Новейший метод секвенирование нового поколения (Next Generation Sequencing, NGS) позволяет выявлять генетическое и эпигенетическое разнообразие хронического лимфолейкоза (ХЛЛ), декодируя мутационный статус и оценивая его влияние на течение заболевания. Учитывая, что тактика лечения пациентов с ХЛЛ претерпела революционные изменения в последние два десятилетия, детекция драйверных мутаций позволит выбирать оптимальные терапевтические схемы и содействовать разработке и дальнейшему внедрению новейших таргетных препаратов и их комбинаций. Риск-адаптированный подход к терапии ХЛЛ, основанный на группах генетического риска, будет являться базисом персонифицированного лечения. Целью настоящей работы является оценка мутационного статуса пациентов сХЛЛ с использованием разработанной лимфоидной таргетной NGS-панели и изучение возможной корреляции мутационного статуса с клиническими характеристиками заболевания. В проспективное исследование было включено 68 пациентов с ХЛЛ: нелеченые (n=56) и рецидивирующие и рефрактерные формы (n=12). С целью апробации «Лимфоидной таргетной NGS панели генов» было выполнено исследование на образцах ДНК пациентов (n=19) с ХЛЛ. Использование NGS позволило выявить генетические аберрации у всех исследуемых пациентов. Общее количество мутаций составило 767. Полученные нами данные пилотного исследования демонстрируют возможность применения технологии NGS в клинической практике. Для оценки корреляции клинико-лабораторных параметров с мутационным статусом пациентов сХЛЛ исследование будет продолжено. Учитывая, что информация о прогностической значимости ряда выявленных мутаций в настоящее время отсутствует, требуются дополнительные исследования с целью определения их влияния на течение ХЛЛ. The newest method - Next Generation Sequencing (NGS) - allows detecting the genetic and epigenetic diversity of chronic lymphocytic leukemia (CLL), decoding the mutational status and assessing its effect on the course of the disease. Considering that the tactics of treatment of patients with CLL have undergone revolutionary changes in the past two decades, the detection of driver mutations will allow choosing the optimal therapeutic regimens and promoting the development and further implementation of the latest targeted drugs and their combinations. A risk-adapted approach to CLL therapy based on genetic risk groups will be the base for personalized treatment. This work aims to assess the mutational status of patients with CLL using the developed “Lymphoid targeted NGS panel” and to study the possible correlation of the mutational status with the clinical characteristics of the disease. The prospective study included 68 patients with CLL: untreated (n=56) and relapse/refractory (n=12). In order to test the “Lymphoid targeted NGS gene panel”, the study was carried out on DNA samples from patients (n=19) with CLL. The use of NGS let to identify genetic aberrations in all patients. The total number of mutations was 767. Our pilot study data demonstrate the possibility of using NGS technology in clinical practice. The study will be continued to assess the correlation of clinical and laboratory parameters with CLL’s mutational status. Considering that there is currently no information on the prognostic significance of several identified mutations, additional studies are required.

Despite the manifestations of primary ciliary dyskinesia (PCD) in the neonatal period or in the first year of life, the diagnosis of this rare disease is usually established at the age of 4–7 years. The aim of the study was to search for reserves for early diagnosis of PCD. Materials and methods of research: 17 patients were observed with PCD, confirmed on the basis of the PICADAR scale, highspeed light video microscopy (all patients) and transmission electron microscopy (in 3 patients) of the mucous biopsy of the respiratory membrane using a genetic study (in one patient). Results: all patients were born full-term; neonatal respiratory distress syndrome (RDS) was observed in 71% of patients; the median duration of mechanical ventilation/oxygen therapy was 14 [7; 21] days; lateralization abnormalities were found in 35% of patients; all patients had persistent nasal congestion and/or rhinorrhea, 94% had chronic or recurrent sinusitis, otitis, recurrent pneumonia, chest x-ray and computed tomography (CT) scans in 71% had atelectasis with constant localization in the middle lobe, 12 of 15 patients, traced in the follow-up, – bronchiectasis (BE), also mainly in the middle lobe (in 9 patients). The average age at diagnosis was 5 [1.75; 7] years, patients with an established diagnosis over the age of 3 years were more often diagnosed with BE. The median of PICADAR scores was 7 [6; 8] points. Conclusions: to establish early diagnosis of PCD, it is important to consider neonatal RDS, lateralization of organs, difficulty in nasal breathing, starting from the first half of life.

