Abstract
Background
In Japan, BRCA genetic testing for the diagnosis of hereditary breast and ovarian cancer is covered by medical insurance. However, other breast cancer (BC) susceptibility genes are rarely examined in these patients, primarily because medical insurance does not cover testing for these genes.
Methods
A multigene panel testing (MGPT) was conducted on patients with BC who met the clinical criteria for BRCA genetic testing.
Results
Of the 494 patients, 45 germline pathogenic variants (GPVs) were identified (9.11%). The BRCA genetic testing criteria items with GPV detection rates > 10% were as follows: “diagnosed at age ≤ 45 years,” “diagnosed with triple-negative breast cancer (TNBC) at age ≤ 60 years” and “≥1 close blood relative within the third degree on the same side of the family with breast, ovarian, or pancreas cancer.”
Conclusions
. MGPT can identify GPVs in genetically high-risk BC patients, including both BRCA1/2 and non-BRCA genes. Thus, MGPT may be an effective approach to support appropriate clinical management for both patients and their relatives.
Keywords: Genetic testing, Breast cancer, Hereditary breast and ovarian cancer, Japan
Introduction
Breast cancer (BC) is the most commonly diagnosed cancer in women [1]. Several factors such as reproductive history, obesity, and hormonal replacement therapy contribute to BC development [2]. Germline pathogenic variants (GPVs) were also identified as a significant factor of BC [3]. According to the use of multigene cancer susceptibility panels, approximately 5%–10% of women with BC carry GPVs [4–7]. Among these, BRCA1 and BRCA2 are well-known genes [8]. In Japan, BRCA genetic testing for the diagnosis of hereditary breast and ovarian cancer (HBOC) has been covered by medical insurance since April 2020 [9]. Although patients who fit the BRCA genetic testing criteria are considered genetically at high-risk, the detection rate of GPVs with BRCA1/2 is approximately 13% [9]. In addition, genetic testing using multigene panel testing (MGPT) is not commonly performed in Japan because it is not covered by medical insurance, resulting in the limited assessment of GPVs in other genes. However, genetic analysis is important in making decisions about therapy and cancer prevention, and facilitates risk management of family members [10, 11]. As the cost of MGPT has decreased currently [12], a recent study reported that an extension of BRCA genetic testing to MGPT in highly developed countries might be justified [13]. Moreover, which individuals are more likely to be offered MGPT must also be considered. In this study, 27 cancer susceptibility genes [14] were sequenced to investigate the prevalence of GPVs in patients undergoing BRCA genetic testing for the diagnosis of HBOC. We also analyzed the association between the BRCA genetic testing criteria and the presence of GPVs.
Methods
Study design
Patients with BC who underwent BRCA genetic testing under medical insurance coverage and consented to additional MGPT between October 2022 and December 2024 were recruited. MGPT was performed concurrently with BRCA genetic testing during the same testing session, rather than sequentially after confirmation of BRCA1/2-negative status. The written informed consent included the possibility of identifying secondary/incidental findings. When such findings were identified, genetic counseling was made available at each participating institution and was provided as appropriate.
The conditions for medical insurance coverage of BRCA genetic testing in Japan are set to include one or more of the following:
Personal history of BC diagnosed at age ≤ 45 years.
Personal history of triple-negative breast cancer (TNBC) diagnosed at age ≤ 60 years.
Personal history of BC diagnosed at any age with ≥ 2 total diagnoses of BC.
Personal history of BC diagnosed at any age with ≥ 1 close blood relative within the third degree on the same side of the family with breast, ovarian, or pancreas cancer.
Personal history of male BC diagnosed at any age.
Personal history of epithelial ovarian (including fallopian tube or peritoneal) cancer diagnosed at any age.
Individuals with cancer who meet the eligibility requirements for supplementary diagnostics related to polyadenosine diphosphate-ribose polymerase (PARP) inhibitors.
In this study, patients who met the criteria (1) to (5) were included. Moreover, 27 cancer susceptibility genes were analyzed, as described previously [14]: APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, EPCAM, HOXB13, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53.
Sequence analysis
A total of 6 mL of whole blood was collected from each patient in EDTA2K tubes (365900, BD) and stored at − 20℃ until use. DNA was extracted from these samples. All coding regions and 2 bp flanking intronic sequences of the 27 genes were analyzed. All coding regions were referred to CCDS v15 or RefSeq Mar. 2020. We applied the multiplex-based targeted PCR methods with 1,087 primer pairs of 4 primer pools to sequence the targeted regions as previously described [15]. The pooled DNA libraries were sequenced using 2 × 150-bp paired-end reads on a NovaSeq 6000 (Illumina). Sequencing reads were aligned to the human reference genome (GRCh37/hg19), and the genetic variants were identified with deposited custom scripts at https://github.com/Laboratory-for-Genotyping-Development/TargetSequence.g.it. In total, 99.97% of the region were analyzed at depths of 20 or higher. All individuals passed QC criteria set at 98% or more of the region at depths of 20 or higher.
Variant classification
The ACMG/AMP guidelines are commonly used for identifying GPVs. However, while guidelines specific to individual genes have been reported, they do not cover all genes analyzed in this study. Therefore, the following patterns were annotated as GPVs, as previously used [14]: variants that resulted in loss of function according to SnpEff version 4.3t or that were designated as pathogenic or likely pathogenic by ClinVar version 2026-03-02.
Statistical analysis
Fisher’s exact test was conducted to compare categorical data between GPVs and BRCA genetic testing criteria. Logistic regression analyses were performed to evaluate the association between BRCA genetic testing criteria and the presence of GPVs in genes other than BRCA1/2. Both univariable and multivariable models were constructed. In the univariable analyses, each criterion was evaluated separately. In the multivariable model, multiple criteria were included simultaneously. In this analysis, the item “personal history of male BC diagnosed at any age” was excluded because of insufficient numbers of cases. Cases with missing or uncertain information, including tumor subtype, were treated as missing values and excluded from the relevant analyses. As this study was exploratory in nature, no formal adjustment for multiple comparisons was performed. P-value < 0.05 was used to consider significance. All analysis was conducted with IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA).
Results
MGPT was offered to 508 patients with BC. One patient who was not Japanese, 6 who underwent testing intended to examine the indication for PARP inhibitors and 7 who had unknown or unregistered reasons for testing were excluded. In total, 494 patients were eligible for this study (Fig. 1).
Fig. 1.
Patient flow diagram. MGPT, multigene panel testing; PARP, polyadenosine diphosphate-ribose polymerase
Genetic testing results
Among the 494 patients with BC, 45 GPVs were identified (9.11%, Table 1). The highest prevalence of GPVs was observed for BRCA2 (n = 21/494, 4.25%). Other positive genes were BRCA1 (n = 11/494, 2.23%), ATM (n = 3/494, 0.61%), RAD51D (n = 3/494, 0.61%), and BRIP1 (n = 2/494, 0.40%). One GPV was identified in each of APC, BARD1, CHEK2, EPCAM, and NF1. One patient had double GPVs in BRCA2 and BRIP1. For gene-level reporting (Table 1), variants were counted separately for each gene. However, for patient-level analyses (Tables 2, 3, 4 and 5), the individual with GPVs in both BRCA2 and BRIP1 was counted once and classified in the “other group”.
Table 1.
Prevalence of germline pathogenic variants
| Gene | Cases (%) (n = 494) |
|---|---|
| Genes associated with high-risk of breast cancer development | |
| BRCA1 | 11 (2.23) |
| BRCA2 | 21 (4.25) |
| CDH1 | 0 |
| NF1 | 1 (0.20) |
| PALB2 | 0 |
| PTEN | 0 |
| STK11 | 0 |
| TP53 | 0 |
| Genes associated with medium-risk of breast cancer development | |
| APC | 1 (0.20) |
| ATM | 3 (0.61) |
| BARD1 | 1 (0.20) |
| BMPR1A | 0 |
| BRIP1 | 2 (0.40) |
| CDK4 | 0 |
| CDKN2A | 0 |
| CHEK2 | 1 (0.20) |
| EPCAM | 1 (0.20) |
| HOXB13 | 0 |
| MLH1 | 0 |
| MSH2 | 0 |
| MSH6 | 0 |
| MUTYH | 0 |
| NBN | 0 |
| PMS2 | 0 |
| RAD51C | 0 |
| RAD51D | 3 (0.61) |
| SMAD4 | 0 |
| Total | 45 (9.11) |
One patient harbored GPVs in both BRCA2 and BRIP1, and therefore was counted once for each gene in this table. High-risk genes represent genes well established to be associated with hereditary breast cancer. The medium-risk category includes other cancer susceptibility genes that were included in the multigene panel analyzed in this study
Table 2.
