Tagging single-nucleotide polymorphisms in candidate oncogenes and susceptibility to ovarian cancer.

Low-moderate risk alleles that are relatively common in the population may explain a significant proportion of the excess familial risk of ovarian cancer (OC) not attributed to highly penetrant genes. In this study, we evaluated the risks of OC associated with common germline variants in five oncogenes (BRAF, ERBB2, KRAS, NMI and PIK3CA) known to be involved in OC development. Thirty-four tagging SNPs in these genes were genotyped in approximately 1800 invasive OC cases and 3000 controls from population-based studies in Denmark, the United Kingdom and the United States. We found no evidence of disease association for SNPs in BRAF, KRAS, ERBB2 and PIK3CA when OC was considered as a single disease phenotype; but after stratification by histological subtype, we found borderline evidence of association for SNPs in KRAS and BRAF with mucinous OC and in ERBB2 and PIK3CA with endometrioid OC. For NMI, we identified a SNP (rs11683487) that was associated with a decreased risk of OC (unadjusted P(dominant)=0.004). We then genotyped rs11683487 in another 1097 cases and 1792 controls from an additional three case-control studies from the United States. The combined odds ratio was 0.89 (95% confidence interval (CI): 0.80-0.99) and remained statistically significant (P(dominant)=0.032). We also identified two haplotypes in ERBB2 associated with an increased OC risk (P(global)=0.034) and a haplotype in BRAF that had a protective effect (P(global)=0.005). In conclusion, these data provide borderline evidence of association for common allelic variation in the NMI with risk of epithelial OC.

Validating genetic risk associations for ovarian cancer through the international Ovarian Cancer Association Consortium.

The search for genetic variants associated with ovarian cancer risk has focused on pathways including sex steroid hormones, DNA repair, and cell cycle control. The Ovarian Cancer Association Consortium (OCAC) identified 10 single-nucleotide polymorphisms (SNPs) in genes in these pathways, which had been genotyped by Consortium members and a pooled analysis of these data was conducted. Three of the 10 SNPs showed evidence of an association with ovarian cancer at P< or =0.10 in a log-additive model: rs2740574 in CYP3A4 (P=0.011), rs1805386 in LIG4 (P=0.007), and rs3218536 in XRCC2 (P=0.095). Additional genotyping in other OCAC studies was undertaken and only the variant in CYP3A4, rs2740574, continued to show an association in the replication data among homozygous carriers: OR(homozygous(hom))=2.50 (95% CI 0.54-11.57, P=0.24) with 1406 cases and 2827 controls. Overall, in the combined data the odds ratio was 2.81 among carriers of two copies of the minor allele (95% CI 1.20-6.56, P=0.017, p(het) across studies=0.42) with 1969 cases and 3491 controls. There was no association among heterozygous carriers. CYP3A4 encodes a key enzyme in oestrogen metabolism and our finding between rs2740574 and risk of ovarian cancer suggests that this pathway may be involved in ovarian carcinogenesis. Additional follow-up is warranted.

Oral contraceptive use and ovarian cancer risk among carriers of BRCA1 or BRCA2 mutations.

Women with mutations of the genes BRCA1 or BRCA2 are at increased risk of ovarian cancer. Oral contraceptives protect against ovarian cancer in general, but it is not known whether they protect against the disease in carriers of these mutations. We obtained self-reported lifetime histories of oral contraceptive use from 451 women who carried mutations of BRCA1 or BRCA2. We used conditional logistic regression to estimate the odds ratios associated with oral contraceptive use, comparing the histories of 147 women with ovarian cancer (cases) to those of 304 women without ovarian cancer (controls) who were matched to cases on year of birth, country of residence and gene (BRCA1 vs BRCA2). Reference ages for controls had to exceed the ages at diagnosis of their matched cases. After adjusting for parity, the odds-ratio for ovarian cancer associated with use of oral contraceptives for at least 1 year was 0.85 (95 percent confidence interval, 0.53-1.36). The risk decreased by 5% (1-9%) with each year of use (P for trend=0.01). Use for 6 or more years was associated with an odds-ratio of 0.62 (0.35-1.09). These data support the hypothesis that long-term oral contraceptive use reduces the risk of ovarian cancer among women who carry mutations of BRCA1 or BRCA2.

Detection of Mycoplasma ribosomal DNA sequences in ovarian tumors by nested PCR.