Background: Rapid progress in next-generation sequencing (NGS) technologies make it possible to spell out the mutational status, the genetic and epigenetic variability of chronic lymphocytic leukemia (CLL). The identification of driver mutations allows us to expand understanding of the pathogenesis of CLL, to identify prognostic groups and to select potential targets for therapy, contributing to the development and implementation of new targeted drugs and its combinations. Nevertheless, the course of CLL does not always correspond to the existing prognostic risk groups, assessed by "standard" cytogenetic and molecular genetic methods. The NGS technology admits establishing markers of an unfavorable course of the disease. Aim: To assess the mutational status of patients (pts) with CLL using the developed Lymphoid Targeted NGS Panel and to study the possible correlation of the mutational status with the clinical characteristics of the disease. Method: In this prospective study were included 24 pts with CLL: treatment-naïve (n=8) and relapsed/refractory (n=16). The diagnosis of CLL was established according to iwCLL criteria (iwCLL, Hallek et al., 2018) and show only typical immunophenotype. The pts were divided into three prognostic groups according to cytogenetic assay: favorable (n=14), neutral (n=2), and unfavorable (n=8). Although, 20/24 pts were divided into two prognostic groups taking into account the data on the mutational status of the immunoglobulin heavy-chain variable (IGHV) region: favorable (n=8) and unfavorable (n=12). Four patients have no available data on IGHV mutational status. All patients had indications for starting treatment: FCR (n=7), RB (n=6), ibrutinib (n=3), venetoclax (n=1), acalabrutinib (n=5), combination of venetoclax and acalabrutinib (n=2). DNA samples were extracted from peripheral B-cell lymphocytes via the standard phenol-chloroform method. Average reading depth of 1000x is produced on a MiSeq platform (Illumina, USA). The 2% threshold of allele frequency (VAF) was used. The clinical significance of mutations was established using the following databases: COSMIC, ClinVar, gnomAD with application in silico analysis (Cscape, Cancer Genome Interpreter, SNPs&Go). The Lymphoid Panel includes 117 genes, part of which is involved in the main 8 cellular signaling pathways underlying the pathogenesis of CLL. We have completed a pilot NGS study using the developed Lymphoid Targeted Gene Panel on DNA samples of six treatment-naïve pts. Results: Genetic aberrations were identified in all DNA samples using NGS. Somatic mutations were detected in 82.9% of cases, in an amount from 15 to 37. In four pts (4/6) with an unfavorable prognosis (cytogenetics and unmutated IGHV), known pathogenic variants of mutations were identified: JAK3 V722L, NOTCH1 P2514fs*4, IDH2 T352P, TP53 Lys120Glu, BRAF D594G. The existing approach to the interpretation of the results does not allow making an unambiguous conclusion about the clinical significance of variants in the IDH2 and JAK3 genes, despite the known pathogenic effect of the variants. The detected variant of the mutation (Lys120Glu) in the TP53 gene is often associated with the presence of a 17p13 deletion, which was confirmed by the FISH assay and correlated with the unfavorable clinical course of the disease in patient CLL-024. Twenty-two mutations were identified, the pathogenicity of which has not yet been determined, in the amount of 2 to 5 (median=3.5) mutations per patient. It should be noted that two patients (CLL-023, CLL-024) with unfavorable prognosis had mutations both in BCR gene and in NOTCH2 gene of unknown significance. Conclusion: The data obtained from a pilot study demonstrate the possibility of using NGS technology in clinical practice. The assessment of the mutational status of pts with CLL using NGS correlates with the clinical parameters of pts. Considering that there is currently no information about prognostic significances of identified mutations, additional research is required. Disclosures Martynkevich: Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Shuvaev:Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Voloshin:Novartis: Honoraria, Speakers Bureau.

