Study in Italy Characteristics in Male Breast Cancer : A Population-based Mutation Status and Tumor BRCA 2 and BRCA 1 Updated Version

To investigate at the population level the impact of BRCA1/BRCA2 gene alterations in male breast cancer, we analyzed a population-based series of 25 male breast cancer cases from Florence, Central Italy. We combined mutational screening with the study of germ-line allele transcript levels and of tumor-associated losses of heterozygosity. Screening by protein truncation test and single-strand conformational polymorphism assay, followed by sequencing, revealed 4 pathogenetic mutations (4 of 25 16%; 95% confidence interval, 5–37%), 1 in BRCA1 and 3 in BRCA2, including mutations recurring in Central Italy (BRCA1 3345delAG and BRCA2 6696delTC). The a priori probability of carrying a mutation, estimated using BRCAPRO software, showed a good agreement between expected and observed mutations (14% versus 16%). A 7-fold association between germ-line mutations and family history of breast-ovarian cancer emerged. To investigate associations between BRCA1/BRCA2 status and clinicopathological characteristics, we analyzed the histopathological and immunophenotypic parameters of the tumors. A significant association emerged between mutation carrier status and high histological grade (P 0.02). Furthermore, one BRCA2 carrier was affected with Paget’s disease, an extremely rare male breast cancer histotype. Overall, BRCA1/2 mutations were observed to be strongly associated with positive c-erbB-2 immunostaining (P 0.004). To evaluate germ-line allele expression, we used primer extension assays targeting frequent BRCA1 and BRCA2 polymorphisms. A BRCA2 allele transcript imbalance was found in one of four heterozygotes tested, all of them negative for germ-line mutations. BRCA1 transcript imbalances were not detected in nine heterozygotes analyzed. Losses of heterozygosity at one or more of nine loci in the BRCA2 region were found in 8 of 22 tumors tested. Interestingly, a case that was negative for BRCA1/BRCA2 germ-line mutations and that had a priori mutation probability <10% showed loss of heterozygosity at all three of the intragenic BRCA2 markers analyzed, which could be related to a somatic involvement of BRCA2. No losses of heterozygosity were detected at BRCA1. In conclusion, constitutional BRCA1/BRCA2 mutations accounted for 16% of the male breast cancer cases in this area of Central Italy. The detection of a BRCA2 germ-line transcript imbalance and of a somatic loss of BRCA2 among the cases that resulted negative for germ-line mutations suggests a role of this gene more relevant than indicated by conventional mutational analysis. A distinct pattern of characteristics indicative of aggressive behavior, including high-grade and c-erbB-2 expression, was evident in tumors from germline BRCA2 mutation carriers. This suggests that phenotypic characteristics may contribute to the identification of hereditary BRCA2-related male breast cancers and that these tumors might share a unique molecular pathway of cancer progression. INTRODUCTION Cancer of the breast is rare in males. In Western countries, in which FBC is the first or second most commonly diagnosed malignancy of women, MBC accounts for less than 1% of all cancers in men. Epidemiological risk factors include pathological conditions associated with primary and secondary hyperestrogenism, radiation exposure, and, particularly, BC FH, which points to a relevant genetic component in disease predisposition (1–2). Furthermore, MBC incidence rates are relatively stable in time, which contrasts with FBC, but tend to vary by race and/or geographical location, suggesting that ethnic and/or locally acting environmental factors may enhance disease risk at the population level (1). Evidence provided by several studies implicate pathogenetic germ-line mutations of BRCA2 and, with lower frequency, of BRCA1 in MBC. In fact, mutations in BRCA1 and BRCA2 were estimated to be responsible for 16 and 76%, respectively, of the MBCs occurring in high-risk breast/ovarian cancer families (3–5). With regard to MBCs unselected for FH, the prevalence of BRCA1/BRCA2 mutations has been investigated in populationand clinic-based series from countries with ethnically diverse populations, such as the United Kingdom, Iceland, continental Europe, the United States, and Israel. Mutation frequencies ranged from 4 to 40% for BRCA2 and up to 4% for BRCA1, being higher in populations with founder mutations (5–15). Overall, these studies suggest that migration patterns and genetic bottlenecks could account for regional differences in the contribution of the two genes to disease. However, the frequency of BRCA1/BRCA2 alterations is probably underestimated in MBCs from populations without highly recurrent founder mutations. In fact, the sensitivity of the most commonly used mutation screening techniques is regarded to be about 70–80% (4). Moreover, extensive genomic deletions, known to account for a subset of BRCA1/2-related breast/ovarian cancers, are missed by standard PCR-based screening strategies, whereas mutations in the promoter regions, in regulatory elements and in introns, which interfere with gene expression or with transcript processing, are currently not investigated (16–21). At the somatic level, MBCs arising in BRCA1/ BRCA2 mutation carriers are anticipated to follow the classic doublehit/loss-of-function mechanism, with conversion to homozygosity often attributable to somatic loss of the wild-type allele (5, 22–24). The phenotypic characteristics of tumors may shed light on the molecular mechanisms implicated in the progression of BRCA1/ BRCA2-related cancers. In fact, studies conducted on FBCs indicate that BRCA1and, to a lesser extent, BRCA2-related tumors tend to manifest specific phenotypic profiles. In particular, BCs arising in female BRCA1 mutation carriers tend to show high histological grade and high proliferative activity, and to be negative for ER/PR and for Received 8/1/02; accepted 11/14/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), coordinated grant “Italian Consortium for Hereditary Breast Cancer,” by MIUR-COFIN 1999 and 2001, and, in part, by a grant from the Italian Ministry of Research to the Center of Excellence on Aging of the University of Chieti. C. D. was a fellow of the Fondazione Italiana per la Ricerca sul Cancro (FIRC). M. F. is a FIRC fellow. 2 To whom requests for reprints should be addressed, at Section of Molecular Pathology, Department of Oncology and Neuroscience, University Gabriele D’Annunzio, Via dei Vestini 31, 66013 Chieti, I. E-mail: r.mariani@istpat.unich. it. 3 The abbreviations used are: BC, breast cancer; MBC, male BC; FBC, female BC; ER, estrogen receptor; PR, progesterone receptor; LOH, loss of heterozygosity; SSCP, singlestrand conformational polymorphism; BIC, Breast Cancer Information Core; NOS, invasive ductal carcinomas not otherwise specified; MSI, microsatellite instability; FH, family history; CI, confidence interval; gDNA, genomic DNA.