ISSN 1866-8836
Клеточная терапия и трансплантация

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Volume 2, Number 1(5)
05/17/2010
Volume 2, Number 1(5)
Editor-in-Chief
Afanasyev B. V. (St. Petersburg, Russia)
Co-Editors-in-Chief
Wagemaker G. (Rotterdam, Netherlands)
Zander A. R. (Hamburg, Germany)
Deputy Editor
Chukhlovin A. B. (St. Petersburg, Russia)
Fehse B. (Hamburg, Germany)
Novik A. А. (Moscow, Russia)
Managing Editor
Claudia Koltzenburg (Hamburg, Germany)
Editorial Board
Aleynikova O. (Minsk, Belarus)
Alyansky A. (St. Petersburg, Russia)
Anagnostou A. (Boston, USA)
Andreeff M. (Houston, USA)
Bacher U. (Hamburg, Germany)
Baуkov V. (St. Petersburg, Russia)
Baranov V. S. (St. Petersburg, Russia)
Barkhatov I. (St. Petersburg, Russia)
Baum C. (Hannover, Germany)
Bilko N. (Kiev, Ukraine)
Borset M. (Trondheim, Norway)
Buechner Th. (Muenster, Germany)
Bykov V. (St. Petersburg, Russia)
Dini G. (Genoa, Italy)
Drize N. (Moscow, Russia)
Egeland T. (Oslo, Norway)
Elstner E. (Berlin, Germany)
Emanuel V. (St. Petersburg, Russia)
Everaus H. (Tartu, Estonia)
Ferrara J. (Ann Arbor, USA)
Fibbe W. (Leiden, Netherlands)
Galibin O. (St. Petersburg, Russia)
Ganser A. (Hannover, Germany)
Granov D. (St. Petersburg, Russia)
Ivanov R. (Moscow, Russia)
Klimko N. (St. Petersburg, Russia)
Kolb H.-J. (Muenchen, Germany)
Konopleva M. (Houston, USA)
Koza V. (Pilsen, Czech Republic)
Kroeger N. (Hamburg, Germany)
Malikov A. (St. Petersburg, Russia)
Mikhailova N. (St. Petersburg, Russia)
Mentkevich G. (Moscow, Russia)
Nagler A. (Tel Hashomer, Israel)
Nemkov A. (St. Petersburg, Russia)
Neth R. (Hamburg, Germany)
Nevorotin A.J. (St. Petersburg, Russia)
Ostertag W. (Hamburg, Germany)
Palutke M. (Detroit, USA)
Roumiantsev A. G. (Moscow, Russia)
Savchenko V. G. (Moscow, Russia)
Smirnov A. V. (St. Petersburg, Russia)
Stamm C. (Berlin, Germany)
Tetz V. (St. Petersburg, Russia)
To B. (Adelaide, Australia)
Totolian A. A. (St. Petersburg, Russia)
Uss A.L. (Minsk, Belarus)
Vilesov A. (St. Petersburg, Russia)
Westenfelder Ch. (Salt Lake City, USA)
Wisloff F. (Oslo, Norway)
Zubarovskaya L. (St. Petersburg, Russia)
Zvartau E. (St. Petersburg, Russia)
In this Issue

Abstracts of the joint EBMT Pediatric Working Party – 3rd Raisa Gorbacheva Memorial Meeting, St. Petersburg, Russia, 2009


The aim of the European Group for Blood and Marrow Transplantation (EBMT) is to promote and facilitate access to state-of-the-art and cutting-edge knowledge in blood and marrow transplantation. The EBMT Paediatric Diseases Working Party (EBMT PDWP) has grown during the last few years and currently has more than 20 European countries and at least three non-European countries participating in its activities. This international profile feeds the background against which most of the actual debate on stem cell transplantation in children takes place.

The PDWP firmly believes that children can only be treated by specially trained and experienced pediatric hematopoietic stem cell transplantation (HSCT) teams.

The upcoming meeting will explore the major issues impacting on HSCT in children and adolescents. Leading experts will present and exchange innovative science and discuss specific clinical problems with the audience.

The EBMT PDWP is very enthusiastic about the joint meeting – which will give a full picture of the interdisciplinary approach – and thanks the organizers for the possibility to come to the wonderful venue of St. Petersburg.

Christina Peters
EBMT Chair Paediatric Working Party

Abstracts (Symposium groups)

A. Leukemia treatment
B. Leukemia research
C. Hematopoietic stem cell sources
D. Minimal residual disease
E. Graft-versus-Host Disease
F. Lymphoproliferated diseases (Lymphoma, Myeloma)
G. Complications in hematology (infections, haemostasis abnormalities, fertility)
H. Autoimmune diseases and aplastic anemia
J. Myeloproliferative diseases
K. Inherited diseases
L. Hemoglobinopathies
M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)
N. Solid tumors
O. Donor registry

Raisa Gorbacheva Memorial Lecture

A. Leukemia treatment

Prognostic value of chimerism in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT)

Maria V. Pryanishnikova, Alexandra A. Sipol, Irina S. Solomonova, Ildar M. Barkhatov, Elena V. Semenova, Liudmila S. Zubarovskaya, Boris V. Afanasyev

Comparing efficacy and toxicity of conditioning regimen “treosulfan+cyclophosphomide” versus “busulfan+cyclophosphomide” in children with hematological malignancies after allogeneic hematopoietic stem cell transplantation

Natalia V. Stancheva, Elena V. Semenova, Natalia I. Zubarovskaya, Yulia G. Vasilieva, Vladimir V. Vavilоv, Irina A. Mushchitskaya, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Survival in adults under 66 years with acute lymphocytic leukemia diagnosed in Norway 2000–2007

Jon-Magnus Tangen1, Jorg Axel Bohl2, Yngvar Floisand3, Einar Haukaas4, Inger Anne Naess5, Tove Skjelbakken6

Epigenetic therapy in childhood AML (pilot study)

Alexander V. Popa1, Elena S. Gorohova2, Elena V. Flejshman3, Larisa G. Fechina4, Vladimir V. Lebedev5, Karapet S. Aslanyan6, Evgenia V. Inyushkina2, Svetlana A. Mayakova1, Irina E. Gavrilova1, Olga P. Chlebnikova4, Elmira G. Boichenko7, George L. Mentkevich1

Efficacy of donor lymphocyte infusions following allogenic stem cell transplantation (allo-HSCT)

Olga A. Slesarchuk, Elena V. Babenko, Maria A. Estrina, Ilya V. Kazantsev, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Is it necessary to treat patients after syngeneic bone marrow transplantation?

Lidia S. Lubimova, Larisa A. Kuzmina, Elena N. Parovichnikova, Inna V. Alexeeva, Valeriy G. Savchenko

Treatment results of childhood acute lymphoblastic leukemia using the COALL-Saint-Petersburg-92 protocol after a 10-year observation period

Elmira G. Boichenko1, Margarita B. Belogurova2, Gritta Janka3, Margarita S. Livshits1, Eleonora M. Petrova1, Marina B. Ivanovskaya1, Irina A. Garbusova1, Galyna G. Radulesku2, Tatjana D. Victorovich2, Emylia D. Tchavpetsova2, Ludmila I. Shats2 

Consequences of ABO incompatibility in allogeneic hematopoietic stem cell transplantation (allo-HSCT)

Maria A. Estrina, Мurat O. Yagmourov, Dmitriy E. Pevtsov, Еvgenia A. Kochina, Natalia E. Ivanova, Alla A. Golovacheva, Alexander L. Alyanskiy, Ludmila S. Zoubarovskaya, Abdulbasir A. Ganapiev, Boris V. Afanasyev

B. Leukemia research

Comparative analysis of acute myeloid leukemia treatment results in the Sverdlovsk Regional Clinical Hospital №1

Alexander V. Vinogradov, Tatiana S. Konstantinova, Alexander F. Tomilov, Vladimir A. Shalaev, Elena V. Chepuryaeva, Julia V. Sveshnikova, Leonid N. Sharov, Natalia V. Vinogradova

Determination of FLT3-ITD and D835Y mutations as a factor of prognosis in immunophenotypically verified diagnosis of patients with acute myeloid leukemia in Saint Petersburg

Konstantin U. Slobodnyuk, Irina Y. Saburova, Margarita V. Gorchakova, Ekaterina B. Rusanova, Antonina V. Kurtova, Mikhail I. Zaraiski, Yekaterina E. Zueva

Expression of angiotensin-converting enzymes (ACE, CD143) and shaperons (BiPs, calnexin, calreticulin) by leukemic dendritic cells (LDCs) in acute myeloid leukemia (AML) patients

Irina V. Galtseva1, Lev M. Pashin1, Elena N. Parovichnikova1, Sergei M. Danilov2, Andrei B. Sudarikov1, Ivan A. Vorobiev1, Valeriy G. Savchenko1

Posttransplantation leukemia relapses: the role of chromosome changes

Nikolay N. Mamaev, Tatyana L. Gindina, Ildar M. Barkhatov, Boris V. Afanasyev

C. Hematopoietic stem cell sources

Features of a stem cell’s cryopreservation

Alexander B. Smolyaninov, Ksenia V. Korovina, Shirzod F. Adylov, Elena A. Kotelevskaya

Virus markers antibodies detection in umbilical cord blood

Olga V. Supilnikova, Irina I. Maslenikova, Alexander B. Smolyaninov

First experience of CD34+ cell selection to high-risk neuroblastoma patients

Yulia A. Yakovleva1,2, Grigory A. Tsaur1,2, Alexander M. Popov1,2,3, Tatyana Yu. Verzhbitskaya1,2, Igor N. Vyatkin1,2, Andrey A. Igumenshev1,2, Natalya G. Maisheva1,2, Anton Yu. Zadoya1,2, Egor V. Shorikov1,2, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

Effectiveness of hematopoietic progenitor cell separation with the help of the Sepax S100 cell separator

Alexander B. Smolyaninov, Dmitry A. Ivolgin, Shirzod F. Adylov, Elena A. Kotelevskaya

Low and high-resolution HLA typing of cord blood samples of register of donors in St. Petersburg

Elena A. Kotelevskaya, Svetlana A. Smirnova, Alexander B. Smolyaninov

Importance of CFU-GM number for prediction of hematological reconstitution after low CD34+ cell dose autotransplantation in children

Yanina I. Isaikina, Valery I. Martinevsky, Nina V. Minakovskaya, Yury S. Strongin, Olga V. Aleinikova

Selection of optimal cryopreservation strategy for hematopoietic stem cells

Leonid I. Savelyev1,2,3, Yulia A. Yakovleva1, Grigory A. Tsaur1,2, Tatyana Yu. Verzbitskaya1,2, Alexander M. Popov1,2,3, Andrey A. Igumenshev1,2, Larisa V. Vakhonina1,2, Igor N. Vyatkin1,2, Egor V. Shorikov1,2, Larisa G. Fechina1,2

D. Minimal residual disease

Algorithm for monitoring minimal residual disease on the basis of patient-associated immunophenotypes

Yekaterina E. Zueva, Margarita V. Gorchakova, Ekaterina B. Rusanova, Konstantin U. Slobodnyuk

Slow fusion gene transcript clearance predicts poor outcome in infants with MLL-rearranged acute lymphoblastic leukemia

Grigory A. Tsaur1,2, Tatyana V. Nasedkina3, Alexander M. Popov1,2,4, Anna S. Ivanova1,2, Yulia A. Yakovleva1,2, Tatyana O. Riger1,2, Egor V. Shorikov1,2, Leonid I. Saveliev1,2,4, Larisa G. Fechina1,2

Detection of minimal residual disease (MRD) by flow cytometry in children with acute lymphoblastic leukemia (ALL)

Margarita G. Bozhieva, Ludmila Y. Grivtsova, Alexander V. Popa, Irina N. Serebryakova, Irina E. Gavrilova, Boris V. Kurdyukov, Rono S. Ravshanova, Georgiy L. Mentkevich, Nikolay N. Tupitsyn

Comparison of MRD data assessed by flow cytometry and RT-PCR of fusion gene transcripts in infants with MLL-rearranged ALL

Alexander M. Popov1,2,3, Grigory A. Tsaur1,2, Anna S. Ivanova1,2, Yulia A. Yakovleva1,2, Tatiana Y. Verzhbitskaya1,2, Tatiana O. Riger1, Egor V. Shorikov1,2, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

E. Graft-versus-Host Disease

The comparative characteristic toxicity and immunosuppressive activity both tacrolimus and cyclosporine in patients undergoing allo-HSCT

Yulia A. Stankevich, Ludmila S. Zubarovskaya, Elena V. Semenova, Sergey M. Alexeev, Elena V. Babenko, Boris V. Afanasyev

Spirometry findings in bone marrow transplant patients

Larisa A. Kuzmina, Lidia S. Lubimova, Valeriy G. Savchenko

Evidence of the “graft-versus-tumor” effect following haploidentical transplantation in a patient with a metastatic relapse of Ewing’s sarcoma

Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, Natalia S. Subbotina, George L. Mentkevich

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Prognostic value of apoptosis of tumor clone bone marrow cells in patients with multiple myeloma

Zhanna V. Chubukina, Ludmila N. Bubnova, Stanislav S. Bessmeltsev

Expression of tumor-associated genes in multiple myeloma patients during high-dose chemotherapy and auto-SCT

Tatiana V. Gaponova, Lidia S. Mendeleeva, Andrey V. Misurin, Elena Yu. Varlamova, Elena N. Parovichnikova,
Valeriy G. Savchenko

Treatment results of children with Hodgkin’s disease (HD) according to protocols DAL-HD and GPOH-HD

Margarita B. Belogurova1,2, Galina G. Radulesku1,2, Tatyana D. Viktorovich1,2, Emilia D. Chavpetsova2, Ludmila I. Shats1,2, Yulia V. Dinikina1

Cellular factors of antitumor immunity in patients with chronic lympholeukemia

Natalya V. Issayeva, Galina A. Zaitseva, Tamara P. Zagoskina

Salvage therapy in relapsed Hodgkin's lymphoma patients

Elena S. Belyaeva, Olga V. Morozova, Andrey I. Slugin, Alexander V. Popa, Georgy L. Mentkevich

Clinical and pathological characterization of the t(14;19)(q32;q13)-positive splenic marginal zone lymphoma

Hunan L. Julhakyan, Tatyana N. Obukhova, Irina B. Kaplanskaya, Rima S. Samoylova, Andrey I. Vorobiev

Association between HLA class I antigens and survival length in patients with multiple myeloma

Elena A. Senkina, Galina A. Zaitseva, Tamara P. Zagoskina, Konstantin A. Martynov

High-dose melphalan versus melphalan plus dexamethasone in patients with AL amyloidosis

Anna G. Smirnova1, Alexey V. Smirnov2, Axel R. Zander3, Boris V. Afanasyev1

The role of p53 polymorphisms in non–Hodgkin’s lymphomas’ susceptibility and prognosis

Elena N. Voropaeva, Tatiana I. Pospelova, Mikhail I. Voevoda, Olga V. Beresina

Virus participation in renal damage in AL patients: histopathological study

Ekaterina G. Vorozheikina1, Vladimir V. Varshavski2, Ekaterina P. Golitsina2, Evgenia N. Glasko1, Irina B. Kaplanskaya1, Ljudmila S. Birjukova1, Elena N. Parovichnikova1, Valeriy G. Savchenko1

Bone marrow angiogenesis is a prognostic value in patients with multiple myeloma undergoing high-dose therapy and autologous stem cell transplantation

Olga S. Pokrovskaya, Lidia S. Mendeleeva, Irina B. Kaplanskaya, Elena Yu. Varlamova, Elena N. Parovichnikova, Sergey M. Kulikov, Valeriy G. Savchenko

Allogeneic stem cell transplantation in multiple myeloma: single centre experience of 112 patients

Liisa Volin, Heli Uotinen, Eeva Juvonen, Anne Nihtinen, Tapani Ruutu

Anemias in lymphomas: pathogenesis mechanisms

Anna S. Lyamkina, Tatiana I. Pospelova

Impact of positron emission tomography on primary staging and changing the therapeutic modality in lymphoma patients

Natalia B. Mikhaylova1, Marina S. Tlostanova2, Yulia N. Vinogradova2, Ekaterina I. Ivanova2, Anna V. Kritskaya2, Sergey E. Korolev1, Anna A. Rats1, Alexander A. Pugachev1, Nikolay V. Ilyin2, Boris V. Afanasyev1, Leonid A. Tyutin2

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Epidemiology of bacterial infections and antibiotic resistance in BMT clinic: single center experience

Vladimir N. Vavilov1, Olga B. Ponomarenko1, Marina O. Popova1, Svetlana S. Emelyanova2, Maria Yu. Averjanova1, Oleg V. Goloschapov1, Ludmila S. Zubarovskaya1, Boris V. Afanasyev1

Management of resistant or recurrent CMV infection following allogeneic SCT

Petr Sedlacek1, Ladislav Krol1, Petr Hubacek1, David Boutolleau2, Tomas Kalina1

C-reactive protein (CRP) concentration in patients with acute myeloblastic leukemia (AML) manifestation

Sofia G. Vladimirova, Lyudmila N. Tarassova, Gulzada N. Mustafina, Valentina V. Cherepanova

Invasive fungal disease (IFD) in patients after allogeneic hematopoietic stem cell transplantation: single center experience

Natalia I. Zubarovskaya1, Yulia G. Vasilieva1, Natalia V. Stancheva1, Elena V. Semenova1, Vladimir N. Vavilov1, Svetlana Emelyanova2, Nikolay N. Klimko3, Boris V. Afanasyev1

Hemostasis in patients with acute myeloblastic leukemia (AML) manifestation

Valentina V. Cherepanova, Sofia G. Vladimirova, Lyudmila N. Tarassova, Gulzada N. Mustafina, Natalia N. Silina

Correlations between reactivation of herpesviruses and common complications of allo-HSCT

Olga S. Pankratova, Alexey B. Chukhlovin, Ludmila S. Zubarovskaya, Boris V. Afanasyev

H. Autoimmune diseases and aplastic anemia

Mesenchymal stem cell transplantation in patents with amyotrophic lateral sclerosis and multiple sclerosis

Miroslav M. Odinak1, Gennadiy N. Bisaga1, Andrey V. Novitsky2, Vadim V. Tirenko2, Dmitriy N. Sculyabin1, Dmitriy G. Polyntsev3, Petr V. Krugliakov3, Anna N. Bilibina3, A. Kolchenko3

Outcomes of hematopoietic cell transplantation (SCT) and combined immunosupression (IST) patients with severe aplastic anemia (SAA) admitted with life-threatening infections

Galina A. Novichkova, Mikhail A. Maschan, Lili A. Khachatrian, Dina D. Baidildina, Olga V. Goronkova, Galina G. Solopova, Elena V. Skorobogatova, Irina P. Shipitsina, Alexey A. Maschan

Alternative method for treating acquired aplastic anemia

Yuri I. Yugov, Nina A. Fedorovskaya, Alexander S. Luchinin

The severity of acquired aplastic anemia (AA) and long-term prognosis: lessons from current results of immunosuppressive treatment (IST)

Alexander D. Kulagin1,2, Igor A. Lisukov1,2, Vyacheslav I. Borisov2, Irina V. Kruchkova2, Vera V. Sergeevicheva2, Svetlana A. Sizikova2, Andrey V. Gilevich2, Vladimir S. Kozhevnikov2, Vladimir A. Kozlov2

Immunoablation followed by autologous stem cell transplantation in refractory autoimmune diseases: clinical outcomes and immune reconstitution

Igor A. Lisukov1,2, Vera V. Sergeevicheva2, Svetlana A. Sizikova2, Alexander D. Kulagin1,2, Irina V. Kruchkova2, Andrey V. Gilevich2, Alexey E. Sizikov2, Lyudmila P. Konenkova2, Elena R. Chernykh2, Vladimir S. Kozhevnikov2, Alexander A. Demin1, Vladimir A. Kozlov2

J. Myeloproliferative diseases

Index granularity of neutrophils in peripheral blood as a new application for screening myelodysplastic syndromes using flow cytometry

Ekaterina B. Rusanova1, Konstantin U. Slobodnyuk1, Margarita V.Gorchakova1, Yekaterina E. Zueva1, Ramon Simon-Lopez2

Case report: combined myeloid and lymphoid lineage disorders in patient with chronic eosinophilic leukemia and T-cell lymphoma

Margarita V. Gorchakova, Ekaterina B. Rusanova, Konstantin U. Slobodnyuk, Irina J. Saburova, Elena A. Stadnik, Michail I. Zaraiski, Yekaterina E. Zueva

K. Inherited diseases

Hematopoietic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID): experiences, possibilities, and prospects

Dmitry N. Balashov, Pavel E. Trakhtman, Elena V. Skorobogatova, Yulia V. Skvortsova, Zarema M. Dyshlevaja, Irina P. Shipitsina, Alexey A. Maschan, Alexander G. Rumyantsev

L. Hemoglobinopathies

Outcomes after addition of rabbit-ATG to the standard Bu+CY preparative regimen for allogeneic matched sibling donor (MSD) hematopoietic stem cell transplantation (HSCT) for hemoglobinopathies in children: a single center experience

Sandeep Soni, MD1, Micah Skeens, RN, MSN1, Thomas E. Gross, MD1, Rajinder P. Bajwa, MD1, Kathryn Klopfenstein, MD2,
K. Scott Baker, MD, MS3, Amanda M.Termuhlen, MD1

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Haploidentical SCT as a salvage therapy in hematological malignancies: A single center experience

Olesya V. Paina, Yulia A. Stankevich, Ilya V. Kazantsev, Natalia V. Stancheva, Alla A. Golovacheva, Elena V. Babenko, Alexander L. Alyanskiy, Natalia E. Ivanova, Elena V. Semenova, Petr V. Krugliakov, Dmitriy G. Polyntsev, Liudmila S. Zubarovskaya, Boris V. Afanasyev

Non-T-cell depleted haploidentical HSCT after RIC in pediatric malignancies

Natalia S. Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich

Properties of cord blood stem cells chromatin

Vladimir S. Tolmachev, Elizaveta I. Borovitskaya, Natalia O. Kudrina, Tatiana G. Kravtsova

Prognostic factors in cord blood stem cell quality

Artur A. Isaev, Alexander V. Prihodko, Sergey L. Kiselev, Maria A. Lagarkova, Ivan V. Potapov, Alexey V. Lundup

Developing new immunological compatible transplants derived from umbilical cord

Artur A. Isaev1, Alexander V. Prihodko1, Irina N. Saburina2, Sergey L. Kiselev1, Varvara S. Melihova2, Ivan V. Potapov1, Alexey V. Lundup1

N. Solid tumors

Aggressiveness of tumor resection improves survival in pediatric malignant gliomas

Ludmila I. Shats1, Margarita B. Belogurova1, Olga G. Zheludkova 2

First experience of treatment of high-risk neuroblastoma patients using autologous peripheral blood stem cells transplantation

Igor N. Vyatkin1,2,  Egor V. Shorikov1,2, Andrey A. Igumenshev1,2, Larisa V. Vakhonina1,2, Natalya G. Maisheva1,2, Yulia A. Yakovleva1,2, Grigory A. Tsaur1,2, Alexander M. Popov1,2,3, Elizaveta R. Semenikhina1, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

Treatment of patients with high-risk axial and pelvic Ewing’s sarcoma (ES). A single-institution experience

Roman I. Pimenov1, Igor S. Dolgopolov1, Vasilii K. Boyarshinov1, Natalia S. Subbotina1, Igor V. Visochin1, Stuart Siegiel2, George L. Mentkevich1

High-dose chemotherapy and autologous stem cell transplantation in children with Ewing’s sarcoma/PNET

Ilya V. Kazantsev1, Tatjana V. Youhta2, Elena V. Morozova1, Svetlana A. Safonova2, Yury A. Punanov2, Liudmila S. Zubarovskaya1, Boris V. Afanasyev1

Restoration of ovarian function after cryopreserved ovarian tissue transplantation in women exposed to complex treatment for gynecological cancer: feasibility of this option in pediatric cancer patients

Alla S. Lisyanskya1,2, Natalia I. Tapilskaya1,3, Margarita B. Belogurova3,4, Yulia V. Dinikina3, Ekaterina V. Tsibatova4, Georgy M. Manikhas1

The outcome of treatment in patients with high-risk (HR) retinoblastoma

Igor S. Dolgopolov1, Tatiana L. Ushakova1, Olga N. Gorovtzova1, Alevtina I. Pavlovskaya2, Roman I. Pimenov1, Vasilii K. Boyarshinov1, Natalia S. Subbotina1, Igor V. Glekov1, Vladimir G. Poliakov1, George L. Mentkevich1

O. Donor registry

Influence of differentiated occurrence of HLA genes and haplotypes on defined clinical parameters in patients after allo–HSCT in association with HLA polymorphism

Alexander L. Alyanskiy, Olesya V. Paina, Natalia E. Ivanova, Alla A. Golovacheva, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Raisa Gorbacheva Memorial Lecture

Treatment of acute myeloid leukemia: Present status and new directions III

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Thomas Büchner

Professor of Medicine and Hematology, University of Münster, Germany


Correspondence:
Thomas Büchner MD PhD, Professor of Medicine and Hematology, University of Münster, Germany
E-mail: buechnr@spam is baduni-muenster.de

A. Leukemia treatment

Reduced-toxicity conditioning in allogeneic stem cell transplantation for malignant hematological disorders

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Tapani Ruutu

Division of Hematology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland


Correspondence
Tapani Ruutu, Division of Hematology, Department of Medicine, Helsinki University Central Hospital, PO Box 340, 00029 HUS, Helsinki, Finland
E-Mail:
tapani.ruutu@hus.fi

Allogeneic stem cell transplantation is an efficient treatment of malignant hematological disorders but considerable morbidity and mortality limit its use. To reduce complications, conditioning regimens with reduced extramedullary toxicity compared to standard regimens – but with a strong cytotoxic effect on hematopoietic cells – can be used. The combination of treosulfan (14 g/m2 x 3) and fludarabine (30 mg/m2 x 5) is such a regimen. It has been used in clinical studies as well as in routine practice in patients too fragile to be given conventional conditioning. In a phase II study in MDS, 45 patients were transplanted using this conditioning. The median age was 50 (range 22–63) years. Of the donors 33% were related and 67% unrelated (MUD). The IPSS risk groups were 7% low, 44% Int-1, 31% Int-2, and 18% high. GVHD prophylaxis was CsA+MTX, and ATG in case of MUD. The graft was PBPC in 89% and BM in 11%. The median follow-up was 25 (range 12–41) months. The median times to neutrophil (> 0.5 x 109/l) and platelet (> 20 x 109/l) engraftment were 18 and 17 days. The cumulative incidence (CI) of complete donor chimerism was 73% on day +28 and 93% on day +100. The toxicity was very modest. The CI of gr II–IV aGVHD was 24% and that of gr III–IV 16%. The CI of cGVHD at 2 years was 59% and that of extensive cGVHD 28%. The CI of non–relapse mortality was 9% at 100 days and 17% at 2 years, and that of relapse/progression 16% at 2 years. The Kaplan-Meier estimates of OS and DFS at 2 years were 71% and 67%. These data confirm the favourable safety and efficacy of treosulfan-based conditioning in MDS. Because of the modest toxicity, allogeneic transplantation using treosulfan-based conditioning can be offered to many patients considered too fragile to receive conventional conditioning.

