Lin, Kevin

Abstract Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Abstract A key resistance mechanism to platinum-based chemotherapies and PARP inhibitors in BRCA-mutant cancers is the acquisition of BRCA reversion mutations that restore protein function. To estimate the prevalence of BRCA reversion mutations in high-grade ovarian carcinoma (HGOC), we performed targeted next-generation sequencing of circulating cell-free DNA (cfDNA) extracted from pretreatment and postprogression plasma in patients with deleterious germline or somatic BRCA mutations treated with the PARP inhibitor rucaparib. BRCA reversion mutations were identified in pretreatment cfDNA from 18% (2/11) of platinum-refractory and 13% (5/38) of platinum-resistant cancers, compared with 2% (1/48) of platinum-sensitive cancers (P = 0.049). Patients without BRCA reversion mutations detected in pretreatment cfDNA had significantly longer rucaparib progression-free survival than those with reversion mutations (median, 9.0 vs. 1.8 months; HR, 0.12; P < 0.0001). To study acquired resistance, we sequenced 78 postprogression cfDNA, identifying eight additional patients with BRCA reversion mutations not found in pretreatment cfDNA. Significance: BRCA reversion mutations are detected in cfDNA from platinum-resistant or platinum-refractory HGOC and are associated with decreased clinical benefit from rucaparib treatment. Sequencing of cfDNA can detect multiple BRCA reversion mutations, highlighting the ability to capture multiclonal heterogeneity. This article is highlighted in the In This Issue feature, p. 151

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance. Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR. Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway. Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance. Citation Format: Rachel M. Hurley, Ksenija Nesic, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Paula A. Schneider, Xiaonan Hou, Cristina Correia, Karen S. Flatten, Giada V. Zapparoli, Alexander Dobrovic, Kevin K. Lin, Thomas C. Harding, Andrea E. Wahner Hendrickson, Elizabeth M. Swisher, Matthew Wakefield, S. John Weroha, Clare L. Scott, Scott H. Kaufmann. Loss of RAD51C promoter hypermethylation confers PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5885.

Purpose Pancreatic cancer has a poor prognosis and limited treatment options. Approximately 9% of pancreatic cancers harbor a germline or somatic BRCA1 or BRCA2 ( BRCA1/2) mutation. Because poly (ADP-ribose) polymerase inhibitors have significant activity in BRCA1/2-mutant ovarian and breast cancers, RUCAPANC investigated the efficacy and safety of rucaparib in BRCA1/2-mutant pancreatic cancer. Patients and Methods RUCAPANC enrolled patients with measurable locally advanced/metastatic pancreatic cancer who had received one to two prior chemotherapy regimens. Patients received oral rucaparib (600 mg twice daily) until disease progression. The primary end point was objective response rate. Results Nineteen patients were enrolled. Sixteen of 19 BRCA1/2 mutations were germline; three were somatic. Patients had received a median of two prior chemotherapy regimens. Four patients achieved a response; two partial responses and one complete response (CR) were confirmed (objective response rate, 15.8%; 3 of 19), with an additional CR unconfirmed. The disease control rate (CR, partial response, or stable disease for ≥ 12 weeks) was 31.6% (6 of 19) in all patients and 44.4% (4 of 9) in those who had received one prior chemotherapy regimen. As prespecified in the protocol, enrollment was stopped because of an insufficient response rate among the first 15 patients. Treatment-emergent adverse events included nausea (63.2%) and anemia (47.4%). Grade ≥ 3 adverse events included anemia (31.6%), fatigue (15.8%), and ascites (15.8%). Secondary resistance mutations were detected in circulating free tumor DNA in two patients with a germline BRCA2 mutation. These mutations are predicted to lead to the reversion of a somatic—not germline—mutation. Conclusion Rucaparib provided clinical benefit to patients with advanced pancreatic cancer and a BRCA1/2 mutation, and demonstrated an acceptable safety profile. Additional trials of rucaparib in this population are warranted.

