Secondary somatic mutations restoring BRCA1/2 predict chemotherapy resistance in hereditary ovarian carcinomas

Abstract

Purpose: Secondary somatic BRCA1/2 mutations may restore BRCA1/2 protein in hereditary ovarian carcinomas. In cell lines, BRCA2 restoration mediates resistance to platinum chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors. We assessed primary and recurrent BRCA1/2-mutated ovarian carcinomas to define the frequency of secondary mutations and correlate these changes with clinical outcomes.

Methods: Neoplastic cells were isolated with laser capture microdissection, and DNA was sequenced at the site of the known germline BRCA1/2 mutation. When secondary mutations were found that restored wild-type sequence, haplotyping was performed using single nucleotide polymorphisms in tumor and paired lymphocyte DNA to rule out retention of the wild-type allele.

Results: There were 64 primary and 46 recurrent ovarian carcinomas assessed. Thirteen (28.3%) of 46 (95% CI, 17.3% to 42.6%) recurrent carcinomas had a secondary mutation compared with two (3.1%) of 64 (95% CI, 1.0% to 10.7%) primary carcinomas (P = .0003, Fisher's exact test). Twelve (46.2%) of 26 (95% CI, 28.7% to 64.7%) platinum-resistant recurrences had secondary mutations restoring BRCA1/2, compared with one (5.3%) of 19 (95% CI, 1.2% to 24.8%) platinum-sensitive recurrences (P = .003, Fisher's exact test). Six (66.7%) of nine (95% CI, 34.8% to 87.8%) women with prior breast carcinoma had a recurrent carcinoma with a secondary mutation, compared with six (17.1%) of 35 (95% CI, 8.2% to 32.8%) with no history of breast carcinoma (P = .007, Fisher's exact test).

Conclusion: Secondary somatic mutations that restore BRCA1/2 in carcinomas from women with germline BRCA1/2 mutations predict resistance to platinum chemotherapy and may also predict resistance to PARP inhibitors. These mutations were detectable only in ovarian carcinomas of women whom have had previous chemotherapy, either for ovarian or breast carcinoma.

Fig 1.

Samples from individual with germline BRCA2 5193delC mutation demonstrating a secondary somatic mutation restoring BRCA2 in her ovarian carcinoma. (A) Lymphocyte DNA sequence of the heterozygous germline BRCA2 5193delC mutation. (B) DNA sequence from her primary ovarian carcinoma. Only mutant 5193delC sequence is seen, consistent with loss of the wild-type BRCA2 allele in neoplastic cells. (C) DNA sequence from recurrent ovarian carcinoma obtained 10 years after initial diagnosis. This patient had multiple chemotherapeutic regimens for recurrent disease, including a poly (ADP-ribose) polymerase (PARP) inhibitor before surgical resection of this recurrence (CS1 in Table 1). An insertion of an A base is seen at nucleotide 5177, which restores the open reading frame disrupted by the 5193delC mutation and restoring full-length BRCA2 protein. Both the original BRCA2 5193delC sequence and the 5177insA/5193delC combination sequence are seen, suggesting that the secondary mutation occurs in just one of a duplicated mutant allele or in just a proportion of the neoplastic cells analyzed. Postoperatively, the recurrent carcinoma was resistant to platinum chemotherapy and subsequently to a trial of a PARP inhibitor. (D) Single nucleotide polymorphism (SNP) haplotyping differentiates the germline mutant and wild-type alleles. Lymphocyte DNA demonstrates an A/G SNP within BRCA2. The primary carcinoma has only G, indicating that the G is on the mutant allele. The recurrent carcinoma also has predominantly G, indicating that the secondary mutation occurred on the mutant allele. (E) Allele diagrams show the relative positions of the SNP, the original mutation, and the secondary mutation.

Fig 2.