Keywords

allogeneic stem cell transplantation, reduced-toxicity conditioning, myelodysplastic syndrome, treosulfan, fludarabine

A. Leukemia treatment

Prognostic value of chimerism in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT)

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Maria V. Pryanishnikova, Alexandra A. Sipol, Irina S. Solomonova, Ildar M. Barkhatov, Elena V. Semenova, Liudmila S. Zubarovskaya, Boris V. Afanasyev

Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

Introduction

For the last 20 years allo–HSCT has been one of the most effective treatments for different hematological malignancies. Hematopoietic chimerism analysis is an important method of monitoring the post-allo-HSCT outcome.

Patients and methods

In the study we included 142 patients with different hematological malignancies allografted between October 2002 and October 2008. The chimerism status was assessed by analyzing short tandem repeat polymorphisms.

Results

Overall survival in patients with complete donor chimerism on day +28 after allo-HSCT was 55% and 17% in patients with mixed chimerism or an absence of chimerism on the same day (p=0.0124). Similar results were observed by analyzing chimerism on day +60 following allo-HSCT. There were no significant differences in overall survival depending on chimerism value on day +28 and +60 in patients with myeloablative and nonmyeloablative conditioning regimen.

Of the patients with mixed chimerism or an absence of chimerism on day +28 after allo-HSCT, 67% had disease relapse, while 33% remained in remission (p=0.023).

Conclusions

Complete donor chimerism on day +28 and day +60 after allo-HSCT is associated with better prognosis. Mixed chimerism at day +28 after allo-HSCT is associated with an increased risk of relapse.

Keywords

chimerism, allo-HSCT, relapse, outcome

A. Leukemia treatment

Comparing efficacy and toxicity of conditioning regimen “treosulfan+cyclophosphomide” versus “busulfan+cyclophosphomide” in children with hematological malignancies after allogeneic hematopoietic stem cell transplantation

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Natalia V. Stancheva, Elena V. Semenova, Natalia I. Zubarovskaya, Yulia G. Vasilieva, Vladimir V. Vavilоv, Irina A. Mushchitskaya, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia


Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: n.stancheva@mail.ru

Background

Allogeneic hematopoietic stem cell transplant (allo HSCT) is a curative approach for children with hematological malignancies but is associated with high treatment-related morbidity and mortality. A conditioning regimen (CR) with reduced toxicity is a potential option used in these cases. Treosulfan is an alkylating agent with high antileukemic, myeloablative activity and low toxicity. We compared the efficacy and toxicity of two conditioning regimens in allo HSCT: treosulfan (TREO)+cyclophosphamide (CY) (first group) and busulfan (BU) +CY (second group).

Patients and methods

The first group – with a follow-up from Feb. 2004 to Nov. 2007 – had 11 patients (pts): 6 boys and 5 girls; a median age of 9 y.o.; ALL – 10 pts and AML – 1 pt;  CR: TREO 30–42 g/m2 and CY 120mg/kg; HSC donors: 2 matched related donors (MRD) and 9 matched unrelated donor (MUD). The second group – with a follow-up from Nov 2000 to Nov 2006 – had 31 pts: 19 boys and 12 girls; a median age of 12 y.o.; AML – 7 pts, ALL – 24 pts; HSC donors: 11 MRD and 20 MUD; CR: BU 16 mg/kg and CY 120mg/kg. Prophylaxis for aGVHD: CsA+MTX. Unrelated allo-HSCT pts received ATG (“Pfizer”) 60 mg/kg.

Results

First vs second group: engraftment on day +17 vs +21, primary non-engraftment 0% vs 6% (p<0.05), hemorrhagic complication 18 vs 68% (p<0.05). Common toxicity criteria (CTC) II/IV: VOD 0% vs 3% (p<0.05), mucositis 18% vs 68% (p<0.05), neurology symptoms 9% vs 23% (p<0.05), hepatic toxicity 18% vs 26%. AGVHD III/IV 36% vs 20%, relapse 27% vs 29%. Three-year overall survival (OS) 37% vs 40%, respectively.

Conclusion

A treosulfan-based conditioning regimen is well tolerable, safe, efficient, and can be used in heavily-pretreated children with severe complications after previous chemotherapy; though in comparing the efficacy of both regimens, there are no differences. 

Keywords

allo HSCT, toxicity, efficacy, treosulfan, busulfan

A. Leukemia treatment

Survival in adults under 66 years with acute lymphocytic leukemia diagnosed in Norway 2000–2007

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Jon-Magnus Tangen1, Jorg Axel Bohl2, Yngvar Floisand3, Einar Haukaas4, Inger Anne Naess5, Tove Skjelbakken6

1Department of hematology, Ulleval University Hospital, Oslo, Norway; 2Hematology section, Haukeland University Hospital, Bergen, Norway; 3Hematology section, Rikshospitalet, Oslo, Norway; 4Department of onco-hematology, Stavanger University Hospital, Stavanger, Norway; 5Hematology section, St.Olav University Hospital, Trondheim, Norway; 6Hematology section, University Hospital of North Norway, Tromso, Norway

In the period 1.1.2000–31.12.2007 129 patients with ALL under 66 years who received treatment with curative intent were registered in Norway (B-ALL 103 T-ALL 26). The median age was 38,6 years (15,6–65,9 years). The majority (110 patients) were treated with a common national protocol consisting of prednisone, vincristine, asparginase, doxorubicin and cyclophosphamide as induction. Consolidation was daunorubicin, cytarabine and  thioguanine, followed by methotrexate i.v. and mercaptopurine per os. Maintenance was prednisone, vincristine and doxorubicin i.v. followed by methotrexate and mercaptopurine per os, given as part of a 3 months cycle repeatedly for 3 ½ years. CNS prophylaxis consisted of i.t. injections of methotrexate. Patients with high risk ALL were elegible for allogenous stem cell transplantation in CR 1.

In all 82,9% reached CR. For patients < 40 years and for patients ≥ 40 years CR rates were 91,4% and 72,9%, respectively. Nine patients received SCT in CR 1 and 2 patients received SCT in CR 2. Five year overall survival was 48,6% (SD 43,0%–54,2%), for patients < 40 years 54,9% (SD 47,3%–62,5%) and for patients ≥40 years 34,9% (SD 26,1%–43,7%).

Keywords

acute lymphatic leukemia, chemotherapy, stem cell transplantation, remission frequency, overall survival

A. Leukemia treatment

Thymoglobulin and ATGAM for prevention of posttransplant complications after HLA-mismatched allogeneic SCT

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Yury R. Zalyalov, Abdulbasir A. Ganapiev, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia


Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: yz21@mail.ru

Allogeneic stem cell transplantation (allo-SCT) is an important therapeutical approach for patients with different malignant and non–malignant disorders. However, the failure to find a proper donor match for all recipients restricts the procedure in most cases due to higher graft-versus-host disease (GVHD) rates. Currently, reports comparing the clinical efficacy of different antithymocyte globulin (ATG) brands regarding their post–transplant prophylaxis value after allo-SCT from HLA-mismatched donors are limited. In this report we have evaluated the outcomes of 24 patients with different hematological malignancies who underwent unrelated SCTs during a period from 2005 to 2008.

The patients’ median age was 15 years (range, 1–48). All transplants had been performed with unrelated grafts carrying one or more mismatches in HLA-А, -В, -С, -DRB1, -DQB1 loci. The post–transplant prophylactic regimens were based on a combination of either cyclosporine A with methotrexate or CellCept with tacrolimus. All transplanted patients received peripheral blood stem cells as stem cell source. The median CD34+ cell dose was 6.6 х106/kg bw (range, 1–18). Myeloablative and nonmyeloablative preparative conditioning had been used in 37% and 63% of all transplants, respectively. Depending on the brand of ATG being used, all patients were subdivided in two groups. The first group (n=7) received thymoglobulin (cum. dose 7.5 mg/kg), while the second group (n=17) ATGAM (cum. dose 60 mg/kg). Both groups were comparable concerning the sex and AB0–blood group mismatch between donor and recipient. 

Neutrophil engraftment rates were similar in both groups: day +15 (range, 13–25) in the thymoglobulin group and day +16 (range, 11–22) in the ATGAM group. All the patients had been successfully engrafted. The trend towards lower acute GVHD II–IV rate had been more noticeably observed in the thymoglobulin group compared to the ATGAM group (28% vs. 70%; p=0.06). The risk of extensive chronic GVHD was also lower in the thymoglobulin group (28% vs. 78%; p=0.05). Overall survival for 1 year (71% vs. 47%; p=0.6) and 1-year TRM (15% vs. 48%; p=0.2) seemed to be better in patients who received thymoglobulin.

In conclusion, our study suggests that the use of thymoglobulin as a post–transplant prophylaxis could be associated with lower acute and/or chronic GVHD rates as well as with better outcomes in recipients of allo-SCT from mismatched unrelated donors as compared to ATGAM.

Keywords

GVHD, thymoglobulin, ATGAM, allo-SCT

A. Leukemia treatment

Epigenetic therapy in childhood AML (pilot study)

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Alexander V. Popa1, Elena S. Gorohova2, Elena V. Flejshman3, Larisa G. Fechina4, Vladimir V. Lebedev5, Karapet S. Aslanyan6, Evgenia V. Inyushkina2, Svetlana A. Mayakova1, Irina E. Gavrilova1, Olga P. Chlebnikova4, Elmira G. Boichenko7, George L. Mentkevich1

1RAMS N.N. Blokhin RCRC, Pediatric Oncology and Hematology Research Institute, Hematology/Oncology Department, Russia; 2Moscow Region Oncology Hospital, Pediatric Oncology Department, Russia; 3RAMS N.N. Blokhin RCRC, Institute of Cancer Genesis, Laboratory of Cytogenetic, Russia; 4Ekaterinburg Region Pediatric Cancer Research Center, Russia; 5Krasnodar Region Pediatric Hospital, Hematology Department, Russia; 6Rostov-na-Donu Region Pediatric Hospital, Hematology/Oncology Department, Russia;
7St. Petersburg Children’s Hospital N1, Russia

Purpose

We presume that a combination of CT and epigenetic therapy (ET) might improve the results in pediatric AML.Methods: Between October 2006 and August 2008, 42 children with AML were enrolled in the study: 25 boys (59.5%) and 17 girls (40.5%); the mean age was 8.3±0.8 ys. Seven pts (16.7%) were found to be standard risk (SR), 15 (34.7%) intermediate risk (IR), and 20 (48.6%) high risk (HR). Standard cytogenetics was performed in 32 patients.

Treatment was carried out according to the NII-DOG-AML 2007 protocol. CT for SR consisted of induction (AIE), consolidation (Ara-C 75mg/m2/d 4 days N4 every week, daunomycin 30mg/m2/m N4 every week, and 6–MP 60mg/m2/d days 1–28), and two courses HAE. For pts with IR and HR, CT had 4 courses: AIE, HAM in timing as induction, and postinduction (FLAG, HAE). ET consisted of ATRA 25mg/m2/d+valproic acid (VPA) 25mg/kg/d. Children with SR got VPA during maintenance until the seventy-eighth week and ATRA for 14 days every 14 days. Pts with IR and HR got VPA during all treatment for 18 mo and ATRA during the first 45 days and for 14 days with every consecutive CT course.

Results

Response was assessed on day 15. CR (M-1) was observed in all 7 SR pts, in 14 IR pts (93.3%), and in 14 HR pts (70%); PR (M-2) was observed in 1  IR pt (6.7%). All 4 children who did not respond on day 15 were in HR. After induction, CR was seen in 39  pts (92.9%). CR was not observed in 3 pts: 1 died from invasive aspergillosis, and 2 never got into CR. The 29 mo EFS was 57.2±8.3%, median follow up 18.8±1.8 mo, DFS 67.7±8.3%, and median follow up 21.1±1.8 mo. ET did not lead to any severe complications.

Conclusion

Chemotherapy combined with epigenetic treatment lead to complete remission in 39 out of 42 pts with AML without any extra toxicity. These results allow us to go on with this study.

Keywords

childhood acute myeloid leukemia, chemotherapy, epigenetic therapy

A. Leukemia treatment

Efficacy of donor lymphocyte infusions following allogenic stem cell transplantation (allo-HSCT)

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Olga A. Slesarchuk, Elena V. Babenko, Maria A. Estrina, Ilya V. Kazantsev, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia


Correspondence
Olga A. Slesarchuk, Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: cadet2002@mail.ru

To evaluate the efficacy of donor lymphocyte infusion (DLI) after allo-HSCT in patients (pts) with acute leukemia.

Patients and Methods

Data from 29 pts given allo-HSCT from HLA-matched related donors (n=12), unrelated donors (n=11), and from haploidentical family member donors (n=6) were retrospectively analyzed. The conditioning regimen was myeloablative in 13 patients and RIC in 16 patients. Underlying malignant diseases were acute myeloid leukemia (AML, n=14) and acute lymphoblastic leukemia (ALL, n=15). The indications for DLI were minimal residual disease (n=2), mixed chimerism (n=3), preemptive treatment (n=1), graft rejection (n=1), and disease relapse (n=22). Fifteen pts with disease relapse received cytoreductive chemotherapy before DLI and 7 pts received DLI alone. The total number of DLI procedures was 56. Cell dose ranged from 3х104 CD3+cells/kg to 1х108 CD3+cells/kg. Fifteen pts received DLI as a bulk dose regimen, 16 pts received an escalating dose regimen. At the moment of DLI all pts had no signs of aGVHD; however, 5 pts had cGVHD./p>

Results

Complete remission (CR) was obtained in 12 pts (41%): 4 (27%) of 15 pts with ALL and 8 (57%) of 14 pts with AML. GVHD grade I–II appeared in 2 (6.8%) pts, grade III–IV in 3 (10%) pts, and in 2 cases it was fatal. Seven pts relapsed after DLI. The duration of CR after DLI ranged from 2 to 11 months. Five pts (17%) after allo-HSCT and DLI are still alive and in CR. Although response rate was greater in AML then in ALL, the 3yr OS was similar for both groups: 5 (36%) and 6 (44%), respectively.

Conclusions

Using DLI is effective in pts with disease relapse after allo-HSCT. However, it is associated with a high risk of aGVHD. Strategies to use a combination of DLI with target agents for efficacy improvement should be investigated in patients after allo-HSCT.

Keywords

relapse post-HSCT, donor lymphocyte infusion, response, GVHD, cell dose

A. Leukemia treatment

RIC hematopoietic stem cells transplantation in patients with acute leukemia

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Boris V. Afanasyev, Elena V. Semenova

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia (EBMT CIC 725)


Correspondence:
Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E–mail: bmt-director@spmu.rssi.ru

In patients with high-risk acute leukemia (AL) hematopoietic stem cell transplantation (HSCT) is an essential part of the treatment strategy. Current trends in HSCT suggest the use of myeloablative conditioning in leukemia patients. However, myeloablative preparative regimens are associated with a risk of treatment-related mortality. Given that the use of reduced intensity conditioning regimens (RIC) decreases TRM rates but appear to be associated with a higher incidence of relapse than observed with more intensive regimens. Data of 109 HSCT performed in patients with acute leukemia were analyzed with purpose to compare outcomes of RIC HSCTs (55 pts) and meyloablative preparative regimens (54 pts). Disease status at the time of transplantation was 1 or 2 CR.

There was no statistically significant difference in OS between RIC HSCT and standard preparative regimen (OS after RIC was 45% versus 46% after myeloablative conditioning). Moreover, statistically significant improvement of OS was noted in patients with ALL. Seven years OS after RIC HSCT was 58% (n=22) versus 32 % (n=34) after myeloablative conditioning. In pediatric ALL 7 years OS after RIC HSCT was 64% (n=14) versus 43% (n=23) after myeloablative conditioning. However, there was no difference in EFS after HSCT performed in pediatric ALL (37% (n=14) after RIC versus 39% (n=23) after myeloablative conditioning).      
Patients after RIC HSCT showed faster engraftment, therefore had lower rate of bacterial complications and received fewer haemotransfusions.

According to our data RIC HSCT is better tolerated by patients, has more favorable short term outcomes than more intensive regimens. Analyzes of HSCT outcomes in pediatric ALL revealed significant improvement in OS after RIC, nevertheless there was no difference in term of EFS. These results encourage us to continue investigations on RIC use. Our researches focus on post HSCT relapse prophylaxis by means of immunadoptive and targeted therapy.

Keywords: acute leukemia, reduced-intensity conditioning, immunoadoptive therapy

A. Leukemia treatment

Is it necessary to treat patients after syngeneic bone marrow transplantation?

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Lidia S. Lubimova, Larisa A. Kuzmina, Elena N. Parovichnikova, Inna V. Alexeeva, Valeriy G. Savchenko

Hematologic Scientific Centre, Russian Academy of Medical Sciences, Moscow, Russia

Aim

To analyze the results of SyBMT in patients (pts) with leukemia.

Materials and methods

We included 11 pts: 7 CML (6 – CP, 1 – AP) and 4 AL (3 in CR, 1 in relapse): M/F 7/4, average age 24.5 years (13–39). In all cases myeloablative conditioning (BU 16 mg/kg+Cph 120 mg/kg) was used. Morphological, cytogenetic and molecular (FISH, RT PCR) investigations were performed.

Results

Of the 7 CML pts, 4 are alive: 2 have been in complete molecular remission (CMR) for 143 and 206 mo, while a third has been in complete cytogenetic remission (CCR) for 17 mo. During the first 3 mo after BMT, the third patient had a very weak expression of the BCR/ABL gene according to an RT PCR. In 6 mo he was negative according to an RT PCR and in 13 and 16 mo BCR/ABL was determined again (0.05–0.04%) accordingly. One pt with AP CML has been in a chronic phase for 136 mo, while 3 pts relapsed and died. Of the 4 pts with AL, 3 are alive – all 3 were in CR before BMT. One pt remained in CMR and 2 pts achieved only CCR, but the persistence of inv (16) – which was detected before BMTaccording to an RT PCR – was revealed. Therefore, of the initial 11 pts, 7 (64%) are alive: 6 of who are in CCR – of which 3 are in CMR. At the same time, 3 pts with CCR – 1 CML and 2 AL – have MRD according to an RT PCR; FISH was negative in all cases.

Conclusion

After SyBMT, long survival of pts is possible in the majority of cases where CCR has been achieved. The RT PCR method was the most informative for the diagnosis of MRD, which was recognized in 3 of the 6 pts. We previously published our first encouraging results of using immunomodulatory therapy after autological BMT: the relapse rate decreased twice in treated pts. At that time a question came up: Is it necessary to treat patients after SyBMT, especially those with MRD? Recently, the use of immunomodulatory therapy after SyBMT was initiated in our clinic: IFN-α in CML and IFN-α + ATRA or IL2 in acute AL. The results will be evaluated in the future.

Keywords

syngeneic bone marrow transplantation, graft-versus-leukemia effects, GVL, acute leukemia, AL, chronic myeloid leukemia, CML, minimal residual disease, MRD

A. Leukemia treatment

Results of treatment of relapsed promyelocytic leukemia in children using chemotherapy and arsenic trioxide (ATO) followed by autologous SCT (ASCT)

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Dina D. Baidildina, Elena V. Samochatova, Michail A. Maschan, Alexey A. Maschan

On behalf of the Russian-Byelorussian Pediatric APL study group

Correspondence:
Elena V. Samochatova, Research Institute for Pediatric Hematology, 117, Leninsky prosp, Moscow, 105062, Russia
E-mail: samochatova@spam is badniidg.ru

The Russian multicenter APL-2003 protocol for pediatric APL demonstrated a non–inferior outcome compared with the APL-93/98 studies despite a reduction of ATRA to 25 mg/m2 and cumulative anthracycline dose to 405 mg/m2: at a median follow up of 35 mo the EFS and OS were 0.79±0.6 and 0.93±0.3. Seven relapses (11.9%) occurred out of 61 patients (pts) at a median of 21 (4–35) mo. Second remission was induced with diverse therapy (Table 1) and consolidated with 14 ATO at 0.15 mg/kg per day;
in 1 pt gemtuzumab ozogamicin (GO) at 6 mg/m2 was added to ATO. All pts achieved 2nd hematological remission and PML/RARα negativity in bone marrow either after induction (3 pts) or after consolidation (4 pts). HDAraC + G–CSF were used for additional “in-vivo purging” and PBSC mobilization. Harvesting was successful in all pts: а median of CD34+ dose 17 (8–40) х 106/kg was achieved after single apheresis. In all cases, apheresis product proved to be PML/RARα negative. AHSCT was performed in 6 pts after conditioning with HDAraC + Mel180 mg/m2 in 4 pts, Bu12mg/kg + Mel 140 mg/m2 in 1 pt and Treosulfan 42 mg/m2 + Mel140 mg/m2 in 1 pt. All pts engrafted at a median of 16 (12–25) d with minimal transplant-related toxicity. Three pts received GO on day +100 after ASCT with minimal toxicity. Two pts with skin involvement received complementary electron beam skin irradiation. At a median of 26 mo 6 pts continued in molecular remission and 1 pt experienced 2nd relapse. We conclude that children with relapsed APL can be treated effectively with chemotherapy, ATO, and ASCT.

Keywords: promylocytic leukemia, arsenic trioxide, autologous hematopoietic stem cell transplantation

A. Leukemia treatment

Treatment results of childhood acute lymphoblastic leukemia using the COALL-Saint-Petersburg-92 protocol after a 10-year observation period

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Elmira G. Boichenko1, Margarita B. Belogurova2, Gritta Janka3, Margarita S. Livshits1, Eleonora M. Petrova1, Marina B. Ivanovskaya1, Irina A. Garbusova1, Galyna G. Radulesku2, Tatjana D. Victorovich2, Emylia D. Tchavpetsova2, Ludmila I. Shats2 

1City Children's Hospital 1, St. Petersburg, Russia; 2City Hospital 31, St. Petersburg, Russia; 3Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Germany

Between January 1999 and August 2008, a total of 272 newly diagnosed patients (pts) up to 18 years of age with ALL were treated according to the COALL-Saint-Petersburg-92 protocol (COALL-S-Pb-92). Since August 2008, the pediatric oncohemathological clinics of St. Petersburg have been affiliated with the all-Russian study Moscow-Berlin 2008.

Methods

Protocol COALL-S-Pb-92 is a modification of the German protocol COALL-92. The intensive phase of treatment lasted  5.5 months in the low risk (LR) group  and 8 months in the high risk (HR) group and consisted of 4 parts: induction, consolidation, CNS-treatment and reinduction. It was followed by maintenance treatment until 2 years from the date of diagnosis. Treatment of presymptomatic CNS disease consisted of i.th. MTX; cranial irradiation (12 Gy) was given additionally  to HR pts with T-ALL and/or primary hyperleukocytosis.

Pts were stratified into LR (n=129, 47%) or HR (n=143, 53%) groups. The criteria for HR were: initial white blood count ≥ 25000/l, primary CNS and/or mediastinal involvement, Т-cell and pre-pre-B-cell immunophenotype, an age ≥ 10 years, Ph–chromosome positivity, and failure to achieve remission at day 28 from the beginning of treatment.

Results

266 pts (98%) achieved complete remission. 6 HR pts died during induction (hemorrhagic and infectious complications, progression of leukemia). There was only the one late responder, no non-responders. 18 pts died of infectious complications while in remission. 47 (17.7%) patients relapsed: 12.4% in the LR group and 22.6% in the HR group. Non-lethal complications – like mucositis, hepatotoxicity, hemorrhagic and infectious complications – were more frequent and severe in HR–group pts. After an observation time of 10 years, the estimate for EFS of all 272 valuable patients is 64.8±3.4% (LR 74.7±4.4%, HR 55.9±4.9%), the estimate of probability of RFS is 75.9±3.4% (LR 82.4±4.1%, HR 69.6±4.8%) and probability of overall survival is 76.0±3.1% (LR 87.5±3.5%, HR 65.3±5.0%).

Conclusions

Treatment results based on the COALL-92 protocol have demonstrated an obvious improvement in comparison with previous results of leukemia chemotherapy in children in St. Petersburg. We are going to continue to follow up our COALL patients in order to be able to compare treatment results in different age and risk groups as well as long-term survival and treatment sequela with different treatment strategies.

Keywords

acute lymphoblastic leukemia, children, intensive chemotherapy

A. Leukemia treatment

Consequences of ABO incompatibility in allogeneic hematopoietic stem cell transplantation (allo-HSCT)

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Maria A. Estrina, Мurat O. Yagmourov, Dmitriy E. Pevtsov, Еvgenia A. Kochina, Natalia E. Ivanova, Alla A. Golovacheva, Alexander L. Alyanskiy, Ludmila S. Zoubarovskaya, Abdulbasir A. Ganapiev, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

Background

ABO incompatibility between donor and recipient is currently considered one of the risk factors for immune-mediated complications, which can influence the outcome of allogeneic hematopoietic stem cell transplantation (allo-SCT).

Patients and methods

We analyzed 140 consecutive allo-HSCT recipients. 124 patients had HLA-compatible donors (39 related and 85 unrelated) and 16 patients had haploidentical donors. Fifty-nine of the donor–recipient pairs were ABO-identical, while 34 had minor incompatibility, 35 major incompatibility, and 12 bidirectional incompatibility. Seventy-eight patients received bone marrow (BM), 46 peripheral blood stem cells (PBSC), and 16 stem cells from both sources. Conventional myeloablative conditioning was used in 64 patients and non–myeloablative regimens in 78 patients. In both groups the same regimen of graft–versus–host disease (GVHD) prophylaxis was administered. In major ABO incompatibility SCT donor stem cells were depleted of RBC (6% hydroxyethyl starch sedimentation), in cases of minor incompatibility donor incompatible plasma was removed, and in cases of bidirectional incompatibility both methods were used.

Results

In all the study groups, we observed no cases of acute hemolysis after PBSC transfusion and only 2 cases after BM transfusion. Engraftment of leukocytes, neutrophils, and platelets was not altered in any of the groups (р=0.45).  Delayed RBC engraftment was more frequent in patients with ABO-incompatible SCTs (р=0.04). There were 4 cases of delayed hemolysis. Incidence and severity of GVHD was higher in the ABO-incompatible allo-SCT group (р=0.005).

Conclusions

ABO incompatibility between donor and recipient can cause acute and delayed hemolysis and it is a risk factor in the development of GVHD. It can delay RBC engraftment, but has no influence on other cell lines. Adequate prophylactic measures allow us to keep the incidence of acute hemolysis low.

Keywords

ABO incompatibility, PBSC, GVHD, engraftment

A. Leukemia treatment

Optimal management of adolescents with acute lymphoblastic leukaemia

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Giorgio Dini, Stefano Giardino, Simone Dini

Dept. of Pediatric Hematology and Oncology, IRCCS “G.Gaslini”, Genoa, Italy

During the last 25 years, improvement in survival for young children with acute lymphoblastic leukaemia (ALL) has been striking: children aged 1 to 9 years have the best outcome. However, children and adolescents aged 10 to 20 years have a markedly worse outcome, which is associated – in part – with genetic abnormalities, a lower incidence of favourable genetic abnormalities such as TEL/AML1 and hyperdiploidy, and a higher incidence of T-cell leukaemia.

Adolescents are less likely to have access to healthcare, are more likely to see providers who are not part of research institutions, and are less likely to be referred to or to join clinical trials, all of which may contribute to worse outcomes. Adolescents are treated by both adult and pediatric teams, following either adult or pediatric protocols. The type of team by which adolescents are treated (pediatric teams, or “combined” teams) may also impact the outcome. An unpublished survey by the EBMT (Dini) showed that 67% of patients aged 14-18 years old who received allogeneic hematopoietic stem cell transplantation (HSCT) for 2nd CR ALL from 1996 to 2005 that was reported to the EBMT registry had been treated by pediatric teams only, while the remaining 33% had been treated by adult teams [1].

