico - 1 Instituto Nacional de Pediatría
PAREDES-AGUILERA RA, RAPA1 *; GALINDO-DELGADO P,
GDP1; GONZALEZ-PEDROZA L, GPL1; ROMERO-GUZMAN LT,
RGLT1
*
Mexico - 1 INSTITUTO NACIONAL DE PEDIATRIA
Introduction:
Hemophagocytic lymphohistiocytosis (HLH) is a rare lifethreatening condition characterized by prolonged high fever, lymphadenopathy, hepatosplenomegaly and jaundice. Laboratory data
show evidence of cytopenia, disseminated intravascular coagulation (DIC), profound elevation of serum triglyceride (TG) lactic dehydrogenase (LDH) and serum ferritin concentration. Bone marrow
examination usually reveals an excessive proliferation of benignlooking histiocytes with prominent hemophagocytosis.
Introduction:
The fulminant hemophagocytic syndromes are aggresive and
often fatal disorders characterized by fever, systemic symp toms,
jaundice, multiple organ failure, coagulopathy, and phagocytosis
of blood elements with cytopenias. Various attempts to control the
diseases have resulted in only transient improvements in most of
the patients. Fatal cases are most common in patients with FHLH,
although in recent years there has been growing concern over the
lethal potential of virus-associated HLH. Recent reports indicate effective control of EBV-related HLH with immunochemotherapy.
Objective:
The purpose of the present study is to evaluate the efficacy
of and induction therapy regimen with etoposide in a group of 34
patients with HLH.
Diagnosis of HLH was made according to the criteria described
by Honter et al and EBV-HLH was diagnosed by serological tests
and by detection of the EBV genome in various biological specimens. Cultures were done on admission and as required during follow up. Patients were classified according to the scheme of clinical
severity of EBV-HLH of Imashuku et al. Patients were treated with
corticosteroids alone, IVIG alone or a combination of these drugs
and since 1999 on a immunochemotherapy protocol.
Results:
An underlying potentially responsible for HLH was identified in
22 patients, five of whom were considered to have FHLH, and two
Griscelli´s syndrome and the remaining 27 cases had secondary
HLH. A triggering organism was identified in 17 of the 34 cases,
seventeen patients had documented infection and the infectious
agents included brucella (two cases) leptospira (one case), HVA
(one case), candida sp. (one case) and serologic evidence of EBV
infection (12 cases). In two patients with inconclusive serological
evidence for active EBV infection, positive hybridization date were
compatible with an active EBV infection. Seven patients ( 20.6%)
did not received treatment, seven patients received mono or combinative therapy and 20 received immunochemotherapy. Overall survival was 0% in the first group, 43% in the second group and 85%
in the third group. In the first group five patients were classified as
high, and seven intermediate risk, in the second group four were
classified as having mild risk and three intermediate risk , and in the
last group nine had mild, nine intermediate and two high risk HLH.
Conclusion:
We conclude that early administration of immunochemotherapy is a highly effective treatment of patients with HLH.
Objective:
In the present study we sequentially monitored blood cytology
parameters and ferritin, lactate dehydrogenase (LDH), and tryglycerides (TG) in patients with HLH during the course of the disease
to evaluate the relationship of these parameters, clinical manifestations, disease activity response to therapy and outcome.
Between 1994 and 2006 we found 34 patients with HLH, of
whom five had primary HLH and two Griscellis Syndrome and the
remaining 27 had secondary HLH. Patients were diagnosed on the
basis of clinical and laboratory features according to the diagnostic
criteria of the FHL Study Group of the Histiocytic Society. All patients were treated with chemoimmunotherapy.
