Clinical and Pathologic Differences Between BRCA1-, BRCA2-, and Non-BRCA-Associated Breast Cancers in a Multiracial Developing Country

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1 DOI /s Clinical and Pathologic Differences Between BRCA1-, BRCA2-, and Non-BRCA-Associated Breast Cancers in a Multiracial Developing Country Cheng-Har Yip Æ N. A. Taib Æ W. Y. Choo Æ S. Rampal Æ M. K. Thong Æ S. H. Teo Ó Société Internationale de Chirurgie 2009 Abstract Background Mutations in BRCA1 and BRCA2 confer an increased risk to breast and other cancers, but to date there have only been limited numbers of studies of BRCA1- and BRCA2-associated cancers among Asians. Malaysia is a multiracial country with three main races: Malays, Chinese, Indians. We determined whether tumor pathologic features and clinical features differ in patients with and without BRCA mutations in this Asian population. Methods We conducted a retrospective review of the medical records of 152 women with breast cancer who underwent genetic testing for BRCA mutations. The patients self-reported ethnicity, age at onset, and clinical stage at diagnosis and tumor pathology were reviewed. Results A total of 31 patients carried germline deleterious mutations (16 BRCA1, 15BRCA2). We found that tumors in BRCA1 carriers were more likely to be estrogen receptor (ER)- negative and progesterone receptor (PR)-negative. HER2 was more likely to be negative in both BRCA1 and BRCA2 subjects compared with non-brca subjects. We found a strong C.-H. Yip (&) N. A. Taib Department of Surgery, University Malaya Medical Centre, Kuala Lumpur, Malaysia yipch@um.edu.my W. Y. Choo S. Rampal Department of Social and Preventive Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia M. K. Thong Genetics Unit, Department of Paediatrics, University Malaya Medical Centre, Kuala Lumpur, Malaysia S. H. Teo Cancer Research Initiatives Foundation, Sime Darby Medical Centre, Jalan SS12/1A, Subang Jaya, Selangor, Malaysia association between triple-negative status and BRCA1 carriers. In addition, tumors in BRCA1 carriers were more likely to be higher grade than those in BRCA2 and non-brca carriers; but the difference was not statistically significant. Conclusions These results suggest that tumors associated with BRCA1 mutations are distinct from those of BRCA2- associated and non-brca-associated breast cancers, and that the tumors associated with BRCA2 mutations are similar to the non-brca-associated breast cancers. Further studies are required to determine if the prognosis is different in each of these groups and the best management strategy for each group. Introduction Malaysia is a multiracial country comprising three main racial groups: Malays (65%), Chinese (26%), and Indians (8%). The incidence of breast cancer in Malaysia is 46.2 per 100,000, with a higher incidence in the Chinese (59.9/ 100,000) followed by the Indians (54.2/100,000) and the lowest in Malays (34.9/100,000). Breast cancer in Malaysia, like that in many Asian countries, is characterized by an early age of onset: Approximately 50% of women present below the age of 50 years, with 16.8% presenting below the age of 40 [1]. Although the early age of onset could be due to genetic predisposition to breast cancer, the younger age at presentation in Asia is more likely to be due to the cohort effect, in that successive generations of women in Asia are exposed to differing risks, with the highest risk in those born after World War II [2]. To date, only a few studies on BRCA-associated breast cancer have been reported in Malaysia and Asia. In a group of 37 women with early-onset breast cancer without a family history, BRCA1 mutations were found in 2.7% and BRCA2

