Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

Recognizing Minor Leukemic Populations with Monocytic Features in Mixed-Phenotype Acute Leukemia by Flow Cell Sorting Followed by Cytogenetic and Molecular Studies: Report of Five Exemplary Cases.

Mixed-phenotype acute leukemia (MPAL), a rare and heterogeneous category of acute leukemia, is characterized by cross-lineage antigen expression. Leukemic blasts in MPAL can be represented either by one population with multiple markers of different lineages or by several single-lineage populations. In some cases, a major blast population may coexist with a smaller population that has minor immunophenotypic abnormalities and may be missed even by an experienced pathologist. To avoid misdiagnosis, we suggest sorting doubtful populations and leukemic blasts and searching for similar genetic aberrations. Using this approach, we examined questionable monocytic populations in five patients with dominant leukemic populations of B-lymphoblastic origin. Cell populations were isolated either for fluorescence in situ hybridization or for clonality assessment by multiplex PCR or next-generation sequencing. In all cases, monocytic cells shared the same gene rearrangements with dominant leukemic populations, unequivocally confirming the same leukemic origin. This approach is able to identify implicit cases of MPAL and therefore leads to the necessary clinical management for patients.

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia.

Retroelements (RE) have been proposed as important players in cancerogenesis. Different cancer types are characterized by a different level of tumor-specific RE insertions. In previous studies, small cohorts of hematological malignancies, such as acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia have been characterized by a low level of RE insertional activity. Acute lymphoblastic leukemia (ALL) in adults and childhood acute leukemias have not been studied in this context. We performed a search for new RE insertions (Alu and L1) in 44 childhood ALL, 14 childhood acute myeloid leukemia, and 14 adult ALL samples using a highly sensitive NGS-based approach. First, we evaluated the method sensitivity revealing the 1% detection threshold for the proportion of cells with specific RE insertion. Following this result, we did not identify new tumor-specific RE insertions in the tested cohort of acute leukemia samples at the established level of sensitivity. Additionally, we analyzed the transcription levels of active L1 copies and found them increased. Thus, the increased transcription of active L1 copies is not sufficient for overt elevation of L1 retrotranspositional activity in leukemia.

Rituximab and reduced-intensity chemotherapy in children and adolescents with mature B-cell lymphoma: interim results for 231 patients enrolled in the second Russian-Belorussian multicentre study B-NHL-2010M.

The value of adding rituximab to chemotherapy in children with aggressive B-cell non-Hodgkin lymphoma (B-NHL) is still insufficiently studied. We enrolled 231 patients [mean age 9 years old (range 2-17); male:female ratio 3·4:1] with Burkitt (BL, 179 patients, 76·7%), diffuse large B-cell (32 patients, 14%), primary mediastinal B-cell (14 patients, 6%), and other (6 patients, 2·6%) B-cell lymphomas in a prospective study of immuno-chemotherapy. Stages were I-II in 32% and III-IV in 68% of the patients. Four doses of 375 mg/m2 rituximab were added to the Berlin-Frankfurt-Munster-NHL-90-like chemotherapy, with methotrexate being reduced or omitted in the first 2 induction blocks. The complete remission rate was 100% in limited-stage and 91·4% in advanced-stage patients. Five advanced-stage patients (2·2%) died in induction and 1 patient with stage 2 B-NHL died in remission; 11 patients in the high-risk group progressed on therapy (3 non-BL are alive after salvage) and 5 relapsed. Sixteen patients (9·7%) with advanced stage disease proceeded to transplant. With a median follow-up of 46 months, 98·5 ± 1% of patients with limited disease and 88·1 ± 2% (88·1% in Risk Group 3; 82·6% in Risk Group 4) in advanced stages are alive. This study confirmed that combined immunochemotherapy for B-lymphomas is highly effective in children, despite reducing the intensity of the induction blocks.