Genomic DNA and amplified fragments are qualified for DNA sequencing
The genomic DNA samples with a size of >5Kb (Fig 1B)isolated from all HGSOC tissues and PBL were fractionally amplified by PCR. First-round PCR amplicons of BRCA2 exon 11 (c.2803-c.6728) (Fig 1A) were amplified from genomic DNA using the 8 pairs of primers; the second-round PCR amplicons covering BRCA2 BRC1~8 repeats and their spacing sequences (c.2915-c.6534) were amplified from the first-round PCR amplicons using the other 8 pairs of specific primers and available for DNA sequencing (Fig 1B and C).
The mutation sites in BRCA2 BRC repeat
No mutation in BRCA2 BRC repeat was identified in PBL of all patients with HGSOC. 44 different nucleotide mutation sites in BRCA2 BRC repeat were identified in tumor tissues of 27 HGSOC patients, inclunding 10 of them were silent mutation, 26 missense mutation and 8 nonsense mutation. Most of them were situated in the spacing sequences between the evolutionary conserved BRC domains. Only 2 (4.5%) missense mutations (c.5076G>A and c.5587A>T) and 2 (4.5%) nonsense mutations (c.5038T>C and c.5608T>C) were located within BRC5 and BRC6 domain, respectively, others (91%) were occurred in the spacing sequences between the BRC4~8 repeat domains(Fig.2A and Table 1). These results showed that all the patients identified were somatic but not germline mutations.
Type and frequencyof BRCA2 BRC repeat mutations
Among 113 patients with HGSOC, silent mutations were identified in 66 (58.4%) cases (c.3623A>G in BRC1~2), 47 (41.6%) cases (c.4034T>C in BRC2~3), 2 (17.7%) cases (c.5430T>C and c.5133A>G in BRC5~6), 1 (0.9%) case (c.4953T>C in BRC4~5 ), 1 (0.9%) case (c.5562T>C in BRC6) and 4 (3.5%) cases (c.6363A>G, c.6513G>C, and c.6462T>C in BRC8~); A relatively high ratio (21.2%, 24/113) of missense and nonsense mutations was found in the BRC4~8 repeats but not in the BRC1~3 repeats. 8 (7.1%) cases with nonsense mutation and 16(14.2%) cases with missense mutation were identified in 113 cases, respectively (Fig. 2B); Among the 24 cases with these mutations, 11 (45.8%)cases were multi-site mutations and 13 (54.2%) cases were single-site mutations (Table 1). Accordingly, the cases having a single nonsense or missense mutation were respectively included in analyses comparing with those without mutation for assessment of clinical outcome.
Nonsense mutation leads to C-terminal truncation of BRCA2 protein
Immunohistochemistry results showed that all patients were positive immunostaining in the N-terminus of BRCA2 protein, but 8 cases with nonsense/frameshift mutation were negative immunostaining in the C-terminus (Fig. 2C), proving that nonsense/frameshift mutation leads to C-terminal truncation of BRCA2 protein.
BRCA2 BRC repeat mutations prolong CFI
HGSOC patients with nonsense or missense in BRCA2 BRC repeat (21.2%, 24/113) had significantly longer CFI and higher sensitivity to platinum-based chemotherapy than non-mutated ones. As shown in Fig. 3A, The median CFI for nonsense (7.1%) and missense (14.13%) mutation were 37 months (95%CI,30.56-43.44 months) and 15 months (95%CI, 12.294-23.972 months), respectively, compared to 8 months (95%CI, 4.657-11.343) for non-mutation (P=0.000 and P=0.044).
BRCA2 BRC repeat mutations contribute to better survival
The median PFS for nonsense and missense mutations were 43 months (95%CI, 36.74-49.26 months) and 21 months (95% CI, 17.573-29.818 months), respectively, compared to 14 months (95%CI,11.55-16.45 months) for non-mutation (P=0.000 and P=0.049) (Fig. 3B). The median OS for nonsense and missense mutation were 56 months (95%CI,38.36-73.64 months) and 38 months (95%CI,33.29-42.71 months), respectively, compared to 31 months (95%CI,28.72-33.28 months) for non-mutation (P=0.002 and P=0.037)(Fig. 3C). The patients with nonsense and missense mutation exhibited significantly higher PFS and OS compared to those with non-mutation. In multivariate analysis, BRCA2 nonsense mutation is an independent factor for longer PFS (HR=0.079, 95%CI, 0.023-0.266, P<0.001) and OS (HR=0.121, 95%CI, 0.029-0.497, P=0.003), and BRCA2 missense mutation is an independent factor only for longer PFS (HR=0.429, 95%CI, 0.206-0.892, P=0.023) (Fig. 3B and C).