Secondary mutations in primary and recurrent carcinomas by platinum sensitivity and a history of previous breast cancer. (A) and (C) refer to primary ovarian carcinomas. (B) and (D) refer to recurrent ovarian carcinomas. (A) One (25%) of four platinum-resistant primary ovarian carcinomas had secondary mutations restoring BRCA1/2, compared with 0 of 56 platinum-sensitive primary ovarian carcinomas (P = .07, Fisher's exact test). Three carcinomas had unknown platinum status and one individual was nonevaluable. (B) Twelve (46.2%) of 26 platinum-resistant recurrent ovarian carcinomas had secondary mutations restoring BRCA1/2, compared with one (5%) of 19 platinum-sensitive recurrent ovarian carcinomas (P = .003). One carcinoma had unknown platinum status. Two carcinomas with secondary mutations are from the same patient (one sensitive and one resistant), and two carcinomas without secondary mutations are from the same patient (one sensitive and one resistant). (C) Two (10.5%) of 19 primary ovarian carcinomas obtained from women with a history of breast carcinoma had secondary mutations restoring BRCA1, compared with 0 of 45 obtained from women with BRCA1/2 mutations and no history of breast carcinoma (P = .08, Fisher's exact test). (D) Six (66.7%) of nine recurrent ovarian carcinomas from women with a history of breast carcinoma before their diagnosis of ovarian carcinoma had secondary mutations restoring BRCA1/2, compared with six (17.1%) of 35 recurrent ovarian carcinomas from women with no prior history of breast carcinoma (P = .007, Fisher's exact test).

Fig 3.

A secondary mutation in the recurrent ovarian carcinoma identified at low levels in the primary carcinoma. (A) Sequences from UW40. Lymphocyte DNA demonstrates the BRCA1 heterozygous germline mutation 2594delC and two heterozygous single nucleotide polymorphisms (SNPs). The primary ovarian carcinoma sequence shows both the 2594delC mutation and wild-type sequence, consistent with a secondary mutation restoring wild-type sequence. SNPs within the primary carcinoma confirm that only one allele is present, indicating that the secondary mutation occurred on the mutant allele, and the wild-type sequence present was not from retained wild-type allele. The recurrent ovarian carcinoma sequence has a 23-bp deletion that, when combined with the 2594delC mutation, restores the open reading frame. The 23-bp deletion is not detectable in the primary carcinoma using conventional sequencing. Adapted with permission from Swisher et al. (B) Mutation-specific primers were designed to amplify sequences with the 23-bp deletion only using polymerase chain reaction. As expected, the 23-bp deletion amplifies well in the recurrent carcinoma (Rec Ca). This 23-bp deletion sequence can also be detected in two separate samples of the primary ovarian carcinoma, from the ovary (Prim Ca Ovary) and from a metastatic lesion in the omentum (Prim Ca Om). Control samples of an unrelated individual's carcinoma (Control DNA), the UW40's lymphocyte DNA (Lymph), and water (H20) do not have detectable sequence.

Fig 4.

Model for acquired resistance to platinum chemotherapy by secondary mutations restoring BRCA1/2. Secondary mutation is hypothesized to occur as a result of DNA-damaging chemotherapy, either from primary adjuvant therapy of the ovarian carcinoma or from previous chemotherapy for a separate carcinoma such as breast. This population of cells with restored BRCA1/2 and improved DNA repair is either induced or selected by adjuvant platinum therapy. Adapted with permission from Sakai et al.

Fig A1.

Distribution of BRCA1 and BRCA2 mutations in primary and recurrent carcinomas by secondary mutation status. (A) Two of 48 BRCA1 mutation carriers with primary carcinomas had secondary mutations restoring BRCA1/2, compared with 0 of 16 BRCA2 mutation carriers (P = 1.00, Fisher's exact test). (B) Six of 31 BRCA1 mutation carriers with recurrent carcinomas had secondary mutations restoring BRCA1/2, compared with six of 13 BRCA2 mutation carriers (P = .13, Fisher's exact test).