Non Germinal-Center B Cell Like Diffuse Large B Cell Lymphoma Results From Repeated Activation of IgM B Cells That Resist Class-Switching.
Authors: Ruminy Philippe , Couronné Lucile , Jardin Fabrice , Parmentier Francoise , Rainville Vincianne , Mauger Camille , Picquenot Jean-Michel , Lenormand Bernard , Cornic Marie , Bertrand Philippe , Tilly Hervé , Bastard Christian 
Published: October 2019

Abstract
Abstract 318
Aberrant junctions involving the switch regions of the IGH locus are frequent in the non-GCB subtype of diffuse large B cell lymphomas (DLBCL), suggesting that some deregulation of the class switch recombination process (CSR) participates in the acquisition of genomic abnormalities in this pathology. To address the impact of this defect on the B cell receptor (BCR) itself we characterized the variable and constant regions of the immunoglobulin heavy chain by RT-PCR in 51 cases. Our results show that, in contrast with most GCB cases, almost all non-GCB DLBCL express an IgM (25/28 vs. 9/23; in non-GCB and GCB cases, respectively, P=0.0002; Fisher's exact test). We addressed the molecular bases of this IgM restriction by studying the configuration of the IGH loci. We found frequent legitimate and illegitimate switch junctions in IgM+ DLBCLs by genomic PCR (in 25/34 cases), showing that CSR is activated during lymphomagenesis but does not modify the isotype of the BCR. Interestingly, fluorescence in situ experiments using a probe directed against the Cμ gene showed that class switch recombination (CSR) occurs symmetrically on the two IGH alleles in IgG+ cases (5/5 tested cases) and only asymmetrically in a majority of IgM+ cases (8/11 tested cases). This finding indicates that the expression of an IgM does not result from a global failure of the CSR machinery but instead from a specific lack of recombination on the productive IGH allele. In line with this hypothesis we observed that the large internal deletions in the switch μ region (Sμ) which are known to be frequent in this pathology are specific to IgM+ cases (15/34 IgM+ vs. 0/15 IgG+ cases, P=0.0018; Fisher's exact test) and cluster on the productive IGH alleles (7/7 cases characterized by allele specific PCR), suggesting that the loss of the Sμ repeats plays a direct role in IgM stabilization by reducing the probability that the Sμ region could be involved in CSR. Finally, we observed that the non-GCB signature is associated with a higher level of somatic mutations in the 5'Sμ region (median mutation rates 4.51% vs. 2.07% in non-GCB and GCB cases, P=0.034, Mann-Whitney Test) with a strong bias toward C>T and G>A transitions suggesting repeated AID activations. We found the same bias it the BCL6 and PAX5 genes but not in VDJ exons, suggesting that the aberrant mutations in multiple oncogenes in this pathology result specifically from repeated CSR attempts rather than from VDJ hypermutation. Together, our data show that, in non-GCB DLBCL, lymphomagenesis occurs in IgM+ B cells which are repeatedly induced to switch but which fail to modify the isotype of their BCR. We hypothesize that the stabilization of this specific isotype is an important contributor to pathogenesis which allows repeated CSR activations in germinal centers and the accumulation of genomic abnormalities.

Disclosures:
No relevant conflicts of interest to declare.