Introduction.In recent times, more and more data are available on the mechanisms of development of primary and secondary resistance to therapy with tyrosine kinase inhibitors (TKIs) in patients with CML. The use of next-generation sequencing (NGS) allows detecting mutations in theBCR-ABLkinase domain (KD) in some patients with resistance to TKI therapy. However, the reasons for resistance in patients without mutations in theBCR-ABLKD are still unclear. Aim.To determineBCR-ABL-independent molecular genetic markers of resistance to TKI therapy in patients with CML using NGS. Materials and methods.We examined blood samples from 15 patients with resistant CP CML (8 men and 7 women) aged 32 to 59 years (Me = 44 years). Resistance to TKI therapy was determined according to the ELN 2013 criteria. In this group: 2 patients received therapy with 2 TKIs, 8 patients with 3 TKIs, 3 patients - 4 TKIs, and 2 patients received 5 TKIs (Me = 3 TKIs). Cytogenetic test revealed trisomy 8 in 3 patients. One patient was diagnosed with the T315I mutation at once. The patients underwent NGS examination of the myeloid panel, which included 55 genes, with an average reading depth of 1000x using a MiSeq device (Illumina). The 2% threshold of variant allele frequency (VAF) was used. The clinical significance of mutations was evaluated using the COSMIC, ClinVar and OMIM databases. Results.By NGS we identified genetic aberrations in all patients: an average of 5 mutations (from 1 to 10 in one patient). A total of 77 mutations with unclear clinical significance were identified: 54 of them were missense mutations, 22 were synonymous mutations, and 1 frameshift mutation. The most common abnormalities were found in theNF1(10),TET2(8),ATRX(7), andSTAG2(6) genes. In 2 patients, the simultaneous presence of mutations in theATRXandNF1genes was found, they had a primary-resistant course of the disease, MMR was not achieved even on the 3rd and 4th lines of targeted therapy. Three patients were found to have simultaneous mutations in theNF1andSTAG2genes, including two patients who also had a primary-resistant course of the disease, and one of them showed resistance to all 5 TKIs. In 2 patients with the simultaneous presence of mutations in theATRXandTET2genes, resistance to 2 TKIs was registered; CCyR without MMR was achieved only with ponatinib. In 3 patients with singleSTAG2mutations, MMR was achieved on the third line of TKI. One patient with 3 mutations inTET2also achieved MMR only on the third line TKI. Mutations inTET2are more common in Ph-negative MPNs. In our study, mutations were most often detected in theNF1,ATRXandSTAG2genes. Although mutations in theNF1andATRXgenes have been described previously, they were found in responding patients with CP CML (Kim et. al. Blood 2017). In our study, mutations in these genes were found in therapy-resistant patients with an unfavorable course of the disease. TheNF1gene encodes a GTPase activating protein (GAP), which is involved in inhibiting the RAS/MAPK signal transduction pathway and is a tumor suppressor. Mutations in this gene are often associated with the development of CMML and JMML, resistance to therapy and decreased overall survival. TheATRXgene encodes a protein involved in chromatin remodeling, regulation of transcription, replication, and maintenance of telomere structure. TheSTAG2gene encodes a protein that is part of the cohesin complex. Possibly, abnormalities in these genes may belong to a clone of leukemic stem cells (Vetrie et. al. Nature 2020), cause their resistance to TKI,BCR-ABL-independent proliferative potential and their genetic instability. In addition, some of these mutations, possibly, are preleukemic - they could precede the emergence of the Ph-clone in such patients by the mechanism of clonal hematopoiesis of indeterminate potential and may cause an unfavorable prognosis. This allowed us to suggest the prognostic and therapeutic value of mutations in theNF1,ATRXandSTAG2genes in CP CML patients with resistance to TKI therapy. Conclusion.The obtained results of a pilot study of NGS use for predictingBCR-ABL-independent resistance to TKI therapy in patients with CML can serve as a basis for further research aimed at developing prognostic models and choosing a treatment method for resistance to targeted therapy. Disclosures Voloshin: Novartis:Honoraria, Speakers Bureau.Fominykh:Novartis:Honoraria, Speakers Bureau;Pfizer:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau.Shuvaev:Novartis:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau;Pfizer:Honoraria, Speakers Bureau.Martynkevich:Pfizer:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau;Novartis:Honoraria, Speakers Bureau.