Our study presents the one of the largest cohorts of breast cancer patients with BRCA1, BRCA2, and PALB2 mutations detected through tumor-only sequencing in Taiwan. We analyzed 924 Thermo Fisher OCP v3 assays from 879 breast cancer patients, dividing them into different groups based on their clinical scenarios. Out of the 924 assays conducted, 281 were positive for mutant genes in 130 patients, with BRCA1, BRCA2, and PALB2 mutations identified in 27 patients (3.1%), 76 patients (8.6%), and 46 patients (5.2%), respectively. Overall, genetic alterations were observed in 14.8% of the assays. The high detection rate of breast cancer susceptibility genes could lead to more patients undergoing germline testing and receiving appropriate treatments.
Genetic Mutations And Variants
Our study uncovered a clinically meaningful prevalence of pathogenic or likely pathogenic (P/LP) variants in three of the most influential breast cancer susceptibility genes from tumor genomic testing. The prevalence of BRCA1, BRCA2, and PALB2 mutations in breast cancer patients varied with the type of testing method and targeted population. In a large literature review of Asian gBRCA1/2 mutated breast cancer patients, the prevalence ranged from 2.3–42% in the gBRCA1 group, and 2.3–11.4% in the gBRCA2 group [44]. In Taiwan, the prevalence of germline BRCA1/2 mutations in an unselected breast cancer cohort was reported as 3.8% [45]. The prevalence of germline PALB2 mutations in Asia has not been widely reported. A comprehensive review of HR-related genes reported that pathogenic variants of germline PALB2 mutations ranged between 0.9% and 3.2% globally [19].
While germline mutations in breast cancer susceptibility genes have been extensively studied, reports of somatic mutations are rare. However, with the advent of tumor-targeted sequencing, we can explore both germline and somatic mutations simultaneously. In 2022, the Dana-Farber/Harvard Cancer Center conducted a tumor-targeted sequencing study across a broad range of malignancies on 7,575 patients, which included 1,514 breast cancer patients. If a patient was identified with any P/LP variants of BRCA1, BRCA2, or PALB2 within the tumor, clinical germline testing (CGT) would be performed. The study found that BRCA1 and BRCA2 mutations were present in 2.5% and 3.7% of breast cancer patients respectively, while PALB2 mutations were present in 0.6% of then. Out of all P/LP variants from tumor-sequencing, 70.5% were confirmed as germline mutations [46].
The study analyzed various genetic variants and identified 176 amino acid (AA) changes. All BRCA1, BRCA2, and PLAB2 mutation information was curated and confirmed by the ClinVAR, the Oncomine™ Knowledgebase Reporter, and the OncoKB™. There were 4 variants that did not have AA changes reported, and 3 of them were novel variants (BRCA1 c.5256 + 1G > A, BRCA1 c.5215 + 1G > A, and BRCA2 c.-38-3CAG > C), which were reported the first time in this Taiwanese cohort. The most common BRCA1 mutation was p.K654fs (c.1960_1961insG), which is a frameshift insertion and deleterious mutation (three cases). Although not recorded by the ClinVAR or the ACMG 73 genes the OncoKB™ has confirmed its clinical implication. The most common BRCA2 mutations were p.N372H (c.1114A > C, 26 cases), p.S2186fs (c.6556_6557insA; 5 cases), p.V2466A (c.7397T > C; 5 cases), and p.X159_splice (c.476-2A > G/c or 476-3C > T; 5 cases). The variant p.N372H was initially reported in 2000 and is one of the common non-synonymous polymorphisms [47. 48]. It has been a research focus in the scientific community and has drawn increasing attention [48–56]. A meta-analysis of 22 studies, involving 22,515 cases and 22,388 controls, found no significant association between the BRCA2 p.N372H polymorphism and breast cancer risk. This suggests that the BRCA2 p.N372H allele may be non-pathogenic. Although p.S2186fs is not annotated by the ClinVAR or listed in the ACMG 73 genes, it is a frameshift insertion and deleterious mutation with clinical implications. This has been confirmed by the OncoKB™. For p.V2466A, the ClinVAR provides conflicting interpretations, with some considering it a benign entity. Regarding p.X159_splice, it is interesting to note that there are two different coding variants being recorded. c.476-3C > T has conflicting implications and has been seen in patients from Central/Eastern Europe. In contrast, c.476-2A > G has been ascertained as pathogenic and has been mentioned in Italian and Chinese population. The most common PALB2 mutation observed was p.I887fs (c.2659_2660delAT), a deleterious frameshift mutation that was observed in 30 patients. Although this mutation is not listed in the ClinVAR or ACMG 73 genes list, its clinical implications have been approved by the OncoKB™.
