B-223 Validation of a Non-invasive DNA NGS Assay for Detecting Somatic Mutations in Normal Appearing Sun Exposed Skin

Abstract Background Ultraviolet radiation (UVR) exposure is a major risk factor for developing nonmelanoma skin cancer and somatic mutations caused by UVR exposure can arise even in normal-appearing skin. Non-invasive identification of UVR induced somatic mutations can provide a quantitative evaluation of the extent of UVR damage. This study describes the development and analytical validation of a noninvasive method for identifying low level somatic variants in sun exposed peripheral skin by ultra-deep Next Generation Sequencing, in specimens collected using adhesive tape designed to non-invasively collect skin samples. Methods Five genomic regions with the highest density of known UVR induced somatic mutations across TP53 and NOTCH1 were amplified by 5-plex PCR primers affixed with Illumina NGS adapters. These amplicons were purified, size selected and then indexed using Unique Dual Indexes. The NGS libraries were pooled to 96-plex and bi-directionally sequenced with 2x150 chemistry on the Illumina iSeq100. Data analysis was performed using the Illumina DRAGEN Somatic Variant Calling pipeline. Titrations between 0.01 and 1 of synthetic controls comprising plasmids containing amplimer inserts with 19 of the most well characterized UVR-induced C > T driver mutations spiked into a wildtype insert were sequenced in duplicate. In addition, 3 cell line controls at input concentrations of a 2-fold serial dilution from 13.2 ng–0.4 ng were spiked into a mutation negative cell line at variant allele frequencies between 0.005 and 1 and sequenced in triplicate across three NGS runs. Finally, 178 specimens collected from the central forehead, nose and cheeks of human donors were sequenced and their cumulative mutation frequency analyzed by linear regression against the age of the participants. Results All samples were sequenced to a mean duplex coverage of >10000×. All expected mutations in the plasmid control titrations were identified for each titration. Both reproducibility and specificity in these samples were 1.0. The cell line controls were sequenced in a matrix of DNA input concentration vs Variant Allele Frequency (VAF). The Limit of Detection for the assay was a function of DNA concentration, with 0.4ng, 0.8ng and 3.3ng DNA required to identify mutations accurately and reproducibly at 0.05, 0.02 and 0.01 VAF respectively. The study found that the mutations detected among the human donor samples strongly correlated cumulative VAF with age (P value = 2.56×10−7). Conclusion This study describes the validation of a method for identifying low frequency variants in sun exposed skin. We demonstrate that the assay is a robust and reliable tool for detecting TP53 and NOTCH1 somatic variants associated with UVR exposure in normal-appearing skin down to 0.01 VAF with >0.8 sensitivity, specificity, and reproducibility. Damage from UV exposure is cumulative and therefore it is expected that older individuals will be more likely to have UVR-induced mutations in their skin, as we observed in this analysis. The assay can detect somatic DNA variations in normal-appearing skin for markers that are associated with UV damage and increased overall risk for certain nonmelanoma skin cancers.