Background. qPCR is widely used to detect JAK2 V617F and MPL W515L/K mutations in myeloproliferative neoplasms. This study evaluated the diagnostic performance of four commercially available Russian qPCR test systems. Methods. 45 positive DNA samples (allelic burden 2—45%) and 18 negative samples were analyzed. Target allele frequencies were established using ddPCR as mean from 5 replicates. Two laboratories tested the kits (manufactured by «Genetechnology» LLC, «Formula gena» LLC, «Inogene» LLC, «Syntol» LLC) as independent participants and they were supplied from a manufacturer’s blinded, centralized purchase. Results. Specificity was 100% for all kits. JAK2 V617F: Sensitivity was 96.7% for three out of four kits, but 86.7% for «Genetechnology». «Syntol» kit showed the largest proportional systematic error (β1=1.63), while «Inogene» kit showed none (β1=1.00). «Genetechnology» kit had the highest coefficient of variation (CV=59.8%); «Syntol» kit had the lowest one (CV=10.2%). For MPL W515L mutation detection « Genetechnology « kit displayed both proportional (β1=0.25) and constant (β0=38.34) systematic errors with CV=14.1%. «Inogene» kit showed only a proportional error β1=0.66 and CV=9.4%. For MPL W515K mutation «Genetechnology» kit had a constant error β0=38.83 and CV=13.5%. «Inogene» kit showed a proportional error β1=1.45 and CV=8.9%. Conclusion. For JAK2 V617F mutation detection, the «Formula gena» kit showed the lowest deviation from the target value. For MPL W515L/K mutations, the tests from «Inogene» and «Genetechnology» provided comparable results.

Translocation t(12;21)(p13;q22)/ETV6::RUNX1 is among the most common genetic aberrations in pediatric B-cell precursor acute lymphoblastic leukekian (BCP-ALL). This translocation is often combined with ETV6 and/or RUNX1 copy number variations. Fluorescence in situ hybridization (FISH) technique, which is widely used to reveal the presence of t(12;21)(p13;q22), also allows the detection of these additional genetic aberrations (FISH patterns). The aim of this study was to provide detailed characteristics of FISH patterns in patients with BCP-ALL and the t(12;21)(p13;q22)/ETV6::RUNX1 translocation. In our study we enrolled 241 patients with t(12;21)-positive ALL who had undergone testing with a dual-color double-fusion FISH assay between 2008 and 2023. This study was approved by the Independent Ethics Committee and the Academic Council of the Research Institute of Medical Cell Technologies (Ekaterinburg). A single FISH pattern (monoclonal cases) was identified in 200 patients (83.0%), 2 or more patterns (polyclonal cases) were detected in 41 (17.0%) patients. The majority of polyclonal cases (n = 39; 95.1%) exhibited 2 patterns. The most common secondary genetic alteration was ETV6 deletion (n = 105; 43.5%). Less common aberrations included an additional copy of RUNX1 (n = 97; 40.2%), a combination of ETV6 deletion and an additional copy of RUNX1 (n = 27; 11.2%), and an additional copy of ETV6 (n = 5; 2.0%). The number of the patients with one FISH pattern that did not contain any additional genetic aberrations as a result of a reciprocal translocation (2F1G1R) was relatively small (n = 35; 17.5%). We identified 5 prognostically unfavorable FISH patterns associated with a high risk of relapse. These included cases with simultaneous presence of ETV6 and RUNX1 additional copies (pattern 2F2G2R), isolated additional copies of RUNX1 (pattern 2F2R-3F2R) or ETV6 (pattern 1F1G2R-2F2G4R), a partial deletion of ETV6 (pattern 2F1Gdim1R) and a non-reciprocal translocation of t(12;21) (pattern 1F1R-1F1G1R). Grouping these unfavorable prognostic FISH patterns together made it possible to predict 6 (46%) out of 13 relapses that occurred in the patients during the study period. It is important to highlight that the number of the patients with unfavorable FISH patterns and initial leukocytosis of 30 × 109 leukocytes/L did not differ significantly from the rest of the group. Another important observation was that the patients with prognostically unfavorable FISH patterns responded well to induction therapy, as assessed both by cytological examination of blood and bone marrow smears on days 8, 15, and 36 of therapy and by MRD response at the end of induction therapy according to the ALL-MB 2015 protocol. A comparison of FISH patterns detected at initial diagnosis and at relapse showed that only 6 (50%) out of 12 cases who had undergone FISH testing at both time points remained stable. In conclusion, BCP-ALL with the translocation t(12;21)(p13;q22)/ETV6::RUNX1 was characterized by a vast variety of secondary genetic aberrations detected by FISH, the most prevalent of which was ETV6 deletion. A group of unfavorable FISH patterns identified in our study warrants further investigation in a larger cohort of ALL patients for their possible re-stratification so that they could receive more intensive treatment.

A decrease in T cell alloreactivity following hematopoietic stem cell transplantation (HSCT) can be achieved via the depletion of TCR_ab⁺ and CD19⁺ lymphocytes or post-transplant therapy with high-dose cyclophosphamide (PTCy). The purpose of this study was to analyze HSCT outcomes according to the type of lymphocyte depletion. In our non-randomized retrospective study, we analyzed the results of 118 HSCTs performed in 107 patients with malignant blood disorders at the Center for Pediatric Oncology and Hematology of the Regional Children’s Clinical Hospital (Yekaterinburg) from 2010 to 2023. The patients were divided into 2 groups: those who had received ex vivo TCR_ab⁺/CD19⁺ depletion using the CliniMACS technology (n = 75; Group 1) and those who had received in vivo depletion using PTCy at a dose of 50 mg/kg (n = 43; Group 2). The study was approved by the Independent Ethics Committee and the Scientific Council of the Institute of Medical Cell Technologies (Yekaterinburg). Informed consent for the research and treatment was obtained from each patient and their legal representatives. Even though the patients from the TCR_ab⁺/CD19⁺ depletion group showed a better engraftment rate (median time: 12 days) than the subjects from the PTCy group (median time: 18 days) (p < 0.0001), there was no statistically significant difference in the cumulative incidence of cytomegalovirus and herpesvirus type 6 infection reactivation post-transplant between the two study groups. The cytomegalovirus reactivation rate was 0.467 and 0.466 in Group 1 and 2 respectively (p = 0.89), while for herpesvirus type 6 the reactivation rate was 0.360 and 0.240 respectively (p = 0.13). There were no significant differences in the cumulative incidence of acute graft-versus-host disease (GVHD) in the two groups by Day +100 (0.294 and 0.419 in Group 1 and 2 respectively; p = 0.77); neither was there any difference in the incidence of severe acute GVHD (0.067 and 0.093 respectively; p = 0.11). The incidence of chronic GVHD was 0.12 in the TCR_ab⁺/CD19⁺ depletion group and 0.26 in the PTCy group (p = 0.11). The event-free survival, overall survival and cumulative incidence of relapse did not differ between the studied groups either. The two methods of the depletion of unwanted T-cell populations showed a comparable effectiveness. The choice between them should be based on factors related to the biological characteristics of the disease and the clinical status of a patient. The availability of different depletion methods at a single transplant center helps to choose the optimal treatment option for patients who are in need of HSCT.