A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC

The most sensitive screening technique for genes that predispose patients for particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested alternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined mutation sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assay with a newly developed technique called denaturing high performance liquid chromatography (DHPLC) to screen the BRCA1gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BRCA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation analysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. Polymorphisms that were present in our samples could be predicted by means of DHPLC testing independently of sequence analysis. We conclude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated. Electronic Publication     

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozygosity in locally advanced laryngeal squamous cell carcinoma

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.     

Clinical implication of p53 mutation in lung cancer

Lung cancer development involves multiple genetic abnormalities leading to malignant transformation of the bronchial epithelial cells, followed by invasion and metastasis. One of the most common changes is mutation of the p53 tumor suppressor gene. The frequency of p53 alterations in lung cancer is highest in small cell and squamous cell carcinomas. A genetic “signature” of the type of p53 mutations has been associated with carcinogens in cigarette smoke. The majority of clinical studies suggest that lung cancers with p53 alterations carry a worse prognosis, and may be relatively more resistant to chemotherapy and radiation. An understanding of the role of p53 in human lung cancer may lead to more rational targeted approaches for treating this disease. P53 gene replacement is currently under clinical investigation but clearly more effective means of gene deliver to the tumor cells are required. Novel approaches to lung cancer therapy are needed to improve the observed poor patient survival despite current therapies.     

Clinical Implications of Rabphillin-3A-Like Gene Alterations in Breast Cancer

For the rabphillin-3A-like (RPH3AL) gene, a putative tumor suppressor, the clinical significance of genetic alterations in breast cancers was evaluated. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) cancers and matching normal tissues. DNA samples were assessed for loss of heterozygosity (LOH) at the 17p13.3 locus of RPH3AL and the 17p13.1 locus of the tumor suppressor, TP53. RPH3AL was sequenced, and single nucleotide polymorphisms (SNPs) were genotyped. RNA samples were evaluated for expression of RPH3AL, and FFPE tissues were profiled for its phenotypic expression. Alterations in RPH3AL were correlated with clinicopathological features, LOH of TP53, and patient survival. Of 121 cancers, 80 had LOH at one of the RPH3AL locus. LOH of RHP3AL was associated with nodal metastasis, advanced stage, large tumor size, and poor survival. Although ~50% were positive for LOH at the RPH3AL and TP53 loci, 19 of 105 exhibited LOH only at the RPH3AL locus. Of these, 12 were non-Hispanic Caucasians (Whites), 15 had large tumors, and 12 were older (>50 years). Patients exhibiting LOH at both loci had shorter survival than those without LOH at these loci (log-rank, P = 0.014). LOH at the TP53 locus alone was not associated with survival. Analyses of RPH3AL identified missense point mutations in 19 of 125 cases, a SNP (C>A) in the 5’untranslated region at -25 (5’UTR-25) in 26 of 104, and a SNP (G>T) in the intronic region at 43 bp downstream to exon-6 (intron-6-43) in 79 of 118. Genotype C/A or A/A of the SNP at 5’UTR-25 and genotype T/T of a SNP at intron-6-43 were predominantly in Whites. Low levels of RNA and protein expression of RPH3AL were present in cancers relative to normal tissues. Thus, genetic alterations in RPH3AL are associated with aggressive behavior of breast cancers and with short survival of patients.     

BRCA1/2 mutation screening and LOH analysis of lung adenocarcinoma tissue in a multiple-cancer patient with a strong family history of breast cancer

BACKGROUND: Germline mutations in BRCA1/2 greatly elevate risks of breast and ovarian cancers, but the role of these genes in tumourigenesis of other cancer types is still being investigated. OBJECTIVE: We report on an investigation of BRCA1/2 mutations and their loss of heterozygosity (LOH) in a patient with a strong family history of breast cancer who was diagnosed with consecutive primary cervical, ovarian and lung carcinomas. METHODS AND RESULTS: BRCA1/2 mutation screening of the proband revealed a common familial breast- and ovarian cancer-associated germline BRCA2 mutation (3034del4bp). We then performed LOH analysis for BRCA2 in lung adenocarcinoma tissue of the patient. Using the laser-capture microdissection (LCM) technique, we obtained pure populations of neoplastic cells from which DNA could be extracted. Mutation analysis by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing revealed loss of the mutant allele in the adenocarcinoma tumour tissue. CONCLUSION: To our knowledge, this is the first report of investigation for LOH for BRCA2 in primary lung adenocarcinoma tissue of a patient with multiple primary tumours related to a familial germline BRCA2 mutation. Interestingly, it was the mutant, not the wild-type, allele which was lost in the lung adenocarcinoma tissue.     