Results:
All patients (100%) had anemia, l5 (44.1%) profound neutropenia (< de 500/mL), 4 (11.7%), 23.5% moderate (de 500 -1000/mL)
and 15 (44.1% ) slight neutropenia (> 1000/ mL), 8 (23.5%) had
< de 10,000 platelet/mL, 14 (41.1%) de 10,000 a 50,000 /mL, 5
(14.7%) de 50,000 a 100,000/mL and 7 (20.5%) > de 100,000. Ferritin levels were increased in 25 of 26 ( 96.1%) patients of whom serum levels were determinated, LDH serum levels were increased in
32 (94.1%) of the patients, and serum triglyceride were increased in
27 of 32 (84.3%) in whom serum levels were measured. Hypofibrinogenemia (< de l.5 g/l) was present in 26 (76.4%) of the patients.
Bone morrow hemophagocytosis was present in all patients.
Conclusion:
Patients who attained complete remission showed normalization of blood cytology, coagulation and enzymatic parameters,
normalization of DHL,TG and ferritin values, and disappearance of
bone marrow hemophagocytosis, TG, DHL, and ferritin measurements were not only important diagnostic laboratory features, but
also important laboratory tests to measure disease activity during
follow-up.
S185
037
039
AN INTERNATIONAL MULTICENTRIC TRIAL WITH
FLUDARABINE PLUS CYCLOSPHOSPHAMIDE
IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA
(CLL) “UP FRONT”: SECOND INTERIM ANALYSIS.
SUBCUTANEOUS ALEMTUZUMAB IN PATIENTS
WITH REFRACTORY/RELAPSED B-CELL CLL
AFTER A FLUDARABINE-BASED REGIMEN. AN
INTERIM ANALYSIS
Bezares, F1 *; Jait, C2; Caviglia, D2; Bar, D2; Rodriguez, A2; Pilnik,
N2; Prates, V2; Lastiri, F2; Cauvi, F3; Carrasco-Yalán, A4; Gabus,
R3; Giralt, S5
*
Argentina - 1 GATLA & LACOHG (Latin American Cooperative
Oncology Hematology Group); 2 GATLA (Grupo Argentino para
el tratamiento de la Leucemia Aguda); 3 LACOHG; 4 Hospital
Edgardo Rebagliati, Lima; 5 MD Anderson CC, USA
Bezares, F1 *; Stemmelin, G2; Argentieri, D3; Lanari, E4; GuyGaray, E5; Campestri, R6; Bortomioli, M7; Garcia, J8; Giralt, S9;
Milone, G10
*
Argentina - 1 GATLA & LACOHG (Latin American Cooperative Oncology Hematology Group); 2 Hospital Británico; 3 Sanatorio Junin;
4
CM Corrientes; 5 CEMIC; 6 Sanatorio Mitre; 7 Hospital Pena Bahia
Blanca; 8 Hospital Privado de Cordoba; 9 MD Anderson CC, USA;
10
FUNDALEU, Argentina
Introduction:
On August 2002 an international multicentric trial on Fludarabine monophosphate (FAMP) plus Cyclophsphamyde (Cy) among
previously untreated B-cell CLL, was activated.
Objective:
Our aim is to evaluate efficacy and toxicity of FAMP plus Cy in
previously untreated B-cell CLL patients (pts). This is the second
interim analysis after four-year
Treatment consists in three consecutive days of oral FAMP 40
mg/m2 (n=84) or i.v. FAMP 25 mg/m2 (n=13) plus i.v. Cy 600 mg/
m2 on day 1 or Cy 250 mg/ m2 from day 1 to 3, every 28 days × 6
cycles. Responses were assessed according to the National Cancer Institute working group criteria after cycle 3 and again after cycle
6. Since August 2002 to March 2006, 109 CLL pts from Argentina
(n=95), Perú (n=11) and Uruguay (n=3) were enrolled for this protocol; eighty-nine were evaluated for response and toxicity. Median
age: 64 years old (range: 44-81); male = 47, female = 42; Binet
staging: A=14, B=45, C=30; median beta-2 microglobuline = 4.00
mg/dL (range: 1.3-9.2); median LDH = 341 UI/L (range: 101-762);
among patients with available data the CD 38 expression more than
10% was 38% (22 of 58 pts). Blood counts at inclusion: median
values (range); Lymphocytes: 32 x109/L (2,7-137), Hb: 120 g/L (50164), platelets: 175x109/L (10-364). Renal and hepatic parameters
within normal range limits. Cytogenetic by banding was available in
27 cases: no alterations (n=17), +12 (n=1), del (6), del (12) (n=1),
lost Y (n=1).
