Comparative evaluation of two NGS-based assays for somatic hypermutation analysis of IGHV genes in chronic lymphocytic leukemia.

[PURPOSE] Somatic hypermutation (SHM) of the immunoglobulin heavy chain variable (IGHV) region is a key prognostic marker in chronic lymphocytic leukemia (CLL). Traditionally, SHM status has been determined using Sanger sequencing (SS); however, next-generation sequencing (NGS) provides an alternative method for assessing both SHM status and clonal rearrangements. This study aimed to compare the performance of two commercially available NGS assays for evaluating IGH clonality and SHM status in CLL.

[METHODS] In this retrospective study, 42 samples from patients diagnosed with CLL were analyzed. Genomic DNA extracted from peripheral blood or bone marrow aspirates was analyzed using two commercial NGS assays: the LymphoTrack® IGHV Leader (Leader) and IGH FR1 (FR1) assays (Invivoscribe, CA, USA). SS was performed as the reference method for SHM assessment.

[RESULTS] The Leader assay identified clonality in 95.2% of cases, whereas the FR1 assay detected clonality in 88.1%. Conclusive SHM status was determined in 90.5% of samples using the Leader assay and in 76.2% using the FR1 assay; when results from both assays were combined, the rate increased to 92.9%. Among samples with conclusive results by both SS and each NGS assay, the Leader assay demonstrated higher concordance with SS (97.1%, 34/35) than the FR1 assay (86.2%, 25/29). Greater variability in clonal detection was observed with the FR1 assay.

[CONCLUSION] These findings indicate that the Leader assay provides a more reliable assessment of SHM status, with higher concordance with SS. Although the FR1 assay may offer additional information regarding clonal patterns, its results should be interpreted cautiously. Given the limited sample size, further studies are warranted to validate these findings. Overall, the Leader assay appears to be more suitable as a primary tool for SHM evaluation, with FR1 results serving a complementary role when interpreted in clinical context.