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Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

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.

An accurate understanding of the clonal origins of tumors is critical for designing effective strategies to treat or prevent cancer and for guiding the field of cancer risk assessment. The intent of this review is to summarize evidence of multiclonal tumor origin and, thereby, contest the commonly held assumption of monoclonal tumor origin. This review describes relevant studies of X chromosome inactivation, analyses of tumor heterogeneity using other markers, single cell sequencing, and lineage tracing studies in aggregation chimeras and engineered rodent models. Methods for investigating tumor clonality have an inherent bias against detecting multiclonality. Despite this, multiclonality has been observed within all tumor stages and within 53 different types of tumors. For myeloid tumors, monoclonal tumor origin may be the predominant path to cancer and a monoclonal tumor origin cannot be ruled out for a fraction of other cancer types. Nevertheless, a large body of evidence supports the conclusion that most cancers are multiclonal in origin. Cooperation between different cell types and between clones of cells carrying different genetic and/or epigenetic lesions is discussed, along with how polyclonal tumor origin can be integrated with current perspectives on the genesis of tumors. In order to develop biologically sound and useful approaches to cancer risk assessment and precision medicine, mathematical models of carcinogenesis are needed, which incorporate multiclonal tumor origin and the contributions of spontaneous mutations in conjunction with the selective advantages conferred by particular mutations and combinations of mutations. Adherence to the idea that a growth must develop from a single progenitor cell to be considered neoplastic has outlived its usefulness. Moving forward, explicit examination of tumor clonality, using advanced tools, like lineage tracing models, will provide a strong foundation for future advances in clinical oncology and better training for the next generation of oncologists and pathologists.

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 B 6 C 3 F 1 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 B 6 C 3 F 1 mice (6 mice/group) were exposed to aerosolized particulate VP by inhalation, 6 h/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 1.0 mg/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.

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.

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.

Few ideas have gained such strong acceptance in the scientific community as the monoclonal origin of tumors; the idea that tumors start with a single mutated cell (or a single clone of cells) that go on to accumulate additional mutations as a tumor develops. The certainty with which this concept is held by the scientific community reflects the length of time it has been unchallenged and the experimental difficulty in obtaining direct evidence to the contrary. Yet, recent findings regarding X chromosome inactivation patch size indicate that the X-linked marker data previously interpreted as evidence of monoclonal tumor origin is actually more consistent with polyclonal tumor origin, a situation where two or more cells or clones of cells interact to initiate a tumor. Although most tumors show homotypy for X-linked markers (as expected given the bias conferred by X chromosome inactivation patch size), the literature contains numerous examples of tumors with X-linked marker heterotypy, examples of which encompass 24 different tumor types. Chimeric models have yielded direct unequivocal demonstrations of polyclonality in rodent and human tumors. Also, mutational data are consistent with polyclonal tumor origin. Methods that analyze levels of tumor-associated oncogene and tumor suppressor gene mutations demonstrate that initiated cells are much more common in normal tissues than previously realized. Also, while tumors have higher levels of mutation than normal tissues, oncogenic mutations frequently are present as subpopulations within tumors, rather than as the pure mutant populations expected to develop from a single initiated cell. Understanding the mutational basis of tumor etiology has important practical significance for assessing cancer risk, as well as in modeling and treating cancer. Therefore, the scientific community needs to re-examine this issue and consider the implications of polyclonal origin for, perhaps, a majority of tumors, encompassing many different tumor types.