Recent European studies have shown better outcomes in adolescents treated with pediatric protocols as compared to those treated with adult ones [2-5].
Adolescents should be referred to research treatment teams that have experience in the management of peediatric ALL, and they should be enrolled in international cooperative studies.  Molecular, genetic, and proteomic evaluation may cast further light on the causes of the rather striking decrease in survival that is seen as the patient progresses from childhood to adolescence.

Keywords

acute lymphoblastic leukemia, adolescents, pediatric ALL protocols, allogeneic hematopoietic stem cell transplantation, allo-HSCT

B. Leukemia research

Comparative analysis of acute myeloid leukemia treatment results in the Sverdlovsk Regional Clinical Hospital №1

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Alexander V. Vinogradov, Tatiana S. Konstantinova, Alexander F. Tomilov, Vladimir A. Shalaev, Elena V. Chepuryaeva, Julia V. Sveshnikova, Leonid N. Sharov, Natalia V. Vinogradova

Ural State Medical Academy, Sverdlovsk Regional Clinical Hospital N1, Ekaterinburg, Russia

Correspondence
Ural State Medical Academy, Sverdlovsk Regional Clinical Hospital N1, Ekaterinburg, Russia, Gurzufskaya Str., 11/1-4, 620086 Ekaterinburg, Russia, Phone: +7 (343) 2683648, Fax: +7 (343) 2120146
E-mail: vinogradov-av@spam is badyandex.ru

Aim

To estimate the prognostic significance of cytogenetic abnormalities in acute myeloid leukemia (AML) patients—who were treated according to the following chemotherapy protocols: RHSC AML 01.99, 01.01, and >60 years old.

Methods

A total of 125 AML pts were treated with RHSC AML 01.99, 01.01 and >60 y.o. protocols from 1999 to 2008. The distribution of pts by protocol was as follows: AML 01.99 – 16, AML 01.01 – 84, and AML >60 y.o. – 25. The cytogenetic variants of AML according to the protocols are presented in Table 1.

Results

The results of the comparative analysis of treatment results are presented in Table 2.

The results of the comparative analysis of treatment according to cytogenetics are presented in Table 3.

The comparative analysis of overall survival (OS) and relapse-free survival (RFS) of AML pts according to cytogenetics is presented in Figures 1 and 2.

Thus, in AML pts treated by RHSC AML 01.99, 01.01, and >60 y.o. protocols, only inv(16) has a favorable prognostic significance. All other cytogenetic subtypes were associated with unfavorable prognosis.

Keywords

acute myeloid leukemia, treatment, cytogenetic abnormalities

B. Leukemia research

Gene therapy of hematopoietic stem cells

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Gerard Wagemaker

Erasmus University Medical Center, Dept. Hematology, Rotterdam, The Netherlands

Correspondence
Gerard Wagemaker, Erasmus University Medical Center, Dept. Hematology, PO Box 2040, 3000 CA Rotterdam, The Netherlands
E-mail: editors@ctt-journal.com

An estimated 24 million people in the European Union alone are affected by an inherited disorder based on a genetic defect. In humans, approximately 7,000 inherited diseases have been identified; in around 1/3 the genetic defect has been mapped and the function of the affected gene elucidated. Human suffering as well as health care costs are immense, and therefore the development of future curative therapy is a humanitarian, medical, societal, and economic necessity.

In patient numbers, the inherited diseases are most frequent in disorders of the blood cell producing system. This provides the rationale for a focus on hematopoietic stem cells as a prime target of gene therapy, solidly rooted in 40 years of experience with the clinical application of HSC transplantation for a variety of acquired and inherited disorders. In those disorders based on a monogenic defect, ex vivo gene correction of the patient’s hematopoietic stem cells and transplantation of those cells is a realistic treatment concept. Due to the specific properties of the progeny of hematopoietic stem cells, curative approaches for disorders in other organ systems also turn out to be accessible to hematopoietic stem cell gene therapy, including disorders that affect the brain. In this overview the results of a European consortium’s (www.gene-therapy.eu) translational research project – in which leading centers collaborated to prepare for the effective and safe gene therapy of selected example diseases that included severe combined immune deficiencies, red cell disorders, and lysosomal enzyme deficiencies – are presented. The research also required an in-depth analysis of the interaction of gammaretroviral and lentiviral vectors with stem cell genome expression levels, revealing new insights in hematopoietic stem cell genomics.

Keywords

inherited diseases, hematopoietic stem cell transplantation, HSCT, gene therapy

B. Leukemia research

Determination of FLT3-ITD and D835Y mutations as a factor of prognosis in immunophenotypically verified diagnosis of patients with acute myeloid leukemia in Saint Petersburg

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Konstantin U. Slobodnyuk, Irina Y. Saburova, Margarita V. Gorchakova, Ekaterina B. Rusanova, Antonina V. Kurtova, Mikhail I. Zaraiski, Yekaterina E. Zueva

Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Purpose

Multicolor flow cytometry has been the “gold standard” in diagnostics of Acute Myeloid Leukemia (AML) since 1995. Different types of AML are associated with different cytogenetic abnormalities, but 40–60% of patients with AML are not determined. FLT3-ITD and D835Y mutations – which are associated with an extremely unfavorable prognosis of AML – have been identified in 20–30% of AML patients with normal karyotypes. The aim was to study a diagnostic significance of detecting FLT3-ITD and D835Y mutations in patients with verified diagnoses of AML.

Materials

The study included 33 patients of St. Petersburg. To verify the diagnosis of AML, we used the method of multicolor flow cytometry (Cytomics FC500, Beckman Coulter), as well as morphology, cytochemistry, and clinics. For the molecular genetic studies of FLT3-ITD and D835Y mutations, we used the standard method of PCR analysis for detection with a double primer scheme of results in a polyacrylamide gel.

Results

Cytogenetic abnormalities were identified among 11 AML patients (33%). In 8 patients (24%) diagnosed with AML, the FLT3-ITD mutation was detected, including the cases with AML M0 (n=1), AML M0-M1 (n=1), AML M2 (n=3), AML M3 (n=1), and AML M4 (n=2). In 25 patients with clinical, immunophenotypic, and morphological verified diagnosis, the mutation is not defined. In only 3 patients (9%) with AML was the D835Y mutation detected, but without the FLT3-ITD mutation including cases with AML M2 (n=1), AML M3 (n=1), AML M4 (n=1).

Summary

Investigation of FLT3-ITD and D835Y mutations may be recommended for inclusion to the standard of diagnosis of acute myeloid leukemia.

Keywords

acute myeloid leukemia, FLT3-ITD, D835Y, mutation, flow cytometry, prognosis

B. Leukemia research

Expression of angiotensin-converting enzymes (ACE, CD143) and shaperons (BiPs, calnexin, calreticulin) by leukemic dendritic cells (LDCs) in acute myeloid leukemia (AML) patients

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Irina V. Galtseva1, Lev M. Pashin1, Elena N. Parovichnikova1, Sergei M. Danilov2, Andrei B. Sudarikov1, Ivan A. Vorobiev1, Valeriy G. Savchenko1

1National Research Center for Hematology, Moscow, Russia; 2University of Illinois, Chicago, USA

Background

DCs play a key role in the immune response. LDCs and DCs are characterized by high levels of co-stimulatory molecules. The difference between LDCs and DCs is a high expression of surface ACEs on DCs.

Aim

To confirm the block of ACE transport to surface LDCs, controlled by shaperons, we quantified the intracellular and surface ACE, BiP, and mRNA expression levels of ACE and BiP, calnexin, calreticulin in DC and LDC were measured.

Methods

Blood samples were collected from 12 AML patients and 10 donors. Mononuclear cells were differentiated into DCs by culturing with 180 n?g/ml calcium ionophore for 4 days at 37°C and 33°C (activated shaperons). Stained surfaces, intracellular ACEs (2 mAbs-clones 1D8, 9B9) and BiPs (mAb KDEL) were analyzed by flow cytometry. MRNA expression levels of ACEs, BiPs, calreticulin, and calnexin were evaluated with RT PCR.

Results

The surface ACEs on LDCs at 37°C was 3±2% (mAb9B9), 2% (mAb1D8), and BiPs 6%, at 33°C 45±10%, 57±8%, and 63±7%, respectively; on DCs at 37°C it was 46±9%,5±2%,  and BiPs 36±4%, at 33°C 34±3%, 6±2%, 21±4%, respectively.

The intracellular ACEs in LDCs at 37°C was 71±9%, 52±8%, and BiPs 92±7%, at 33°C 6±2%, 5±2%, 52±6%, respectively; in DCs at 37°C was 50±10%, 27±7%, BiPs 69±7%, at 33°C 40±10%, 33±7%, 88%, respectively.

Gene expression was measured by RT PCR using the TaqMan procedure and calculated in relative units using GAPDH expression for normalization. The expression of ACEs increased in 4 out of 7cases in DCs at 33°C compared to DCs at 37°C, and in 8/9 cases of LDCs at 33°C compared to LDCs at 37°C. The expression of BiPs increased in 6/8 cases in DCs, and in 3/6 cases of LDCs. For calnexin, appropriate measures showed 6/8 for DCs, 3/6 for LDCs; for calreticulin 2/8 for DCs, and 3/6 for LDCs, respectively.

Conclusion

The data demonstrates the block of ACE transport to the surface of LDCs at 37°C and the different reactions of LDCs and DCs in stress conditions (33°C increased surface ACEs, BiPs, and ACE gene expression by LDCs).

Keywords

acute myeloblastic leukemia, AML, dendritic cells

B. Leukemia research

Posttransplantation leukemia relapses: the role of chromosome changes

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Nikolay N. Mamaev, Tatyana L. Gindina, Ildar M. Barkhatov, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Nikolay Mamaev, Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: nikmamaev@spam is badrambler.ru

Aim

To study the cytogenetic profile of leukemia cells in patients with relapses after hematopoietic stem cell transplantation (HSCT).

Patients and methods

The group enrolled 5 patients with acute myeloid (3) and acute lymphoblastic (2) leukemias. The standard G-banding cytogenetic technique supplemented by rtPCR was used.

Results

Modal classes of chromosomes ranged from 44 up to 50 (Table). Four patients were associated with multiple chromosome changes and 1 with translocation (9;11)(p22;q23). Of the 5 tested patients, 3 (60%) contained the whole or partial loss of chromosome number 9. Several patients lost chromosomes 13 and 17, while they gained chromosome number 8. The following structural chromosome rearrangements are important: a) translocations (1;6)(p10;q10), (1;18)(p10;p10), (4;12)(q25;q13), and (11;14)(p13;q11); and b) deletions of 7q22 and 3p. 

Conclusion

Multiple chromosome changes and the loss of 9/9p- are the most common findings in patients with posttransplantation relapse.

Keywords

leukemia, hematopoietic stem cell transplantation, relapse, chromosome changes

C. Hematopoietic stem cell sources

Features of a stem cell’s cryopreservation

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Alexander B. Smolyaninov, Ksenia V. Korovina, Shirzod F. Adylov, Elena A. Kotelevskaya

Stem Cell Bank Pokrovski, St. Petersburg, Russia

Correspondence
Alexander B. Smolyaninov, Stem Cell Bank Pokrovski, St. Petersburg, 199106, Russia, Bolshoi Prospekt V.O., 85
Phone: +7 (812) 908-77-27
E-mail: stemcellbank@inbox.ru

Stem cells are the unique source for the renewal of all kinds of tissue. The umbilical cord blood (UCB) is well known to be a rich source of stem cells with practical and ethical advantages, but it can be received only at childbirth. That is what raised the question about the preservation of stem cells from cord blood.

To solve this problem, cryopreservation in liquid nitrogen is used. The storage of biopreparations – after freezing – is performed in cryobanks in liquid nitrogen with cryoprotectant. DMSO is used as a cryoprotectant to prevent cell destruction at low temperatures. The received cells are poured  into cryobags (20 ml) or cryoampules (4.5 ml), frozen with DMSO, and placed in liquid nitrogen (-196°С) for long-term storage. The process of cryoconservation consists of three stages: cooling from +20ºС to +1ºС using Coolmix (Biosafe, Switzerland), then freezing from +1ºС to -100ºС using Planer (UK), and finally transferring into storage with a temperature of -196ºС.

For optimal freezing conditions, Planer controller freezer (Planer, UK) was used. The freezing program has been optimized for stem cells and the speed of freezing is from 1 to 3 K per minute, which  avoids cell damage.

To avoid cross contamination, all samples of cord blood are placed in a specialized Dewar quarantine vessel until the results of an analysis on infections are received. Then samples are removed to “clear” Dewar vessels. There are currently more than 150 samples in the cryobank at the Stem Cell Bank Pokrovski.

According our results, after unfreezing the cell decreases only by 21.35±2.64% (p<0,001). The duration of UCB samples stored in a frozen state was 1 day and 1, 6, and 12 months; there was no difference in viability that depended on the storage duration. Therefore, the selected method of cryoconservation is rather effective and suitable for stem cells from umbilical cord blood. As a result, at the Stem Cell Bank Pokrovski the method of cord blood cryopreservation was optimized and adopted successfully.

Keywords

cryopreservation, stem cell, controller freezer, viability, crystallization point

C. Hematopoietic stem cell sources

Virus markers antibodies detection in umbilical cord blood

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Olga V. Supilnikova, Irina I. Maslenikova, Alexander B. Smolyaninov

Stem Cells Bank Pokrovski, Saint Petersburg, Russia

In accordance with the Ministry of Health’s Order № 325 – “About the developing of cells technologies,” 25 June 2003 – 120 samples of cord blood were tested at the Stem Cell Bank Pokrovski for the presence of infectious agents (anti-HIV 1 and 2, p24 HIV1, anti-HTLV I and II, anti-НВcorAg, HBsAg, anti-HCV, anti-CMV, anti-Toxoplasma gondii, and RW) using the ELISA method.

We detected none of the listed markers in 29.2% of samples. НВcorAg antibodies were detected in 6.6% of samples, HCV antibodies in 1.6%, CMV antibodies in 62.5%, and T. gondii antibodies in 20% of samples. The absence of the positive detection of HIV antibodies, р24 HIV1 antigen, and HbsAg depends on the isolation of the epidemic cause of mothers infected with HIV and HBV.

We also detected  T. pallidum antibodies in 2 cases with negative RW using the ID-PaGIA method. Those patients had a history of treating the disease caused by T. pallidum. HTLV I/II antibodies were not detected in the explored samples. Simultaneous detection of 2, rarer to 3 causative agents have been observed. Simultaneous presence of antibodies have been detected to CMV – 5 cases of 120, anti-НВcorAg and anti-HCV – 1 case of 120. However, T. gondii antibodies were not detected together with НВcorAg, HCV, and T. pallidum antibodies.

Thus, considering that cord blood derived from stem cells can be placed in long-term storage on condition of negative results of testing for the presence of anti-HIV 1 and 2, anti-HTLV I and II, anti-НВcorAg, HbsAg, and anti-HCV, 110 samples from the explored samples were deemed fit for storage and usage.

Keywords

hematopoietic stem cells, umbilical cord blood

C. Hematopoietic stem cell sources

First experience of CD34+ cell selection to high-risk neuroblastoma patients

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Yulia A. Yakovleva1,2, Grigory A. Tsaur1,2, Alexander M. Popov1,2,3, Tatyana Yu. Verzhbitskaya1,2, Igor N. Vyatkin1,2, Andrey A. Igumenshev1,2, Natalya G. Maisheva1,2, Anton Yu. Zadoya1,2, Egor V. Shorikov1,2, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

1Regional Children Hospital 1, Ekaterinburg, Russia; 2Research Institute of Medical Cell Technologies, Ekaterinburg, Russia;
3Ural State Medical Academy, Ekaterinburg, Russia

Objective

To estimate the applicability of CD34+ cell selections using CliniMACS plus equipment (Milteni Biotec).

Methods

From June 2007 to April 2008 eight selections were performed on 7 children with high-risk neuroblastoma (stage 4). In 4 patients peripheral blood stem cells (PBSC) were mobilized with G-CSF (10 µg/kg/day) for 6 days and leukapheresis was performed on days 5 and 6 of G-CSF administration by the “Cobe Spectra” cell separator (Gambro). In 2 patients bone marrow (BM) was used for the CD34+ cell selection. In 1 patient PBSC and BM were used consecutively. The total number of nucleated cells was measured by Sysmex KX 21 (Roche) before and after CD34+ cell selections. The number and viability of cells were assessed by the “FACSCanto II” flow cytometer (BD, USA).

Results

Before CD34+ selection, the median percentage of CD 34+ cells in harvested cells was 1.64% (0.23–5.5). The median dose of CD34+ cells per 1 kg of patients body weight was 10.64*106 (1.61–42.37*106). After CD34+ selection, the median of selected CD34+ cells per 1 kg of weight was 4.93*106 (1.42–14.5*106) with a median purity of 96.74% (88.2–97.7%).
The viability of isolated CD34+ cells was 91.8% (71.6–99.6%). The median time to achievement of 500/µl ANC was 34 days (28–40).

Conclusion

Immunomagnetic selection of CD34+ cells by CliniMACS allows the obtainment of viable and highly purified autologous CD34+ cells. CD34+ selection leads to delayed hematopoietic recovery in our patients.

Keywords

high-risk neuroblastoma, children, CD34+ cell selection

C. Hematopoietic stem cell sources

Effectiveness of hematopoietic progenitor cell separation with the help of the Sepax S100 cell separator

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Alexander B. Smolyaninov, Dmitry A. Ivolgin, Shirzod F. Adylov, Elena A. Kotelevskaya

Stem Cells Bank Pokrovski, Bolshoi Prospekt V.O., Saint Petersburg, Russia

Correspondence
Alexander B. Smolyaninov, Stem Cells Bank Pokrovski, Bolshoi Prospekt V.O., 85, 199106, Saint Petersburg, Russia,
Phone: +7 (812) 908-77-27
E-mail: stemcellbank@inbox.ru

Cord blood processing for hematopoietic progenitor cell (HPC) separation had a quantum jump through the last few years. Only 5 years ago the cord blood bank in Singapore, which separated buffy coat practically by hand, had been accredited by the AABB. But now there is equipment that simplifies this step of cord blood processing. Confirmation of the effectiveness of the method of HPC separation using the Sepax S100 cell separator is our study purpose.

Using the established Stem Cells Bank Pokrovski procedure, we added the HES solution to the cord blood harvest and then placed it into the Sepax S100. We tested the leukoconcentrate after processing and before cryopreservation.

We processed 135 samples of cord blood using this technique. The average volume of processed cord blood was 89.45±23.52 ml. The average volume of leukocytal fraction was 21.35±5.35 ml (we used different protocols according to the cord blood harvest volume). The total amount of leucocytes in the concentrate was equal to 34.6±4.87*106cell/ml and the CD34+ cell fraction was 0.072±0.014 cell/ml – 0.2% of mononuclear cells on average. The vitality of HPC amounted to 91.2±8.4% on average. The tests had been carried out using a Beckman Coulter flow cytometer. We also found bacterial contamination in 4 samples (2.9%).

Our conclusion is that the effectiveness of cord blood processing using the Sepax S100 is equal to and – in some cases – better than other popular methods of cell separation. The comfort, processability, and accordance with international standards of that technique can significantly simplify cord blood processing. The Sepax S100 is capable of making this processing more available for our developing branch of stem cell banking and cell therapy.

Keywords

cord blood, hematopoietic progenitor cells, cell separation

C. Hematopoietic stem cell sources

Low and high-resolution HLA typing of cord blood samples of register of donors in St. Petersburg

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Elena A. Kotelevskaya, Svetlana A. Smirnova, Alexander B. Smolyaninov

Stem Cell Bank Pokrovski, St. Petersburg, Russia

Umbilical cord blood (CB) contains hematopoietic stem cells (HSC) and can be used as an alternative to bone marrow transplantation in certain cases. Currently, the first register of cord blood donors is be created by the Stem Cell Bank Pokrovski. It aims to resolve the problem of deficiency the of HSC units in the country. The most important factor of effective transplantation is the degree of HLA matching. The target of this work is to HLA type each cord blood unit by low-resolution and then transition to high-resolution typing by sequence based typing (SBT).

During the work, 20 samples of cord blood were analyzing by polymerase chain reaction (PCR) with sequence specific primers (PCR-SSP). DNA was isolated from 0.7 ml of the entire quantity of blood by the PROTRANS DNA Box 500 (PROTRANS, Germany) DNA extraction column. HLA-A, HLA-B, and HLA-DRB1 loci were analyzed using the PROTRANS HLA-A*, -B*,
-DRB1* (PROTRANS, Germany) Cyclerplate System according to the manufacturer’s instructions. HLA-DRB1 high-resolution genotyping of 5 units was performed by the PROTRANS S4 system (PROTRANS, Germany), specially designed for the Beckman Coulter SEQ 8000 Genetic Analyzer (Beckman Coulter, USA). The program software Sequence Pilot for sequence result analyzing was also provided by PROTRANS.

The most frequently identified alleles were: HLA-A*02 (25%), *01 (20%), *24 (17.5%); HLA-B*08 (17.5%), *18 (12.5%), *35 (12.5%); HLA-DRB1*15 (17.5%), *07 (15%), *03 (12.5%).

The results of low-resolution HLA typing could be applied to the primary selection of donors when an HSC transplant HSC is required. Also they significantly simplify the high-resolution typing operation. The advantages of a national HLA-identified cord blood register in Russia are accessibility and lower costs of samples. But the major positive is the increased likelihood of HLA-matching in the same population.

Keywords

hematopoietic stem cells, umbilical cord blood, register of cord blood donors, transplantation, HLA typing

C. Hematopoietic stem cell sources

Importance of CFU-GM number for prediction of hematological reconstitution after low CD34+ cell dose autotransplantation in children

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Yanina I. Isaikina, Valery I. Martinevsky, Nina V. Minakovskaya, Yury S. Strongin, Olga V. Aleinikova

Belarusian Research Centre for Pediatric Oncology and Hematology, Minsk, Belarus

Correspondence
Isaikina Yanina I., Belarusian Research Center for Pediatric Oncology and Hematology, Pos. Lesnoe-2, 223040, Minsk reg., Belarus, Phone: +375-17-265-40-89, Fax: +375-17-265-42-22
E–mail: yaninai@mail.ru

Objective

The insufficiency of CD34+ cell numbers at the time of peripheral blood stem cell (PBSC) collection is frequently observed in children after multiple blocks of chemotherapy.

Aim

To detect the additional parameter of transplant quality for the prediction of successful hematological reconstitution after auto-PBSC transplantation with low doses of CD34+ cells in graft.

Methods

Forty-one children with malignances – who had received CD34+cells/kg <2х106 – were included in our study evaluating the correlations between CD34+cell/kg and CFU-GM/kg at different dose levels ≥2х105/kg and <2х105/kg and the time of hematopoietic recovery. A Mann-Whitney U test and multivariate correlation method were used to evaluate the importance of each graft parameter for the prediction of engraftment.

Results

There was no correlation between CD34+ cell dose and the time of engraftment in these pts. A high level of correlation was detected between the number of CFU-GM/kg infused and the time of  neutrophil (r=-0.67, p<0.05), platelet (r=-0.3, p<0.05), and/or first reticulocyte ›2‰ (r=-0.66, p<0.05) reconstitution. Infusion of CFU-GM/kg ≥2х105/kg (n=26) resulted in rapid short–term neutrophil >0.5x109/l and platelet >20x109/l recovery (10 days (5–13) and 22 days (8–57), respectively) compared with CFU-GM/kg <2х105/kg (n=15) (13 days (11–21) and 25 days (10–95), respectively) (p<0.05).

Long-term hematopoietic recovery platelet counts >50x109/l were 31 days (range: 9–81) in CFU–GM/kg ≥2х105/kg infusion.

We conclude that the number of CFU-GM/kg was the most important parameter for predicting the auto-PBSC with CD34+ cell dose <2x106/kg engraftment. The minimal recommended number of CFU-GM is 2x105/kg for the efficacy of hematopoietic recovery after auto-PBSCT.

Keywords

autotransplantation, CFU-GM, insufficiency of CD34+ cells, engraftment

C. Hematopoietic stem cell sources

Selection of optimal cryopreservation strategy for hematopoietic stem cells

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Leonid I. Savelyev1,2,3, Yulia A. Yakovleva1, Grigory A. Tsaur1,2, Tatyana Yu. Verzbitskaya1,2, Alexander M. Popov1,2,3, Andrey A. Igumenshev1,2, Larisa V. Vakhonina1,2, Igor N. Vyatkin1,2, Egor V. Shorikov1,2, Larisa G. Fechina1,2

1Regional Children’s Hospital №1, Yekaterinburg, Russia; 2Research Institute of Medical Cells Technologies, Yekaterinburg, Russia;
3Ural State Medical Academy, Yekaterinburg, Russia

Background

The results of hematopoietic stem cell (HSC) transplantation depend primarily on transplant quality. The temperature compensation provided during controlled rate preservation for the release of latent heat results in improved post-cryopreservation cell viability. The major variables involved are the rate of chamber temperature decrease, hold temperature and duration, the rate of temperature increase, and the temperature at which chamber cooling is reinitiated.

Aim

To create – on the basis of international recommendations – an optimal program of freezing for HSC using cryoprotective compounds – available in Russia – and the IceCube 15M programmed freezer.

Methods

Peripheral blood mononuclear cells (PBMC) were used as a test model. The cryoprotective solution contained high–molecular weight dextran, autologous serum, and DMSO with a final concentration of 1.6%, 5%, and 10%, respectively. Results were estimated by the shape of freezing curves and cells’ viability with 7-AAD staining on FacsCanto II.

Results

We tested 2 different volumes (185 ml and 100 ml) of PBMC. After adjusting freezing programs to the selected volume, the 2 different freezing curves gave optimal results. Afterwards created programs were used for the programmed freezing of patients’ HSC (24 samples). Thirteen samples were thawed for autologous transplantation. The median viability of HSC after thawing was 94% (range 77.5–98.9%) and the median time to hematopoietic recovery was 10 days (range 9–13).

Conclusion

It was shown that various volumes of PBMC/HSC need different freezing conditions. Thus, 2 different freezing curves were constructed for volumes less than 100 ml and more than 100 ml, respectively, that gave optimal results when assessed by viability and time to hematopoietic recovery (“take”).

Keywords

hematopoietic stem cells, cryopreservation, program of freezing, transplantation

C. Hematopoietic stem cell sources

Is young matched unrelated donor comparable or superior to older sibling donor? An analysis of 76 allografted patients aged above 50 years from the Czech Acute Leukaemia Clinical Register (ALERT)

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Vladimir Koza1, Pavel Jindra1, Karel Indrak2, Fedor Alsabty3, Sarka Hrabetova4

1Dpt. Haematology & Oncology, Univ. Hosp., Pilsen, Czech Republic; 2Dpt. Haematology & Oncology, Univ. Hosp., Olomouc, Czech Republic; 3Dpt. Haematology, Univ. Hosp., Bratislava, Slovak Republic; 4Dpt. Haematology, Faculty Hosp., Prague, Czech Republic

Allo-SCT can be the only curative option even in the elderly. However, the siblings of elderly pts are naturally older and often ineligible for stem cell collection. The question is whether younger unrelated donors could compensate for the higher immune incompatibility.

Method

We retrospectively analyzed 76 AML pts aged above 50 (median 55y) from 5 Czech and Slovak centres allografted either with related (SIB group, n=47) or unrelated, 9–10/10 HLA allele-level matched donors (MUD group, n=29). Groups were comparable in terms of age, remission status pre-SCT, cytogenetic risk, donor sex, CMV D-/R- status, and conditioning. As expected, the MUDs were significantly younger and received more CD34+.

