Higher Order Restrictions Of The Immunoglobulin Repertoire In CLL Stereotyped Subsets #2 And #169. NGS immunoprofiling of CLL subsets #2 and #169

Stereotyped subset #2 (IGHV3-21/IGLV3-21) is the largest subset in CLL (~3% of all patients). Assignment to subset #2 is clinically relevant since these patients experience an aggressive disease irrespective of the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene. Low-throughput evidence suggests that stereotyped subset #169, a minor CLL subset (~0.21% of all CLL), resembles subset #2 at the immunogenetic level. In specific: (i) the clonotypic heavy chains (HC) of subset #169 are encoded by the IGHV3-48 gene, closely related to the IGHV3-21 gene; (ii) both subsets carry VH CDR3 of 9-amino acids (aa) with a conserved aspartic acid (D) at VH CDR3 position 3; and, (iii) both subsets bear light chains (LC) encoded by the IGLV3-21 gene with restricted VL CDR3. Here we assessed more thoroughly the ontogenetic relationship of CLL subsets #2 and #169 as evidenced in their respective immunogenetic signatures. Using next-generation sequencing methodologies, intraclonal diversification due to ongoing SHM was identified in both heavy and light chain genes of both subsets, albeit more intense in the latter. Furthermore, we identified the presence of common light chain clonotypes which also displayed shared SHMs, with some remarkable examples of virtually ubiquitous SHMs in all analyzed cases at either clonal or subclonal level, strongly suggestive of common antigenic drive. This phenomenon was apparent in the heavy chain as well, where, despite using different, albeit phylogenetically related genes, identical SHMs were detected between CLL subsets #2 and #169. The present high-throughput immunogenetic evidence cements the immunogenetic relatedness of CLL stereotyped subsets #2 and #169, further highlighting the role of antigen selection throughout their natural history.