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.


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.

Author notes


Asterisk with author names denotes non-ASH members.