Exploring co-occurring POLE exonuclease and non-exonuclease domain mutations and their impact on tumor mutagenicity.

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer (CRC) and endometrial cancer (EC), leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated CRCs, ECs, and ovarian cancers (OC) classified as TMB-High ≥10 mut/Mb (TMB-H) or TMB-Low <10 mut/Mb (TMB-L). TMB was significantly highest in tumors with 'POLE ExoD driver plus POLE Variant' (CRC and EC, p<0.001; OC, p<0.05). TMB increased with additional POLE variants (p<0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE-models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the 'POLE ExoD driver plus POLE Variant' tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes.