Advances in precision medicine have maintained pace with the development of genetic profiling technologies. Diseases that were previously classified into only a few types have gradually been subdivided into specific diagnoses, particularly in oncology, leading to an era of personalized treatment. Various therapeutic alternatives to conventional chemotherapy, such as targeted therapy and immunotherapy, have been established and meticulously investigated [1,2].

Next-generation sequencing (NGS) has markedly changed the way breast cancer is identified and treated. In addition to molecular subtyping, physicians should consider numerous genetic factors to combat breast cancer. Testing germline BRCA1/2 (gBRCA1/2) plays a pivotal role in assessing the hereditary risk of breast cancer and in guiding treatment decisions, particularly with the development of poly(ADP-ribose) polymerase (PARP) inhibitors [3,4].

Although gBRCA1/2 aberration must be confirmed under specific clinical conditions, additional costs and the necessity of blood sampling present practical hurdles to this process. Attempts have been made to infer the result of germline mutations obtained from tissue NGS results, but accuracy remains a concern. In addition, although the clinical and pathological characteristics of gBRCA-related breast cancer have been well-acknowledged over several decades [5,6], the genetic aspects of these characteristics have not been fully revealed at the NGS level.

Based on these considerations, this study was conducted to assess the consistency of gBRCA1/2 aberrations between blood germline testing and tumor tissue sequencing and to characterize the genomic landscape of breast cancer according to gBRCA1/2 pathogenicity

In this study, we analyzed breast cancer genomic landscape according to germline BRCA1/2 pathogenicity. The germline BRCA1/2 test using DNA extracted from blood samples and tumor BRCA1/2 test using tumor samples and NGS showed fair consistency (94.3%). Patients harboring the gBRCA1/2p mutation showed lower TMB values and fewer SNVs than patients with gBRCA1/2w. Although considerable differences were observed for PIK3CA and PTEN mutations, these were not statistically significant. Higher number of genes related to homologous recombination (HR) repair were mutated more frequently in the gBRCA1/2w group than in the gBRCA1/2p group, and the differences were remarkable when limited to non-BRCA1/2 HR-DDR genes. Moreover, the number of non-BRCA1/2 HR-DDR gene aberrations correlated with the number of reported SNVs.

In the current era of precision medicine, tumor NGS is frequently performed to identify potential therapeutic targets. The major goal of tissue NGS is to detect somatic mutations for actionable targets; however, information beyond somatic SNVs should also be acknowledged. Considerable efforts have been undertaken to determine germline mutations by sequencing clinical tumor samples [10]. However, discrepancies and inaccuracies in identifying germline mutations using tissue NGS have been discussed continuously. A previous study showed that tumor-only sequencing without matched normal samples could not definitively confirm germline aberrations, resulting in high false positivity [11]. Although some discrepancy is inevitable, it is proposed that the number of well-characterized pathogenic genes or their variants should be considered in tumor sequencing. BRCA1/2, MLH1, MSH2, and MSH6 are often included in cancer panels and are highly associated with inheritance of pathogenicity [12]. Our study revealed a high consistency rate (94.3%) between blood gBRCA1/2 testing and tumor tissue sequencing, suggesting that it is possible to infer germline BRCA1/2 aberration from tumor biopsy samples in clinical settings. The two inconsistent cases of our study, S01005 and S04259, presented fair quality of NGS (mean depth 794.59, 442.08 and on-target rate 90.83%, 95.72%). The inconsistency might not be due to low-quality or reference gene diversity. The only possibility is unknown human error in reporting, but clear explanation is not yet determined.

TMB is another key biomarker that can be indirectly inferred by clinical NGS. TMB is defined as the number of base alterations and indels, usually calculated by whole exome sequencing (WES). TMB calculation algorithms used in our study and their clinical efficacy were validated by comparing with TMB calculated by WES, which proved its feasibility with R² of 0.71 [13]. Moreover, it is comprehensive with previous studies and demonstrated a fine correlation between TMB by targeted sequencing and WES (R²=0.74) [14,15]. Similarly, in our study, we assessed the matched relationship between TMB and the number of reported SNVs from a panel of tumor samples (Pearson’s correlation coefficient, 0.378; p=0.001).