OBJECTIVE: The aim of this study was to investigate the reported association between mycoplasma infection and ovarian cancer by screening ovarian tumor tissues for the presence of mycoplasma DNA. METHODS: Forty-six benign and malignant ovarian tumors were obtained from patients undergoing pelvic surgery at a regional cancer center. DNA was isolated from snap-frozen tumor tissues, and commercial nested polymerase chain reaction (PCR) kits were used to detect the presence of 12 species of mycoplasma in tumor DNA samples. PCR products were isolated from ethidium bromide-stained agarose gels, and sequenced with an automated DNA sequencer. Species were identified through nucleotide sequence similarity searches using the National Center for Biotechnology Information BLAST program. RESULTS: Mycoplasma DNA was detected in 6 (13.0%) of the 46 tumor DNA samples. Nucleotide sequence similarity searches of nested PCR products revealed that one Mycoplasma salivarium and five M. arginini DNA sequences were amplified from the ovarian tissues. CONCLUSIONS: Since M. salivarium and M. arginini are frequently encountered laboratory contaminants that do not have a recognized role as human pathogens, our findings do not support an association between human mycoplasma pathogens and ovarian cancer.

Primary ovarian dysgerminoma in a patient with a germline BRCA1 mutation.

Germline mutations in the BRCA1 tumor suppressor gene are associated with increased risk for the development of ovarian cancer. All such cancers thus far reported have been of the epithelial histologic type. We identified an ovarian dysgerminoma in a 16-year-old woman (proband) with a family history of ovarian cancer during a review of histopathologic characteristics of ovarian cancers from women enrolled in the Gilda Radner Familial Ovarian Cancer Registry. Mutation analysis of DNA from this patient's peripheral blood leukocytes revealed a germline BRCA1 mutation (3312insG). The mutation was also present in the mother with breast cancer, a maternal aunt and a distant cousin with ovarian cancer, and a maternal grandfather and an uncle with skin cancer. The development of the proband's dysgerminoma may be unrelated to her germline BRCA1 mutation. Alternatively, such dysgerminomas may be caused by BRCA1 mutations, but occur so infrequently compared with epithelial cancers that they are seldom identified. Analysis of a larger series of ovarian germ cell tumors may resolve this question.

Histopathology of familial ovarian tumors in women from families with and without germline BRCA1 mutations.

Breast cancers from patients with germline BRCA1 mutations show characteristic histopathologic features. However, similar studies of BRCA1-associated ovarian cancers have reported inconsistent findings. Interobserver differences in histopathologic classification are a significant source of variation, and most studies have obtained histopathologic information from pathology reports rather than from review of histopathology slides. We therefore reviewed the histopathology slides and pathology reports to determine histologic type, grade, and stage for cancers of the ovary or peritoneum in 217 women from 126 families enrolled in the Gilda Radner Familial Ovarian Cancer Registry. Peripheral blood DNA from at least 1 affected member of each family was analyzed for BRCA1 mutations, and tumors from BRCA1 mutation-positive families were compared with those from BRCA1-negative families. Of 66 patients from 36 BRCA1-positive families, 64 had ovarian carcinoma, 1 had an ovarian carcinoma in situ, and 1 had a dysgerminoma. Of 151 patients from 90 BRCA1-negative families, 135 had ovarian carcinoma, 10 had ovarian borderline tumors, 3 had ovarian sex cord/stromal tumors, and 3 had primary peritoneal carcinoma. There were fewer grade 1 (P <.001) and stage I (P =.10) cancers in patients from BRCA1-positive families than in patients from BRCA1-negative families. Neither mucinous nor borderline tumors were found in the BRCA1-positive families. Ovarian cancers arising in women from BRCA1-positive families are more likely to be high grade and nonmucinous than cancers arising in women from BRCA1-negative families. The absence of borderline tumors in patients from BRCA1-positive families adds to accumulating evidence that BRCA1 mutations do not play a role in the development of these tumors. HUM PATHOL 31:1420-1424.

Comparative study of ovarian cancer histopathology by registry pathologists and referral pathologists: a study by the Gilda Radner Familial Ovarian Cancer Registry.

OBJECTIVE: The aim of this study was to evaluate whether there is a significant difference in the pathology diagnoses of women in the Gilda Radner Familial Ovarian Cancer Registry between the two expert Registry pathologists and the referral pathologist. Inaccuracies in verification that ovarian cancer did occur in family members could lead to unnecessary prophylactic surgery or genetic testing. METHODS: A retrospective review was performed of (1) site of malignancy; (2) histopathology of malignancy; (3) grade of malignancy; and (4) the presence or absence of malignancy between the Registry and referral pathologists. RESULTS: There was 95.3% complete agreement between the Registry and the referral pathologist on site of origin with a major difference in only 1.0% of the cases. In comparison of histopathology, there was a 61.7% complete agreement, and only 1.0% were considered major differences. There was 68.8% complete agreement in grade of the malignancy, whereas 2.3% were considered major differences. CONCLUSION: When constructing a family pedigree, it is important to obtain pathology reports to confirm the index case diagnosis of the presence or absence of ovarian cancer. However, because of the small percentage of major differences in diagnosis between the two Registry pathologists and the multiple referral pathologists, we believe genetic counselors and treating physicians can rely, in most instances, on the original histopathology report of verification of ovarian cancer without review of the original histopathology slides when recommending surveillance, genetic testing, and/or prophylactic surgery.

Ovarian carcinoma in situ with germline BRCA1 mutation and loss of heterozygosity at BRCA1 and TP53.

BACKGROUND: The two-hit hypothesis for the genesis of cancer predicts that cancer can develop when the wild-type allele of a tumor suppressor gene is lost in an individual with a germline mutation in that gene. Neither loss of heterozygosity (LOH) for BRCA1 nor mutations of the TP53 (also known as p53) gene have been documented prior to invasion in ovarian cancers arising in women with germline BRCA1 mutations. Such documentation is difficult because lesions are rarely identified in ovarian epithelium. We, therefore, looked for LOH at microsatellite polymorphisms linked to the BRCA1 and TP53 tumor suppressor loci in an incidental carcinoma in situ of the ovary removed prophylactically from a woman with a germline BRCA1 mutation. METHODS: By use of laser-capture microdissection, we obtained pure populations of atypical ovarian epithelial cells and normal stromal cells. DNA was extracted, amplified with primers flanking polymorphic microsatellites linked to BRCA1 (D17S855 and D17S579) and TP53 (TP53 and D17S786), and analyzed for LOH at these microsatellites. We also tested for p53 expression in the abnormal epithelium by immunohistochemistry. RESULTS: Both of the markers linked to TP53 showed LOH, as did an intragenic BRCA1-linked marker (D17S855). The other microsatellite marker for BRCA1 was uninformative. Immunohistochemical staining with an antibody to p53 showed strong immunoreactivity confined to the atypical epithelium. CONCLUSIONS: BRCA1, as well as TP53, can undergo LOH prior to stromal invasion in BRCA1-associated ovarian cancer. Strong immunoreactivity for p53 suggests the presence of mutated p53 in these cells as well. These findings suggest that loss of function of these two tumor suppressor genes occurs early in ovarian carcinogenesis in BRCA1 mutation carriers.

Microsatellite instability in ovarian and other pelvic carcinomas.

Twenty-six cases of ovarian carcinoma and six cases of other pelvic neoplasms were analyzed for microsatellite instability (MSI) using frozen specimens, fluorescence technology, and four selected markers (D2S123 on chromosome 2, D18S58 on chromosome 18, BAT26 on chromosome 2, and BAT40 on chromosome 1). This procedure also allowed the detection of loss of heterogeneity (LOH) at the four selected loci. One of the cases of ovarian carcinoma exhibited MSI and this was evident at three loci. Of 44 informative loci, 7 exhibited LOH representing 3 cases of ovarian carcinoma, 3 of 4 cases of primary peritoneal carcinoma, and one case of unknown primary. These data support other findings that MSI is not a frequent occurrence in ovarian cancer; however, LOH is a more frequent event and may be a target for the development of diagnostic/prognostic procedures for ovarian and primary peritoneal carcinoma.

Classification of IVS1-10T-->C as a polymorphism of BRCA1.

