Germline Mutations of BRCA1 and BRCA2 in Korean Ovarian Cancer Patients

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1 ORIGINAL STUDY Germline Mutations of BRCA1 and BRCA2 in Korean Ovarian Cancer Patients Finding Founder Mutations Min Chul Choi, MD,* Jin-Hyung Heo, MD,Þ Ja-Hyun Jang, MD, PhD,þ Sang Geun Jung, MD, PhD,* Hyun Park, MD, PhD,* Won Duk Joo, MD, PhD,* Chan Lee, MD, PhD,* Je Ho Lee, MD, PhD,* Jun Mo Lee, MD, PhD,* Yoon Young Hwang, MD, PhD,* and Seung Jo Kim, MD, PhD* Objectives: To investigate and analyze the BRCA mutations in Korean ovarian cancer patients with or without family history and to find founder mutations in this group. Methods/Materials: One hundred two patients who underwent a staging operation for pathologically proven epithelial cancer between January 2013 and December 2014 were enrolled. Thirty-two patients declined to analyze BRCA1/2 gene alterations after genetic counseling and pedigree analysis. Lymphocyte specimens from peripheral blood were assessed for BRCA1/2 by direct sequencing. Results: BRCA genetic test results of 70 patients were available. Eighteen BRCA1/2 mutations and 17 unclassified variations (UVs) were found. Five of the BRCA1/2 mutations and 4 of the UVs were not reported in the Breast Cancer Information Core database. One BRCA2 UV (8665_8667delGGA) was strongly suspicious to be a deleterious mutation. BRCA1/2 mutations were identified in 11 (61.1%) of 18 patients with a family history and in 7 (13.5%) of 52 patients without a family history. Candidates for founder mutations in Korean ovarian cancer patients were assessed among 39 BRCA1/2 mutations from the present study and from literature reviews. The analysis showed that 1041_1043delAGCinsT (n = 4; 10.2%) and 3746insA (n = 4; 10.2%) were possible BRCA1 founder mutations. Only one of the BRCA2 mutations (5804_5807delTTAA) was repeated twice (n = 2; 5.1%). Conclusions: The prevalence of BRCA1/2 mutations in Korean ovarian cancer patients irrespective of the family history was significantly higher than previously reported. Possible founder mutations in Korean ovarian cancer patients were identified. Key Words: BRCA mutation, Korean ovarian cancer, Founder mutation Received February 24, 2015, and in revised form June 7, Accepted for publication June 8, (Int J Gynecol Cancer 2015;00: 00Y00) *Comprehensive Gynecologic Cancer Center, Departments of Obstetrics and Gynecology and Pathology, CHA Bundang Medical Center, CHA University, Seongnam-si, Gyeonggi-do, Republic of Korea; and GreenCross Laboratories, Yongin City, Republic of Korea. Address correspondence and reprint requests to Jun Mo Lee, MD, PhD, CHA Bundang Medical Center, Yatap-dong, Bundang-gu, Seongnam-si, Gyeonggi-do , Republic of Korea. leejm@catholic.ac.kr. The authors declare no conflicts of interest. Copyright * 2015 by IGCS and ESGO ISSN: X DOI: /IGC wo high-susceptibility genes for familial breast-ovarian can- have been identified: BRCA1 by Miki et al 1 in 1994 and Tcer BRCA2 by Wooster et al 2 in Individuals who inherit germline mutations in these genes have a high lifetime risk of ovarian and breast cancers. The cumulative risk for ovarian cancer for carriers of BRCA1 or BRCA2 mutation by 70 years of age has been reported to be 39% and 11%, respectively. 3 Many investigators from different countries have reported many disease-associated BRCA1 and BRCA2 (BRCA1/2) mutations. Several founder mutations have been identified in specific ethnic groups, including Icelanders, Ashkenazi Jews, Russians, and Israelis. Ethnicity might play a significant role in International Journal of Gynecological Cancer & Volume 00, Number 00, Month