2 mutations in 5.4%, which is comparable to other Asian studies [3]. Another study on 187 women with early-onset, with or without family history, and bilateral breast cancer, found 27 deleterious mutations in BRCA1 and BRCA2 (15%) in this high-risk group [4]. Notably, the latter study reported that there were equal numbers of BRCA1 and BRCA2 carriers among the Chinese but a larger number of BRCA2 than BRCA1 carriers among the Malays [4]. Most of the studies in Asia have focused on the incidence and prevalence of BRCA1 and BRCA2 mutations in high-risk women and their families [5 7], with few studies on the clinicopathologic features of the tumors in these women [8, 9]. BRCA1-associated breast cancers often occur in younger women, and such tumors are usually of high grade and lack hormone receptors [10], with a high proportion being triple negative: negative for estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER2) receptor) [11]. Although each of these features is associated with a poor prognosis, evidence on the effect of BRCA1 and BRCA2 mutation on the prognosis and overall survival is inconsistent, with some studies reporting poor results [12, 13] and other reporting similar outcomes [14]. The objective of this study was to compare the clinicopathologic features of BRCA1-associated with BRCA2- associated breast cancer and also BRCA1- and BRCA2- associated breast cancers with non-brca-associated breast cancers in this Asian population Methodology Recruitment of breast cancer patients The recruitment of patients with breast cancer started in 2003 at the University Malaya Medical Centre in Kuala Lumpur. Patients with breast cancer who had completed treatment for breast cancer and were followed up at the Breast Clinic were first approached by clinicians responsible for their care to see if they would participate in a research study to determine the genetic factors that increase the risk of breast cancer in Malaysia. Thereafter, individuals interested in the project were approached by a member of the research team, who explained to them the nature and objectives of the research project. Index patients signed a consent form, and a blood sample was obtained. A family history was recorded and the pedigree analyzed. The study was approved by the Ethics Committee at University Malaya Medical Centre. Inclusion criteria for analysis of BRCA1 and BRCA2 Patients were included if they had either early-onset breast cancer (B40 years) and one or more cases of breast cancer in first- or second-degree relatives; breast cancer (C40 years) and two or more additional cases of breast cancer in first- or second-degree relatives; bilateral breast cancer; or a personal or family history of ovarian cancer. All patients had at least a 10% a priori chance of having a deleterious mutation in BRCA1 or BRCA2. Mutation analysis Blood from the index cases was separated into two 10-ml EDTA tubes, and DNA was extracted using standard methods. Samples were analyzed using direct DNA sequencing of all coding regions and intron exon junctions and using multiple ligation dependent probe amplification (MLPA), as previously described [4]. Confirmation tests were performed on a second blood sample. Naming and interpreting the sequence analyses were performed as previously described, and all patients were classified as having a deleterious mutation if BRCA1 or BRCA2 protein terminated prematurely at least 10 or 110 amino acids, respectively, from the C-terminus. In total, 31 patients were found to carry deleterious germline mutations (16 in BRCA1, 15 in BRCA2). These data were compared with those of 121 women in the same group in whom no pathogenic BRCA mutations were identified. Assessment of clinicopathologic features The ERs, PRs, histologic grade (Bloom and Richardson classification), and HER2 status were obtained from pathology reports. The ER status and PR status were determined by immunoperoxidase staining of the tumor. ER and PR were considered negative if staining of the tumor cell nuclei was \10%. HER2 was assessed through immunohistochemistry (IHC) and, based on a staining intensity, was classified as negative, 1?,2?,or3?.Fluorescenceinsituhybridization (FISH) was not carried out as it was not available at our institution. Although full sequencing was conducted for 191 patients, 39 patients who did not have the grade or the ER, PR, or HER2 assay results recorded were excluded from the study, leaving 152 patients for analysis. The grade, ER, PR, and HER2 were compared among the BRCA1, BRCA2, and non- BRCA group, respectively. A triple negative breast cancer is characterized by the lack of ER, PR, and HER2. Statistical analysis The data were analyzed using SPSS software (SPSS, Chicago, IL, USA). Continuous data were summarized using means ± standard deviation (SD), and any differences between the three groups were tested using one-way analysis of variance (ANOVA). Categorical data were presented as proportions; and differences between groups

