The incidence of breast cancer (BC) has been rising and approaching 36.1/100,000 in China (1). Actionable genetic mutations account for 5-10% of BC occurrence (2–4). Genetic testing for BC patients might change traditional management paradigms to encompass personalized treatment strategies (5, 6). For instance, genetic testing for germline BRCA1/2 mutations has evolved to be a part of the standard clinical practice in patients with triple negative breast cancer (TNBC) (5, 7, 8). As a consequence, patients harboring deleterious mutations of BRCA1/BRCA2 genes are identified who may be highly sensitive to treatment with DNA-damaging agents such as platinum-based chemotherapy agents, topoisomerase II inhibitors and recent poly (ADP‐ribose) polymerase (PARP) inhibitors (7, 9). Multiple genes are associated with hereditary BC, most of which are involved in DNA damage repair pathways such as homologous recombination repair (HRR) and mismatch repair (MMR) (4, 9–11). Mutation carriers of BRCA1, BRCA2, CDH1, CHEK2, MSH2, and ATM account for 10% of all BC patients (2).

It is becoming increasingly evident that germline BC mutations may vary across ethnicities (12, 13). For example, founder mutations in BRCA1 (187delAG and 5385insC) and BRCA2 (6174delT) constitute more than 90% of mutations in Ashkenazi Jews, but occur less frequently in other populations (14–16). Distinct BRCA1 c.5470_5477delTGCCCAAT and BRCA1 c.981_982delAT occur frequently in Chinese individuals, suggesting that they are potential founder mutations in the Chinese population (5, 10, 13). The frequencies of gene mutations also differ by ethnic groups. The frequency of BRCA1/2 gene mutations in Ashkenazi Jews is approximately 11.1% (16), but is lower in other populations, with an estimate of 5.3% in unselected Chinese patients (4) and 6.0% in unselected European patients (8), respectively. The frequencies in other breast cancer susceptibility genes such as CHEK2 and ATM are lower in East Asian populations than that of European populations (8, 10).

Currently the genetic epidemiology of BC is mainly based on data from Caucasian populations (17). Being the largest non-European population, the Chinese population is further diversified across regions (18). Whether currently available genetic information can guide clinical practice in the Chinese population remains underexplored. To elucidate the landscape of germline mutations in Chinese BC patients, we retrospectively analyzed the clinical and genetic data from patients treated at the Department of Breast Oncology, Peking University Cancer Hospital from Jan. 2013 to Dec. 2019. Gene mutations, age of onset, family history, phenotype and clinical outcomes were analyzed.

In this study, NGS was used to profile genomic DNA collected from real world Chinese patients diagnosed with advanced breast cancer in order to identify the presence of cancer-associated germline mutations. Our analysis detected 87 pathogenic or likely pathogenic germline mutations across 77 patients. The most frequently mutated genes were BRCA2 (4.8%) and BRCA1(2.2%), consistent with previous data reported for Asian populations. Xie et al. reported that the prevalence rate of BRCA1/2 gene mutations in breast cancer patients in the Chinese population was 5.3% overall (3.5% in BRCA2 and 1.8% in BRCA1) (4). Similar frequencies were also previously reported in unselected Japanese breast cancer patients, with BRCA2 mutations observed in 2.71%, and BRCA1 mutations observed in 1.45% of the patients (17). These rates are lower than that observed in Ashkenazi Jewish patients (11.1%) (5) and Arab patients (10.2%) (19). Most of these deleterious mutations were nonsense (30) or frameshift (34) mutations, leading to premature proteins. In keeping with previous reports by others, pathogenic mutations were more commonly nonsense and frameshift mutations but less frequently missense mutations (5, 8, 10). As previously reported, around 80% of (likely) pathogenic mutations generated a premature termination codon truncating the encoded protein and 10% were missense variants encoding a stable mutant protein (20). Missense mutations classified as pathogenic tend to occur in limited regions of the BRCA proteins, including the RING domain and tandem BRCT domain in BRCA1, or the OB folds and helical domain in BRCA2 (20). In this study, we also detected a pathogenic missense mutation in BRCA1 (p. Cys64Trp) located in the RING domain ( Figure 5B ), and a pathogenic missense mutation in BRCA2 (p.Arg2336His) located in the upstream region of the helical domain ( Figure 5C ). Thus, in keeping with previous reports, the pathogenic BRCA1/2 mutations identified in this study were mostly located within the functional domains of these genes.

