High Expression of Activation-Induced Cytidine Deaminase and in Vivo Class Switch Recombination in Mantle Cell Lymphoma: Further Support for Antigen Involvement in Lymphomagenesis

Abstract 1538

According to the WHO 2008 Classification, the cellular origin of mantle cell lymphoma (MCL) is traced to a peripheral B cell of the inner mantle zone, mostly of naïve pre-germinal center type. This notion, however, is seriously challenged by both the remarkable restrictions of the immunoglobulin gene repertoire in MCL and, furthermore, by the fact that the great majority of cases exhibit imprints of somatic hypermutation (SHM) in rearranged IGHV genes, ranging from (mostly) minimal to pronounced. These findings support an antigen-driven origin for MCL, at least for a substantial fraction of the entire cohort. Activation-induced cytidine deaminase (AID) is induced in B cells following contact with antigen and is critically implicated in both somatic hypermutation (SHM) and class switch recombination (CSR). Although the available information about AID expression and in vivo CSR in MCL is limited and contradictory, at least some MCL cases have been reported to express AID and undergo ongoing CSR. With this in mind, here we investigated AID-mRNA isoform expression and isotype switch events in a large series of MCL cases and explored possible associations with IGHV gene repertoire and SHM status. Overall, 107 cases were included in the study and tumor-involved diagnostic tissue samples of different types were evaluated, including: fresh-frozen lymph nodes (LN, n=53), peripheral blood (PB, n=42), spleen (n=5), bone marrow biopsies (n=3) and other (n=4). The neoplastic lymphocytic infiltration ranged from 52–98% (median 80%). Thirty-five of 107 cases (32.7%) carried IGHV genes with 100% identity to the germline (GI) whereas the remaining 72 cases bore some imprint of SHM: in particular, 48/107 cases (44.9%) carried IGHV genes with 97–99.9% GI and, finally, 24/107 cases (22.4%) carried IGHV genes with <97% GI. In keeping with the literature, the IGHV gene repertoire of the present cohort was remarkably biased, with the IGHV3–21, IGHV4–34, IGHV3–23 and IGHV1–8 genes accounting for 55.1% of cases. Profiling of AID mRNA expression was performed by RQ-PCR for the full-length AID (AID-FL) as well as the most frequent splice variants, namely AID-ΔE4a (lacking the first 30 nucleotides from exon 4), and AID-ΔE4 (loss of the entire exon 4). AID transcript levels were calculated as the percentage of AID copy number divided by the copy number of the reference transcript (c-ABL). AID-FL transcripts were detected in 104/107 (97%) cases whereas the AID-ΔE4a and AID-ΔE4 splice variants were detected in 72/107 (67.3%) and 107/107 cases (100%), respectively. The median values for AID-FL, AID-ΔE4a and AID-ΔE4 transcripts were 4.45%, 0.133% and 0.918%, respectively. AID transcript levels varied between different cases by up to 5-log for AID-FL transcripts and 4-log for splice variants. Not unexpectedly, the median transcript levels in LN samples were higher (up to 1-log) compared to PB samples. A highly significant (p<0.001) association was noted between medium-to-high AID-FL transcript levels (AID-FL/ABL○1%) and IGHV GI 100%. Given the difference in tissue origin of our samples, we also performed a separate analysis for LN samples only and found that cases with 100% IGHV GI expressed high AID-FL transcript levels (AID-FL/ABL○10%) significantly (p=0.04) more frequently than cases carrying mutated IGHV genes. Isotype switch events were investigated in 41 cases: overall, 4 cases (9.7%), all with GI<100%, carried alternative tumor-derived Cγ (n=1) or Cα (n=3) transcripts. In conclusion, the present analysis documents AID expression in the vast majority of MCL, thus corroborating our previous hypothesis for antigen involvement in MCL ontogeny. Ongoing CSR events appear to be a feature of MCL, further supporting an activated status, at least for subset of cases.