Prevalence and clinical characterization of BRCA1 and BRCA2 mutations in Korean patients with epithelial ovarian cancer

Abstract This study was performed to investigate the prevalence, clinical characteristics, and treatment response according to BRCA1 and BRCA2 (BRCA) mutations in Korean patients with epithelial ovarian cancer (EOC). Two‐hundred and ninety‐eight Korean women diagnosed with high‐grade serous and/or endometrioid EOC from 2010 to 2015 were tested for germline and 86 specimens for somatic BRCA mutations, regardless of the family history. Clinical characteristics including survival outcomes were compared in patients with and without BRCA mutations (NCT02963688). A total of 43 different germline BRCA mutations were identified in 78 patients among 298 patients (26.2%). Somatic BRCA mutations were identified in 11 (12.8%) patients among patients without germline BRCA mutations. Haplotype analysis demonstrated no founder mutations in our Korean patient cohort. Insignificant differences in age at diagnosis, primary site, and residual disease after surgery were observed between patients with and without BRCA mutations. In multivariate analysis for overall survival (OS), the presence of BRCA mutation was significantly associated with OS (P = .049) in addition to platinum sensitivity (P < .001), indicating it is an independent prognostic factor for survival regardless of platinum sensitivity to first‐line chemotherapy. In addition, a higher response rate to subsequent chemotherapy after recurrence was observed in EOC patients with BRCA mutations resulting in better OS. In the current study, the prevalence of BRCA mutations in Korean patients with EOC was higher than previously reported in other ethnic groups. We demonstrated characteristics and treatment response in Korean EOC patients with BRCA mutations. These findings may provide valuable information to be considered in future clinical trials including Asian patients.


| INTRODUC TI ON
Epithelial ovarian cancer (EOC) is the second leading cause of death related to gynecologic cancers and the eighth most common leading cause of death from cancer in women worldwide. 1 The standard therapy for EOC consists of surgical cytoreduction and adjuvant treatment with taxane and platinum-based chemotherapeutic agents.
Despite a positive response to taxane and platinum-based chemotherapeutic agent in patients with advanced EOC, most patients experience relapse. 2 Repeated chemotherapy in recurrent EOC patients diminishes effectiveness and leads to cumulative increased toxicity. 3 Recent trends in treatment for gynecological cancer fields include incorporation of molecular target therapy in chemotherapy. PARPi (olaparib, rucaparib, and niraparib) showed its efficacy in EOC patients for the maintenance therapy of relapsed platinum-sensitive EOC. [7][8][9] In addition, PAPRi, in single or multiple combinations including anti-angiogenic agents and immune check-point inhibitors, is under extensive investigation in clinical trials in patients with firstline setting of EOC as well as in recurrent settings. However, there is still a need for investigation of predictive molecular biomarkers and the best combination for PARPi in the treatment of EOC patients.
Approximate frequency of gBRCAm in EOC widely vary from 5% to 30%, 10,11 and less frequent occurrence of sBRCAm (2%-8% [12][13][14] has been reported in patients with EOC. EOC patients with gBRCAm were reported to have better survival outcome compared to patients without gBRCAm, and the mechanism underlying this benefit was hypothesized as a high response rate to platinum agents, particularly in patients with high-grade serous type. 10,15 The reason is that patients with gBRCAm are impaired in their ability to repair doublestranded DNA breaks through homologous recombination (HR). 16 Despite the discovery of BRCA more than 20 years ago, almost all the available data are from women in the United States, Europe, and Australia, and there have only been a few studies, mostly with small sample size, in Asian populations. 12,[17][18][19][20][21][22] Recently, many international clinical trials have been recruiting Asian patients and several clinical trials have exhibited different outcomes between European and Asian EOC patients, for example pazopanib maintenance therapy 23,24 and dose dense weekly paclitaxel plus tri-weekly carboplatin chemotherapy in first-line treatment in ovarian cancer. 25,26 BRCAm has recently become known as a prognostic factor in EOC, therefore it is necessary to understand more fully its prevalence and clinical characteristics, including the treatment response of EOC patients with BRCAm in Asian populations, through a larger scale study and comprehensive analysis of its clinical characterization.
Thus, we evaluated the clinical characterization and treatment response of EOC patients with the BRCA mutation in 298 Korean patients with high-grade serous and/or endometrioid EOC.