Gene-gene Interactions
The study revealed a significant tendency of co-occurrence between BRCA1/2, BRCA1-PALB2, and BRCA2-PALB2 mutations. It should be noted that 17 samples were discarded due to missing values in at least one of the interrogated genes, preventing analysis of mutual exclusivity. Constructing a gene-gene interaction (GGI) network is important for understanding breast carcinogenesis, as single gene or protein alterations are not sufficient to induce cancer. Rather, the interactions with other genes or microenvironment play a key role. A large-scale study on the interaction between genes was conducted on European Non-Small Cell Lung Cancer (NSCLC) risk, using a total of 445,221 participants from various projects [57]. The study found important gene-gene interactions in the 5p15.33 and 6p21.32 regions, which can be used to improve lung cancer screening models.
It was found that BRCA1 interacts with RAD51 to play a role in DNA repair, while BRCA2 co-localizes with RAD51 and BRCA1, indicating a similar function [58]. PALB2, first reported by Xia et al. in 2006 [59], plays a fundamental role in HR. It acts as a bridging molecule that connects the BRCA complex (BRCA1-PALB2-BRCA2-RAD51) and facilitates the function of RAD51, a protein that is vital for strand invasion during HR [18, 19].
A large-scale mutational analysis in 7,325 individuals identified four interactions between mutations in the breast cancer susceptibility genes [60]. These interactions include ATM and CHEK2 with BRCA1 and BRCA2, ATM and BRCA, CHEK2 and BRCA1/BRCA2 combined, and CHEK2 and BRCA1 or BRCA2. The results show a lower risk of breast cancer than that predicted by the multiplicative product of the constituent risks. These findings likely reflect functional relationships between the encoded proteins in DNA repair and have important implications for models of disease predisposition and clinical translation.
Currently, limited studies have addressed the gene-gene interaction among BRCA1, BRCA, and PALB2 in real-world settings of large-scale breast cancer gene analysis. Our results may shed light on exploring the association between breast cancer susceptibility genes and the possibility of creating a genetic panel for predicting and prognosing hereditary breast cancer.
Clinical Features
The BRCA1 mutation cohort has a higher proportion of advanced-stage disease compared to others, which may be due to various reasons. One possible explanation is that patients with BRCA1 mutations are more likely to have triple-negative breast cancer [61], which is associated with a higher risk of distant recurrence [62, 63]. Another possible reason is that BRCA1 mutations may be associated with a higher likelihood of developing bilateral breast cancer, which could increase the risk of disease progression [64]. Additionally, patients with BRCA1 mutations are more likely to develop breast cancer at a younger age, when the disease may be more aggressive and therefore more likely to be diagnosed at an advanced stage [65, 66]. Despite having more advanced disease, evidence is mixed as to whether BRCA-associated breast cancer has poorer outcomes. Some studies have shown that carriers of a BRCA1 mutation have worse overall survival [67], while others have found no significant difference in outcomes [68].
Clinical Implications And Practice Of Tumor-targeted Sequencing
Nowadays, tumor-only targeted sequencing has gained increasing attention. One advantage of tumor-only testing is that it can reveal P/LP variants in genes associated with cancer predisposition and potential therapeutics with a higher level of coverage. Identification of mutations through tumor-only sequencing may lead to reflex germline testing, which can identify individuals at an increased risk for cancer and allow for early detection and intervention.
