Allele-specific Competitive Blocker PCR Research Articles

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.

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.

Lifespan Kras mutation levels in lung and liver of B6C3F1 mice.

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.

Variation in organ-specific PIK3CA and KRAS mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas.

Large‐scale sequencing efforts have described the mutational complexity of individual cancers and identified mutations prevalent in different cancers. As a complementary approach, allele‐specific competitive blocker PCR (ACB‐PCR) is being used to quantify levels of hotspot cancer driver mutations (CDMs) with high sensitivity, to elucidate the tissue‐specific properties of CDMs, their occurrence as tumor cell subpopulations, and their occurrence in normal tissues. Here we report measurements of PIK3CA H1047R mutant fraction (MF) in normal colonic mucosa, normal lung, colonic adenomas, colonic adenocarcinomas, and lung adenocarcinomas. We report PIK3CA E545K MF measurements in those tissues, as well as in normal breast, normal thyroid, mammary ductal carcinomas, and papillary thyroid carcinomas. We report KRAS G12D and G12V MF measurements in normal colon. These MF measurements were integrated with previously published ACB‐PCR data on KRAS G12D, KRAS G12V, and PIK3CA H1047R. Analysis of these data revealed a correlation between the degree of interindividual variability in these mutations (as log10 MF standard deviation) in normal tissues and the frequencies with which the mutations are detected in carcinomas of the corresponding organs in the COSMIC database. This novel observation has important implications. It suggests that interindividual variability in mutation levels of normal tissues may be used as a metric to identify mutations with critical early roles in tissue‐specific carcinogenesis. Additionally, it raises the possibility that personalized cancer therapeutics, developed to target specifically activated oncogenic products, might be repurposed as prophylactic therapies to reduce the accumulation of cells carrying CDMs and, thereby, reduce future cancer risk. Environ. Mol. Mutagen. 58:466–476, 2017. © 2017 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

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.

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 1mg/m3. 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/m3) 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.

Low-frequency KRAS mutations are prevalent in lung adenocarcinomas.

This study quantified low-frequency KRAS mutations in normal lung and lung adenocarcinomas, to understand their potential significance in the development of acquired resistance to EGFR-targeted therapies. Allele-specific Competitive Blocker-PCR was used to quantify KRAS codon 12 GAT (G12D) and GTT (G12V) mutation in 19 normal lung and 21 lung adenocarcinoma samples. Lung adenocarcinomas had KRAS codon 12 GAT and GTT geometric mean mutant fractions of 1.94 × 10-4 and 1.16 × 10-3, respectively. For 76.2% of lung adenocarcinomas, the level of KRAS mutation was greater than the upper 95% confidence interval of that in normal lung. KRAS mutant tumor subpopulations, not detectable by DNA sequencing, may drive resistance to EGFR blockade in lung adenocarcinoma patients.

Abstract 4267: Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma

Abstract Data within the Catalogue of Somatic Mutations in Cancer indicates that a number of different hotspot oncogene mutations occur infrequently in breast tumors as compared to other tumor types (e.g., colon and lung). We hypothesized that greater sensitivity is required to detect somatic mutations in breast tumors, due to their highly-heterogeneous clonal structure. Therefore, the Allele-specific Competitive Blocker PCR method (with a sensitivity of 10-5), was used to quantify mutations in DNA isolated from normal human breast and ductal carcinoma (DC) samples (n = 10). The specific mutations quantified were: KRAS codon 12 GGT to GAT (G12D) and GGT to GTT (G12V), HRAS codon 12 GGC to GAC (G12D), BRAF codon 600 GTG to GAG (V600E), and PIK3CA codon 1047 CGT to CAT (H1047R). In addition, comparison of normal and malignant tissue was employed in order to define the lower bound on abnormal levels of mutation, as tumors that possess a mutant fraction (MF) greater than the upper 95% confidence interval of that present in normal breast. Interestingly, different patterns of mutation were observed for the different hotspot point mutations. We previously demonstrated that significantly higher levels of KRAS mutation are present in colon and lung tumors relative to the cognate normal tissue; however, the frequencies of KRAS codon 12 GGT to GAT and GGT to GTT mutation in normal breast and in DCs were quite similar (∼10-5 for the KRAS codon 12 GGT to GAT mutation in both tissues). Only one normal sample and two DCs had BRAF codon 600 GTG to GAG MFs >10-5. With respect to HRAS codon 12 GGC to GAC mutation, however, ∼80% of DCs had a MF greater than the upper 95% confidence interval of that present in normal breast tissue. Importantly, no breast tumor had a HRAS codon 12 GGC to GAC MF ≥10-1 (i.e., that detectable by DNA sequencing). Unexpectedly high levels of PIK3CA codon 1047 CGT to CAT mutation (>10-2) were observed in 4/10 normal breast samples and 4/10 DCs, suggesting that preexisting PIK3CA mutations in normal breast contribute to breast cancer susceptibility. In summary, all DC samples analyzed contained somatic mutations, none of which would be detectable by DNA sequencing. Consequently, these results indicate somatic mutations are more prevalent in breast cancer than is currently recognized. Ongoing analyses of additional tumors, corresponding to different breast cancer subtypes, will provide a better understanding of the role of somatic mutations in breast cancer. Citation Format: Malathi Banda, Meagan B. Myers, Karen L. McKim, Yiying Wang, Barbara L. Parsons. Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2014-4267