| Clinical characteristics assessment
Epithelial ovarian cancer patients routinely underwent primary cytoreductive surgery followed by six to nine cycles of adjuvant intravenous taxane and platinum-based chemotherapy, or patients were treated with three cycles of neoadjuvant chemotherapy followed by interval debulking surgery and an additional three to six cycles of taxane and platinum-based chemotherapy according to the physician's clinical decision. After initial treatment, patients were routinely followed up every 3 months for the first year, then every 6 months for up to 5 years, and annually thereafter. Patients were monitored based on clinical symptoms, laboratory tests, and imaging examinations. For treatment for recurrent EOC, a taxane and platinum-based regimen was used for the platinum-sensitive group and a nonplatinum-based chemotherapy regimen (topotecan/ belotecan, pegylated liposomal doxorubicin, etoposide, and others) was used for the platinum-resistant group. Since PARPi was not used as standard treatment during the period in which the study subjects were being treated, chemotherapy was the main treatment. There was no difference in treatment regimen for recurrence between patients with and without BRCAm in this study.
Clinical and pathologic data were collected from the KGOG 3019 database and patient medical records: family cancer history for second-degree relevant, personal cancer history, age at diagnosis, surgical outcome at primary cytoreductive surgery/interval debulking surgery, tumor histology, stage of disease, chemotherapy regimen (first and subsequent lines of treatment), and response assessments including CA-125 level/imaging results.
Surgical staging was assessed according to the International Federation of Gynecologist and Obstetricians (FIGO) staging system when diagnosed. Surgical outcomes were categorized as residual disease of 0-10 mm as optimal and >10 mm as suboptimal after debulking surgery. Platinum sensitivity was classified as two groups: platinum sensitive and platinum resistant. Regarding sensitivity for platinum chemotherapy, platinum sensitive was defined as more than 6 months of platinum-free interval and platinum resistance was

| Bioinformatic analysis and mutation prioritization
Data analysis including signal processing, base calling, read alignment, and coverage analysis was done using Torrent Suite software be/BRCA1/, http://www.umd.be/BRCA2/) were categorized as known mutations. 31,32 The "A" of the ATG translation initiation codon is described as position number 1 in the BRCA1 (NM_007294.2) and BRCA2 (NM_000059.3).

| Sanger sequencing
All of the prioritized mutations were validated with independent Sanger sequencing. The mutated DNA was amplified by PCR using primer pairs designed with Primer3 software. Relevant regions were sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems Foster City).

| RE SULTS
A flowchart outlining the study is shown in Figure 1. A total of 565 patients were enrolled to the KGOG 3019 from 2010 to 2015, and 298 patients were eligible for the current study: patients with EOC, peritoneal or fallopian tube primary tumor with high-grade serous or endometrioid cell type and consent to examine BRCA genotyping.
Median follow-up for all patients was 28.1 months.

| Patient characteristics
The patient characteristics of the current study and BRCAm status are shown in Table 1  In the present study, regarding optimality, described as residual disease after debulking surgery, a residual tumor less than 1 cm after primary debulking surgery was observed in 80.2% of patients.

| Prevalence of BRCA1/2 mutations
In the present study, germline mutation was observed in 26.2% of patients and somatic mutation in gBRCAm(-) specimens was observed in 12.8% (Table 1). All 298 patients were tested for gBRCAm.
A total of 49 different mutations were identified in 78 patients. The germline mutation profile of 78 patients is shown in

| Survival outcomes
For more a homogenous group to assess the accurate results for survival analysis and treatment response, we excluded patients who   Figure S1).

| Treatment response
The number of patients by the chemotherapy regimen type (platinum-based/non platinum-based) in BRCAm(+) and BRCAm(-) group is shown in Table S3. There was no difference in treatment regimen between patients with BRCAm(+) and BRCAm(-) for the second (P = .272) and third (P = .316) regimens in this study. The ORR of first-line to third-line chemotherapy of patients was assessed by BRCAm status (Figure 3 and Table S3). There was no statistical difference in ORR in first-line chemotherapy between BRCAm(+) and

