P594: increased frequencies of igh locus suicide recombination points on chronic lymphocytic leukemia with low rate of aid related somatic mutations, myc overexpression and short telomeres

Background: In normal B-cells, Activation Induced-cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and IGHV somatic hypermutation (SHM). AID is also implicated in another IgH rearrangement, the Locus Suicide Recombination (LSR). LSR occurs in activated B-cells and recombines the IgH locus between the switch µ (Sµ) region and one 3’a2 regulatory region (3’a2RR) of the IgH locus. LSR results in the complete deletion of the cluster of IgH constant genes. When LSR hits the active IgH locus, it induces the loss of BCR expression and the death of the concerned B cell. Chronic lymphocytic leukemia (CLL) is an indolent non-Hodgkin B-cell lymphoma. Tumor CLL B-cells weakly express a B cell receptor (BCR) on the surface which is composed, in the vast majority of cases, of immunoglobulins (Ig) of the mu (µ) and delta (δ) isotypes and Ig class-switched CLL are rare, raising the question of abnormalities in the Ig gene recombination machinery in this B-cell cancer. Aims: Searching for abnormalities of IgH locus recombination in CLL, we investigated CSR and LSR in CLL. Methods: In this study, we used different molecular biology technics as high throughput Sequencing (HTS) to analyze CSR and LSR junctions, IgHV, and PIM-1 mutation. Quantitative reel time PCR (quantification of AID, cMYC and IgH locus transcripts). Samples are from CLL patients (N=47) with more than 98% blood tumor cell infiltration and controls consisted in healthy volunteers (HV) (N=9). Bioinformatics uses CSReport tool. Statistics are from graph pad or R. Results: CSR levels were lower in CLL than in HV samples contrariwise to LSR that was found at comparable levels in both HV and CLL groups (Fig. A, B). Moreover, some patients exhibited increased LSR counts. Because distribution of LSR counts was bimodal with a valley at 27, that value being also the mean of LSR counts in HVs, we separated CLL patients in two groups so called LSR-High and LSR-Low with that threshold of 27 LSR count per sample. We analyzed the diversity of the LSR junctions using Shannon Index. LSR junction diversity was significantly higher in LSR-High than in LSR-Low CLLs and HVs. This result is in opposition with the IgHV mutation rate since LSR-High CLLs exhibited a stronger homology to IgHV reference sequences (unmutated CLLs). We also found that LSR-High CLLs exhibited a marked lowest rate of AID off-target PIM1 mutations (Fig. C). AID expression was low and at comparable levels in both LSR-Low and LSR-High CLLs. Because diversity of LSR junctions in LSR-High CLLs is evocative of an on-going process, we analyzed the expression of productive and non-productive transcripts of the constant part of IgH locus, which we found increased in LSR-High when compared to LSR-Low CLLs. We also observed shorter telomere lengths and c-Myc overexpression in the LSR-High group. Both shorter telomeres and c-Myc overexpression are very likely to reflect an increased number of DNA replication cycles in the history of the CLL tumor cell (Fig. D, E). Consistently, Kaplan Meyer curves of Treatment Free Survival (TFS) show that TFS of LSR-High CLLs was significantly shorter than the one of LSR-Low counterpart, an indication of a more rapidly evolving CLLs (Fig. F). Image:Summary/Conclusion: Altogether, these results indicate the accessibility of IgH locus and the proliferation in CLL patients with high rate and increased diversity of LSR junctions could be increased in Myc dependent manner resulting in shorter survival and pointing on an AID independent mechanism of IgH recombination.