3 were tested using the chi-squared test. Alternatively, Fisher s exact test was used when the chi-squared test was violated. Results Of the 152 patients who were included in this study, most were Chinese (61.6%) followed by Malays (25.8%) and then Indians (12.6%), which reflected the demographic distribution of the patients treated in this single center. The mean age of diagnosis of breast cancer was 39.8 ± 9.03 years in BRCA1 carriers, 41.9 ± 9.18 years in BRCA2 carriers, and 44.2 ± 11.6 years in those without deleterious mutations (Table 1). Of the 31 BRCA carriers, 16 had deleterious mutations in BRCA1 and 15 in BRCA2. Our data indicate that there may be some differences in the prevalence of BRCA1 versus BRCA2 mutations among the Malays, Chinese, and Indians. Among the Malays there were more BRCA2 than BRCA1 carriers (5 BRCA2 vs. 2 BRCA1); among the Chinese there was an equal number (8 BRCA1 vs. 9 BRCA2); and among the Indians there were more BRCA1 than BRCA2 carriers (6 BRCA1 vs. 1 BRCA2) (Table 1). We found that the breast tumors in BRCA1 carriers were more likely to be of a high grade: 69.2% of BRCA1 carriers developed grade 3 tumors, compared with 40% of BRCA2 carriers and 37.5% of the non-brca carriers. However, the difference was not statistically significant (Tables 2, 3, 4). In the subset of patients in whom the ER status was reported, BRCA1 status was significantly associated with ER negativity: non-brca vs. BRCA1, p = 0.001; non- BRCA vs. BRCA2, p = 0.736; BRCA1 vs. BRCA2, p = 0.002). Similarly, in the subset of patients in whom the PR status was reported, BRCA1 status was significantly associated with PR negativity (100% of BRCA1 carriers, 50% of BRCA2 carriers, 45.9% of non-brca carriers). In the subset of patients in whom the HER2 receptor was assessed, we found that HER2 was more likely to be negative in tumors in BRCA1 carriers and in BRCA2 Table 1 Race and age and its association with BRCA status Parameter Non-BRCA (n = 121) BRCA1 (n = 16) BRCA2 (n = 15) Age (years) 44.2 (11.6) 39.8 (9.03) 41.9 (9.18) Race Malays 32 (26.4) 2 (12.5) 5 (33.3) Chinese 76 (62.8) 8 (50.0) 9 (60.0) Indians 12 (9.9) 6 (37.5) 1 (6.7) Eurasian 1 (0.9) 0 0 Results are the mean and SD (in parentheses) Table 2 Association between pathologic features and non-brca and BRCA1 status Feature Non-BRCA BRCA1 p 1 7 (8.7) (53.8) 4 (30.8) 3 30 (37.5) 9 (69.2) ER Positive 68 (56.7) 1 (6.7) Negative 52 (43.3) 14 (93.3) PR Positive 40 (54.1) 0 Negative 34 (45.9) 11 (100) HER2 a Negative 54 (52.9) 11 (84.6) Positive 48 (47.1) 2 (15.4) Triple negative \0.001 Yes 13 (18.3) 10 (100) No 58 (81.7) 0 ER estrogen receptors, PR progesterone receptors, HER2 human epidermal growth factor recepter-2 Table 3 Association between pathologic features and non-brca and BRCA2 status Feature Non-BRCA BRCA2 p 1 7 (8.7) (53.8) 6 (60.0) 3 30 (37.5) 4 (40.0) ER Positive 68 (56.7) 8 (66.7) Negative 52 (43.3) 4 (33.3) PR Positive 40 (54.1) 5 (50.0) Negative 34 (45.9) 5 (50.0) HER2 a Negative 54 (52.9) 8 (80.0) Positive 48 (47.1) 2 (20.0) Triple negative Yes 13 (18.3) 3 (33.3) No 58 (81.7) 6 (66.7) carriers compared to non-brca carriers. However, there is no difference between BRCA1 and BRCA2 carriers (Table 4)

4 Table 4 Association between pathologic features and BRCA1 and BRCA2 status Feature BRCA1 BRCA2 p (30.8) 6 (60.0) 3 9 (69.2) 4 (40.0) ER Positive 1 (6.7) 8 (66.7) Negative 14 (93.3) 4 (33.3) PR Positive 0 5 (50.0) Negative 11 (100) 5 (50.0) HER2 a Negative 11 (84.6) 8 (80.0) Positive 2 (15.4) 2 (20.0) Triple negative Yes 10 (100) 3 (33.3) No 0 6 (66.7) In cases where all three molecular markers were known, we found a significant association between BRCA1 status and triple-negative breast cancers (all 10 BRCA1 carriers developed triple-negative tumors, 33.3% of BRCA2 carriers, and 18.3% of non-brca carriers). Collectively, our data suggest that BRCA1 carriers tend to develop breast cancers that have poorer prognostic outcomes, given that they are associated with the prognostic markers of higher grade and triple negativity. Discussion The discovery of the BRCA1 and BRCA2 genes has transformed the management of women who are at high risk of developing breast and ovarian cancer, but there remain gaps in our understanding of the implications for many Asian populations in which there have only been a limited number of