Results:
On March 2006, 56.2% (50 pts) had completed 6 cycles and
97.8% (87 pts) had undergone at least 3 cycles. OR: 92% = 81 pts
(CR: 39% = 35 pts; PR: 52% = 46 pts); treatment failure: 9% = 7 pts.
Evaluation for toxicity: 89 episodes of haematological toxicity and 7
episodes of infection grade 3-4 were reported after 436 cycles. 13
pts died: 7 died of infectious complications due to prolonged hematologic toxicity, 1 pt. died due to Tumoral Lysis Syndrome and 1 pt
died due hemoptysis associated with Lung Cancer. The estimated
DFS at 36 months: 70%, (SE 7.6%). Estimated OS was 76% at 36
months (SE 7.4%). The median OS was not achieved in responders
Conclusion:
FAMP plus Cy combination as front-line treatment is effective
in B-cell CL.
Introduction:
Alemtuzumab is the only single agent immunotherapy that
demonstrated to be effective at treating patients with B-cell chronic
lymphocytic leukemia (B-CLL), who have relapsed from or are refractory to Fludarabine.
Objective:
The optimized schedule for alemtuzumab that achieves maximal efficacy with manageable toxicity is still being explored. Here,
we report the second interim analysis of alemtuzumab administered
subcutaneously (SC) in refractory/relapsed B-CLL
Alemtuzumab was dose escalated from 10 to 20 mg during the
first week, 30 mg twice week during the second and third weeks,
and 30 mg once weekly during Weeks 4, 6, 8, 10, 12, 16, 20, 24,
28, 34, and 40. Antiviral prophylaxis included TMP/SMX bid 3 times
a week and acyclovir 200 mg three-times daily.
Results:
Of the 38 patients, 12 (31.6%) were refractory and 26 (68.4%)
had relapsed from prior therapy. Patients had a median age of 66.5
years (range, 43-86 years), 30 were male (79%), and 45% and 53%
had Binet stage B and C, respectively. The median number of prior
therapies was 1 (range: 1-4). The median duration of therapy was
7 weeks (range, 2-24 weeks), with a median cumulative alemtuzumab dose of 457 mg (range, 120-1,080 mg). Among the 35 patients who were evaluable for response, the overall response rate
was 88.6%: 45.8%complete response (CR), 2.9% unconfirmed CR,
42.8% partial response (PR). Four patients ( 11.5%) did not respond
to therapy. Of the 9 patients with refractory disease, 1 achieved a
CR, 6 a PR and 2 did not respond. Median follow up was 13 months
and median overall survival was not achieved. Minimal residual disease (MRD) was measured by flow cytometry in 6 patients who
achieved a CR: 4 patients had <0.5% of CD5/CD19+ cells, 1 patient
had <5% of CLL cells, and 1 patient had <10% CLL cells. According
to WHO toxicity criteria, from the 38 evaluable patients, 4 (10,6%)
experienced grade 3/4 infection, 2 (5.2%) had grade 2/3 granulocytopenia/thrombocytopenia, 1 patient (2.6%) had cytomegalovirus
(CMV) reactivation without CMV disease, and 1 patient (2.6%) developed EBV with prolonged bone marrow hypoplasia.
Conclusion:
Results of this second interim analysis suggest that a less intense regimen of alemtuzumab is feasible, effective, and safe for
patients with refractory/relapse B-CLL after fludarabine therapy.
S186
056
067
IMMUNOPHENOTYPE AND IMMUNOGLOBULIN
VARIABLE GENES (IGVH) MUTATION STATUS
IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL).
