Demographic features
A total of 52 Asian male patients harboring BRCA mutations were identified in the SYSUCC cohort. The median age at the time of diagnose was 63.5 years (range, 4-86 years). 69.2% (36/52) of the cases were at the age of 50-69 years. 28.8% (15/52) had a family history of tumor, and only 3.8% (2/52) demonstrated multiple primary tumors. Prostate was the most common site of tumor disease in this cohort (19/52, 36.5%), followed by colorectum (13/52, 25.0%), stomach (7/52, 13.5%), and lung (4/52, 7.7%). Only one male patient with breast cancer harboring germline BRCA mutation was observed. The majority were at stage III (15/52, 28.8%) and stage IV (28/52, 53.8%). 16 cases (30.8%) were tested positive for only BRCA1 mutation, of which 3 were germline, and 13 were somatic. 33 (63.5%) patients harbored only BRCA2 mutation, of which 4 were germline, 26 were somatic, and 3 cases displayed both germline and somatic BRCA2 mutations. We also find that three patients harbored both BRCA1 and BRCA2 somatic mutations. Besides, 13.5% (7/52) of the cases showed a microsatellite instability-high (MSI-H) phenotype, while 30.7% showed moderate to high TMB (≥10 muts/Mb). The clinical characteristics of SYSUCC cohort are summarized in Table 1.
The incidence of BRCA mutation in male patients was 6.0% (294/4871) in the TCGA cohort, and the majority were white. The median age at the time of diagnose was 65 years (range, 34-88 years), and the patients were predominantly at the age of 50-79 years. Carcinomas of lung (57/294, 19.4%), bladder (46/294, 15.6%), stomach (35/294, 11.9%), head and neck (25/294, 8.5%), colorectum (24/294, 8.2%), as well as cutaneous melanoma (48/294, 16.3%) were the frequent tumors demonstrating BRCA mutation. However, prostatic cancer was only observed in 11 cases, and three patients had breast cancer. The majority were at stage II (86/294, 29.3%) and stage III (28/52, 53.8%). Similar to the findings in SYSUCC cohort, 81 cases (27.6%) had a BRCA1 mutation, while 193 cases (65.6%) had a BRCA2 mutation. Patients with both BRCA1 and BRCA2 mutations (20/294, 6.8%) showed a median total mutation counts of 2390 muts (range, 264-15832 muts), which was significantly higher than in patients with either BRCA1 or BRCA2 mutation. The clinical characteristics of TCGA cohort are summarized in Table 2.
Clinical characteristics between males and females with BRCAm
The clinical characteristics were compared between male and female patients with BRCAm, as shown in Table 2. The incidence of BRCAm (6.0 vs. 6.6%), as well as the age at diagnosis of tumor between male and female patients was similar, and no statistical difference was observed (p=0.275 and p=0.15, respectively). However, the spectrum of tumors that occurred in males was obviously different from that in females. Carcinomas of breast, ovary, uterus, and cervix were frequently occurred in females with BRCAm, while a higher incidence of carcinomas in lung, bladder, stomach, head and neck, as well as cutaneous melanoma, was found in male BRCAm carriers (Figure 1A). Adenocarcinoma was the most common histological type in both groups, while there were more cases with melanoma, urothelial carcinoma, and squamous cell carcinoma in males (Figure 1B). Besides, stage III (24.5 vs. 11.5%) and stage IV (13.3% vs. 2.3%) disease were more common in males than in females. Interestingly, individuals with both BRCA1 and BRCA2 mutations were more frequently observed in females (14.1% vs. 6.8%).
