Quantification of cancer driver mutations in human breast and lung DNA using targeted, error‐corrected CarcSeq

Abstract

There is a need for scientifically-sound, practical approaches to improve carcinogenicity testing. Advances in DNA sequencing technology and knowledge of events underlying cancer development have created an opportunity for progress in this area. The longterm goal of this work is to develop variation in cancer driver mutation (CDM) levels as a metric of clonal expansion of cells carrying CDMs because these important early events could inform carcinogenicity testing. The first step toward this goal was to develop and validate an error-corrected next-generation sequencing method to analyze panels of hotspot cancer driver mutations (hCDMs). The "CarcSeq" method that was developed uses unique molecular identifier sequences to construct single-strand consensus sequences for error correction. CarcSeq was used for mutational analysis of 13 amplicons encompassing >20 hotspot CDMs in normal breast, normal lung, ductal carcinomas, and lung adenocarcinomas. The approach was validated by detecting expected differences related to tissue type (normal vs. tumor and breast vs. lung) and mutation spectra. CarcSeq mutant fractions (MFs) correlated strongly with previously obtained ACB-PCR mutant fraction (MF) measurements from the same samples. A reconstruction experiment, in conjunction with other analyses, showed CarcSeq accurately quantifies MFs ≥10 −4. CarcSeq MF measurements were correlated with tissue donor age and breast cancer risk. CarcSeq MF measurements were correlated with deviation from median MFs analyzed to assess clonal expansion. Thus, CarcSeq is a promising approach to advance cancer risk assessment and carcinogenicity testing practices. Paradigms that should be investigated to advance this strategy for carcinogenicity testing are proposed.

Antiprogestins reduce epigenetic field cancerization in breast tissue of young healthy women

“…Somatic TP53 mutations are highly prevalent in TNBCs [ 13 ]. Their presence was also recently documented in a variety of normal tissues [ 49 – 53 ] including the breast [ 54 ]. In order to test whether the TP53 mutation frequency changes as a function of exposure to mifepristone, we applied ultra-accurate duplex sequencing to samples from those volunteers of Clinical Trial 2 (‘ The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations ’) which we deemed to respond to mifepristone based on the reduction in the WID-Breast29 index (DNAme Set 3) [ 55 ].…”

Error-corrected Duplex Sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter-laboratory consistency

“…To circumvent this problem, error-corrected sequencing (ecNGS) techniques have been developed to improve accuracy and specificity for detecting true, low frequency mutations [6][7][8] . The highest resolution of ecNGS methods, such as Duplex Sequencing (DuplexSeq; TwinStrand Biosciences, Seattle, WA), involves labeling both strands of a DNA duplex with one or more forms of a unique molecular index (UMI) that both relates and distinguishes the sequences of the two strands from each other and from those of other molecules.…”

Application of Genomics-Based Methodologies in Genetic Toxicology to Advance In Vitro Chemical Assessment

“…However, the application of NGS has been hindered by the high per base pair rate of technical errors (on the order of 1 in 10 3 ) leading to an overestimation of up to 100,000 times the true MF in cells. To circumvent this problem, error-corrected sequencing (ecNGS) techniques have been developed to improve accuracy and specificity for detecting true, low frequency mutations (Kennedy et al, 2014;Harris et al, 2020;You et al, 2020).…”