Germline breast cancer susceptibility gene mutations and breast cancer outcomes

Multigene panel testing is increasingly adopted for managing breast cancer susceptibility in high risk individuals suspected of having hereditary breast cancer, but the evidence-based practice guidelines remain far from being comprehensive. The advent of next generation sequencing (NGS) technologies is making multigene panel testing easier and affordable [1‐4]. In addition, multigene panel testing could identify up to 50% more individuals with cancer susceptibility gene mutations in comparison with testing only for BRCA1 and BRCA2 (BRCA) [5]. Most of these additional mutations are from moderate risk genes, many of which could result in alterations of cancer risk estimation and clinical action [5, 6]. However, arriving at consistent and optimal clinical recommendations on the basis of the interpretations of the multigene panel testing and associated variants of uncertain significance (VUS) could be challenging due to lack of comprehensive understanding on the consequences of the genetic alterations [7]. As the multigene panel testing is becoming widely adopted, studies are needed to develop evidence-based practice guidelines.

In addition to risk assessment of breast cancer susceptibility in germline mutation carriers, understanding prognosis after breast cancer diagnosis will also impact practice guidelines for breast cancer. With the efficacy of poly (ADP-ribose) polymerase (PARP) inhibitors in controlling BRCA mutation positive tumors, many clinical trials are now underway evaluating their use in breast cancer [8]; their incorporation into systemic therapy in clinical practice is highly anticipated. It has not been established whether BRCA or any cancer susceptibility gene mutation is an independent prognostic factor after breast cancer diagnosis. Despite the suspicion for a more aggressive tumor phenotype, most studies have fallen short of showing differences in clinical outcomes in BRCA mutation carriers [9‐12]. Systematic reviews with larger pooled sample size have yielded conflicting conclusions, possibly due to variability of included studies [13‐15]. Consequently, conventional decisions regarding systemic therapy for BRCA mutation-associated breast cancer have been based on disease characteristics rather than BRCA mutation status. As such, it would be informative to discover causal or statistical correlations of germline breast cancer susceptibility gene mutations to breast cancer prognosis.

A panel of 20 known and candidate breast cancer susceptibility genes were selected herein for the multigene panel testing study. Among the 20 genes, BRCA1, BRCA2, PALB2, TP53, CDH1, PTEN, ATM, CHEK2, BARD1, STK11, NBN have been well established as breast cancer susceptibility genes [7, 16, 17]. Some are part of rare high-penetrance cancer predisposing syndromes (e.g. BRCA1, BRCA2, TP53, CDH1, PTEN, STK11, PALB2) while others are moderate-penetrance genes (e.g. ATM, NBN, CHEK2, BARD1). The impacts of the mutations in RAD50, RAD51C, and RAD51D on breast cancer susceptibility and survival are controversial: Mutations in RAD50 have been found not associated with breast cancer risk [17]. Also mutations in RAD51C have not been found to increase the risk of breast cancer [18‐20] and mutations in RAD51D have been associated with high risk of ovarian cancer but not with breast cancer [21]. Nevertheless, other studies have indicated that mutations in RAD51C [22] and RAD51D [17, 23, 24] contribute to the risk of both breast and ovarian cancer, and that RAD50 is an intermediate-risk breast cancer susceptibility gene [25]. Although germline mutations in the DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2) have been mostly associated with Lynch syndrome, evidence has been established to support the connections between the mutations in the DNA mismatch repair genes and the risk or survival of breast cancer [26‐29]. Similarly, whether BRIP1 is a breast cancer susceptibility gene remains controversial [30], and perhaps is dependent on the ethnicity of the cohort studied [22]. NF1 mutations have been known to associate with increased risk of breast cancer in younger population [31] and poor breast cancer survival [32]. To clarify the controversies, we included in the panel the potentially relevant genes above to explore the germline mutation-dependence of breast cancer predisposition and outcomes in our local high risk population.

Different ethnic populations need respective studies on cancer risks pertinent to germline mutations. In the western populations, about 5% of the breast cancer patients may carry heritable cancer susceptibility gene mutations [33, 34]. BRCA1 and BRCA2 account for the majority of these gene mutations, with BRCA1 being the most common [34, 35]. However, studies in Asian populations have indicated somewhat different conclusions: available results show that BRCA mutation rates in Asians are lower than those in Whites, and that the distributions of the gene mutations are also different [36‐43]. It is imperative to enrich mutation databases on different ethnic populations, so as to better interpret ethnically specific germline mutations and better manage cancer risks among corresponding ethnic populations.

We showed that BRCA germline mutation carriers in this large ethnic Chinese cohort were more likely to be diagnosed with breast cancer already spread to regional lymph nodes, and their breast cancer related outcomes were significantly worse. The 5-year disease-free survival rate was only 73.3% for BRCA mutation carriers, in contrast to 91.1% for non-mutation carriers. The BRCA mutation status was an independent prognostic factor with an adjusted hazard ratio of 3.04 (95% CI 1.40–6.58) for cancer recurrence or death. The poor clinical outcome in BRCA mutation carriers mainly resulted from recurrence as distant metastasis, therefore excluding the contribution by new primary cancer in the ipsilateral or contralateral breast, of which the risk had been known to be elevated in BRCA mutation carriers. Our result implied the more aggressive nature of breast tumors in BRCA germline mutation carriers. Most previous studies on clinical outcomes of BRCA mutation carriers have failed to show a significant prognostic effect by BRCA mutation [9‐11, 13, 14]. However, a recent systematic review showed that both BRCA1 and BRCA2 mutation carriers had significantly worse breast cancer specific survival [15]. The discrepancy in these studies may in part result from limitations due to small sample sizes, lack of adjusting for disease characteristics, variations in mutation assay techniques, mutation types, cancer treatment modalities, or lengths of follow-up. Our study was conducted in an all-Chinese cohort where all study participants underwent the same NGS-based complete sequencing of the coding regions of BRCA genes among other genes. The majority of the mutations were in the BRCA2 gene. The follow-ups were extensive in terms of length, with median duration over 5 years, and completeness. The tumor characteristics and outcome data had been collected in a prospective manner in a breast cancer registry as well as after the participants were enrolled. The majority of the breast cancer patients in the cohort (94%) had undergone treatment in a single cancer center where cancer characteristic-based treatment guideline was consistently adhered to. The homogeneity on data collection may have strengthened the validity of the prognostic analysis.