In this patient cohort, BRCA1 mutations were significantly associated with the triple negative phenotype relative to other BC subtypes. BRCA2 mutations were also more frequent in the triple negative breast cancer and HR+ HER2- subtypes, although these trends were not significant. These findings are consistent with data reported by others showing that triple negative breast cancers had the highest prevalence of deleterious gene mutations among the four molecular breast cancer subgroups (4), and that mutation rates of BRCA1 and BRCA2 were higher in TNBC compared to other breast cancer subtypes (10). Due to lack of known targets, TNBC is treated primarily with chemotherapy and is associated with poor survival (5). TNBC is a heterogeneous subtype (21), which Shao et al. has further classified into four transcriptome-based subtypes: (1) luminal androgen receptor (LAR), (2) immunomodulatory, (3) basal-like immune-suppressed (BLIS), and (4) mesenchymal like (22). These four TNBC subtypes exhibit different responses to treatment and survival (22). BLIS patients have been characterized with higher HRD scores compared with patients of other subtypes and therefore might benefit from DNA-damaging agents (22). Xie et al. reported that the frequency of BRCA1/2 mutations in TNBC was 11.2%, with BRCA1 in 7.4% and BRCA2 in 3.8% of patients respectively (4). In another prior study, BRCA1-positive mutation status was also strongly associated with TNBC (8). Moreover, 66-70% of BRCA1 carriers develop TNBC compared to 19-23% of BRCA2 carriers and 24% of non-carriers (7, 21), while 76% of BRCA2 mutation carriers develop HR+ HER2- disease (7). PALB2, and FANCM gene mutations have also previously been reported in association with the TNBC phenotype (21). Thus, the observed enrichment of BRCA1/2 germline mutations in TNBC, and BRCA2 germline mutations in HR+ patients in the present study of Chinese patients is consistent with previously reported findings across other patient groups. No other significant associations were observed between BC subtypes and any other genes in this study.

BRCA1/2 mutation status was also associated with age in our study. The average age of the patient cohort was 48.8 years, and a high proportion of deleterious BRCA2 mutations (23.5%) was detected in patients under 30 years. This finding is in keeping with other studies reporting that breast cancer patients with early onset age (under 40 years) were more likely to harbor deleterious mutations in BRCA1/2 (6.4%) and other DNA repair genes (4.5%) than those diagnosed at the age of 40 or later (4, 9). Moreover, the onset age in BC patients with DNA repair gene mutations has been reported to be significantly younger than that of non‐carriers (9). Breast cancer patients carrying BRCA1 or BRCA2 germline mutations have a mean onset age of 40 and 43 years, respectively, and patients carrying PALB2 mutations have a mean onset age of 53 years (21). Thus, similar to other populations, BRCA1/2 mutations were more prevalent in younger patients in our Chinese cohort.

Our study also found that BRCA1 tumors are more frequently high-grade, in keeping with previous publications reporting BRCA1 mutation carriers had tumors characterized for high histological grade and higher proportion of Ki67-positive cells than noncarriers (7, 8). In contrast to a previous report showing that axillary node involvement was more frequent in BRCA2 carriers (7), in our study BRCA2 mutations were more frequently found in patients without lymph node metastasis ( Table 2 ).

In the present study, patients with family history of cancers were also more likely to harbor BRCA1/2 mutations (7.7%/13.3%) than patients without family history (1.6%/5.3%). Xie et al. reported that the rate of BRCA1/2 mutations in patients with familial breast cancer was 18.1% (4, 9). Other studies have revealed that relative to other types of cancer, BC is more commonly associated with a positive family history, followed by ovarian, colorectal, gastric, and cervical cancer (10). In one study, the proportion of cancers associated with positive family histories was 22.0% for all cancers, 7.2% for breast cancer, and 6.5% for breast and/or ovarian cancer (8). Moreover, a Japanese study found that for breast cancer patients, the frequency of family history of different cancer types associated with hereditary breast cancer syndromes was: 11.8% breast, 1.2% ovary, 3.5% pancreas, 2.9% prostate, and 0.8% thyroid cancer (17). Thus, our observation that BRCA1/2 germline mutations are enriched in Chinese breast cancer patients with a positive family history of cancer is consistent with patterns observed in other studies.

For breast cancer patients, there are conflicting results regarding the prognostic and predictive value of BRCA1/2 germline mutations (7). In a large unselected Chinese population, BRCA1 mutation carriers had significantly worse disease-free survival and disease-specific survival than noncarriers, while no significant difference in survival was found between BRCA2 mutation carriers and non-carriers (4). Other studies showed that Her2- BC patients with BRCA1/2 mutation had improved survival due to better response to treatment (5, 6, 23). In this cohort, two patients harboring BRCA1/2 mutation treated with platinum-containing adjuvant treatment, and obtained longer DFS. Germline mutations in BRCA1/2 and other DNA repair genes are associated with an increased risk of breast, ovarian, prostate and/or pancreatic cancer (24). Germline heterozygous mutations affecting BRCA2 also significantly elevate the risk of cancers of the pancreas, male breast, prostate, and other tissues (20). BRCA1/2 mutation carriers with different cancers shows different survival outcomes. Among ovarian cancer patients, BRCA1 and especially BRCA2 carriers respond better than non-carriers to platinum-based chemotherapy and have prolonged survival (7, 21, 25). BRCA1/2 mutation carriers also benefit from PARP inhibitors, and our previous study demonstrated that fluzoparib, a PARP inhibitor, had antitumor activity in BC and ovarian cancer, particularly in BRCA1/2-mutated patients (26).

