Rationale and Roadmap for Developing Panels of Hotspot Cancer Driver Gene Mutations as Biomarkers of Cancer Risk

Harris, Kelly L.; Myers, Meagan B.; McKim, Karen L.; Elespuru, Rosalie K.; Parsons, Barbara L.

doi:10.1002/em.22326

Cited by 16 publications

(13 citation statements)

References 156 publications

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“…Drugs and chemicals which are not mutagenic will not produce a signal in mutagenesis assays-either conventional or sequencing-based. However, as shown here, it appears possible to use ecNGS to infer carcinogenesis via detection of clonal expansions carrying oncogenic driver mutations as a marker of a neoplastic phenotype (43). This concept is more complex to design, insofar as it necessitates some a priori knowledge about the common drivers that are operative in different tissues in response to different classes of carcinogens.…”

Section: Discussionmentioning

confidence: 99%

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Valentine

Young

Fielden

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10−3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10−7. Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.

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Section: Discussionmentioning

confidence: 99%

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Valentine

Young

Fielden

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Those which are not mutagenic would not produce a signal in conventional mutagenesis assays. We have shown that Duplex Sequencing, in addition to measuring mutagenesis, can also infer carcinogenesis via detection of clonal expansions carrying oncogenic driver mutations as a marker of a neoplastic phenotype 42 . We illustrated this phenomenon in response to a mutagenic chemical and follow-up efforts will be needed to demonstrate the same with non-genotoxic carcinogens.…”

Section: Discussionmentioning

confidence: 99%

Direct Quantification of in vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

Valentine¹,

Young

Fielden

et al. 2020

Preprint

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ABSTRACTThe ability to accurately measure mutations is critical for basic research and identification of potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and don’t fully represent other species such as humans. Next generation sequencing (NGS) is a conceptually attractive alternative for mutation detection in the DNA of any organism, however, the limit of resolution for standard NGS is poor. Technical error rates (~1×10−3) of NGS obscure the true abundance of somatic mutations, which can exist at frequencies ≤1×10−7. Using Duplex Sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by 3 carcinogens in 5 tissues of 2 strains of mice within 31 days following exposure. We observed a strong correlation between mutation induction measured by Duplex Sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified clear early signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by apparent clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses.SIGNIFICANCE STATEMENTError-corrected next generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic impact of environmental exposures or endogenous processes in any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, as well as cross-species and cross-locus applicability of ecNGS compared to existing methods make it a powerful new tool for mutational research, regulatory safety testing, and emerging clinical applications.

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“…SEER breast cancer incidence data was used to calculate a cumulative risk for each age (c), as the cumulative sum of incidence observed at the current and all previous years. Finally, the sum of PIK3CA and TP53 MFs ≥10 −4 in normal breast were plotted relative to the cumulative risk expected based on the tissue donor's age (d) magnitude of interindividual variability in CDMs is reflective of clonal expansion driven by CDMs in individuals, which occurs in a stochastic manner that mirrors the carcinogenic process itself (Harris et al, 2019). Another way to state this is that the more heterogeneity there is in normal tissue, the more potential there is for clonal selective advantage leading to carcinogenesis.…”

Section: Discussionmentioning

confidence: 99%

“…An approach that enables prediction of tumorigenic responses due to lifetime rodent exposures from shorter‐term rodent studies (28 days to 6 months) would be invaluable (Parsons 2018). Hotspot cancer driver mutations (hCDMs) have potential as biomarkers for use in carcinogenicity testing and assessing potential cancer risks associated with exogenous exposures, whether therapeutic, occupational, environmental, genotoxic, or non‐genotoxic (Harris et al ., 2019). Advantages of hCDMs as biomarkers of cancer risk include their relevance to carcinogenesis in both rodents and humans, their known roles in oncogenesis, and their ability to confer a growth advantage to a neoplastic cell in the microenvironment of the tissue in which the cancer arises, leading to clonal expansion of cells carrying cancer driver mutations (CDMs) (Figure 1) (Stratton et al ., 2009).…”

Section: Introductionmentioning

confidence: 99%

“…Advantages of hCDMs as biomarkers of cancer risk include their relevance to carcinogenesis in both rodents and humans, their known roles in oncogenesis, and their ability to confer a growth advantage to a neoplastic cell in the microenvironment of the tissue in which the cancer arises, leading to clonal expansion of cells carrying cancer driver mutations (CDMs) (Figure 1) (Stratton et al ., 2009). hCDMs have been assessed primarily as DNA based measurements, meaning analyses can be performed on any tissue from any species from which DNA can be isolated (Harris et al ., 2019). When using justifiable assumptions of mutant zygosity, cancer driver (CD) mutant fraction (MF) can be translated into mutant cell numbers or proportions, providing information useful in mathematical modeling of carcinogenesis (Soh et al ., 2009).…”

Section: Introductionmentioning

confidence: 99%

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

Harris

Walia

Gong

et al. 2020

Environ and Mol Mutagen

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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.

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Rationale and Roadmap for Developing Panels of Hotspot Cancer Driver Gene Mutations as Biomarkers of Cancer Risk

Cited by 16 publications

References 156 publications

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Direct Quantification of in vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

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

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