Meagan B. Myers

Oncomutations as biomarkers of cancer risk

Cancer risk assessment impacts a range of societal needs, from the regulation of chemicals to achieving the best possible human health outcomes. Because oncogene and tumor suppressor gene mutations are necessary for the development of cancer, such mutations are ideal biomarkers to use in cancer risk assessment. Consequently, DNA‐based methods to quantify particular tumor‐associated hotspot point mutations (i.e., oncomutations) have been developed, including allele‐specific competitive blocker‐PCR (ACB‐PCR). Several studies using ACB‐PCR and model mutagens have demonstrated that significant induction of tumor‐associated oncomutations are measureable at earlier time points than are used to score tumors in a bioassay. In the particular case of benzo[a]pyrene induction of K‐Ras codon 12 TGT mutation in the A/J mouse lung, measurement of tumor‐associated oncomutation was shown to be an earlier and more sensitive endpoint than tumor response. The measurement of oncomutation by ACB‐PCR led to two unexpected findings. First, oncomutations are present in various tissues of control rodents and “normal” human colonic mucosa samples at relatively high frequencies. Approximately 60% of such samples (88/146) have mutant fractions (MFs) >10−5, and some have MFs as high as 10−3 or 10−4. Second, preliminary data indicate that oncomutations are present frequently as subpopulations in tumors. These findings are integrated into a hypothesis that the predominant preexisting mutations in particular tissues may be useful as generic reporters of carcinogenesis. Future research opportunities using oncomutation as an endpoint are described, including rodent to human extrapolation, dose‐response assessment, and personalized medicine. Environ. Mol. Mutagen., 2010. Published 2010 Wiley‐Liss, Inc.

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.

ACB-PCR measurement of H-ras codon 61 CAA→CTA mutation provides an early indication of aristolochic acid I carcinogenic effect in tumor target tissues

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI‐induced carcinogenesis through analysis of a tumor‐relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI‐induced toxicity. H‐ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele‐specific competitive blocker‐PCR (ACB‐PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA‐induced rodent forestomach tumors. Treatment‐related differences were observed, with the H‐ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle‐treated controls (Fishers exact test, P < 0.05). Statistically significant correlations between dA‐AAI adduct levels (measured previously in the same animals) and induced H‐ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H‐ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor‐associated mutations is a useful tool for elucidating the mechanisms underlying the tissuespecificity of carcinogenesis. Environ. Mol. Mutagen. 2012.

Temporal Changes in K-ras Mutant Fraction in Lung Tissue of Big Blue B6C3F1 Mice Exposed to Ethylene Oxide

Ethylene oxide (EO) is a genotoxicant and a mouse lung carcinogen, but whether EO is carcinogenic through a mutagenic mode of action remains unclear. To investigate this question, 8-week-old male Big Blue B6C3F₁ mice (10 mice/group) were exposed to EO by inhalation-6 h/day, 5 days/week for 4 weeks (0, 10, 50, 100, or 200 ppm EO) and 8 or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for levels of 3 K-ras codon 12 mutations (GGT→GAT, GGT→GTT, and GGT→TGT) using ACB-PCR. No measureable level of K-ras codon 12 TGT mutation was detected (ie, all lung mutant fractions [MFs] ≤ 10⁻⁵). Four weeks of inhalation of 100 ppm EO caused a significant increase in K-ras codon 12 GGT→GTT MF relative to controls, whereas 50, 100, and 200 ppm EO caused significant increases in K-ras codon 12 GGT→GAT MF. In addition, significant inverse correlations were observed between K-ras codon 12 GGT→GTT MF and cII mutant frequency in the lungs of the same mice exposed to 50, 100, or 200 ppm EO for 4 weeks. Surprisingly, 8 weeks of exposure to 100 and 200 ppm EO caused significant decreases in K-ras MFs relative to controls. Thus, the changes in K-ras MF as a function of cumulative EO dose were nonmonotonic and were consistent with EO causing early amplification of preexisting K-ras mutations, rather than induction of K-ras mutation through genotoxicity at codon 12. The possibility that these changes reflect K-ras mutant cell selection under varying degrees of oxidative stress is discussed.

Hotspot oncomutations: implications for personalized cancer treatment

Understanding the extent to which specific tumor mutations impact or mediate patient response to particular cancer therapies has become a rapidly increasing area of research. Recent research findings regarding four predominant mutational targets (KRAS, BRAF, EGFR and PIK3CA) show that these tumor mutations have predictive power for identifying which patients are likely to respond to particular therapies, and have prognostic significance irrespective of treatment. However, in this regard, the literature is frequently nuanced and sometimes contradictory. This lack of clarity may be due, at least in part, to the utilization of mutation detection methods with varying sensitivities across studies of different patient populations. Nevertheless, considerable evidence suggests minor tumor subpopulations may be contributing to inappropriate patient stratification, development of resistance to treatment, and the relapse that often follows treatment with molecularly targeted therapies. Consequently, mutant tumor subpopulations need to be considered in order to improve strategies for personalized cancer treatment.

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.

Using ΦX174 DNA as an exogenous reference for measuring mitochondrial DNA copy number

Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction efficiency of nDNA and mtDNA and variation in the ploidy of experimental samples. Here, we report that the ratio of mtDNA to nDNA varies in repeated DNA extractions but that PhiX174 DNA, added before DNA extraction, is extracted with a similar efficiency to mtDNA, making it a suitable alternative reference for quantifying mtDNA copy number.

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium

Summary We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.

Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas

Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measureable levels of at least three of the five mutations. HRAS G12D was significantly increased in DCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10− 3 to 4.6 × 10− 2) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047R mutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells.

Ovarian effects of prenatal exposure to benzo[a]pyrene: Roles of embryonic and maternal glutathione status

Females deficient in the glutamate cysteine ligase modifier subunit (Gclm) of the rate-limiting enzyme in glutathione synthesis are more sensitive to ovarian follicle depletion and tumorigenesisby prenatal benzo[a]pyrene (BaP) exposure than Gclm+/+ mice. We investigated effects of prenatal exposure to BaP on reproductive development and ovarian mutations in Kras, a commonly mutated gene in epithelial ovarian tumors. Pregnantmice were dosed from gestational day 6.5 through 15.5 with 2mg/kg/day BaP or vehicle. Puberty onset occurred 5 days earlier in F1 daughters of all Gclm genotypes exposed to BaP compared to controls. Gclm+/- F1 daughters of Gclm+/- mothers and wildtype F1 daughters of wildtype mothers had similar depletion of ovarian follicles following prenatal exposure to BaP, suggesting that maternal Gclm genotype does not modify ovarian effects of prenatal BaP. We observed no BaP treatment or Gclm genotype related differences in ovarian Kras codon 12 mutations in F1 offspring.