Karen L. McKim

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.

Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation.

This study investigated whether Kras mutation is an early event in the development of lung tumors induced by inhalation of particulate vanadium pentoxide (VP) aerosols. A National Toxicology Program tumor bioassay of inhaled particulate VP aerosols established that VP-induced alveolar/bronchiolar carcinomas of the B6C3F1 mouse lung carried Kras mutations at a higher frequency than observed in spontaneous mouse lung tumors. Therefore, this study sought to: (1) characterize any Kras mutational response with respect to VP exposure concentration, and (2) investigate the possibility that amplification of preexisting Kras mutation is an early event in VP-induced mouse lung tumorigenesis. Male Big Blue B6C3F1 mice (6 mice/group) were exposed to aerosolized particulate VP by inhalation, 6h/day, 5 days/week for 4 or 8 weeks, using VP exposure concentrations of 0, 0.1, and 1 mg/m(3). The levels of two different Kras codon 12 mutations [GGT → GAT (G12D) and GGT → GTT (G12V)] were measured in lung DNAs by Allele-specific Competitive Blocker PCR (ACB-PCR). For both exposure concentrations (0.1 and 1.0mg/m(3)) and both time points (4 and 8 weeks), the mutant fractions observed in VP-exposed mice were not significantly different from the concurrent controls. Given that 8 weeks of inhalation of a tumorigenic concentration of particulate aerosols of VP did not result in a significant change in levels of lung Kras mutation, the data do not support either a direct genotoxic effect of VP on Kras or early amplification of preexisting mutation as being involved in the genesis of VP-induced mouse lung tumors under the exposure conditions used. Rather, the data suggest that accumulation of Kras mutation occurs later with chronic VP exposure and is likely not an early event in VP-induced mouse lung carcinogenesis.

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.

Dose and temporal evaluation of ethylene oxide-induced mutagenicity in the lungs of male big blue mice following inhalation exposure to carcinogenic concentrations: Evaluation of EO-Induced Mutagenicity in Male BB Mice

Ethylene oxide (EO) is a direct acting alkylating agent; in vitro and in vivo studies indicate that it is both a mutagen and a carcinogen. However, it remains unclear whether the mode of action (MOA) for cancer for EO is a mutagenic MOA, specifically via point mutation. To investigate the MOA for EO‐induced mouse lung tumors, male Big Blue (BB) B6C3F1 mice (10/group) were exposed to EO by inhalation, 6 hr/day, 5 days/week for 4 (0, 10, 50, 100, or 200 ppm EO), 8, or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for cII mutant frequency (MF) at 4, 8 and 12 weeks of exposure; the mutation spectrum was analyzed for mutants from control and 200 ppm EO treatments. Although EO‐induced cII MFs were 1.5‐ to 2.7‐fold higher than the concurrent controls at 4 weeks, statistically significant increases in the cII MF were found only after 8 and 12 weeks of exposure and only at 200 ppm EO (P ≤ 0.05), which is twice the highest concentration used in the cancer bioassay. Consistent with the positive response, DNA sequencing of cII mutants showed a significant shift in the mutational spectra between control and 200 ppm EO following 8 and 12 week exposures (P ≤ 0.035), but not at 4 weeks. Thus, EO mutagenic activity in vivo was relatively weak and required higher than tumorigenic concentrations and longer than 4 weeks exposure durations. These data do not follow the classical patterns for a MOA mediated by point mutations. Environ. Mol. Mutagen. 58:122–134, 2017.

Abstract 1739: The prevalence of KRAS, PIK3CA, and BRAF mutant subpopulations in tumors may be impacting the success of personalized cancer treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tumor mutations are being used as predictive biomarkers of response, in order to select the most effective treatment for individual cancer patients. Currently, this is being done without sufficient characterization of relevant oncogene mutations as quantitative biomarkers. The goal of the current study was to define normal and pathological levels of the most prevalent hotspot mutations in the KRAS, PIK3CA, and BRAF genes, to establish the frequency with which the mutations occur as subpopulations, and what diagnostic sensitivity is needed to detect defined percentages of tumors carrying mutant subpopulations. Therefore, the sensitive Allele-specific Competitive Blocker-PCR (ACB-PCR) was used to quantify the levels of specific hotspot point mutations in a panel of normal human tissues and tumors. The mutations examined have established significance in terms of personalized cancer treatment, specifically KRAS G12D, KRAS G12V, BRAF V600E, and PIK3CA H1047R. The tissues examined included lung, colon, pancreas, and thyroid. In colon tumors, the 5th, 25th, 50th, 75th, and 95th percentiles of KRAS G12D mutant fraction (MF) are 1.7 x 10−5, 7.4 x 10−5, 3.0 x 10−4, 2.8 x 10−2, and 8.4 x 10−1, respectively. In lung tumors, the 5th, 25th, 50th, 75th, and 95th percentiles of KRAS G12V MF are 7.0 x 10−6, 1.1 x 10−5, 3.3 x 10−5, 2.3 x 10−2, and 1.2 x 10−1, respectively. Based on the data across these tissue types, 67.5% of tumors carry KRAS G12D or G12V mutation at a subpopulation frequency higher than that observed in normal tissue. Only 18.1% of tumors had a KRAS MF α10−1 (i.e., that detectable by DNA sequencing). From these data it was determined a diagnostic with a sensitivity of 10−2 or 10−3 would detect 27.7% or 43.4% of these tumors, respectively. Surprisingly, analysis of KRAS mutation in papillary thyroid tumors showed KRAS G12V mutations were present above normal thyroid levels, but as subpopulations in 42.1% of papillary thyroid tumors, even though the COSMIC database indicates this mutation occurs in only 0.15% of papillary thyroid tumors. The occurrence of these KRAS G12V mutations was positively correlated with percent tumor necrosis. For PIK3CA H1047R mutation in colon tumors, the 5th, 25th, 50th, 75th, and 95th percentiles are 1.2 x 10−6, 5.3 x 10−4, 7.6 x 10−4, 1.1 x 10−3, and 4.2 x 10−2, respectively. Data on BRAF V600E shows it occurs primarily as large subpopulations in papillary thyroid tumors. For effective development of personalized cancer treatment, quantitative and sensitive analyses of tumor mutations are needed to establish the effect of mutant subpopulations on patient response and/or relapse. Because so many tumors carry KRAS mutation, therapies targeting KRAS mutant cells are needed for use in conjunction with therapies directed against other targets. The views presented do not necessarily reflect those of the US FDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1739. doi:1538-7445.AM2012-1739

