ISSN 1866-8836
Клеточная терапия и трансплантация

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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

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

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

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

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

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

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