Analysis of BRCA1 and BRCA2 Mutations and Gene Expression in Hematological Malignancies - Implications for Differential Involvement in Pathogenesis of Lymphoid Neoplasms

Background: BRCA1 and BCRA2 (breast cancer 1 and 2) are tumor suppressor genes involved in repair of DNA double-strand breaks. Loss of BRCA1/2 activity by (germline) mutations (mut) or downregulation (DR) of gene expression (GE) correlates with an increased risk of breast and ovarian cancer. Mut in or reduced GE of BRCA1/2 have also been implicated in some hematological malignancies, although data are so far scarce. Moreover, PARP1 inhibitors, known to induce synthetic lethality in tumors harboring BRCA1/2 mut were shown to affect FLT3(ITD)-positive AML (acute myeloid leukemia) cells.

Aims: Investigation of BRCA1/2 mut and GE in 3340 patients with different hematological malignancies by whole genome sequencing (WGS) and RNA sequencing.

Patient cohorts and methods: WGS was performed for 3340 patients with different hematological malignancies: 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. All reported p-values are two-sided and were considered significant at p<0.05. For GE analysis, estimated gene counts were normalized and the resulting log2 counts per million were used as a proxy of GE in each sample.

Results: We identified 14/3340 patients diagnosed with a variety of hematological neoplasms with a pathogenic (according to ClinVar) BRCA1/2 mut (BRCA1: n=11; BRCA2: n=3; incidence: 0.42%). Analysis of the mut frequencies of 1067 cancer associated genes revealed RNF213 (6/14, 43%), ARID1B, CREBBP (5/14, 36%, respectively), JAK3, TET2, NUMA1, POLD1, ZFHX3 (4/14, 29%), ATM, KMT2D and POLE (3/14, 21%) as most frequently mutated genes in cases with BRCA1/2 mut. Three of them (ATM, POLD1, POLE) are known to be directly involved in DNA repair. Comparison of mut frequencies of these 11 genes in cases with/without BRCA1/2 mut revealed JAK3 and POLD1 to be significantly more abundant in BRCA1/2 mut (JAK3: 29% vs. 9%, p=0.028; POLD1: 29% vs. 8%, p=0.026). GE analyses of BRCA1/2 showed DR of BRCA1 and/or BRCA2 in several lymphoid malignancies: for BRCA1, the strongest reduction was detected in PPBL (persistent polyclonal B-cell lymphocytosis; normalized BRCA1 expression: 1.5; control: 5.2), CLL (chronic lymphocytic leukemia; 1.6), T-PLL (T-cell prolymphocytic leukemia; 1.7) and MZL (marginal zone lymphoma; 2.2). DR of BRCA2 was less pronounced (control: 6.3; PPBL: 3.5; HCL, hairy cell leukemia: 4.0; T-PLL: 4.1; MZL: 4.6). In myeloid malignancies reduction was less (see Figure). For further analysis, the 50 lymphoid cases with the lowest and highest BRCA1/2 GE (BRCA1/2 high/low) were selected: these included for BRCA1 low (median GE: 0.8) mainly CLL (33/50), T-PLL (4/50) and PPBL (4/50), whereas BRCA1 high (median: 5.4) comprised B-ALL (23/50), T-ALL (15/50) and Burkitt lymphoma (7/50). BRCA2 low (median: 3.4) included only 2/50 CLL cases, as mainly PPBL (14/50), T-PLL (10/50), multiple myeloma (9/50) and HCL (9/50) cases showed a low BRCA2 GE. BRCA2 high (median: 6.6) included T-ALL (21/50), B-ALL (16/50) and Burkitt lymphoma (4/50). Regarding molecular mut, all entities with a strong DR of BRCA1/2 show a high mut frequency of ATM (analyzed cohort: PPBL, 22%; MZL, 24%; CLL, 25%; HCL, 28%, T-PLL: 74%); BRCA1 (p=0.023) and BRCA2 (p<0.001) GE was significantly lower in ATM mut cases. Comparison of BRCA1/2 low/high revealed several significant differences in detected gene mut: for BRCA1 low vs. high, JAK3 was more frequently mutated in low (18% vs. 4%, p=0.05), whereas KAT6B (4% vs. 24%, p=0.008), BCL11B (0% vs. 18%, p=0.003) and PHF6 (0% vs. 18%, p=0.003) were more abundant in high. For BRCA2, results were similar (JAK3: 20% vs. 4%, p=0.028; BCL11B: 2% vs. 18%, p=0.025; PHF6: 0% vs. 24%, p<0.001), moreover BRAF (20% vs. 2%, p=0.008) and STAT5B (16% vs. 0%, p=0.006) were more frequently mutated in low.

Conclusions: 1) The incidence of BRCA1/2 mut in patients with hematological malignancies is comparable to the frequency in a normal population. 2) Mut in JAK3 and POLD1 are significantly more frequent in cases with BRCA1/2 mut. 3) GE analyses revealed a strong DR of BRCA1 in CLL, T-PLL and MZL. 4) T-ALL and B-ALL were not found to show a BRCA1/2 DR. 5) DR correlates with a high mut frequency of ATM and JAK3, for BRCA2 also of BRAF and STAT5B. 6) Thus, not molecular mut in, but DR of BRCA1/2 in CLL, T-PLL and MZL could play a more important role in pathogenesis than so far expected.

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