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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.
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.
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).
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.
Ten percent to 15% of ovarian carcinomas occur in women with heterozygous germline mutations in BRCA1 and BRCA2 (BRCA1/2).1,2 BRCA1/2 are tumor suppressor genes, and BRCA1/2 proteins are instrumental in repairing damaged DNA, through homologous recombination.3,4 The vast majority of primary carcinomas in patients with BRCA1/2 mutations have deletions of the wild-type BRCA1/2 allele,5–7 leading to a neoplasm deficient in BRCA1/2 protein. BRCA1/2-deficient carcinomas have decreased capacity to repair DNA and are sensitive to chemotherapy with DNA cross-linking agents such as cisplatin and carboplatin (platinum agents)8–10 and are susceptible to synthetic lethality from poly (ADP-ribose) polymerase (PARP) inhibitors.11–13 Women with BRCA1/2-associated ovarian carcinoma have improved survival compared with those with sporadic ovarian carcinomas when treated with platinum agents.14–16 Despite this, the majority of women with BRCA1/2-mutated ovarian carcinomas ultimately develop recurrent disease that is resistant to platinum agents.
Our group and another have recently outlined a novel mechanism for resistance to platinum chemotherapy using BRCA2-deficient cancer cell lines.17–19 Secondary mutations that restore functional BRCA2 protein can be induced by exposing BRCA2-deficient pancreatic or ovarian cancer cell lines to cisplatin or PARP inhibitors. The resulting clones with restored BRCA2 are resistant to both cisplatin and PARP inhibitors.17–19 Furthermore, secondary mutations restoring wild-type sequence or a functional reading frame have been seen in a small number of ovarian neoplasms from both BRCA1 and BRCA2 mutation carriers.17–20
This study aimed to define the frequency of secondary mutations that restore BRCA1/2 function within the ovarian carcinomas of BRCA1/2 mutation carriers and correlate these changes with therapeutic outcomes.
Patients with primary and/or recurrent ovarian, peritoneal, or fallopian tube carcinomas and a confirmed BRCA1/2 mutation were identified using the University of Washington and Cedars-Sinai Women's Cancer Institute Gynecologic Oncology Tissue Banks, as approved by the Human Subjects Committee of the institutional review board at each institution, as well as one patient from Edinburgh Cancer Research Centre with institutional review board approval through Fred Hutchinson Cancer Research Center. Neoplastic samples were most often formalin-fixed paraffin embedded blocks, with some samples frozen in optimal cutting-temperature compound. One neoplastic sample was isolated from a malignant pleural effusion, using the CELLection Epithelial Enrich protocol (Invitrogen, Carlsbad, CA).
Neoplastic cells in blocks were isolated from surrounding normal cells by laser capture microdissection using a Veritas system (Moelcular Devices, Sunnyvale, CA). Neoplastic DNA was extracted using the PicoPure DNA extraction kit (Arcturus; Applied Biosystems, Foster City, CA), and neoplastic and normal DNA (from lymphocytes) was amplified by polymerase chain reaction (PCR), using primers flanking the known mutation in BRCA1 or BRCA2. PCR products were purified and sequenced with BigDye Terminator v3.1 (Applied Biosystems) using the ABI 3100 Genetic Analyzer (Applied Biosystems). Sequences at the mutation site were analyzed for relative ratios of wild-type and mutant sequences by comparing fluorescent peak heights. In cases with suspected secondary mutations, intragenic heterozygous single nucleotide polymorphisms (SNPs) were identified in lymphocyte DNA and compared with neoplastic DNA to rule out contamination with non-neoplastic cells or retention of the wild-type allele. If a heterozygous SNP was not identified, microsatellite testing was used.
Platinum sensitivity was defined by the response to platinum chemotherapy after the carcinoma was sampled (rather than the usual definition using the treatment-free interval before the recurrence). Carcinomas that responded to platinum chemotherapy and were in remission for at least 6 months after chemotherapy were defined as platinum sensitive; all others were labeled platinum resistant.