Mutations inactivating the tumor suppressor gene BRCA1 may be responsible for disease for up to 80% of familial ovarian cancer cases. In this syndrome, tumorigenesis classically initiates from an inherited mutation in one allele followed by somatic deletion of the normal allele. Sequencing of BRCA1 amplified from genomic DNA of lymphocytes and microdissected ovarian tumor cells of a familial ovarian cancer patient revealed three, rare heterozygous DNA variations (2418delA, 233G-->A, and IVS1-10T-->C) in both tumor and constitutional (lymphocyte) DNA. Thus, both copies of BRCA1 were retained in tumor. Haplotype analysis of the patient and four siblings assigned 2418delA to one copy of BRCA1 and 233G-->A and IVS1-10T-->C to the other. The DNA change, 2418delA, is considered a mutation that inactivated one BRCA1 allele because it caused a frameshift and generation of a premature stop codon, resulting in synthesis of a truncated peptide as evidenced by an in vitro protein truncation test. The DNA variation, 233G-->A, does not result in an amino acid change, and is considered a benign polymorphism. IVS1-10T-->C is a unique BRCA1 change that occurs in the last nucleotide of a consensus sequence for a branch site critical for RNA splicing. Therefore, we investigated whether IVS1-10T-->C deleteriously affected BRCA1 splicing or expression, and thereby inactivated the other BRCA1 allele. Using the technique of reverse transcription-polymerase chain reaction (PCR) with RNA isolated from lymphoid cell lines of the patient and of controls, no evidence was found that IVS1-10TC abnormally disrupted mRNA splicing or caused the absence of BRCA1 mRNA. Thus, IVS1-10T-->C is not harmful to BRCA1 function, and is classified a benign polymorphism. Retention of the normal BRCA1 allele in the tumor with the heterozygous germline BRCA1 mutation, 2418delA, indicated that mutational inactivation of both BRCA1 alleles was not required for tumorigenesis. It is possible that the normal allele may be functionally inactivated by a nonmutational mechanism.

Mutation analysis of BRCA1, TP53, and KRAS2 in ovarian and related pelvic tumors.

Cancer may be viewed as a genetic disease resulting from critical mutations that disrupt normal cell growth. To characterize the involvement of the BRCA1 and TP53 tumor suppressor genes and of the KRAS2 protooncogene in gynecologic cancer, mutation analysis of these genes was conducted in pelvic tumors of 85 patients that included 49 epithelial ovarian carcinoma cases. The 85 pelvic tumors contained 5 tumors with BRCA1 mutations, 33 with TP53 mutations, and 1 with a KRAS2 mutation. Each of the BRCA1 and KRAS2 mutations, and 25 of the TP53 mutations, were in ovarian carcinomas. Four of the BRCA1 mutations were germline and 1 was somatic. The 4 patients with germline BRCA1 mutations had an early age of disease onset (33-48 years) relative to the mean age of onset (58 years) of all 49 ovarian carcinoma patients, and 3 of these 4 patients had a family history of ovarian or breast cancer. None of the 4 tumors with germline BRCA1 mutations had a KRAS2 mutation or a TP53 mutation, despite a 51% frequency of TP53 mutations in the 49 ovarian carcinomas. Three of the 4 tumors with germline BRCA1 mutations retained a wild-type BRCA1 allele. The tumor with the somatic BRCA1 mutation contained a TP53 mutation and had no evidence for wild-type BRCA1 and TP53 alleles. These data suggest that both BRCA1 and TP53 were inactivated in 1 of 49 ovarian carcinomas. Moreover, mutational inactivation of both BRCA1 and TP53 did not occur in 4 tumors with a germline BRCA1 mutation. It has been proposed that tumorigenesis in cells with a heterozygous BRCA1 mutation requires inactivation of the wild-type BRCA1 and TP53 alleles, which results in genomic instability and acquisition of mutations in protooncogenes. Clearly, mutational inactivation of TP53 and the wild-type BRCA1 allele in ovarian tumors with a heterozygous, germline BRCA1 mutation is not an absolute requirement for tumor formation. It is possible that these alleles may be inactivated by nonmutational mechanisms or that other tumor formation pathways exist.

Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

Correlation of TP53 mutations and p53 expression in ovarian tumors.

