Highly Recurrent Copy Number Alterations and Insight Into The Origins Of 6q Deletions In Waldenström’s Macroglobulinemia Revealed By Whole Genome Sequencing

Over 90% of patients with Waldenström's Macroglobulinemia (WM), and 50-80% of patients with the precursor condition, IgM MGUS, express MYD88 L265P. These findings suggest that other mutations may support progression of IgM MGUS to WM. Chromosomal aberrations including large losses in 6q are commonly present in WM patients, though the gene loss accounting for WM pathogenesis remains unclear. We therefore sought to delineate copy number alterations (CNA) and structural variants using whole genome sequencing (WGS) in order to more clearly define other important gene alterations in WM.

DNA from CD19⁺ bone marrow lymphoplasmacytic lymphoma cells (LPC) and CD19-depleted peripheral blood mononuclear cells from 10 WM patients was used for paired tumor/germline analysis by WGS. Coverage in the tumor sample was divided by the coverage in the paired germline sample for each matching position, resulting in coverage ratios for each 100Kb window. Statistically significant windows within each genome were then analyzed across the cohort by randomizing the coverage positions to assess the probability of observing the given frequency of a CNA by random chance. TaqMan quantitative polymerase chain reaction (PCR) copy number assays was used to validate findings. Translocations were validated by Sanger sequencing across the breakpoint including flanking sequences.

Functional annotation for identified CNAs was undertaken using Ingenuity Pathway Analysis that revealed a significant enrichment for pathways dysregulated in B-cell malignancies (Table 1). Iteratively randomizing the genomic position of CNAs not related to the chromosome 6 deletions revealed a greater than 3 fold increase in the targeting of COSMIC genes than expected by chance (p< 0.001). Affected genes in the COSMIC census were BTG1 (9/10; 90%), FOXP1 (7/10; 70%), FNBP1 (7/10; 70%), CD74 (7/10; 70%), TOP1 (6/10; 60%), MYB (5/10; 50%), CBLB (5/10; 50%), ETV6 (5/10; 50%), TNFAIP3 (5/10; 50%), FBXW7 (5/10; 50%), PRDM1 (5/10; 50%), TFE3 (4/10; 40%), JAK1 (4/10; 40%), MAML2 (4/10; 40%), FAM46C (4/10; 40%), EBF1 (4/10; 40%), STL (4/10; 40%), and BIRC3 (4/10; 40%). Other affected genes of interested included PRDM2 (8/10; 80%), HIVEP2 (8/10; 80%), ARID1B (7/10; 70%) as well as LYN (7/10; 70%).

Table 1

Functional pathway annotations derived from the list of CNA affected genes filtered only for unique functional annotations and sorted by statistical significance.

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There were no singular regions of statistical significance in 6q to denote a minimally deleted region though neither of the previously suspected target genes for 6q loss, PRDM1 and TNFAIP3, were included in the regions of highest statistical significance. Losses in HIVEP2 (8/10; 80%) as well as ARID1B (7/10; 70%) and BCLAF1 (7/10; 70%) constituted the most common deletions in chromosome 6, and were present in patients with and without the large-scale losses in 6q. While no recurrent translocations were noted in this study, 2 or the 5 (40%) of the 6q deletions corresponded with translocation events. In one case, this was a result of chromothripsis focused on 6q while in the other case, a t(6;X) translocation linked to the amplification of Xq was identified.

Validation studies confirmed presence of somatic deletions in BTG1 (4/5; 80%) at Chr. 12q21.33, HIVEP2 (4/5; 80%) at 6q24.2, LYN (3/5 60%) at 8q12.1, PLEKHG1 (3/5; 60%) at 6q25.1, ARID1B (3/5 60%) at 6q25.1, PDRM2 (2/5; 40%) at 1p36.21, FOXP1 (2/5; 40%) at 3p13, and MKLN1 (2/5 40%) at 7q32. As some CVAs were subclonal, we validated the correlation between the PCR relative copy number and WGS coverage predictions (rho = .926; p =2.2x10^-16).

Highly recurrent CNAs are present in WM LPCs that include genes with critical regulatory roles in lymphocytic growth and survival signaling.

No relevant conflicts of interest to declare.