Clinical characteristics between males with and without BRCAm
The mean age at the time of diagnose was 61 years (range, 14-86 years) in male patients without BRCAm, which was younger than those with BRCAm (p<0.001). No difference was observed in ethnic distribution between groups. It is noteworthy that, compared to non-BRCAm carriers, cutaneous melanoma (16.3 vs. 5.0%), lung cancer (19.4 vs. 11.8%), bladder cancer (15.6 vs. 5.6%), and stomach cancer (11.9 vs. 5.5%) accounted for a significantly higher proportion of the tumor diagnosed in BRCAm group. There were fewer cases that had a diagnosis of renal cell carcinoma (4.8 vs. 12.3%) or prostatic carcinoma (3.7 vs. 10.6%) in BRCAm group than in non-BRCAm group (Table 2, Figure 1A). Histologically, adenocarcinoma was the most common type in both groups, but melanomas and urothelial carcinomas were more commonly found in BRCAm group (Figure 1B). Patients with BRCAm were also likely to present a more advanced stage of tumor disease (p=0.007) and much more total mutation counts than those without BRCAm (median, 322 vs. 63 muts).
The mutational profiles of BRCA1/2 in males
The characteristics of BRCA1/2 mutation between male and female patients with a diagnosis of tumor are demonstrated in Table 3. A total of 438 BRCA1/2 variations were identified in males within TCGA cohort (detail in Supplementary Table 1) and
SYSUCC cohort (detail in Supplementary Table 2). Mutations affecting BRCA1 and BRCA2 was accounted for 29.0% (127/438) and 71.0% (311/438), respectively. For BRCA1, 21.8% (29/127) of the mutations were deleterious or likely deleterious. Similarly, 28.6% (89/311) of BRCA2 variations were identified as deleterious or likely deleterious. 17.3% (60/346) of the male patients harbored more than one BRCA1/2 mutation individually. Missense mutation was the predominant mutational type of both BRCA1 and BRCA2, accounting for 70.1% (89/127) and 69.5% (216/311), followed by nonsense and frameshift mutations.
As shown in Figure 1C, BRCA2 mutation was more frequently detected than BRCA1 regardless the location of tumors in males, which accounted for 56.7-100.0% of cases. Mutations simultaneously affecting both BRCA1 and BRCA2 were mainly occurred in head/neck squamous cell carcinoma (18.8%) and cutaneous melanoma (12.5%). However, in terms of pathogenicity (Figure 1D), only a few BRCA1/2 mutations in cutaneous melanomas (8/48, 16.7%) and tumors located in head and neck (5/27, 18.5%) and CNS (1/7, 12.5%) were deleterious. None of the BRCA1/2 mutated in renal cell carcinomas included in this study was deleterious (0/14). More frequent deleterious mutations were identified in carcinomas of colorectum (23/37, 62.2%), prostate (13/30, 43.3%), and stomach (18/42, 42.9%). Notably, all BRCA mutations in male breast cancer were deleterious (4/4). Compared to BRCA1/2 mutations in female patients, fewer deleterious (or likely deleterious) mutations affecting BRCA1 were detected in male (21.8 vs. 34.7%, p=0.032), while those affecting BRCA2 showed no difference between groups (28.6 vs. 25.8%, p=0.454). Besides, male patients displayed fewer frameshift mutations of BRCA1 than female (5.5 vs. 10.4%, p=0.013). On the contrary, the frameshift mutations of BRCA2 were likely more frequently found in male (15.1% vs. 9.5%), while no statistic difference was observed.