Recent research has shown that tumor-targeted sequencing may have more potential benefits than germline mutation testing alone. The Dana-Farber/Harvard Cancer Center conducted a study on the prevalence of P/LP mutations in BRCA1, BRCA2, and PALB2 using tumor-targeted sequencing and subsequent CGT across various malignancies [46]. They found that patients with BRCA mutations were more likely to undergo CGT than those without. Interestingly, over half (52.9%) of the tumor-identified P/LP patients did not meet any personal or family history criteria for CGT. Additionally, 32.7% of patients with BRCA1/2 or PALB2 P/LP variants did not have any other clinical indication for germline testing. Nonetheless, 70.5% of P/LP variants identified through CGT were germline origin. These results show the potentiality of tumor-only sequencing in detecting P/LP mutations in cancer predisposition genes across malignancies. Furthermore, they highlighted the necessity of expanding the indications for CGT beyond traditional criteria. A significant proportion of patients with these mutations may not have any personal or family history of cancer, which may have a significant impact on cancer risk assessment, surveillance, and treatment decisions in the future. Before universal germline and tumor sequencing becomes feasibility, tumor-targeted sequencing seems to be a reasonable choice for personalized therapy.
Both germline and somatic alterations can affect treatment decisions and outcomes. For breast cancer patients, the results of the TBCRC-048 and TBB trials suggested further exploration of PARP inhibitors in metastatic or advanced breast cancers with HR-associated mutations beyond BRCA1 and BRCA2 [16, 17]. Identifying additional biomarkers to expand this treatment in somatic BRCA1/2-mutant or HR-related-gene-mutant advanced breast or ovarian cancers could significantly benefit patients who would otherwise receive chemotherapies as the only regimen. These efforts may reveal a patient population that would benefit from targeted therapy, improving patient outcomes and reducing the complications associated with cytotoxic chemotherapy.
Annotation And Curation
In the study, 60.2% (106) of the reported AA changes were not documented in either the ClinVAR or the commercial the Oncomine™ Knowledge base Reporter. However, when using the OncoKB™ for annotation, 171 AA changes were found to have clinical implications. Nearly half (N = 80, 45.5%) of these variants with AA changes were due to frameshift deletions or insertions, which were all clinically significant according to the OncoKB™. Interestingly, half of missense mutations without clinical implications (4 out of 8) were deemed insignificant, and 23.1% (3 out of 13) of AA changes not recorded in the ClinVAR or the Oncomine™ Knowledge base Reporter were not considered clinically relevant. Regarding the 40 splice site mutations, 62.5% (25) were not reported as deleterious mutations by the Oncomine™ Knowledgebase Reporter, but the ClinVAR identified 60% (15 out of 25) of these mutations as P/LP variants. Furthermore, the OncoKB™ identified that 85% (34 out of 40) of all splice site mutations to be clinically significant.
Accurate interpretation of genetic variants is critical in both clinical and research settings. Before reporting detected variants, appropriately trained and certified molecular diagnostic procedures must be carefully carried out in the context of clinical scenarios, including histologic features [69]. However, the classification criteria can vary between submitters, and the evidence for a particular variant may be conflicting, leading to difficulties in an unbiased interpretation. Numerous studies have explored potential indicators for reinterpreting pathogenic variants within specific databases, as well as across distinct platforms. For example, in a recent comparison of the RefSeq and Gencode human gene databases, only 27.5% of transcripts annotated in the Gencode are shared by the RefSeq [70]. Whiffin et al. selected 43 variants from the ClinVAR classified as P/LP, which were not rare enough in at least one of the Exome Aggregation Consortium populations [71]. Their analysis showed that 42 of these variants should be considered variants of uncertain significance (VUS) instead of P/LP. Xiang et al. analyzed common P/LP variants in the ClinVAR database and identified indicators associated with reclassification, indicating missing opportunities due to misinterpretation [72]. The study selected 217 variants in 173 genes for manual interpretation according to guidelines, with 40% of which downgraded to benign or VUS, while 2% identified as more likely risk alleles. Inappropriate classification was associated with low-rank, older annotation, higher allele frequency, and collection through methods other than clinical testing. It is important to note that the reinterpretation of cancer predisposition genes requires a multidisciplinary effort involving clinicians, genetic counselors, bioinformaticians, and researchers [73].
In summary, the reinterpretation of cancer predisposition genes due to annotation inconsistencies among distinct database is an important issue that requires careful consideration and multidisciplinary collaboration. The use of updated annotation and rigid guideline follow-up and the integration of multiple types of genomic data can help improve the accuracy of cancer risk assessment and inform personalized prevention and treatment strategies.