ALBERT

Description: The genetic hallmark of mantle cell lymphoma (MCL) is the t(11;14)(q13;q32). Fluorescence in situ hybridisation (FISH) and microarray based comparative genomic hybridisation (array CGH) experiments have demonstrated that additional genetic clonal alterations occur in the majority of MCL and may have prognostic or pathological relevance. We previously developed and validated by CGH or FISH an inexpensive and sensitive genomic PCR assay (Multiplex PCR of Short Fluorescent Fragments, QMPSF) to detect gene copy number abnormalities in diffuse large B-cell lymphoma and chronic lymphocytic leukemia (Haematologica 2008,93:543-Leukemia, 2007,21:1460). In the aim to determine the incidence and clinical relevance of recurrent additional genomic number abnormalities, we specifically designed a single QMPSF assay dedicated for MCL and correlated results with pathological and clinical data. For this purpose, a series of 42 newly diagnosed MCL cases with available frozen-and paraffin-embedded lymph node tissues and clinical features were selected [median age=67y; median MCL international prognostic index (MIPI)=6.1; low risk 21%, intermediate risk 33%, high risk 45%; 3-year overall survival (OS) rate=38%]. The assay was designed according to the most frequent gene copy number abnormalities reported in MCL, allowing simultaneous analysis of 8 relevant genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, and MDM2) and 2 reference genes (SEM4F and CECR1). DNA copy number gains of MYC, CDK2, CDKN1B (p27kip1) and MDM2 are observed in an equal frequency (10%). Losses of DNA copies of RB1, CDNK2A, ATM, or TP53 are observed in 38, 31, 26, and 10 % of cases respectively. Some genes are almost exclusively gained (MYC,CDK2), deleted (RB1, ATM, TP53, CDNK2A) or both (CDK2, CDKN1B). Deletions of ATM and RB1 appear strongly associated (21% of cases, p=.001). CDKN2A (p14arf and p16ink4a) homozygous deletions and CDKN1B gains are more frequently observed in blastoid variants (p=.04 and .005 respectively). According to the MIPI score, the number of gene copy abnormalities tends to increase in the high/intermediate risk group (median = 1, range 0–6), as compared to the low risk group (median=0, range 0–3, p=.07). More specifically, MYC gain is exclusively observed in the high risk group (p=.04) and CDKN2A deletions are observed in patients with the highest MIPI. The prognostic relevance of the assay was tested in 42 patients. With a median follow-up of 22 months, CDK2 (3y OS=0%) or MDM2 (3y OS=0%) gains and CDKN2A (3y OS=20%) or TP53 (3y OS=25%) losses correlate to a shorter OS (p

Description: 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.