A novel next-generation sequencing capture-based strategy to report somatic hypermutation status using genomic regions downstream to immunoglobulin rearrangements

The somatic hypermutation (SHM) status of the clonotypic, rearranged immunoglobulin heavy variable (IGHV) gene is an established prognostic and predictive marker in chronic lymphocytic leukaemia (CLL). Analysis of SHM is generally performed by PCR-amplification of clonal IGHV-IGHDIGHJ gene rearrangements followed by sequencing to identify IGHV gene sequences and germline identity. Targeted-hybridisation next-generation sequencing (NGS) can simultaneously assess clonality and other genetic aberrations, however it has limitations for SHM analysis due to sequence similarity between different IGHV genes and mutations introduced by SHM, which can affect alignment efficiency and accuracy. We have developed a novel SHM assessment strategy using a targeted-hybridisation NGS approach (EuroClonality-NDC assay) and applied it to 331 samples of lymphoproliferative disorder (LPDs). Our strategy focuses on analysing the sequence downstream to the clonotypic, rearranged IGHJ gene up to the IGHM enhancer (IGHJ-E) which provides more accurate alignment. Overall, 84/95 (88.4%) CLL cases with conventional SHM data showed concordant SHM status, increasing to 91.6% when excluding borderline cases. Additionally, IGHJ-E mutation analysis in a wide range of pre- and post-germinal centre LPD showed significant correlation with differentiation and lineage status, suggesting that IGHJ-E analysis is a promising surrogate marker enabling SHM to be reported using NGS-capture strategies and whole genome sequencing.