studies focusing on the incidence of mutations among familial or early-onset breast cancer patients. We report that, at least among women in Malaysia with an a priori 10% chance of having a mutation in BRCA1 and BRCA2, there is no significant difference in the average age of onset of breast cancer among BRCA1 carriers (40 years old) and BRCA2 carriers (42 years old) compared to that in a predominantly Caucasian population living in a developed country (average age of onset 40 and 41 years in BRCA1 and BRCA2 carriers, respectively) [15]. This suggests that the differences in genetic and nongenetic factors, may not contribute significantly to a difference in average age of onset in Asian versus Caucasian women. There are some differences between the tumors that develop in BRCA1 and BRCA2 carriers. Our study has demonstrated that in the Asian population there is a significant association between triple negativity and BRCA1 but not BRCA2 status and a trend toward a higher percentage of grade 3 cancers in the BRCA1 carriers but not in the BRCA2 carriers. All 10 BRCA1 patients in our study for whom all three markers are known were triple negative, which is also associated with high grade, younger age, and poorer outcome [16 18]. Histologically and transcriptionally, triple-negative breast cancers have many similarities to BRCA1-associated breast cancers, which suggests that dysfunction in BRCA1 or related pathways occurs in this subset [18, 19]. Notably, the association between BRCA1 carriers and triple-negative breast cancers and higher-grade tumors has also been reported in other populations [9, 11, 20]. We also report that a significant proportion of the BRCA1 tumors were ER-negative. Almost all the BRCA1 cancers were ER-negative (14/15 cases), and the ERpositive rate in BRCA2 and non-brca patients were similar (66.7% and 56.7%, respectively, p = 0.5). These data are consistent with those reported in several other studies in both Caucasian and Asian populations [8, 10, 20]. Notably, in a review of breast cancers in 208 BRCA1 mutation carriers, 88 BRCA2 mutations carriers, and 804 women with no known mutations, the BRCA1 mutations carriers were more likely to have ER-negative tumors than the other two groups. The ER negativity of BRCA1 carriers was unrelated to the age at diagnosis or the high grade but is an intrinsic property of BRCA1-associated cancers. The ER-negative status of these tumors may reflect the cell of origin of BRCA1-related breast cancers [21]. Similarly, PR was negative in all the BRCA1 carriers in our study, and in BRCA2 and non-brca carriers it was similar (50.0% and 54.1%, respectively, p = 0.8). BRCA1-associated breast cancers have histopathologic features associated with a poor prognosis, whereas those with BRCA2 mutations are similar to the non-brca group. Ongoing studies seek to determine whether these histopathologic features may also aid in identifying Asian women with germline mutations in BRCA1 [22]. BRCA1 has been reported to be more prevalent than BRCA2 in selected high-risk cohorts living in western Asia (e.g., Pakistan) [23] and among early-onset breast cancer patients in many parts of Asia, including China [7]. BRCA1 and BRCA2 have been reported at similar incidences in high-risk cohorts among the Chinese in Malaysia [4] and Singapore [24] and, notably, in Korea among sporadic

5 breast cancer patients [8]. BRCA2 has been reported to be more prevalent than BRCA1 in high-risk cohorts in Southeast Asia, including the Philippines [25], Indonesia [5], and the Malay population in Malaysia [4]. By contrast, in most published studies in the Caucasian population, BRCA1 is more prevalent than BRCA2 [21]. Given that the clinicopathologic features of BRCA2 tumors appear indistinguishable from those of non-brca-associated tumors and that BRCA2 mutations may be more common at least in some Asian populations compared to others, there remain major challenges in improving our ability to improve risk prediction models for the identifying BRCA2 carriers in Asians. Conclusions In Malaysia, BRCA1-associated breast cancer has features associated with a poor prognosis (i.e., triple-negative breast cancer and high grade) and appears to be distinct from BRCA2-associated breast cancers, which are similar to the non-brca group. 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