OP0179 Molecular documentation of the clonal evolution of a diffuse large b-cell lymphoma out of clonally expanded rheumatoid factor-expressing b cells in a sjÖgren’s syndrome patient

Background Sjogren’s syndrome (SS) is the autoimmune disease with the highest risk of lymphoma development. SS patients develop most frequently MALT lymphoma and to a lesser extent diffuse large B-cell lymphoma (DLBCL). Previously, we have shown that at least 40% of salivary gland MALT lymphomas express groups of near identical (also called stereotypic) B-cell receptors, which display in vitro mono-specific rheumatoid factor (RF) reactivity1. Recently, we have analysed the B-cell immunoglobulin heavy variable (IGHV) repertoire in 4 SS labial salivary glands. In 2 out of 4 salivary glands only one minor stereotypic RF B-cell clone was detected. Interestingly, in one salivary gland of patient SG22 a highly expanded stereotypic RF-expressing B-cell clone was present, which was also detected in peripheral blood. Twenty six months later, a clonally-related DLBCL was diagnosed2. Objectives To study the molecular alterations present in the RF-expressing B cell clones of salivary gland, peripheral blood and in the DLBCL of SS patient SG22. Methods From peripheral blood 4 RF-expressing immortalised B-cell clones with identical IGHV rearrangements were isolated. These 4 RF-clones were analysed by whole exome sequencing and the identified non-synonymous exome mutations were traced in the salivary gland and in the DLBCL, using targeted next generation sequencing. Results In total we identified 56 exome mutations in the 4 RF B-cell clones. Twelve non-synonymous mutations were shared between all 4 RF-clones, of which one was a missense mutation in CARD11, a well-known oncogenic mutation of DLBCL. In the salivary gland all shared 12 non-synonymous mutations were detected, whereas in the DLBCL only 4 of these mutations were detected. Remarkably, the CARD11 mutation was not detected in the DLBCL. The exome mutations were analysed for typical characteristics of induction by the B-cell specific mutator AID. This revealed that the large majority of mutations (80%) were (i) not located within 1,5 kB of the transcription initiation site of the genes, (ii) did not concern C/G nucleotides in AID hotspots (WRC/GYW) and (iii) were not in genes expressed by B cells. This lack of evidence for AID involvement, suggests that the exome mutations of the RF-clones were due to replication errors, obtained during their clonal expansion. Interestingly, as compared to the RF-clones of peripheral blood, the DLBCL accumulated 44 extra somatic mutations together in IGHV and IGKV. As expected, these new IGHV/IGKV mutations showed the characteristics of AID involvement, indicating that in an ancestor RF-clone, AID activity was induced. Conclusions We have documented the clonal outgrowth and diversification of a RF-specific B-cell clone in a SS patient and the evolution of this clone into a DLBCL. Our analysis are indicative for AID activity in the lymphoma cells, represented by accumulation of IGHV/IGKV mutations. However, we have no evidence that AID is instrumental in the accumulation of the non-IG mutations in the RF-clones from peripheral blood. References [1] Bende, et al. J. Exp. Med2005;201:1229–1241. [2] Bende, et al. Arthritis Rheumatol2015;67:1074–1083. Disclosure of Interest None declared