To characterize the involvement of the TP53 tumor suppressor gene in ovarian cancer, mutation analysis of exons 2-11 of TP53 and immunodetection of its protein product, p53, were done in 48 ovarian tumors. Normally, p53 is not immunodetectable. Missense TP53 mutations have been reported to result in p53 accumulation and detection, but mutations generating premature stop codons have not. Mutations were identified in 19 of 41 malignant tumors but not in 5 benign tumors and 2 tumors of low malignant potential. Fifteen of the 19 tumors with mutations also stained positively by immunohistochemistry or Western blot or both. They included 11 missense mutations, 1 in-frame duplication (474ins6), and 3 frameshift mutations generating premature stop codons. The three tumors with frameshifts also had a wild-type TP53 allele and displayed normal size but not truncated p53 by Western blot. This indicates that these tumors express wild-type p53. The significance of TP53 mutations in the development of the three tumors is questionable unless there is a mechanism for inactivating wild-type p53. Nine of the 19 mutations found here, including the 3 frameshifts, were previously not reported in ovarian cancer. Thirteen of the 19 mutations were single nucleotide substitutions with 6 transitions and 7 transversions. The ratio of transversions to transitions (1.2) was different from literature reports (0.5) (P < 0.01). Thus, the spectrum of TP53 mutations in our study differed from other ovarian tumor reports. This difference may be due to population-based differences in the molecular epidemiology of TP53 mutations.

Identical, unique p53 mutations in a primary ovarian mucinous adenocarcinoma and a synchronous contralateral ovarian mucinous tumor of low malignant potential suggest a common clonal origin.

The origin of malignant ovarian epithelial tumors is uncertain and has been the subject of considerably controversy. Some favor the theory of origin in precursor lesions such as benign cystadenomas or tumors of low malignant potential (LMP; borderline tumors), whereas others favor the concept of an independent origin of carcinomas from the ovarian surface epithelium or inclusion cysts. Recently, the demonstration of identical molecular alterations in morphologically benign and malignant areas within the same ovarian tumor have suggested the possibility that the malignant epithelium had undergone differentiation to a benign appearance. Because both areas were present in the same tumor, however, the possibility of progression of the morphologically benign component could not be excluded. We present a case of simultaneous mucinous carcinoma and contralateral tumor of LMP which exhibited identical, unique mutations of the p53 gene, suggesting a clonal origin. Because these were separate and distinct tumors, we believe this case provides strong support for the differentiation hypothesis. We also provide evidence for markedly different levels of p53 expression in areas with identical p53 mutations.

Expression of the p53 induced tumor suppressor p21waf1/cip1 in ovarian carcinomas: correlation with p53 and Ki-67 immunohistochemistry.

Mutations of the p53 gene are the most common genetic alteration in malignant human tumors, including ovarian carcinomas of surface epithelial origin. A cyclin-dependent kinase inhibitor, p21waf1/cip1, is thought to be an important mediator of p53-induced cell cycle arrest. Although numerous studies have reported p53 expression and mutation in ovarian tumors, none have correlated p53 expression with that of its downstream effector, p21waf1/cip1. We studied p53 and p21waf1/cip1 expression by immunohistochemistry in 44 ovarian carcinomas of different histologic types and correlated these findings with each other and with proliferation as measured by expression of the Ki-67 nuclear antigen. Fifty percent of tumors expressed p53, whereas 34% expressed p21waf1/cip1. Clear cell carcinomas expressed p21waf1/cip1 significantly more often than other histologic types, and tumors with squamous differentiation showed higher p21waf1/cip1 expression in these areas. There was no correlation of p21waf1/cip1 expression with p53 expression, p53 mutation, or Ki-67 expression. p21waf1/cip1 appears to be induced independently of p53 in these tumors and may be associated with differentiation rather than proliferation.

Somatic and germline mutations of the BRCA2 gene in sporadic ovarian cancer.

The breast and ovarian cancer susceptibility gene BRCA2 has recently been isolated. A role for BRCA2 in sporadic breast and ovarian cancer has been suggested by loss of heterozygosity (LOH) studies which show frequent LOH in the BRCA2 region at chromosome 13q12. In addition, the observation of nonrandom loss of the wild-type chromosome in a breast/ovarian cancer family which shows linkage to BRCA2 suggests it may act as a tumor suppressor gene. To determine the extent of somatic alteration involving BRCA2 in sporadic ovarian cancer, 50 tumors were analyzed for mutations throughout the entire BRCA2 coding region. Mutations predicted to result in truncation of the BRCA2 protein were detected in four tumors. Analysis of germline DNA revealed two of these alterations to be of somatic origin. In addition, all four tumors exhibited loss of the second BRCA2 allele as predicted by Knudson's hypothesis for a tumor suppressor gene. These results suggest that, as is the case with BRCA1, somatic mutations of BRCA2 are infrequent in sporadic ovarian cancer, despite the relatively high frequency of LOH detected around the BRCA2 locus.