The number of patients with a secondary mutation was too small to adequately assess a survival difference between those with and without secondary mutations.
Statistical analysis was performed using Instat or Prism software (Graphpad, San Diego, CA). Contingency tables were made for comparison of categorical variables, and P values were derived using Fisher's exact test.
We analyzed 64 primary (48 BRCA1, 16 BRCA2) and 46 recurrent (32 BRCA1, 14 BRCA2) ovarian, fallopian tube, and primary peritoneal carcinomas (subsequently collectively referred to as ovarian carcinomas). The 46 recurrent carcinomas were from 44 women, as two women had two separate recurrences analyzed. Seven primary and 15 recurrent carcinomas were previously published.17,18,20 All carcinomas had deletion of the wild-type BRCA1/2 allele, as determined by no wild-type sequence being detected or by confirmation with SNPs or microsatellite testing.
Secondary mutations restoring BRCA1/2 were more common in recurrent ovarian carcinomas (13 [28.3%] of 46; 95% CI, 17.3% to 42.6%) than in primary carcinomas (two [3.1%] of 64; 95% CI, 1.0% to 10.7%; P = .0003, Fisher's exact test). Secondary mutations were more common in recurrent ovarian carcinomas of BRCA2 mutation carriers (six [46.2%] 13; 95% CI, 23.0% to 71.1%,), compared with those of BRCA1 mutation carriers (six [19.4%] 31; 95% CI, 9.2% to 36.4%), but this did not achieve statistical significance (P = .13; Appendix Fig A1, online only).
Of the 46 recurrences, previous chemotherapy regimen information was complete on 42. Twenty-five recurrences were sampled after only one previous chemotherapy regimen, and four had secondary mutations (16%; 95% CI, 6.5% to 34.9%). In contrast, 17 recurrences were sampled after two or more chemotherapy regimens, and eight contained secondary mutations restoring BRCA1/2 (47%; 95% CI, 26.0% to 69.2%; P = .04, Fisher's exact test). Of the four recurrent cases with secondary mutations sampled after only one chemotherapy regimen for ovarian carcinoma, three had received chemotherapy previous to their ovarian cancer diagnosis for breast carcinoma.
Cases with secondary mutations are summarized in Table 1. Two secondary mutations were insertions or deletions that restored a functional reading frame, 11 were reversions to wild-type sequence, one was a synonymous mutation restoring the wild-type amino acid sequence, and one was loss of the mutant BRCA2 allele. An example of sequences from a patient with a secondary mutation is shown in Figure 1.
Of 64 primary ovarian carcinomas, four (6.3%) were primarily platinum resistant, 56 were platinum sensitive, three were of unknown platinum sensitivity, and one was nonevaluable for response. One of four platinum-resistant primary ovarian carcinomas had a secondary mutation restoring BRCA1 (25%; 95% CI, 5.3% to 71.4%), as compared with 0 of 56 platinum-sensitive primary carcinomas (95% CI, 0% to 6.3%; P = .07, Fisher's exact test; Fig 2A).
In contrast, of 46 recurrent ovarian carcinomas, 26 (56.5%) were platinum resistant (defined as recurrence within 6 months of stopping secondary chemotherapy after the recurrent carcinoma was sampled), 19 were platinum sensitive, and one was unknown. Twelve of 26 platinum-resistant recurrences had secondary mutations restoring BRCA1/2 (46.2%; 95% CI, 28.7% to 64.7%), as compared with one of 19 platinum-sensitive recurrences (5.3%; 95% CI, 1.2% to 24.8%; P = .003, Fisher's exact test, Fig 2B).