K-ras codon 12 GGT-->GAT and GGT-->GTT mutations are the most frequently observed K-ras point mutations in human and rodent tumors and therefore are implicated in carcinogenesis for many tissues. Measurement of these mutations in rat models and human tissue could facilitate a more logical extrapolation of rodent tumorigenesis data to human disease. We have developed allele-specific competitive blocker PCR (ACB-PCR) assays for rat K-ras codon 12 GGT-->GTT and GGT-->GAT mutations that parallel the already published assays for human K-ras codon 12 mutations. Liver K-ras codon 12 mutant allele fractions were measured in vehicle-treated and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-treated Big Blue rats. The average K-ras codon 12 GGT-->GTT mutant fraction (MF) for four control rats was 50 x 10(-6) (95% CI: 27 x 10(-6), 95 x 10(-6)) and for four treated rats was 165 x 10(-6) (95% CI: 87 x 10(-6), 312 x 10(-6)), indicating a 3.3-fold increase with treatment (95% CI: 1.3-8.1). The average MF of K-ras codon 12 GGT-->GAT for control rats was 1320 x 10(-6) (95% CI: 498 x 10(-6), 3500 x 10(-6)) and for treated rats was 8450 x 10(-6) (95% CI: 3180 x 10(-6), 22 400 x 10(-6)), indicating a 6.4-fold increase with treatment (95% CI: 1.6-25.4). These transgenic rats were part of a study that included analysis of liver lacI mutations. Although data from lacI determinations show that this compound induces mostly G-->T mutations, using the ACB-PCR method both K-ras codon 12 GGT-->GTT and GGT-->GAT MFs were significantly increased in treated rats versus control rats. This data raises the possibility that N-OH-AAF may not only induce mutations by a genotoxic mechanism, but also by amplification of both de novo and pre-existing K-ras mutation.

Deficiencies in DNA mismatch repair (MMR) result in predisposition to neoplasia in both rodents and humans. Pms2 is one of the several proteins involved in the eukaryotic MMR system. In order to determine the effect of Pms2-deficiency on mutation, we measured mutant frequencies in the endogenous Hprt gene of lymphocytes from male Pms2 −/− , Pms2 +/− , and Pms2 +/+ mice. Spleens were removed from mice of various ages and lymphocytes isolated from spleens were cultured to determine the frequency of 6-thioguanine-resistant mutants. Mean mutant frequencies in Pms2 −/− mice at 6, 10, 18, and 34 weeks of age [42.6 × 10 −6 ( n = 6), 38.5 × 10 −6 ( n = 6), 58.2 × 10 −6 ( n = 9), and 49.1 × 10 −6 ( n = 5), respectively] were significantly higher than those of comparably aged Pms2 +/+ and Pms2 +/− mice (all less than 3 × 10 −6 ). Mutant clones from the mice were expanded, RNA extracted, and Hprt cDNA amplified by RT-PCR. DNA sequencing analysis of 221 mutant cDNAs from the three different Pms2 genotypes identified 182 clones with independent mutations, including five clones that contained multiple mutations. When compared to the mutational spectrum observed in Pms2 +/+ and Pms2 +/− mice, the mutational spectrum for Pms2 −/− mice was significantly different. The Pms2 −/− mutational analysis indicated that loss of the Pms2 protein causes increases in the frequencies of strand-slippage-type frameshift mutations and of A:T → G:C transitions in the Hprt gene. The absolute frequencies of A:T → G:C transitions in MMR-deficient mice suggest increases in this mutation may be a common feature of MMR-deficient mice, not just of Pms2-deficient mice, and may be related to the cancer predisposition that results from loss of MMR function.

DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

The mechanisms underlying the susceptibility of neonatal mice to genotoxic carcinogens were investigated by analyzing the DNA adducts and mutations induced in the livers of neonatal and adult Big Blue transgenic mice by 4-aminobiphenyl (4-ABP), a potent human and rodent carcinogen. Neonatal and adult mice were treated with a regimen of 4-ABP known to induce tumors in neonatal mice. Animals were sacrificed 1 day after the last treatment for DNA adduct analysis and 8 weeks after the last treatment for analysis of lacI and cII mutant frequency (MF). N-(Deoxyguanosin-8-yl)-4-ABP was the major DNA adduct identified in the livers of the 4-ABP-treated mice and levels of this adduct were significantly higher in treated animals than in the controls for both the neonates and adults. Adduct levels for adult females (44.0 ± 4.8 adducts/106 nucleotides) were higher than in neonatal females (25.9 ± 2.2 adducts/106 nucleotides), while adduct levels in adult males (13.5 ± 2.0 adducts/106 nucleotides) were lower than in neonatal males (33.8 ± 4.1 adducts/106 nucleotides). 4-ABP treatment significantly increased the liver cII MFs in both sexes of neonatal mice but not in adult mice. Sequence analysis of cII mutant DNA revealed that 4-ABP induced a unique spectrum of mutations in neonatal mice, characterized by a high frequency of G:CT:A transversion, while the mutation spectrum in 4-ABP-treated adults was similar to that of control mice. Our results indicate that DNA adduct formation by 4-ABP depends as much on sex as it does on age, whereas the conversion of DNA adducts into mutations differed with animal age. These observations suggest that neonates are more sensitive than adults to genotoxic carcinogens because the relatively high levels of cell division in the developing animal facilitate the conversion of DNA damage into mutation. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html © 2005 Wiley-Liss, Inc.

Allele-specific competitive blocker PCR (ACB-PCR) amplification and quantification was developed for mouse p53 codon 270 CGTTGT base substitution and codon 244/245 AAC/CGCAAT/TGC tandem mutation. PCR products corresponding to p53 mutant and wild-type DNA sequences were generated. These DNAs were mixed in known proportions to construct samples with defined mutant fractions and the allele-specific detection of each mutation was systematically optimized. Each assay was used to analyze eight simulated solar light (SSL)-induced tumors. By analyzing mutant fraction (MF) standards in parallel with PCR products generated from tumor samples, p53 mutants could be quantified as subpopulations within the tumors. All eight tumors contained detectable levels of p53 codon 270 CGTTGT mutation. Three tumors had p53 MFs between 10−4 and 10−3. Five tumors had p53 MFs between 10−3 and 10−2. None of the eight mouse skin tumors had measurable levels of p53 codon 244/245 tandem mutation. Frequent detection of p53 codon 270 CGTTGT mutation provides additional evidence that a pyrimidine dinucleotide overlapping a methylated CpG site (PyrmeCG) is a susceptible target for SSL-induced mutagenesis. The absence of p53 codon 244/245 mutation in tumors may be explained by its mutant p53 phenotype and/or indicate that this site is not methylated. These initial results indicate that p53 codon 270 CGTTGT mutation may be a sensitive biomarker for SSL- or UV-induced mutagenesis. This mutational endpoint may be useful for evaluating the co-carcinogenicity of compounds administered in combination with UV or SSL. Environ. Mol. Mutagen., 2005. Published 2005 Wiley-Liss, Inc.

The effects of deficiency in the DNA mismatch repair (MMR) protein Pms2 were investigated using the endogenous mouse Hprt and Tk genes as reporters of intragenic mutation and loss of heterozygosity (LOH). Pms2(-/-)Tk(+/-), Pms2(+/+)Tk(+/-), Pms2(+/-)Tk(+/-) and Pms2(-/-)Tk(-/-) mice were bred from Pms2(+/-)Tk(+/-) mice. At 2 months of age, the body weight and splenic T lymphocyte yields were significantly lower in Pms2(-/-)Tk(-/-) mice than in littermates of the other genotypes. The mice were evaluated for their spontaneous mutant frequencies in the Hprt and Tk genes of splenic lymphocytes and their frequency of micronuclei in polychromatic erythrocytes from bone marrow. The cloning efficiency of lymphocytes derived from Pms2(-/-)Tk(-/-) animals was 12-fold lower than that of animals of the other genotypes. Compared with Pms2(+/+) and Pms2(+/-) mice, Pms2(-/-) mice had a 21- to 69-fold increase in the Hprt mutant frequency. The Hprt mutant frequency was equally high in Pms2-deficient, Tk(+/-) and Tk(-/-) mice. No significant Pms2-dependent change in mutant frequency was detected using the Tk mutational target. When individual Tk mutants were analyzed for LOH mutation by allele-specific genotyping, the fraction of LOH mutants was lower in Pms2-deficient than in Pms2-proficient mice (29.2 and 43.6%, respectively). The frequency of bone marrow micronuclei was significantly higher in Pms2(-/-)Tk(-/-) mice than in Pms2(-/-)Tk(+/-) mice. These observations suggest that the simultaneous occurrence of Tk and Pms2 deficiencies may cause a decrease in cell viability that diminishes the Tk mutational response, making it impossible to discern clearly the effect of Pms2 deficiency on LOH-type mutation using the Tk reporter system. The interaction between Tk deficiency and a component of the MMR system suggests that Tk-deficient cells may have higher levels of DNA polymerase misincorporation or endogenous DNA damage than Tk-proficient cells.

