Thomas S. Frank

Clinical Characteristics of Individuals With Germline Mutations in BRCA1 and BRCA2: Analysis of 10,000 Individuals

PURPOSE To assess the characteristics that correlate best with the presence of mutations in BRCA1 and BRCA2 in individuals tested in a clinical setting. PATIENTS AND METHODS The results of 10,000 consecutive gene sequence analyses performed to identify mutations anywhere in the BRCA1 and BRCA2 genes (7,461 analyses) or for three specific Ashkenazi Jewish founder mutations (2,539 analyses) were correlated with personal and family history of cancer, ancestry, invasive versus noninvasive breast neoplasia, and sex. RESULTS Mutations were identified in 1,720 (17.2%) of the 10,000 individuals tested, including 968 (20%) of 4,843 women with breast cancer and 281 (34%) of 824 with ovarian cancer, and the prevalence of mutations was correlated with specific features of the personal and family histories of the individuals tested. Mutations were as prevalent in high-risk women of African (25 [19%] of 133) and other non-Ashkenazi ancestries as those of European ancestry (712 [16%] of 4379) and were significantly less prevalent in women diagnosed before 50 years of age with ductal carcinoma in situ than with invasive breast cancer (13% v 24%, P =.0007). Of the 74 mutations identified in individuals of Ashkenazi ancestry through full sequence analysis of both BRCA1 and BRCA2, 16 (21.6%) were nonfounder mutations, including seven in BRCA1 and nine in BRCA2. Twenty-one (28%) of 76 men with breast cancer carried mutations, of which more than one third occurred in BRCA1. CONCLUSION Specific features of personal and family history can be used to assess the likelihood of identifying a mutation in BRCA1 or BRCA2 in individuals tested in a clinical setting.

Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk.

PURPOSE Previous studies of mutations in BRCA1 or BRCA2 have used detection methods that may underestimate the actual frequency of mutations and have analyzed women using heterogeneous criteria for risk of hereditary cancer. PATIENTS AND METHODS A total of 238 women with breast cancer before age 50 or ovarian cancer at any age and at least one first- or second-degree relative with either diagnosis underwent sequence analysis of BRCA1 followed by analysis of BRCA2 (except for 27 women who declined analysis of BRCA2 after a deleterious mutation was discovered in BRCA1). Results were correlated with personal and family history of malignancy. RESULTS Deleterious mutations were identified in 94 (39%) women, including 59 of 117 (50%) from families with ovarian cancer and 35 of 121 (29%) from families without ovarian cancer. Mutations were identified in 14 of 70 (20%) women with just one other relative who developed breast cancer before age 50. In women with breast cancer, mutations in BRCA1 and BRCA2 were associated with a 10-fold increased risk of subsequent ovarian carcinoma (P = .005). CONCLUSION Because mutations in BRCA1 and BRCA2 in women with breast cancer are associated with an increased risk of ovarian cancer, analysis of these genes should be considered for women diagnosed with breast cancer who have a high probability of carrying a mutation according to the statistical model developed with these data.

Interpreting epidemiological research: blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1

While sequence analysis is considered by many to be the most sensitive method of detecting unknown mutations in large genes such asBRCA1, most published estimates of the prevalence of mutations in this gene have been derived from studies that have used other methods of gene analysis. In order to determine the relative sensitivity of techniques that are widely used in research on BRCA1, a set of blinded samples containing 58 distinct mutations were analysed by four separate laboratories. Each used one of the following methods: single strand conformational polymorphism analysis (SSCP), conformation sensitive gel electrophoresis (CSGE), two dimensional gene scanning (TDGS), and denaturing high performance liquid chromatography (DHPLC). Only the laboratory using DHPLC correctly identified each of the mutations. The laboratory using TDGS correctly identified 91% of the mutations but produced three apparent false positive results. The laboratories using SSCP and CSGE detected abnormal migration for 72% and 76% of the mutations, respectively, but subsequently confirmed and reported only 65% and 60% of mutations, respectively. False negatives therefore resulted not only from failure of the techniques to distinguish wild type from mutant, but also from failure to confirm the mutation by sequence analysis as well as from human errors leading to misreporting of results. These findings characterise sources of error in commonly used methods of mutation detection that should be addressed by laboratories using these methods. Based upon sources of error identified in this comparison, it is likely that mutations inBRCA1 and BRCA2are more prevalent than some studies have previously reported. The findings of this comparison provide a basis for interpreting studies of mutations in susceptibility genes across many inherited cancer syndromes.

Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR

DNA was extracted from unstained 5-μm sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 103 nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.

Incidence of non-founder BRCA1 and BRCA2 mutations in high risk Ashkenazi breast and ovarian cancer families

Inherited predisposition to cancer is a major contributor to the breast and ovarian cancer burden among people of Ashkenazi ancestry. Approximately 2.5% of all people of Ashkenazi Jewish descent carry one of three ancient (founder) mutations in BRCA1 or BRCA2 (185delAG or 5382insC in BRCA1 and 6174delT in BRCA2) .1–3 In a recent population based study, 29% of Jewish women with ovarian cancer were shown to carry one of these three founder mutations.4 In a series of 220 high risk Ashkenazi breast cancer families, a founder BRCA mutation was detected in 44%. If ovarian cancer was present in the kindred, 73% of families segregated a founder BRCA mutation.5 Despite the high proportion of hereditary breast and ovarian cancer attributed to founder mutations of BRCA1 or BRCA2 in this population, some Ashkenazi families with histories highly suggestive of an inherited cancer predisposition have been shown to segregate other (non-founder) mutations of BRCA1 6 or BRCA2 .7 Counselling of families considering full sequence BRCA genotyping is complicated by the limited information available regarding the incidence of these non-founder mutations in the Ashkenazi population. We present a series of Ashkenazi Jewish kindreds at hereditary risk for breast and ovarian cancer who do not segregate one of the three Ashkenazi founder mutations and who have undergone full sequencing of the coding regions and flanking intronic regions of BRCA1 and BRCA2 . Using the BRCAPRO algorithm, we have estimated whether the prevalence of non-founder BRCA1 and BRCA2 mutations in a genetic isolate (Ashkenazim) is consistent with the background rate in an admixed population, or if selective or other effects have led to a non-founder mutation rate lower than would be expected. Records of all patients seen by the Clinical Genetics Service at Memorial Sloan-Kettering Cancer Center (MSKCC) from 1.6.95 to …

Detection and Characterization of Atypical Mycobacteria by the Polymerase Chain Reaction

The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65-kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogens Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, and Mycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, including M. kansasii and Mycobacterium leprae, in formalin-fixed paraffin-embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin-embedded tissue sections.

Synchronous dual primary ovarian and endometrial carcinomas

OBJECTIVES: The synchronous occurrence of carcinoma confined to the ovary and endometrium presents a diagnostic and therapeutic dilemma. These tumors have been variously staged as FIGO Stage IIA ovarian carcinoma, Stage III endometrial carcinoma, or synchronous dual primary carcinomas. Accumulating evidence suggests such patients have a favorable outcome. This retrospective study was undertaken to review our experience with these fascinating tumors. METHODS: The clinical records and the pathologic findings of 16 patients with synchronous dual primary ovarian and endometrial carcinomas were reviewed. RESULTS: The median age was 51 years. Abnormal uterine bleeding was the most common presenting symptom (70%). All patients had Stage I ovarian and endometrial carcinomas. Fourteen patients (88%) had endometrioid carcinoma in both sites, while two patients (12%) had dissimilar histology. For 15 patients (94%), the grade of both tumors was identical. Only three (19%) patients had myometrial invasion, with less than 50% involvement in each case. All patients underwent surgical staging, 11 (70%) of whom received adjuvant radiation or chemotherapy. The five patients treated with surgery alone had Grade 1 endometrioid tumors. The only relapse occurred in a patient with a clear cell component in both sites. No patient has died of disease. CONCLUSIONS: Patients with synchronous dual primary carcinomas appear to have a more favorable prognosis than that expected with Stage IIA ovarian or Stage III endometrial carcinoma (100% vs. 63% or 42% survival at 3 years, respectively). The excellent survival for patients with Grade 1 dual endometrioid tumors treated with surgery alone suggests that adjuvant therapy may not be necessary for this sub‐group.