One primary carcinoma with a secondary mutation occurred in a 0.8-mm stage IA fallopian tube carcinoma diagnosed at the time of risk-reducing salpingo-oophorectomy, in a carrier of a germline BRCA1 120A>G mutation who was previously treated with chemotherapy for breast cancer (UWF35, Table 1). She received three cycles of docetaxel and carboplatin for the tubal carcinoma and has no evidence of recurrence 2 years later. She was considered nonevaluable for response given her lack of residual disease after initial surgery. The other patient with a secondary mutation in a primary carcinoma had a germline BRCA1 2594delC mutation and stage IV primary platinum-resistant disease (UW40, Table 1).20 She also was treated previously with chemotherapy for breast cancer (unknown agents). She subsequently experienced recurrence within 3 months of completing primary platinum-containing chemotherapy.
The patient with a secondary mutation in a platinum-sensitive recurrent carcinoma (UW200) initially presented with stage IIIB ovarian carcinoma, treated with primary surgery and six cycles of carboplatin and paclitaxel. She then experienced recurrence 3 years later, when the recurrent sample with a secondary mutation was obtained. This was a small-volume recurrence that was removed with no visible residual disease. She was treated with intraperitoneal cisplatin and intravenous paclitaxel for six cycles and did not experience recurrence again for 13 months after completing that regimen. Her recurrence at that time was also found to have a secondary mutation and was platinum resistant with disease progression within 2 months of completing platinum-based therapy. Interestingly, her recurrent carcinoma has subsequently had a partial response on a clinical trial combining carboplatin, gemcitabine, and a PARP inhibitor. However, the lack of a complete response to platinum is consistent with platinum resistance.
Six patients with platinum-resistant recurrent ovarian carcinomas were treated with PARP inhibitors (Table 2). Three carcinomas had secondary mutations restoring BRCA1/2 (including the one just described), two did not respond, and one had a partial response. Two patients who did not have secondary mutations were noted to have a complete response to PARP inhibition, and one had a partial response.
A history of breast carcinoma occurring before the primary ovarian carcinoma was present in 19 (29.7%) of 64 patients who had primary carcinomas sequenced. Two (10.5%) of 19 (95% CI, 3.2% to 31.7%) had secondary mutations in their primary ovarian carcinoma after a previous breast carcinoma, compared with 0 of 45 with no history of breast carcinoma (95% CI, 0 to 7.7%; P = .08, Fisher's exact test; Fig 2C). Data regarding prior treatment with chemotherapy was incomplete; however, at least 11 of 19 had chemotherapy for breast carcinoma before diagnosis with ovarian carcinoma. Interestingly, the two primary carcinomas with secondary mutations that restored BRCA1 had received their chemotherapy for breast carcinoma 10 and 11 years before their diagnosis of fallopian tube or ovarian carcinoma, respectively.
Of 44 women with recurrent ovarian carcinomas (two women had two recurrences), nine (20.5%) had been previously treated for breast carcinoma. Six of nine recurrent ovarian carcinomas in women with a history of breast carcinoma had secondary mutations (66.7%; 95% CI, 34.8% to 87.8%), compared with six of 35 recurrent ovarian carcinomas in women with no history of breast carcinoma (17.1%; 95% CI, 8.2% to 32.8%; P = .007, Fisher's exact test; Fig 2D). Information on chemotherapy for breast carcinoma was incomplete; however, at least four of nine women had chemotherapy for breast carcinoma before their primary ovarian carcinoma, and three of four had secondary mutations restoring BRCA1/2. To date, no secondary mutation has been identified in a primary or recurrent ovarian carcinoma in an individual without any history of prior chemotherapy. Of women with breast carcinoma and a secondary mutation, the range of time from breast cancer diagnosis to ovarian cancer diagnosis was 60 to 156 months (median, 115.5 months).
Five women received neoadjuvant chemotherapy immediately before surgery for their primary ovarian carcinoma (range, three to four cycles), and none had detectable secondary mutations in their primary carcinomas.
