Biallelic Loss of 7q34 (TRB) and 9p21.3 (CDKN2A/2B) in Adult Ph-Negative Acute T-Lymphoblastic Leukemia.

Tumor cells of acute lymphoblastic leukemia (ALL) may have various genetic abnormalities. Some of them lead to a complete loss of certain genes. Our aim was to reveal biallelic deletions of genes in Ph-negative T-ALL. Chromosomal microarray analysis (CMA) was performed for 47 patients with de novo Ph-negative T-ALL, who received treatment according to RALL-2016m clinical protocol at the National Medical Research Center for Hematology (Moscow, Russia) from 2017 to 2023. Out of forty-seven patients, only three had normal molecular karyotype. The other 44 patients had multiple gains, losses, and copy neutral losses of heterozygosity. Biallelic losses were found in 14 patients (30%). In ten patients (21%), a biallelic deletion of 9p21.3 involved a different number of genes, however CDKN2A gene loss was noted in all ten cases. For seven patients (15%), a biallelic deletion of 7q34 was found, including two genes-PRSS1, PRSS2 located within the T-cell receptor beta (TRB) locus. A clonal rearrangement of the TRB gene was revealed in 6 out of 7 cases with 7q34 biallelic loss. Both biallelic deletions can be considered favorable prognostic factors, with an association with 9p21 being statistically significant (p = 0.01) and a trend for 7q34 (p = 0.12) being observed.

Kinetics of Recovery of Naïve and Memory T Cells in Acute Leukemia Patients after Allogeneic Stem Cell Transplantation Depending on Different GVHD Prophylaxis Regimens.

Background: Memory T cells are a heterogeneous population of immune cells that provide adaptive immunity. Its full recovery seems essential for graft-versus-tumor reactions that provide an opportunity for biological cure in patients with acute leukemia. The use of mismatched or haploidentical donors has increased, which has become possible because of modifications in graft versus host disease (GVHD) prophylaxis. Materials and Methods: Sixty-five leukemia patients (acute myeloid leukemia - 40, acute lymphoblastic leukemia - 25), median age 33 (17-61) years, underwent allo-HSCT from 2016 to 2019 in the National Research Centre for Hematology. Patients were divided into three groups based on the impact of GVHD prophylaxis on T cell recovery: horse antithymocyte globulin (ATG)-based regimen (n=32), horse ATG combined with posttransplant cyclophosphamide (PT-Cy) (n=18), and ex vivo T cell depletion (n=15). Results: The early period after transplantation (before day +100) was characterized by significantly lower absolute numbers of T naïve, memory stem and T central memory cells in peripheral blood in patients after ATG+PT-Cy-regimen or ex vivo T cell depletion than after ATG-based prophylaxis (p<0.05). Moreover, strong depletion of naïve T and memory stem cells prevents the development of GVHD, and determining the absolute number of CD8+ naïve T and memory stem cells with a cutoff of 1.31 cells per microliter seems to be a perspective in assessing the risks of developing acute GVHD (p=0.008). The dynamics of T cell recovery showed the involvement of either circulating or bone marrow resident T effector cells shortly after allogeneic transplantation in all patients, but the use of manipulated grafts with ex vivo T cell depletion requires the involvement of naïve and memory stem cells. There was no significant effect of T cell recovery on leukemia relapse after allogeneic transplantation. Conclusion: These experimental outcomes contribute to providing the best understanding of immunological events that occur early after transplantation and help in the rational choice of GVHD prophylaxis in patients who will undergo allogeneic transplantation. Our study demonstrated the comparable immunological effects of posttransplant cyclophosphamide and ex vivo T cell depletion and immunological inefficiency of horse ATG for GVHD prevention.

DNA Copy Number Alterations and Copy Neutral Loss of Heterozygosity in Adult Ph-Negative Acute B-Lymphoblastic Leukemia: Focus on the Genes Involved.

The landscape of chromosomal aberrations in the tumor cells of the patients with B-ALL is diverse and can influence the outcome of the disease. Molecular karyotyping at the onset of the disease using chromosomal microarray (CMA) is advisable to identify additional molecular factors associated with the prognosis of the disease. Molecular karyotyping data for 36 patients with Ph-negative B-ALL who received therapy according to the ALL-2016 protocol are presented. We analyzed copy number alterations and their prognostic significance for CDKN2A/B, DMRTA, DOCK8, TP53, SMARCA2, PAX5, XPA, FOXE1, HEMGN, USP45, RUNX1, NF1, IGF2BP1, ERG, TMPRSS2, CRLF2, FGFR3, FLNB, IKZF1, RUNX2, ARID1B, CIP2A, PIK3CA, ATM, RB1, BIRC3, MYC, IKZF3, ETV6, ZNF384, PTPRJ, CCL20, PAX3, MTCH2, TCF3, IKZF2, BTG1, BTG2, RAG1, RAG2, ELK3, SH2B3, EP300, MAP2K2, EBI3, MEF2D, MEF2C, CEBPA, and TBLXR1 genes, choosing t(4;11) and t(7;14) as reference events. Of the 36 patients, only 5 (13.8%) had a normal molecular karyotype, and 31 (86.2%) were found to have various molecular karyotype abnormalities-104 deletions, 90 duplications or amplifications, 29 cases of cnLOH and 7 biallelic/homozygous deletions. We found that 11q22-23 duplication involving the BIRC3, ATM and MLL genes was the most adverse prognostic event in the study cohort.