2 Choi et al International Journal of Gynecological Cancer & Volume 00, Number 00, Month 2015 hereditary ovarian cancer through its association with founder mutations. Most previous studies of BRCA1/2 genes have been performed on white populations; however, there have been recent efforts to determine the prevalence of mutations and to find the presence of founder mutations in Asian populations. 4Y10 In Korea, possible hereditary breast cancer cases are actively investigated by the Korean Hereditary Breast Cancer (KOHBRA) study as a nationwide effort. 3 However, no such study has been established to investigate Korean ovarian cancer (KoOC). The purpose of the present work was to investigate and analyze BRCA1/2 mutations in KoOC patients with or without a family history (FH). The secondary purpose was to try to find founder mutations in KoOC subgroups. MATERIALS AND METHODS Study Participants This study was approved by the institutional review board. All study participants were recruited between January 2013 and December A total of 102 patients who underwent a staging operation were enrolled. Epithelial ovarian cancer (EOC) was diagnosed pathologically. Thirty-two (31.4%) of them declined to analyze BRCA1/2 gene alterations after genetic counseling and pedigree analysis. Informed consent was obtained from all participants at the time of peripheral blood sampling. Direct Sequencing DNA extraction from peripheral blood was performed using automated magnetic bead technology (chemagic Magnetic Separation Module I [PerkinElmer, Waltham, MA]). Primers for polymerase chain reaction amplification and sequencing were designed by the authors to include the whole exons and the flanking intronic sequences of the BRCA1/2 genes. Polymerase chain reaction amplification was performed with an ABI 2720 cycler (Life Technologies, Foster City, CA) using the following protocol: initial denaturation for 5 minutes at 94-C; and then 35 cycles of extension at 94-C for 30 seconds, 60-C for 40 seconds, and 72-C for 1 minute; followed by 5 minutes of final extension at 72-C. Each reaction was performed in a 50-KL reaction volume containing 5 KL of 10 buffer, 400 nm of each primer, 200 KM of each deoxyribonucleotide, and 1.25 units of Taq DNA polymerase (Takara Bio Inc, Shiga, Japan). Direct sequencing was performed with Big Dye Terminator V3.1 Cycle Sequencing Kit (Life Technologies) and ABI 3500xl (Life Technologies). Variations were described following the nomenclature system of the Human Genome Variation Society ( and the conventional nomenclature system from the Breast Cancer Information Core (BIC; RESULTS The clinical characteristics of the study participants are shown in Table 1. BRCA1/2 genetic test results of 70 patients were available. The mean age at diagnosis of ovarian cancer was 52.0 years, ranging from 24 to 77 years. The most common histopathologic type was serous (n = 44; 62.8%), 2 TABLE 1. Clinical characteristic of patients n = 70 % Age, y 52.0 (24Y77) Histology Serous Mucinous Seromucinous Clear Endometrioid Grade I II-III Stage I II III IV followed by mucinous (n = 9; 12.8%), clear cell type (n = 9; 12.8%), endometrioid (n = 6; 8.6%), and seromucinous cell type (n = 2; 2.8%). After grade assessment, 10 (14.3%) cases were grade I and 60 (85.7%) cases were grade II or III. Based on International Federation of Gynecology and Obstetrics staging, 19 (27.1%) cases were determined to be stage I, 9 (12.8%) cases were stage II, 40 (57.1%) cases were stage III, and 2 (2.8%) cases were stage IV. Ovarian cancer patients with FH were defined as patients with primary breast cancer or patients with first-degree relatives (FDRs) diagnosed as having