In the present study, we observed a trend of favorable overall survival for BRCA1/2 carriers, most notably in TNBC patients, although this trend was not statistically significant. However, DDR pathway deficiency, encompassing alterations in a wider index of DNA damage response genes was significantly associated with shorter DFS and OS in carriers. Common deleterious mutations in other DDR genes besides BRCA1/2 including ATM, RAD50 and BARD1were also detected. However, these deleterious mutations were only detected in less than 2% of these patients. Mutations of non-BRCA1/2 genes in the HRR, DNA damage response and mismatch repair pathways have been reported to have medium-to-high penetrance of hereditary BC (8, 10). The rate of non-BRCA1/2 gene repair mutations is 6.5% vs 8.5% (8, 10). Most of these mutations are found in the ATM, CHEK2, PALB2, TP53, RAD50, RAD51D and BRIP1 genes (9, 10). Non-BRCA1/2 cancer-associated gene (RAD51D, TP53, MSH6, CHEK2, APC, and FANCC) mutation rate is 2.7% in Ashkenazi Jewish (16). PALB2 (1.2%) was the most commonly mutated gene other than BRCA1/2 in Chinese breast cancer patients, while CHEK2 (2.82%), ATM (1.06%), and PALB2 (0.87%) were the most commonly mutated genes other than BRCA1/2 in European populations (8). In this study, the most commonly mutated genes other than BRCA1/2 were ATM (6%), RAD50 (6%) and BARD1 (3%), which was not completely consistent with the data previously reported. This may be explained by the low rate of the mutations in these genes and the relatively small number of patients in this study. We also found DDR deficiency patients had shorter DFS ( Figure 5 ), and these patients may benefit from treatment with PARP inhibitors. Overall, it appears that BRCA1 carriers have poorer survival, probably due to the fact that they frequently develop TNBC, whereas BRCA2 germline mutations were not found to have a prognostic impact. This poses a warrant of genetic testing for TNBC patients where PARP inhibitors can be added to improve treatment efficacy and prolong the survival of patients with DDR mutations. Based on our knowledge and understanding from testing results of this study, we designed some clinical trials using PARP inhibitors. There is an ongoing phase I trial of “Niraparib Plus Anlotinib in Advanced Solid Tumors with Homologous Recombination Repair (HRR) Gene Mutations (NCT04764084)” (27); and a “phase I trial to evaluate the safety and efficacy of fluzoparib in combination with apatinib in patients with OC or BC (NCT03075462)” has been finished and the result will publish soon.

i)We do not treat patients depend on the phenotype of the tumor varies whether it is a BRCA1 (mainly TNBC) or a BRCA2 (mainly HR positive) mutations; ii) Adjuvant therapy is a standard systemic treatment and depends on tumor stage, grade, and molecular subtypes, but not gene mutation variants.

This was a comprehensive cancer-related genetic profiling analysis of a large cohort of Chinese patients with advanced breast cancer. Distinct distributions of pathogenic mutations in breast cancer subtypes and differential associations between mutation status and clinical features were observed. The most prevalent germline mutations were BRCA1/2 mutations, followed by ATM and RAD50 mutations. Collectively, our findings confirm the presence of BRCA1/2 and other DDR gene alterations in a substantial proportion of the Chinese breast cancer population and demonstrate their association with poor patient outcomes. Our observations verify the enrichment of these alterations among cancer patients diagnosed with TNBC and high-grade tumors, as well as in patients who are diagnosed at young age and/or have a positive family history of cancer. Our findings support the use of mutational profiling of Chinese breast cancer patients with these characteristics to assess the presence of germline mutations in BRCA1/2 and other DDR pathway genes in order to identify patients who may benefit from treatment with PARP inhibitors.

The data analyzed in this study was obtained from raw sequencing database call files (.BCL), the following licenses/restrictions apply the NGS data cannot be uploaded to a repository policy from Human Genetic Resource Administration of China(HGRAC). Requests to access these datasets should be directed to Xiaomei Wu (wuxiaomeihx@163.com).

The studies involving human participants were reviewed and approved by IRB of Peking University Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.

JZ, NW, and TZ contributed equally to this work. All authors contributed to the article and approved the submitted version.

Authors TZ, TL, YH, FX, YZ, SJ and JY were employed by Huidu Shanghai Medical Sciences.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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