NEW PROGNOSTIC PATTERNS DETECTION AND
TARGET THERAPY.
TUMOR NECROSIS FACTOR-A (TNF-A) AND NOT
VASCULOENDO-THELIAL GROWTH FACTOR
(VEGF) PREDICTS SURVIVAL IN A GROUP
OF PATIENTS WITH LYMPHOPROLIFERATIVE
DISEASES.
Gabus, R1 *; Pritsch, O2; Landoni, AI1; Canessa, C1; Bianchi, S2;
Tiscornia, A2; Borelli, G1; Uturubey, F1; Bodega, JE1
*
Uruguay - 1 Hematology. Hospital Maciel. Montevideo; 2 Molecular
Pathology Unit. Medicine Public University.Montevideo. Uruguay
Villela, L.1 *; Caballero, R.1; Ruiz, R.1; Rojas, A.1; Borbolla, J.R.2;
García-Herrera, H.1; Mejía, M.D.1
*
Mexico - 1 Centro Médico ISSEMyM; 2 Instituto Tecnológico de
Monterrey
Introduction: The IgVH mutational profile is a strong prognostic indicator in CLL patients (pts.), particularly in early stages,
where clinical staging fails to accurately predict outcomes.
Objetivo: To evaluate peripheral blood inmunophenotype including CD38 and the IgVH and correlate these parameters with
clinical evolution and treatment requirements, in non treated newly
diagnosed CLL patients, below 75 years old.
Materials and method: We recruited 20 pts. with stage Binet
A, 10 pts. Binet B and 10 pts. Binet C. Ig nucleotide sequence
was carried out at the Medicine Public University in Montevideo.
A watch and wait strategy for Binet A and indolent Binet B was adopted whatever IgVH mutation status and fludarabine based regimen as standard first line therapy for progressive Binet A and B and
all Binet C .
Results: From June 2003 to June 2006, 40 untreated CLL pts:
Twenty-two males and 18 females, median age of 61 years (range:
38-73). Median lymphocytes counts at diagnosis was 53.100 per
mm3; for Binet A 25.460 mm3, for Binet B 47.880 mm3 and for Binet
C 113.820 mm3. Eight-five percent had typical immunophenotype
pattern (score 4-5) and 15% had atypical one. Eighty percent were
CD38 negative (cut off level: 30%). Among the 33 patients tested for
IgVH: 48% (16/33) were mutated and 52% (17/33) were unmutated
(cut off label 2% changes of naive pattern). All of the 12 Binet A
IgVH mutated pts. are in watch and wait strategy without signs of
progression, but 5 over 6 Binet A IgVH unmutated pts. had criteria
of progression and started into a Fludarabine regimen strategy. We
failed to find a significant correlation between IgVH mutation status
and CD38 expression,particularly in advanced stages. Median PFS
in the untreated group (18 pts.) was 37 months and for the treated
one (22 pts.) was 22 months. Median PFS for IgVH mutated and
unmutated pts. were 39 and 22 months respectively; and median
OS for IgVH mutated and unmutated pts were 45 and 28 months
respectively. In Binet-A median OS for those mutated and untreated
pts. was 47 months and for unmutated and treated pts. the median
PFS and OS were 34 and 39 respectively. Median OS for Binet B
and C were 23 and 21 months respectively
Conclusion: Unmutated patients display a worse prognosis
when compared to mutated ones. This is particularly true is early
stages where the presence of an unmutated status results in short
PFS and treatment requirement in most cases.
Introduction: VEGF levels appear to be able to predict the
mortality in aggressive lymphomas (AL) and Hodgkin Disease (HD).
There are no reports about the levels of TNF-a and VEGF together.
Objective: To evaluate the correlation of the mortality in a
group of patients (p) with LPS and the levels of VEGF y TNF-a.
