Search

KRAS mutations in colorectal and lung cancers predict failure to respond to therapies that target the EGFR. Significant percentages of patients with KRAS wild-type tumors also fail to respond to these therapies. Relapse occurs in patients with KRAS wild-type and mutant tumors, with moderately longer progression-free survival in patients with KRAS wild-type tumors. Colon and lung tumors frequently carry KRAS mutant tumor subpopulations not detected by DNA sequencing. This suggests detected and undetected KRAS mutant subpopulations in colon and lung tumors are undermining the efficacy of anti-EGFR therapies. Therefore, consideration should be given to combining therapies that target KRAS mutant cells with those that downregulate EGFR signaling. As tumors are frequently polyclonal in origin and comprised of distinct clonal populations carrying complementing genetic and/or epigenetic lesions, preclinical models that assess the efficacy of combination therapies in the context of heterogeneous tumor cell populations will be essential for progress in this area.

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 patient's 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, KRAS PIK3CA 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.

Cancer driver mutations (CDMs) are being developed as theranostic biomarkers. This study used high-sensitivity quantification by ACB-PCR, with a sensitivity of 10−5, to elucidate the tissue-specific properties of CDMs. We employed the paradigm of comparing levels of specific hotspot CDMs in normal human tissues and tumors. Specifically, we measured KRAS G12D, KRAS G12V, PIK3CA E545K, and PIK3CA H1047R mutant fractions (MFs) in normal breast, colon, lung, and thyroid and quantified the same mutations in mammary ductal carcinomas (DCs), colonic adenocarcinomas, lung adenocarcinomas, and papillary thyroid carcinomas. Three major findings were: 1) these mutations occur in normal human tissues at relatively high frequencies, 2) there is considerable variability in CDM MFs in normal tissues across individuals, and 3) tumors frequently carry CDMs at levels greater than that present in normal tissue, but below that detected by standard DNA sequencing. Understanding the prevalence of mutant subpopulations is important because they can drive resistance to molecularly-targeted therapies. The PIK3CA H1047R profile for normal breast and DC were similar, with large mutant subpopulations present in both tissue types. Because the PIK3CA H1047R mutation is the most prevalent point mutation reported in breast cancer, we conclude that it can drive breast carcinogenesis as a subpopulation, potentially through a paracrine mechanism, and may be a useful biomarker of breast cancer susceptibility. Other properties of CDMs elucidated were gender differences, the accumulation of mutation with donor age, and the correlation between MF and maximum tumor dimension (MTD). Male lung had significantly greater PIK3CA H1047R MFs than female lung. The PIK3CA H1047R MF in normal breast was positively correlated with donor age, and the mutation in DCs was positively correlated with MTD. By contrast, KRAS MFs showed non-significant decreases in normal lung with increasing donor age. For colorectal adenocarcinomas, KRAS G12V MF was negatively correlated with MTD, consistent with previous findings that KRAS G12V MF decreases during adenoma to adenocarcinoma progression. This suggests that the selective advantage provided by KRAS G12V mutation is context-dependent and can be either positive or negative. Across the four mutational targets and four tissue types, a significant positive correlation was observed between the variability (Log10 standard deviation) of MF measured in normal tissues and the prevalence with which the mutation reportedly occurs in tumors, as per The Cancer Genome Atlas database. This suggests that level and inter-individual variation in CDMs can be used to identify promising tissue-specific, mutational biomarkers of cancer susceptibility. This is not a formal dissemination of information by FDA and does not represent agency position or policy. Citation Format: Barbara L. Parsons, Meagan B. Myers, Karen L. McKim, Malathi Banda. Characterization of the tissue-specific properties of cancer driver mutations suggests spontaneous mutation in normal tissues drives tumor susceptibility. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5147.

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.

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

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

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

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.

The p53 codon 270 CGT to TGT mutation was investigated as a biomarker of sunlight-induced mutagenesis and carcinogenesis. The relationship between tumor development and abundance of this hotspot mutation was analyzed in mouse skin tumors induced by chronic exposure to simulated solar light (SSL). The 24 tumors analyzed had similar growth kinetics, with an average doubling time of approximately 16.4 d. Levels of the p53 codon 270 mutation were quantified in the 24 mouse skin tumors using allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). All tumors contained measurable amounts of the mutation. The p53 codon 270 CGT to TGT mutant fraction (MF) ranged from 2.29 x 10(-3) to 9.42 x 10(-2), with 3.26 x 10(-2) as the median. These p53 MF measurements are lower than expected for an initiating mutation involved in the development of tumors of monoclonal origin. There was no evidence of a correlation between p53 codon 270 MF and either tumor area or an estimate of tumor cell number. Thus, the data do not support the idea that p53 mutation accumulates linearly during tumor development. To investigate how p53 mutation was distributed within tumors, 19 needle biopsies from seven different tumors were analyzed by ACB-PCR. This analysis demonstrated that p53 codon 270 mutation is heterogeneously distributed within tumors. The long-term goal of this research is to combine morphological and p53 MF measurements from tissues corresponding to the various stages of tumor development, in order to derive mathematical models relating the p53 codon 270 mutation to the development of SSL-induced skin tumors.

Little is known about the role of low-frequency somatic mutations in breast cancer, a cancer that is quite variable in terms of clinical features, prognosis and response to treatment. Because undetected mutant subpopulations have the potential to cause resistance to molecularly-targeted cancer therapies, this study investigated whether different subtypes of ductal carcinomas (DCs) encompass subpopulations of cells with cancer-driver mutations known to impact response to targeted therapies. Using the sensitive and quantitative ACB-PCR approach PIK3CA H1047R and BRAF V600E point mutations were quantified in 82 DCs, consisting of ER/PR+/HER2- (n = 20), ER/PR+/HER2+ (n = 20), ER/PR-/HER2+ (n = 21), and ER/PR-/HER2- (TNBC; n = 21) subtypes. Considering all subtypes together, the geometric mean mutant fraction (MF) measurements [provided along with (5th to 95th percentiles)] were: PIK3CA H1047R, 6.8 × 10−6 (2.5 × 10−8 to 6.3 × 10−2) and BRAF V600E, 1.9 × 10−5 (5.1 × 10−6 to 1.6 × 10−4). The PIK3CA H1047R and BRAF V600E mutations were detected (i.e., had MFs > 10−5) in 41% and 37% of DCs, respectively. In addition, normal breast tissues were analyzed by ACB-PCR to define the background levels of these mutations. For normal breast (n = 10), the geometric mean MF measurements [provided along with (5th to 95th percentiles)] were: PIK3CA H1047R, 8.0 × 10−4 (1.6 × 10−5 to 3.8 × 10−2) and BRAF V600E, 2.0 × 10−6 (9.7 × 10−7 to 1.7 × 10−4). DC subtype analyses revealed ER/PR+/HER2- and TNBC subtypes had significantly greater BRAF V600E MFs relative to that measured in normal breast. The ACB-PCR data indicated 15% of DCs carry BRAF V600E mutation at a level greater than the upper 95% confidence interval for the mutation in normal breast. Interestingly, for PIK3CA H1047R mutation, ER/PR+/HER2-, ER/PR+/HER2+ and TNBC subtypes had significantly less PIK3CA H1047R mutation relative to that measured in normal breast. We hypothesize that pre-existing PIK3CA mutation can cooperate with other clones to drive tumor initiation and early progression, but is lost or outgrown by clones carrying other cancer driver mutations in advanced breast cancers. Data from the COSMIC database supports this idea that the level of PIK3CA mutation decreases as disease progression ensues, with PIK3CA mutation detected in 47% of hyperplasia, 29% of DCIS, and 21% in ductal carcinomas. Furthermore, TNBCs had significantly less PIK3CA H1047R mutation relative to that measured in each of the other subtypes, suggesting individuals with these tumors may have a lower response to inhibitors of the PI3K/AKT/mTOR pathway. Analyses of other cancer driver hotspot point mutations in the different DC subtypes is ongoing and will provide additional information regarding the role of low-frequency somatic mutations in breast cancer. Citation Format: Meagan B. Myers, Malathi Banda, Karen L. McKim, Barbara L. Parsons. Subclonal structure of breast cancer subtypes determined by quantitative analyses of activating mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1119. doi:10.1158/1538-7445.AM2015-1119

Methods that detect rare base substitutions within populations of DNA molecules are valuable tools for studying the DNA-damaging effects of chemicals and for pool screening for single-nucleotide polymorphisms. Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) uses a mutant-specific PCR primer with more 3'-terminal mismatches to an abundant or wild-type sequence than to a rare or mutant sequence in order to amplify specifically an allele that differs from the wild-type by a single base pair. ACB-PCR reactions include a blocker primer to reduce the amount of background signal generated from the abundant wild-type template. The nonextendable blocker primer preferentially anneals to the wild-type DNA sequence, thereby excluding the annealing of the extendable mutant-specific primer to the wild-type sequence. Inclusion of single-strand DNA binding protein in the ACB-PCR reaction and use of the Stoffel fragment of Taq DNA polymerase both significantly increase allele discrimination. The concurrent analysis of mutant fraction standards and equivalent PCR products amplified from genomic DNA samples makes ACB-PCR a quantitative method that can detect a base pair substitution in the presence of a 105-fold excess of wild-type DNA. Methods for the ACB-PCR measurement of the mouse H-ras codon 61 CAA --AAA mutation are presented.

Methods that detect rare base substitutions within populations of DNA molecules are valuable tools for studying the DNA-damaging effects of chemicals and for pool screening for single-nucleotide polymorphisms. Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) uses a mutant-specific PCR primer with more 3'-terminal mismatches to an abundant or wild-type sequence than to a rare or mutant sequence in order to amplify specifically an allele that differs from the wild-type by a single base pair. ACB-PCR reactions include a blocker primer to reduce the amount of background signal generated from the abundant wild-type template. The nonextendable blocker primer preferentially anneals to the wild-type DNA sequence, thereby excluding the annealing of the extendable mutant-specific primer to the wild-type sequence. Inclusion of single-strand DNA binding protein in the ACB-PCR reaction and use of the Stoffel fragment of Taq DNA polymerase both significantly increase allele discrimination. The concurrent analysis of mutant fraction standards and equivalent PCR products amplified from genomic DNA samples makes ACB-PCR a quantitative method that can detect a base pair substitution in the presence of a 105-fold excess of wild-type DNA. Methods for the ACB-PCR measurement of the mouse H-ras codon 61 CAA --> AAA mutation are presented.

