Classification of patients with chronic lymphocytic leukemia (CLL) based on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene, excluding the VH CDR3. This may underestimate the true impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions i.e. the VH CDR3. Here we investigated whether SHM may also be present within the VH CDR3 of cases bearing ‘truly unmutated’ IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a ‘truly unmutated’ CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3’IGHV, IGHD, 5’IGHJ regions of the VH CDR3 in both the major IGHV-IGHD-IGHJ gene clonotype and its variants. The identified SHMs were shared between different patients of the same subset, supporting that they represent true events rather than artefacts; moreover, they located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, very likely attributed to SHM, in CLL patients carrying ‘truly unmutated’ IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-focused SHM in U-CLL.
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