Clonal Selection in the Ontogeny and Evolution of Splenic Marginal Zone Lymphoma Confirming the Existence of Distinct Molecular Subtypes
Authors: Bikos Vasilis , Stalika Evangelia , Karypidou Maria , Baliakas Panagiotis , Davis Zadie , Algara Patricia , Kanellis George , Dagklis Antonis , Traverse-Glehen Alexandra , Hockley Sarah , Mollejo Manuela , Tsaftaris Athanasios , Anagnostopoulos Achilles , Ponzoni Maurilio , Berger Francoise , Felman Pascale , Gonzalez David , Matutes Estella , Piris Miguel Angel , Papadaki Theodora , Ghia Paolo , Oscier David , Belessi Chrysoula , Darzentas Nikos , Stamatopoulos Kostas 
Published: October 2019

Abstract
Abstract 1556
Splenic marginal-zone lymphoma (SMZL) exhibits biased immunoglobulin (IG) heavy and light chain gene repertoires, alluding to selection by antigens and/or superantigens in lymphomagenesis. We recently reported that immunogenetic analysis of the IG receptors enables the identification of molecular subtypes of SMZL, since, remarkably, greater than 30% of cases can be assigned to a single subgroup defined by usage of the *04 polymorphic variant of the IGHV1–2 gene. The IGHV1–2*04 receptors carry long and often positively charged heavy complementarity-determining region 3 with restricted motifs and show biased associations with certain light chain genes (IGKV3–20, IGKV1–8, IGLV2–14). Furthermore, they exhibit low-level, yet non-randomly targeted somatic hypermutation (SHM), likely reflecting a distinctive process of immune interaction with antigen(s). Preliminary work from our group indicated a continued influence of antigen on SMZL clones expressing IGHV1–2*04 receptors reflected in intraclonal diversification (ID) of rearranged IG heavy variable genes. Here we significantly extend our ID studies and explore whether IGHV1–2*04 SMZL are distinctive also in terms of ID compared to SMZL utilizing other IGHV genes. To this end, we performed a comprehensive subcloning analysis of IGHV-IGHD-IGHJ gene rearrangements in a total of 728 subcloned sequences from 39 SMZL cases, including 20 cases expressing IGHV1–2*04 receptors with a median identity to the germline (GI) of 98.7% and 19 cases utilizing other IGHV genes, namely IGHV1–18 (n=3), IGHV3–23 (n=6), IGHV3–30 (n=30), IGHV4–34 (n=3), IGHV4-4 (n=1) and IGHV5–51 (n=3), with a median GI of 97.9%. All non-ubiquitous sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. In order to compare mutation counts between different rearrangements, mutations were normalized to both the nucleotide length and the number of subcloned sequences for each rearrangement. ID was identified in 17/20 (85%) IGHV1–2*04 rearrangements, with confirmed nucleotide substitutions ranging from 1 to 21 per case for a total of 77 unique substitutions. Of these, 51 resulted in the replacement of the germline-encoded amino acid (Replacement mutations, R), whereas the remaining 26 were silent (S). Certain codons (e.g: VH CDR1–36, VH FR2–39, VH FR3–87) were highly targeted for novel changes leading to ID. Interestingly, identical confirmed mutations were shared by subclones of different rearrangements confirming a remarkable restriction in the ongoing SHM profiles in that subclones shared identical confirmed mutations. In fact, 12 amino acid replacements were confirmed in the subclones of at least two rearrangements and were characterized as ”recurrent”. In contrast to IGHV1–2*04 rearrangements, only 8/19 (42%) rearrangements utilizing other IGHV genes showed imprints of ID (p=0.005). Moreover, the ID profiles of non-IGHV1–2*4 cases were different, as evidenced by both the lower numbers of confirmed mutations and the lack of shared mutations (per group of cases utilizing the same IGHV gene). In conclusion, continuous stimulation by antigen seems to be relevant not only for SMZL development but also for selection leading to clonal evolution, at least for a substantial proportion of cases. The extensive and distinctive ID observed in IGHV1–2*04 cases compared to cases utilizing other IGHV genes further supports the hypothesis that the former might constitute a distinct subgroup with important implications for both SMZL sub-classification and future clinical and biological research.

Disclosures:
No relevant conflicts of interest to declare.