Patient UW40 had a 23 base-pair (bp) deletion downstream from her BRCA1 2594delC mutation that restores a functional reading frame (Fig 3A), as previously described.20 Primers were designed to PCR amplify only the sequence containing the secondary 23-bp deletion. Using Sanger sequencing, this 23-bp deletion was detectable only in the recurrent carcinoma and not in the paired primary carcinoma from the same individual. However, PCR amplification using the mutation-specific primers detected the secondary mutation in the patient's primary carcinoma (Fig 3B), indicating that the secondary mutation in the recurrent carcinoma was present in a minority of neoplastic cells at the time of diagnosis.
In this large series of BRCA1/2-mutated ovarian, tubal, and peritoneal carcinomas, the presence of secondary somatic mutations that restore BRCA1/2 in recurrent carcinomas was significantly correlated with response to platinum-based chemotherapy, with 12 (92%) of 13 carcinomas with restored BRCA1/2 proving to be platinum-resistant (95% CI, 66.1% to 98.2%). When treating ovarian carcinomas, oncologists traditionally have used the interval from completion of platinum-based chemotherapy to disease progression to predict platinum sensitivity or resistance. When the treatment-free interval is greater than 6 months before progression, the patient is thought to have a platinum-sensitive recurrence and is often re-treated with platinum-based chemotherapy. In our series, the presence of a secondary mutation restoring BRCA1/2 was a better predictor of platinum resistance than interval since last treatment (Table 1). Of 26 recurrent carcinomas that proved to be platinum resistant, 12 (46.2%) of 26 (95% CI, 28.7% to 64.7%) could have been predicted by detection of a secondary mutation restoring BRCA1/2, as opposed to only four (15.4%) of 26 by treatment-free interval (95% CI, 6.3% to 33.7%; P = .03, Fisher's exact test, two-tailed). Additionally, secondary mutations were significantly more common in recurrent carcinomas sampled after more than one chemotherapy regimen than those sampled after a single chemotherapy regimen. The ability to predict response to platinum chemotherapy is clinically valuable, as it would allow the avoidance of unnecessary toxicity and earlier use of alternate agents. Although our data support the clinical utility of testing for secondary mutations in BRCA1/2-mutated ovarian carcinomas to predict platinum resistance, the clinical implementation may be challenging because this is a technically demanding test, requiring careful microdissection to obtain a pure neoplastic cell population. Additionally, the differentiation of a secondary reversion mutation from contamination with wild-type sequence from non-neoplastic cells requires careful allelic identification with SNP haplotyping or microsatellite testing.
There was a marked association between prior history of breast carcinoma and secondary mutations in either the primary or recurrent ovarian carcinomas, with the breast carcinoma often preceding the ovarian carcinoma by many years. Cumulative chemotherapy exposure is the most likely contributor to these secondary genetic changes. Cyclophosphamide, commonly used in patients with breast carcinoma, is a DNA cross-linking agent and theoretically would induce or select for BRCA1/2 restoration in similar fashion to platinum compounds.21 The occurrence of secondary mutations in ovarian carcinomas more than a decade after chemotherapy for breast cancer is not easily explainable. If breast cancer chemotherapy induced a secondary mutation that restored wild-type sequence in a non-neoplastic ovarian or tubal epithelial cell, such a cell would be homozygous for wild-type BRCA1/2 and presumably be subsequently protected from neoplastic transformation. Alternatively, hypothesizing that the secondary mutation occurs in a cell that is already BRCA1/2 deficient (secondary to deletion of the wild-type allele) implies that the earliest neoplastic cell exists many years before clinical diagnosis. A better understanding of the timing of secondary mutations will shed light on the natural history and molecular progression of hereditary ovarian carcinomas. The association of breast carcinoma with secondary mutations in subsequent ovarian carcinomas could also be unrelated to chemotherapy and perhaps reflects a more severe phenotype in those patients.
