A number of sequencing studies identified the prognostic impact of somatic mutations in myelodysplastic syndrome (MDS). However the majority of them focused on methylation regulation, apoptosis and proliferation genes. Despite the number of experimental studies published on the role of micro-RNA processing and checkpoint genes in the development of MDS, the clinical data about mutational landscape in these genes is limited. We performed a pilot study which evaluated mutational burden in these genes and their association with common MDS mutations. High prevalence of mutations was observed in the genes studied: 54% had mutations in DICER1, 46% had mutations in LAG3, 20% in CTLA4, 23% in B7-H3, 17% in DROSHA, 14% in PD-1 and 3% in PD-1L. Cluster analysis that included these mutations along with mutations in ASXL1, DNMT3A, EZH2, IDH1, RUNX1, SF3B1, SRSF2, TET2 and TP53 effectively predicted overall survival in the study group (HR 4.2, 95%CI 1.3–13.6, p = 0.016). The study results create the rational for incorporating micro-RNA processing and checkpoint genes in the sequencing panels for MDS and evaluate their role in the multicenter studies.

There is evidence that relapses of acute myeloid leukemia (AML) are closely related to heterogeneous population of leukemic precursors. At least, two classes of the leukemia-initiating cells (LIC) may be discerned, according to recent experimental studies with hematopoietic cell transplants to immunodeficient mice. The main class of LICs is presented by immature precursors with CD34 + CD38immunophenotype which, in turn, are capable of selective expression of BAALC gene. The second class of LICs is presented by relatively mature precursors with more differentiated immunophenotypes. According to indirect findings, they are able of WT1 gene expression, along with blast cells. Since both BAALC and WT1 mRNAs may be quantitatively evaluated by means of standardized quantitative polymerase reaction in real time (qRT-PCR), this approach may be effective for specifying the mechanisms of relapses and resistance to therapy in AML patients. The aim of this work was to perform simultaneous dynamic evaluation of BAALC and WT1 genes expressions along with determination of blast numbers in the tested bone marrow samples in 14 AML patients treated at our Center with Gemtuzumab ozogamicin (GO, Mylotarg), which was combined with high-dose chemotherapy (ChT), followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our preliminary results are as follows: a) superior 3-year overall survival (OS) in general group of patients with normal or nearly-normal karyotypes, and FLT3-mutated AML variants as compared to those with more complex karyotypes and EVI1 gene overexpression (85.7% vs 16.7%; p=0.032); b) highly sensitive response of immature BAALC-expressing precursors to combined ChT and GO treatment; c) hypothetical participation of some mature precursors, along with blast cells, in WT1 gene expression; d) real evidence for switching hematopoietic regulation from immature BAALC-expressing precursors to more mature WT1-expressing progeny. These results suggest diagnostic utility of combined BAALC/ WT1/blast counts panel for quantitative studies and assessment of distinct precursors in AML progression and emergence of relapses.

The presented data are showing the crucial role and great prognostic significance of BAALC-expressing leukemic precursors in origin and development of posttransplant relapses (PTR) in acute myeloid leukemia patients with different cytological variants. For evidence simultaneous serial measurements of BAALC and WT1 transcript copy numbers by means of quantitative real time polymerase chain reaction were used at diagnosis, before conditioning regimen as well as at PTR in 50 patients treated with allogeneic hematopoietic stem cell transplantation (alloHSCT). Thirty-eight of them were adults and twelve pediatric patients aged 1-60 years (median – 25.8 years). It was shown, that BAALC gene overexpression to be presented in all studied cytological and being combined with increased level of WT1 expression at PTR in most of them. This combination was prognostically poor since it is largely associated with increased cumulative incidence of PTR (p<0.0001), and shortened event free (p<0.0001) and overall survival (p=0.002).

