Germline BRCA Mutation and Clinical Outcomes in Breast Cancer Patients Focusing on Survival and Failure Patterns: A Long-Term Follow-Up Study of Koreans

Background and Objectives: This study aimed to evaluate the effect of a BRCA mutation on survival and failure patterns, focusing on the risk of ipsilateral recurrence and contralateral breast cancer in patients. Materials and Methods: We retrospectively reviewed medical records of 300 patients with breast cancer who underwent genetic screening for BRCA1/2 genes and were treated at Samsung Medical Center between 1 January 2000 and 31 December 2010. Ultimately, clinical outcomes of 273 patients were analyzed. Results: The median follow-up duration was 102 months (range, 1 to 220 months). Patients with BRCA1/2-mutated tumors had a shorter 10-year disease-free survival (DFS) rate compared to those with non-mutated tumors (62.8% vs. 80.0%, p = 0.02). Regarding failure patterns, patients with BRCA1/2-mutated tumors showed a higher incidence of contralateral breast cancer than those with non-mutated tumors (BRCA1/2 non-mutated vs. mutated tumors: 4.9% vs. 26.0%, p < 0.001). BRCA mutation status remained a significant prognostic factor for contralateral breast recurrence-free survival (HR: 4.155; 95% CI: 1.789–9.652; p = 0.001). Korean patients with a BRCA mutation showed inferior DFS compared to those without a BRCA mutation. Conclusions: BRCA mutation status is a strong predictor of recurrence in contralateral breast cancer. Strategies such as prophylactic treatment and active surveillance should be discussed with breast cancer patients who have a BRCA mutation.


Introduction
A BRCA mutation is a mutation in either the BRCA1 or BRCA2 gene, both of which are tumor-suppressor genes. A harmful mutation in these genes might provoke a hereditary breast-ovarian cancer syndrome in affected persons. BRCA1 and BRCA2 mutations have been found in 5-10% of all breast cancers and in up to 20-25% of tumors in patients with a family of breast and/or ovarian cancer [1]. However, the impact of these gene mutations on women might be more profound.
According to previous studies on BRCA mutations, BRCA1 mutation carriers are more likely to be triple negative with a lower estrogen receptor level, higher histological grade, and higher proliferation index than patients who have no such mutation. On the other hand, BRCA2 mutation carriers are more likely to be estrogen-receptor positive, similar to those with sporadic tumors [2][3][4][5]. Whether a BRCA mutation in breast cancer is associated with poor prognosis remains controversial. Some studies have demonstrated that BRCA1/2 mutation carriers have a worse survival outcome [6], while others Medicina 2020, 56, 514 2 of 11 have shown that BRCA1/2 mutation carriers have similar or better survival than non-carriers [7][8][9]. In meta-analysis, BRCA1 mutation carriers show decreased overall survival (OS) and progression-free survival (PFS) while BRCA2 mutation carriers do not. In terms of failure patterns, several studies have suggested that the recurrence rate in BRCA1/BRCA2 mutation carriers is not increased compared to that in non-carriers [10][11][12][13]. Other studies have compared ipsilateral and/or contralateral breast recurrence in BRCA1 and BRCA2 mutation carriers and patients with sporadic cancers. These studies have consistently found an elevated risk of contralateral breast cancer in BRCA mutation carriers [14][15][16]. However, whether the risk of ipsilateral recurrence is higher in women with a BRCA mutation remains controversial. Current treatment for BRCA mutation-associated breast cancer is not different from that for sporadic breast cancer.
The purpose of this study was to evaluate the effect of a BRCA mutation on survival and recurrence rate, focusing on risk of ipsilateral recurrence and contralateral breast cancer in breast cancer patients who underwent genetic screening for a BRCA1/2 mutation and were treated at the Samsung Medical Center.