Abstract Background: PARP inhibitors (PARPis) are active in cancers with homologous recombination (HR) defects. Preclinical studies have shown that secondary mutations or alterations in gene expression (e.g., downregulation of 53BP1, Ku70, Ku80 or DNA-PKcs) restore HR and confer PARPi resistance. In addition, low PARP1 expression can diminish PARP trapping and cause PARPi resistance. ARIEL2 Part 1 is a phase 2 study of the PARPi rucaparib in platinum sensitive, relapsed high grade serous or endometrioid ovarian cancer (OC). Pretreatment OC biopsies were previously assayed for HR gene mutations and loss of heterozygosity (LOH), a genomic scar that reflects HR deficiency. Two tumor groups (BRCA wildtype [wt]/LOH high and BRCA1 or BRCA2 mutant) have objective response rates of 29.3% and 80%, respectively, as well as progression free survival (PFS) of 5.7 and 12.8 months, respectively, on rucaparib (E. Swisher et al., Lancet Oncol., in press). Common AEs included nausea (80%), asthenia/fatigue (78%), constipation (46%), and vomiting (44%). The present studies tested the hypotheses that i) lower PARP1 expression and/or ii) lower expression of NHEJ components 53BP1, DNA-PKcs, Ku80, Ku70, or LIG4, may correlate with diminished response rate as well as PFS in pts treated with rucaparib on ARIEL2. Methods: Immunohistochemical assays were developed for 53BP1, DNA-PKcs, Ku80, Ku70, LIG4, and PARP1 and validated in formalin fixed, paraffin embedded cell lines differing in analyte expression. Available pretreatment OC biopsies from ARIEL2 Part 1 were stained and scored for % of tumor nuclei that were negative (0), weak (1+), moderate (2+) or strong (3+). Modified H-scores were correlated with clinical characteristics and outcome measures. Results: Pretreatment biopsies from 62-68 pts were successfully stained for each repair protein. Across the samples, PARP1 H-scores varied from 0 to 300 (median 160). Focusing on the BRCA wt/LOH high group (n = 38), there was no significant difference in PFS of pts with low (<100), intermediate (100-200) or high (>200) PARP1 H-score (p = 0.57). Expression of DNA-PKcs, Ku70, Ku80, and LIG4 was generally lower (median H-scores 20-60) and did not individually correlate with response or PFS. In contrast, there was a trend toward improved PFS in BRCA wt/LOH high OC pts expressing intermediate or high 53BP1 (H-score ≥ 100, n = 10) compared to pts with low 53BP1 (H-score <100, n = 25), with median PFS 20.7 vs. 5.5 months (p = 0.073), respectively. Staining of additional pretreatment biopsies is planned. Conclusions: In the BRCA wt/LOH high group, pretreatment PARP1 expression does not correlate with rucaparib response. In contrast, BRCA wt/LOH high OC pts with low 53BP1 have a trend toward shorter PFS with rucaparib, suggesting that 53BP1 downregulation might correlate with clinical PARPi resistance in BRCA wt/LOH high OC. Citation Format: Andrea E. Wahner Hendrickson, Kevin K. Lin, Daniel W. Visscher, Rachel M. Hurley, Mitch Raponi, Thomas C. Harding, Linda M. Murphy, Jill M. Wagner, Heidi Giordano, Iain McNeish, Elizabeth M. Swisher, Scott Kaufmann. DNA repair protein expression and response of homologous recombination deficient ovarian cancer to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib in the ARIEL2 Part 1 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4676. doi:10.1158/1538-7445.AM2017-4676

Abstract Germline and somatic mutations in BRCA1 and BRCA2 (BRCA) confer PARP inhibitor sensitivity. Promoter hypermethylation is an alternate mechanism of gene down-regulation, and BRCA1 promoter methylation is relatively common in sporadic ovarian cancer. The clinical significance of BRCA1 methylation is less clear than for mutations, as the Cancer Genome Atlas (TCGA) and others have failed to show improved survival in ovarian carcinomas with BRCA1 methylation. No one has previously tested whether BRCA1 methylation confers in vivo sensitivity to PARP inhibitors in patients with ovarian cancer. ARIEL2 is a phase 2 study of the PARP inhibitor rucaparib in patients with recurrent platinum sensitive high-grade ovarian, peritoneal or fallopian tube carcinoma. At enrollment, ARIEL2 required pre-treatment tumor biopsies with the goal of developing tissue predictors of PARP inhibitor sensitivity other than BRCA mutations. The number of women with known germline mutations was capped at 15 patients in order to predominantly enroll BRCA wildtype cases. As presented at ASCO 2016, in cases with no BRCA mutations, a high fraction of genomic loss of heterozygosity (LOH) significantly predicted a better progression-free survival (the primary endpoint), longer duration of response, and a higher fraction of responders compared to cases with low LOH. We assessed BRCA1 and RAD51C promoter hypermethylation using methylation-sensitive polymerase chain reaction in paired archival and pre-treatment biopsies from patients on ARIEL2. Of 165 cases for which methylation analyses were completed, 21 (12.7%) were methylated at the BRCA1 promoter and four (2.4%) at the RAD51C promoter. Methylation of BRCA1 and RAD51C was mutually exclusive with mutation in BRCA or other homologous recombination genes. All four cases with RAD51C methylation and 15/19 (78.9%) with BRCA1 methylation were associated with high LOH. In 90 paired samples archival and pre-treatment tissues, RAD51C methylation was 100% concordant and BRCA1 methylation was highly concordant (p<0·001). For 13 cases with BRCA1 methylation in the archival specimen, 4 (30·8%) were unmethylated in the paired pretreatment tumor, but for 77 unmethylated archival specimens, gain of methylation in the pretreatment biopsy was observed just once. Confirmed RECIST responses were seen in 52.4% (11/21) BRCA1 methylated and 75.0% (3/4) RAD51C methylated cases. In conclusion, BRCA1 and RAD51C methylation in ovarian carcinomas correlates with a high response rate to PARP inhibitors. If methylation was to be used as a predictor of PARP inhibitor sensitivity, it would need to be assessed in a pre-treatment (not archival) specimen. The loss of BRCA1 methylation in recurrent ovarian carcinoma, which was common even in these platinum sensitive cases, could explain why BRCA1 methylation is associated with similar survival to methylated cases, despite initial improved therapeutic sensitivity. Citation Format: Elizabeth Swisher, Maria Harrell, Kevin K. Lin, Clare Scott, Sandra Goble, Amit Oza, Robert L. Coleman, Gottfried Konecny, Anna V. Tinker, David M. O'Malley, Rebecca Kristeleit, Ling Ma, James Brenton, Katherine Bell-McGuinn, Ana Oaknin, Alexandra Leary, Elaina Mann, Heidi Giordano, Mitch Rapon, Iain McNeish, Scott H. Kaufmann. BRCA1 and RAD51C Promoter Hypermethylation Confer Sensitivity to PARP Inhibitors in Patients with Platinum Sensitive Ovarian Carcinoma [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP28.

Abstract High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984–98. ©2017 AACR. See related commentary by Domchek, p. 937. See related article by Quigley et al., p. 999. See related article by Goodall et al., p. 1006. This article is highlighted in the In This Issue feature, p. 920