CLL-174: NGS Approach for IGHV Rearrangement Analysis in Complicated CLL Cases

• Published in: Clinical lymphoma, myeloma and leukemia Vol. 21; p. S321
• Main Authors: Biderman, Bella, Dumpis, Sergey, Likold, Ekaterina, Julhakyan, Hunan, Sudarikov, Andrey
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.09.2021
• Subjects: CLL; IGHV; NGS; rearrangement analysis; CLL; NGS; IGHV; rearrangement analysis
• ISSN: 2152-2650; 2152-2669
• DOI: 10.1016/S2152-2650(21)01755-9

Mutational status of the IGHV genes and B-cell receptor stereotype are the key prognostic factors for CLL. However, the routinely used Sanger sequencing technique may fail for cases with an expansion of more than one tumor B-cell clones, especially with rearranged genes of the same IGHV family or in cases with low tumor burden. To evaluate the robustness of NGS technology for IGHV analysis in complicated CLL cases. DNA samples from 76 CLL patients for whom we previously failed to obtain IGHV rearrangement data by Sanger sequencing were included in the study. IGHV loci were PCR amplified as described in the BIOMED-2 protocol and by Campbell et al. Sequencing libraries were prepared with Nextera XT kit and were analyzed with MiSeq Genome Analyzer (Illumina, USA). Sequencing data were processed via Mixcr software (Bolotin et al.https://github.com/milaboratory/mixcr). The mutational status and receptor stereotype were identified using the IMGT and ARRest databases. In 52 cases, two clonal rearrangements were identified; in 49 cases, IGHV genes of the same family were used in both clones, and in 3 cases, IGHV families were different. In 50 cases, only one rearrangement was productive, and two patients had two productive IGHV rearrangements. Herewith, the mutational status did not differ within the rearrangement pairs. Thus, we determined U-CLL for 36 patients and M-CLL for 16 patients. In addition, four patients with stereotyped receptor CLL#2 and three patients with CLL#1 were found. Twenty patients had only one productive clonal rearrangement. The heterogeneity of Sanger sequencing in these cases was presumably related to a low tumor burden in the sample compared with a high polyclonal background or with intraclonal heterogeneity. Four patients had three or more clonal rearrangements. In two cases, all rearrangements were productive and highly mutated. Two other patients had IGHV rearrangements with different mutational status; this could be possible in the presence of additional lymphoproliferative disease. Though Sanger sequencing is still more suitable for routine IGHV gene analysis in CLL, a significant number of complicated cases might benefit from NGS technology, which allows definite diagnosis and assignment of appropriate treatment.