TMB has emerged as a promising biomarker in the context of immunooncology, particularly in melanoma and non–small cell lung cancer [16–18]. High TMB can predict the clinical response to immune checkpoint inhibitors [19,20], but the correlation is not clear in breast cancer [21]. Approximately 20% of metastatic breast cancer cases demonstrate a high TMB [21]. In our study, 14 of 77 patients (18.2%) presented high TMB (> 16 Mb). However, the prevalence sharply increased to 27.9% (12/43) when only the gBRCA1/2 wild-type group was assessed. Taking these findings into account, further translational studies may reveal whether certain germline pathogenicity is predictive of the therapeutic effect of immunotherapy.

Hypermutated breast cancer can be driven by multiple mechanisms, including homologous recombination deficiency (HRD), and breast cancer with HRD has the highest median TMB among groups with dominant signatures [15]. In our study, the genes were selected for academic purposes and were different from those widely used commercially. With the limited resource and practical barriers with targeted sequencing, we could not clearly present HRD score. Although their important role in HR-DDR were well acknowledged in preclinical studies, genes which had not been included in approved commercial panel, such as ARID1A and NBN, should be interpreted with caution. Although BRCA1/2 are two of the essential HR-DDR genes, our data showed frequent HR-DDR aberrations and higher mean TMB in the gBRCA1/2w group than in the gBRCA1/2p group. Previous studies reported contradictory results about relationship between germline mutation and TMB. Somatic mutation of BRCA1/2 was associated with higher mutation burden and higher TMB comparing with wild type in The Cancer Genome Atlas–based analysis [22,23]. However, when the patients were classified according to germline BRCA1/2 mutation, differences of TMB was insignificant or even lower in gBRCA1/2-mutated group [24,25]. With these evidences and results of our study, we can infer that germline pathogenic variation in BRCA1/2 might contribute less to TMB than variations in HRD genes in breast cancer. As with tremendous effort to detect HRDness in solid cancer and applying the results to choosing effective treatment, the role of TMB in breast cancer should be further evaluated. The results of clinical trials evaluating the efficacy of PARP inhibitors in patients with HRDness are anticipated [26].

In our study, we noted more PIK3CA and PTEN mutations in the gBRCA1/2w group than in the gBRCA1/2m group. As two of the major cancer driver mutations, our findings provide an evidence for their mutual exclusiveness, consistent with previous studies [24,27]. A recent study also showed that breast cancer with altered phosphoinositide 3-kinase pathway harbors a significantly low rate of homologous recombination co-alterations [28]. In the future, we intend to explore the correlation between germline and somatic mutations using more genomic data from a larger sample size.

In clinical settings, many of the genes known to cause hereditary cancer syndrome are included in most cancer panels. In addition, because of convenience of its application and short turnaround time, NGS panels are increasingly being used to identify germline aberrations. Use of comprehensive genetic analysis based on WES and whole genome sequencing (WGS) is expected to increase as their accessibility has improved. These advances have expanded the possibilities of discovering novel germline mutations. Therefore, further functional genomic studies based on WES and WGS aimed at determining clinical implications of genetic variants and their effects on drug sensitivity are needed.

However, our study has several limitations. First, the sample size was too small to ensure statistical power. There had been number of patients who did their NGS analysis with blood, but they could not be included because the study was performed to investigate concordance rate of germline variants between tumor tissue and peripheral blood mononuclear cell. The statistical results should be interpreted with caution considering selection bias and relatively low prevalence of gBRCA1/2 mutation in the study cohort. Some patients were enrolled at the early stage of breast cancer, whereas others were enrolled at an advanced stage, making the total genomic profile heterogeneous. In addition, we could not verify the clinical significance of HR-DDR aberration or high TMB in terms of therapeutic targets or predictive markers. As none of the Food and Drug Administration–approved PARP inhibitors are currently reimbursed for patients with metastatic breast cancer under the Korean national health scheme, platinum-based chemotherapy remains the most affordable treatment option.

In conclusion, we herein describe the consistency between gBRCA1/2 status based on blood testing and tissue sequencing and the differences in genetic landscape according to germline BRCA1/2 gene variations. The concordance rate of gBRCA1/2 results by tissue NGS was 94.3%, and a significant difference was observed in TMB value and aberrated non-BRCA1/2 HR-DDR genes according to germline BRCA1/2 pathogenicity in patients with breast cancer. In the future, our data should be validated in a larger cohort, and the clinical impact on survival outcomes should be further elucidated.