Materials and method: The determination of VEGF and TNFa was performed by ELISA with a commercial reagent.The patients
were divided in 6 groups: (A) DLCL (B and T; 13p), (B) MM (8 p),
(C) ALL (B and T; 10p), (D) Indolent NHL (FL 3p; MCL 2p; MALT 2p;
4 CLL-B), (E) HD (3p) y (F) LPS atypical (1p).The value to consider
high the levels of VEGF was = 600 ng/dL and TNF-a <20 pg/dL. The
differences between VEGF (<600 vs. =600) y TNF-a (<20 vs. =20)
were evaluated with the crosstable and validated by the Fisher’s exact test*, the risks for each group were also considered [p validated
by Mantel-Haenszel** (p<0.05)]. The Kaplan-Meier survival curve
and p validated by log rank test (p<0.05) were also included.
Results: 46 patients were evaluated with a median follow-up
of 9 months (range: 1-19).
All patients
VEGF(N=46)
<600
≥600
TNF-α (N=42)
<20
≥20
Alive(%) Dead(%) p*
HR
56.5
8.70
26.10
8.70
95%CI
p**
0.42
2.16 0.46 to 10 0.32
50.0
16.7
04.4
28.9
0.0001 18.0 3.2 to 101 0.001
When the risk of death was evaluated in each group accord VEFG
(< 600 vs. =600) and TNF-a (<20 vs. =20) the HR observed in the
group (A), (B), (C), (D) and (E) were not significant. The survival for
the group of VEGF (<600 vs. = 600) was 14.39 months vs. 12.46
(95% Cl: 12.35 to 16.44 vs. 8.13 to 16.8, p=N.S.) for the group of
TNF-a (<20 vs. = 20) was 17.09 vs. 9.75 months (95%Cl: 15.33 to
18.85 vs. 6.27 to 13.22, p=0.0001).
Conclusion: The TNF-a high levels determined before the
treatment predict with more accuracy the mortality of patients with
LPS than the VEGF levels.
156
FLUDARABINE MONOPHOSPHATE AS FIRST
LINE THERAPY FOR CHRONIC LYMPHOCYTIC
LEUKEMIA (CLL) - URUGUAY 1995-2006
Novoa, J.E.1 *; Rojo, A.L.1; Beñaran, B.1; Draper, R.1; Calvo, H.1;
Iriondo, N.1; Cabrera, A.1; Pebet, M.1; Brignoni, S.1; Luongo, A.1;
† De Bellis, R.
1
*
Uruguay - 1 Servicio de Hematología. Hospital Policial
Background: fludarabine (F) has become the standard first line
therpay for chronic lymphoid leukemia (CLL) in younger patients.
Objectives: to assess the efficacy, safety and quality of life of F in
previously untreated LLC(B) in a Group of Medical Institutions in
Uruguay from 1995 to 2006. Methods:168 patients in the period
1995 - 2006 were evaluated.120 of them received the intravenous
formulation and 48 the oral one. Age: 48 to 85 years old, media 67
years old. Gender:male 90, female 78. Inclusion criteria for CLL-B
S187
was Binet stages B, C and A progressive (Ap), 18 to 85 years old,
non multiorganic failure, performance status 0 to 2 (WHO), informed
consent. First condition was non previous treatment. Staging: CLLB: Binet Ap 12/168, B 116/168 & C 40/168. Treatment: as first line
therapy all the patients received (minimum): 6 cycles of Fludarabine
(Fludara®, Schering) 25 mg/m2/daily (5 days) e/ 30 days or Oral
Fludarabine, 40 mg/m2/daily (5 days), 6 cycles. Results: The overall
response rate (ORR) was 78%.Safety: on the 1100 cycles ithe toxicity was: 1 AIHA, 2 pancytopenia, 3 plaquetopenia. Infection 1,3% ;
degree 3 and 4. No alopecia was observed.Kaposi sarcoma (0,7%).
Mortality rate: 1,7%.Causes of death: Richter 12%, sepsis 5%, associated disease 34%, second malignancy 17% and others 30%.