Clinicopathological characteristics
| Cases (% cohort) | |||
|---|---|---|---|
| Other group (n = 13) |
BRCA group (n = 31) |
Non-carrier group (n = 450) |
|
| Age at diagnosis, median (range), years | |||
| 44.0 (33–62) | 43.0 (30–80) | 45.0 (20–83) | |
| Age distribution, years | |||
| ≤ 29 | 0 | 0 | 5 (1.1) |
| 30–39 | 2 (15.4) | 9 (29.0) | 78 (17.3) |
| 40–49 | 6 (46.2) | 13 (41.9) | 213 (47.3) |
| 50–59 | 4 (30.8) | 8 (25.8) | 95 (21.1) |
| 60–69 | 1 (7.7) | 0 | 36 (8.0) |
| ≥ 70 | 0 | 1 (3.2) | 23 (5.1) |
| Personal history of cancer | |||
| Breast cancer only | 13 (100) | 30 (96.8) | 432 (96.0) |
| + Ovarian cancer | 0 | 1 (3.2) | 1 (0.2) |
| + Any non-breast or non-ovarian cancer | 0 | 0 | 14 (3.1) |
| Unknown | 0 | 0 | 3 (0.7) |
| Family history of cancer within the second-degree relative | |||
| No known family history of cancer | 0 | 2 (6.5) | 64 (14.2) |
| Breast cancer | 7 (53.8) | 20 (64.5) | 226 (50.2) |
| Ovarian cancer | 2 (15.4) | 1 (3.2) | 19 (4.2) |
| Any non-breast or non-ovarian cancer | 11 (84.6) | 22 (71.0) | 291 (64.7) |
| Unknown | 0 | 0 | 10 (2.2) |
| Clinical stage | |||
| 0 | 2 (15.4) | 1 (3.2) | 78 (17.3) |
| 1 | 3 (23.1) | 11 (35.5) | 190 (42.2) |
| 2 | 5 (38.5) | 14 (45.2) | 144 (32.0) |
| 3 | 2 (15.4) | 5 (16.1) | 25 (5.6) |
| 4 | 0 | 0 | 5 (1.1) |
| Unknown | 1 (7.7) | 0 | 8 (1.8) |
| Biological subtype | |||
| ER positive (+), HER2 negative (−) | 5 (38.5) | 14 (45.2) | 308 (68.4) |
| ER positive (+), HER2 positive (+) | 2 (15.4) | 6 (19.4) | 41 (9.1) |
| ER negative (−), HER2 positive (+) | 0 | 0 | 17 (3.8) |
| TNBC | 5 (38.5) | 9 (29.0) | 51 (11.3) |
| Not classified | 1 (7.7) | 2 (6.5) | 33 (7.3) |
Clinicopathological characteristics of patients stratified by GPV status. “Other group” indicates patients with GPVs except BRCA1/2, “BRCA group” indicates patients with BRCA1 or BRCA2 GPVs, and “non-carrier group” indicates patients without GPVs. One patient had double GPVs in BRCA2 and BRIP1. This case was counted once and classified in the other group
ER estrogen receptor, HER2 human epidermal growth factor receptor 2, TNBC triple-negative breast cancer; GPV, germline pathogenic variant
Table 3.
Detection rate to each BRCA genetic testing criteria item
| Cases | Detection rate (%) | |||||
|---|---|---|---|---|---|---|
| Other group (n = 13) |
BRCA group (n = 31) |
Non-carrier group (n = 450) |
GPVs (other+BRCA1/2) |
GPVs (other) |
GPVs (BRCA1/2) |
|
| Diagnosed at age ≤ 45 years (n = 262) | ||||||
| 8 | 19 | 235 | 10.3 | 3.1 | 7.3 | |
| Diagnosed with TNBC at age ≤ 60 years (n = 56) | ||||||
| 4 | 8 | 44 | 21.4 | 7.1 | 14.3 | |
| ≥ 2 total diagnoses of breast cancers in patients (n = 76) | ||||||
| 1 | 4 | 71 | 6.6 | 1.3 | 5.3 | |
| ≥ 1 close blood relative within the third degree on the same side of the family with breast, ovarian, or pancreas cancer (n = 316) | ||||||
| 9 | 22 | 285 | 9.8 | 2.8 | 7.0 | |
| Male breast cancer (n = 3) | ||||||
| 0 | 0 | 3 | 0 | 0 | 0 | |
“Other group” indicates patients with GPVs except BRCA1/2, “BRCA group” indicates patients with BRCA1 or BRCA2 GPVs, and “non-carrier group” indicates patients without GPVs. One patient had double GPVs in BRCA2 and BRIP1. This case was counted once and classified in the other group
GPVs germline pathogenic variants, TNBC triple-negative breast cancer
Table 4.
Relative risk of GPVs in genes other than BRCA1/2 for each BRCA genetic testing criteria item
| Univariate odds ratio (95% CI) |
P-value | Multivariable odds ratio (95% CI) |
P-value | |
|---|---|---|---|---|
| Diagnosed at age ≤ 45 years | ||||
| 1.46 (0.47–4.54) | 0.51 | 1.83 (0.56–5.97) | 0.32 | |
| Diagnosed with TNBC at age ≤ 60 years | ||||
| 4.02 (1.19–13.59) | 0.03 | 4.92 (1.41–17.14) | 0.01 | |
| ≥ 2 total diagnoses of breast cancer in patients | ||||
| 0.45 (0.06–3.48) | 0.44 | 0.52 (0.06–4.30) | 0.54 | |
| ≥ 1 close blood relative within the third degree on the same side of the family with breast, ovarian, or pancreas cancer | ||||
| 1.30 (0.40–4.30) | 0.66 | 1.66 (0.48–5.75) | 0.43 | |
GPVs germline pathogenic variants, CI confidence interval, TNBC triple-negative breast cancer
Table 5.
Number of BRCA genetic testing criteria met and positivity rate
| Cases | Positivity rate (%) | P-value | ||
|---|---|---|---|---|
| Other group (n = 13) |
Non-carrier group (n = 450) |
|||
| 1 item met | 5 | 283 | 1.7 | 0.149 |
| 2 items met | 7 | 147 | 4.5 | |
| ≥ 3 items met | 1 | 20 | 4.8 | |
Number of BRCA genetic testing criteria met stratified by GPV status. “Other group” indicates patients with GPVs except BRCA1/2, and “non-carrier group” indicates patients without GPVs. One patient had double GPVs in BRCA2 and BRIP1. This case was counted once and classified in the other group
GPV germline pathogenic variant
Among the 494 patients, 32 carried GPVs in BRCA1 or BRCA2. When the analysis was restricted to patients without BRCA1/2 GPVs (n = 462), GPVs in other genes were identified in 12 patients (2.60%).
Clinicopathological characteristics
All participants tested were > 20 years of age. The median age at the diagnosis of patients who had GPVs, except BRCA1 /2 (hereinafter called the “other group”), was 44.0 (range, 33–62) years, that of patients with BRCA1 or BRCA2 GPVs (hereinafter called the “BRCA group”) was 43.0 (range, 30–80) years and that of patients without GPVs (hereinafter called the “non-carrier group”) was 45.0 (range, 20–83) years.
Positive personal history of cancer (except BC) was reported in 15 (3.3%) of patients in the non-carrier group, including 1 (0.2%) ovarian cancer and 14 (3.1%) other cancers. In the BRCA group, one patient had a history of ovarian cancer (3.2%). The other group had a personal history of BC alone.
Most patients had a family history of BC within the second-degree, including 7 (53.8%) in the other group, 20 (64.5%) in the BRCA group, and 226 (50.2%) in the non-carrier group. A positive family history of ovarian cancer was reported in 15.4% (n = 2/13), 3.2% (n = 1/31), and 4.2% (n = 19/450) of patients in the other, BRCA and non-carrier groups, respectively. Any non-breast or non-ovarian cancer was reported in 11 (84.6%), 22 (71.0%), and 291 (64.7%) patients in the other, BRCA, and non-carrier groups, respectively. All patients in the other group had family history of cancer.