Immunophenotype of Measurable Residual Blast Cells as an Additional Prognostic Factor in Adults with B-Cell Acute Lymphoblastic Leukemia.

Measurable residual disease (MRD) is a well-known independent prognostic factor in acute leukemias, and multicolor flow cytometry (MFC) is widely used to detect MRD. MFC is able not only to enumerate MRD accurately but also to describe an antigen expression profile of residual blast cells. However, the relationship between MRD immunophenotype and patient survival probability has not yet been studied. We determined the prognostic impact of MRD immunophenotype in adults with B-cell acute lymphoblastic leukemia (B-ALL). In a multicenter study RALL-2016 (NCT03462095), 267 patients were enrolled from 2016 to 2022. MRD was assessed at the end of induction (day 70) in 94 patients with B-ALL by six- or 10-color flow cytometry in the bone marrow specimens. The 4 year relapse-free survival (RFS) was lower in MRD-positive B-ALL patients [37% vs. 78% (p < 0.0001)]. The absence of CD10, positive expression of CD38, and high expression of CD58 on MRD cells worsened the 4 year RFS [19% vs. 51% (p = 0.004), 0% vs. 51% (p < 0.0001), and 21% vs. 40% (p = 0.02), respectively]. The MRD immunophenotype is associated with RFS and could be an additional prognostic factor for B-ALL patients.

High expression of granzyme B in conventional CD4+ T cells is associated with increased relapses after allogeneic stem cells transplantation in patients with hematological malignancies.

Granzyme B is known to be a serine protease contained in granules of cytotoxic T cells. We have previously reported an influence of granzyme B expression in T regulatory cells (Tregs) on the risk of acute graft versus host disease (GVHD) onset. However, it is still unknown if conventional T cells (Tcon) use the granzyme B pathway as a mechanism of alloimmunity. We hypothesized that granzyme B in Tcon may affect recurrence within the first 6 months after allogeneic transplantation (allo-HSCT). A total of 65 patients with different hematological malignancies were included in this study. Blood samples were collected on day +30 after allo-HSCT. The percentage of granzyme B positive conventional T cells in patients who developed relapse in the first 6 months after allo-HSCT was 11.3 (4.5-35.3) compared to the others in continuous complete remission-1.3 (3.65-9.7), р = 0.011. The risk of relapse after allo-HSCT was in 3.9 times higher in patients with an increased percentage of granzyme B positive conventional T cells. The findings demonstrated that the percentage of granzyme B positive conventional T cells on day +30 after allo-HSCT could be a predictable marker of relapse within the first 6 months after allo-HSCT.

Diagnostic significance of flow cytometry scales in diagnostics of myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) can present a challenge for clinicians. Multicolor flow cytometry (MFC) can aid in establishing a diagnosis. The aim of this study was to determine the optimal MFC approach for MDS. METHODS: The study included 102 MDS (39 low-grade MDS), 83 cytopenic patients without myeloid neoplastic disorders (control group), and 35 healthy donors. Bone marrow was analyzed using a six-color MFC. Analysis was conducted according to the "Ogata score," "Wells score," and the integrated flow cytometry (iFC) score. RESULTS: The respective sensitivity and specificity values were 77.5% and 90.4% for the Ogata score, 79.4% and 81.9% for the Wells score, and 87.3% and 87.6% for the iFC score. Specificity was not 100% due to deviations of MFC parameters in the control group. Patients with paroxysmal nocturnal hemoglobinuria (PNH) had higher levels of CD34+ CD7+ myeloid cells than donors. Aplastic anemia and PNH were characterized by a high proportion of CD56+ cells among CD34+ precursors and neutrophils. The proportion of MDS-related features increased with the progression of MDS. The highest number of CD34+ blasts was found in MDS with excess blasts. MDS with isolated del(5q) was characterized by a high proportion of CD34+ CD7+ cells and low granularity of neutrophils. In 39 low-grade MDS, the sensitivities were 53.8%, 61.5%, and 71.8% for Ogata score, Wells score, and iFC, respectively. CONCLUSION: The results support iFC as a useful diagnostic tool in MDS.

Level of Granzyme B-positive T-regulatory cells is a strong predictor biomarker of acute Graft-versus-host disease after day +30 after allo-HSCT.

Acute Graft-versus-host-disease (aGVHD), the major complication and one of the main causes of poor outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays there are no widely accepted cell, plasma or another biomarker that can be used for aGVHD prediction. We hypothesized that a level of Granzyme B-positive T regulatory (GZMB-positive Treg) cells on day+30 after allo-HSCT could be the measure of immune response suppression and could predict aGVHD development after day +30. We applied a widespread and easy-to-perform method of multicolor flow cytometry to measure level of GZMB-positive Treg cells. Levels of GZMB-positive Tregs on day +30 after allo-HSCT were significantly higher in those patients who never developed aGVHD in comparison with the other group of patient with aGVHD after day +30 (p=0.0229). We conclude that the level of GZMB-positive Treg cells is a strong predictor of acute Graft-versus-host disease after day +30 after allo-HSCT.