RNA sequencing detects more challenging cases of somatic hypermutation in the IgHV gene in patients with chronic lymphocytic leukemia.

BACKGROUND/AIM: Chronic lymphocytic leukemia (CLL) exhibits marked biological heterogeneity, and immunoglobulin heavy chain variable (IgHV) mutation status remains a key prognostic biomarker. We evaluated patient IgHV status via RNA sequencing (RNA-seq) and examined real-life clinical outcomes in a retrospective CLL cohort.

MATERIALS AND

METHODS

Eighty-seven adults diagnosed with CLL between 2020 and 2022 at three centers were included. Total RNA from peripheral blood underwent anchored multiplex polymerase chain reaction with the Immunoverse BCR-IGH IGKL v1.0 kit (Integrated DNA Technologies) and was sequenced on the MiSeq platform (Illumina). IgHV mutation status was determined according to ERIC 2022 guidelines, specifically regarding classification of "challenging" double-rearranged cases, with discordant productive rearrangements (one mutated, one unmutated) categorized as unmutated (u-IgHV) to ensure prognostic reliability. Clinical characteristics, treatment patterns, and survival outcomes were analyzed and overall survival (OS) was estimated by the Kaplan-Meier method.

RESULTS

Median age at diagnosis was 60 years and 64% of patients were male. RNA-seq identified double IgHV rearrangements in 73% (n = 63) of patients, 63% of whom harbored two productive rearrangements without evidence of biclonality. Unmutated IgHV (u-IgHV) was observed in 53% (n = 46). Compared to mutated IgHV (m-IgHV), patients with u-IgHV more frequently required first-line therapy (66% vs. 34%) and proceeded to second-line treatment more rapidly. Deletion 17p was significantly more common in the u-IgHV group. After median follow-up of 37.5 months, the 3-year OS rate was 91.9%, with no significant difference in OS between the m-IgHV and u-IgHV cohorts.

CONCLUSION

RNA-seq provided detailed insight into IgHV mutation status and revealed a higher incidence of challenging double-rearranged cases. These real-life findings underscore the clinical utility of RNA-based IgHV profiling and support its use. Prospective studies comparing RNA- and DNA-based assays are warranted to validate concordance and inform risk-adapted management strategies in the treatment of CLL.