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Functional characterization of VUS mutations found in patients' cell-free circulating tumor DNA (ctDNA) using Precision Cancer Analysis System (PCAS).
Background: A major advancement in patient diagnosis and monitoring is the use of ctDNA analysis using next-generation sequencing. Sequencing of patient samples using Guardant Health’s 70-gene panel identifies Variants of Unknown Significance (VUS). Here, we investigate the functional significance of these variants by utilizing the NovellusDx PCAS, which monitors the activity level of signaling pathways by means of a transfected cell-based assay. As a functional platform, PCAS reveals activated pathways regardless of the type of mutation, i.e., whether it is a known mutation or a VUS Methods: We investigated the VUS mutations that appear most frequently in a cohort of 10,168 patients that were tested with the Guardant360 ctDNA panel at its CLIA-certified, CAP-accredited clinical laboratory. The test is sensitive to a single DNA fragment of mutated ctDNA in a 10 ml blood sample (analytic specificity for SNVs of > 99.999 %). We synthesized the mutated genes’ and mapped them to their major signaling pathways. Using the PCAS platform we monitored the functional significance of the chosen mutations within these pathways and compared them to mutations of known significance Results: Using the Guardant360 test we found 14 frequent VUS mutations in EGFR, ERBB2, MET, KIT, FGFR1 and FGFR2 that occurred a total of 402 times in the cohort. Frequency of occurrence ranged from 10-80 patients, with a mean of 28 in 342 tests on 328 patients (3.2% prevalence). Of patients with one of the frequent VUS mutations, 27% did not have another actionable mutation. The PCAS platform was able to analyze all the frequent VUS mutations, identifying varying degrees of activity levels, as well as unique pathway specific patterns Conclusions: The high prevalence of VUS mutations in the patient population highlights the need to characterize their functional relevance. Using the PCAS platform offers a way to interpret novel mutations and identify actionable mutations. The findings expand our understanding of deleterious mutations, and creates targeted therapy treatment opportunities for patients who previously would not have had such treatment options