The mutations on linear BRCA1 or BRCA2 protein and its domains were mapped as shown in Figure 2. In accordance with the previous studies, there was no obvious hotspot mutantional region in either BRCA1 or BRCA2. In male BRCA1 mutation carriers, only 2 of 127 mutations (1.6%) located in RING domain that associates with BRCA1-associated RING domain protein 1 (BARD1) and catalyses protein ubiquitylation. 6.3% (8/127) were mutated at the BRCA1 C-terminus (BRCT) domain and 6.3% (8/127) were mutated at the serine-rich domain associated with BRCT, which facilitates phospho-protein binding, checkpoint activation and DNA repair. Similarly, in female BRCA1 mutation carriers, 3.1% (6/193) mutations located in RING domain. 6.2% (12/193) and 4.7% (9/193) were mutated at BRCT domain and serine-rich domain associated with BRCT, respectively. BRCA1 fusion mutations containing preferred partner, including NF1, FAM134C, BECN1, and LSM12, were more frequent detected in male carriers, while mutations affecting E111 (E111*, E111Gfs*3) or R1443 (R1443*, R1443Q) site were more commonly found in BRCA1 in female patients. In male BRCA2 mutation carriers, a total of 19 mutaions (6.1%) were detected in the eight centrally located BRC repeats in BRCA2, which has been suggested to mediate binding and regulation of RAD51 on resected DNA substrates. 20.3% (63/311) of the mutations were located at the BRCA2 DNA-binding domain, of which 5.1% (16/311) at helical domain, 14.2% (44/311) at oligonucleotide binding folds, and 1.0% (3/311) at tower domain, which may affect BRCA2 binding to both single-stranded DNA and double-stranded DNA. In female BRCA2 mutation carriers, 6.3% (25/399) of the mutations affected BRC repeats and 18.3% were located at the BRCA2 DNA-binding domain, of which 5.0% (20/399) at helical domain, 11.3% (45/399) at oligonucleotide binding folds, and 2.6% (8/311) at tower domain. Besides, frameshift mutations of BRCA2 at N1784 (N1784Kfs*3, N1784Tfs*3) were frequently observed in both male and female patients. Recurrent BRCA2 mutations at P606L/S, E832K/G, and T3033Lfs*29 were shown in male, while R1512C/H, K1691Nfs*15, S1882*, R2842C/H, E3342K, and K3416Nfs*11 appeared repeatedly in female BRCA2 mutation carriers.
Prognostic implications of BRCAm in males
The male BRCAm carriers with a diagnosis of malignancy were followed-up for 0.3-151.3 months, with a median of 25.9 months. For comparisons, those male patients with no BRCAm were followed-up for 0.1-357.4 months, with a median of 22.9 months. Moreover, the female patients with BRCAm were followed-up for 0.1-302.1 months, with a median of 26.2 months. Survival analyses were conducted in both unmatched and matched cohorts. The survival curves among three groups are shown in Figure 3. BRCAm in males was associated with a decrease in overall survival (OS) and progression-free survival (PFS) when comparing to female patients with BRCAm in both unmatched and matched cohorts (Figure 3A-D; p<0.001). No statistic difference of OS was observed between male patients with and without BRCAm in either unmatched (p=0.698; Figure 3A) or matched cohort (p=0.191; Figure 3E), but those with deleterious BRCAm displayed a significant increased OS than non-BRCAm carriers in both 1:3 (p=0.021; Figure 4A) and 1:6 matched models (p=0.042; Supplementary figure 1). Furthermore, among male patients with BRCAm, after matching for age and TNM stage, those with deleterious BRCAm also displayed an increased OS than patients with undetermined significant BRCAm (p=0.027; Figure 4C). However, no difference of PFS was found in any comparison in males between groups (Figure 3B, 3F, 4B, and 4D). Subgroup survival analysis was also conducted among male patients with BRCA1, BRCA2, and both mutations was conducted, but no difference in survival was found (Supplementary figure 2).
The associations between BRCAm and the survival outcomes in male patients in the subgroups of different tumor types are demonstrated in Figure 5 . BRCAm was associated with hazard ratios for OS of 0.61 (95% CI 0.39-0.94; p=0.05) and 0.60 (95% CI 0.39-0.94; p=0.05) in the subgroups of patients with bladder cancer and stomach cancer, respectively. On the contrary, the hazard ratio of patients with BRCAm for OS was 3.07 (95% CI 1.45-6.53; p<0.01) in glioma/glioblastoma. Besides, BRCAm was associated with hazard ratios for PFS of 2.42 (95% CI 1.22-4.81; p=0.01) and 3.24 (95% CI 1.15-9.08; p=0.02) in patients with prostatic cancer as well as head and neck squamous cell carcinoma, respectively. The survival curves of OS and PFS between groups in these tumors are shown in Figure 6.