| D ISCUSS I ON
This study was a large-scale nationwide next-generation sequencing (NGS)-based germline and somatic BRCA mutation prevalence study with clinical outcomes including treatment response in Korean ovarian cancer patients regardless of family history and age at diagnosis. We conducted germline and somatic BRCAm testing with whole blood and fresh frozen samples, respectively, using NGS analysis.
The combined prevalence of BRCA germline and somatic mutation in patients with EOC differed depending on the sample cohort and detection method. The prevalence of germline or somatic BRCA mutations in epithelial ovarian cancer with all histologic types is known to vary from 5% to 29% by ethnicity and country. 37 The reported frequency of BRCAm in patients with high-grade serous EOC varies between 19% and 30%. 13  proportion of BRCA mutations than BRCA2. 18,19,21,22,43,44 The high ratio for BRCA1 to BRCA2 mutation (4.6:1) in this study was interesting, but a larger study may be needed in the future. Founder mutations were shown in various ethnic groups, including Ashkenazi Jews, Icelanders, Russians, and Israelis. [45][46][47] Until recently, no founder mutations had been reported in Asian populations. [17][18][19][20]22,43 In this study, we also did not identify founder mutations. Therefore, genetic testing of BRCA1m and BRCA2m using full sequencing may  (Table 3 and Figure 2B), similar to previous reports. 10,11,15,36,51 BRCAm is known to be associated with better platinum response. 10,36,52 Several studies reported higher platinum response in the gBRCAm patient group even for first-line chemotherapy 10,36 and this effect may persist in subsequent lines of chemotherapy. Interestingly, our results were slightly different. In first-line chemotherapy, there was no difference platinum response between BRCAm(+) and BRCAm(-) patients ( Figure 3).
In second-line treatment, the platinum response of BRCAm(+) was There were several limitations in our study, including a number of limitations regarding gene analysis. First, we did not analyze a large genomic rearrangement (LGA). 12 Hasmad et al 43 reported that LGA was discovered in patients with negative for BRCAm using the sequencing mutation test, and the proportion of LGA was approximately 11% of the total BRCAm. An increasing number of LGA are being identified. 53 Second, analysis was not done F I G U R E 3 Comparison of overall response rate (ORR) in subsequent chemotherapy between BRCAm(+) and BRCAm(-) patients on whether BRCA gene inactivation was by epigenetic silencing. 13 Third, other genes associated with HR deficiency in patients with EOC were not analyzed. 11,13 Consequently, there is a possibility that a proportion of patients with BRCAm(-) might have HR-related gene alterations and they might show similar clinical outcomes, such as platinum sensitivity, to BRCAm(+) patients. Fourth, somatic BRCA mutation was tested only in patients without gBRCAm and with presence of available tissue. The family histories of the patients in this study were retrieved not from a genetic counselling interview but from questionaire and medical records. In addition, underestimation of the presence of actual familial history cannot be excluded due to separation or loss of families. Therefore, the real proportion of family history in Korean EOC patients may be higher than results in the current study. 22 Finally, the duration of follow-up of patients (28.1 months) was too short to provide accurate survival outcome, and not all enrolled patients were used in survival analysis.
The theory has been persistently raised that the penetrance and biology of BRCAm in Asian patients with EOC differ from that in the Caucasian population, resulting in different presentation of EOC. Furthermore, there is a suggestion that this depends on ethnicity and country. Currently, there are not sufficient data on the risk of EOC among Asian BRCAm carriers, but the risk of breast cancer seems to be lower than in Caucasians. 54 In this study, we demonstrated several differences to Caucasian group, such as comparable or higher prevalence of germline and/or somatic mutations, younger age at diagnosis of ovarian cancer resulting in no difference between age at diagnosis of EOC with or without BRCAm, high ratio of BRCA1m to BRCA2m, and a pattern of platinum sensitivity in the BRCAm group. However, the survival benefit and improved overall platinum sensitivity in the BRCAm group were similar to previous reports involving other ethnicities. Risk for cancer is affected by a number of factors, such as population incidence of cancer, birth cohort, genetic background, and reproductive and lifestyle factors, and these may alter the risk and presentation of EOC in Asian women. 54 There is therefore is a need to determine the risk of cancer and calibrate risk assessment methods for Asian women. The current study provides valuable insight and information about the role and presentation of BRCAm in Asian as well as Korean patients with EOC. In a future international clinical trial for high-grade serous EOC, ethnicity including BRCAm and its clinical significance should be considered since this may affect the clinical outcome of investigational treatments.

ACK N OWLED G M ENTS
We thank the patients and all investigators involved. This study was funded by AstraZeneca.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.