Grigory Tsaur

A Novel Three-Colour Fluorescence in Situ Hybridization Approach for the Detection of t(7;12)(q36;p13) in Acute Myeloid Leukaemia Reveals New Cryptic Three Way Translocation t(7;12;16)

The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20–30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting.

Prospective investigation of applicability and the prognostic significance of bone marrow involvement in patients with neuroblastoma detected by quantitative reverse transcription PCR

Detection of bone marrow (BM) involvement in patients with neuroblastoma is crucial for staging and defining prognosis. Furthermore, the persistence of residual tumor cells in the BM is associated with an unfavorable outcome.

SIGNIFICANCE OF ETV6-RUNX1 FUSION GENE TRANSCRIPT DETECTION IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA WITH TRANSLOCATION t(12;21)(p13;q22)

Introduction. Translocation t(12;21)(p13;q22) is one of the most common structural genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). It cannot be detected by conventional G-banding, so a reverse-transcriptase polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization are used for this purpose. The aim of the study was to evaluate the prognostic significance of qualitative and quantitative detection of ETV6-RUNX1 fusion gene transcript at various time points in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Materials and methods. ETV6-RUNX1 fusion gene transcript was revealed by both reverse-transcriptase PCR and quantitative real-time PCR (RQ-PCR) in 34 out of 166 (20.5 %) children with BCP-ALL. Qualitative ETV6-RUNX1-positivity at days 36 and 85 led to unfavorable outcome (lower event-free survival –EFS and higher cumulative incidence of relapse – CIR). While ETV6-RUNX1 status at day 15 did not allow to divide patients with different outcomes. By ROC curve analysis we determined threshold levels (TL) for ETV6-RUNX1/ABL1 ratio at days 0, 15, 36 and 85. Afterwards we adjusted obtained results to 10-fold scale. Results. So practically applicable TL were as follows 500.0 %, 1 %, 0.1 % и 0.01 % for days 0, 15, 36 and 85, respectively. EFS and CIR were both worse in patients with ETV6-RUNX1/ABL1 ratio equal or above defined TL. Moreover, initial ratio ≥500,0 % corresponded to delayed blast clearance at days 15 and 36. We showed good qualitative (84.8 %) and quantitative (R 2 = 0.953) concordance between ETV6-RUNX1/ABL1 ratio and MRD data obtained by flow cytometry at days 15, 36, 85. Of note, defined TL for ETV6-RUNX1/ABL1 at days 15, 36, 85 were equal to prognostically important levels for flow cytometry MRD. Conclusion. Thus, qualitative detection and quantitative value of ETV6-RUNX1 fusion gene transcript showed prognostic significance in the course of treatment in children with BCP-ALL. Based on these results we propose standardization approaches for Moscow – Berlin ALL study group.

Rare cases of laboratory tests discrepancies in diagnostics of pediatric Burkitt lymphoma/leukemia

Introduction. The main features of bone marrow blasts cells in Burkitt lymphoma/leukemia (BL) are L3 morphology, mature immunophenotype of blasts with surface IgM expression, and presence of typical MYC gene rearrangements. The aim of the study was to show discrepancy examples in laboratory signs of BL. Patients and methods. 10 patients (8 boys and 2 girls) aged 1 to 18 years were included in the present study. The inclusion criterion was the identification of discrepancies between flow cytometric, morphological and cytogenetic data. Results. In 2 cases there were no rearrangements of the MYC gene. In 2 patients, the L2 morphological variant went against the presence of typical MYC gene rearrangements. In one case, undifferentiated blasts cells were described by morphology together with presence of surface IgM, and atypical genetics. In 8 patients, there was no expression of surface IgM. Of these, patients with absence of cytomorphological data cytometric and genetic data were controversial. Сonclusion. The cases presented in this study and the cases described in the literature demonstrate the importance of an attentive and comprehensive approach in evaluating the results of laboratory tests in the diagnosis of BL.

RUSSIAN-BELARUSIAN MULTICENTER GROUP STANDARD GUIDELINES FOR CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA FLOW CYTOMETRIC DIAGNOSTICS

Background. Flow cytometry is one of the key technologies for acute lymphoblastic leukemia (ALL) diagnostics. Nevertheless lack of technological standards hampers implementation of immunophenotyping data in treatment protocols. Objective: development of harmonized guidelines for flow cytometric diagnostics of childhood ALL. Materials and methods. Three reference laboratories of the immunophenotyping group “Moscow–Berlin” took part in the development and standardization of approaches to the cytometric determination of the minimal residual disease. The sequence of steps for staining with monoclonal antibodies, selection of reagents for immunophenotyping, flow cytometer adjustment algorithms were discussed. Results. We developed and implemented in multicenter setting the harmonized approach for immunophenotyping flow cytometric childhood ALL. Successful integration of this protocol in the multicenter group has shown good level of our approach reproducibility. Conclusion . These results will allow implementing diagnostic standards in stratification system of pediatric ALL treatment protocols of russianbelarusian multicenter group.

Clinical significance of cytogenetic changes in childhood T-cell acute lymphoblastic leukemia: results of the multicenter group Moscow–Berlin (MB)

Abstract The prognostic significance of genetic lesions in T-cell ALL still needs to be elucidated. Karyotyping and FISH were performed in samples from 120 patients with T-cell ALL registered in the trial Moscow–Berlin 2008. Most frequent rearrangements were TLX3 (N = 29; 24%) and TAL1 (N = 18; 15%), followed by KMT2A (N = 6; 5%), TLX1 (N = 5; 4.2%), and 11p13-15 (N = 5; 4.2%). In 16.7% of patients, the karyotype was normal, and in 30.8% ‘other’ aberrations were seen. Patients with a normal karyotype, TAL1, or KMT2A rearrangements had the most favorable outcome (probability of event free survival (pEFS): 82% ± 6%), while prognosis for patients with TLX3 and TLX1 rearrangements and ‘other’ aberrations was less favorable (pEFS: 62% ± 6%). Worst outcome was observed for five patients with 11p rearrangements (pEFS: 20% ± 18%). In summary, three subgroups of patients with T-cell ALL with significantly different outcomes could be defined by cytogenetic profiling.

Transplantation of hematopoietic stem cells in the Regional Children’s Clinical Hospital № 1 in Ekaterinburg. Results of work for the period from 2006 to 2016

This article represents results of work of Center of pediatric oncology and hematology of Regional pediatric clinical hospital № 1 of Yekaterinburg on Hematopoietic stem cell transplantation (HSCT) during the period 2006–2016. One hundred seventeen HSCT performed during this time including 70 autologous, 33 allogenic, 14 haploidentical at patients with solid tumors (n = 55), acute leukemias (n = 27), lymphomas (n = 9), non-malignant diseases (n = 11). Results of treatment depending on type of disease and HSCT were shown. It was indicated that presence of minimal residual disease (MRD) before HSCT in case of leukemia and neuroblastoma was indicated as negative prognosis. Possible way of treatment of persistent MRD after HSCT with the help of blinatumomab and donor lymphocytes infusion showed.

Influence of mixed chimerism upon outcomes of allogeneic stem cell transplantation (allo-SCT) in patients with non-malignant diseases

AlloSCT is the only curative option for treatment of hematological disorders with suppressed hematopoiesis and primary immune deficiencies. Nonmyeloablative conditioning (MAC) regimens lead to long persistence of mixed chimerism (MC) in majority of the patients. Purpose of our study was to estimate relationships between the type of hematopoietic chimerism and development of GVHD following alloSCT being performed in patients with non-malignant diseases.