Low-Frequency Mutational Heterogeneity of Invasive Ductal Carcinoma Subtypes: Information to Direct Precision Oncology.

Information regarding the role of low-frequency hotspot cancer-driver mutations (CDMs) in breast carcinogenesis and therapeutic response is limited. Using the sensitive and quantitative Allele-specific Competitor Blocker PCR (ACB-PCR) approach, mutant fractions (MFs) of six CDMs (PIK3CA H1047R and E545K, KRAS G12D and G12V, HRAS G12D, and BRAF V600E) were quantified in invasive ductal carcinomas (IDCs; including ~20 samples per subtype). Measurable levels (i.e., ≥ 1 × 10-5, the lowest ACB-PCR standard employed) of the PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E mutations were observed in 34/81 (42%), 29/81 (36%), 51/81 (63%), 9/81 (11%), 70/81 (86%), and 48/81 (59%) of IDCs, respectively. Correlation analysis using available clinicopathological information revealed that PIK3CA H1047R and BRAF V600E MFs correlate positively with maximum tumor dimension. Analysis of IDC subtypes revealed minor mutant subpopulations of critical genes in the MAP kinase pathway (KRAS, HRAS, and BRAF) were prevalent across IDC subtypes. Few triple-negative breast cancers (TNBCs) had appreciable levels of PIK3CA mutation, suggesting that individuals with TNBC may be less responsive to inhibitors of the PI3K/AKT/mTOR pathway. These results suggest that low-frequency hotspot CDMs contribute significantly to the intertumoral and intratumoral genetic heterogeneity of IDCs, which has the potential to impact precision oncology approaches.

A subset of papillary thyroid carcinomas contain KRAS mutant subpopulations at levels above normal thyroid.

The molecular pathogenesis of papillary thyroid carcinoma (PTC) is largely attributed to chromosomal rearrangements and point mutations in genes within the MAPK pathway (i.e., BRAF and RAS). Despite KRAS being the 6th most frequently mutated gene for all cancers, the reported frequency in thyroid cancer is only 2%. This may be due, in part, to the use of insensitive mutation detection methods such as DNA sequencing. Therefore, using the sensitive and quantitative ACB-PCR approach, we quantified KRAS codon 12 GGT → GAT and GGT → GTT mutant fraction (MF) in 20 normal thyroid tissues, 17 primary PTC, 2 metastatic PTC, and 1 anaplastic thyroid carcinoma. We observed measurable levels of KRAS codon 12 GAT or GTT mutation in all normal thyroid tissues. For PTCs, 29.4% and 35.3% had KRAS codon 12 GAT and GTT MF above the 95% upper confidence interval for the corresponding MFs in normal thyroid. The highest observed KRAS codon 12 GTT MFs were associated with tumors with follicular characteristics and relatively high levels of tumor necrosis. The results indicate KRAS mutant subpopulations are present in a large number of thyroid tumors, a fact previously unrecognized. The presence of KRAS mutation may indicate a tumor with an aggressive phenotype, thus directing the course of clinical treatment.

Evaluation of clonal origin of malignant mesothelioma.

BACKGROUND: The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy. METHODS AND RESULTS: To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM. CONCLUSIONS: Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic "field effect" of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.

CarcSeq detection of lorcaserin-induced clonal expansion of Pik3ca H1047R mutants in rat mammary tissue.

Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a nongenotoxic rat carcinogen, which induced mammary tumors in male and female rats in a 2-yr bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 wk. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary, and liver samples using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as nongenotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations, namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error-corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, P < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 wk as compared with 12 wk. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a nongenotoxic carcinogen using a treatment duration as short as 3 months.

Repeat treatment of organotypic airway cultures with ethyl methanesulfonate causes accumulation of somatic cell mutations without expansion of bronchial-carcinoma-specific cancer driver mutations.

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Assessment of K-Ras mutant frequency and micronucleus incidence in the mouse duodenum following 90-days of exposure to Cr(VI) in drinking water.