Results

After a median follow-up of 18 months, 39 pts (51%) are alive. The 3-y
Kap.-Meier OS and EFS probabilities were 42% and 40%, respectively, while overall NRM and relapse rates were 25% (19/76) and 26% (20/76), respectively. After splitting the pts into SIB vs. MUD, the figures were as follows: number of surviving pts 24/47 vs. 15/29, resulting in median OS probabilities for SIB and MUD of 16 and 21 months (p=0.817), respectively. The relapse rates were not significantly higher in the SIB group (30% vs. 21%, p=0.433); whereas the NRM rate was similar (23% vs. 28%, p=0.77). The incidences of aGVHD were identical both for SIB and MUD, comparable results were also reported for extensive cGVHD incidences.

Conclusions

Unrelated donors did not adversely affect the SCT outcome in AML patients aged >=50, and the younger allele-matched unrelated donor is – at least – the equivalent of an older HLA-identical sibling. We should not be reluctant with MUD in older pts, and molecularly-matched young unrelated donors should be tested in appropriately designed trials with the larger patient’s cohort.

Keywords

allo-SCT, donor, age, HLA compatibility

D. Minimal residual disease

Algorithm for monitoring minimal residual disease on the basis of patient-associated immunophenotypes

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Yekaterina E. Zueva, Margarita V. Gorchakova, Ekaterina B. Rusanova, Konstantin U. Slobodnyuk

Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Yekaterina E. Zueva, Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: yekaterina.zueva@spam is badgmail.com

Aims

Algorithm for monitoring MRD on the basis of patient-associated immunophenotypes.

MRD monitoring aims to identify cells with aberrant or rare phenotypes. It is appropriate to use patient-associated panels for MRD monitoring, which include: (i) phenotypic markers of transformed cells identified at the date of primary diagnostics, and (ii) markers often occurring in a particular variant of AL. Immunological MRD monitoring is an individualized laboratory diagnostic, since individual patients could have a complete preservation of aberrant phenotypes of transformed cells, as well as changes in the expression of some markers.

Methods

A multicolor flow cytometry was used.

Results

Seventy-five cases of primary pediatric AL were studied. Among them were 53 cases of B-ALL, 8 cases of T-ALL, and 13 cases of AML. Monitoring of MRD was performed in 23 B-ALL cases, 6 T-ALL cases, and 5 AML cases.

During MRD monitoring, we observed the complete preservation of phenotypes of transformed cells in 11 cases of B-ALL (48% of cases) and 4 cases of a change in expression (17%). In other cases, MRD of B-ALL was not detected (35%). Complete preservation of phenotypes of transformed cells was observed in 3 cases of T-ALL (50%) and in 3 cases MRD was not identified (50%). Complete preservation of cell phenotypes was observed in 3 cases of AML (60%), while in 2 cases (40%) a change in expression was revealed.

Summary

An algorithm for monitoring MRD on the basis of patient-associated immunophenotypes allows the verification of MRD through the presence of malignant cells with a sensitivity up to 10-5. This algorithm provides full information to clinicians, and thus the possibility to plan transplantations.

Keywords

MRD, flow cytometry, ALL, patient-associated algorithm, monitoring

D. Minimal residual disease

Slow fusion gene transcript clearance predicts poor outcome in infants with MLL-rearranged acute lymphoblastic leukemia

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Grigory A. Tsaur1,2, Tatyana V. Nasedkina3, Alexander M. Popov1,2,4, Anna S. Ivanova1,2, Yulia A. Yakovleva1,2, Tatyana O. Riger1,2, Egor V. Shorikov1,2, Leonid I. Saveliev1,2,4, Larisa G. Fechina1,2

1Regional Children Hospital 1, Ekaterinburg, Russia; 2Research Institute of Medical Cell Technologies, Ekaterinburg, Russia; 3Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; 4Ural State Medical Academy, Ekaterinburg, Russia

Correspondence
Grigory Tsaur, Regional Children Hospital №1, Pediatric Oncology & Hematology Center
S. Deryabina Street 32, 620149 Ekaterinburg, Russia, Phone: +7-343-216-2517, Fax: +7-343-216-2517
E-mail: grigory.tsaur@spam is badgmail.com

Objective

To find out a single time point (TP) for fusion gene transcript (FGt) monitoring by RT-PCR that clearly predicts the outcome in infants with MLL-rearranged ALL. All patients (pts) were treated by MLL-Baby protocol, containing all-trans retinoic acid (ATRA).

Methods

The monitoring of FGt was performed in 18 infants with defined MLL translocation partner genes and more than 4 follow-up samples. MRD-negativity was defined as an absence of FGt in nested RT-PCR with sensitivity of 1E-5. The median of follow-up was 25 months. There were
11 MLL-AF4-positive pts, 3 MLL-MLLT10-positive, 2 MLL-EPS15-positive, 1 MLL-MLLT1-positive patient, and 1 MLL-MLLT3-positive patient. Bone marrow samples were obtained at diagnosis, on day 15 of remission induction (TP1), end of remission induction (TP2), one week after first ATRA administration (TP3). In MLL-AF4-positive pts following TPs (TP4–TP9) were scheduled before each HR block. In all other pts the presence of FGt was estimated before each reinduction (TP4-TP6).

Results

FGt elimination speed does not correlate to any known prognostic factors. Retrospectively, patients were divided into 2 groups. The first group included 14 patients who achieved molecular remission by TP4, while 2 relapses occurred (MLL-AF4-positive and MLL-EPS15-positive). The second group consisted of 4 MLL-AF4-positive pts who did not achieve molecular remission by TP4; there were 3 relapses. The number of relapses in this group was significantly higher (p=0.04). The 70-month RFS in the first group was 0.84, in the second group 0.25 (p=0.02). The cumulative incidence of relapse in the first group was 0.15, in the second group 0.75 (p=0.02)

Conclusions

The presence of FGt by TP4 helps to identify pts with a high risk of relapse.

Keywords

ALL, infants, MLL rearrangements, fusion gene transcripts, MRD monitoring

D. Minimal residual disease

Detection of minimal residual disease (MRD) by flow cytometry in children with acute lymphoblastic leukemia (ALL)

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Margarita G. Bozhieva, Ludmila Y. Grivtsova, Alexander V. Popa, Irina N. Serebryakova, Irina E. Gavrilova, Boris V. Kurdyukov, Rono S. Ravshanova, Georgiy L. Mentkevich, Nikolay N. Tupitsyn

Pediatric Oncology and Hematology Research Institute, N. N. Blokhin RCRC RAMS, Moscow, Russia

Background

The initial response of leukemia cells to treatment has consistently been shown to be a reliable prognostic indicator, and its evaluation has been significantly enhanced by methods that allow detection of submicroscopic levels of leukemia.

Methods

From January 2007 to April 2009, 45 patients with newly-diagnosed ALL were enrolled in ALL-IC BFM 2002 studies at our institution and had residual disease studies by flow cytometry on day 15 and day 33 of treatment. Of the 45 patients, 37 patients (82.2%) had a BCP-ALL and 8 had T-ALL (17.8%). Analyses were performed on a FACScan (Becton Dickinson). We used several standardized antibody combinations to screen ALL samples at diagnosis for leukemia-associated aberrations as well as to investigate BM.

Results

In 40 samples (88.9%) on day 15 we identified at least 0.01% leukemic cells (0.01% – <0.1% in 12 (26.7%), 0.1% – <1% in 16 (35.6%), and  ≥ 1% in 12 (26.7%). Of the 45 bone marrow samples studied by flow cytometry on day 15 of remission–induction therapy, 11 (24.4%) had leukemic lymphoblasts identifiable by morphologic analysis. In all the 11 morphologically positive samples, at least 0.1% cells expressing leukemia-specific immunophenotypes were detected by flow cytometry (median 17.2%, range 0.37% to 82.2%). Among the 34 (75.6%) samples without leukemic lymphoblasts recognizable by their morphologic features, 29 (64.5%) had detectable cells expressing leukemia-associated immunophenotypes (median 1.4%, range 0.01% to 25%).

Conclusion

The proportion of samples that were MRD+ was high on day 15 and decreased to 59.6% on day 33. Children with ALL who achieved an early clearance of leukemic cells had an excellent outcome.

Keywords

acute lymphoblastic leukemia, children, minimal residual disease, flow cytometry

D. Minimal residual disease

Comparison of MRD data assessed by flow cytometry and RT-PCR of fusion gene transcripts in infants with MLL-rearranged ALL

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Alexander M. Popov1,2,3, Grigory A. Tsaur1,2, Anna S. Ivanova1,2, Yulia A. Yakovleva1,2, Tatiana Y. Verzhbitskaya1,2, Tatiana O. Riger1, Egor V. Shorikov1,2, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

1Pediatric Oncology and Hematology Center, Regional Children’s Hospital, Ekaterinburg, Russia; 2Research Institute of Medical Cell Technologies, Ekaterinburg, Russia; 3Ural State Medical Academy, Ekaterinburg, Russia

Correspondence
Alexander M. Popov, Regional Children’s Hospital, Pediatric Oncology & Hematology Center, S. Deryabina Street 32, 620149 Ekaterinburg, Russia, Phone: +7 (908) 9037736, Fax: +7 (343) 2162517
E-mail: uralflow@spam is badgmail.com

Aim

To evaluate qualitative and quantitative concordance between MRD data assessed by flow cytometry (FC) and fusion gene transcript (FGt) copy number (CN) measured by real-time quantitative PCR (RQ-PCR) in infants with primary and relapsed MLL-rearranged ALL during treatment.

Methods

A tandem application of multicolor FC for MRD detection and RQ-PCR for FGt CN was performed in 72 follow-up bone marrow samples from 15 infants with MLL-rearranged B-lineage ALL. Twenty samples were obtained during remission induction, 36 during postinduction, and 16 during relapse treatment.

Results

Of the 72 samples, 7 (9.72%) were MRD-negative by both methods, while 2 (2.78%) were negative by FC but positive by RQ-PCR. The remaining 63 samples (87.50%) were MRD-positive by both methods. High qualitative concordance (97.22%) between FC and RQ-PCR was obtained. In contrast, low quantitative concordance was found (r=0.54). The significant quantitative difference in FC and RQ-PCR data could be associated with variability of FG expression during treatment that does not correspond to the cell number. Moreover, during FC the MRD detection percentage of tumor blasts among all nucleated cells is calculated, while the MRD value in RQ-PCR of FGt corresponds to the initial FGt and control gene levels. FC appears to be better for a quantitative MRD assessment; however FGt detection in RQ-PCR is more appropriate for MRD qualitative analysis because of its higher sensitivity. Hence, FC is more applicable for early treatment stratification and RQ-PCR of FGt for later time points.

Conclusion

A tandem application of FC at early time points and FGt detection by RQ-PCR at later time  points seems to be a useful tool for MRD monitoring in infants with MLL rearranged ALL.

Keywords

MRD, MLL, infants, flow cytometry, fusion gene transcript

E. Graft-versus-Host Disease

The comparative characteristic toxicity and immunosuppressive activity both tacrolimus and cyclosporine in patients undergoing allo-HSCT

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Yulia A. Stankevich, Ludmila S. Zubarovskaya, Elena V. Semenova, Sergey M. Alexeev, Elena V. Babenko, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: juliastan@spam is badmail.ru

Background

Graft-versus-host disease (GVHD) still remains one the major obstacles to successful allogeneic hematopoietic cell transplantation (HSCT). GVHD is a major cause of morbidity and mortality even when the recipient and donor are matched at the major histocompatibility complex. In its acute and chronic forms, GVHD has an important impact on survival and the recipient’s quality of life. The ability to prevent GVHD – i.e., the application of successful prophylaxis – is the cornerstone of success. Immunosupression with pharmacological agents such as cyclosporine (CsA) or tacrolimus (Tx) is more often used for GVHD prophylaxis. Because they share a common intracellular target, the toxicity profiles of CsA and Tx are very similar. We found Tx to be superior for the prophylaxis of acute GVHD, but more toxic. While hypertension and myelosupression is more commonly seen with CsA, neurological, nephrology, and gastrointestinal side effects are more often associated with the administration of Tx.

Patients and methods

From 2000 to December 2008, 171 patients (pts) received allogeneic HSCT. In two patients allo-HSCT was repeated because of relapse of disease, so the total number of transplantations was 173. Patients and donor characteristics, type of allo-HSCT, and preparing regimens are shown in Table 1.

Results

There was no significant difference in survival. The 6-month overall survival rate was 53% for the Tx group and 65% for the CsA group; the 6-month relapse-free survival rate was 42% (Tx) vs. 50% (CsA). There was no difference in rates of relapse between the two groups.

Infections were the most common cause of death, accounting for 33% of deaths. Other causes of death included acute GVHD (31%), relapse (20%), and multisystem organ failure (8%). The incidence of infections was similar in the two study groups.

Although a significantly greater proportion of patients was treated with cyclosporine, the incidence of grades II-IV acute GVHD was significantly lower in patients who received tacrolimus (29% vs. 54% in related HSCT and 32% vs. 60% in unrelated HSCT).

Despite the fact that Tx proved to be superior as a prophylaxis of acute GVHD, it was found to be more toxic. The incidence of hypertension was significantly higher in the CsA group (38% vs. 24%), but neurological complications (headache, tremor, paresthesia) were significantly higher in the Tx group (44% vs. 30%). Gastrointestinal disturbances were more common in the Tx group (46% vs. 36%, NS), as was hyperglycemia (24% vs. 16%), nephrotoxicity (19% vs. 15%), and metabolic disturbances (11% vs. 7%).

Conclusion

The use of Tx seems to be preferable in groups with a higher risk of developing GVHD because it has a more potent immunosuppressive effect. But Tx has more toxic complications such as neurological, nephrology, gastrointestinal, and metabolic disturbances. On the other hand, hypertension and myelosuppression is more commonly seen with CsA.

Keywords

graft-versus-host disease, allogeneic hematopoietic cell transplantation, immunosuppressive therapy

E. Graft-versus-Host Disease

Eye graft-versus-host disease in patients with allogeneic hematopoietic stem cell transplantation (updated data)

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Alexandra L. Mikhaylova1, Yuri S. Astakhov2, Boris V. Afanasyev1

1Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia; 2Chair of Ophthalmology of St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

Graft versus-host-disease (GVHD) is a complication caused by allogeneic hematopoietic stem cell transplantation (HSCT). It can cause dry eye syndrome.

Objectives

To evaluate the probability of eye acute and chronic GVHD (aGVHD and cGVHD) occurrence and response to topical therapy, including drops with cyclosporine 0.05%.

Methods

Two hundred and twelve patients with different hematological malignancies were treated with allogenic HSCT and underwent ophthalmological examination (2006–2009). Dry eye disease stages were diagnosed according to clinical symptoms, positive staining with fluorescein, Schirmer’s I test results, and a tear film break-up time. The treatment schedules were: Stage 1: artificial tears b.i.d. or t.i.d.; Stage 2: artificial tears t.i.d. and dexamethasone 0.1% t.i.d. topical; Stage 3: artificial tears 4–6 times/day, dexamethasone 0.1% t.i.d. topical and cyclosporin A 0.05% 1 drop/eye b.i.d. The Wilcoxon pair matched test, Spearman test, and Pearson test were used for statistical analysis.

Results

Ophthalmologic complications were observed in 48.5% (103/212) of HSCT patients. Eye GVHD was revealed in 25% of patients. Forty-four patients were eligible for assessment of system cGVHD. Among them, 70% (31/44) of patients had signs of dry eye syndrome. All patients with aGVHD received systemic GVHD prophylaxis and eye component of aGVHD responded quickly to local dexamethasone 0.1% treatment. The median day of onset of eye cGVHD was D+145±29. The Table 1 represents eye cGVHD treatment results.

There was a strong correlation between both tests (p=0.004). Three patients could not tolerate cyclosporine topical treatment due to intensive local conjunctive irritation.

Conclusion

About half of treated patients had signs of different eye complications. The frequency of eye cGVHD was 70%. Cyclosporine local therapy proved to be an effective treatment of Stage 3 dry eye syndrome caused by cGVHD.

Keywords

graft-versus-host disease, GVHD, dry eye syndrome, cyclosporin, hematopoietic stem cell transplantation, HSCT

E. Graft-versus-Host Disease

Applying mesenchymal stem cells for the prevention and treatment of steroid-resistant GVHD in children in Belarus

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Nina V. Minakovskaya, Yanina I. Isaikina, Olga V. Aleinikova

Belarusian Research Center for Pediatric Oncology and Hematology, Minsk, Belarus

Correspondence
Nina V. Minakovskaya, Belarusian Research Center for Pediatric Oncology and Hematology, Minski distr., Pos. Lesnoe, Minsk, Belarus, 223053, Phone: +375 17 265 40 89, Fax: +375-17-265-42-22
E-mail: minakovskaya@tut.by

Objective

We analyzed the clinical effect of MSC infusion on day +30 after
HSCT as a GVHD prophylaxis and the application of MSCs as a treatment of severe steroid–resistant GVHD.

Patients and methods

Nine pts after allogeneic hematopoietic stem cell transplantation (HSCT) underwent MSC infusions (median age was 11 years, 6 males, 3 females) between 2006 and 2009.

Diagnoses

ALL – 4, AML – 1, AA – 3, MDS – 1. GVHD prophylaxis for pts with ALL and MDS consisted of CSA and MTX 10 mg/m2 (n=3); for pts with AA, CSA+MMF; for pts with AML, CSA and MTX 10 mg/m2 (n=4). For the treatment of GVHD all pts received methylprednisolone 1–2 mg/kg. Five pts received MSCs once and 4 pts, twice. For 4 pts MSCs were used as a GVHD prophylaxis on day +30 after HSCT; the median dose was 1.0 (0.7–1.5)x10^6/kg. Five pts received MSCs for the treatment of steroid–resistant GVHD with a medium time of MSC infusion after HSCT of 126 (110–151) days and a mean dose of 2.2 (1.3–3.7)x10^6/kg.

Results

There was no evidence of early or late side effects of MSC infusion. One patient died from pulmonary GVHD 1 month after MSC infusion, while eight pts are alive. All pts (n=4) who received MSCs on day +30 as a GVHD prophylaxis developed grades II–IV GVHD and needed a secondary MSC infusion; the median time between MSC infusions was 120(90–150) days.

Four pts out of five with steroid-resistant GVHD showed significant improvement of the clinical signs of GVHD; this allowed the reduction of immunosuppressive therapy and the cessation of steroids.

Conclusion

Our experience demonstrates the absence of a positive GVHD prophylactic efficacy when MSC infusion was carried out on day +30. However, we observed a decrease of GVHD grades III–IV to 0–II, when MSCs were used as a treatment of steroid-resistant GVHD.

Keywords

GVHD, MSC, allogeneic HSCT

E. Graft-versus-Host Disease

Spirometry findings in bone marrow transplant patients

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Larisa A. Kuzmina, Lidia S. Lubimova, Valeriy G. Savchenko

National Haematologic Scientific Centre, Russian Academy of Medical Sciences Moscow, Russia

Correspondence
Larisa A. Kuzmina, National Haematologic Scientific Centre, Russian Academy of Medical Sciences, Novozykovski pr., 4, 125167, Moscow, Russia, Phone: +7 (916) 148-71-31
E-mail: Kuzlara@rambler.ru

Aim

To investigate the frequency, character of spirometry findings, determine the prognostic of unfavorable alterations, and analyze responses on immunosuppressive therapy (IT) according to the character of spirometry findings in bone marrow transplantation (BMT) recipients.

Methods and materials

The spirometry tests were performed using the national spirometer SMP-21/01–«R-D» with a value of 15 standard tests.  We included 54 recipients of allogeneic BMT: M/F 28/26, aged 17–45, AL – 30, and CML – 24. Myeloablative conditioning (MC) (BU 16 mg/kg+Cph 120 mg/kg) was used. Before BMT spirometry tests were performed in 28 pts during the first year, then 31, and later 32 pts. The total number of investigations was 185. 

Results

The pulmonary functional alterations seen in BMT pts were as follows: restrictive, obstructive, and combined. After BMT the following alterations were revealed in 20 pts (32%): restrictive in 7, obstructive in 6, and bots (combined) in 7. During the first year restrictive alterations (50%) predominated. After 12 mo we generally observed obstructive and combined alterations (80%) in 70% of cases in pts with chronic graft versus host disease (GVHD). There were no adequate correlations between the spirometry results before BMT and the early period after BMT and the later development of  severe lung complications. In those patients that presented restrictive alterations, significant clinical symptoms of pulmonary insufficiency in the majority of pts was not observed: IT was successful. In those patients that presented obstructive alterations, we only observed the improvement or the stabilization of the process in a minority of pts by using a combined long IT. In 6 of the 8 pts with the most severe clinical symptoms – i.e., apnea and oxygen dependence – there were combinate alterations; IT was unsuccessful.

Conclusion

Spirometry is an important method for the definition of the IT strategy in accordance with recognized alterations, especially after 6 mo when GVHD prophylaxis is over. Regular examinations are of great importance, especially in patients with chronic GVHD.

Keywords

spirometry, bone marrow transplantation, BMT, pulmonary complications/ mortality, myeloablative conditioning, MC, graft-versus-host disease, GVHD, immunosuppressive therapy, IT

E. Graft-versus-Host Disease

Chronic graft-versus-host disease in children and adolescents

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Anita Lawitschka

St. Anna Children’s Hospital, Vienna, Austria

Chronic graft-versus-host disease (cGVHD) remains the major late complication of hematopoietic stem cell transplantation (HSCT) and is the main cause of non–relapse mortality (NRM) and morbidity in the long-term follow up. Reported pediatric incidence rates range from 23%–40%, with rising numbers as a result of the increasing use of peripheral blood stem cells, mismatched donors, and adoptive cellular therapies. Compared to adults, considerable fewer studies have been performed in children due to the lower number of patients and because reported data are very heterogeneous. Both cGVHD and therapy may have a profound deleterious impact on organ functions and organ development, hormone balance, and psychomotoric development of the growing child.

Here, we summarize pediatric aspects of risk factors, clinical presentation, and treatment options including supportive and topical therapy. The recently recommended standards for diagnosis and staging are also briefly described. The early identification of high-risk patients, perception of multisystem comorbidities, and exact response evaluation may lead to individualized management of cGVHD patients.

Keywords

pediatric chronic GVHD, diagnosis, staging, treatment, comorbidities, response evaluation

E. Graft-versus-Host Disease

Evidence of the “graft-versus-tumor” effect following haploidentical transplantation in a patient with a metastatic relapse of Ewing’s sarcoma

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Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, Natalia S. Subbotina, George L. Mentkevich

Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia

A 16-year-old girl was admitted with a metastatic lung relapse of Ewing’s sarcoma six months after completion of multimodal therapy for a localized left shoulder tumor. She received second line ICE chemotherapy with no decrease in tumor volume. She had surgery and 2 and 3 metastases were removed from the right and left lung respectively. No histological response was seen. The appearance of 2 new foci in the right lung was observed within the 2 successive weeks. She received reduced-intensity conditioning, including fludarabine 180 mg/m2, busulfan 8 mg/kg and ATG at 40 mg/kg. She also received 10.8 Gy of whole lung irradiation as part of the conditioning. The donor was the patient's 4/6 HLA-mismatched mother.
The girl was grafted on d0 and +1 with 2.2x106 CD34/kg and 6.7x108CD3/kg after 30’ incubation of inoculum with vincristine and methylprednisolone. GvHD prophylaxis consisted of short courses of methotrexate and cyclosporine A. The level of WBC >1.0x109/l was reached on day +12.
The girl required no PLT transfusion. A complete donor chimerism was observed on d+90. In the early post-transplant period grade I skin GvHD was observed. A cyclosporine-A related encephalopathy occurred on d+60 and the GvHD consisted of MMF and methylprednisolone at that time.
The recovery of immunity was prompt and fast. The decrease in the size of metastases was observed to occur progressively from d+30 until their complete disappearance by day +90. At this time the girl is disease-free and well, with no evidence of GvHD.

As far as we know this is the first report of a successful family-mismatched transplantation with evidence of “graft-versus-tumor” effect in a patient with relapsed Ewing’s sarcoma refractory to second-line therapy.

Keywords

graft-versus-tumor effect, Ewing’s sarcoma, haploidentical transplantation, reduced-intensity conditioning

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Prognostic value of apoptosis of tumor clone bone marrow cells in patients with multiple myeloma

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Zhanna V. Chubukina, Ludmila N. Bubnova, Stanislav S. Bessmeltsev

Russian Research Institute of Hematology and Transfusiology, St. Petersburg, Russia

Purpose

To study the apoptosis of tumor cells (plasma cells, PC) in bone marrow of patients with multiple myeloma (MM) and to evaluate the possibility of using this parameter as a predictor of treatment efficacy.

Materials and Methods

The study comprised 34 patients with active stage MM. Myelogram: PC 11.4–86.4 %. Apoptosis of PC was detected by binding Annexin V-FITC with a membrane marker of early apoptosis–phosphatidylserine. The analysis was performed using the Flow Cytometer “Cytomics FC 500 Beckman Coulter”, USA. Apoptosis of PC was evaluated before and after a course of specific therapy.

Results

It was demonstrated that initially spontaneous apoptosis of PC in all MM patients was on average 25.4 ± 3.3%. But in 32% (11) of patients apoptosis was significantly lower than average (15.6 ± 2.9%),while in 68% (23) of patients PC apoptosis was significantly higher than average (35.2 ± 3.0%). After treatment there was a 2.5-fold (39.1 ± 4.1%) increase of apoptosis index in those patients with an initial decrease. This coincided with an occurrence of clinical–hematological remission in this group. In patients with a higher initial spontaneous apoptosis, its increase had not been noted in any case (34.6 ± 2.8%), and clinical therapeutic effect was also absent.

Conclusion

Initially decreased apoptosis of bone marrow PC is a predictor of a more favorable course of disease and of therapeutic efficacy. On the contrary, a higher level on initial spontaneous PC apoptosis is prognostically an unfavorable marker, because tumor cells remain refractory to chemotherapy and the induction of the apoptotic process is insufficient.

Keywords

multiple myeloma, apoptosis, plasma cells

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Expression of tumor-associated genes in multiple myeloma patients during high-dose chemotherapy and auto-SCT

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Tatiana V. Gaponova, Lidia S. Mendeleeva, Andrey V. Misurin, Elena Yu. Varlamova, Elena N. Parovichnikova,
Valeriy G. Savchenko

National Research Center for Hematology, Moscow, Russia

High-dose therapy enables further improvement in the outcome of multiple myeloma (MM) patients. However, it is still necessary to determine prognostic factors that may influence treatment results and provide additional criteria for the precise selection of treatment approaches. There are several tumor-associated genes (MAGE, LAGE, GAGE, PRAME) that are over-expressed in different malignancies including MM. These genes are believed to modulate cancer properties and should be taken into account during treatment. Their significance as prognostic factors is under investigation.

The aim of our study was to analyze the expression levels of PRAME, WT1, and XIAP genes in MM patients at diagnosis and during high-dose chemotherapy followed by auto-SCT and therapy of proteasome inhibitors.

Our study included 34 primary MM patients; all of them gave informed consent. The median age was 48 years (range 31–62). IgG MM was diagnosed in 28 cases, IgA MM in 2 and light chain MM in 4. Initial treatment consisted of 3 cycles of VAD. If CR or PR were not achieved, the treatment was changed to bortezomib at 1.3 mg/m2 on days 1,4,8, and 11 and dexamethasone (dex) at 40 mg daily on days 1–4 (4–6 cycles). If CR or PR was attained, stem cell mobilization was performed with cyclophosphamide 6 mg/m2+G-CSF. Melphalan at 200 mg/m2 was given before auto-SCT. Investigation was performed before therapy (n=34), after VAD (n=20), after bortezomib (n=10), and after auto-SCT (n=9). Results were normalized against the expression of the ABL gene, which was used as an internal control.