Comparing oral with intravenous formulation in overall survival the
results were: CLL 34% vs 36% (p= NS). Conclusions: fludarabine
monofosfate (Fludara®) looks like an effective and safe treatment
for CLL-B.. The oral and intravenous formulations have a similar
response rate in elderly and young patients. The challenge remains
to integrate new information to apply novel therapies in a diseasespecific and risk-adapted maner. A longer follow up and a larger
trial, might be needed to confirm these results.
S188
POSTER SESSION
06.2 HEMOSTASIS AND THROMBOSIS
Chairman: Raúl Altman
Chairman: Nelson Hamerschlak
160
091
ANTISS2 GLYCOPROTEIN 1 &RECURRENT FETAL
LOSS (APS)
THROMBIN GENERATION AMONG SUDANESE
ESSENTIAL HYPERTENSION PATIENTS.
Novoa, J.E.1 *; Steffano, B.1; Guillermo, C.1; Quevedo, E.1; Briosso, J.1; Godoy, W.1
*
Uruguay - 1 FEMI, COMERI & CEDITH
Hassan, Fathelrahman1 *; Hamid, Maria2
*
Sudán - 1 Sudan University; 2 University of Khartoum
Background: antiphopspholipid syndromes (APS) are diagnosed by the coincidence of clinical events of venous and arterial
thrombosis or recurrent miscarriage and abnormal specific laboratory tests.Objective: to study the prevalence and clinical significance
of elevated A-ß2GP1 in 100 women with history of recurrent fetal
loss (more than 2). The relationship between the clinical history,
atiphospholipid antibodies (APA), ACA (IgG & IgM), lupus anticoagulant (LA) and A-ß2GP1, mainly related to clinical events.Material
and methods: 100 women were included on this study.Their ages
ranged from 23 to 45 years old and were studied in the period november 2005 - september 2006. The blood samples were obtained
from venous puncture. The A-ß2GP1 were screened using an ELISA assay, repeated 6 - 12 weeks later. The results were divided in
positive, light positive or negative. Results: 65% of women with RFL
were reactive for anti-beta2glycoprotein 1; 44% showed reacivity
for APA, ACA (IgG or IgM). In two cases with reactivity for all the
tests, the women have had history of deep vein thrombosis (2%).
Table 1 shows the results of the markers evaluated on this study.
Table 1-Antiphospholipid Syndrome and Biologic Markers
All patients
Alive(%)
VEGF(N=46)
56.5
<600
8.70
≥600
TNF-α (N=42)
50.0
<20
16.7
≥20
Dead(%) p*
HR
26.10
8.70
95%CI
0.42
2.16 0.46 to 10
04.4
28.9
0.0001 18.0 3.2 to 101
p**
Background: The conversion of prothrombin to thrombin is a
central event in the coagulation cascade. Prothrombin fragment 1+2
(F1+2) is a polypeptide released from the prothrombin during its activation to thrombin by the prothrombinase complex. Measurement
of circulating levels of F1+2 has been considered a specific marker
of thrombin generation in vivo1,2. Elevated TAT measurements may
be accompanied by increased levels of prothrombin fragment 1+2
for detecting of deep venous thrombosis3.
Objectives: To check the feasibility of assessing hypercoagulability in hypertensive patients by measuring thrombin generation, and
determine the possible thrombosis of these Sudanese patients.
Materials and Methods: This is a descriptive, prospective analytical case-control based study conducted in Khartoum State teaching hospitals during the period of October 2003 to February 2006
to determine the thrombin generation markers among Sudanese
hypertensive patients. 200 patients and 50 controls were studied.
patients were those who fulfilled the clinical diagnosis of hypertension of either sex, on or off treatment. The controls were normal,
non-hypertensive individuals of either sex. Both patients and control
were above 40 years of age. patients (male and female ) without
previous history of venous or arterial thrombosis, diabetes mellitus.
TAT and F1+2 levels were determined by enzyme-linked immunosorbent assay.
