Abstract
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Background: Pathogenic POLE (p POLE) variants interfere with the exonuclease activity of POLε during DNA replication, resulting in somatic ultramutation and conferring susceptibility to ICI. Historical classification of p POLE relies on localization to the exonuclease domain (ED). We present the landscape of p POLE variants in CRC according to a proposed phenotypic classification model. Methods: Comprehensive genomic profiling was performed on tissue (TBx) and liquid (LBx) biopsies during the course of clinical care. A POLE-specific variant classification model including tumor mutational burden (TMB), mutational signatures, and germline frequency was used to differentiate p POLE from benign POLE (b POLE) variants. Results: POLE variant status was evaluated in 455,965 TBx and 41,804 LBx pan-tumor. POLE alterations, including p POLE and variants of unknown significance, were detected in 3.9% of samples with 7,404 discrete alleles observed. After applying the classification model, 30 variants were classified as p POLE, representing 3.6% (751/20,601) of observed POLE variants. All p POLE were localized to the ED and a majority were missense substitutions (29/30). There were an additional 620 ED variants categorized as b POLE. CRC had the second highest prevalence of p POLE variants (0.5%, 302/60,547). p POLE variants were associated with ultramutation in CRC where the median tissue TMB was 183.0 mut/Mb, 1-2 orders of magnitude higher than for CRC harboring b POLE variants (7.8 mut/Mb) or without POLE variants (3.5 mut/Mb). 32.2% of p POLE CRC also had evidence of high microsatellite instability (MSI-H). Combined p POLE + MSI-H was associated with markedly elevated TMB compared to either signature alone (344.4 versus 53.1 and 151.3 mut/Mb for b POLE + MSI-H and p POLE + MS-Stable, respectively). A small minority of CRC tumors with p POLE variants (7/298, 2.3%) had low TMB (<10 mut/Mb). However, this was associated with a low p POLE variant allele frequency (VAF; median 2.8% versus 24.0% for tumors w/ TMB ≥10, p < .0001) pointing to low tumor purity and likely an underestimation of TMB. While targetable alterations (e.g., homologous recombination repair [HR] gene variants) were enriched in p POLE tumors, these tumors had a low rate of HR deficiency (HRD) as determined by a computational algorithm (HRDsig), suggesting these are likely incidental monoallelic variants. Conclusions: A POLE phenotype-specific classification model identified p POLE variants associated with ultramutation in CRC. Co-occurrence of p POLE and MSI-H in CRC is common and has a synergistic effect leading to even higher TMB. We hypothesize that p POLE detection predicts ultramutation and response to ICI even when TMB is under-detected due to low tumor purity. Caution is advised for therapy selection based on co-alterations in the setting of p POLE due to an abundance of incidental variants.