In primary MM patients, PRAME gene expression was found in 62% (n=21), WT1 in 20% (n=7) of patients, all of who were PRAME positive. Median expression levels were 0.3% (0.001–132%) for PRAME and 0.01% (0.002–2.54%) for WT1. In primary MM patients the XIAP expression was found in 100%. The meaning of XIAP/ABL*100% varied between the range of 5 to 5382% (median 28%). Of the MM patients, 41% (n=14) demonstrated XIAP hyperexpression (XIAP/ABL*100%>40%). We subdivided MM patients into two groups according to XIAP/ABL*100%, meaning that I<40%, II>40%. In the control group of healthy donors (n=9) PRAME gene expression was found in 50% (median expression 0.003%), WT1 in 12% (median 0.0017%), and XIAP in 62% (median 2%). PRAME, WT1, and XIAP expression did not correlate with tumor bulk and was independent of the levels of M–protein, beta-2M, and albumin.

In the group with primary XIAP hyperexpression (II) we observed that the decrease of XIAP expression paralled tumor reduction (from more then 40% to 5–20%). By contrast, in group I the XIAP gene expression increased right after chemotherapy initiation to extremely high levels of 2425%. However, after high-dose melphalan and auto-SCT, the XIAP level significantly decreased (22–157%) along with the attainment of CR or very good PR. The level of the XIAP gene expression changed depending on the response of the bortezomib treatment. In the MM patients who achieved CR+PR after 4–6 courses of bortesomib+dex (n=6), the expression of XIAP reduced from 11–325% (median 66%) to 1–123% (median 20%). In the MM patients with a poor response after 4–6 courses of bortesomib+dex (n=4), we observed an increased expression of XIAP from 16–127% (median 36%) to 22–528% (median 121%).

The expression of PRAME significantly decreased after 3 VAD cycles to 0.001–207% (n=10): at the moment of auto-SCT it was 0.001–6.1% (n=6) and after auto-SCT it was 0.004–4.9% (n=9). However, for WT1we observed an increase of WT1 expression after 3 VAD cycles to 0.004–0.07% (n=5); at the moment of auto-SCT it was 0.001–0.4% (n=6), and after auto-SCT it was 0.0193–2.03% (n=7). In the patients with high primary PRAME expression (>median expression) the frequency of CR+PR was significantly lower than in PRAME–negative primary patients and in patients with low (<median expression) primary PRAME expression (27% vs 69). It should be stressed that during treatment in a small number of initially negative PRAME and WT1 gene patients, we demonstrated detection by PCR. We detected the appearance of gene expression during therapy at low levels in 1 of 13 initially negative PRAME patients (6.1%) and in 8 of 26 initially negative WT1 patients (range of level 0.01–1.03%). The detection of gene expression did not correlate with disease status. All these patients achieve CR+VGPR. In one of the secondary positive patients – who acquired PRAME and WT1 – relapse occurred, with high and low expression of PRAME (104%), WT1 (0,62%), and XIAP (1264%).

Conclusion

The expression of the PRAME gene was found in 62% of primary patients, and the level of PRAME decreased with tumor reduction. A high expression level of PRAME turned out to be a factor of unfavorable prognosis. The expression of WT1 was found in 20% of MM patients – all of who were PRAME positive. WT1 expression increased during treatment in a small group of patients. Some initially negative patients acquired PRAME and WT1 expression during treatment, but clinical relevance of this is so far unclear. In the MM patients at diagnosis, the level of XIAP expression was high. The decrease of XIAP expression correlates with the chemotherapy and proteasome inhibitor treatment efficicacy. XIAP expression comes to the normal values at the time of CR and PR achievement.

Keywords

multiple myeloma, prognostic factors, tumor associated genes

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Supporting therapy of natural zeolites at patients with lymphoproliferative diseases

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Anna S. Chernyshova, Lev E. Panin

Institute of Biochemistry, Siberian Division, Russian Academy of Medical Science, Novosibirsk, Russia

The aim of this study was to investigate liver functionin patients with lymphoproliferative diseases who had received zeolites along with polychemotherapy.

Materials and methods

Research consisted of 68 patients with T- and B-cells non–Hodgkin’s lymphomas. All patients received a polychemotherapy course of cytostatic drugs with hepatotoxic, nephrotoxic, and cardiotoxic side effects. The investigative group (IG) of 28 patients received natural zeolites between chemotherapy courses as supporting therapy. The comparison group (CG) – consisting of 40 patients of identical sex, age, and IPI– received treatment according to standard protocol.

Results

After the polychemotherapy course 6 patients (8.8%) were found to have cytolysis syndrome confirmed by an increase in alanine aminotransferase (ALT) activity. Also, the median of ALT activity in IG patients was 28.3 ed/l (21.3; 47.8), and 29.7 ed/l (22.3; 44.4) in CG patients. It was noticed later that ALT activity decreased by 18.5 ed/l (11.1;25.3) (р=0.021) during the zeolite course, in contrast with ALT activity of 29.95 ed/l (21.2;41.8) (р=0.0004) in the CG. After the introduction of cytostatic medication the mean value of the whole protein content in the IG and the CG was 71.38±1.09 and 71.95±1.42 g/l respectively. After the zeolite treatment the concentration of serum protein in the IG increased up to 73.88±0.82 g/l (р=0.026), which was higher than in the CG (71.00±0.96 g/l, р=0.033).
The fibrinogen content – after the zeolites course – in the IG increased up to 3.6 g/l (р=0.048), which is lower than that in the CG (р=0.008) by 0.84 g/l.

Conclusion

The use of natural zeolites noticeably reduces the hepatotoxicity of chemotherapeutic drugs used for the treatment of lymphoproliferative diseases, allowing the utilization of zeolites as hepatoprotectors.

Keywords

non-Hodgkin’s lymphoma, supporting therapy, liver, natural zeolites, polychemotherapy

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Treatment results of children with Hodgkin’s disease (HD) according to protocols DAL-HD and GPOH-HD

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Margarita B. Belogurova1,2, Galina G. Radulesku1,2, Tatyana D. Viktorovich1,2, Emilia D. Chavpetsova2, Ludmila I. Shats1,2, Yulia V. Dinikina1

1St. Petersburg State Pediatric Medical Academy, Division of Oncology with a course of radiodiagnostics and radiotherapy;
2City Clinical Hospital №31, Department of Pediatric Oncology and Hematology, St. Petersburg, Russia

Correspondence

Margarita B. Belogurova, City Clinical Hospital №31, Department of Pediatric Oncology and Hematology, St. Petersburg, 197110, Russia
E-mail: deton.hospital31@inbox.ru

According to the data of the population-based Cancer Registry 2008, the structure of oncopediatric morbidity in SPb in the age group of 10–14 years (y) is most frequently malignant lymphoma  (30.7%), both for girls and boys.

Methods

Sixty-four primary patients (pts) with HD were treated from 1992 to 2002 according to the following protocols: DAL-HD-90, GPOH-HD-95 (60 pts) and GPOH-HD-2002 (4 pts). The median age was 14 y. Distribution according to stages was as follows: Stages I–II were diagnosed in 46 pts (72%), 18 pts (28%) had advanced HD. The ratio of boys to girls in the group up to 11 y was 3.5:1; after 11 y both sexes were affected equally. Diagnosis was confirmed by histology (100%) and immunohistochemistry (52%). In our pts nodular sclerosis was predominant (81%). In children under 7 y a mixed cellularity variant was more frequent. In GPOH-HD-2002 protocol procarbazine was replaced by dacarbazine to decrease the adverse effects on the male reproductive system.

Results

The CR was achieved in 61 pts (95.3%).  In terms of medical observation from 8 to 210 months OS - 94.8% ± 2.89 and did not change after 29 months. RFS and EFS were 96.5% ± 2.37 and 92.1 ± 3.39 respectively. All events were registered within a period of 11 months.

During chemotherapy early toxic effects were minimal: mucositis, duodenal ulcer, and neuropathy. Late complications of radiotherapy – muscle atrophy in area of irradiation (2 cases) and tumors of thyroid gland (2 cases) – were revealed in terms of 2 to 8 years.

Conclusions

Hodgkin’s disease in children can be considered as a practically curable disease with moderate intensive treatment and low toxicity.

Keywords

Hodgkin’s disease, children, chemotherapy, fertility

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Cellular factors of antitumor immunity in patients with chronic lympholeukemia

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Natalya V. Issayeva, Galina A. Zaitseva, Tamara P. Zagoskina

FSE, Kirov Research Institute of Hematology and Blood Transfusion of FMBA of Russia, Kirov, Russia

The purpose of the study was to analyze a number of cellular factors of antitumor immunity in patients with chronic lympholeukemia (CLL) at the beginning of the disease. Using the method of flow cytometry, the absolute content of the following lymphocyte subpopulations in the peripheral blood were assessed as the main antitumor factors in 59 CLL patients: NC-cells (CD3-/CD(16+56)+), NC-cells with a high cytotoxic activity (CD3-/CD8+), Т-NK-lymphocytes (CD3+/CD(16+56)+), CD3+/CD8+-lymphocytes including T-lymphocytes with cytotoxic properties. The count of lymphocytes functioning as antitumor immunity activators was also taken into account, i.e., all T-lymphocytes (CD3+/CD19-) and T-helpers (CD3+/CD4+).

An expressed spread in the values of the indices studied was revealed in the result of the investigation. A decrease in the content of cells with the markers CD3-/CD(16+56)+, CD3-/CD8+, CD3+/CD(16+56)+, and CD3+/CD8+ was seen in 10%, 10.8%, 38.3%, and 19% of patients, respectively. A combined decrease in two or three studied effectors of antitumor immunity was noted in 52% of patients. A quantitative deficiency of antitumor immunity activators – all T-lymphocytes and T-helpers – was less commonly observed.

The conclusion was that laboratory assessment of the main effectors for antitumor protection of CLL patients with the aim to reveal patients with their depression is advisable and can be taken into consideration when stratifying the patients.

Keywords

chronic lympholeukemia, antitumor immunity, T-lymphocytes, NC-cells

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Salvage therapy in relapsed Hodgkin's lymphoma patients

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Elena S. Belyaeva, Olga V. Morozova, Andrey I. Slugin, Alexander V. Popa, Georgy L. Mentkevich

N. N. Blokhin RCRC RAMS, Pediatric Oncology and Hematology Research Institute, Moscow, Russia

The treatment of children with refractory or early relapsed Hodgkin's lymphoma remains one of the most important problems in pediatric oncology. In order to improve the outcome of this group of patients we used the ViGePP regimen. We present the results of the ViGePP regimen for children with relapsed/refractory LH.

Method

Between 2008 and 2009, 9 pts with relapsed/refractory LHwere enrolled in this study. Of the 9, 4 pts had refractory LH. The median age was 15.1 y. CR (from 6–48 mo) lasted for a median of 21 mo in 5 pts.

Induction therapy consisted with 2–5 cycles of ViGePP (gemcitabine – 1000 mg/m2/d 1,8,15 d., vinorelbine – 30 mg/m2/d 1,8 d, procarbazine 100 mg/m2/d 1–7 d., prednisolone 30 mg/m2/d 1–15 d).

Results

The response rate was assessed after 2 cycles of ViGePP: CR – 3, PR – 6. After the end of treatment the status was CR – 7, PR – 1. One pt continues treatment. Two pts have been in CRII for 15 and 12 mo. One has been in CRIII for 2 mo. Two pts had a fourth relapse 7 and 9 mo after the end of treatment. Two pts in CR have been allocated for consolidation SCT. One pt died from sepsis after ASCT.

Hematological toxicity: leukopenia grade 3 (n=4), grade 4 (n=3), thrombocytopenia grade 4 (n=2), grade 3 (n=5), anemia grade 3 (n=4).

Non–hematological toxicity: mucositis grade 2 (n=2), grade 1 (n=2), gastrointestinal grade 3  (n=1), grade 2 (n=1). Liver toxicity (ALT, AST) grade 3 (n=4), grade 2 (n=3). Heart and kidney toxicity > grade 1 were not observed.

Conclusion

The ViGePP regimen was effective in 6/9 LH pts. Toxicity was acceptable. Future studies are warranted to evaluate larger cohorts of pts with relapsed and refractory LH.

Keywords

Hodgkin’s lymphoma, children, relapse, therapy, ViGePP

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Clinical and pathological characterization of the t(14;19)(q32;q13)-positive splenic marginal zone lymphoma

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Hunan L. Julhakyan, Tatyana N. Obukhova, Irina B. Kaplanskaya, Rima S. Samoylova, Andrey I. Vorobiev

Hematology Research Center of the Russian Academy of Medical Science, Moscow, Russia

Splenic marginal zone lymphoma (SMZL) is a B-cell neoplasm which is characterized by splenomegaly, lymphocytosis, and involvement of bone marrow and, sometimes, an M–component. The most frequent findings in SMZL involve chromosomes 1, 3, 7 (usually deleted in 7q), and 18. The t(14;19)(q32;q13) is a rare cytogenetic abnormality with a bcl-3 rearrangement that has been reported in B-cell lymphomas and leukemia.

Aim

The aim of the study was to characterize the clinical and morphological changes in patients with t(14;19)(q32;q13)-positive SMZL.

Patients, methods and results

In our center between January 2005 and February 2009, 3 cases of SMZL with t(14;19)(q32;q13) were identified. All the patients (males, median age 58 years, range 51–67 years) presented with lymphoma with B-symptoms, a high level of lactate dehydrogenase (LDH), hepatosplenomegaly, and with splenic hilar lymphadenopathy only. The median hemoglobin was 110 g/l (range 92–122 g/l). All patients had a normal leukocyte count with an absolute lymphocytosis (median lymphocyte count 72.3 x 109 g/l, range
58–83 x 10
9/l) and thrombocytopenia. A morphological examination of the peripheral blood and bone marrow lymphocytes showed that all lymphocytes were atypical with a wide cytoplasm and nuclear indentation. In all cases bone marrow involvement was nodular and composed of heterogeneous mixture cells, with the majority being medium sized. The results of the immunophenotypic analysis were the expression of mature
B-cells antigens and an absence of
СD10-, CD23-, CD5-, CD43-, CyclinD1-.

In debut two patients were to receive chemotherapy (CHOP–regimen). However there was progression: enlargement of the spleen and a decrease in the thrombocyte count. All patients underwent a splenectomy. The median weight of the spleen was 2083 g (range 1800–2850 g). The splenic section generally showed massive nodular patterns (involving the white and red pulp) associated with diffuse invasion of the sinuses. In each case, high Ki-67 was discovered. Progression occurred 3–6 months after the splenectomies, and was characterized by an increase in the leukocyte count (range
45.4–101.8 x 10
9 /l), a high level of LDH, and an appearance of peripheral and visceral lymph nodes. In consideration of increasing the leukocyte count and the presence of lymphadenopathy in all patients, alkylating agents were used (in one patient chlorambucil, and in two cyclophosphamide). There was a normal peripheral blood index, LDH level , and an absence of lymphadenopathy in all cases after 4–6 months of treatment with alkylating agents. Median observation was 19+ months (range 17–22 months). All patients were alive.

Conclusion

SMZL with t(14;19)(q32;q13) is a distinct variant that is characterized by transient progression after splenectomy and high efficiency of alkylating agents.

Keywords

splenic marginal zone lymphoma, t(14;19)(q32;q13), morphology, spleen, splenectomy, alkylating agents

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Effects of targeted therapy of multiple myeloma according to the City Hematological Center of Novosibirsk

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Natalia V. Skvortsova, Tatiana I. Pospelova, Sofia J. Kovalchuk

State Medical University, City Hematological Center Novosibirsk, Russia

Aim

The aim of this research was to estimate the efficiency and toxicity of bortezomib in the treatment of patients with multiple myeloma (MM) as first-line and second-line therapies.

Methods

Fifty-five patients with MM were treated; the patients’ median age was 66.3 years with a range of 38 to 87 years. Bortezomib was used as a second-line therapy in 41 patients with MM, who had earlier received at least 4 courses of therapy. All patients were tolerant to the last scheme of therapy. Bortezomib was applied in the form of monotherapy in a dose of 1.3 mg/m2 in 21 patients (41.2%) and was also applied in combination to dexamethasone in a dose of 20 mg p.o. in 20 patients (39.2%). As a first-line therapy bortezomib has been appointed in combination with МР 10 (19.6%) by the patient. The response to bortezomib was evaluated according to the criteria of the European Group for Blood and Marrow Transplantation (EBMT). Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria, version 3.0.

Results

The overall response rate to bortezomib as a second-line therapy was 76.5%: 15 patients (29.4%) had a complete response and near complete response (CR/nCR); 21 patients (41.2%), partial response (PR); and 3 patients (5.9%), minimal response  (МR). The overall clinical response to bortezomib in firstline therapy was 80% (6 CR and 2 nCR). The most common adverse effects were neuropathy (58%), fever (25%), and asthenic syndrome (45%). Other frequent effects were: herpes Zoster (33% of cases), thrombocytopenia (45%), and anorexia (40%). All other adverse effects – such as a diarrhea and skin rashes – were documented in less than 10% of cases. The settlement median of the general survival rate was not reached, and the 5-year-old survival rate was 90%.

Conclusion

Bortezomib is a highly effective medical drug and plays an important role in the treatment of MM as a first and second-line therapy.

Keywords

multiple myeloma, bortezomib, adverse effects, chemotherapy

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Association between HLA class I antigens and survival length in patients with multiple myeloma

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Elena A. Senkina, Galina A. Zaitseva, Tamara P. Zagoskina, Konstantin A. Martynov

FSE Kirov Research Institute of Hematology and Blood Transfusion of FMBA of Russia, Kirov, Russia

Aim

The aim of the investigation was to determine the association between human leukocyte antigens (HLA) class I phenotype and the length of survival in patients with multiple myeloma.

Methods

HLA class I (loci A and B) antigens were tested using the standard lymphocytotoxicity test with reagents produced by “Gisans”, St. Petersburg. The HLA phenotype was identified in 120 patients with multiple myeloma. There were 53 (41.2%) males and 67 (55.8%) females among them. The median age at diagnosis was 60 years. The diagnosis of multiple myeloma was established according to standard criteria. Patients received the following chemotherapy programs: MP, M2, VAD, and PAD. Survival was calculated from the date of diagnosis using curves Kaplan-Meier (“Statistica 6.0”). Survival lengths of patient groups were compared using the log-rank method (“Biostat”).

Results

A statistically significant difference was obtained in the comparison of survival curves of patients with HLA-B5 and without (p<0.05). Patients who had HLA-B5 in their HLA phenotype, had a higher risk of death than others, with a median survival length of 29 months compared to 79 months.

Conclusion

Poor prognosis and low survival length in patients with multiple myeloma are associated with HLA-B5.

Keywords

multiple myeloma, HLA, survival

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

High-dose melphalan versus melphalan plus dexamethasone in patients with AL amyloidosis

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Anna G. Smirnova1, Alexey V. Smirnov2, Axel R. Zander3, Boris V. Afanasyev1

1Chair of Hematology, Transfusiology, and Transplantology, Raisa Gorbacheva Memorial Institute of Children Hematology and Transplantation; 2Chair of Internal Medicine, Pavlov State Medical University, St. Petersburg, Russia; 3Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Correspondence
Anna G. Smirnova, Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: dr.annasmirnova@gmail.com

Purpose

AL amyloidosis is a clonal plasma cell dyscrasia related to multiple myeloma, in which monoclonal light chains transform to amyloid deposit in various tissues and lead to progressive organ dysfunction. The prognosis is poor and median survival is about 1 year in untreated patients. This study assessed whether high-dose melphalan followed by autologous hematopoietic stem-cell transplantation (AHSCT) improves the outcome of these patients.

Patients and methods

Between June 2002 and February 2009, 36 patients with AL amyloidosis were included: 20 males, 16 females. The median age was 57 y.o. The number of involved organs ranged from 1 to 5, 17 (47%) patients had more than 2 organs involved and 26 (72%) had cardiac involvement. All patients younger than 70 y.o. with a performance status of 0–2 by ECOG were aimed to AHSCT. Patients ineligible for ASCT received melphalan and dexamethasone. 

Seventeen patients received AHSCT (conditioning regimen was melphalan 100–200 mg/m2), 11 chemotherapy (melphalan+dexamethasone), and 8 died before treatment.

Results

After a median follow-up of 36 months, the 3-year overall survival (OS) was 70% and 30% in the AHSCT and chemotherapy groups, respectively (p=0.00) (Fig. 1). Progression-free survival (PFS) of 1.5 was also better in the AHSCT group: 40% and 10%, respectively (p=0.007). The main factors that decreased OS were cardiac involvement and a performance status of more than 2 by ECOG at the time of diagnosis. Age, number of involved organs, and melphalan dose did not have an impact on OS and PFS in our study.

Figure 1.

2009-5-en-Smirnova-et-al-Figure-1.JPG

Conclusion

AL amyloidosis is usually associated with poor health status, rapid disease progression, and involvement of multiple organs. Survival depends on:
•    cardiac involvement
•    the general health of the patient
•    the intensity of the treatment

Keywords

AL amyloidosis, treatment, melphalan, hematopoietic stem-cell transplantation

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

The role of p53 polymorphisms in non–Hodgkin’s lymphomas’ susceptibility and prognosis

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Elena N. Voropaeva, Tatiana I. Pospelova, Mikhail I. Voevoda, Olga V. Beresina

State Medical University, Scientific Research Institute of Therapy, The Russian Academy of Medical Sciences, Novosibirsk, Russia

Purpose

The purpose of our research was to estimate genotype distribution of p53 polymorphisms in NHL patients as well as the association of them with
non-Hodgkin’s lymphoma (NHL) risk and prognosis.

Patients and methods

Ninety-nine unrelated patients from the Novosibirsk City Hematological Center with different histological NHL entities were investigated. Genotyping was carried out with use of PCR and PCR-PLRF. The genotype and alleles distribution of Arg72Pro ex4, dup16bp in3 and G/C in6 gene p53 in patients were compared with the distribution in healthy, white Russian subjects from Novosibirsk and the Novosibirsk region.

Results

Statistically significant (P < 0.05) heterogeneity was observed between the patients with indolent and aggressive variants and different histological NHL entities.  In addition, our data suggests that Pro-carrying genotypes of the p53 codon 72 (OR, 3.67; CI, 1.00–13.44; P < 0.05) and m-allele of the p53 in3 (OR, 5.56; CI, 2.25–13.7; P < 0.05) increased the risk of B non-small lymphocytic lymphoma, compared with population controls. At the same time, the patients with G-allele in6 gene p53 showed a significantly increased odds ratio (OR, 2.9; CI, 1.01–8.31; P < 0.05) of having B small lymphocytic lymphoma, compared with normal controls without G-allele. We further investigated the association of p53 polymorphisms with prognosis, according to the International Prognostic Index in NHL patients. Patients with an association of minor alleles in3, in6, and ex4 gene p53 tended to have poorer prognosis than those with normal homozygous genotype in these loci.

Conclusion

Our data suggested that Arg72Pro ex4, dup16bp in3, and G/C in6 gene p53 polymorphisms may play a role in NHL susceptibility and prognosis.

Keywords

polymorphism, p53, non-Hodgkin’s lymphoma, susceptibility

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Virus participation in renal damage in AL patients: histopathological study

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Ekaterina G. Vorozheikina1, Vladimir V. Varshavski2, Ekaterina P. Golitsina2, Evgenia N. Glasko1, Irina B. Kaplanskaya1, Ljudmila S. Birjukova1, Elena N. Parovichnikova1, Valeriy G. Savchenko1

1National Research Center of Hematology, Moscow, Russia; 2I.M. Sechenov’s Moscow Medical Academy, Russia

Correspondence
National Research Center of Hematology, Novozykovski Pr 4., 125167 Moscow, Russia
Phone: +7 (495) 612 4592, Fax: +7 (495) 6124252 
E-mail: vekgen@mail.ru

Aim

To investigate the morphology and electron microscope characteristics of kidney damage in patients (pts) with AL.

Patients and Methods

Forty-one patients with AL from 2003 to 2008 were observed. RB was performed in 11 pts with functional renal insufficiency (3-ALL, 8-AML); the median age was 38 years (19–67). Autopsy data were studied in 30 pts with renal insufficiency and acute renal failure. Light microscopy, immunohistochemistry, and electron microscopy was used.

Results

In 30 kidney autopsies, tubular interstitial changes with sclerosis prevalence were found. In 2 pts mesangioproliferative glomerulonephritis was revealed. Other changes included  the low proliferation of mesangiocytes, arteriolosclerosis of vessels (18pts), and the presence of leukemic infiltration (7pts). Tubular-interstitial changes were found in all patients who underwent RB; in 3 cases glomerulonephritis was found and leukemic infiltration in 2. An immunohistochemical study showed that in all cases tubular epithelium cells and interstice-infiltrating lymphocytes were EBV and HSV ½ positive. CMV was found in 1 case and herpes infection also in 1 case. Increased antiviral IgD levels were found in 10 patients, DNA probes were negative. Renal structural changes analyzed by electron microscopy were difficult to interpret, but in all cases viruses were found in different cell types. We can suppose that renal structural damage was to some extent associated with the presence of viruses.

Conclusions

Kidney damage in acute leukemia is characterized mostly by tubular interstitial changes. We also proved – using electron miscopy and an immunohistochemical study – the massive viral invasion of renal tissue.

Keywords

acute leukemia, AL, kidney, renal biopsy, RB, autopsy, viruses

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Bone marrow angiogenesis is a prognostic value in patients with multiple myeloma undergoing high-dose therapy and autologous stem cell transplantation

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Olga S. Pokrovskaya, Lidia S. Mendeleeva, Irina B. Kaplanskaya, Elena Yu. Varlamova, Elena N. Parovichnikova, Sergey M. Kulikov, Valeriy G. Savchenko

Research Center of Hematology, Moscow, Russia

Background

Angiogenesis plays an important role in hematological malignancies. An increase of angiogenesis – measured as bone marrow (BM) microvessel density (MVD) and as an increase in angiogenic factors – has been detected in patients with acute and chronic leukemia, myelodysplastic syndromes, and multiple myeloma (MM). Angiogenesis is a constant hallmark of multiple myeloma (MM) progression. It has also been reported that bone marrow angiogenesis is a predictive factor of poor survival in newly-diagnosed myeloma. The goal of our study was to investigate the dynamics of BM MVD in patients undergoing high-dose therapy (HDT) and autologous stem cell transplantation (ASCT).

Patients and methods

Our study included 36 patients (pts) with newly-diagnosed MM (22 in stage II and 14 in stage III according to Salmon and Durie) – 21 males and 15 females, median age – 51 ys (range 31–67). IgG MM was diagnosed in 18 cases, IgA MM in 6 cases, IgD MM in 1 case, light chain MM in 9 cases, and nonsecretory MM in 2 cases. All patients underwent HDT that included 3–4 cycles of induction therapy (VAD), stem cell mobilization with cyclophosphamide 6 g/m2 and G-CSF 5 mcg/kg, EDAP, and 1 or 2 ASCT with melphalan 200 mg/m2. The bone marrow biopsies for histological and immunohistochemical analysis were performed at the time of diagnosis, after the induction, after stem cell mobilization, before the 1st ASCT and after the end of therapy (5 times during the treatment). The control group was constituted by the healthy donors of BM, (7 male and 3 female, median age 29, age range 17–59). In healthy donors BM biopsy was performed during BM harvesting for allo-BMT. Blood vessels were highlighted by immunostaining of endothelial cells with a monoclonal antibody to CD34 (Novocastra Lab Ltd). The MVD was calculated in 10 fields of view using a 40x objective and 16x ocular lens.