0.32
178
0.001
Recurrent fetal loss were associated with sustained antibeta2
glycoprotein1 (p< 0.01), but deep vein thrombosis was not. Antiß2GP1
showed the maximum frequency of positivity and can help to
confirm the diagnosis of APS of pregnancy (APSp) in the cases of
recurrent fetal loss and ACA/APA/LA negative.Conclusions: antiß2GP1 is highly associated with recurrent fetal loss and must be
considered as a very good predictor of antiphosphlipid syndrome
in pregnancy.
VON WILLEBRAND DISEASE. FOUR YEARS
EXPERIENCE AT THE PEDIATRIC HSPITAL CENTER
IN MONTEVIDEO , URUGUAY
Boggia, B.1 *; Mezzano, R.1; Raffo, C.1; Rodriguez Grecco, I.1
*
Uruguay - 1 Centro Hospitalario Pereira Rossell
OBJECTIVE: Von Willebrand’s disease is the most frequent
clotting pathology, although it shows the most difficult diagnostic
confirmation. We show our experience in the creation of a clinic for
clotting pathologies, where various specialists refer patients with
personal and/or familial antecedents of bleeding as well as alterations in their basic coagulation studies requested preoperatively.
METHODS:Population of children/adolescents (0 to 15 years)
attended in this hospital, the registry comprises 237 primary consultations from 2000 - 2004.A Clinical History is completed: anamnesis
and paraclinics according to the proposed algorithm.
RESULTS: One clotting pathology was found in 82 patients
(39.59%), 43 (18.14%) underwent surgery and 9 (3.7%) showed
major bleeding perioperatively, requiring reposition with a commer-
cial factor. No major complications appeared and an adequate haemostatic management was achieved.
DISCUSSION: We would like to point out that there was a major bleeding in otorrhino-laryngologic surgeries (inter- and post-operatively) in 3 patients who underwent surgery in other hospitals of
the country who were referred to our Hospital Center.We focused
our efforts on the prevention of these complications, working in an
inter-disciplinary team: anesthetist, surgeon, hemotherapist, laboratory technician, performing the right assessment of the patient. It
is stressed the significance of the anamnesis with reference to the
personal and familial antecedents of the patient.As a national reference center in pediatrics, we are preparing guidelines-protocols,
which might be implemented in all Health Centers for a better care
of these children, as well as a national pediatric registry for clotting
pathologies.
145
FACTOR V LEIDEN (G1691A) ALLELE AMONG
PATIENS WITH DEEP VENOUS THROMBOSIS AND
IN THE GENERAL POPULATION IN CUBA
Pérez, G.1 *; Torres, W.1; Carnot, J.1; Muñio, J.1; De Castro, R.1;
Martinez, C.1; Hernandez, C.1; Pérez, D.1
*
Background: Factor V Leiden is associated to a higher risk
of deep venous thrombosis in different countries whereas the
prevalence is diverse according to the ethnic component of
the people Objective: To assess the frequency of factor V Leiden
(G1691A) allele in patients with deep venous thrombosis (DVT)
and in the general population in Cuba. Material and methods: A
case-control study was conducted where 100 healthy blood donors
and116 patients with DVT were genetically analyzed for the presence of this polymorphism.. The diagnosis of DVT was confirmed
by phlebography or Doppler ultrasonography, depending on the
case. Alllele frequency and the DVT risk associated with this mutation was estimated. Results: Allele frequency of factor V Leiden
(G1691A) was 0.028 in patients with DVT and 0.010 in the general
population (p=0.235). After adjustment for age and gender, the odds
ratio for DVT associated with the presence of G1691A allele was
statistically significant (95% confidence intervals 0.63-9.19). Conclusions: The knowledge of genetical causes of venous thrombosis
is very important in order to avoid them and to have useful strategies of treatment.
Key words: Deep venous thrombosis, Factor V Leiden allele.