Comparative Analysis of Histologic Homologues of Endometrial and Ovarian Carcinoma

We compared molecular alterations in histologically homologous ovarian and uterine carcinomas, including the prevalence of allelic loss of markers on 17q (within and distal to the familial breast-ovarian cancer gene BRCA1), mutations of codon 12 of Ki-ras and immunohistochemical expression of the p53 and c-erbB2 gene products in endometrioid and papillary serous carcinomas occurring in the uterus and ovary. A total of 86 uterine and 28 ovarian endometrioid carcinomas, as well as 8 uterine and 26 ovarian papillary serous carcinomas, were evaluated. The prevalence of p53 gene product immunoreactivity was similar in papillary serous carcinomas occurring in the uterus (6 of 8, 75%) and ovary (16 of 26, 62%). Allelic loss on 17q also was seen in similarly high proportions of uterine (3 of 7, 43%) and ovarian (16 of 25, 64%) papillary serous carcinomas. In contrast, expression of the p53 gene product was seen in significantly more endometrioid tumors of the ovary (14 of 28, 50%) than in those occurring in the uterus (4 of 86, 5%) (p < 0.0001). Allelic loss on 17q also was present in significantly more ovarian (19 or 27, 70% than in uterine (2 of 72, 3%) endometrioid carcinomas (p < 0.0001). Immunohistochemical expression of c-erbB2 and mutations of codon 12 of Ki-ras were present in a minority of carcinomas. Endometrioid tumors of the ovary and endometrium, although histologically similar, may arise from different genetic events, whereas uterine papillary serous carcinoma shares with its ovarian counterpart several molecular alterations that may account for its aggressive clinical behavior.

Hepatitis B and alterations of the p53 tumor suppressor gene in hepatocellular carcinoma

In areas of the world where hepatitis B and aflatoxin ingestion are common, alterations of the p53 tumor suppressor gene have frequently been reported in hepatocellular carcinoma (HCC). In particular, G-to-T transversions at codon 249 of the p53 gene have been consistently observed in hepatocellular carcinomas in China and sub- Saharan Africa. The goal of this study was to determine the frequency and relationship of p53 gene alterations and hepatitis B in formalin-fixed, paraffin-embedded HCCs resected in the United States. Since immunoreactivity for p53 correlates closely with the presence of missense mutations in the p53 gene, we performed immunohistochemical staining with the monoclonal antibody PAbl801. Only seven of 37 cases (19%) demonstrated nuclear accumulation of p53 gene product, in contrast to 10 of 20 cases (50%) of colon carcinoma metastatic to the liver. Staining was not observed in seven liver cell adenomas, 10 cases of focal nodular hyperplasia, or eight cases of cirrhosis. DNA was extracted from formalin-fixed paraffin sections for additional analysis with use of the polymerase chain reaction (PCR). G-to-T transversions of the third nucleotide of codon 249 were demonstrated in only four of 37 cases (11%), three of which had stained with PAbl801. Of 13 patients for whom there was information about a restriction fragment length polymorphism (RFLP) for BstUl within the fourth exon of the p53 gene, allelic loss of p53 was demonstrated in only two cases (15%), both of which stained with PAbl801. Because of previous reports specifically associating hepatitis B with p53 mutations in HCC, we performed nested PCR for hepatitis B virus DNA. Five of 37 cases (14%) contained hepatitis B virus DNA, two of which stained diffusely for p53 and three of which had codon 249 mutations. Our findings indicate that alterations in the p53 gene, particularly at codon 249, are uncommon in HCCs in the United States, and when present are associated with hepatitis B. Since hepatitis B is infrequently associated with HCC in our patient population, the role of p53 alterations in hepatocellular carcinogenesis may not be as significant as in other parts of the world where hepatitis B and aflatoxin are more prevalent

A region of interstitial 17q25 allelic loss in ovarian tumors coincides with a defined region of loss in breast tumors

Chromosomal regions of allelic imbalance in tumors are predicted to define the general location of tumor suppressor genes. We previously localized a putative breast tumor suppressor gene to a 3 cM region on 17q25 by deletion mapping of microsatellite markers in breast tumors. To determine if the same 17q25 region of loss is important in the genesis of other tumor types, 32 ovarian tumors and 24 prostate tumors, as well as 33 additional breast tumors, were analysed with 17q25 polymorphic microsatellite markers. While no significant loss was observed in prostate tumors, greater than half of ovarian tumors exhibited loss coincident with the candidate region previously defined in breast tumors. These results suggest that one or more novel tumor suppressor genes exist on 17q25 within a concordant region of interstitial loss defined in both breast and ovarian neoplasms.