Most patients in the non-carrier group presented with estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative BC (68.4%, n = 308/450), and 11.3% (n = 51/450) had TNBC. In contrast, the other group had higher rates of TNBC (38.5%, n = 5/13) and the BRCA group had 29.0% (n = 9/31). Patients with TNBC in the other group had GPVs with BARD1, BRIP1, EPCAM, RAD51D, or NF1. In this study, many cases were not classified with a biological subtype because the patients were in clinical stage 0 and had HER2 IHC 2+, and fluorescence in situ hybridization testing had not been performed. One (7.7%) in the other group, 2 (6.5%) in the BRCA group, and 33 (7.3%) in the non-carrier group were categorized as “not classified”. Of these, patients with ER-positive and undetermined HER2 status at clinical stage 0 accounted for 1 (7.7%), 0 (0%), and 24 (5.3%) patients, respectively; at clinical stage 1 or higher, 0 (0%), 2 (6.5%), and 1 (0.2%) patients, respectively; and completely unknown subtype accounted for 0 (0%), 0 (0%), and 8 (1.8%) patients, respectively. Patients with ER-positive and undetermined HER2 status were classified as non-TNBC and were therefore included as not meeting the criterion “personal history of TNBC diagnosed at age ≤ 60 years” in the following analysis. All study clinicopathological characteristics are summarized in Table 2.
Association between genetic testing results and BRCA genetic testing criteria
Personal history of BC diagnosed at age ≤ 45 years
The detection rate of GPVs in patients who met this criterion was 10.3% (Table 3). Moreover, 8 (61.5%) in the other group, 19 (61.3%) in the BRCA group, and 235 (52.2%) in the non-carrier group met this criterion (other vs. non-carrier, P = 0.582). The odds ratio for the detection rate of this criterion was 1.46 (95% CI, 0.47–4.54) in univariate analysis and 1.83 (95% CI, 0.56–5.97) in multivariable analysis for the other group (Table 4).
Personal history of TNBC diagnosed at age ≤ 60 years
The detection rate of GPVs in patients who met this criterion was 21.4%. Moreover, 4 (30.8%) patients in the other group, 8 (25.8%) in the BRCA group, and 44 (9.8%) in the non-carrier group met this criterion (other vs. non-carrier, P = 0.038). The odds ratio for the detection rate of this criterion was 4.02 (95% CI, 1.19–13.59) in univariate analysis. In multivariable analysis, the association remained significant (adjusted OR 4.92, 95% CI, 1.41–17.14). This item indicated a significantly higher risk of detecting GPVs in genes other than BRCA1/2. However, given the small number of events and the presence of missing subtype data, this result should be interpreted with caution.
Personal history of BC diagnosed at any age with ≥ 2 total diagnoses of BC
The detection rate was 6.6%. Specifically, 1 (7.7%) patient in the other group, 4 (12.9%) in the BRCA group, and 71 (15.8%) in the non-carrier group met this criterion (other vs. non-carrier, P = 0.702). The odds ratio for the detection rate of this criterion was 0.45 (95% CI, 0.06–3.48) in univariate analysis and 0.52 (95% CI, 0.06–4.30) in multivariable analysis for the other group.
Personal history of BC diagnosed at any age with ≥ 1 close blood relative within the third degree on the same side of the family with breast, ovarian, or pancreas cancer.
The detection rate was 9.8%. Specifically, 9 (69.2%) in the other group, 22 (71.0%) in the BRCA group, and 285 (63.3%) in the non-carrier group met this criterion (other vs. non-carrier, P = 0.776). The odds ratio for the detection rate of this criterion was 1.30 (95% CI, 0.40–4.30) in univariate analysis and 1.66 (95% CI, 0.48–5.75) in multivariable analysis for the other group.
Personal history of male BC diagnosed at any age
No patients in the other and BRCA groups met this criterion, and the detection rate was 0%. Specifically, 3 (0.7%) patients in the non-carrier group met this criterion (other vs. non-carriers, P = 1.000).
Number of BRCA genetic testing criteria met and positivity rate
The number of items among the five test criteria fulfilled by patients in the other and non-carrier groups and the positivity rates are shown in Table 5. The median number of the testing criteria met was 2.0 in the other group and 1.0 in the non-carrier group. Concerning the positivity rate, 1.7% of patients who met only one of the five criteria had GPVs in genes other than BRCA1/2. This increased to 4.5% when two items were met. This rate increased as more items were met; 4.8% when ≥ 3 items were met (P = 0.149). Although a numerical increase in positivity rates was observed with a greater number of criteria met, this trend did not reach statistical significance.
Discussion
MGPT was performed in patients with BC considered genetically high risk. This study revealed that 9.11% of the patients carried GPVs in cancer susceptibility genes, including 6.5% with BRCA1/2 GPVs. Although the tested genes varied, previous studies have shown that 5.73% (BRCA1/2, 4.16%) of patients in Japan [16], 6.2% (BRCA1/2, 2.7%) in the United States [7], 7.4% (BRCA1/2, 4.4%) in Canada [6], and 4.55% (BRCA1/2, 2.12%) in Cyprus [4] have GPVs. These four studies included all patients with BC. In contrast, the present study focused on patients who had genetically high-risk BC and underwent BRCA genetic testing; thus, the GPV positivity rate was probably higher than those in the other four studies, particularly BRCA1/2 GPVs. As expected, the BRCA genetic testing criteria appear to be specialized when identifying GPVs in BRCA1/2. Furthermore, the prevalence of GPVs increases among patients who meet the hereditary cancer testing criteria of the National Comprehensive Cancer Network [7]. Consistent with this, the present findings indicate that genetically high-risk individuals are more likely to carry GPVs.
The use of MGPT has expanded rapidly in the evaluation of hereditary cancer susceptibility, enabling the simultaneous assessment of multiple cancer predisposition genes beyond BRCA1 and BRCA2 [17, 18]. In this study, GPVs were also identified in several non-BRCA genes, including ATM, RAD51D, BRIP1, BARD1, CHEK2, and NF1. Although some genes included in the panel are not primarily associated with BC risk, they are related to hereditary cancer syndromes for which established surveillance and risk reduction strategies exist [19]. In terms of clinical utility, identification of GPVs in BC–associated genes may inform BC risk assessment, surveillance strategies, and therapeutic decision-making. In contrast, identification of GPVs in other hereditary cancer genes may primarily inform individualized cancer risk management for other cancer types associated with the respective hereditary cancer syndromes and may guide referral to specialized genetic counseling services [20]. Current clinical practice guidelines, such as the NCCN Genetic/Familial High Risk Assessment Guidelines, recommend consideration of MGPT and tailored management strategies in individuals at elevated risk for hereditary cancer syndromes [19]. Furthermore, such findings may have implications for family members, as cascade testing can enable the identification of at-risk relatives who may benefit from tailored cancer surveillance and preventive interventions [20]. These considerations highlight the potential clinical value of MGPT in hereditary cancer evaluation, even when the strength of gene–disease associations varies across genes [17, 18].
Moreover, the association between the BRCA genetic testing criteria and the presence of GPVs was investigated. Commonly, ≥ 10% probability is cited as a reference point when determining who should undergo genetic testing [21]. In Australia, Medicare-funded genetic testing is offered to people with newly diagnosed BC if the likelihood of a GPV finding, based on family history and tumor pathology, is at least 10% [22]. In this study, the detection rate of GPVs exceeded approximately 10% among patients who met at least one of the following criteria: “diagnosed at age ≤ 45 years,” “diagnosed with TNBC at age ≤ 60 years,” or “≥1 close blood relative within the third degree on the same side of the family with breast, ovarian, or pancreas cancer.” Therefore, MGPT should be offered to patients meeting any of these criteria, as it is likely to be effective in identifying hereditary cancer predisposition and beneficial not only for the patients but also for the health management of their relatives. However, some patients who did not meet any of these three criteria were also found to carry GPVs. In this study, the GPV detection rate in patients who meet the criteria “≥2 total diagnoses of BCs” was < 10%, although patients with this item are generally considered appropriate candidates for BRCA genetic testing [9]. Several studies have indicated that the risk of contralateral or bilateral BC in patients with GPVs in genes other than BRCA1/2 is not significantly different from that of the general BC population [6, 23]. Further investigation is needed to determine whether MGPT is warranted for these patients. In male BC cases, a reliable assessment could not be performed owing to the small number of cases.