ovarian or breast cancer. Of 70 patients who underwent genetic testing, 18 patients had an FH. BRCA1/2 mutations were identified in 11 (61.1%) of the 18 patients with FH. Ten of the mutations were in the BRCA1 gene and 1 was in the BRCA2 gene (Table 2). Of the 52 patients without FH, BRCA1/2 mutations were identified in 7 (13.5%). Five of these mutations were in the BRCA1 gene and the other 2 mutations were in the BRCA2 gene (Table 3). Therefore, 18 mutations were detected in 70 EOC patients (25.7%) regardless of FH. Of the 15 BRCA1 and 3 BRCA2 mutations, 4 of the BRCA1 and1ofthebrca2 mutations were not reported in the BIC. Two of them were novel mutations (3033G 9 TinBRCA1, 3496_3497delATinsG in BRCA2). Three of the BRCA1 mutations (509C 9 A, 1041_1043delAGCinsT, and 2552delC) and 1 BRCA2 mutation (5804_5807delTTAA) were recurrent (Tables 2 and 3). A total of 17 unclassified variations (UVs) were identified in the 70 EOC patients. Of the 6 BRCA1 and 11 BRCA2 UVs, 4 BRCA2 UVs had not been previously reported in the BIC. Two of them were novel variations (2344A 9 G and 599 T 9 C in BRCA2). One BRCA2 UV (8665_8667delGGA; inframe deletion) was strongly suspicious to be a deleterious mutation rather than an UV after confirming co-segregation with her sister, who had breast cancer (Table 2). Mutations were most prevalent in serous cell-type EOC patients with FH (78.6%; Table 4). No mutation was noted * 2015 IGCS and ESGO

3 International Journal of Gynecological Cancer & Volume 00, Number 00, Month 2015 BRCA Mutation in Korean Ovarian Cancer TABLE 2. BRCA1 and BRCA2 germline mutations in ovarian cancer patients with FH Case Age, y Gene Site Mutation Mutation Type Citation No. in BIC Double Primary* FH FH of Other Cancer Pathogenic mutations CHAOv BRCA1 Exon _1043delAGCinsT NS 2 V Ovary (mother), breast (sister) CHAOv BRCA1 Exon G 9 T NS V, novel Br Ovary (sister), breast (mother) Colon (mother) CHAOv BRCA1 Exon IVS G 9 A SP 5 Br Cervix (mother) CHAOv BRCA1 Exon delC FS 11 V Ovary (mother, grandmother, nephew) Neuroblastoma (daughter) CHAOv BRCA1 Exon C 9 T NS 36 V Breast (sister) Pancreas (mother) CHAOv BRCA1 Exon 7 509C 9 A NS 1 V Breast (mother) CHAOv BRCA1 Exon A 9 T NS 2 V Ovary (sister), Thyroid (patient) breast (aunt) CHAOv BRCA1 Exon IVS G 9 A SP 5 V Ovary (mother, grandmother) CHAOv BRCA1 Exon _1043delAGCinsT NS 2 Br Stomach (brother) CHAOv BRCA2 Exon _3497delATinsG FS V, novel Br Pancreas (father), stomach (grandfather) CHAOv BRCA1 Exon 7 509C 9 A NS 1 V Breast (sister) UVs CHAOv BRCA1 Exon T 9 C UV 96 V Ovary (mother, grandmother, nephew) Neuroblastoma (daughter) CHAOv BRCA2 Exon T 9 A UV 1 Br Pancreas (father), stomach (grandfather) CHAOv BRCA2 Exon _8667delGGA IF V V Breast (sister) Lung (father) *Breast cancer history of the patient. Strongly suspicious to be a deleterious mutation after co-segregation from her sister with breast cancer. FS, frameshift; IF, in-frame deletion; NS, nonsense; SP, splicing; UV, variants of unknown clinical significance. * 2015 IGCS and ESGO 3

4 Choi et al International Journal of Gynecological Cancer & Volume 00, Number 00, Month 2015 TABLE 3. BRCA1 and BRCA2 germline mutations in ovarian cancer patients without FH Case Age, y Gene Site Mutation Mutation Type Citation No. in BIC Double Primary FH FH of Other Cancer Pathogenic mutations CHAOv BRCA1 Exon dupA FS V V Ovary (aunt) Pancreas (grandfather), Stomach (grandmother, uncle) CHAOv BRCA1 Exon delC FS 11 V V CHAOv BRCA1 Exon delG FS V V V Cervix (patient), brain (father) CHAOv BRCA1 Exon 7 509C 9 A NS 1 V V CHAOv BRCA1 Exon C 9 T NS V V V CHAOv BRCA2 Exon _5807delTTAA FS 29 V Breast (aunt) Lung (father), colon (mother) CHAOv