187
A MILD HEMOPHILIA A PATIENT CARRIER, WITH
AN INHIBITOR OF HIGH RESPONSE.
Rodriguez Grecco, I.1 *; Boggia, B.1; Mezzano, R.1; Pissano, S.1
*
Uruguay - 1 Centro Hospitalario Pereira Rossell
Introduction:The development of inhibitors in mild Hemophilia
A (factor VIII> 5%) is not a frequent complication with a percentage of appearance that goes from 3 to 13%. The mechanism and
incidence of the development of the inhibitor in mild hemophilia A
is unknown.
Patient and Method.In this work we will present the clinic history of an eight year old patient, without any family antecedent to
highlight; he was diagnosed mild hemophilia A at the age of two
(factor VIII 17%), after a frontal haematoma; he was treated with
commercial factor VIII. In 2001 he consulted the doctors because of
a right knee haemarthrotic, with a bad response to the reposition
therapy. As a result, an inhibitor’s investigation was done having a
positive result with a title of 1400 Bethesda Units (BU).
S189
During this period of time, the patient had some bleeding episodes which were controlled using local measures.
The evolution of the inhibitor is shown in table Nº1:
Inhibitor’s dosage 2001 2002 2005 200
UB 1400 3200 3846 200
In August 2006, the patient was sent to the province of Durazno in Uruguay, having a traumatism in the lumbar and left flank’s
regions. Through a Computerized Axial Tomography, we could diagnose a great haematoma situated in the psoas-illiac muscle and
also a renal haematoma. The patient received treatment in the intermediate care unit, and he was treated with rfVIIa (NovoSeven) and
prothombin complex (Octaplex).
Result.It was possible to stop the bleeding with the use of rFVIIa (4,8 mg each two hours, 2 doses), continuing with the reposition of Octaplex 50-100 UI/Kg for a period of 15 days.
The patient had a good evolution from the clinic and tomography point of view.
Conclusions.
It has been a great challenge for the medical team to have
discharged this patient from hospital in the best medial conditions.
However, we feel worried about the evolution of this child considering his pathology as well as the reality of our health system, taking
special consideration to the economic resources available in our
country.
102
INHERITED COAGULATION DISORDERS IN
CENTRAL PART OF IRAN
Mojtabavi Naini, M.1 *; Derakhshan, F.1; Makarian, F.1; Hoorfar,
H1; Derakhshan, R.1
*
Iran - 1 Isfahan University of Medical Sciences
Background: the incidence of hereditary coagulation disorders
may vary according to the country and ethnic origin. Demographic
datasets are vital in setting priorities, allocation of resources, measurement of outcomes, and comparison of alternate approaches.
Aim: The aim of this study was to document the epidemiological features, disease severity and complications associated with
inherited coagulation disorders in central part of Iran.
Methods: A comprehensive survey was undertaken in January
2006. Clinical history, Laboratory and treatment data, and long term
complications of all cases (553 persons) diagnosed with inherited
coagulation disorders, were studied in Hematology-Oncology Department, Isfahan University of Medical Sciences.
Results: 465 male and 88 female with Mean±SD age of
23.4±12.9 were studied. Hemophilia A was found in 341(61.7%), 48
(8.7%) had hemophilia B, 74 (13.4%) had Von Willebrand disease,
and 34(6.1%) had platelet dysfunctions. The rare coagulation disorders (n=88) include 30 patients with FV deficiency, 23 with FVII,
13 with afibrinogenaemia, 10 with FX. Among them 19 (3.4%) had
combined FVIII and FV deficiency. 228 (41.2%) patients had severe hemophilia. The most common complications were Epistaxis
(n=59), Hemartrosis (n=51) and Hemophilic Arthropathy (n=49).
None of the patients were human immunodeficiency virus positive
but 125 (22.6%) were hepatitis C virus positive and 2 (0.4%) were
hepatitis B positive. Replacement therapy primarily relied on Cryoprecipitate and Fresh Frozen P

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