Patients
After obtaining approval from our Institutional Review Board (IRB), we retrospectively reviewed medical, pathology, and radiotherapy records of 300 patients with breast cancer who underwent genetic screening for BRCA1/2 mutation and were treated at Samsung Medical Center between 1 January 2000 and 31 December 2010. Genetic screening was performed for those who met the criteria of National Health Insurance System of Korea, including breast cancer with family history, bilateral breast cancer, breast cancer with family history of ovarian cancer, male breast cancer, and diagnosed before 40 years old. Among these, patients with bilateral breast cancer (25 patients) and those with history of previous or concurrent malignancy except for thyroid cancer at the time of diagnosis (n = 2) were excluded from this study. Ultimately, clinical outcomes of 273 patients were analyzed, with emphasis on the recurrence rate, including risks of ipsilateral recurrence and contralateral breast cancer according to BRCA mutation status. All tumors were staged based on the seventh edition of the American Joint Committee on Cancer (AJCC) tumor staging criteria. Revised Response Evaluation Criteria In Solid Tumors (RECIST) guidelines (version 1.1) were used for tumor response evaluation.

Treatment Scheme
After genetic screening, 218 (79.9%), 42 (15.4%), and 13 (4.7%) patients underwent breast-conserving surgery (BCS), modified-radical mastectomy (MRM), and total mastectomy (TM), respectively. Sentinel lymph node biopsy (SLNB) and axillary lymph node dissection (ALND) were performed in 141 (51.6%) and 121 (43.3%) patients, respectively. The remaining 11 (5.1%) patients who were diagnosed with ductal carcinoma in situ underwent BCS only without lymph node sampling. In the current study, none of the patients underwent prophylactic contralateral mastectomy. The median number of sampled lymph nodes (LNs) was 8 (range, 1-49). Neoadjuvant chemotherapy was delivered to 17 (6.2%) patients while adjuvant chemotherapy was delivered to 190 (69.6%) patients except for three patients who were indicated but refused further treatment. The most common regimen was anthracycline plus cyclophosphamide, followed by taxane. All patients who were hormone receptor positive at initial clinical diagnosis (n = 193, 70.7%) received adjuvant hormone treatment. Among patients who were HER2/neu receptor positive at initial diagnosis (n = 37, 13.6%), 27 received adjuvant herceptin treatment. Adjuvant radiotherapy was delivered to 234 (85.7%) patients after surgery or at the end of chemotherapy. RT dose delivered to the whole breast or chest wall was most frequently at 50 Gy in 25 fractions. Later, the primary tumor bed was boosted most commonly with 9-15 Gy in 3-5 fractions according to the surgical resection margin status.
BRCA mutation analysis was conducted mainly at the Department of Laboratory Medicine and Genetics at Samsung Medical Center with the cooperation of three other DNA testing laboratories, all of which are certified annually by the Korean Institute of Genetic Testing Evaluation. Genomic DNA was extracted and purified from peripheral blood leukocytes. The whole exons and the flanking intrinsic sequences of the BRCA1 gene or BRCA2 gene were amplified by polymerase chain reaction. The amplified products were directly sequenced, and the sequences were then compared with reference sequences using Sequencher software (Gene Codes Co., Ann Arbor, MI, USA). The nomenclature for BIC (Breast Cancer Information Core) traditional mutations was used, based on U14680 (BRCA1) and U43746 (BRCA2). In addition, all mutations were described according to HUGO-approved systematic nomenclature (nomenclature for the description of sequence variations, Human Genome Variation Society. http://www.hgvs.org/mutnomen/). HUGO-approved mutation nomenclature of BRCA1 (GenBank accession no. NP_009225.1) and BRCA2 (GenBank accession no. NP_000050.2) defined the A of the ATG translation initiation codon as nucleotide +1. Splicing-defect mutations in intronic region were described at the genomic DNA level using GenBank genomic reference sequence NC_000017.10 (BRCA1) and NC_000013.10 (BRCA2). In addition, variants of unknown significance were excluded. Genetic testing of high-risk breast cancer patients was approved by the IRB of Samsung Medical Center (2010-09-006-001).

Statistical Analysis
Overall survival (OS) was defined as the time from the date of the surgery until the date of death from any cause or the latest documented follow-up. Disease-free survival (DFS) was defined as the time from the date of the surgery until the date of the first documented recurrence or the latest follow-up. To compare clinicopathologic characteristics according to BRCA mutation status, Chi-squared or Fisher's exact tests was used. Survival rates were estimated using the Kaplan-Meier method and compared using log-rank tests. Factors that showed a probability value of <0.1 and those that were thought to be relevant were entered into a Cox proportional hazard regression analysis to determine independent prognostic factors. A p ≤ 0.05 was regarded as indicative of statistical significance in two-tailed tests. Statistical analysis was performed using SPSS software, standard version 24.0 (IBM Corporation, Armonk, NY, USA).

