450 Sun exposed individual normal keratinocytes tolerate multiple cancer-causing mutations in vivo

Abstract

The burden of somatic mutation in sun-exposed normal skin has been described to be as high as visceral carcinomas using bulk DNA sequencing approaches. Mutations in so called keratinocyte carcinoma “driver genes”, such as TP53 and NOTCH1, are also frequently detected. Here we use a single colony sequencing approach to determine the mutation profiles of individual normal keratinocytes isolated from the periphery of multiple separate patient squamous cell carcinomas (SCC). We find the burden of mutation in individual normal sun-exposed keratinocytes ranges orders of magnitude, from 0.3 single nucleotide variants (SNV) per mega-base (Mb) of exome-captured DNA to 30.9 SNV / Mb, similar to those reported in keratinocyte carcinomas (ranging 1 to over 100 SNV / Mb). Forty-five percent of single sun-exposed keratinocytes harbored mutations in at least one known SCC driver gene: NOTCH1 (30% of cells), TP53 (15%), NOTCH2 (10%), NOTCH3 (5%), and FAT1 (5%). Surprisingly some single cells harbored mutations in up to three separate driver genes, a number not often identified in keratinocyte carcinomas for these defined driver genes. Although multiple single cell derived colonies were sequenced from individual patients, we found no evidence of clonal expansion of cells harboring NOTCH or TP53 mutations as reported from bulk sequencing approaches. In contrast, we found that a high proportion of single colony clones sequenced from normal laryngeal keratinocytes adjacent to a laryngeal SCC were populated by three separate clones, providing evidence of so-called “field cancerization” in larynx but not in skin. Mutation signature analysis demonstrated presence of UV mutation in sun-exposed skin and presence of tobacco mutation in larynx. Collectively these data have implications on both keratinocyte stem cell biology in the skin and larynx as well as the differential ability of normal stratifying epithelia to tolerate somatic mutation.