ACB‐PCR measurement of spontaneous and furan‐induced H‐ras Codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H-ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H-ras codon 61 CAA to AAA mutation increased in furan-treated mice. Allele-specific competitive blocker PCR (ACB-PCR) has been used previously to quantify early, carcinogen-induced increases in tumor-associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre-existing H-ras mutant cells in B6C3F1 mouse liver. H-ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H-ras codon 61 CTA or AAA mutant fraction (MF) greater than 10(-5) . Several furan-treated mice had H-ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically-significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H-ras mutant cells.

No Evidence for JAK2V617F Mutation in Monoclonal B Cells in 2 Patients with Polycythaemia Vera and Concurrent Monoclonal B Cell Disorder

Occurrence of Philadelphia chromosome-negative myeloproliferative neoplasms (Ph^– MPN) and lymphoproliferative disorders, like B cell chronic lymphocytic leukaemia (B-CLL), in the same patient is rare. JAK2^V617F mutation was recently introduced as a powerful diagnostic tool for Ph^– MPN. JAK2^V617F mutation is not present in B-CLL. In 4 previously reported patients with JAK2^V617F-positive Ph^– MPN and B-CLL there was no definitive proof of JAK2^V617F mutation in B-CLL cells, although this was suggested in 1 patient. We present 2 patients with JAK2^V617F-positive polycythaemia vera who subsequently developed a monoclonal B cell disorder. The granulocytes were separated from the mononuclear cells by centrifugation on density gradient. Using an ARIA-SORP sorter, the CD20+/CD5+ B cells were separated from the CD20+/CD5– B cells, T cells, NK cells and monocytes. On each of the fractions JAK2^V617F mutation was analysed by allele-specific competitive blocker-PCR. In both patients JAK2^V617F mutation was present in granulocytes confirming the clinical diagnosis of polycythaemia vera. We did not detect the JAK2^V617F mutation in the CD20+/CD5+ B cells but detected it in CD20+/CD5– B cells, T and NK cells, indicating a lymphoid subdifferentiation of the JAK2^V617F MPN clonality. JAK2^V617F MPN and monoclonal B cell disorder can coexist but there is no evidence that the proliferative behaviour of these B cells is mediated through the JAK2^V617F mutation.

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 (Fisher's 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 tissue specificity of carcinogenesis.

