Somatic Mutation and Isotype Switch Events Outside Igv Gene Loci Provide New Evidence for a Role of Antigenic Challenge in Mantle Cell Lymphoma Etiology

Mantle cell lymphoma (MCL) represents 5-10% of B-cell lymphomas and generally exhibits an aggressive clinical behavior. t(11;14) translocation, the genetic hallmark and early initiating event of MCL pathogenesis, lead to the constitutive CCND1 expression and consequently to cell-cycle deregulation. However, t(11;14) is also present in the blood from rare healthy individuals indicating that alone the t(11;14) is not sufficientfor malignant transformation. It has long been assumed in MCL that t(11;14)⁺ cells were actually carried by circulating naive and antigen-inexperienced B-cells. Yet, several reports, including long-term persistence of t(11;14)⁺ cells in lymphoma free-patients, biased immunoglobulin heavy variable chain gene (IgV_H) repertoire and evidence of subset of cases with mutated IgV_H genes led us to question this model and further investigate the role of antigenic challenge in MCL etiologic origin.

As a first approach, we sought to determine the class-switch recombination (CSR) status of t(11;14)⁺ cells in MCL patients. CSR, as somatic hypermutation (SHM), are diversification mechanisms of the B cell receptor induced upon antigen encounter in the germinal center (GC) reaction, and absent in naive cells. Evidence for ongoing switch events in MCL has been previously reported with detection of mature switch transcripts, however in MCL cells, sIgM(D) expression remains clearly evident suggesting that effective deletional switch events occur infrequently on the functional allele. To characterize CSR in t(11;14)⁺ cells, we thus took advantage of their unique BCL1/IGH translocation signature assuming that BCL1/IGH fusions do not prevent CSR to occur on the downstream constant region of the IGH locus. Using 2 long-range PCR assays designed to amplify unswitched BCL1/Sµ and switched BCL1/Sγ regions, DNA from 30 MCL patients differing by their IgV_H status were tested. Of the MCL cases, we confirmed that 7 of 30 (23%) were unmutated (UM, 100% germline homology) and 23 of 30 (77%) were mutated (M), considering as mutated all cases exhibiting any level of SHM (ranging from minimal: >0.3% to high rate: 7.0%). Although mutated BCRs were common in most MCL cases, we found that only 1 out of 30 MCL cases underwent CSR to IgG on the non-productive, translocated allele. Accordingly, this case carried a mutated IgV_H gene with 97.5% germline identity in line with a GC/antigen-experienced signature of the tumor cells. Contrary to SHM events of the IgV_H gene region, CSR deletional events appeared very infrequent in the bulk of MCL cells both on the functional and translocated allele, contrasting from findings in other GC-derived non-Hodgkin lymphomas where the same selective pressure to maintain a sIgM is accompanied by deletional switch events on the non-functional allele. To get more insights into a role of antigenic drive in MCL and AID targeting outside the V_H gene loci, we next evaluated the clonal evolution of t(11;14)⁺ cells from 11 MCL cases (2 UM and 9 M) by analyzing SHM in the Sμ region on the translocated allele using sequencing of LR-PCR amplicons (with 2 subclones/patient). We found that 63% (7 out of 11) of BCL1/IGH clones display significant levels of SHM in the Sµ together with intra-Sµ indels (2/11) compatible with off-target AID-mediated events linked to chronic(?) antigen encounter. In addition, we found a good correlation between mutated BCR and the SHM levels in Sμ regions for a given patient. Importantly, in one instance subclones from a MCL case displayed intraclonal diversification (despite the fact that the expected rate of SHM is much lower in the Sµ region than in the IgV_H region), indicative of an ongoing AID activity at least in a subset of MCL cases. In conclusion, we found that CSR events, although detectable in some MCL cases, do not appear to participate predominantly or to be selected during MCL lymphomagenesis. By contrast, analysis of SHM profiles both on the functional and the non-functional translocated allele provide evidence for an active AID activity in MCL cells inside and outside V-gene loci, highlighting the propensity of some t(11;14)⁺ clones to acquire a complementary oncogenic hit over time as a result of recurrent antigen challenge.