Coexisting trisomies of chromosomes 12 and 19 define a cytogenetic subgroup of IgG+ CLL enriched for BIRC3 mutations

Abstract Introduction: Coexisting trisomies of chromosomes 12 and 19 define a cytogenetic subgroup of CLL (+12+19 CLL) with shared clinicobiological characteristics. +12+19 CLL cases often (~70%) also carry trisomy 18; most cases (~85%) are CD38+ with high percentages of CD38+ cells; all cases with available FACS data express surface IgG with mutated IGHV genes; and, the clinical course is very indolent. Here, we sought to better understand +12+19 CLL through a thorough genomic and transcriptomic analysis. Methods and Results: Whole-genome sequencing in +12+19 CLL (n=10, 7 also harbored +18) revealed 3 cases with BIRC3 pathogenic variants: 2 with an identical c.1639delC frameshift deletion and 1 with a stop-gain SNV. We sought to validate this finding in a series of 47 independent +12+19 CLL cases (29/47 also harbored +18) using a target enrichment panel including 205 genes known as putative drivers in hematologic malignancies, including CLL, as well as a backbone of SNPs across the genome allowing the detection of copy-number alterations. We found 24/46 (52%) cases harboring BIRC3 pathogenic variants, for a total of 8 different variants that were located within the intervening region between the CARD and RING domains. The most frequent was c.1639delC, p.Q547Nfs*21 (VAF range: 3.9-42%), present in 19/24 (79%) BIRC3mut cases; in 7 cases it coexisted with other BIRC3 variants. To investigate the functional consequences of BIRC3 c.1639delC, we lentivirally expressed it in MEC1 cells using a doxycycline-inducible expression vector (pCW57-GFP-2A-MCS), resulting in >96% GFP+ cells. Western analysis confirmed a lower molecular weight band below the endogenous full-length protein, consistent with the predicted truncated form (p.Q547Nfs*21). The truncated protein was overexpressed compared to the full-length WT protein, suggesting dominant mutant expression. RNA-seq of MEC1 cells (biological triplicates) expressing BIRC3 c.1639delC versus empty-vector identified 2525 significantly deregulated genes (padj<0.05), 1547 up- (log₂FC>2) and 978 down-regulated (log₂FC<–2). Pronounced disruption of cell cycle regulatory networks was noted, with downregulation of key G1/S transition drivers (e.g. CCND1, CCNE1, CCNE2, CDC6, E2F1, E2F2) and core mitotic regulators (e.g. CDK1, CDK6, MAD2L1, PLK1), alongside upregulation of the cyclin-dependent kinase inhibitor CDKN1A. BIRC3mut MEC1 cells also showed upregulation of anti-apoptotic regulatory genes (BCL2L10, BCL2L1) and downregulation of pro-apoptotic mediators (FAS, CASP7, CASP8AP2). To better understand the hierarchy of genomic aberrations in relation to IG class switching, we profiled high-purity (>98%) IgM+ and IgG+ cell fractions obtained by FACS-sorting from 11 CLL cases with +12+19 CLL (5/11 also carried +18) using the gene panel mentioned above. In all cases, IgM+ cells represented a very minor population, ranging from 0.1-1.6% of total CD19+ cells. Eight cases (73%) harbored identical trisomies in both fractions, while 3/11 were negative for trisomies in the IgM+ fraction. BIRC3 c.1639delC was detected in both fractions of 4 cases, with higher VAF in IgG+ cells; in only IgG+ cells of 3 cases; and, in only IgM+ cells in 1 case. Clonally related mu and gamma IGHV-IGHD-IGHJ transcripts were detected in the IgM+ and IgG+ fractions, respectively, of 3 cases with available RNA. We also investigated the transcriptome of +12+19 CLL (n=16, 13 also harbored +18) by RNA-seq using as a comparator CLL stereotyped subset #4 (n=7) on the grounds that these two subgroups share IgG expression, mutated IGHV genes and indolent clinical courses. Compared to subset #4, +12+19 CLL displayed upregulation of genes associated with signaling, inflammation, cytokines, transcription, translation and metabolism. Prompted by this result, we studied the signaling capacity of +12+19 CLL (n=10) through BcR crosslinking with anti-IgG. However, this stimulation did not induce changes in either the phosphorylation status of ERK and PLCG2 or the calcium influx in B cells, as assessed by flow cytometry, indicating attenuated BcR signaling. Conclusion: We report a remarkable enrichment of BIRC3 mutations in +12+19 CLL. In this cytogenetic CLL subgroup, IgG+ cells coexist with infrequent IgM+ cells bearing shared genomic aberrations. On these grounds, class switching to IgG was apparently selected for in the clonal progenitors.