Chronic exposure to high concentrations of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) in drinking water induces duodenal tumors in mice, but the mode of action (MOA) for these tumors has been a subject of scientific debate. To evaluate the tumor-site-specific genotoxicity and cytotoxicity of SDD in the mouse small intestine, tissue pathology and cytogenetic damage were evaluated in duodenal crypt and villus enterocytes from B6C3F1 mice exposed to 0.3-520mg/L SDD in drinking water for 7 and 90 days. Allele-competitive blocker PCR (ACB-PCR) was used to investigate the induction of a sensitive, tumor-relevant mutation, specifically in vivo K-Ras codon 12 GAT mutation, in scraped duodenal epithelium following 90 days of drinking water exposure. Cytotoxicity was evident in the villus as disruption of cellular arrangement, desquamation, nuclear atypia and blunting. Following 90 days of treatment, aberrant nuclei, occurring primarily at villi tips, were significantly increased at ≥60mg/L SDD. However, in the crypt compartment, there were no dose-related effects on mitotic and apoptotic indices or the formation of aberrant nuclei indicating that Cr(VI)-induced cytotoxicity was limited to the villi. Cr(VI) caused a dose-dependent proliferative response in the duodenal crypt as evidenced by an increase in crypt area and increased number of crypt enterocytes. Spontaneous K-Ras codon 12 GAT mutations in untreated mice were higher than expected, in the range of 10(-2) to 10(-3); however no treatment-related trend in the K-Ras codon 12 GAT mutation was observed. The high spontaneous background K-Ras mutant frequency and Cr(VI) dose-related increases in crypt enterocyte proliferation, without dose-related increase in K-Ras mutant frequency, micronuclei formation, or change in mitotic or apoptotic indices, are consistent with a lack of genotoxicity in the crypt compartment, and a MOA involving accumulation of mutations late in carcinogenesis as a consequence of sustained regenerative proliferation.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Aristolochic acid-induced carcinogenesis examined by ACB-PCR quantification of H-Ras and K-Ras mutant fraction.

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen associated with the development of urothelial cancer in humans. AA induces forestomach, kidney and urothelial tract tumours in rats and mice. This study was conducted to characterise AA's carcinogenic mechanism of action and compare allele-specific competitive blocker-polymerase chain reaction (ACB-PCR)-based early detection of carcinogenic effect using two different tumour-relevant endpoints. H-Ras codon 61 CAA→CTA mutation was analysed because it is found in rodent forestomach tumours and A:T→T:A transversion is the predominant mutational specificity induced by AA. K-Ras codon 12 GGT→GAT mutation was analysed because it is a common spontaneous mutation present in various rodent tissues and may be a useful generic biomarker for carcinogenic effect. DNA samples from Big Blue rats treated with 0, 0.1, 1.0 or 10.0 mg AA/kg body weight (bw) by gavage, 5 days/week for 12 weeks were used in ACB-PCR in order to examine the induction of the two specific mutations. A significant dose-dependent induction of H-Ras mutant fraction (MF) was observed in liver and kidney. Statistically significant correlations were observed between AA-induced DNA adduct levels or cII mutant frequencies (previously measured in the same rats) and H-Ras MF measurements. No correlation between AA dose and K-Ras MF was found in liver or kidney, although there was a significant induction of K-Ras mutation in kidneys exposed to 0.1 mg/kg bw AA relative to controls. Thus, the data establish a straightforward dose-related increase in H-Ras MF due to fixation of AA-induced DNA adducts, whereas the common spontaneous K-Ras mutation showed a non-monotonic dose-response, consistent with loss of non-targeted mutation at cytotoxic doses.

p53 codon 271 CGT to CAT mutant fraction does not increase in nasal respiratory and olfactory epithelia of rats exposed to inhaled naphthalene.

A 2-year rat tumor bioassay testing whole body exposure to naphthalene (NA) vapor found a significant increase in nasal respiratory epithelial adenomas in male rats and in olfactory epithelial neuroblastomas in female rats. To obtain mechanistic insight into NA-induced nasal carcinogenesis, NA dose-response was characterized in nasal epithelium using a tumor-relevant endpoint. Specifically, levels of p53 codon 271 CGT to CAT mutation were measured in nasal respiratory and olfactory epithelium of NA-exposed male and female rats by allele-specific competitive blocker-PCR (ACB-PCR). Male and female, 8-9 week-old F344 rats (5 rats/group) were exposed to 0, 0.1, 1.0, 10, and 30ppm NA vapor for 13 weeks (6h/day, 5 days/week). The geometric mean p53 mutant fraction (MF) levels in nasal epithelium of control treatment groups ranged between 2.05 × 10(-5) and 3.05 × 10(-5). No significant dose-related changes in p53 mutant fraction (MF) were observed in the olfactory or respiratory epithelia of female rats. However, statistically significant treatment-related differences were observed in male respiratory and olfactory epithelium, with the p53 MF in the respiratory epithelium of male rats exposed to 30ppm NA significantly lower than that in controls. Further, a significant trend of decreasing p53 MF with increasing dose was observed in the male respiratory epithelium. Of the tissue types analyzed, respiratory epithelium is the most sensitive to the cytotoxic effects of NA, suggesting cytotoxicity may be responsible for the loss of p53 mutation. Because ACB-PCR has been used successfully to detect the effects of known mutagenic carcinogens, the absence of any significant increases in p53 MF associated with NA exposure adds to the weight of evidence that NA does not operate through a directly mutagenic mode of action.