Lifespan Kras mutation levels in lung and liver of B6C3F1 mice: Kras Mutation in Aging Mouse Lung and Liver

Somatic mutations accumulate in the human genome and are correlated with increased cancer incidence as humans age. The standard model for studying the carcinogenic effects of exposures for human risk assessment is the rodent 2‐year carcinogenicity assay. However, there is little information regarding the effect of age on cancer‐driver gene mutations in these models. The mutant fraction (MF) of Kras codon 12 GGT to GAT and GGT to GTT mutations, oncogenic mutations orthologous between humans and rodents, was quantified over the lifespan of B6C3F1 mice. MFs were measured in lung and liver tissue, organs that frequently develop tumors following carcinogenic exposures. The MFs were evaluated at 4, 6, 8, 12, 21, and 85 weeks, with the 12‐week and 21‐week time points being coincident with the conclusion of 28‐day and 90‐day exposure durations used in short‐term toxicity testing. The highly sensitive and quantitative Allele‐specific Competitive Blocker PCR (ACB‐PCR) assay was used to quantify the number of mutant Kras codon 12 alleles. The mouse lung showed a slight, but significant trend increase in the Kras codon 12 GAT mutation over the 85‐week period. The trend with age can be equally well‐fit by several non‐linear functions, but not by a linear function. In contrast, the liver GAT mutation did not increase, and the GTT mutation did not increase for either organ. Even with the slight increase in the lung GAT MFs, our results indicate that the future use of Kras mutation as a biomarker of carcinogenic effect will not be confounded by animal age. Environ. Mol. Mutagen. 59:715–721, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Abstract A15: Establishing an ex-vivo, tumor spheroid culture system to assess molecular-targeted therapies for personalized treatment of non-small cell lung cancer

Molecularly-targeted therapeutics are being developed for personalized cancer treatment based on the ideas that treatments should be determined by specific characteristics of an individual patient9s tumor and that treatments should target tumor cells without damaging normal cells. Accumulating evidence indicates tumor heterogeneity can lead to acquired resistance to molecularly-targeted therapies. Currently, preclinical investigations rely heavily on the use of tumor cell lines and mouse xenograft models. The high failure rate in oncology drug development suggests that such test systems do not adequately model tumor heterogeneity. Therefore, more predictive preclinical models are needed to ensure that only the most promising molecularly-targeted, combination therapies are assessed in clinical studies. Here we established a novel 3D lung adenocarcinoma tissue-originating spheroid model, which may be used to investigate the efficacy of molecularly-targeted cancer therapeutics. Fresh lung adenocarcinomas were minced and either digested with Liberase DH enzyme (5.6 units) or homogenized to generate cancer tissue-originating spheroids. Spheroids were plated in Matrigel and cultured in stem cell media, with 0.01, 0.1, 1 or 10 μM erlotinib or the DMSO vehicle. Erlotinib is an epidermal growth receptor inhibitor commonly used to treat non-small cell lung cancer (NSCLC). Using an ImageXpress Micro Widefield High Content Screening System (Molecular devices), z-stack bright field images were collected 1, 7, and 14 days after plating. Composite z-stack images were analyzed using LASX (Leica Microsystems) software. Total spheroid area (in pixels) was measured for replicate cultures exposed to different erlotinib concentrations at each time point. The total area of spheroids for 7 and 14 days were normalized to day 1 measurements and cell death was evaluated as the decrease in total area of the spheroids in culture. Our results indicate that direct homogenization of lung adenocarcinomas generated at least ten times greater yield of spheroids than was achieved using digestion with the Liberase DH. The yield and appearance of spheroids varied significantly. Spheroids prepared from some lung adenocarcinomas were more spherical and aggregated than those prepared from other tumors. There was time-dependent decrease in total area of spheroids following 7 and 14 days in culture. Also spheroids prepared from different lung adenocarcinomas responded differently to different concentrations of erlotinib. High-sensitivity quantification of specific mutations (EGFR, KRASPIK3CA and BRAF) will be conducted before and after erlotinib treatments to determine whether this model can be used to assess the potential outgrowth of drug-resistant tumor subpopulations. Citation Format: Malathi Banda, Karen L. McKim, Barbara Parsons. Establishing an ex-vivo, tumor spheroid culture system to assess molecular-targeted therapies for personalized treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A15.