Results

At diagnosis in all MM pts MVD was extremely high compared to healthy donors (152±8 vs 74±4). A significant decrease of BM MVD under each phase of therapy was registered: after the induction therapy MVD was 124±6, before the 1st ASCT – 109±5, and at the end of treatment – 97±3. There was a statistically significant increase of MVD after stem cell mobilization due to G-CSF (143±4). Despite the evident decrease of BM MVD in pts with CR or VGPR, it stayed, nevertheless, significantly higher compared with the control group (p<0,001).

The analysis of probability of CR or VGPR duration after ASCT – according to MVD at different phases of therapy – showed that MVD at diagnosis and before the 1st ASCT are important prognostic factors. The probability of duration of CR or VGPR was 63% in the group with low MVD before the 1st ASCT compared with 15% in the group with high MVD (p<0.02). MVD was revealed to be a more powerful prognostic factor for progression free survival (PFS) than CR or VGPR achievement.

We found a good relationship between BM MVD and  the MM stage (p<0.05). The correlation between BM angiogenesis and albumin levels – as with β2-microglobulin levels – was not significant. There was no correlation between BM MVD before chemotherapy and plasma cell infiltration in these patients.

Conclusion

BM angiogenesis highly increased in patients with MM. Our data demonstrates a highly significant decrease in BM MVD in patients who achieved remission after HDT. But even after the end of HDT and ASCT, MVD is higher than in the control group. There is a statistically significant increase of MVD after stem cell mobilization with cyclophosphamide and G-CSF. MVD at the time of diagnosis and before the 1st ASCT are important prognostic factors for overall survival and PFS after ASCT. MVD before the 1st ASCT seems to be a more powerful prognostic factor for PFS than remission rate.

Our results reveal the decrease of MVD to be a powerful prognostic parameter for PFS in pts with MM, and they underscore the importance of angiogenesis in this disease.

Keywords

angiogenesis, multiple myeloma, autologous stem cell transplantation, microvessel density

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Allogeneic stem cell transplantation in multiple myeloma: single centre experience of 112 patients

Liisa Volin, Heli Uotinen, Eeva Juvonen, Anne Nihtinen, Tapani Ruutu

Department of Medicine, Division of Hematology, Helsinki University Central Hospital, Helsinki, Finland

At Helsinki University Central Hospital 112 patients with multiple myeloma (MM) have been treated with allogeneic stem cell transplantation (SCT) since 1995. The conditioning was at first myeloablative (MA), but reduced intensity conditioning (RIC) after autologous SCT has mostly been used since 1999. However, MA conditioning has been considered in young patients with aggressive disease. Of the patients 54 were male and 58 female. The median age at SCT was 50 (27–64) years. The median number of chemotherapy lines was 2 (range 1–7) before allogeneic SCT. In addition, prior autologous SCT had been performed in 71 patients. The median time from diagnosis to allogeneic SCT was 12 (4­–170) months, and the time between autologous and allogeneic SCT, 6 (2–142) months. At the time of allogeneic SCT 13 patients were in CR, 84 in PR, 4 had SD, and 11 were in progression. Eighty-six patients had a sibling donor and 26 patients had an HLA-matched unrelated donor. The conditioning was MA in 49 and RIC in 63 patients. Thirty patients received a BM graft and 82 patients a PB graft. The median follow-up time from allogeneic SCT was 43 (1–166) months. The OS was 50% at 95 months post-SCT and there were no deaths after 96 months (11 patients at risk). One hundred-day transplant-related mortality was 3.6% and that of the whole follow-up time was 10.8%. Thirty-eight patients have died. The cause of death was MM in 25 patients, GVHD in 7, and infection in 4. The present results show that with appropriate patient selection, allogeneic transplantation can be carried out in myeloma with low transplant-related mortality and encouraging survival rates. 

Keywords

myeloma, allogeneic stem cell transplantation, myeloablative and reduced intensity conditioning

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Unusual B-cell lymphoproliferative disorders and the new WHO classification

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Margarita Palutke

Wayne State University School of Medicine, Detroit, Michigan USA

There is a group of chronic B-cell lymphoproliferative disorders that at this time are difficult to classify, many of which are thought to originate in the spleen. The terms B-cell lymphoma with villous lymphocytes, hairy cell leukemia variant, and splenic diffuse red pulp small lymphocytic lymphoma have been used. The WHO puts some of these entities into the splenic B-cell lymphoma/leukemia unclassifiable category.

We have studied a group of such disorders-which have come to us for consultation – with a variety of diagnoses, including: hairy cell leukemia, hairy cell leukemia variant, splenic marginal zone leukemia, prolymphocytic leukemia, and atypical chronic lymphocytic leukemia. In addition to morphological evaluations and flow cytometric immunophenotyping, immunohistochemical and cytogenetic studies were performed in an attempt to achieve a clinically useful diagnosis.

Keywords

chronic lymphoproliferative disorders, splenic lymphomas

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Cases of secondary acute myeloid leukemia and myelodysplastic syndrome in patients with multiple myeloma

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Alexander S. Luchinin

Research Institute of Hematology and Blood Transfusion as a Federal State establishment under Rosmedtechnologies

Standard chemotherapy using alkylating agents both during the induction of remission and its maintenance therapy had been applied for 30 years in treatment of multiple myeloma (MM). But after a long treatment utilizing alkylating preparations the risk of secondary leukemias becomes unjustified.

We analysed 329 MM cases during the years 1994 to 2008. The males numbered 128 (39%) and the females 201 (61%). The median overall survival (OS) rate for all patients was 31 months. Secondary leukemias were diagnosed in 5 MM patients and were diagnosed as follows: AML (M2 subtype according to FAB) in 2 patients, refractory anemia with excess blasts (RAEB-1 according to WHO) in 2 patients and refractory anemia with excess blasts (RAEB-2 according to WHO) in 1 patient. The median period before a second tumor appeared was 7.3 years. The actuary risk of secondary leukemia after 5 years of MM was 1.3%. The general occurrence of secondary AML and MDS equaled 1.7%. Low occurrence of this phenomenon is due to the fact that the mean period for development of a secondary tumor is 3 times greater than the median life expectancy for MM patients. The median OS rate after the diagnosis of a second leukemia was 2 months.

Thus, secondary AML and MDS are rare complications of chemotherapy in MM patients. But if they appear and develop, it leads to a rapid reduction of life expectancy for the patients because of resistance to the therapy carried out. Patients with smoldering myeloma and a high life expectancy of 5 to 10 years die of a qualitatively new leukemia as a result of a long chemotherapy using alkylating agents. That is why the risk of a second tumor cannot be considered as justified.

Keywords

multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Anemias in lymphomas: pathogenesis mechanisms

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Anna S. Lyamkina, Tatiana I. Pospelova

The State Medical University, City Hematological Center, Novosibirsk, Russia

Aims

To spot the value of blood serum cytokines (IL-1β, IL-6, TNF-α, IF-γ) in the development of an anemic syndrome in patients with lymphoproliferative diseases.

Methods

We inspected 88 patients with lymphoproliferative diseases: T and B–cellular non–Hodgkin’s lymphomas and multiple myeloma. The level of serum interleukins (IL-1β, IL-6, TNF-α, IF-γ) was identified.

Results

Patients with lymphoproliferative diseases and an anemia had average levels of IL-1β, TNF-α, IF-γ in their blood serum that were 3.4, 2.8, and 14.4 times higher respectively than in patients with lymphomas without an anemia (p<0.05). The return correlation between levels of IL-1β, TNF-α, IF-γ and the level of erythropoietin serum (r=–0.79) confirms the importance of
IL-1β, TNF-α, IF-γ in an anemia pathogenesis at hemoblastoses. For 8.3% of patients with lymphomas and an anemia,  myelodysplasia tags, recorded levels of IL-1β and TNF-α were twice as high as patients with normal erythron. This indicates a probable role of the researched cytokines in the development of secondary myelodysplasia in patients with lymphomas.

The return correlation between IL-6 and erythron of bone marrow was available in patients with multiple myeloma (r=–0.96). IL-6 raises a proliferation of myeloma cells, which, in turn, displace and depress a proliferation of remaining sprouts of myelopoiesis, including erythroid.

Conclusions

The cited data bears the importance of cytokines IL-1β, IL-6, TNF-α, and
IF-γ in a pathogenesis of an anemic syndrome for patients with lymphoproliferative diseases.

Keywords

cytokines, lymphoproliferative diseases, anemia, non-Hodgkin’s lymphoma, multiple myeloma

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Genetic polymorphisms of CYP1A1, GSTM1, GSTT1, and GSTP1 genes in В-cellular chronic lymphocytic leukemia (B-CLL)

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Vanik A. Ovsepyan, Vitaliy A. Rosin

Federal State Institution, Kirov Hematology and Blood Transfusion Research Institute under Federal Medicobiological Agency, Kirov, Russia

Aim

The aim of this study was to examine the relationship between genetic polymorphisms in GSTM1, GSTT1, GSTP1, and CYP1A1 genes and their susceptibility to B–CLL.

Methods

The DNA for our research was isolated from the leukocytes of venous peripheral blood taken from 146 patients with B-cell CLL and 221 healthy individuals, using a standard phenol-chloroform extraction method. GSTM1 and GSTT1 genotypes were determined by a multiplex PCR technique that detects homozygous deletions of the mentioned genes and the β-globin housekeeping gene as an internal control. CYP1A1 mutation A4889G (Ile462Val) and GSTP1 polymorphisms A1578G (Ile105Val) and T2293C (Аla114Val) were identified by the PCR-RFLP approach.

Results

For the first time it was shown that the mutant genotype (GSTP1-105IleVal+GSTP1-105ValVal) occurs significantly more often in B-CLL patients than that of healthy individuals (65.1% ps. 53.4%, χ2 = 4.92, p<0.05; OR=1.63, 95% CI=1.06÷2.50), whereas the "wild" genotype GSTP1-105IleIle was significantly underrepresented in patients with CLL compared with those of healthy individuals (34.9% ps. 46.6%, χ2 = 4.92, p<0.05; OR=0.62, 95% CI=0.40÷0.95).

At the same time the difference in distribution of homozygous deletion of the GSTM1 and GSTT1 genes and also of the CYP1A1-Ile462Val and GSTP1-Ala114Val genotypes between B-CLL patients and healthy individuals was statistically not significant (p>0.05).

Conclusions

The results of the current study suggested that the GSTP1 polymorphism may influence the risk of B-CLL developing.

Keywords

B-CLL, genetic susceptibility, CYP1A1 polymorphism, GSTM1 polymorphism, GSTT1 polymorphism, GSTP1 polymorphism

F. Lymphoproliferated diseases (Lymphoma, Myeloma)

Impact of positron emission tomography on primary staging and changing the therapeutic modality in lymphoma patients

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Natalia B. Mikhaylova1, Marina S. Tlostanova2, Yulia N. Vinogradova2, Ekaterina I. Ivanova2, Anna V. Kritskaya2, Sergey E. Korolev1, Anna A. Rats1, Alexander A. Pugachev1, Nikolay V. Ilyin2, Boris V. Afanasyev1, Leonid A. Tyutin2

1Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia; 2Russian Research Centre for Radiology and Surgical Technologies, St. Petersburg, Russia

Objectives

We investigated the usefulness of FDG-PET in initial staging and its contribution in changing the therapeutic plan in lymphoma patients.

Methods

More than 200 PET scans were performed in 95 lymphoma patients (42-HL, 53-NHL) between 2004 and 2009. In 54 cases PET was performed at diagnosis and after completion of first-line therapy. In 41 patients the first PET study was done after first-line therapy or at the relapse of the disease. Patients with metabolic activity in nodes after first-line therapy received salvage therapy consisting either of conventional chemotherapy (CT) with PET positive node irradiation or high-dose CT with HSCT.

Results

In 20% of NHL patients PET was the only method that revealed at least one pathological lesion not confirmed by computer tomography at diagnosis. In 71.2% of patients full metabolic response was achieved after first-line therapy; 27.8% of patients remained PET positive. Additional cycles of conventional CT and radiation converted PET-positivity to PET-negativity in 19.5% of cases, compared with 16% after ASCT. Early relapse was observed in 30% of patients with metabolic activity after first-line therapy compared with 20% in PET negative patients. Seventy-one percent of patients with PET-negative status did not relapse during the time of observation, whereas only 35% of patients with metabolic activity after first-line treatment remained in remission. The death rate was 23% in the PET-positive group, while no deaths were recorded in  the PET-negative group. The overall survival rate was calculated in 41 patients and is represented in Fig. 1.

Figure 1.

2009-5-en-Mikhayliva-et-al-Figure-1.JPG

Conclusion

FDG-PET is useful at primary diagnosis. Approximately 20% of lymphoma patients could benefit from therapy changing initiated by PET status.

Keywords

lymphomas, prognosis, positron emission tomography

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Epidemiology of bacterial infections and antibiotic resistance in BMT clinic: single center experience

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Vladimir N. Vavilov1, Olga B. Ponomarenko1, Marina O. Popova1, Svetlana S. Emelyanova2, Maria Yu. Averjanova1, Oleg V. Goloschapov1, Ludmila S. Zubarovskaya1, Boris V. Afanasyev1

1Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia; 2Petersburg Nuclear Physics Institute, Russian Academy of Science, St. Petersburg, Russia

Correspondence
Vladimir Vavilov, Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia
E-mail: vladimir_vavilov@mail.ru 

Background

High-dose chemotherapy, hematopoietic stem cell transplantation (HSCT) and “graft-versus-host” disease treatment are associated with greatly increasing the risk of the most frequent and most life–threatening infectious complications.

Aim

The aim of this single-center study was to evaluate the epidemiology and antibiotic resistance of bacterial infections in oncohematological and HSCT patients.

Patients and methods

Three hundred consecutive, positive cultures were sampled from 79 high-risk patients. Cultures were provided with BacT/Alert® and Vitek® technologies.

Results

The organisms that most commonly caused infectious complications were
S. epidermidis, E. faecalis, E. faecium, S. aureus, K. pneumoniae,
Enterobacter spp., Acinetobacter spp.,
and P. aeruginosa.

The agents that most commonly caused bacteremia were S. epidermidis,
K. pneumoniae, P. aeruginosa, E. coli,
and E. faecalis. Compared with causes of bacteremia in 2006–2007, the study of 2008–2009 shows the superior but decreasing role of Gram-positive cocci and increasing rates of bacteremia episodes caused by Gram-negative agents.

Analysis of antibiotic resistance shows high efficacy of vancomycin and linezolid, cefoperazone/sulbactam and piperacillin, and moderate efficacy of carbapenems, quinolones, and aminoglycosides. In contrast, increasing resistance to some third generation cephalosporins (only ceftriaxone and ceftazidime) was observed.

We observed increasing rates of multiresistant strains of K. pneumoniae,
P. aeruginosa
and Acinetobacter spp. At the same time there is no increasing rates of Gram-positive cocci: only 12.5% of S. aureus were resistant to oxacillin (ORSA). Low rates of vancomycin resistant enterococci (VRE) infections were observed.

Conclusions

Our study shows the emerging role of Gram-negative bacteria with increasing rates of multiresistant strains. Analysis of antibiotic sensitivity shows that the principles of antimicrobial agents use in patients with hematological and oncological diseases treated in BMT clinics should be changed.

Keywords

stem cell transplantation, acute leukemia, infectious complications, antibiotic resistance

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Management of resistant or recurrent CMV infection following allogeneic SCT

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Petr Sedlacek1, Ladislav Krol1, Petr Hubacek1, David Boutolleau2, Tomas Kalina1

1Department of Pediatric Hematology and Oncology, HSCT Unit, Prague, Czech Republic; 2UPMC University Paris and Service de Virologie, GH Pitié-Salpêtrière, Paris, France

Despite the strategy of preemptive treatment of CMV infections, CMV disease is still a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Patients who failed to respond to ganciclovir (GCV) may be rescued by other virostatics or by cellular therapy. Conversely, by using preemptive strategies a lot of patients might be unnecessarily overtreated. Other tools have to be exploited in order to improve post-transplant management of CMV infection.  

We monitor CMV quantity in whole blood using real-time PCR technology. If resistance to GCV is suspected, we change the therapy and we indicate PCR detection of the most frequent CMV resistant strains. Detection of CMV-specific immune response is based on a polychromatic flow cytometry cytokine staining method. We evaluate the ability of CD4+ and CD8+ T-cells to produce interferon-γ and interleukin-2, to express activation marker CD40L, and/or to mobilize degranulation marker CD107a in response to CMV antigens.

In almost half of about 200 pediatric patients we detected CMV DNA in the sample; about 30% of them were indicated for preemptive therapy. Despite preemptive therapy CMV disease developed in 10 children (8 deceased). Clinical suspicion for CMV resistant strains was observed in 22 children and known resistance mutation was proved in 4 of them. 

Implementation of methods that allow CMV-specific T-cell reconstitution monitoring may allow us to define a subgroup of patients who are able to resolve a CMV infection without virostatics. These patients could be spared from virostatic toxicity. Inefficient reconstitution of immunity, infection with ganciclovir resistant CMV strains, and inadequate intensity of therapy are factors responsible for treatment failure.

Acknowledgements

Supported by GAUK-47807/2007, IGA ČR -NS/9996-4 and NR/9418-3.

Keywords

CMV disease, allogeneic stem cell transplantation, resistance, reconstitution of immunity, PCR monitoring

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

C-reactive protein (CRP) concentration in patients with acute myeloblastic leukemia (AML) manifestation

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Sofia G. Vladimirova, Lyudmila N. Tarassova, Gulzada N. Mustafina, Valentina V. Cherepanova

Kirov Research Institute of Hematology and Blood Transfusion, Russia

Correspondence
Sofia G. Vladimirova, Kirov Research Institute of Hematology and Blood Transfusion, Krasnoarmeyskaya str., 72, Kirov, 610027, Russia, Phone: +7 (8332) 54-51-83, Phone/Fax: +7(8332) 54-97-31
E-mail: vlsg@mail.ru

The aim of the study was to estimate the dependence of CRP concentration from the presence of an infection and the amount of leukocytes and blasts in patients with AML manifestation. We observed 24 patients with AML at the stage of diagnosis of the disease (12 male, 12 female; aged 20–76 years; median age, 49).  FAB classification variants of AML were: М0–2, М1–2, М2–14, М4–5 and М5–1. CRP concentration was measured by immunoturbidimetric assay. The statistical difference between groups was assessed by Dann’s test. Spearman’s rank correlation coefficient was calculated to measure the degree of association between CRP concentration and the amount of leukocytes and blasts. All patients were divided into 3 groups: 1 – patients without fever and infection (n=10); 2 – patients with fever and infection focal (n=7); 3 – patients with fever but without the visible focal of infection (n=7).  CRP levels in group 1 ranged from 0 to 0.043g/l (median – 0.000g/l); in group 2, from 0.007 to 0.383g/l (median – 0.044g/l); and in group 3, from 0.015 to 0.260g/l (median – 0.160g/l). When comparing groups 1 vs. 2 and 1 vs. 3, differences were significant (р<0.05); between groups 2 and 3, difference was absent (p>0.05). The correlation between CRP concentration and the amount of leukocytes and blasts was observed in the patients in group 1 (rs 0.718 and 0.676 accordingly; р<0.05). These parameters did not correlate in the patients with infectious complications (groups 2 and 3). Thus, in patients with AML manifestation the increase in the number of leukocytes and blasts in peripheral blood influences CRP production in patients with AML manifestation. Its concentration increases even more with the presence of an infection. The results enable us to consider this parameter as a marker of infection during AML manifestation.

Keywords

acute myeloblastic leukemia, AML, C-reactive protein, CRP, infection complications

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Invasive fungal disease (IFD) in patients after allogeneic hematopoietic stem cell transplantation: single center experience

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Natalia I. Zubarovskaya1, Yulia G. Vasilieva1, Natalia V. Stancheva1, Elena V. Semenova1, Vladimir N. Vavilov1, Svetlana Emelyanova2, Nikolay N. Klimko3, Boris V. Afanasyev1

1Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia; 2Petersburg Nuclear Physics Institute, Russian Academy of Science, St. Petersburg, Russia; 3Academy of postgraduate education, St. Petersburg, Russia

Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: bmt@spmu.rssi.ru

Aim

The objective was to evaluate the incidence of IFD, to determine risk factors of IFD, and to study its influence on OS in patients undergoing allo-HSCT.

Patients and methods

The study included 221 post-allo-HSCT patients (pts) between October 2000 and July 2008. The age range was 1–66 years old (median 21 y.o.) There were 150 AL pts; 27 CML, CMF and CLL pts; 11 AA pts; 8 congenital disorder pts; 17 NHL/HD pts; 6 MDS pts; and 3 solid tumor pts. Eighty-six pts underwent myeloablative conditioning, while 135 pts underwent non–myeloablative conditioning (RIC) followed by allo-HSCT. At the time of allo-HSCT, 134 pts were in CR, and 87 in relapse. Types of donor: 77 MRD pts, 135 MUD pts, and 9 haploidentical pts. Sources of HSC: 142 PBSC pts, 67 BM pts, and 12 BM+PBSC pts. Median: CD34+- 8 х106/kg. Acute GVHD prophylaxis was standard.

Results

The incidence of IFD until D+100 was 27%; after D+100, 36%. Possible IFDs were detected in 27 pts, probable IFDs in 37 pts, and proven IFDs in 2 pts. The onset of IFD was on D+1-940 (median D+86). In a multivariable analyses the following risk factors were revealed: age older than 10 years (RR=1.2; 95%CI, 1.01–1.6), usage of ATG in conditioning in pts older than 21 y.o. (RR=0.7; 95% CI, 0.59–0.88, Р<0.05), mucositis I–IV (RR=2.1; 95% CI, 1.2–3.8), chGVHD extensive form in patients younger than 21 y.o. (RR= 2.1; 95% CI, 1.3–3.4, Р<0.05). Conditioning, time of engraftment, stage of disease, and source of HSC were not independent risk factors for IFD. In pts younger than 21 y.o., synergistic effect of the following factors was detected: relapse, myeloablative conditioning, and BM as a source of HSC (RR=0.4; 95% CI, 0.21–0, 76, Р<0.05). In pts older than 21 y.o. with MRD after RIC and a source of HCS PBSC, the incidence of IFI was lower (RR=1.59; 95% CI, 1.01–2.51, Р<0.05). Overall survival (OS) in pts undergoing HSCT with IFD versus without IFD is 25% vs 53% (p<0.005).

Conclusion

The rates of IFD after HSCT remain high and impair OS.

Keywords

related allo-HSCT, unrelated allo-HSCT, allo-HSCT invasive fungal disease

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Hemostasis in patients with acute myeloblastic leukemia (AML) manifestation

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Valentina V. Cherepanova, Sofia G. Vladimirova, Lyudmila N. Tarassova, Gulzada N. Mustafina, Natalia N. Silina

Kirov Research Institute of Hematology and Blood Transfusion, Kirov, Russia

Bleeding and infections in acute leukemia (AL) are the most frequent and dangerous complications. Autopsy results of 65 patients treated in our clinic from 1992 to 2000 testify the importance of studying blood coagulation disorders in AL: hemorrhages are cited as the cause of death in 28.6% of cases, thrombosis in 10.8% of cases. The aim was to estimate the hemostasis in patients with AML manifestation. A total of 74 patients with AML manifestation were observed (39 males and 35 females, aged 16–77 years, median age of 42). FAB classification variants of AML were as follows: М1 – 18, М2 – 39, М4 – 8, and М5 – 9. Of the 74 patients, 67 had complications, 27 of them infectious, and 46 hemorrhagic. Type I hemorrhagic syndrome was observed in 10%, II in 28%, III in 9% and IV in 20% (classification of Dabberha Nafa, 1992). A mild degree of thrombocytopenia was noticed in 25% of patients, moderate in 34% and deep in 11% (classification of Agranenko VA, 1998). The mean of APTT corresponded to the norm, but was prolonged in 17.6% of patients. Fibrinogen concentration was increased. Its decrease is revealed only in 13.5% of patients. Increase of intravascular coagulation markers was observed, such as soluble fibrin monomer by o-phenanthroline (25.7%), by ethanol tests (74.3%), and D-dimers (9.5%). There was plasminogen activation in all patients. Hageman-dependent euglobulin clot lysis on average was considerably prolonged, that in the case of hypofibrinogenemia did not exclude fibrinolysis activation. It is confirmed not only by literature data but also by our patients: in 7 patients its shortening was noted. Simultaneous antithrombin III activation and protein C consumption were established. Thus, in patients with AML manifestation high frequency of hemorrhagic complications was revealed. The complications are related not only to thrombocytopenia but also to changes in plasma coagulation.

Keywords

acute myeloblastic leukemia, AML, hemostasis, bleeding complications, hemorrhagic syndrome

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Correlations between reactivation of herpesviruses and common complications of allo-HSCT

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Olga S. Pankratova, Alexey B. Chukhlovin, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Patients and methods

We studied 145 patients with different hematological malignancies, including ALL (n=51), AML (n=37), CML (n=15), lymphomas (n=9), and MDS (n=6). They underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). The patients received bone marrow (35%), or peripheral blood stem cells (65%). Allo-HSCT from an unrelated donor occurred in 64% of cases. CMV-, HSV-, and EBV-specific DNA in leukocytes was detected weekly – with commercial PCR kits – up to 100 days after allo-HSCT. Cases of pneumonia, neurological signs, herpetic rushes, gut mucositis, hemorrhagic cystitis, and aGVHD grade were registered.

Results

HSV-, EBV-, and CMV-specific DNAs in post-HSCT blood samples revealed rates of 51%, 57%, and 45%, respectively (2.3 to 2.5-fold exceeding pretransplant rates). Types of underlying malignancy and gender did not influence these ratios. Following myeloablative versus reduced conditioning regimens, HSV positivity (>2 positive findings) was found in 36% and 21% of cases (p=0.02), respectively. Similar differences with CMV and EBV positivity were expressed only as tendencies.

Meanwhile, the incidence of HSV and CMV findings was age-dependent, i.e., minimal frequencies of PCR positivity were observed from 1–4 years of age, followed by increased viral reactivation from the age of 10–20 years. Among young patients (<21 years old), a correlation was found between neurological symptoms and multiple HSV positivity (p=0.002). Similarly, mucositis severity was associated with the persistence of either HSV, or CMV (p values, 0.02 and 0.008, respectively). Risk of intestinal aGVHD proved to correlate with EBV positivity (p=0.008). Similarly, a risk for posttransplant cystitis was dependent on EBV reactivation (p=0.01).

Conclusion

The study suggests that there is a correlation between repeated positivity of HSV, EBV, CMV, and some common complications after allo-HSCT. The significance of herpesvirus infection after allo-HSCT may be tested in further studies.

Keywords

hemopoietic cells, transplantation, complications, herpesviruses, reinfection

G. Complications in hematology (infections, haemostasis abnormalities, fertility)

Risk factors of invasive candidiasis in children with leukemia

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Elmira G. Boichenko, Alexey S. Kolbin

City Children's Hospital № 1, St. Petersburg, Russia

Methods

512 children with leukemia were analysed: acute lymphoblastic leukemia (ALL) – 310, acute myeloid leukemia (AML) – 76, relapse of leukemia – 126 children. The median age was 7 years. There were 341 boys (67%) and 171 girls (34%).