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

Abstract 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

Abstract A23: Rare allele enrichment by DNA melting analysis

Abstract Since the sequencing of the human genome and the development of high throughput genotyping techniques, many genes have been identified that are associated with a variety of diseases through their SNPs and mutations. For example, in lung cancer, mutations in the gene encoding epidermal growth factor reporter (EGFR) can confer either positive therapeutic response or drug resistance to tyrosine kinase inhibitors. Additionally, mutations in the BRAF gene are strongly associated with cancerous thyroid nodules. In the early and post-treatment stages of cancer or in prenatal diagnostics, the proportion of the mutant allele is typically low within a background of wild-type. Even after amplification by standard PCR, the low levels of the minority allele that are present in tumor tissue or in plasma circulating DNA are not sufficient to permit genotyping or sequencing. Mutation detection plays a key role in several areas of medicine including diagnosis, treatment and prognosis. In cancer-related gene mutation diagnostics, low-level (&amp;lt;10%) mutations cannot be sequenced by standard PCR or genotyping techniques. We have developed methods of rare allele enrichment and melting curve detection using Snapback primers and unlabeled probes. A snapback primer is a tail at the 5′ end of a primer that is complementary to its own extension product. When intramolecular hybridization is favored over intermolecular pairing, a hairpin is formed whose stem melting behavior depends on sequence. Rapid cycle PCR with a short extension time (0 sec) and an extension temperature lower than the Tm of the hairpin of the wild-type, perfectly matched allele obstructs primer extension while mismatched alleles denature, thus providing the competitive advantage and preferential amplification of minority alleles. Minority alleles down to 0.1% (1:1000) can be detected. Even greater detection sensitivity can be achieved using allele specific PCR, probe blocking and melting analysis. Unlabeled probes, dual hybridization probes, or molecular beacons can be used. Allele-specific competitive blocker PCR (ACB-PCR) was modified by using asymmetric PCR and melting analysis to increase the sensitivity. The probe serves as both a blocker and produces the probe melting curve to implement detection. Mutation detection by probe melting curve analysis is more reliable than PCR quantification because only the specifically amplified allele produces the melting curve associated with the target. Quantitative PCR with dsDNA dyes cannot distinguish the intended PCR product from primer dimers and other non-specific PCR products. In contrast, when using probe melting curves to detect mutations, the melting temperature and overall melting profile of probe melting curves specifically identify the known mutant target. Additionally, based upon probe melting temperatures of wild-type and mutant hybrids, a precise annealing temperature between these Tms guarantees that the PCR selectively amplifies the minority allele. Low allele specific primer concentrations (asymmetric PCR) further increase the sensitivity and increase the probe signal allowing a sensitivity of 0.001% (1:105) mutant to wild-type genomic DNA, or just a single copy in a 500 ng genomic DNA. Methods that do not require labeled probes (snapback primer and unlabeled probes) can be used to selectively amplify minority alleles by methods that are rapid (less than 20 minutes), easy to design, inexpensive and reliable. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A23.

Abstract 3179: ACB-PCR quantification of PIK3CA codon 1047 CAT to CGT mutant fraction in human tumor and non-tumor tissues

Abstract Phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) is a proto-oncogene encoding the p110 catalytic subunit of phosphoinositide 3-kinase (PI3K). A variety of tumors carry PIK3CA mutations. These mutations are thought to increase PI3K activity, increase cell proliferation, and may contribute to anti-cancer drug resistance. Three specific hotspot mutations within the PIK3CA gene are localized within the helical and kinase domains and account for &amp;gt;90% of all reported mutations in PIK3CA. One PIK3CA mutation in the kinase domain (codon 1047 CAT to CGT) accounts for more than 30% of all reported mutations. Because PIK3CA mutation may have clinical significance in terms of patient prognosis and treatment selection, we are using a sensitive allele-specific amplification approach called allele-specific competitive blocker-PCR (ACB-PCR) to analyze the PIK3CA codon 1047 CAT to CGT mutant fractions (MFs) in various human tissues. Specific goals of this study are to: 1) define normal and pathological levels of PIK3CA mutation in various human tissues, and 2) obtain data on the co-occurrence of PIK3CA mutation with other mutations (e.g., the KRAS codon 12 GAT and GTT mutations). To date, PIK3CA codon 1047 CAT to CGT MF has been measured in 50 human colon tissue samples, including 17 normal-appearing colonic mucosa samples, 13 normal tumor-adjacent samples, 10 adenomas, and 10 adenocarcinomas. The PIK3CA codon 1047 CAT to CGT mutation was present at measurable levels in all normal appearing colonic mucosa samples. The PIK3CA geometric mean MF in normal colonic mucosa ranged from 3.24 × 10-4 to 6.48 × 10-4, with an average (geometric mean) MF of 5.12 × 10-4. The PIK3CA geometric mean MFs measured in normal tumor-adjacent samples, adenomas, and adenocarcinomas were 6.45 × 10-4, 7.32 × 10-4, and 3.45 × 10-4, respectively. All colon tumors were found to carry PIK3CA codon 1047 CAT to CGT mutations as mutant subpopulations. The levels of PIK3CA mutation in normal-appearing colonic mucosa samples are similar to the levels of KRAS GAT mutation (∼10-4) measured previously in the same samples. Analyses of PIK3CA mutation in breast, pancreas, and thyroid are ongoing. In conjunction with analyses of other mutational targets, these types of data should aid in the identification of the best mutational biomarkers for particular tissues types (those mutations with consistently higher levels in tumors than in normal tissues). Also, by describing the prevalence of different molecular lesions within functional pathways related to carcinogenesis, the most promising molecular targets for therapeutic intervention will be identified. The contents of the abstract do not necessarily reflect the views or policies of the U.S. FDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3179. doi:10.1158/1538-7445.AM2011-3179

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 30 ppm NA vapor for 13 weeks (6 h/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 30 ppm 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.