Genotypic selection methods detect rare sequence changes in populations of DNA molecules. These methods have been used to investigate the chemical induction of mutation and for the detection and diagnosis of cancer. The possible use of genotypic selection for improving current risk assessment practices is based on the premise that the frequency of somatic mutation is of critical importance in understanding and modeling carcinogenesis. If genotypic selection can measure the induction of specific mutations that disrupt normal cell/tissue homeostasis, then it could provide key mechanistic information for cancer risk assessment. For example, genotypic selection data might support a particular low-dose extrapolation method or characterize the relationship between rodent and human cancer risk. Strategies for evaluating the use of genotypic selection in cancer risk assessment include the concept of developing a battery of targets that detect a range of agent-specific effects. Ideal targets to examine by genotypic selection are the oncogene and tumor suppressor gene mutations frequently detected in human tumors because these are thought to represent tumor-initiating events. The most commonly occurring basepair (bp) substitutions within the ras and p53 genes are identified. Also, the battery of genotypic selection methods is defined in terms of the most important mutational specificities to include. In theory, the major basepair substitution mutations induced by 29 of 31 chemical carcinogens could be detected by analyzing three different mutations: G:C-->T:A, G:C-->A:T, and A:T-->T:A. Genotypic selection will have the greatest impact on risk assessment if measurement of spontaneous mutation is possible. Data from phenotypic selection assays suggest this corresponds to detection of mutant fractions of approximately 10(-7), and this would necessitate examining DNA samples containing >10(7) target molecules. Despite its apparent potential, considerable development and validation is needed before genotypic selection data can be applied to cancer risk assessment.

The detection of rare mutations has many important applications, including risk assessment of drugs and chemicals, measuring environmental exposures to genotoxins, and cancer cell detection. A sensitive genotypic selection method has been developed that combines two different mutant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evaluated for the detection of a CAA AAA mutation at codon 61 of the mouse H-ras gene. The MutEx enrichment is based on MutS binding to a mismatched basepair in heteroduplex DNA. The bound MutS protects the mutant allele from degradation during subsequent exonuclease treatment. ACB-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mutant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 107. This sensitivity was achieved with the thermostable Thermus aquaticus MutS protein but not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate ± the standard error of the mean for this particular basepair was estimated to be 8 ± 3 × 10−7 errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods for the detection of mutation. Environ. Mol. Mutagen. 32:200–211, 1998 © 1998 Wiley-Liss, Inc.

The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H- ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA). The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences were amplified by PCR. When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50 000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.

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.

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.

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.

Azoxymethane (AOM) administration to F344 male rats is a widely used model of human colon carcinogenesis. The current study investigates quantitatively the accumulation of K-Ras codon 12 mutations following AOM exposure. Male, 6-week-old F344 rats were treated subcutaneously with 30 mg/kg body weight of AOM, and colon tissue was collected at 1, 8, 24, and 32 weeks after treatment. The K-Ras codon 12 GGT to GAT and GGT to GTT mutant fractions (MFs) were measured using allele-specific competitive blocker polymerase chain reaction (ACB-PCR). Between 1 and 32 weeks after AOM treatment, the K-Ras codon 12 GGT to GAT geometric mean MF in the rat colon increased significantly from 12.9 × 10−5 to 145 × 10−5, and the GGT to GTT geometric mean MF increased significantly from 5.26 × 10−5 to 180 × 10−5. K-Ras codon 12 GGT to GAT MF also increased significantly in AOM-treated rat colon tissue at 1 week compared to controls (4.44 × 10−5). The accumulation of the GGT to GAT MF long after the DNA adduct repair phase suggests that a portion of cells containing this mutation have a proliferative advantage, allowing them to accumulate as nascent tumors progress. Also, the GGT to GAT background MF increased in untreated rats, indicating that there is accumulation with age. The ACB–PCR assay generates quantitative data of cancer-related mutations and thus provides insight into pathological processes following carcinogen exposure. Environ. Mol. Mutagen., 2011. © Published 2011 Wiley-Liss, Inc.