To determine a statistical significance of risk factors of IC development the following computing methods and  importance of distinctions criteria were applied: odds ratio (OR), a confidential interval (CI), criterion р, median (Ме).

Results

The frequency of IC was 4.9% (25 patients). The statistically significant risk factors of IC development were the following: relapse of leukemia (OR 0.24, 95% CI 0.06–0.89, p <0.001), treatment of relapse (OR 0.24, 95% CI 0.06–0.91, p <0.001), duration of neutropenia (< 0.5x109/l) more than 14 days (OR 0.35, 95% CI 0.08–1.51, p=0.008), use of carbapenems (OR 0.18, 95% CI 0.06–0.59, p=0.002) and vancomycin (OR 0.23, 95% CI 0.07–0.72, p=0.014).

Conclusions

1. Risk factors of IC development in children with leukemia were the following: relapse of leukemia, treatment of the relapse, duration of neutropenia more than 14 days, the use of carbapenems and vancomycin for the antibacterial treatment. 2. Patients with risk factors of IC should receive systemic antifungal prophylaxis to prevent the development of life-threatening infection.

Keywords

leukemia, children, invasive candidiasis, risk factors

H. Autoimmune diseases and aplastic anemia

Mesenchymal stem cell transplantation in patents with amyotrophic lateral sclerosis and multiple sclerosis

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Miroslav M. Odinak1, Gennadiy N. Bisaga1, Andrey V. Novitsky2, Vadim V. Tirenko2, Dmitriy N. Sculyabin1, Dmitriy G. Polyntsev3, Petr V. Krugliakov3, Anna N. Bilibina3, A. Kolchenko3

1Department of Neurology and 2Department of  Hematology and Clinical Immunology of the Military Medical Academy, St. Petersburg; 3Trans–technologies Ltd., St. Petersburg, Russia

Correspondence
Andrey V. Novitsky, Dept. of Neurology of the Medical Military Academy, Lesnoy prospekt 2, 194044, St. Petersburg, Russia
Phone: +7812292-33-37, Fax +7812542-88-19
E-mail: anov1@mail.ru

Background

Stem cell transplantation is suggested to be the most promising therapeutic strategy for degenerative and progressive diseases, among which the most significant are amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Human mesenchymal stem cells (MSCs) are thought to be multipotent cells that have the potential to differentiate to lineages of mesenchymal tissues including bone, muscle, neurons, and oligodendrocytes.

Materials and methods

In this study autologous MSCs were isolated from the bone marrow of 10 patients with ALS (5F, 5M, aged 40–63 ys, duration 18–57 mths, 32–20 points of ALSSS scale) and 3 patients with MS (1F, 2M, aged 30–37 ys, duration 4–14 ys, SP course (2 pts) and RR course (1 pt), EDSS 3.5–6.5). Growth kinetics, immunophenotypes, and karyotypes were evaluated during in vitro expansion. The in vitro expanded MSCs did not show any bacterial or fungal contamination, hemopoietic cell contamination, chromosomic alterations, or early cellular senescence. The patients received monthly intravenous injections of autologous MSCs in doses of 2 x 106 cells/kg for 3–20 months. No signs of abnormal cell proliferation were observed.

Results

No significant acute or late side effects were evidenced. Minor adverse events were headaches and transient hyperthermia after MSC infusion in two ALS patients, reversible after 10 hours. Four ALS patients died 3–6 mths after start of therapy due to conditions not directly related with MSC therapy: 1 pt committed suicide (had not had any positive results from therapy), 2 pts died of cardiac arrest (both had a long history of ischemic cardiac disease),
1 pt died of disease progression and of refusing mechanical ventilation. Three ALS patients show a significant (for 4–10 mths), 2 –doubtful (for 1–3 mths), and 5 lack of progression slowing.  The EDSS scores of all 3 MS patients show a decrease in 1 point despite repeated relapses every 3 mths in 1 pt.

Conclusion

Our results seem to demonstrate that MSCs represent a good chance for stem cell therapy in MS, but are considerably less preferable in ALS patients. MSC therapy was safe for the observed treatment period. 

Keywords

stem cell, cell therapy, multiple sclerosis, lateral sclerosis, transplantation

H. Autoimmune diseases and aplastic anemia

Outcomes of hematopoietic cell transplantation (SCT) and combined immunosupression (IST) patients with severe aplastic anemia (SAA) admitted with life-threatening infections

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Galina A. Novichkova, Mikhail A. Maschan, Lili A. Khachatrian, Dina D. Baidildina, Olga V. Goronkova, Galina G. Solopova, Elena V. Skorobogatova, Irina P. Shipitsina, Alexey A. Maschan

Federal Research Center for Pediatric Hematology, Oncology, and Immunology, Moscow, Russia

Introduction

Infections remain a dominant cause of treatment failure in children with AA. Often severe infections present at admission are considered as contraindications to curative therapy.

Patients

Among 164 pts admitted with AA between 1998–2005, 35 (21%) had serious infections. There were 23 males and 12 females (med age 10.6 y) with SAA (2 pts) or very SAA (33 pts). Twelve pts were affected by bacterial sepsis, 9 by invasive mycoses (invasive aspergillosis (IA) 7, candidiasis 2), 4 by mixed fungal and bacterial infections, 2 had pneumonia, 6 severe stomatitis, 3 sepsis of unknown origin and 1 had cellulitis. One pt died of Gram (-) septic shock – aged 24 hours – after admission, while in the other 34 pts either SCT (5 pts) or combined IST with ATG+Cyclosporine A (29 pts) were carried out. Med interval from admission was 28 d for SCT and 10 d for IST. Intensive appropriate antimicrobial therapy was given to all patients immediately after admission.

Results

In all 5 SCT recipients complete resolution of infections (all IA) was achieved after bone marrow recovery. One pt died on day 176 from GVHD, CMV, and graft failure; the other 4 pts are alive. Of the 29 recipients of IST 14 (48%) are alive in complete (12 pts) or partial (2 pts) remission, whereas 15 died. Death occurred during the first, second, and third mo in 1, 2 and 1 pts respectively.  In 8 nonresponders to IST the main cause of death was primary infection, whereas in 7 it resolved completely and they died from secondary infections or hemorrhages. As of 09/2008, 18 (51%) of the original 35 pts are alive: the OS rate is 51%±8% and failure-free survival is 31%±7%.

Conclusion

Intensive antimicrobial therapy allows the safe performing of SCT or IST in severely infected pts with SAA. Most of them survive the early post-treatment period, while half are long term survivors.

Keywords

severe aplastic anemia, infections, hematopoietic cell transplantation, combined immunosupression

H. Autoimmune diseases and aplastic anemia

Alternative method for treating acquired aplastic anemia

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Yuri I. Yugov, Nina A. Fedorovskaya, Alexander S. Luchinin

Research Institute of Hematology and Blood Transfusion, Kirov, Russia

Acquired aplastic anemia (AA) is an autoimmune disorder which is characterized by pancytopenia and hypocellular bone marrow. Combined immunosuppressive therapy (CIT) including the use of antithymocyte globulin (ATG), cyclosporine-A (CS) and methylprednisolone (MP) is the main therapeutic method. However, responding patients will frequently have relapses, become dependent on CS, or develop a secondary clonal disease.

In 1987, researchers at our hematology clinic formulated the aim of scientific investigations in this direction. The purpose of their work was to create an alternative protocol of CIT AA that would be no less effective than existing protocols. Cyclophosphamide (CPh) was used as the main immunosupppressive drug. Moreover, splenectomies (SE), MP and cyclosporine (CS) were principally used in this scheme. Protocol was being constantly modified. The final protocol version is given below.

MP was administered intravenously at 500–250 mg on days 1–7, and after that orally at 36 mg per day with a subsequent gradual cancellation over 1.5 months. CS was administered at 5mg/kg per os daily from day 1 and continued for 12–28 weeks. SE was carried out on days 4–7 from the beginning of MP. CPh was administered intravenously at 200 mg per day. №5 started on days 4–10 after SE, then №5 every other day, then two times a week until transfusion independence was achieved. However, the general dose was not allowed to exceed 4 g.

This protocol for CIT was applied to 13 patients aged 13 to 43 years (7 males, 6 females). Nonsevere AA was diagnosed in 2 patients, severe AA in 8 and very severe AA in 3 cases. Remission was achieved in 11 patients, 2 patients died during therapy, 1 patient had a relapse, and 1 developed myelodysplasia. Of the 13 patients, 8 are alive and in complete remission.

Keywords

aplastic anemia, treatment, cyclophosphamide, splenectomy

H. Autoimmune diseases and aplastic anemia

The severity of acquired aplastic anemia (AA) and long-term prognosis: lessons from current results of immunosuppressive treatment (IST)

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Alexander D. Kulagin1,2, Igor A. Lisukov1,2, Vyacheslav I. Borisov2, Irina V. Kruchkova2, Vera V. Sergeevicheva2, Svetlana A. Sizikova2, Andrey V. Gilevich2, Vladimir S. Kozhevnikov2, Vladimir A. Kozlov2

1Novosibirsk State Medical University, Novosibirsk, Russia; 2Institute of Clinical Immunology SB RAMS, Novosibirsk, Russia (EBMT CIC 375)

Correspondence
Alexander D. Kulagin, Yadrintsevskaya str, 14, 630047, Novosibirsk, Russia
E-mail: kulagingem@rambler.ru

Objectives and Methods

We retrospectively analyzed the outcome of 65 patients (39 M and 26 F, median age 21) with moderate (MAA 22), severe (SAA 26) and very severe (VSAA 17) AA treated with ATG and CsA+/-Daclizumab (44) or with CsA alone (21) from October 1995 to May 2009. 

Results

Twenty-two (34%) and 25 (38%) patients achieved CR and PR, respectively (according to strict response criteria of B. Camitta, 2000). The quality of response was higher in the VSAA group (CR/PR=11/1) than in the SAA (9/10, p=0.02) and MAA (2/14, p=0.001) groups.

There were 2 early and 7 late deaths. Four patients (8.5%) relapsed and 3 responded again after re-treatment with ATG and CsA. Late events included MDS/AML (n=2), rectal cancer (n=1) and hemolytic PNH (n=5). The 5-year overall survival was 82 ± 6% without any difference between the MAA (81.6 %), SAA (82.1 %), and VSAA (82.4 %).  Surprisingly, there was a marked trend towards better failure-free survival (FFS) in more severe disease (32.6 ± 12.6%, 45.2 ± 11.8% and 68.8 ± 11.7% in MAA, SAA, and VSAA respectively), which was also observed even when the CsA group alone was excluded from analysis. 

The results of the telomere length measurement in PB cells by flow-FISH analysis in 35 patients gave a possible explanation for better quality of response and FFS in VSAA. The responders with VSAA have a less marked telomere loss than in MAA and SAA patients, probably due to lack of intrinsic stem cell defects and early intensive IST.    

Conclusions

Modern IST provides a high rate of hematological response and long-term survival in more than 80% of AA patients. Nevertheless, different treatment-failure events remain a crucial problem. The severity of AA is a controversial prognostic factor. Our data indicates that more severe AA predicts a better long-term prognosis.

Keywords

aplastic anemia, severity, ATG, cyclosporine, response, telomere, prognosis  

H. Autoimmune diseases and aplastic anemia

Immunoablation followed by autologous stem cell transplantation in refractory autoimmune diseases: clinical outcomes and immune reconstitution

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Igor A. Lisukov1,2, Vera V. Sergeevicheva2, Svetlana A. Sizikova2, Alexander D. Kulagin1,2, Irina V. Kruchkova2, Andrey V. Gilevich2, Alexey E. Sizikov2, Lyudmila P. Konenkova2, Elena R. Chernykh2, Vladimir S. Kozhevnikov2, Alexander A. Demin1, Vladimir A. Kozlov2

1Novosibirsk State Medical University, Novosibirsk, Russia; 2Institute of Clinical Immunology SB RAMS, Novosibirsk, Russia

Correspondence
Alexander D. Kulagin, Institute of Clinical Immunology SB RAMS, Yadrintsevskaya str, 14, 630047, Novosibirsk, Russia
E-mail: kulagingem@rambler.ru

High-dose immunosuppression and autologous hemopoietic stem cell transplantation (ASCT) has been proposed as an investigational therapy for patients with refractory autoimmune diseases (AD). We report the results of a single-center study of ASCT in 15 patients with refractory systemic lupus erythematosus (SLE), 7 patients with progressive multiple sclerosis (MS),
1 patient with autoimmune thrombocytopenia (ITP) relapsed after splenectomy, and 1 patient with pure red cell aplasia (PRCA) in our institution from 1998 to 2009.

Methods: Autologous HSC were collected from bone marrow (n=4) or mobilized from peripheral blood with either granulocyte colony-stimulating factor (G-CSF) (n=2) or Cy and G-CSF (n=18).

Results

Three SLE patients died due to transplant-related complications, 1 MS patient died due to t-AML 4 years after ASCT, 2 SLE and 1 PRCA patients died after 8, 7 and 2 years respectively due to relapses. At a median follow up of 54 (16–124) months 7 patients are in complete remission (4 SLE, 2 MS,
1 ITP). Three MS patients are in a stabilization phase, while 1 MS patient relapsed 5 years after ASCT. CR observed in SLE patients is accompanied by a disappearance of anti–ds DNA and ANA antibodies and an increasing number of CD4+CD45RA+ T cells, CD4+CD25+bright T cells and CD4+Foxp3+ cells. We also demonstrated the increase of CD4+ and CD8+ T cells in the S/G2M phase of the cell cycle until1 year after ASCT.

Conclusion

ASCT in refractory AD can induce stable long-term remissions; however, the majority of patients relapse. The assessment of immune reconstitution can be important to understanding the mechanisms of self-tolerance
re-establishment. International clinical trials would be required to clarify these questions. 

Keywords

immunosuppression, autologous hematopoietic stem cell transplantation, lupus erythematosus, multiple sclerosis, autoimmune thrombocytopenia

J. Myeloproliferative diseases

Decitabine for treatment of relapsed myelodysplastic syndrome after allogeneic stem cell transplantation: Case report

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Yulia V. Rudnitskaya, Elena V. Morozova, Boris V. Afanasyev

Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Elena V. Morozova, Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: dr_morozova@mail.ru

Introduction

The only curative treatment available for myelodysplastic syndromes (MDS) is hematopoietic stem cell transplantation (HSCT). There are two types of conditioning regimens followed by HSCT: reduced-intensity conditioning (RIC) and standard (myeloablative) conditioning. RIC regimens were designed to perform allogeneic HSCT in patients who had a high risk of death due to toxicity of standard preparative chemotherapy regimens (elder patients, patients with severe comorbid conditions, or heavy pretreated patients). However, the use of RIC can increase the risk of relapse after HSCT. We report the single case of treatment with decitabine of relapsed MDS after RIC matched related allograft stem cell transplantation.

Patient characteristics

The patient was a 49-year-old female, who had been diagnosed with MDS, refractory anemia with an excess of blasts II, and IPSS-3, which was verified in September 2007 based on anemia, leucopenia, blasts in bone marrow >10%, multiple dysplastic signs, and multiple chromosomes’ disorders (46,хх,del(2)(p22),add(3)(q29)[15] / 46,хх,del(2)(p22) [3] / 46,xx [2]).

SCT and follow up

Due to high risk MDS (IPSS-3), it was decided to perform allograft HSCT from an HLA identical sibling as the first line treatment (day 0, 18 January 2008). The conditioning regimen was RIC (Fludarabin+Busulfan). GVHD prophylaxis was CSA + MTX.

The engraftment was carried out on day +16. On day +30 the patient had mixed chimerism (50%); 46,xy; and complete disappearance of chromosomes’ disorders and no evidence of acute GVHD.

However, disease relapse was revealed on day +68 (15% of blasts in bone marrow, cytopenia, multiple chromosomes’ disorders). On day +72 CSA was stopped. On day +82 a donor lymphocytes infusion (DLI) №1 was performed. Nevertheless, on day +93 there were still signs of disease progression (22% of blasts in bone marrow, mixed chimerism 30%).

Decitabine 20 mg/m2/day, 1-hour IV infusion for 5 days, was started with the main goal to reduce the blasts quantity in the bone marrow. DLI №2 and DLI №3 were performed on days +110 and +136, respectively. Since day +154 patient has had chronic GVHD of the skin.

We achieved complete remission: normal blood counts, normal account of blasts in bone marrow, full donor’s chimerism, and 46,xy.

Conclusion

Combined treatment (Decitabine and DLI) showed clinical activity and can be used for treatment of relapsed myelodysplastic syndrome after allogeneic HSCT.

Keywords

myelodysplastic syndrome, reduced-intensity conditioning, allogeneic HSCT, relapsed myelodysplastic syndrome, donor lymphocytes infusion, decitabine

J. Myeloproliferative diseases

Index granularity of neutrophils in peripheral blood as a new application for screening myelodysplastic syndromes using flow cytometry

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Ekaterina B. Rusanova1, Konstantin U. Slobodnyuk1, Margarita V.Gorchakova1, Yekaterina E. Zueva1, Ramon Simon-Lopez2

1Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia; 2Ramon Simon-Lopez, Beckman Coulter, Nyon, Switzerland


Correspondence
Ekaterina B. Rusanova, Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
Phone/Fax: +7 (812) 233 9726, E-mail: katerina.rusanova@gmail.com

Purpose

Myelodysplastic syndrome (MDS) is a group of clonal bone marrow disorders. The list of features used commonly for diagnosis of MDS includes several morphological cell characteristics, such as the presence of hypogranulation in the neutrophils. We have observed in our daily experience that the NE Mean scatter (SS) was lower in MDS than in normal cases and the scatter of lymphocyte SS is quite constant in normal cases and in the majority of MDS.

Methods

Twenty-four diagnosed with MDS and 16 normal blood donors as a control group were enrolled into this study. The protocol of study included evaluation of blood cell populations with the GenS (BC) hematological analyzer, smears microscopy, and flow cytometry for identifying neutrophils granularity levels (FC500, BC). We created a single tube protocol for immunophenotyping of blood cells with multiparametric flow cytometry using a panel of monoclonal antibodies: CD14-FITC, CD16-PE, CD33-PC5, and CD45-PC7 (BC). In order to standardize the NE mean SS we also used an index granularity of neutrophils (IGN), which was calculated by dividing the neutrophil mean SS by lymphocyte mean SS.

Results

The results show that IGN was significantly lower in MDS than in the normal group (p<0.001). We obtained AUC=0.874 (significance level 0.0001) with a cut-off of <=8.34, a sensitivity of 87.5% with a specificity of 81.5% using the IGN to detect MDS.

Conclusions

The information about hypogranularity of the neutrophils is a well-known feature of MDS. Using IGN may be a useful tool to detect or flag neutrophil hypogranularity and could facilitate the differential diagnosis of MDS.

Keywords

myelodysplastic syndrome, dysplasia, hypogranularity, flow cytometry, screening, peripheral blood

J. Myeloproliferative diseases

Case report: combined myeloid and lymphoid lineage disorders in patient with chronic eosinophilic leukemia and T-cell lymphoma

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Margarita V. Gorchakova, Ekaterina B. Rusanova, Konstantin U. Slobodnyuk, Irina J. Saburova, Elena A. Stadnik, Michail I. Zaraiski, Yekaterina E. Zueva

Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia


Correspondence
Margarita V. Gorchakova, Department of Clinical Laboratory Diagnostics, Center for Laboratory Diagnostics, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: Rita.gorchakova@gmail.com 

Aims

We present a rare clinical case of combined myeloid and lymphoid lineage disorders in patients with chronic eosinophilic leukemia and T-cell lymphoma.

Materials

The patient (female, 51) complained of general weakness, a permanent hyperthermia up to 38.5–39ºC, constant painful dry cough, and also she expressed redness, peeling and itchy skin on her face. An external inspection showed a significant peripheral lymphadenopathy. A clinical blood analysis revealed the following: leukocytes – 21.9 *10^9 /L, segmented neutrophils – 47%, eosinophilia – 17% (3.7 *10^9 /L), lymphocytes – 28% (6.1 *10^9 /L), monocytes – 8%, hemoglobin – 96 g /L, and ESR – 32 mm /h. Up to 44% of eosinophils were found in the myelogram.

Results

The bulk of lymphoid bone marrow cells were represented by a homogeneous population of CD2+CD3+CD4+CD5+ T-cells. A significant portion of the T-cells had an early thymic phenotype CD4+CD8+CD3+. The eosinophils phenotype was represented by markers CD13+CD15+CD33+ with a lack of CD117 and CD23. Molecular biological studies identified chimerical gene transcripts FIP1L1/PDGRFα and monoclonal T-cell receptors. The level of mRNA expression of the interleukin–5 gene was negative.

Summary

T-lymphocytes represented a clone of transformed cells confirmed by the molecular genetics data (receptor clonality) and flow cytometry (aberrant phenotype). The presence of mutations of the gene FIP1L1/PDGRFα showed the clonal nature of hypereosinophilia. A lack of increase in IL5 proved the lymphoproliferative variant of HES to be incorrect. Thus, using molecular genetics and immunological techniques we verified the presence of two diseases: chronic eosinophilic leukemia and T-cell lymphoma unspecified IVA.

Keywords

hypereosinophilic syndrome, T-cell lymphoma, case report, chronic eosinophilic leukemia

K. Inherited diseases

Hematopoietic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID): experiences, possibilities, and prospects

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Dmitry N. Balashov, Pavel E. Trakhtman, Elena V. Skorobogatova, Yulia V. Skvortsova, Zarema M. Dyshlevaja, Irina P. Shipitsina, Alexey A. Maschan, Alexander G. Rumyantsev

Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow, Russia

Correspondence
Dmitry N. Balashov, Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, 117/2, Leninsky prosp, Moscow, Russia, 105062
Phone: +7(495) 9369023, Fax +7(495)9369120
E-mail: bala8@yandex.ru

HSCT is the standard, and an effective method of therapy of SCID patients. Unfortunately, the poor somatic status of children and/or infectious complications are often grounds for the modification of the existing standard approach to HSCT in SCID. In some cases, decreasing the intensity of conditioning regimens seems to be the cause of mixed chimerism, delayed immunological reconstitution, long-term (or life-long) demand for IVIG transfusions, and an increase in morbidity and mortality after HSCT. Currently there are many publications about gene therapy. It’s a new and promising approach to the treatment of SCID patients. Perhaps it will be a good alternative for the standard HSCT method in the future.

Between 1997 and 2009, twelve patients (1 female and 11 males) with a median age of 6 months (range 2–12 months) received standard HSCT for SCID at the Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology. In 7 patients we found genetic mutations, and confirmed X1-SCID (n=5), ADA-deficient-SCID (n=1), and Omenn's syndrome (n=1). In other cases we did not discover any genetic mutations, but patients showed clinical signs and immunological phenotypes that match SCID. One patient was transplanted from an HLA-compatible sibling donor, other cases were treated with haploidentical HSCT from their parents. At present, the median follow-up is 76 months (range 3–164 months); 7 children are alive and have full donor chimerism, and five of them demonstrate full immunological reconstitution. 

Currently, gene therapy is not available for most patients, but HSCT is an effective method of treatment for SCID patients.

Keywords

SCID, transplantation, conditioning, gene therapy, children

L. Hemoglobinopathies

Outcomes after addition of rabbit-ATG to the standard Bu+CY preparative regimen for allogeneic matched sibling donor (MSD) hematopoietic stem cell transplantation (HSCT) for hemoglobinopathies in children: a single center experience

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Sandeep Soni, MD1, Micah Skeens, RN, MSN1, Thomas E. Gross, MD1, Rajinder P. Bajwa, MD1, Kathryn Klopfenstein, MD2,
K. Scott Baker, MD, MS3, Amanda M.Termuhlen, MD1

1Nationwide Children’s Hospital, Columbus, OH, USA 43205; 2Department of Pediatrics, East Tennessee State University, Johnson City, Tennessee, USA; 3Seattle Cancer Care Alliance, Seattle, WA, USA 98109

Introduction

Allogeneic HSCT for hemoglobinopathies following myeloablative conditioning in a matched sibling donor (MSD) setting has resulted in excellent outcomes in children (90–95% EFS). Though the outcomes are encouraging, there is still a 7%–10% incidence of rejection, 15–20% incidence of acute GVHD, and 12%–15% incidence of chronic GVHD. Also, there is a high incidence (15–30%) of non-compliance and lack of adherence with oral medications in this population due to multiple factors. The addition of anti-thymocyte globulin (ATG) leads to in-vivo T-cell depletion improving engraftment. We proposed the hypotheses that using a medium dose of r-ATG (10 mg/kg; Thymoglobulin, Genzyme, Cambridge, USA; equivalent to ~100 mg/kg of Fresenius r-ATG) during the peritransplant period will eliminate the risk of rejection and GVHD, especially in this psychosocially-challenged patient population where non-compliance with GVHD prophylactic medications is very high.

Methods

Patients of severe sickle cell disease (SCD) with sequelae and transfusion dependent beta thalassemia – who had appropriately matched sibling donors – have been enrolled in an IRB approved study since January 2003. All patients have received IV busulfan (16 mg/kg over 4 days with targeted AUC 900–1350 µM/mt, based on first-dose pharmacokinetics), cyclophosphamide (50 mg/kg for 4 days), and r-ATG (Thymoglobulin, 2.5 mg/kg/day from days -6 to -3). GVHD prophylaxis was with cyclosporine (dose adjusted to maintain a nadir serum level of 200–300 ng/ml for 6 months) and a standard short course of methotrexate (15 mg/kg followed by 10 mg/kg IV) given on days +1, +3, and +6. All patients have received levetiracetam for seizure prophylaxis.

Results

Ten patients (8 severe SCD and 2 beta thalassemia) have received the MSD HSCT using the described preparative regimen. Median age of patients’ was 4 years (18 months–18 years) and there were 6 females and 4 males. The regimen was very well tolerated. All patients developed mucositis grade II–III. One patient developed mild VOD that responded to supportive care. Most of the patients developed hypertension secondary to cyclosporine use and required aggressive anti-hypertensive management. There was no incidence of seizures or reversible posterior leukoencephalopathy syndrome. One patient developed CMV reactivation on day +17 and responded to IV foscarnet. No other viral (CMV, EBV, HHV-6, etc.) or fungal infections were detected.

All patients have received BM grafts from MSD with the median cell dose of 3.5 x 108 TNC /kg (1.8–10.1 x 108/kg). One hundred percent have engrafted with ANC >500 by a median of day +15 (range: 10–23 days). Serial peripheral blood donor chimerism analysis post-HSCT has revealed stable engraftment in all patients ranging from 51–100%. None of the patients have developed acute GVHD. One patient developed limited chronic GVHD of the skin requiring only topical treatment. All patients assessed >1 year post-HSCT have reconstituted their immune system. All SCD patients have shown complete resolution of disease related symptoms. All thalassemia patients are transfusion free and end organ function is stable in all patients >1 year post-HSCT.

Conclusions

Addition of r-ATG to the standard Bu+CY myeloablative regimen has further reduced the incidence of rejection and acute/chronic GVHD without increasing the toxicity or incidence of viral infections in HSCT for hemoglobinopathies.

Keywords

HSCT, hemoglobinopathies, sickle cell disease, sibling donor, rabbit ATG

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Haploidentical SCT as a salvage therapy in hematological malignancies: A single center experience

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Olesya V. Paina, Yulia A. Stankevich, Ilya V. Kazantsev, Natalia V. Stancheva, Alla A. Golovacheva, Elena V. Babenko, Alexander L. Alyanskiy, Natalia E. Ivanova, Elena V. Semenova, Petr V. Krugliakov, Dmitriy G. Polyntsev, Liudmila S. Zubarovskaya, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia, Phone: +7 (812) 232-81-20
E-mail: bmt-registry@spmu.rssi.ru

Background

The usage of haploidentical donors is one curative option for patients (pts) with acute leukemias without a donor – related or unrelated – match.