Patients diagnosed with TNBC at age ≤ 60 years had a higher likelihood of harboring GPVs in genes other than BRCA1/2. This observation is consistent with previous findings, which indicate that genetic predisposition contributes to the phenotype [24, 25]. BRCA1, BRCA2, BARD1, BRIP1, PALB2, RAD51C, and RAD51D are associated with an increased risk of TNBC [6, 24–27]. In our cohort, TNBC was observed in patients with GPVs in BRCA1, BRCA2, BARD1, BRIP1, EPCAM, NF1, or RAD51D. These genes, except for EPCAM and NF1, have been previously reported to be associated with TNBC. Although the therapeutic management of TNBC remains a significant challenge [28], patients with GPVs may obtain clinically meaningful information to guide treatment strategies [29]. It proposes a potential for broader clinical utility of MGPT in this patient population.
An increase in the number of fulfilled criteria for BRCA genetic testing has been associated with a higher positivity rate of BRCA1/2 GPVs [30]. Similarly, in this study, the GPV positivity rate in genes other than BRCA1/2 increased with the number of applicable criteria. Although the association was not significant, this finding underscores the importance of multiple matching items to enhance GPV detection, highlighting the need for further investigation.
Several limitations should be acknowledged. First, the sample size was not large enough to detect GPVs that are expected to occur at relatively higher frequencies, such as PALB2 [16, 24]. This limitation is partly attributable to patient preferences, as some individuals elected to undergo BRCA genetic testing only and were reluctant to proceed with MGPT. Second, some cases meeting the criteria for BRCA genetic testing were not identified as such. Given that a family history of cancer was based on patient-reported information, any family history unknown to the patient could not be captured. Third, the number of non-BRCA1/2 GPV events was small (n = 13), which may have resulted in unstable estimates in the logistic regression analyses, as reflected by the wide confidence intervals. Therefore, these findings should be interpreted with caution. Fourth, cases with missing or uncertain information including tumor subtype regarding BRCA genetic testing criteria were excluded from the regression analyses. This approach may have introduced selection bias and affected the robustness and generalizability of the findings. Fifth, given the limited number of events, we did not perform sensitivity analyses or apply penalized regression methods, which may further limit the reliability of the estimates. Sixth, this study was exploratory in nature, and no formal adjustments for multiple comparisons were performed. Therefore, the findings should be considered hypothesis-generating. Finally, we did not analyze copy number variants or large genomic rearrangements in this study. Therefore, this study might underestimate the carrier frequency of GPVs. In addition, we relied on ClinVar to annotate clinical significance. Therefore, we might miss non-loss-of-function pathogenic variants not registered in ClinVar. Taken together, these limitations increase the possibility that our findings are not broadly generalizable, emphasizing the need for additional cases and large-scale validation studies in Japan.
Acknowledgements
We would like to thank all investigators and patients who were involved in this study and the Genomics and Transcriptomics Unit, Advanced Multi-Omics Technology Division, RIKEN Center for Integrative Medical Sciences (IMS). We thank Enago (www.enago.jp) for the English language editing.
Author contributions
Conceptualization, S.N.; data curation, M.T., R.S., and M.E.; writing – original draft, M.K.; writing – review & editing, M.I., R.M., M.S., K.K., J.T., A.N., T.H., Y.H., T.U., C.W., Y.K., and Y.M.; supervision, K.T. All authors reviewed and approved the final manuscript draft before publication.
Funding
Open Access funding provided by SHOWA Medical University. This work was supported by the “Strategic International Collaborative Research Program” (SICORP; 2021–2024) research program, granted by the Japan Agency for Medical Research and Development (AMED) [grant number JP24jm0210089]. This program included four academic and cancer hospitals in Japan: Showa Medical University Hospital, St. Luke’s International Hospital, Keio University Hospital and Cancer Institute Hospital of Japanese Foundation for Cancer Research.
Data availability
The datasets generated and/or analyzed during the current study are not publicly available due to patient privacy but are available from the corresponding author on reasonable request.
Declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethical approval and consent to participate
This study was approved by the Showa University Research Ethics Review Board [approval No, 22-066-A]. Informed consent was obtained from all participants involved in the study.
Consent for publication
Not applicable.
Footnotes
Seigo Nakamura Deceased. Contribution completed prior to death.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and/or analyzed during the current study are not publicly available due to patient privacy but are available from the corresponding author on reasonable request.