BRCA2 Exon _5807delTTAA FS 29 V V Unspecified uterine (grandmother) UVs CHAOv BRCA2 Exon G 9 A UV 4 V V CHAOv BRCA2 Exon G 9 T UV 24 V V Thyroid (patient) CHAOv BRCA2 Exon C 9 T UV 1 V V CHAOv BRCA2 Exon G 9 T UV 24 V V CHAOv BRCA1 Exon T 9 C UV 1 V V Thyroid (patient) CHAOv BRCA2 Exon A 9 G UV V, novel V V Stomach (mother) CHAOv BRCA2 Exon A 9 C UV V V V CHAOv BRCA1 Exon A 9 G UV 4 V V CHAOv BRCA1 Exon T 9 C UV 96 V V Melanoma (uncle) CHAOv BRCA1 Exon IVS delG UV 2 V V CHAOv BRCA1 Exon T 9 C UV 18 V V CHAOv BRCA2 Exon T 9 C UV V, novel V V CHAOv BRCA2 Exon A 9 G UV 47 V V CHAOv BRCA2 Exon A 9 G UV 47 V V FS, frameshift; NS, nonsense; UV, variants of unknown clinical significance. 4 * 2015 IGCS and ESGO

5 International Journal of Gynecological Cancer & Volume 00, Number 00, Month 2015 BRCA Mutation in Korean Ovarian Cancer TABLE 4. BRCA mutations in variable risk FH in FDR FH in SDR Serous Type* Nonserous Type Early Onset (G45 y) Serous + FH in FDR Total n BRCA BRCA Total (%) 11 (61.1) 13 (61.9) 18 (40.9) 0 4 (23.5) 11 (78.6) 18 (25.7) *All high-grade cancer. SDR, second-degree relative. in any nonyserous cell-type EOC in the present study. All mutations were identified in high-grade serous types of EOC. A total of 21 BRCA1/2 mutations that had available specific mutational information from literature reviews of KoOC patients (with or without breast cancer) from previously reported studies, including case reports, were collected. 11Y15 We tried to find a founder mutation among these 21 mutations and the 18 BRCA1/2 mutations from the present study (a total of 39 mutations). The results of our analysis indicate that the 1041_1043delAGCinsT BRCA1 mutation (n = 4; 10.2%), which was repeated twice in the present study, and the 3746insA BRCA1 mutation (n = 4; 10.2%) were considered the most potential founder mutations in KoOC (Table 5). The latter mutation was reported 3 times in Lim et al 11 and once in Kim et al 13 was not reported in the present study. In addition, the 2552delC (n = 3; 7.7%) and 509C 9 A (n = 3; 7.7%) were also considered a candidate for founder mutation. Among the BRCA2 mutations, only the 5804_5807delTTAA mutation was repeated twice (n = 2; 5.1%). DISCUSSION In the current study, we found 18 BRCA1/2 mutations and 17 UVs in 70 KoOC patients who underwent genetic testing. Five of the 18 BRCA1/2 mutations were not reported in the BIC, and 2 of these were novel mutations. Four of the 17 UVs were also not reported in the BIC, and 2 of them were novel. One BRCA2 UV (8665_8667delGGA) was strongly suspicious to be a pathogenic mutation after co-segregation in FDRs with breast cancer (Table 2). The prevalence of BRCA1/2 mutations in sporadic ovarian cancer is known to vary by ethnicity and country. 16 Some studies have reported the prevalence of sporadic KoOC to be 2.7% to 9.0%. 11,13 In the present study, sporadic KoOC was 13.5% (7/52), which is more comparable to the prevalence of ovarian cancer in Europe than to the prevalence in Korea. The prevalence of BRCA1/2 mutations in KoOC patients with FH has been reported to be 33% to 43%, 11,12 and 55% in Japanese patients. 10 In the present study, however, the prevalence of BRCA1/2 mutations in this group was 61.1% (11/18), which is higher than ever reported before. Because this is a small study, there might be effects of selection bias; nevertheless, the prevalence of BRCA1/2 mutations in KoOC patients is not lower than the prevalence in Western countries. In a large population-based series from North America, 17 the pathogenesis of high-grade serous ovarian cancer is likely to be related to BRCA1/2 genetic mutations. Eighteen BRCA1/2 mutations were found in the present study, all from patients with high-grade serous ovarian cancer (Table 4). We expect that if more studies are performed, BRCA1/2 mutations will also be found in nonyserous-type ovarian cancer patients, similar to the findings of other studies. 