Pathologic Characteristics According to BRCA Mutation Status
Pathologic characteristics according to BRCA mutation status are described in Table 1. BRCA1/2-mutated tumors and molecular subtypes showed significant correlations (p = 0.001).

Discussion
Patients with deleterious mutations in either BRCA1 or BRCA2 have about five times higher risk of breast cancer that those without such mutations. However, the effect of BRCA mutation on survival and the recurrence rate with emphasis on the risk of ipsilateral recurrence and contralateral breast cancer in breast cancer patients remains controversial. Through this single institutional study with an overall median follow-up of 8.5 years, we showed that BRCA1/2-mutated tumors were associated with negative hormonal and HER2/neu and positive Ki-67 receptor status, and higher histologic grade compared to non-mutated tumors. A significant correlation between BRCA mutation and the risk of secondary ovarian cancer was also found (non-mutated vs. BRCA1/2-mutated tumors: 2.2% vs. 14.0%, p = 0.001).
In the aspect of BRCA mutation status as a prognostic factor, many previous studies have shown conflicting results. Specifically, Robson et al. [6] have reported that 10-year breast-cancer-specific survival is significantly worse in BRCA1 mutation carriers than that in non-carriers (62% vs. 86%, p < 0.001), but not in BRCA2 mutation carriers (84% vs. 86%, p = 0.76). However, BRCA1 mutation status was predictive of a worse outcome in those who did not receive chemotherapy. Similarly, Goodwin et al. [13] have proved that the survival of BRCA1 carriers who are administered chemotherapy is similar to that of non-carriers, although the survival of BRCA1 carriers is worse in the absence of chemotherapy (HR: 1.97; 95% CI: 0.65 to 5.94). In contrast, Rennert et al. [7] have shown no difference in 10-year survival rates among BRCA1 mutation carriers, BRCA2 mutation carriers, and non-carriers. Brekelmans et al. [8,9] have also reported that breast-cancer-specific survival is not different between BRCA1-mutation carriers and sporadic controls (HR: 1.29, 95% CI: 0.85-1.97). Later, Huzarski et al. [18] performed a study to estimate 10-year OS rates for patients with early-onset breast cancer with or without BRCA1 mutation and identify prognostic factors among those with BRCA1-positive breast cancer. They concluded that the survival rate among women with BRCA1 mutation was similar to that of patients without BRCA1 mutation (p = 0.41). Among BRCA1 mutation carriers, positive lymph node status was a strong predictor of mortality (adjusted HR: 4.1; 95% CI: 1.8 to 8.9; p < 0.001). A recent prospective cohort study [19] has shown that breast cancer patients with young-onset who carry a BRCA mutation have similar survival to non-carriers. Results from the Danish Breast Cancer Group [20] also confirmed that BRCA mutation was not associated with OS (adjusted HR: 1.98, 95% CI: 0.87-4.52, p = 0.10) while BRCA1 breast cancer patients had shorter ten-year DFS than BRCA2 BC patients. Based on these studies, BRCA mutation status was not regarded as an independent predictor for clinical outcome of breast cancer. Against this background, Wang et al. [21] reported that BRCA mutation carriers were more likely to be diagnosed with breast cancer with lymph node involvement (66.7% vs. 42.6%; p = 0.011) and had significantly worse breast-cancer-specific outcomes. The 5-year disease-free survival was 73.3% for BRCA mutation carriers and 91.1% for non-carriers (hazard ratio for recurrence or death 2.42, 95% CI 1.29-4.53; p = 0.013). After adjusting for clinical prognostic factors, BRCA mutation remained an independent poor prognostic factor for cancer recurrence or death (adjusted hazard ratio 3.04, 95% CI 1.40-6.58; p = 0.005). Non-BRCA gene mutation carriers did not exhibit any significant difference in cancer characteristics or outcomes compared to those without detected mutations. In the current study, we confirmed that 10-year OS rate did not show significant difference according to BRCA mutation status (non-mutated versus BRCA1/2-mutated tumors: 96.2% vs. 98.0%, p = 0.844), similar to previous studies. However, we proved that non-mutated tumors and BRCA1/2-mutated tumors showed significant difference in 10-year DFS rates (80.0% vs. 62.8%, p = 0.02, Figure 1). Specifically, BRCA1 mutation was associated with decreased DFS (non-mutated versus BRCA1-mutated tumors: 78.6% vs. 62.1%, p = 0.031), while BRCA2 mutation was not (p = 0.197). As a prognostic factor, clinical N stage remained a significant prognostic factor for DFS (HR: 2.078; 95% CI: 1.043-4.143; p = 0.038) on multivariate analysis (Table 4). BRCA mutation status remained a significant prognostic factor for contralateral breast RFS (HR: 4.155; 95% CI: 1.789-9.652; p = 0.001) on multivariate analysis (Table 4).