CarcSeq Measurement of Rat Mammary Cancer Driver Mutations and Relation to Spontaneous Mammary Neoplasia.

The ability to deduce carcinogenic potential from subchronic, repeat dose rodent studies would constitute a major advance in chemical safety assessment and drug development. This study investigated an error-corrected NGS method (CarcSeq) for quantifying cancer driver mutations (CDMs) and deriving a metric of clonal expansion predictive of future neoplastic potential. CarcSeq was designed to interrogate subsets of amplicons encompassing hotspot CDMs applicable to a variety of cancers. Previously, normal human breast DNA was analyzed by CarcSeq and metrics based on mammary-specific CDMs were correlated with tissue donor age, a surrogate of breast cancer risk. Here we report development of parallel methodologies for rat. The utility of the rat CarcSeq method for predicting neoplastic potential was investigated by analyzing mammary tissue of 16-week-old untreated rats with known differences in spontaneous mammary neoplasia (Fischer 344, Wistar Han, and Sprague Dawley). Hundreds of mutants with mutant fractions ≥ 10-4 were quantified in each strain, most were recurrent mutations, and 42.5% of the nonsynonymous mutations have human homologs. Mutants in the mammary-specific target of the most tumor-sensitive strain (Sprague Dawley) showed the greatest nonsynonymous/synonymous mutation ratio, indicative of positive selection consistent with clonal expansion. For the mammary-specific target (Hras, Pik3ca, and Tp53 amplicons), median absolute deviation correlated with percentages of rats that develop spontaneous mammary neoplasia at 104 weeks (Pearson r = 1.0000, 1-tailed p = .0010). Therefore, this study produced evidence CarcSeq analysis of spontaneously occurring CDMs can be used to derive an early metric of clonal expansion relatable to long-term neoplastic outcome.

Assessment of Clonal Expansion Using CarcSeq Measurement of Lung Cancer Driver Mutations and Correlation With Mouse Strain- and Sex-Related Incidence of Spontaneous Lung Neoplasia.

Quantification of variation in levels of spontaneously occurring cancer driver mutations (CDMs) was developed to assess clonal expansion and predict future risk of neoplasm development. Specifically, an error-corrected next-generation sequencing method, CarcSeq, and a mouse CarcSeq panel (analogous to human and rat panels) were developed and used to quantify low-frequency mutations in a panel of amplicons enriched in hotspot CDMs. Mutations in a subset of panel amplicons, Braf, Egfr, Kras, Stk11, and Tp53, were related to incidence of lung neoplasms at 2 years. This was achieved by correlating median absolute deviation (MAD) from the overall median mutant fraction (MF) measured in the lung DNA of 16-week-old male and female, B6C3F1 and CD-1 mice (10 mice/sex/strain) with percentages of spontaneous alveolar/bronchioloalveolar adenomas and carcinomas reported in bioassay control groups. A total of 1586 mouse lung mutants with MFs >1 × 10-4 were recovered. The ratio of nonsynonymous to synonymous mutations was used to assess the proportion of recovered mutations conferring a positive selective advantage. The greatest ratio was observed in what is considered the most lung tumor-sensitive model examined, male B6C3F1 mice. Of the recurrent, nonsynonymous mouse mutations recovered, 55.5% have been reported in human tumors, with many located in or around the mouse equivalent of human cancer hotspot codons. MAD for the same subset of amplicons measured in normal human lung DNA samples showed a correlation of moderate strength and borderline significance with age (a cancer risk factor), as well as age-related cumulative lung cancer risk, suggesting MAD may inform species extrapolation.

ACB-PCR quantification of K-RAS codon 12 GAT and GTT mutant fraction in colon tumor and non-tumor tissue.

K-RAS mutation is being developed as a cancer biomarker and tumor K-RAS is being used to predict therapeutic response. Yet, levels of K-RAS mutation in normal and pathological tissue samples have not been determined rigorously, nor inter-individual variation in these levels characterized. Therefore, K-RAS codon 12 GAT and GTT mutant fractions were measured in colonic mucosa of individuals without colon cancer, tumor-distal mucosa, tumor-proximal mucosa, normal tumor-adjacent tissues, colonic adenomas, and carcinomas. The results indicate K-RAS codon 12 GAT mutation is present at measurable levels in normal appearing mucosa. All tumors carried K-RAS mutation, in most cases as a mutant subpopulation.

