Reversion of BRCA1/2 Germline Mutations Detected in Circulating Tumor DNA From Patients With High-Grade Serous Ovarian Cancer

Abstract

Purpose Germline BRCA1 or BRCA2 mutations in patients with high-grade serous ovarian cancer (HGSC) are associated with favorable responses to chemotherapy. However, secondary intragenic (reversion) mutations that restore protein function lead to clinically significant rates of acquired resistance. The goal of this study was to determine whether reversion mutations could be found in an unbiased manner in circulating cell-free DNA (cfDNA) to predict treatment response in HGSC. Patients and Methods Plasma and tumor samples were obtained from 30 patients with HGSC with either BRCA1 or BRCA2 germline mutation. Two cohorts were ascertained: patients with a malignancy before undergoing primary HGSC debulking surgery (n = 14) or patients at disease recurrence (n = 16). Paired tumor and plasma samples were available for most patients (24 of 30). Targeted amplicon, next-generation sequencing was performed using primers that flanked germline mutations, whose design did not rely on prior knowledge of reversion sequences. Results Five patients were identified with intragenic mutations predicted to restore BRCA1/2 open reading frames, including two patients with multiple independent reversion alleles. Reversion mutations were only detected in tumor samples from patients with recurrent disease (five of 16) and only in cfDNA from patients with a tumor-detected reversion (three of five). Findings from a rapid autopsy of a patient with multiple independent reversions indicated that reversion-allele frequency in metastatic sites is an important determinant of assay sensitivity. Abundance of tumor-derived DNA in total cell-free DNA, as measured by TP53 mutant allele frequency, also affected assay sensitivity. All patients with reversions detected in tumor-derived DNA were resistant to platin- or poly ADP ribose polymerase inhibitor-based chemotherapy. Conclusion Reversion mutations can be detected in an unbiased analysis of cfDNA, suggesting clinical utility for predicting chemotherapy response in recurrent HGSC.

Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer

“…We first established the potential of the assay for detecting ctDNA in patients with ovarian cancer, as prior studies suggested low rates of mutation detection in ctDNA of ovarian cancer patients (46). Massively parallel sequencing analysis of germline DNA from peripheral blood leukocytes, microdissected tumor and plasma DNA of 19 cases ( BRCA1 , n=12; BRCA2 , n=7), using previously validated methods (18,20), yielded median depths of coverage of 1,569x (range 852x–2,272x), 823x (range 272x–2,328x) and 1,978x (range 1,287x––4,157x), respectively (Supplementary Table S2).…”

“…BRCA1 and BRCA2 reversion mutations have been documented as potential mechanisms of resistance to platinum-based chemotherapy and PARP inhibitors in cell line models and patient samples (4,13,14,21,46). Here we report on the detection of putative BRCA1 and BRCA2 reversion somatic mutations in the cfDNA of platinum-based chemotherapy and/or PARP inhibitor resistant/ refractory ovarian and breast cancer patients harboring germline BRCA1 or BRCA2 germline mutations.…”

Homology-Directed Repair and the Role of BRCA1, BRCA2, and Related Proteins in Genome Integrity and Cancer

“…In both high-grade serous ovarian cancer and metastatic prostate cancer, multiple secondary mutations in both germ-line and somatically mutated BRCA2 and PALB2 were identified in cfDNA from patients with recurrent disease (Christie et al 2017, Goodall et al 2017, Quigley et al 2017). Interestingly, in the case of prostate cancers treated with PARPi (either olaparib or talazoparib), analysis of mutations in cfDNA revealed evidence of multiclonal heterogeneity of BRCA2 reversion mutations, information that would be difficult to glean from a single tumor biopsy sample (Quigley et al 2017).…”

Analysis of Circulating Cell-Free DNA Identifies Multiclonal Heterogeneity of BRCA2 Reversion Mutations Associated with Resistance to PARP Inhibitors

“…Multiple mechanisms of resistance to PARPi have been proposed (reviewed in [11]). Amongst these, secondary reversion mutations in the BRCA1/2 genes have been associated with clinical resistance to platinum or olaparib (12–14). However, the mechanism of resistance in prostate cancer, and to PARPi other than olaparib, is unknown.…”