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 × 10−4 in controls, indicating an average of 1 mutation in every ∼1,288 lung cells. The K-Ras codon 12 TGT geometric mean MFs were as follows: 3.56 × 10−4; 6.19 × 10−4; 2.02 × 10−3, and 3.50 × 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 × 10−7, 1.47 × 10−6, 2.19 × 10−6, 5.71 × 10−6, and 8.99 × 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. Environ. Mol. Mutagen. 2010. Published 2009 Wiley-Liss, Inc.

A modified allele-specific competitive blocker PCR (ACB-PCR) has been developed as an approach for genotypic selection, the detection of a rare mutant allele based solely upon its altered nucleotide sequence. ACB-PCR genotypic selection operates through the preferential PCR amplification of mutant DNA using a primer that has more mismatches to the wild-type allele than the mutant allele. In addition, a blocker-primer with a 3'-terminal dideoxynucleotide and more mismatches to the mutant allele than the wild-type allele is incorporated to reduce the background and increase sensitivity. Using ACB-PCR, the CAA-->AAA base substitution at codon 61 of the mouse H-ras gene was detected regularly at mutant fractions of 10(-5). To accurately quantify the occurrence of this particular mutation, an internal amplification standard (AS) DNA was constructed. The H-ras and AS DNAs were subject to the same genotypic selection but were amplified using different upstream primers to give PCR products that can be distinguished by size. Defined mixtures of mutant and wild-type AS DNAs were used to study the effects of various components of the ACB-PCR. The concentration of dNTPs, blocker primer and Perfect Match Polymerase Enhancer, as well as the choice of thermostable DNA polymerase and annealing temperature were examined. Conditions were identified for the concurrent detection of the CAA-->AAA mutation in the H-ras and AS DNAs. Using the identified conditions, approximately equal signals were obtained from equivalent amounts of the two DNA templates over a wide range of mutant fractions (1 in 10 to 1 in 10(5)). This ACB-PCR method can be used for any application where it is necessary to quantify relatively small mutant fractions.

Genotypic selection enriches a particular DNA sequence relative to another closely-related DNA sequence based only on a change of one or a few bases. This review is a survey of the genotypic selection methods that have the sensitivity to detect rare point mutations. These methods are primarily being used to study mutations caused by environmental mutagens; however, the ability to detect and measure very minor DNA sequence populations is likely to further research efforts in many fields. The approaches for allele-selection have intrinsic strengths and weaknesses, and vary greatly in sensitivity. The most sensitive method is Restriction Fragment Length Polymorphism/Polymerase Chain Reaction (RFLP/PCR) by which mutant fractions as low as 1 mutant allele in 10(8) wild-type alleles can be detected. The RFLP/PCR approach is presented as a prototype genotypic selection method. Genotypic selection methods are categorized in terms of those that (1) selectively destroy the abundant or wild-type allele, (2) selectively amplify the rare or mutant allele, or (3) spatially separate the alleles. Issues relevant to the further development of genotypic selection methods include initial DNA pool size, strategies to eliminate the bulk of extraneous DNA, the use of an internal copy number standard in quantitative PCR, the fidelity of thermostable DNA polymerases, and the effective use of PCR in linking two or more genotypic selection techniques. We conclude that proficient genotypic selection requires more than one allele-enrichment technique with at least one of these preceding a high-fidelity PCR amplification step.