Measurement of tumor-associated mutations in the nasal mucosa of rats exposed to varying doses of formaldehyde.

This study examined the potential induction of tumor-associated mutations in formaldehyde-exposed rat nasal mucosa using a sensitive method, allele-specific competitive blocker-PCR (ACB-PCR). Levels of p53 codon 271 CGT to CAT and K-Ras codon 12 GGT to GAT mutations were quantified in nasal mucosa of rats exposed to formaldehyde. In addition, nasal mucosa cell proliferation was monitored because regenerative cell proliferation is considered a key event in formaldehyde-induced carcinogenesis. Male F344 rats (6-7 weeks old, 5 rats/group) were exposed to 0, 0.7, 2, 6, 10, and 15 ppm formaldehyde for 13 weeks (6 h/day, 5 days/week). ACB-PCR was used to determine levels of p53 and K-Ras mutations. Although two of five untreated rats had measureable spontaneous p53 mutant fractions (MFs), most nasal mucosa samples had p53 MFs below 10(-5). All K-Ras MF measurements were below 10(-5). No dose-related increases in p53 or K-Ras MF were observed, even though significant increases in bromodeoxyuridine incorporation demonstrated induced cell proliferation in the 10 and 15 ppm formaldehyde-treatment groups. Therefore, induction of tumor-associated p53 mutation likely occurs after several other key events in formaldehyde-induced carcinogenesis.

ACB-PCR Quantification of Low-Frequency Hotspot Cancer-Driver Mutations.

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive and quantitative approach for the selective amplification of a specific base substitution. Using the ACB-PCR technique, hotspot cancer-driver mutations (tumor-relevant mutations in oncogenes and tumor suppressor genes, which confer a selective growth advantage) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant-specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules. A blocker primer having a non-extendable 3'-end and a 3'-penultimate mismatch relative to the wild-type DNA sequence, but a double 3'-terminal mismatch relative to the mutant DNA sequence is included in ACB-PCR to selectively repress amplification from abundant wild-type molecules. Consequently, ACB-PCR can quantify the level of a single base pair substitution mutation in a DNA population when present at a mutant:wild-type ratio of 1 × 10-5 or greater. Quantification of rare mutant alleles is achieved by parallel analysis of unknown samples and mutant fraction (MF) standards (defined mixtures of mutant and wild-type DNA sequences). The ability to quantify specific mutations with known association to cancer has several important applications in evaluating the carcinogenic potential of chemical exposures in rodent models. Further, the measurement of cancer-driver mutant subpopulations is important for precision cancer treatment (selecting the most appropriate targeted therapy and predicting the development of therapeutic resistance). This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human PIK3CA codon 1047, CAT→CGT (H1047R) mutation.

Outgrowth of erlotinib-resistant subpopulations recapitulated in patient-derived lung tumor spheroids and organoids.

A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.

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

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 long-term 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.

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

Cancer driver mutations (CDMs) are necessary and causal for carcinogenesis and have advantages as reporters of carcinogenic risk. However, little progress has been made toward developing measurements of CDMs as biomarkers for use in cancer risk assessment. Impediments for using a CDM-based metric to inform cancer risk include the complexity and stochastic nature of carcinogenesis, technical difficulty in quantifying low-frequency CDMs, and lack of established relationships between cancer driver mutant fractions and tumor incidence. Through literature review and database analyses, this review identifies the most promising targets to investigate as biomarkers of cancer risk. Mutational hotspots were discerned within the 20 most mutated genes across the 10 deadliest cancers. Forty genes were identified that encompass 108 mutational hotspot codons overrepresented in the COSMIC database; 424 different mutations within these hotspot codons account for approximately 63,000 tumors and their prevalence across tumor types is described. The review summarizes literature on the prevalence of CDMs in normal tissues and suggests such mutations are direct and indirect substrates for chemical carcinogenesis, which occurs in a spatially stochastic manner. Evidence that hotspot CDMs (hCDMs) frequently occur as tumor subpopulations is presented, indicating COSMIC data may underestimate mutation prevalence. Analyses of online databases show that genes containing hCDMs are enriched in functions related to intercellular communication. In its totality, the review provides a roadmap for the development of tissue-specific, CDM-based biomarkers of carcinogenic potential, comprised of batteries of hCDMs and can be measured by error-correct next-generation sequencing. Environ. Mol. Mutagen. 61:152-175, 2020. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.