18 Analyses to identify possible founder mutations in KoOC were conducted (Table 5). The 1041_1043delAGCinsT BRCA1 mutation (n = 4; 10.2%), 3746insA BRCA1 mutation (n = 4; 10.2%), 11, delC (n = 3; 7.7%), and 509C 9 A BRCA1 mutations (n = 3; 7.7%) were considered as candidates for founder mutation in KoOC. The KOHBRA study has proposed the 7708C 9 T BRCA2 mutation to be the most possible founder mutation in Korean patients with breast cancer (18/148; 12.2%) in their study. 3 This mutation has been reported only once (1/ 39; 2.6%) in KoOC. 11 Other overlapping mutations are as follows: 509C 9 A (11/148; 7.4%), 3746insA (4/148; 2.7%), 5804_5807delTTAA (4/148; 2.7%), and 1041_1043delAGCinsT TABLE 5. Possible candidates for Korean founder mutation (total n = 39) Gene Site Mutation Mutation Type n % BRCA1 Exon _1043delAGCinsT Nonsense BRCA1 Exon insA Frameshift BRCA1 Exon delC Frameshift BRCA1 Exon 7 509C 9 A Nonsense BRCA1 Exon IVS G 9 A Splicing BRCA2 Exon _5807delTTAA frameshift BRCA1 Exon insT Frameshift * 2015 IGCS and ESGO 5

6 Choi et al International Journal of Gynecological Cancer & Volume 00, Number 00, Month 2015 (3/148; 2%). 3 It would seem that the possible founder mutations between the 2 types of cancer exhibit different aspects. Studies to find founder mutation are underway in various Asian countries 4Y10 ; however, specific mutations that account for a high frequency of cases, such as that seen in Europeans and the Jews, 16 have not yet been discovered. Identification of founder mutations is an important and primary step toward the improvement of genetic counseling. It also makes it possible to use a more specific approach to molecular testing that is more cost-effective. If a larger number of cases are analyzed, it might provide highly accurate information about the frequency of founder mutations. Evidence of differences in susceptibility and in age of onset of cancer among carriers of a specific founder mutation could also make it possible to define the role of riskreducing management. There are some limitations of the current study. This is a single institutional study that has a limited number of patients (n = 70). Thirty-two (31.4%) of 102 patients declined the test for several reasons: (1) economic status, (2) afraid of results, (3) disbelief, (4) refusal by family, (5) no children, and (6) no certainty. One report emphasized the barriers to participating in genetic counseling and BRCA testing even in Western country and indicated the testing rate as 81%. 19 Compared with the testing rate, the lower 68.6% (70/102) in present study seems to be resulted in immature public awareness of genetic tests. Because there might be some selection bias and referral bias, larger prospective studies are necessary to identify the definite prevalence of BRCA1/2 mutations in the Korean population. This is, therefore, a very preliminary report in the field of Korean BRCA1/2 mutation research. Unlike KOHBRA, the study for Korean hereditary ovarian cancer is being conducted at a very basic level. There has been some research about the association of KoOC with BRCA1/2 mutations, 11Y13 but there are no convincing data yet. Therefore, many future research directions in this field exist, such as (1) application of poly (ADP-ribose) polymerase inhibitor (olaparib) to patients with BRCAness ovarian cancer, 20 (2) management of the risk to unaffected BRCA carriers, (3) BRCA