Abstract Purpose Tumor mutation load is an emerging prognostic and diagnostic marker for many cancers. Sensitivity to immunotherapeutic agents, which stimulate an antitumor immune response by selectively inhibiting immunosuppressive cell surface ligands, is known to correlate with high mutation load in the tumor. Numerous somatic and germline defects can cause genomic instability, including alterations affecting the mismatch repair pathway, DNA polymerases, and cell cycle regulators. We describe here the discovery of previously unreported mutations in the promoter of the PMS2 gene that are associated with significantly increased tumor mutation load. Methods Comprehensive genomic profiling by hybridization capture of exonic regions from either 236 or 315 cancer-related genes and select introns from 19 genes commonly rearranged in cancer was used to characterize more than 60,000 clinical FFPE cancer specimens. At least 50 ng of extracted DNA was analyzed per sample and the constructed libraries were sequenced to high, uniform median coverage (>500x). Samples were assessed for base substitutions, short insertions and deletions, copy number alterations and gene fusions/rearrangements. Mutation load is assessed as the number of somatic coding point mutations per megabase of targeted territory. Results Mutation load from targeted cancer gene analysis recapitulates previous results evaluating whole exome and whole genome mutation load in tumors and cell lines. A novel mutation hotspot was identified in the promoter of PMS2, which codes for the PMS2 protein, a dimerization partner of MLH1 and integral to the DNA mismatch repair complex. Promoter mutations were found in 7.5% of melanoma specimens (n = 101/1348) and 17% of skin squamous cell carcinomas (n = 30/175). In both diseases, PMS2 promoter mutations are the most significant genomic correlate of high mutation load. In melanoma, PMS2 promoter mutant specimens have 4x the median mutation load of the general melanoma population. Skin squamous cell carcinomas show a 2.5-fold increase. Functional characterization is underway to support the hypothesis that these mutations lead to modified PMS2 transcriptional activity, which is known to cause hypermutation. Conclusions The growing corpus of cancer genome data continues to enable novel discoveries in cancer biology. Non-coding and regulatory mutations have not been the target of focused study, and our findings extend the small set of regulatory mutations thought to affect tumor development. Our discovery highlights the power of large-scale genomic analysis to uncover additional disease mechanisms, and furthers our understanding of how cells maintain genome integrity. With the documented association of mutation load and immunotherapy sensitivity, investigation into the response of PMS2 promoter mutant tumors is warranted. Citation Format: Zachary R. Chalmers, Franklin W. Huang, Laurie M. Gay, Siraj M. Ali, Roman Yelensky, Juliann Chmielecki, Jeffery S. Ross, Vincent A. Miller, Philip J. Stephens, Levi A. Garraway, Garrett M. Frampton. Broad analysis of recurrent somatic mutations in cancer reveals a common novel non-coding mutation in the promoter of PMS2 associated with greatly increased tumor mutation load. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3576.