Additive effects of variants of unknown significance in replication repair-associated DNA polymerase genes on mutational burden and prognosis across diverse cancers

Defects in replication repair-associated DNA polymerases often manifest an ultra-high tumor mutational burden (TMB), which is associated with higher probabilities of response to immunotherapies. The functional and clinical implications of different polymerase variants remain unclear.Targeted next-generation sequencing using a 425-cancer gene panel, which covers all exonic regions of three polymerase genes (POLE, POLD1, and POLH), was conducted in a cohort of 12,266 patients across 16 different tumor types from January 2017 to January 2019. Prognostication of POL variant-positive patients was performed using a cohort of 4679 patients from the The Cancer Genome Atlas (TCGA) datasets.The overall prevalence of somatic and germline polymerase variants was 4.2% (95% CI 3.8% to 4.5%) and 0.7% (95% CI 0.5% to 0.8%), respectively, with highest frequencies in endometrial, urinary, prostate, and colorectal cancers (CRCs). While most germline polymerase variants showed no clear functional consequences, we identified a candidate p.T466A affecting the exonuclease domain of POLE, which might be underlying the early onset in a case with childhood CRC. Low frequencies of known hot-spot somatic mutations in POLE were detected and were associated with younger age, the male sex, and microsatellite stability. In both the panel and TCGA cohorts, POLE drivers exhibited high frequencies of alterations in genes in the DNA damage and repair (DDR) pathways, including BRCA2, ATM, MSH6, and ATR. Variants of unknown significance (VUS) of different polymerase domains showed variable penetrance with those in the exonuclease domain of POLE and POLD1 displaying high TMB. VUS in POL genes exhibited an additive effect as carriers of multiple VUS had exponentially increased TMB and prolonged overall survival. Similar to cases with driver mutations, the TMB-high POL VUS samples showed DDR pathway involvement and polymerase hypermutation signatures. Combinatorial analysis of POL and DDR pathway status further supported the potential additive effects of POL VUS and DDR pathway genes and revealed distinct prognostic subclasses that were independent of cancer type and TMB.Our results demonstrate the pathogenicity and additive prognostic value of POL VUS and DDR pathway gene alterations and suggest that genetic testing may be warranted in patients with diverse solid tumors.