The timing of when secondary mutations develop during the treatment of ovarian carcinoma is clinically important. Secondary mutations may be induced by DNA-damaging chemotherapy either for breast carcinoma or primary ovarian carcinoma. Alternatively, secondary mutations could be present in the primary carcinoma due to genomic instability and then selected by chemotherapy. Data from patient UW40 (Fig 3) reveals that secondary mutations can be present in rare cells in the primary carcinoma. Her secondary mutation may have been induced by chemotherapy for breast carcinoma. Whether secondary mutations restoring BRCA1/2 exist in primary ovarian carcinomas in women with no previous chemotherapy exposure is not known. In chronic myelogenous leukemia, BCR-ABL mutations that confer resistance to imatinib therapy in some cases are detectable by PCR in rare neoplastic cells before imatinib exposure.22 If secondary mutations that restore BRCA1/2 are already present in a small percentage of cells in a primary ovarian carcinoma similarly, this could have therapeutic implications. It is possible that the use of PARP inhibitors for prolonged consolidation after primary therapy could accelerate the selection of BRCA1/2-restored neoplastic cells and consequently accelerate the development of platinum resistance after recurrence.
These data add to the current understanding of BRCA1/2 carcinogenesis. Loss of heterozygosity in BRCA1/2 leads to a carcinoma deficient in BRCA1/2 protein. This carcinoma with decreased ability to repair DNA then becomes highly sensitive to DNA cross-linking agents such as platinum drugs. Secondary mutations restoring DNA repair function of BRCA1/2 can occur within the carcinoma, likely as a consequence of prior chemotherapy or perhaps from genomic instability and spontaneous mutation. This restoration of DNA repair can then lead to acquired resistance to platinum and likely resistance to PARP inhibition (Fig 4).
Not all platinum-resistant recurrent carcinomas had detectable secondary somatic mutations, indicating that other mechanisms of acquired resistance occur. Possibly, we missed some larger insertions or deletions that restore the BRCA1/2 reading frame,19 as these alterations would be missed with the relatively short amplicons generated by our PCR of formalin-fixed tissues. Additionally, our retrospective series does not represent all women with BRCA1/2-associated ovarian carcinoma as we selected those cases who had known mutations as opposed to prospectively testing all cases. BRCA1/2 mutation carriers selected in this fashion are likely to include a higher proportion of women with two primary cancers as well as women with better survival who are more likely to undergo clinical genetic testing.
Secondary mutations in BRCA1/2 could also confer resistance to PARP inhibitors. Neoplastic cells with restored BRCA1/2 are resistant to PARP inhibitors,17–19 and platinum-resistant BRCA2-mutated pancreatic cancer clones without secondary BRCA2 mutations remain sensitive to PARP inhibition in vitro.18 Response data from our small number of cases treated with PARP inhibitors support the in vitro data that secondary BRCA1/2 mutations predict PARP inhibitor resistance. However, larger studies are needed to determine whether testing for secondary BRCA1/2 mutations could be used to determine which patients will benefit from PARP inhibition.
We thank Simon Langdon, PhD, for providing tumor and clinical information for sample PE01. We thank Mary-Claire King, PhD, Piri Welcsh, PhD, and Tom Walsh, PhD, for guidance and assistance.
Supported by National Institutes of Health Grants No. P50CA83636 and R01CA125636, Marsha Rivkin Center Pilot Study Program Grant, and Howard Hughes Medical Institute.
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
The author(s) indicated no potential conflicts of interest.
Conception and design: Barbara Norquist, Toshiyasu Taniguchi, Elizabeth M. Swisher
Financial support: Toshiyasu Taniguchi, Elizabeth M. Swisher
Administrative support: Elizabeth M. Swisher
Provision of study materials or patients: Elizabeth M. Swisher
Collection and assembly of data: Barbara Norquist, Kaitlyn A. Wurz, Christopher C. Pennil, Rochelle Garcia, Jenny Gross, Wataru Sakai, Beth Y. Karlan, Toshiyasu Taniguchi, Elizabeth M. Swisher
Data analysis and interpretation: Barbara Norquist, Toshiyasu Taniguchi, Elizabeth M. Swisher
Manuscript writing: All authors
Final approval of manuscript: All authors