Mutation analysis for systemic mastocytosis

Mast cells from over 80% of adult systemic mastocytosis (SM) patients carry an activating mutation at codon 816 of the KIT gene. In fact, a KIT mutation constitutes one of the minor WHO 2008 classification criteria for SM. The most common is the c.2447A>T: p.Arg816Val (D816V) mutation. The KIT gene encodes a type III transmembrane receptor tyrosine kinase whose ligand is stem cell factor. Activation of KIT mediates proliferation and maturation signalling. D816 V causes constitutive activation of the KIT tyrosine kinase activity, which also results in resistance to the tyrosine kinase inhibitor imatinib mesylate. Therefore knowing the mutational status of KIT codon 816 is important for diagnosis and treatment strategy. The population of mutant mast cells is often low in relation to normal cells and so a sensitive molecular detection technique is required. Several have been reported in the literature, the most sensitive include RT-PCR + RFLP, PNA-mediated PCR and allele-specific PCR. Within the diagnostic setting the Peter MacCallum uses the rapid and highly sensitive allele-specific competitive blocker PCR (ACB-PCR) assay with a sensitivity level of between 1 and 0.1%. Between Jan 2008 and Oct 2010, 338 samples were tested, of which 58% were positive.

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.

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 (6–7weeks old, 5 rats/group) were exposed to 0, 0.7, 2, 6, 10, and 15ppm formaldehyde for 13weeks (6h/day, 5days/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 15ppm formaldehyde-treatment groups. Therefore, induction of tumor-associated p53 mutation likely occurs after several other key events in formaldehyde-induced carcinogenesis.

K‐Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

K-Ras mutant fraction (MF) was measured to examine the default assumption of low-dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of 10 male A/J mice (7- to 9-weeks old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P and were sacrificed 28 days after treatment. K-Ras codon 12 TGT and GAT MFs in lung DNAs were measured using Allele-specific Competitive Blocker-PCR (ACB-PCR). The K-Ras codon 12 TGT geometric mean MF was 3.88 x 10(-4) in controls, indicating an average of 1 mutation in every approximately 1,288 lung cells. The K-Ras codon 12 TGT geometric mean MFs were as follows: 3.56 x 10(-4); 6.19 x 10(-4); 2.02 x 10(-3), and 3.50 x 10(-3) for the 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The 5 and 50 mg/kg dose groups had TGT MFs significantly higher than did controls. Although 10(-5) is considered as the limit of accurate ACB-PCR quantitation, K-Ras codon 12 GAT geometric mean MFs were as follows: 8.38 x 10(-7), 1.47 x 10(-6), 2.19 x 10(-6), 5.71 x 10(-6), and 8.99 x 10(-6) for the 0, 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The K-Ras TGT and GAT MFs increased in a B[a]P-dose-dependent manner, with response approximately linear over the 0.05 to 5 mg/kg dose range. K-Ras MF increased with B[a]P adduct burden measured for identical doses in a separate study. Thus, ACB-PCR may be useful in characterizing the shape of a dose-response curve at low doses and establishing relationships between DNA adducts and tumor-associated mutations.

Simulated solar light‐induced p53 mutagenesis in SKH‐1 mouse skin: A dose–response assessment

Sunlight and ultraviolet-induced mutation of the p53 gene is a frequent, possibly obligate step in skin cancer development, making quantitative measurement of p53 mutation an ideal biomarker for sunlight-induced skin carcinogenesis. To understand how the appearance of p53 mutation relates to skin tumor development, SKH-1 hairless mice were exposed 5 d per week to one of four different doses of simulated solar light (SSL; 0, 6.85, 13.70, 20.55 mJ x CIE/cm(2)) previously characterized for their tumorigenic potential. Allele-specific competitive blocker-PCR (ACB-PCR) was used to measure levels of p53 codon 270 CGT to TGT mutation within DNA isolated from dorsal skin of exposed mice. For each dose, p53 mutant fraction (MF) was measured after 4, 16, and 28 wk of exposure. Significant dose- and time-dependent increases in p53 MF were identified. All p53 MF measurements were integrated by relating the observed p53 MF to the cumulative dose of SSL. The increase in the logarithm of p53 MF was described by the linear function: log(10) MF = alpha + 0.0016 x d, where alpha is the spontaneous log(10) MF after a particular time point and d is the dose of SSL in mJ x CIE/cm(2). The p53 MF induced in nontumor bearing skin by 28 wk of exposure at the high dose of SSL was significantly lower than that found in skin tumors induced by approximately 32 wk of exposure to the same dose of SSL. p53 MF showed a strong negative correlation with tumor latency, suggesting this quantitative biomarker has the potential to predict tumorigenicity.