Comparative analysis of somatic and germline polymerase proofreading deficiencies in cancer: molecular and clinical implications

Polymerases ε and δ maintain genome integrity through exonuclease proofreading. Germline and somatic pathogenic variants (PVs) in the exonuclease domain (ED) of POLE and POLD1 impair proofreading, causing hypermutated tumors. Despite shared mutational features that make these tumors highly immunogenic, molecular and clinical distinctions between POLE and POLD1 mutations, and between somatic and germline variants, remain incompletely understood. We compared molecular and clinical characteristics of POLE and POLD1 ED PVs (n=31), assessing their location, pathogenicity, clinical phenotypes, mismatch repair (MMR) status, tumor mutational burden (TMB) and signatures. We analyzed 360 proofreading-deficient tumors (TCGA/COSMIC) and 70 families (249 individuals) with polymerase proofreading-associated polyposis (PPAP). All germline and somatic PVs had high AlphaMissense scores (0.87-1), and clustered within or near Exo motifs. Recurrent, non-founder germline PVs, POLE L424V and POLD1 S478N, showed low/modest REVEL scores. Somatic variants occurred mainly in endometrial cancers (75% of proofreading-deficient TCGA cancers), while colorectal cancer predominated in PPAP (56% of carriers). Cancer risks and tumor spectra differed between POLE and POLD1 PV carriers. Aggressive hereditary phenotypes were linked to either specific POLE PVs (e.g., S297F, V411L, P436R, M444K, A456P, S4611T) or to the co-occurrence of germline ED PVs with germline MMR gene PVs. Distinct hypermutator profiles were confirmed for polymerase ε and polymerase δ proofreading deficiencies via unique mutational signatures (Polymerase ε: SBS10a/b, SBS28; Polymerase δ: SBS10c/d). Tumors with combined proofreading and MMR deficiencies had significantly higher TMB and a shift in the associated mutational spectra. Unlike POLE, POLD1 ED PVs exhibited haplosufficiency, typically requiring a somatic second hit (e.g., loss of heterozygosity) or MMR deficiency to drive hypermutation. In conclusion, differences between POLE and POLD1, and between somatic and germline mutations, influence clinical presentation, mutagenic potential, and reliance on cooperating defects in tumorigenesis. These insights advance the understanding of proofreading-deficient cancers, with implications for diagnostics, genetic counseling, and precision oncology.