Maintaining genome integrity is a dynamic process that involves many different cellular pathways, including methylation maintenance, homologous recombination, DNA replication, DNA repair, and cell cycle control. A defect in one or more of these pathways can compromise genome integrity and result in cancer. A number of DNA repair proteins have been identified that also impact the regulation of the cell cycle and/or apoptosis (1, 2). Individuals carrying germ-line mutations in such genes, like MLH1 or BRCA1, are predisposed to develop cancer (2). In this issue of PNAS, Cortellino et al. (3) provide evidence that MED1 belongs to this critical group of multifunction proteins. They show that MED1 impacts three processes necessary for the maintenance of genome integrity: base excision repair (BER), DNA mismatch repair (MMR), and the cell cycle response to DNA damage. Their work adds significant information to the growing body of research on the function of MED1 in maintenance of genome integrity (Fig. 1). Fig. 1. Summary of known MED1 functions and the cellular processes impacted by MED1 functions. The multifunctional nature of MED1, also known as MBD4, was apparent from the time of the molecule's discovery. The molecule was identified by using two different screening methods. The human and mouse MBD4 genes were isolated based on DNA sequence homology to the methyl-binding domain of MeCP2, a human 5-methylcytosine (5-meC) binding protein and transcriptional repressor (4). It was shown that the MBD4 gene product could bind 5-meC both in vitro and in vivo . Thus, the first functional description …

Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.

Regulation of three cDNA clones (pT52, pT53, and pT58) was analyzed in terms of wounding alone and wounding in conjunction with developmental and environmental cues (ripening, ethylene, and light) in tomato fruit tissue. The pT52-specific transcript level is induced by wounding in early-red and red stage fruit and by ethylene. The pT58-specific transcript level is also induced by wounding and ethylene in early-red stage fruit but is not induced by wounding in red fruit. The pT53-specific transcript level is repressed by wounding in early-red and red stage fruit. Like the pT52- and pT58-specific transcripts, the pT53-specific transcript is induced by ethylene. Furthermore, the level of the pT52-specific transcript is regulated by light. Analysis of unwounded tissue showed that the abundance of each cDNA-specific transcript changes during fruit ripening and that each of the transcripts is present in other plant organs as well. This analysis provides information about the interactions between developmental and environmental factors affecting these genes.

The telomeres of orthopoxvirus DNAs consist largely of short repeated sequences organized into at least two separate sets. Although the sequence composition of the orthopoxvirus telomeres is highly conserved, these regions do not appear to encode any proteins. At late times during infection, the telomeres of vaccinia virus are transcribed. A promoter in the region between the two sets of repeats directs transcription towards the hairpin-loop end of the viral DNA. This promoter resembles the promoters of other poxvirus late genes, and directs the synthesis of RNAs whose structure is consistent with the presence of 5′ poly(A) sequences typical of late RNAs. The lengths of these late transcripts suggest that some transcription extends through the hairpin-loop region. This might occur either when the genome is in a monomeric form or when the genome is in the concatemeric form of the DNA replication intermediate. The function of late transcription of the telomeres is unclear, but similar transcription of the telomeres of vaccinia virus, cowpox virus, and raccoonpox virus suggests that such transcription may have a role in viral replication.

The sequence of 15,441 nucleotides from the adenovirus-2 genome has been determined and includes the regions between coordinates 0-32% and 89-100%. These regions contain the early (E) transcription units E1A, E1B, E2B, and E4, the genes for polypeptides IVa2 and IX, the COOH terminus of fiber polypeptide, as well as the two virus-associated RNAs and the leader sequences for the major late mRNAs. Analysis of tryptic peptides from the terminal protein and its precursor (Smart, J. E., and Stillman, B. W. (1982) J. Biol. Chem. 257, 13499-13506) has allowed the gene for the precursor terminal protein to be positioned between coordinates 28.0 and 23.5 on the 1-strand. A minimum Mr = 74,500 is predicted. A second, longer open reading frame is also found on the 1-strand between coordinates 22.9 and 14.2 and predicts a polypeptide of at least Mr = 120,000. Many open reading frames longer than 10,000 exist within this sequence although less than half of them can be assigned to previously characterized polypeptides. As with other viral genomes, the available coding information is highly compressed. Intergenic distances are very short and examples are found of genes which overlap either on the same strand or the complementary strand.

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.