Osteopetrosis is a group of rare inheritable disorders of the skeleton characterized by increased bone density. The disease is remarkably heterogeneous in clinical presentation and often misdiagnosed. Therefore, genetic testing and molecular pathogenicity analysis are essential for precise diagnosis and new targets for preventive pharmacotherapy. Mutations in the CLCN7 gene give rise to the complete spectrum of osteopetrosis phenotypes and are responsible for about 75% of cases of autosomal dominant osteopetrosis. In this study, we report the identification of a novel variant in the CLCN7 gene in a patient diagnosed with osteopetrosis and provide evidence for its significance (likely deleterious) based on extensive comparative genomics, protein sequence and structure analysis. A set of automated bioinformatics tools used to predict consequences of this variant identified it as deleterious or pathogenic. Structure analysis revealed that the variant is located at the same “hot spot” as the most common CLCN7 mutations causing osteopetrosis. Deep phylogenetic reconstruction showed that not only Leu614Arg, but any non-aliphatic substitutions in this position are evolutionarily intolerant, further supporting the deleterious nature of the variant. The present study provides further evidence that reconstructing a precise evolutionary history of a gene helps in predicting phenotypical consequences of variants of uncertain significance.

Osteopetrosis is a group of rare inheritable disorders of the skeleton characterized by increased bone density. The disease is remarkably heterogeneous in clinical presentation and often misdiagnosed. Therefore, genetic testing and molecular pathogenicity analysis are essential for precise diagnosis and new targets for preventive pharmacotherapy. Mutations in the CLCN7 gene give rise to the complete spectrum of osteopetrosis phenotypes and are responsible for about 75% of cases of autosomal dominant osteopetrosis. In this study, we report the identification of a novel variant in the CLCN7 gene in a patient diagnosed with osteopetrosis and provide evidence for its significance (likely deleterious) based on extensive comparative genomics, protein sequence and structure analysis. A set of automated bioinformatics tools used to predict consequences of this variant identified it as deleterious or pathogenic. Structure analysis revealed that the variant is located at the same &ldquo;hot spot&rdquo; as the most common CLCN7 mutations causing osteopetrosis. Deep phylogenetic reconstruction showed that not only Leu614Arg, but any non-aliphatic substitutions in this position are evolutionarily intolerant, further supporting the deleterious nature of the variant. The present study provides further evidence that reconstructing a precise evolutionary history of a gene helps predicting phenotypical consequences of variants of uncertain significance.

No abstract

The role of prophylactic TKIs after allogeneic stem cell transplantation in Ph-positive acute lymphoblastic leukemia (ALL) remains controversial. We performed a retrospective study in 106 adult patients subjected to allogeneic hematopoietic stem cell transplantation (allo-HSCT) from matched related donors (MRD, 26%), matched unrelated donors (MUD/MMUD, 60%), and haploidentical donors (14%) in complete remission (CR1, 59%), CR2 (14%), and advanced disease (27%). Among them, 60 (57%), received 1 st -or 2 nd -generation TKIs as prophylaxis after allo-HSCT. In multivariate analysis of RFS, the following factors were associated with reduced risk of relapse or death: allo-HSCT after 2012 (HR=0.46, 95%CI 0.26-0.83, p=0.009), any MRD status of the disease before allo-HSCT except active disease with relatively similar HR in the context of post-transplant TKI prophylaxis. Allo-HSCT from haploidentical donor was associated with increased risk of relapse or death (HR=2.71, 95% CI 1.20-6.13 p=0.016). We were unable to demonstrate the significance of chronic GvHD when performing landmark analysis on day+180 and day+270, as based on available data (HR=0.43, 95% CI 0.13-1. 45, p=0.17 and HR=0.5, p=0.161, respectively), under the conditions of maintaining TKI therapy after allo-HSCT. This relatively large study in unfavorable group of patients confirms an importance of TKIs prophylaxis for adult patients with Ph-positive ALL after allo-HSCT. A larger group of patients is required to formulate strong clinical recommendations in this cohort.

Detection of molecular markers characteristic to malignant cells is the key task of molecular genetic studies in oncohematology, aiming for timely diagnosis of the diseases and evaluation of their progression, like as relapse risks. Exosomes are among the most promising objects for such studies. They represent a fraction of extracellular membrane particles ranging from 30 to 150 nm in size, presumably containing such tumor markers. These vesicles are found in blood serum and other biological fluids, being important participants in cell-cell interactions capable of transferring various biopolymers, which can also mediate the reprogramming of the functions of the recipient cells and influence the development of resistance to therapy. On the basis of exosomes, one may optimize such a novel diagnostic approach as fluid biopsy which is minimally invasive, and, therefore, can also be used as a specific monitoring tool for prediction of tumor relapse development.