Studies have shown that tumor cells with BRCA mutations may show different response to different chemotherapy agents; they may have enhanced sensitivity to platinum while more resistant to taxanes. However, clinical studies on comparison of chemotherapy regimens in the BRCA mutation populations are limited [12]. For the breast cancer patients in our cohort, less than 10% received cisplatin in the neoadjuvant setting and none in the adjuvant setting, while about a third received a taxane (docetaxel) in the adjuvant setting. There were no significant differences in chemotherapy choice between the groups with and without BRCA mutation. Despite a higher rate of BRCA mutation carriers receiving chemotherapy, they had poorer cancer outcomes. Further prospective studies are needed to determine the optimal chemotherapy for BRCA mutation carriers. With the anticipated efficacy of incorporation of PARP inhibitors in the treatment of BRCA mutation associated breast tumors, knowledge of BRCA mutation status prior to initial cancer treatment becomes even more crucial.

In this Chinese cohort of high risk individuals for hereditary breast cancer, we found an overall prevalence of 13.5% for carriers of germline mutations in 11 of the 20 breast cancer susceptibility genes. In contrast to western populations, BRCA2 mutations (52.3%) were much more common than BRCA1 mutations (9.2%) in our cohort, similar to findings in other studies in the Asian population [34]. Non-BRCA genes contributed to 38.5% of the mutation carriers, with PALB2 (13.8%), RAD51D (9.2%), and ATM (4.6%) being the majority. PALB2 is particularly important since lifetime risk for breast cancer can reach 58% in those with family history [51], and NCCN guideline recommends consideration of risk-reducing mastectomy [52]. Among the 8 cases with RAD51C and RAD51D mutations, 6 (75%) were triple negative breast cancer, in agreement with the recent studies suggesting that mutations in these two genes may confer higher risks of triple-negative or basal subtypes of breast cancer [23, 24]. We also found 2 individuals with protein-truncating mutations in TP53 and PMS2 genes, which are high-penetrance cancer predisposing genes and would result in significantly high risk for other cancers. These results showed that testing more than BRCA1 and BRCA2 increased the detection rate of clinically actionable high and moderate risk gene mutations, therefore may be an important strategy in the Chinese population. In a study by Thompson et al., significant excess of mutations was only observed for PALB2 and TP53 in familial breast cancer cases compared to cancer-free controls [6]. We similarly only found a small number of genes contributing to the majority of mutation carriers. To overcome the challenge of high rates of VUS and questionable clinical actionability, we recommend limiting cancer susceptibility multigene panel in clinical testing to include only a handful of genes with high clinical impact.

Among the six risk factors for hereditary breast cancer in this cohort of all high risk individuals, only bilateral breast cancer showed a statistically significant odds ratio of 3.27 for having a germline mutation in multivariate analysis. In addition, having more risk factors was also associated with a high detection rate of mutations (OR 2.07 for having more than one risk factor). These results suggest that these known risk factors were helpful in identifying individuals for genetic testing and we may need to pay particular attention to those with bilateral breast cancer, even in the absence of family history or young age of onset. Larger cohorts are needed to clarify the significance of ovarian cancer and male breast cancer on breast cancer susceptibility gene mutations in the Asian population. Results from the correlation analysis done with the few high penetrance genes were similar to those done with all the studied genes, suggesting that the correlations were driven by these high penetrance genes including BRCA1, BRCA2, PALB2, and TP53, which was expected since they represented the majority of the pathogenic variants.

There were some limitations in our study. First, we did not conduct experiments to detect large genome rearrangement (LGR) in all study participants, but used bioinformatics analytical tools to detect copy number variations on the NGS data. This could underestimate the prevalence of LGR in this cohort. However, LGRs have not been shown to contribute significantly to germline mutations in BRCA genes in East Asian populations [53, 54]. Second, we were conservative in classifying variants as pathogenic and limited those to protein-truncating variants, which were without ambiguity in assignment of pathogenicity. There were two missense variants classified as likely pathogenic in the ClinVar database, and many missense variants deemed damaging by multiple in silico models. However, we did not assign those as pathogenic mutations in this study. We could therefore have underestimated the prevalence of pathogenic mutations. Further studies are underway to evaluate variant segregation with cancer in families, and the accuracy of in silico models.

In this high risk ethnic Chinese cohort, 13.5% had a germline pathogenic mutation in one of twenty breast cancer susceptibility genes, and 8.3% had a BRCA1or BRCA2 mutation. BRCA mutation carriers, when diagnosed with breast cancer, were more likely to have lymph node involvement. Their breast cancer specific outcomes were significantly worse even after adjusting for clinical prognostic factors, suggesting BRCA mutation to be an independent factor for poor prognosis. Our results highlighted the importance of early testing for breast cancer susceptibility genes, not only for prevention and earlier diagnosis of breast cancer, but also for optimal treatment and surveillance strategies after breast cancer is diagnosed. Further studies are needed to evaluate different treatment approaches for breast cancer in BRCA mutation carriers to improve outcome.