mutation prevalence in other pelvic cancers (fallopian tubal cancer, peritoneal carcinoma, among others), and (4) other hereditary mutations in BRCA-negative patients with FH. 21 A nationwide, multicenter collaboration study is needed for further research. We hope that this work will become a cornerstone in the research on BRCA1/2 in KoOC. REFERENCES 1. Miki Y, Swensen J, Shattuck-Eidans D, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994;226:66Y Wooster R, Bignell G, Lancaster J, et al. Identification of the breast cancer susceptibility gene BRCA2. Nature. 1995;378:789Y Han SA, Park SK, Ahn SH, et al. The Korean Hereditary Breast Cancer (KOHBRA) study: protocols and interim report. Clin Oncol. 2011;23:434Y De Leon Matsuda, Liede A, Kwan E, et al. BRCA1 and BRCA2 mutations among breast cancer patients from the Philippines. Int J Cancer. 2002;98:596Y Hedau S, Jain N, Husain SA, et al. Novel germline mutations in breast cancer susceptibility genes BRCA1, BRCA2 and p53 gene in breast cancer patients from India. Breast Cancer Res Treat. 2004;88:177Y Khoo US, Chan KY, Cheung AN, et al. Recurrent BRCA1 and BRCA2 germline mutations in ovarian cancer: a founder mutation of BRCA1 identified in the Chinese population. Hum Mutat. 2002;19:307Y Yassaee VR, Zeinali S, Harirchi I, et al. Novel mutations in the BRCA1 and BRCA2 genes in Iranian women with early-onset breast cancer. Breast Cancer Res. 2002;4:R6. 8. Rashid MU, Zaidi A, Torres D, et al. Prevalence of BRCA1 and BRCA2 mutations in Pakistani breast and ovarian cancer patients. Int J Cancer. 2006;119:2832Y Ikeda N, Miyoshi Y, Yoneda K, et al. Frequency of BRCA1 and BRCA2 germline mutations in Japanese breast cancer families. Int J Cancer. 2001;91:83Y Sekine M, Nagata H, Tsuji S, et al. Mutational analysis of BRCA1 and BRCA2 and clinicopathologic analysis of ovarian cancer in 82 ovarian cancer families: two common founder mutations of BRCA1 in Japanese population. Clin Cancer Res. 2001;7:3144Y Lim MC, Kang S, Seo SS, et al. BRCA1 and BRCA2 germline mutations in Korean ovarian cancer patients. J Cancer Res Clin Oncol. 2009;135:1593Y Kang HC, Kim IJ, Park JH, et al. Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families. Hum Mutat. 2002;20: Kim YT, Nam EJ, Yoon BS, et al. Germline mutations of BRCA1 and BRCA2 in Korean sporadic ovarian carcinoma. Gynecol Oncol. 2005;99:585Y Kim HS, Lee SW, Choi YJ, et al. Novel germline mutation of BRCA1 gene in a 56-year-old woman with breast cancer, ovarian cancer, and diffuse large B-cell lymphoma [online publication ahead of print]. Cancer Res Treat doi: / crt Yim SH, Lee KH, Lee AW, et al. One Korean patient with a family history of BRCA1-associated ovarian cancer. J Genet Med. 2009;6:179Y Ramus SJ, Gayther SA. The contribution of BRCA1 and BRCA2 to ovarian cancer. Mol Oncol. 2009;3:138Y Pal T, Permuth-Wey J, Betts JA, et al. BRCA1 and BRCA2 mutations account for a large proportion of ovarian carcinoma cases. Cancer. 2005;104:2807Y Lakhani SR, Manek S, Penault-Llorca F, et al. Pathology of ovarian cancers in BRCA1 and BRCA2 carriers. Clin Cancer Res. 2004;10:2473Y Kathryn JS, Herman FJ, Carla CW, et al. Barriers to participating in genetic counseling and BRCA testing during primary treatment for breast cancer. Genet Med. 2007;9:766Y Suh DH, Kim JW, Kang S, et al. Major clinical research advances in gynecologic cancer in J Gynecol Oncol. 2014;25:236Y Minion LE, Dolinsky JS, Chase DM, et al. Hereditary predisposition to ovarian cancer, looking beyond BRCA1/ BRCA2 [online publication ahead of print]. Gynecol Oncol pii: S (15) doi: / j.ygyno * 2015 IGCS and ESGO

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