Ongoing Antigen Interactions In Splenic Marginal Zone Lymphoma: Revelations From The Analysis Of Intraclonal Diversification In Immunoglobulin Light Chain Genes

Immunogenetic studies have made seminal contributions towards understanding the pathogenesis of splenic marginal zone lymphoma (SMZL) by documenting a highly skewed immunoglobulin (IG) gene repertoire with molecular features strongly implicating selection by antigen(s) in disease ontogeny and evolution. Indeed, a major subset of SMZL, roughly 30% of the entire cohort, is defined by the expression of IG receptors with heavy variable domains (VH) utilizing the IGHV1-2*04 gene. These VH domains exhibit low-level, yet non-randomly targeted somatic hypermutation (SHM), carry long and often positively charged heavy complementarity-determining region 3 with restricted motifs, and also show biased associations with certain light IG genes, namely IGKV3-20, IGKV1-8 and IGLV2-14. We recently documented that the great majority of SMZL, especially IGHV1-2*04 cases, exhibit intraclonal diversification (ID) in IGHV genes, reflecting ongoing interactions with antigen(s). In this study we further extend the analysis of ID in SMZL focusing on IG light genes. To this end, we performed a comprehensive subcloning analysis of IGKV-IGKJ and IGLV-IGLJ gene rearrangements from 11 SMZL cases; two patients were studied at two different time-points. A total of 311 subcloned sequences (10-38/sample, median, 25) were obtained from 9 IGKV-IGKJ and 4 IGLV-IGLJ rearrangements. Multiple alignment of the subcloned sequences revealed that: (1) only one of 13 studied samples (7.8%) carried identical subclones (no ID); (2) 6/13 (46.1%) carried only unconfirmed mutations (UCMs, mutations in single subclones; unconfirmed ID); and, (3) 6/13 (46.1%) carried confirmed mutations (CMs, identical mutations in at least 2 subclones; confirmed ID). Both IGHV1-2*04 cases of the present series belonged to the confirmed ID category and both displayed intense ID with extensive subclone formation. Among cases positive for ID, the number of nucleotide substitutions introduced by ongoing SHM ranged from 1-21. A total of 66 unique substitutions were identified in 42 positions of the variable domain; 44 of these resulted in the replacement of the germline-encoded amino acid (R), while the remaining 22 were silent (S). The distribution of replacement and silent CMs and UCMs in CDRs and FRs was compatible with a canonical SHM process in that R/S ratios were higher in CDRs, except CDR2. In general, ID was more pronounced in IGKV-IGKJ versus IGLV-IGLJ rearrangements, regardless of the overall mutational load of each rearrangement. The analysis of the same rearrangement at different time-points revealed a consistent ID profile: one case carried only UCMs at both time-points, while the other (IGHV1-2*04 case) exhibited a dynamic pattern of appearing and disappearing CMs. Overall, the presence of mutations in the context of ID was independent of the mutational status, as ID was observed even in minimally mutated cases (98-99.9% germline identity). Moreover, in 3/6 cases with confirmed ID, subclones were classified in ≥2 main mutational groups, indicating early “branching” of the clonal population in subpopulations with distinct mutational profiles. In conclusion, the present study complements the immunogenetic profile of SMZL, offering novel molecular evidence for crosstalk with the microenvironment mediated through the clonotypic B-cell receptors. Furthermore, it underscores the significance of IG light chains in the immune pathogenesis of SMZL.