KRAS tumor mutational status predicts response to therapies that target the epidermal growth factor receptor (EGFR) and is being used to make treatment decisions for individual colon cancer patients. There is evidence showing that undetected KRAS mutant tumor subpopulations can subvert durable responses, lead to drug resistance, and drive relapse in colon cancer patients treated with EGFR-targeted therapies. Patients with adenocarcinomas of the lung are treated with EGFR thymidine kinase inhibitors and, theoretically, the efficacy of these treatments could be influenced by KRAS mutational status, even though KRAS mutational status is not currently evaluated prior to treatment of lung cancer patients. In order to assess the potential for undetected KRAS mutant subpopulations to impact response in lung adenocarcinoma patients treated with EGFR thymidine kinase inhibitors, the sensitive allele-specific competitive blocker polymerase chain reaction (ACB-PCR) method was used to quantify KRAS codon 12 GGT to GAT and GGT to GTT mutant fraction (MF) in 19 normal lung samples and 21 primary lung adenocarcinomas. ACB-PCR is a type of allele-specific amplification that can measure 3 mutant alleles in a background of 300,000 normal alleles and gives a quantitative readout, rather than reporting the presence/absence of mutation. ACB-PCR was used to quantify KRAS mutation in normal lung and lung tumor tissue in order to empirically define pathological levels of KRAS mutation as those above the upper 95% confidence interval of that present in normal lung. Measurable levels (≥10-5) of KRAS codon 12 GTT and/or GAT mutations were observed in 17/19 (89%) of normal lung samples. The KRAS codon 12 GAT and GTT geometric mean MFs in normal lung were 3.25 x 10-5 and 3.15 x 10-5, respectively. Normal colonic mucosa and normal lung tissue contain similar levels of KRAS codon 12 GAT mutation, however normal lung tissue has a significantly higher level of the KRAS codon 12 GTT mutation than colonic mucosa. All lung adenocarcinomas had measurable levels of both mutations. Adenocarcinomas from male smokers had significantly greater KRAS codon 12 GTT MFs than adenocarcinomas from female smokers. The highest observed KRAS codon 12 GAT MFs were associated with tumors characterized as having relatively low necrosis and tumor cell content. Importantly, 47.6% and 57.1% of lung adenocarcinomas had KRAS codon 12 GAT and GTT MFs greater than the upper 95% confidence interval for MF in normal lung (1.02 x 10-4 and 9.16 x 10-5, respectively). In addition, 28% of lung adenocarcinomas had abnormally-high levels of both mutations. Extrapolation of these results to include additional KRAS hotspot point mutations leads to the conclusion that most, if not all, lung adenocarcinomas possess greater KRAS mutant cell content than normal lung tissue. These results suggest there is a strong possibility that treatment of lung adenocarcinoma patients with therapies that target EGFR may select for the outgrowth of KRAS mutant subpopulations and contribute to the development of drug resistance and relapse. Citation Format: Meagan B. Myers, Karen L. McKim, Fanxue Meng, Barbara L. Parsons. High sensitivity characterization of KRAS mutant subpopulations in normal lung tissue and adenocarcinomas of the lung. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B56. doi: 10.1158/1557-3125.RASONC14-B56

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

Tumors are generally thought to be monoclonal in origin, although this paradigm stemmed decades ago mostly from the study of hematopoietic malignancies and sarcomas. The clonal origin of malignant mesothelioma, a deadly cancer resistant to the current therapies, has never been investigated before. We used the HUMARA (human androgen receptor) assay to examine the pattern of clonality in 14 sporadic and 2 familial Malignant Mesotheliomas (MM). Of 16 specimens studied, 14 were informative and nearly all (13/14) revealed two electrophoretically distinct methylated HUMARA alleles, indicating a polyclonal origin for these tumors. This discovery has important clinical implications, in that an accurate assessment of tumor clonality is key to the design of novel molecular strategies for the treatment of MM. Citation Format: Sabahattin Comertpay, Rosanna Mezzapelle, Mika Tanji, Oriana Strianese, Harvey I. Pass, Tracey Weigel, Joseph Friedberg, Paul Sugarbaker, Thomas Krausz, Ena Wang, Giovanni Gaudino, Haining Yang, Amy Powers, Barbara Parsons, Sandra Pastorino, Michele Carbone. Evaluation of clonal origin of malignant mesothelioma/polyclonal origin of malignant mesothelioma. [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 3188. doi:10.1158/1538-7445.AM2014-3188

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 …

The deduced protein sequence of a partial tomato cDNA clone is rich in the amino acid glycine and contains repeats of the amino acid sequence, (Gly)5Tyr(Gly)4-5Tyr(Gly)3ArgArgGlu. This protein sequence has significant similarity to a sorghum glycine-rich protein [S1, Cretin and Puigdomenech (1990) Plant Mol. Biol. 15: 783] and a maize embryo, abscisic acid-induced glycine-rich protein [Gomez et al. (1988) Nature 334: 262]. Tissue printing was used to localize the glycine-rich protein transcript in tomato fruit, stem and hypocotyl sections. The transcript is present throughout the tomato fruit pericarp but is enriched in the vascular bundles. In tomato hypocotyl tissue prints, the glycine-rich protein transcript as well as rRNA were localized within the vascular tissue. This shows that differential loading of RNA may occur when using the tissue printing technique on hypocotyl sections. Direct isolation and comparison of RNA from vascular-containing and non-vascular stem tissue confirmed, however, that the glycine-rich protein transcript is accumulated abundantly in the vascular tissue of tomato stem.

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 >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

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.