Patients and methods

Twenty-four very high-risk pts underwent haploidentical SCT: ALL –10 (42%) pts, AML – 11 (44%) pts, JMML – 1pt, CML – 1pt, and resistant neuroblastoma – 1pt. The total number of resistant/in progression pts was 16 (66%), while 8 (33%) pts were in remission. Nineteen (79%) pts were children (aged 1–18), and 5 (21%) were adults (aged 21–47). In each case reduced intensity conditioning regimens (RIC) were used: Flu and ATG with the addition of different alkylating agents (busulphan, melphalan or thiotepa). Sources of HSC-PBSC and bone marrow. For PBSC CD34+ positive selection was performed with CliniMACS. The mean CD34+ count was 12.8x106 /kg (1.6–30.7). In 20 pts aGVHD prophylaxis consisted of CsA and a short course of MTX with or without MMF. In 4 pts tacrolimus and MMF were used. In 2 pts in D-1, mesenchymal stem cells (MSC) from third–party donors were used to prevent aGVHD; while in 3pts, MSC was used for the treatment of aGVHD.

Results

The incidence and severity of aGVHD were no greater than in other types of allo-HSCT: 6 (25%) pts had grade III–IV aGVHD with skin and gut involvement and 1 pt died. In the case of MSC usage in the conditioning regimen only aGVHD stage I was observed. The treatment of aGVHD with MSC was successful: of 3pts, 2 achieved CR. The toxicity of the conditioning regimen was acceptable: 6 (25%) developed grade II–III organ toxicity, 5 (21%) pts had invasive aspergillosis, and 8 (33%) pts had CMV reactivation. The 1-year OS was 62%, with median observation terms of 4.6 months (1 to 12 months). Five pts died of relapse and 3 in CR (1pt from infection and another from engraftment and aGVHD of the gut).

Conclusions

Haploidentical HSCT with RIC is characterized by acceptable toxicity, aGVHD control, and stable engraftment. It proved to be a good option for the group of pts with poor prognosis. Randomized clinical trials are necessary for an estimation of the therapeutic effect of MSCs in haploidentical HSCT pts.

Keywords

haplo-SCT, alternative donor source, salvage therapy

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Non-T-cell depleted haploidentical HSCT after RIC in pediatric malignancies

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Natalia S. Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich

Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia

Correspondence
Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia
Phone: +7 (903) 1990666
E-mail: irdolg@rambler.ru

The main purpose of this study was to evaluate the potential outcomes of haploidentical hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning regimen (RIC) in the management of pediatric solid and hematological malignancies.

Our protocol, based (mostly) on fludarabine, ATG, and busulfan, was used in 40 pediatric patients with refractory hematological (n=28) or solid (n=12) malignancies. Diagnoses were: AML: 10, ALL: 4, CML: 4, JMML: 5, MDS: 1, NHL: 4, NB: 7, Ewing's S: 4, and melanoma: 1. The median age of patients (pts) was 8.5 yrs (1–18). HLA compatibility was: 3/6:  62.5%, 4/6: 27.5%, and 5/6: 10%. In vitro graft T-depletion with vincristine and methylprednisolone was the only procedure performed.

Three pts with leukemia progression at the time of transplantation didn’t recover and died. Four pts with JMML/MDS rejected and relapsed less than 2 months after transplantation. Thirty-three pts (82.5%) recovered, and achieved full donor chimeras after transplantation. Toxicity was mild in all but one case. Incidence of acute GVHD II–IV in first 100 days was 53%
(gr IV=0). Incidence of chronic GVHD was 52%. Relapse rate 1 yr after transplantation was 75% and 33% for solid tumors and hemoblastoses respectively. In two pts with hemoblastoses the second transplantation was successful. In the entire group the results were as follows: 9 pts (22.5%) now alive, with median follow-up of 41 months (8.1–88.5); 20 (50%) pts died due to relapse; and 11 (27.5%) died of other causes: 2 pts (5%) of acute GVHD, 7pts (17.5%) of chronic GVHD, and 2 pts (5%) of infection.

Haploidentical HSCT after RIC in children can provide long-term anti-leukemia/lymphoma effect without significant complications. Patients with JMML/MDS and solid tumors need additional therapeutic modalities.

Keywords

anti-tumor effect, reduced intensity, haploidentical hematopoietic stem cell transplantation, children

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Properties of cord blood stem cells chromatin

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Vladimir S. Tolmachev, Elizaveta I. Borovitskaya, Natalia O. Kudrina, Tatiana G. Kravtsova

Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan

Correspondence
Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan
E-mail: lab_cyto_kz@mail.ru

We conducted a cytochemical analysis of the structure and function of chromatin found in cord blood mononuclear cells (MNC). In the cellular cycle, 97.9% MNC were in stage G0-G1, 1.1% in the phase of synthesis, 0.9% in G2-M, and proliferation 1.9%. The expression of receptors CD 34+ and CD 45+ was changed by the cultivation of MNC: chromatin was destabilized according to the data of histone and non-histone proteins. The growth factors were approved in the culture of MNC. The level of MNC with apoptosis in an unfrozen sample increased. The changes of the supramolecular structure of MNC chromatin were displayed in acridine orange fluorochromes and rivanol SO2 with DNA phosphate groups binding diminishing, and in an increase of gistons primulin and durable green coloration. The condensation of MNC chromatin in cord blood was twice as high as in peripheral blood lymphocytes. The reduced ability of MNC chromatin to bind to an ethidium bromide and the acridine orange was saved through deproteinization, before affecting 0.45 M NaCl on cells. The methods of erythrocytes’ predecessors receipt was studied from the culture of cord and peripheral blood MNC for the treatment of newborns with low weight, anemia, and hypoxia. The bases of fetal hypoxia diagnostics and prognosis of the early postnatal period were developed by specific markers of apoptosis: complex DNA-Ca2+ and DNA fragmentation by lymphocytes of the mother.

Keywords

mononuclear cells, cord blood, cellular cycle, apoptosis, ethidium bromide, acridine orange, complex DNA-Ca2+, DNA fragmentation

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Ex vivo expanded hematopoietic progenitor cells (HPC) from cord blood in clinical trials for patients with hematological malignancies

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Yael Margolin, David Snyder, Tony Peled

Gamida Cell, Ltd., Tel Aviv, Israel

Cord blood transplantation (CBT) is an acceptable treatment for patients with hematological malignancies who have no matched bone marrow graft. However, low cell dose has limited the use of CBT.

To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation.

We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x107 cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34+–fold expansion in culture of 76 (range 9–149). Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study. One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days.

Keywords

cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Prognostic factors in cord blood stem cell quality

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Artur A. Isaev, Alexander V. Prihodko, Sergey L. Kiselev, Maria A. Lagarkova, Ivan V. Potapov, Alexey V. Lundup

Human Stem Cells Institute, Gemabank, Moscow, Russia

Cord blood (CB) is a safe and rich source of hematopoietic stem cells (HSC) for oncohematology. The main indicators allowing the assessment of transplant feasibility is a number of nucleated (NC) and CD34+ cells.

A number of HSC is directly related to CB volume, depending on the processing technique and cryopreservation conditions. This study aims to isolate, identify, and quantify NC and CD34+ cells, as well as to evaluate cryopreservation on HSC viability and identify factors affecting HSC numbers in CB.

The average volume of collected CB was 71.48±3.8 ml, NC number was 0.82±0.05x109, viability was 93.47±0.37%. CD34+ and NC numbers corresponded directly to cord blood volume, whereas CB volume is reflected in newborn weight. Cryoconservation has no effect on the number of viable HSC. Immunocytochemical analysis did not reveal any effects of cryoconservation on the number of viable CD34+; although, it might be necessary to apply more accurate methods of analysis. The maximum number of nuclear and CD34+ cells that can be isolated from cord blood volume is 70 ml from a newborn with a weight of 3500 g or more.

Keywords

cord blood, cryoconservation, viability, HSC

M. Alternative sources of hematopoietic stem cells (haploidentical relative donors, cord blood)

Developing new immunological compatible transplants derived from umbilical cord

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Artur A. Isaev1, Alexander V. Prihodko1, Irina N. Saburina2, Sergey L. Kiselev1, Varvara S. Melihova2, Ivan V. Potapov1, Alexey V. Lundup1

1Human Stem Cells Institute, Moscow, Russia; 2Laboratory of Cell Technology, Moscow, Russia

Rejection, immunosuppression, and tissue and organ compatibility selection are crucial problems in current transplantology. One promising approach is autologous cell transplantation. Therefore investigators and clinicians at present time focus their attention on autologous cell and tissue banking and their storage.

The umbilical cord is the structure containing mesenchymal cells, e.g. fibroblast-like cells (FLC) and umbilical vein endothelial cells (HUVEC). FLC are known as a source for skin burns and scars treatment, while HUVEC can be used for ischemic tissues revascularization. Therefore, developing new isolation protocols, cell culture expansion, and cell banking methods from the umbilical cord are important issues for immunologically compatible transplants in contemporary clinical use.

Here we present our method of consecutive isolation, cultivation, and cryopreservation of two cell types – HUVEC and FLC – from one umbilical cord sample. Plastic-adherent cell cultures that contained diploid cells with high proliferative activity were observed for 4–6 passages. FLC expressed vimentin, collagen types 1 and 4, alfa-actin, possessed low MHC expression, and did not express CD45, CD11b, or CD14. Cultivated HUVEC demonstrated endotheliocyte-like phenotype, expressed the Von Willebrand factor, and formed tubular structures. Both FLC and HUVEC were cryopreserved for a long period and thawed without a lack of morphofunctional properties.

Thus, the proposed method of consecutive isolation provides the possibility to isolate and preserve two cell types from one umbilical cord sample without changing traditional cord blood banking protocols. These cells can be used for autotransplantation in the treatment of numerous diseases and expand the role of personal cell banking as a “biological insurance.”

Keywords

umbilical cord, fibroblast-like cells, HUVEC, banking

N. Solid tumors

Aggressiveness of tumor resection improves survival in pediatric malignant gliomas

Ludmila I. Shats1, Margarita B. Belogurova1, Olga G. Zheludkova 2

1City Hospital № 31, St. Petersburg, Russia; 2Clinic Research Center of Hematology, Oncology, and Immunology, Moscow, Russia

Correspondence
City Hospital № 31, pr. Dinamo, 3, St. Petersburg, Russia
Phone/Fax: +7 (812) 235 07 21, E-mail: deton.hospital31@inbox.ru

Over a 13-year period (1994–2007) 48 children between 3 and 18 years of age with anaplastic astrocytoma (26 pts) and glioblastoma multiforme
(22 pts) were treated with surgery, local radiation therapy, and different regimens of chemotherapy. The retrospective analysis of survival was provided.  In our study there was a statistically significant difference in overall survival (OS) between patients with malignant gliomas who underwent a greater-than-90% resection of the tumor and those who underwent a less-than-90% tumor’s resection (5-year OS 42% vs 20% P=0.013). This difference held up stronger for patients with anaplastic astrocytoma who underwent total and subtotal resection (5-year OS 88.8% vs 43% of P=0.0036). A greater-than-90% surgical resection of newly-diagnosed malignant gliomas in children confers a statistical survival advantage when followed by local field irradiation and any kind of chemotherapy.

Figure 1.

2009-5-en-Shats-et-al-Figure-1.JPG

Keywords

anaplastic astrocytoma, glioblastoma multiforme, pediatric malignant glioma, neurosurgery, children

N. Solid tumors

First experience of treatment of high-risk neuroblastoma patients using autologous peripheral blood stem cells transplantation

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Igor N. Vyatkin1,2,  Egor V. Shorikov1,2, Andrey A. Igumenshev1,2, Larisa V. Vakhonina1,2, Natalya G. Maisheva1,2, Yulia A. Yakovleva1,2, Grigory A. Tsaur1,2, Alexander M. Popov1,2,3, Elizaveta R. Semenikhina1, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

1Regional Children Hospital 1, Ekaterinburg, Russia; 2Research Institute of Medical Cell Technologies, Ekaterinburg, Russia; 3Ural State Medical Academy, Ekaterinburg, Russia

Correspondence
Egor Shorikov, S. Deryabina Street 32, 620149 Ekaterinburg, Russia, Regional Children Hospital 1, Pediatric Oncology & Hematology Center, Phone: +7 (343) 216-6881, Fax: +7 (343) 216-6887
E-mail: cohc@spam is badbk.ru

Objective

To investigate the efficacy of auto-PBSCT in patients (pts) with high-risk (HR) neuroblastoma.

Methods

From June 2007 to August 2008 five pts with HR neuroblastoma were recruited. There were 3 boys and 2 girls aged from 11 to 113 months (median 52). Three pts with stage 4 and 2 pts with relapsed neuroblastoma (4 in CR and 1 in PR) received auto-PBSCT with a regimen of Melphalan 135 mg/m2+etoposide 40 mg/kg+carboplatin 1500 mg/m2. In all patients PBSCs were mobilized with G-CSF 10 µg/kg/day for 6 days and leukapheresis was performed on days 5 and 6 of G-CSF administration by cell separator. In 3 out of 5 cases positive immunomagnetic selection of CD34+ harvested cells was carried out. The median purity and viability of selected CD34+ cells was 97.1% (range 88.2%–97.4%) and 91.8% (range 71.6%–99.6%), respectively. The median number of administered CD34+ cells was 11.3×106/kg (range 5.1 to 41.4×106/kg).

Results

All children demonstrated engraftment. The median time for take of neutrophils and platelets after auto-PBSCT was 11 days (range 9–40) and 40 days (range 13–79), respectively. No patients experienced organ toxicities greater than WHO grade II or life-threatening infections. Progression disease was documented in 1 patient and 2 relapses were registered at the median time of 223 days (range 188–390); 2 patients remained in CR for 295 and 534 days. Both relapsed patients died; the other 3 are alive.

Conclusion

These preliminary results suggest that auto-PBSCT can be considered a curative therapeutic approach in patients with HR neuroblastoma.

Keywords

neuroblastoma, PBSCT, children, CD34+ cell selection

N. Solid tumors

Treatment of patients with high-risk axial and pelvic Ewing’s sarcoma (ES). A single-institution experience

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Roman I. Pimenov1, Igor S. Dolgopolov1, Vasilii K. Boyarshinov1, Natalia S. Subbotina1, Igor V. Visochin1, Stuart Siegiel2, George L. Mentkevich1

1Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia; 2CHLA, Los-Angeles, USA

Correspondence
Roman I. Pimenov, 24, Kashirskoye shosse, 115476, Moscow, Russia, Phone: +7 (903) 1991909
E-mail: onco@spam is badlist.ru

This study was initiated in an attempt to improve DFS rates in patients (pts) with high-risk axial and pelvic ES by using autoPBSCT. From January 1997 to December 2007 35 pts (M/F–15/21) with high-risk ES (localized 25 pts; metastatic 10 pts (lungs-only: 2, combined or other sites: 8) received HDCT with autologous transplantation. The median primary tumor volume was 739 cm3. CT consisted of 5 courses: numbers 1, 3, and 5 included cyclophosphamide 2100 mg/m2 day on days 1 and 2, doxorubicin 37.5 mg/m2 as a 24-hour infusion on days 1 and 2, and vincristine 1.5 mg/m2 on days 1, 8, and 15. Cycles 2 and 4 consisted of ifosfamide 2400 mg/m2/day on days 1–5 and VP-16 100 mg/m2/day on days 1–5. No G-CSF was given routinely between cycles. RT was given after the fifth cycle of CT at a median dose of 52 Gy (range, 50–56). Patients with lung involvement received lung irradiation after the second cycle of CT at a dose of 10.8-12 Gy. All pts received busulfan 16 mg/kg, melphalan 140 mg/m2-based HDCT (n=9); with the addition of thiotepa (TT) 600-900 mg/m2 (n=11) or VP16 1400 mg/m2 (n=15), followed by autologous stem cell recovery (6.4 (1.9–25.3) x 106 CD34+ cells/kg). The median number of days to WBC>1.0x109/l, Plt>20, and 50x109/l was 10 (8–14), 16 (0–52) and 28 (11–66) days, respectively. TRM was 6% (2 pts out of 35). Six pts relapsed. DFS at 5 yrs was 50% in metastatic pts vs 88% in pts with localized tumor (p>0.05). EFS and DFS were 69% and 75% with a median follow-up of 103 and 112 months, respectively. DFS in the TT, VP16, and BuMel groups was 83%, 67%, and 79%, respectively (p<0.05).

We conclude that pts with high-risk ES may benefit from BuMel-based regimens, and that an addition of any drug to BuMel regimen increases toxicity with no influence on DFS.

Keywords

Ewing’s sarcoma, high-dose chemotherapy, autologous bone marrow transplantation

N. Solid tumors

High-dose chemotherapy and autologous stem cell transplantation in children with Ewing’s sarcoma/PNET

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Ilya V. Kazantsev1, Tatjana V. Youhta2, Elena V. Morozova1, Svetlana A. Safonova2, Yury A. Punanov2, Liudmila S. Zubarovskaya1, Boris V. Afanasyev1

1Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia; 2Federal State Institution N.N. Petrov Research Institute of Oncology, Saint Petersburg

Correspondence
Ilya V. Kazantsev, Comissara Smirnova str., 10a, 32, St. Petersburg, 194044, Russia, Phone: +7 (921) 317-47-49
E-mail: Ilya_Kazantsev@spam is badinbox.ru

The clinical course of Ewing’s sarcoma/PNET in children is characterized by a rapid increase of tumor mass and early metastases. In this context a substantial part of the affected pediatric population – children with clinically detectable metastatic disease or heavy tumor burden – has a poor prognosis. Although contemporary therapy programs – those that combine systemic chemotherapy with local control measures – achieve  5-year event-free survival (EFS) in 70–80% of children with localized disease, the results in high-risk groups are still to be improved with a 5-year EFS rate of 20–30%. Some research data provides evidence of high-dose chemotherapy (HDCT) with autologous hematopoietic stem cell transplantation (auto-HSCT) being a promising option for treatment of these high-risk patients.

Patients and methods

From 2003 to December 2008 a total of 17 pediatric patients with Ewing’s sarcoma/PNET received HDCD with auto-HSCT. There were 11 males and 6 females (male-female ratio of 1.8:1) with a median age at diagnosis of 13.4 yrs (range 3–18 yrs). In all the cases, diagnosis was histologically proved. Of the 17 patients, 13 had metastatic disease and 4 had localized disease. In all the patients with localized disease the initial tumor volume exceeded 200 sm3. In all the cases the therapy program included 6 courses of polychemotherapy (ifosfamide, vincristine, doxorubicin and etoposide), surgical treatment or local irradiation of primary disease sight (48–56 Gy). There were 8 patients who received 8 courses of maintenance chemotherapy (vincristine, dactinomycin, ifosfamide). In 9 cases the maintenance therapy was substituted for HDCT with auto-HSCT. We used the following conditioning regimens: oral busulfan 16 mg/kg and melphalan 140 mg/m2 in 7 patients and melphalan 140–200 mg/m2 in 2 patients. We used different stem cell sources: bone marrow (BM) in 5 patients, peripheral blood stem cells (PBSC) in 2 patients, and BM and PBSC – due to the poor yield of SC – in 2 patients. A mean dose of CD34+ cells in the graft amounted to 3.26 x 106 (range 1.0–8.9 x 106).

Results

In the maintenance therapy group (n=8) 5 patients had a relapse of metastatic disease and died of tumor progression, 2 patients had a local relapse and received salvage therapy – in one case, HDCT with auto-HSCT – and both are presently disease–free, while 1 patient died of overwhelming sepsis.

In the HDCT group (n=9) 8 of the patients are alive – 1 died of disease progression – 4 patients remain in remission, 2 patients failed to achieve remission and received allogeneic HSCT, and 2 patients relapsed 2–8 months after HDCT with auto-HSCT. All children transplanted in partial remission or disease progression later relapsed. All of the children that are at the moment disease-free were diagnosed stage IV Ewing’s sarcoma– distant metastases at the time of diagnosis – but achieved complete remission (CR) at the end of the induction chemotherapy course. The toxicity of HDCT regimens was moderate: all of the patients had grade II–III mucositis, 6 patients had febrile neutropenia, and 2 patients had toxic hepatitis.

Conclusions

HDCT with auto-HSCT in high-risk group children with Ewing’s sarcoma/PNET has acceptable toxicity and allows improved PFS for patients in CR after standard therapy regimens.

Keywords

Ewing's sarcoma, PNET, autologous hematopoietic stem cell transplantation, maintenance therapy

N. Solid tumors

Restoration of ovarian function after cryopreserved ovarian tissue transplantation in women exposed to complex treatment for gynecological cancer: feasibility of this option in pediatric cancer patients

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Alla S. Lisyanskya1,2, Natalia I. Tapilskaya1,3, Margarita B. Belogurova3,4, Yulia V. Dinikina3, Ekaterina V. Tsibatova4, Georgy M. Manikhas1

1City Clinical Oncology Dispensary; 2AVA-Peter Clinic; 3St. Petersburg State Pediatric Medical Academy, Department of Oncology, Radiodiagnostics and Radiotherapy Section; 4City Clinical Hospital №31, Department of Pediatric Oncology and Hematology Saint Petersburg, Russia

Correspondence
Alla S. Lisyanskya, City Clinical Oncology Dispensary, pr. Veteranov 56, 198255 St. Petersburg, Russia
Phone: +7 (812) 3768917, E-mail: deton.hospital31@inbox.ru

Objective

To appreciate the possibilities for preserving ovarian function and improving quality of life in women of a reproductive age exposed to complex treatment for gynecological cancer.   

Due to increasing pediatric cancer survival rates attention has turned to the long-term sequelae of treatment.  Possibilities to preserve fertility in these pts are widely discussed.

Materials and methods

Sixteen pts with gynecological cancer participated in our research. Before undergoing gonadotoxic treatment, a laparoscopic collection of ovarian cortical tissue – which was then cryopreserved – was carried out in all pts. After the completion of a patient-specific treatment, hormonal blood tests confirmed premature ovarian failure. After heterotopic transplantation was performed, ovarian function was continuously monitored by hormonal blood tests; follicular development was assessed by ultrasonography.

Results

Normal hormonal status was recovered in 15 pts due to transplantation procedure, while in 10 pts regular cyclic follicular development was observed. In 1 patient received a mature ovule after a follicle puncture. According to the psychological tests taken in all pts exposed to ovarian tissue transplantation, an increase in their life quality was registered.   

Conclusions

Autotransplantation of ovarian tissue in reproductive-aged women with oncogynecological disease can preserve fertility and “stop” the menopause. Cryopreserved ovarian tissue – transplanted into a heterotopic site – may lead to the production of mature ovules that admit the possibility to conceive a child. These methods are reasonable in oncopediatric pts who have a realistic chance of long-term survival and whose scheduled treatment carries a high risk of gonadal damage.

Keywords

oncogynecology, children, chemotherapy, fertility, cryopreservation

N. Solid tumors

The outcome of treatment in patients with high-risk (HR) retinoblastoma

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Igor S. Dolgopolov1, Tatiana L. Ushakova1, Olga N. Gorovtzova1, Alevtina I. Pavlovskaya2, Roman I. Pimenov1, Vasilii K. Boyarshinov1, Natalia S. Subbotina1, Igor V. Glekov1, Vladimir G. Poliakov1, George L. Mentkevich1

1Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia; 2Institute of Clinical Oncology

From 2001 to 2008 16 patients (8 female, 8 male) with high-risk retinoblastoma (RB) were treated in our center. HR was defined as microscopic residual tumor after enucleation (stage II, n=8); regional extension (stage III, n=4): a) overt orbital disease (n=3) b) involvement of regional or cervical lymph nodes (n=1); metastatic disease (stage IV): a) metastasis (multiple lesions (n=1)), b) CNS extension: prechiasmatic lesion (n=3). The median age was 32 (8–115) years and the median body weight was 13.8 (9.8–32) kg. Two patients (pts) had bilateral RB (1st case: operated on left eye and relapsed in the right eye with orbital involvement 3 months after surgery; second: metastatic disease with prechiasmatic lesion). The induction phase included 4 courses of CT including cyclophosphamide, VP-16 and carboplatin, surgery and external beam RT at a dose of 50 Gy. PBSCs were harvested after the first course of CT. Conditioning included busulfan 16 mg/kg and melphalan 140 mg/m2, followed by autologous SCT. Twelve pts received a median number of 3.5 (2.2–4.8)x106 CD34+ cells/kg. Toxicity was moderate in all but one pt (stage III), who died on d+8 from sepsis caused by Klebsiella pneumonia. The median number of days to WBC >1.0x109/l, Plt >20, and 50x109/l was 10 (9–11), 14 (10–20), and 17 (14–35) days respectively. Three pts died due to relapse: in CNS (n=2), and one with metastatic disease experienced a progression of disease in bone, BM, testis and lymph nodes 8 months after HDCT. Eight patients are alive and well at follow-up of 55 months. EFS and DFS were 64% and 68% respectively. Three patients refused HDCT. Two out of 3 (both with prechiasmatic lesion) relapsed in CNS and died. One with stage II is alive and disease free. One pt with overt orbital disease progressed when on treatment and later died.

Keywords

high-risk retinoblastoma, high-dose chemotherapy, autologous stem cell transplantation

O. Donor registry

Influence of differentiated occurrence of HLA genes and haplotypes on defined clinical parameters in patients after allo–HSCT in association with HLA polymorphism

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Alexander L. Alyanskiy, Olesya V. Paina, Natalia E. Ivanova, Alla A. Golovacheva, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Alexander L. Alyanskiy, Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia
E-mail: bmt-registry@spam is badspmu.rssi.ru

Background

In the Memorial R.M. Gorbacheva Institute of Children Hematology and Transplantation of St. Petersburg Pavlov State Medical University (SPSMU) more than 170 allo-HSCTs from unrelated donors were performed between 2000 and 2009. The International Registers network facilitated the finding of HLA unrelated donor matches for approximately 50% of Russian patients. Nevertheless, the number of Russian recipients has not yet matched the number of unrelated donors in the BMDW database.

Materials and methods

We have analyzed the distribution frequency of HLA-А, -B, -C, -DRB1, and -DQB1 genes and haplotypes for SPSMU patients in comparison to donors listed in the BMDW and SPSMU registries and the influences of differentiated occurrence of HLA genes and haplotypes on defined clinical parameters – such as acute GVHD and TRM – in patients after allo-HSCT. The HLA-typing was performed in an SPSMU HLA typing laboratory using PCR SSP “PROTRANS” technology. The donors’ high resolution confirmatory typing (CT) was performed in an EFI-accredited HLA-typing laboratory in the EU. 

Results

As a result of the conducted research the difference in occurrence of certain genes and haplotypes in Russian patients and donors in comparison to donors listed in the BMDW database is authentically proved. A certain dependence between differentiated occurrence of HLA genes and haplotypes and defined clinical parameters in patients after allo-HSCT has been revealed.

Conclusion

There are certain differences in the occurrence of separate HLA genes and haplotypes in the group of Russian donors and recipients in comparison to donors listed in the BMDW database. Differentiated relative occurrence of HLA genes and haplotypes in patients and donors can be considered as one of the factors influencing defined clinical parameters in patients after allo-HSCT.

Keywords

HLA genes, haplotypes, polymorphism, MUD search