Genome wide DNA copy number analysis of serous type ovarian carcinomas identifies genetic markers predictive of clinical outcome

Ovarian cancer is the fifth leading cause of cancer death in women. Ovarian cancers display a high degree of complex genetic alterations involving many oncogenes and tumor suppressor genes. Analysis of the association between genetic alterations and clinical endpoints such as survival will lead to improved patient management via genetic stratification of patients into clinically relevant subgroups. In this study, we aim to define subgroups of high-grade serous ovarian carcinomas that differ with respect to prognosis and overall survival. Genome-wide DNA copy number alterations (CNAs) were measured in 72 clinically annotated, high-grade serous tumors using high-resolution oligonucleotide arrays. Two clinically annotated, independent cohorts were used for validation. Unsupervised hierarchical clustering of copy number data derived from the 72 patient cohort resulted in two clusters with significant difference in progression free survival (PFS) and a marginal difference in overall survival (OS). GISTIC analysis of the two clusters identified altered regions unique to each cluster. Supervised clustering of two independent large cohorts of high-grade serous tumors using the classification scheme derived from the two initial clusters validated our results and identified 8 genomic regions that are distinctly different among the subgroups. These 8 regions map to 8p21.3, 8p23.2, 12p12.1, 17p11.2, 17p12, 19q12, 20q11.21 and 20q13.12; and harbor potential oncogenes and tumor suppressor genes that are likely to be involved in the pathogenesis of ovarian carcinoma. We have identified a set of genetic alterations that could be used for stratification of high-grade serous tumors into clinically relevant treatment subgroups.     

Oncogene and tumor suppressor gene alterations in nasal tumors

The development of nasal tumors in humans and rodents is likely mediated through the accumulation of genetic alterations in genes that regulate cell proliferation, cell death and differentiation (oncogenes and tumor suppressor genes). By examination of the relationship between genetic alterations that are known to occur in human cancers with those induced in rodent tumors with defined carcinogenic exposures, biologically relevant mechanistic linkages of molecular events leading to tumors in rodents and humans can be established. Molecular genetic studies on nasal squamous cell carcinomas (SCC) in rats thus far have indicated the presence of oncogenes unrelated to the ras oncogene family and that p53 mutation occurs at a high frequency among the nasal SCC examined. The finding of p53 mutations in rat nasal SCC and the high prevalence of p53 mutations among human SCC, indicates that a common molecular alteration is shared between rodent and human SCC.     

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines

Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.     

Loss of heterozygosity and K-ras gene mutations in gastric cancer

In order to identify relevant genetic lesions in gastric carcinoma, we searched for tumor suppressor gene inactivation and K-ras gene mutations by analyzing tumor and control DNAs from 34 patients. These were from an epidemiologically defined area of Italy characterized by one of the world's highest incidences of stomach cancer. Allele losses were investigated by the Southern blotting procedure at 16 polymorphic loci on 11 different chromosomes. Our data demonstrate that chromosomal regions 5q, 11p, 17p and 18q are frequently deleted, and that 7q and 13q chromosome arms are also involved, although at a lower frequency. Loss of heterozygosity (LOH) at region 11p was not found during other surveys carried out on patients of different geographic origins. No specific combination of allelic losses could be recognized in the samples analyzed, the only exception being that tumors with 17p allelic loss also showed LOH on the 18q region. When matching frequent LOH events and the stage of progression of the tumors, we observed a trend of association between advanced stages and allelic losses on 17p and 18q chromosome arms. The analysis of K-ras, carried out by the polymerase chain reaction and denaturing gradient gel electrophoresis, demonstrated transforming mutations in only 3 out of 32 cases. Colorectal tumorigenesis proceeds by the accumulation of genetic alterations, including K-ras mutations and inactivation of tumor suppressor genes on the 5q, 17p and 18q regions. Our data indicate that, although gastric and colorectal neoplasias share common genetic alterations, they probably progress through different pathways.     

Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene

BACKGROUND: Alterations in the p53 tumor suppressor gene constitute one of the most frequent genetic events associated with the development of human cancers. Determination of an individual's p53 status may be of value in early diagnosis, prediction of response to treatment, and for the detection of minimal residual cancer. Recent studies have also revealed that specific mutations affecting the p53 gene are associated with a poor outcome. The majority of tumor biopsies that are sent for study in the laboratory contain neoplastic cells intermingled with stroma, such that the detection of alterations in the p53 gene requires a tumor enrichment technique and/or highly sensitive mutation detection technologies. Thus, it is desirable that a clinically useful assay for detecting point mutations in the p53 gene function in the presence of significant quantities of wild-type sequence and identify the critical sequence aberrations. MATERIALS AND METHODS: We utilized molecular beacons in a real-time allele-specific PCR format to obtain reference data on samples of quantitatively known p53 mutation status. These data have been statistically analyzed and the results used to detect p53 mutations, indicating the presence of occult tumor. RESULTS: We describe validation of a simple, rapid, sensitive, and quantitative ARMS assay for identifying the levels of 80 point mutations within the p53 gene that, when mutated, constitute at least 1% of the total p53 sequences. CONCLUSIONS: The assay successfully identifies rare p53 gene mutations in clinical samples and overcomes many of the limitations of current technologies.     

Gain of Function of p53 Cancer Mutants in Disrupting Critical DNA Damage Response Pathways

Loss of the tumor suppression activity of p53 is required for the progression of most human cancers. In this context, p53 gene is somatically mutated in about half of all human cancers; in the rest human cancers, p53 is mostly inactivated due to the disruption of pathways important for its activation. Most p53 cancer mutations are missense mutations within the core domain, leading to the expression of full-length mutant p53 protein. The expression of p53 mutants is usually correlated with the poor prognosis of the cancer patients. Accumulating evidence has indicated that p53 cancer mutants not only lose the tumor suppression activity of WT p53, but also gain novel oncogenic activities to promote tumorigenesis and drug resistance. Therefore, to improve current cancer therapy, it is critical to elucidate the gain-of-functions of p53 cancer mutants. By analyzing the humanized p53 mutant knock-in mouse models, we have identified a new gain of function of the common p53 cancer mutants in inducing genetic instability by disrupting ATM-mediated cellular responses to DNA double-stranded break (DSB) damage. Considering that some current cancer therapies such as radiotherapy kills the cancer cells by inducing DSBs in their genome DNA, our findings will have important implications on the treatment of human cancers that express common p53 mutants.     

ND4 mutation in transitional cell carcinoma: Does mitochondrial mutation occur before tumorigenesis?

To investigate how mitochondrial mutation occurs in cancers, we analyzed ND4 mutation in 53 transitional cell carcinomas (TCCs) of the upper urinary tract and the normal counterpart (perirenal soft tissue). Three methods, i.e., DNA sequencing, restriction fragment length polymorphism (RFLP), and denaturing high-performance liquid chromatography (DHPLC), were employed because of their different sensitive of detecting mutation. The results of sequencing and RFLP showed that ND4 mutations were only found in 24.5% (13/53) of tumor. However, 11 of these mutations could also be identified in the normal tissue by DHPLC, indicating that most mitochondrial mutations identified in tumors preexist as minor components, which are too low in quantity to be detected by less sensitive methods such as DNA sequencing. The result suggests that mtDNA mutation occurs before tumorigenesis and become apparent in cancer cells.     

Evaluation of arrayed primer extension for TP53 mutation detection in breast and ovarian carcinomas

Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2-9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.     

Combined p53/Bax mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability

Gastric cancer is highly refractory to DNA-damaging therapies. We therefore studied both gene mutation and protein expression of p53 and Bax in a cohort of 116 patients with gastric cancer who underwent R0-resection with a curative intent. Bax mutation was independent from severe microsatellite instability (MSI), that is, global mismatch repair deficiency as determined by analysis of BAT-25/BAT-26 microsatellite markers. Thus, Bax-frameshift mutation is a feature of tumors with low MSI. In contrast and as expected, no p53 mutations were observed in the microsatellite instable tumors. p53 Mutation or p53 overexpression did not have an impact on disease prognosis. p53-Inactivation was, however, associated with an extremely poor prognosis in the subgroup of patients with Bax-mutated tumors. Thus, we show for the first time that the combined mutation of p53 and Bax, two key regulators of the mitochondrial apoptosis pathway, results in an extremely aggressive tumor biology and poor clinical prognosis.     

brca2 and tp53 Collaborate in Tumorigenesis in Zebrafish

Germline mutations in the tumor suppressor genes BRCA2 and TP53 significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for BRCA2 or TP53 often display loss of the wildtype allele. In addition, BRCA2-associated cancers often exhibit mutations in TP53. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for BRCA2 and TP53 in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous tp53^M214K background had a high incidence of malignant tumors. The brca2^Q658X mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for tp53 occurred in the majority of malignant tumors from brca2 wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both brca2 and tp53 frequently displayed LOH for both genes. In contrast, LOH for tp53 was uncommon in malignant tumors from brca2 homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in tp53 and brca2 typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable BRCA2 and TP53 mutations.     

APC mutations and other genetic and epigenetic changes in colon cancer

Relationships between adenomatous polyposis coli (APC) mutations, BRAF V600E mutations, and the CpG island methylator phenotype (CIMP) in colon cancer have not been explored. In addition, controversies exist about the proportion of tumors with APC mutations in the mutation cluster region (MCR); how commonly APC, Ki-ras, and p53 mutations occur in the same tumor; and whether APC mutations occur in sporadic microsatellite-unstable tumors. The APC gene was therefore sequenced in 90 colonic adenocarcinomas previously evaluated for CIMP, microsatellite instability, BRAF, Ki-ras, and p53. APC mutations were inversely related to BRAF mutations (P = 0.0003) and CIMP (P = 0.02) and directly related to p53 and Ki-ras mutations (P = 0.04). Slightly more than half of APC mutations occurred outside of the MCR, and frameshift mutations were more likely than nonsense mutations to occur in the MCR (21 of 28 versus 12 of 40, P = 0.0003). APC mutations were found in sporadic microsatellite-unstable tumors and were more likely to be frameshifts in short nucleotide repeats (P = 0.007). The occurrence of APC, Ki-ras, and p53 mutations together in the same tumor was uncommon (11.1%). In conclusion, an analysis restricted to the MCR will miss more than half of APC mutations as well as mischaracterize their mutational spectrum. The conventional wisdom that most colon cancers contain APC, Ki-ras, and p53 mutations is incorrect. Microsatellite instability may precede acquisition of APC mutations in sporadic microsatellite-unstable tumors. The relationships of APC mutations to other genetic and epigenetic alterations add to the already impressive genetic heterogeneity of colon cancer.     

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.     

Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC)

Spontaneous renal cell carcinoma (RCC) occurs with a high frequency in Eker rats carrying a germline alteration of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene. To determine the frequency with which the wild-type allele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect loss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-embedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(Ek/+)) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitation of peak areas of PCR products specific for the mutant and wild-type Tsc-2 alleles. For normal DNA isolated from heterozygous animals, the allele ratio (AR) of mutant to wild-type PCR products was empirically determined to be 1.5+/-0.3 (n=30) and LOH was defined as >2 standard deviations away from this mean, i.e. any AR >2.1. Analysis of 15 spontaneous frozen RCC samples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibited an even higher frequency of LOH, with 6/6 paraffin-embedded, formalin-fixed tumors exhibiting LOH. 100% concordance was observed between the results obtained by DHPLC and traditional methodologies. Therefore, LOH appears to occur with a high frequency in both spontaneous and carcinogen-induced RCC in this animal model and DHPLC is a sensitive and high throughput methodology for detecting this type of genetic alteration.     

TP53 codon 72 and intron 3 polymorphisms and mutational status in gastric cancer: an association with tumor onset and prognosis

Although TP53 alterations have been studied in human tumors, data considering the role of two common TP53 polymorphisms (Pro72Arg in codon 72 and Ins16bp in intron 3) and their associations with TP53 mutations in gastric cancer are very limited. Thus, we analyzed these parameters taking into consideration the clinicopathological data. DNA from 106 gastric tumor samples was available for TP53 Pro72Arg and TP53 Ins16bp polymorphism genotyping by PCR-RFLP and PCR, respectively. The mutational status of the TP53 exons 5-7 was assessed by the single-strand conformational polymorphism test. The TP53 72ArgArg genotype was statistically associated with patients aged ≥65 years (p = 0.039), and the intron 3 A2A2 genotype was correlated with late-stage tumors (III and IV; p = 0.043). Considering both polymorphisms, a negative correlation between the TP53 Pro-A1 haplotype and age <65 years (r = -0.211; p = 0.030) was found. Taking into account the TP53 mutations, the Pro/Pro genotype was positively correlated with the presence of exon 7 mutations (p = 0.049), and a correlation between this genotype and the number of mutations in TP53 was observed (p = 0.019). This study corroborates the understanding of TP53 polymorphisms in gastric carcinogenesis, especially regarding the genetic features in tumor onset and prognosis.