In terms of failure patterns, many studies have compared ipsilateral and/or contralateral tumor recurrence in BRCA1 and BRCA2 mutation carriers and patients with sporadic cancers. Studies have consistently identified an elevated risk of contralateral breast cancer in BRCA mutation carriers. Haffty et al. [15] have reported a recurrence rate of 42% in carriers versus 9% rate in non-carriers (p = 0.001) at 12 years. They suggest that the high risk of contralateral breast cancer in BRCA1/BRCA2 mutation carriers must be taken into account when choosing treatment. In contrast, reports regarding whether the risk of ipsilateral recurrence is higher in women with a BRCA mutation have shown conflicting results. Kirova et al. [22] investigated whether mutation status can influence the rate of ipsilateral and contralateral breast cancers after breast-conserving treatment. They found no significant difference in rates of ipsilateral tumors among mutation carriers, non-carriers, and controls (p = 0.13). On multivariate analysis, age was the most significant predictor for ipsilateral recurrence (p < 0.001). The rate of contralateral cancer was significantly higher in familial cases: 40.7% (mutation carriers), 20% (non-carriers), and 11% (controls) (p < 0.001). After 13.4 years of follow-up, the rate of ipsilateral tumors was not higher in mutation carriers than that in non-carriers or controls. The present study also confirmed that BRCA1/2-mutated tumors showed a higher incidence of contralateral breast recurrence compared to non-mutated tumor (non-mutated versus BRCA1/2-mutated tumors: 4.9% vs. 26.0%, p < 0.001). Statistically-significant difference in 10-year contralateral breast RFS rates was found between non-detected and detected groups (95.0% vs. 73.7%, p < 0.001). However, no significant difference was found in the incidence of ipsilateral breast recurrence (4.5% vs. 6.0%, p = 0.649) or 10-year ipsilateral breast RFS rates (93.8% vs. 93.0%, p = 0.926). Additionally, there was no difference in the rate of regional recurrence (p = 0.991) or distant metastasis (p = 0.657) between the two groups.
Our current study has several limitations. First, it was a retrospective study and there might have been selection bias. Second, the sample size was too small to determine the statistical significance of associations between the two groups. Nonetheless, our current study demonstrated that patients having a BRCA mutation, especially BRCA1 mutation, showed inferior DFS, although it did not reach statistical significance. BRCA mutation status was associated with higher risk of contralateral breast cancer. It was also a significant prognostic factor for contralateral breast RFS, although the rate of ipsilateral recurrence was not higher. Thus, clinicians should inform about the role of prophylactic contralateral mastectomy, prophylactic oophorectomy, tamoxifen administration, and/or active surveillance with close radiological surveillance to breast cancer patients with a BRCA mutation. Recently, PARP inhibitors have been investigated not only as chemo/radiotherapy sensitizers, but also as single agents to selectively kill cancers defective in DNA repair, specifically cancers with mutations in BRCA1/2 genes.

Conclusions
Korean patients having a BRCA mutation, especially BRCA1 mutation, showed inferior DFS, despite such interiority not reaching statistical significance. BRCA1/2-mutated tumors herald a higher risk of contralateral breast cancer. BRCA mutation status was a strong predictor of recurrence in contralateral breast. Strategies such as prophylactic treatment and active surveillance should be discussed with BRCA mutation-associated breast cancer patients. To determine the effect of BRCA1 or BRCA2 mutation on breast cancer survival, further study with larger scale is needed.