Characteristics of breast cancer patients tested for germline BRCA1/2 mutations by next‐generation sequencing in Ramathibodi Hospital, Mahidol University

Abstract Introduction Germline mutations in BRCA1/2 are the most common cause of hereditary breast and ovarian cancer (HBOC) syndrome. Few studies published during the past decade reported the prevalence of germline BRCA mutations in Asian patients with breast cancer. We aimed to assess the prevalence and characteristics of Thai patients with breast cancer with germline BRCA1/2 mutations. Methods We retrospectively reviewed all breast cancer patients who were tested for germline BRCA1/2 mutations during 2014–2018. BRCA mutations were detected using next‐generation sequencing and confirmed using Sanger sequencing. We analyzed the characteristics of patients with or without BRCA mutations. Disease‐free survival (DFS) and the associated factors were determined. Results Among 67 patients, 12 (18%) were BRCA1/2 carriers (6 each), 4 (6%) harbored variants of uncertain significance, and 51 (76%) were non‐carriers. We discovered two novel BRCA2 frameshift mutations (c.2380delA and c.8855dupT). Mean ages at breast cancer diagnosis of BRCA1, BRCA2, and non‐carriers were 39.8, 46.2, and 42.0 years, respectively. The 12 tumors of BRCA carriers were mainly the luminal‐B subtype. Two of these tumors were HER2‐positive luminal‐B, and the triple‐negative subtype was not detected. After adjusting for stages and luminal subtypes, BRCA carriers experienced worse 3‐year DFS than non‐carriers (81.5% vs. 90.3%, HR 2.04 [0.64–6.49], p = .229). The stage at diagnosis was the sole factor significantly associated with 3‐year DFS (100%, 84.8%, and 72.7%; stages I, II, and III, respectively). Conclusion Thai patients with breast cancer with BRCA1/2 mutations were mainly the luminal‐B subtypes with worse prognosis than those without mutations.


| INTRODUCTION
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females in most countries. 1 In Thailand, breast cancer is the third most frequent cancer and the third most common cause of cancer death of both sexes. 2 Breast cancer, which primarily occurs in women and 1% of men, is typically acquired through multistep accumulations of somatic mutations, whereas 5%-10% of breast cancers are inherited through germline mutations. Germline mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are the most common causes of hereditary breast and ovarian cancer (HBOC) syndrome. 3 A large prospective study found that women who inherit deleterious germline mutations of BRCA1 or BRCA2 have very high cumulative risks for developing breast cancer (e.g., risks to 80-year-olds are 72% and 69% for BRCA1 and BRCA2 carriers, respectively). 4 The main characteristics of patients with breast cancer with BRCA mutations include a family history of breast cancer, younger age at diagnosis, male breast cancer, or multiple tumors (bilateral breast cancer or breast and ovarian cancer) in the same patient. 5 Furthermore, patients with BRCA1 mutations are most commonly younger at diagnosis and associated with the triple-negative breast cancer (TNBC) subtype. In contrast, patients with BRCA2 mutations are mainly associated with estrogen receptor (ER)-positive breast cancer. [6][7][8] Furthermore, a more advanced stage at diagnosis and the presence of multiple foci of breast cancers are more common in patients with BRCA1 and BRCA2 mutations than in patients with sporadic tumors.
Abundant data show that patients with breast cancer with BRCA1/2 mutations (BRCA carriers) experience higher mortality rates than noncarriers. 5,8 BRCA1 and BRCA2 are tumor suppressor genes located at chromosomes 17q21 and 13q12.3, respectively. 9-12 BRCA1 and BRCA2 proteins are essential for repairing DNA-double strand breaks (DSBs) by homologous recombination, cell growth regulation, and control of cell division. 3,13,14 Genetic alterations of these genes occur in 5% of all breast cancers and 15%-25% of familial breast cancers worldwide. 7,15 The prevalence and phenotype of BRCA mutations vary according to country and race. The prevalence of BRCA mutations depends on the risk of breast cancer development; namely, a low prevalence in patients with sporadic breast cancer but higher in selected high-risk cases such as breast cancer with a strong family history, bilateral breast cancer, and multiple organ cancer. Only a few studies have investigated the prevalence of BRCA mutations in Asian patients with breast cancer. Data from Korea shows a BRCA1/2 prevalence of 8.9% in high-risk patients without family history and 22.3% in patients with family history. 16 In China, the prevalence of BRCA1/2 in high-risk patients is 9.1%, but only 3.5% in sporadic breast cancer patients. 17

| Analysis of germline mutations in BRCA1 and BRCA2 detected by NGS
Genomic DNA was extracted from the peripheral blood of patients.
Analyses of germline mutations of BRCA1 and BRCA2 were performed using NGS at the CMG. All coding regions in BRCA1 and BRCA2 were sequenced, and aligned with the Homo sapiens genome assembly GRCh37 (hg19) published by the Genome Reference Consortium. 23,24 Sequence variants were classified in decreasing order of clinical importance as "Pathogenic," "Likely pathogenic," "Variants of Uncertain Significance (VUS)," "Likely benign," or "Benign," according to the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines. 25 Selected variants identified as "Pathogenic" or "Likely pathogenic" were verified using Sanger sequencing. All Sanger sequencing results confirmed that the selected Pathogenic/ Likely pathogenic variants were genuinely present in the samples.

| Statistical analysis
Descriptive statistical analysis was used to describe patients' charac-   in four of 67 (6.0%) patients. All were missense mutations, and one of the patients carried two VUS. VUS were not detectable in BRCA1 ( Figure 2).

| CONCLUSION
In summary, this study describes the characteristics, treatment outcomes, and prognostic factors of patients with breast cancer treated at a single university hospital in Thailand. We found that breast cancer in BRCA carriers was significantly associated with the luminal-B subtype. For clinical outcome, the difference of DFS between BRCA1, BRCA2 carriers, and non-carriers cannot be demonstrated from this small sample size with a short follow-up cohort. A longer follow-up of this study is required to determine the long-term survival outcomes.

AUTHOR CONTRIBUTIONS
Methodology, Formal analysis, Visualization, Writing-initial draft, and Conceptualization, Writing-Review and editing, and contributed to later drafts of the manuscript, R.P. All authors analyzed the results, read, and approved the manuscript for submission.