Swisher, Elizabeth M.

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.

Abstract Purpose: We hypothesized that mutations in homologous recombination repair (HRR) genes beyond BRCA1 and BRCA2 improve outcomes for ovarian carcinoma patients treated with platinum therapy and would impact the relative benefit of adding prolonged bevacizumab. Experimental Design: We sequenced DNA from blood and/or neoplasm from 1,195 women enrolled in GOG-0218, a randomized phase III trial in advanced ovarian carcinoma of bevacizumab added to carboplatin and paclitaxel. Defects in HRR were defined as damaging mutations in 16 genes. Proportional hazards models were used to estimate relative hazards for progression-free survival (PFS) and overall survival (OS). Results: Of 1,195 women with ovarian carcinoma, HRR mutations were identified in 307 (25.7%). Adjusted hazards for progression and death compared with those without mutations were lower for women with non-BRCA HRR mutations [HR = 0.73; 95% confidence interval (CI), 0.57–0.94; P = 0.01 for PFS; HR = 0.67; 95% CI, 0.50–0.90; P = 0.007 for OS] and BRCA1 mutations (HR = 0.80; 95% CI, 0.66–0.97; P = 0.02 for PFS; HR = 0.74; 95% CI, 0.59–0.94; P = 0.01 for OS) and were lowest for BRCA2 mutations (HR = 0.52; 95% CI, 0.40–0.67; P < 0.0001 for PFS; HR = 0.36; 95% CI, 0.25–0.53; P < 0.0001 for OS). A test of interaction showed no difference in the effect of bevacizumab on PFS between cases with and without mutations. Conclusions: HRR mutations, including non-BRCA genes, significantly prolong PFS and OS in ovarian carcinoma and should be stratified for in clinical trials. The benefit of adding bevacizumab was not significantly modified by mutation status. Clin Cancer Res; 24(4); 777–83. ©2017 AACR.

Abstract Purpose: To determine the dose limiting toxicities (DLT), maximum tolerated dose (MTD), and recommended phase II dose (RP2D) of veliparib in combination with weekly topotecan in patients with solid tumors. Correlative studies were included to assess the impact of topotecan and veliparib on poly(ADP-ribose) levels in peripheral blood mononuclear cells, serum pharmacokinetics of both agents, and potential association of germline repair gene mutations with outcome. Experimental Design: Eligible patients had metastatic nonhematologic malignancies with measurable disease. Using a 3 + 3 design, patients were treated with veliparib orally twice daily on days 1–3, 8–10, and 15–17 and topotecan intravenously on days 2, 9, and 16 every 28 days. Tumor responses were assessed by RECIST. Results: Of 58 patients enrolled, 51 were evaluable for the primary endpoint. The MTD and RP2D was veliparib 300 mg twice daily on days 1–3, 8–10, and 15–17 along with topotecan 3 mg/m2 on days 2, 9, and 16 of a 28-day cycle. DLTs were grade 4 neutropenia lasting >5 days. The median number of cycles was 2 (1–26). The objective response rate was 10%, with 1 complete and 4 partial responses. Twenty-two patients (42%) had stable disease ranging from 4 to 26 cycles. Patients with germline BRCA1, BRCA2, or RAD51D mutations remained on study longer than those without homologous recombination repair (HRR) gene mutations (median 4 vs. 2 cycles). Conclusions: Weekly topotecan in combination with veliparib has a manageable safety profile and appears to warrant further investigation.

Abstract Background: In vivo synergy with concurrent PI3-Kinase inhibition and PARP inhibition has been observed in BRCA-deficient and BRCA-proficient preclinical models of triple negative breast cancer (TNBC) and ovarian cancer (OC). A phase I trial of the oral pan-class I PI3-Kinase inhibitor BKM120 and the PARP inhibitor olaparib demonstrated anti-cancer activity in TNBC and OC, both in patients with and without germline BRCA1 and BRCA2 (BRCA) mutations. However, CNS toxicity (depression) and liver function test abnormalities limited dose escalation of BKM120 prompting evaluation of the alpha specific PI3-Kinase inhibitor BYL719 (which has no CNS toxicity) in combination with olaparib. Methods: Olaparib was administered twice daily (tablet formulation) and BYL719 daily on a 28-day cycle, both orally. A 3 + 3 dose-escalation design was employed with primary objectives of defining the maximum tolerated dose (MTD) and recommended phase 2 dose of the combination of BYL719 and olaparib, and secondary objectives of defining toxicity, activity, and pharmacokinetic profiles of both agents. Eligibility included recurrent TNBC or high grade serous (HGS) OC, or any histology OC or breast cancer (BC) with presence of a known germline BRCA mutation, performance status of 0-1 and measurable/evaluable cancer. Patients with platinum sensitive or resistant or refractory OC were eligible and prior PARP inhibitor use was allowed. Dose-expansion cohorts at the MTD were enrolled for both BC and OC. Results: 46 patients (16 BC and 30 OC) have been enrolled in the study; 28 patients participated in the dose escalation portion of the study (4 BC and 24 OC). Two patients with OC did not receive study drugs because of ineligibility. MTD was defined as BYL719 200mg once daily and olaparib 200mg twice daily. Dose limiting toxicities included hyperglycemia, rash and fever with decreased neutrophil count. Four patients (3 OC and 1 BC) discontinued protocol therapy because of toxicity (2 for hyperglycemia, 1 for nausea and 1 for allergic reaction). Most common toxicities included nausea, hyperglycemia, fatigue, diarrhea and vomiting. At the MTD, 6 patients with OC and 12 patients with BC were enrolled into a dose expansion cohort. The OC expansion cohort has completed enrollment, while the BC cohort is still enrolling. Among patients with OC who received study drugs (28 patients, 26 (93%) with platinum resistant disease), objective response rate (ORR) by RECIST 1.1 was 36% (10/28 patients, all partial responses (PRs)). Median duration of response was 167 days (range 16-398 days); 5 of 10 patients with PR remain on treatment. ORR was 33% for patients with germline BRCA mutations and 31% for patients without germline BRCA mutations. Among patients without germline BRCA mutations with platinum resistant OC, ORR was 29%. Conclusions: Combined BYL719 and olaparib is feasible, and similar clinical benefit was observed in patients with and without germline BRCA mutations. The activity of this combination in OC patients without germline BRCA mutations and with platinum resistant disease was higher than expected from olaparib monotherapy and warrants further investigation. This work was funded in part by the Stand Up To Cancer Ovarian Dream Team. Clinical trial: NCT01623349. Citation Format: Panagiotis A. Konstantinopoulos, William T. Barry, Michael Birrer, Shannon N. Westin, Sarah Farooq, Karen Cadoo, Christin Whalen, Weixiu Luo, Hui Liu, Carol Aghajanian, David B. Solit, Gordon B. Mills, Barry S. Taylor, Helen Won, Michael F. Berger, Sangeetha Palakurthi, Joyce F. Liu, Lew Cantley, Scott H. Kaufmann, Elizabeth M. Swisher, Alan D. D'Andrea, Eric Winer, Gerburg M. Wulf, Ursula A. Matulonis. Phase I study of the alpha specific PI3-Kinase inhibitor BYL719 and the poly (ADP-Ribose) polymerase (PARP) inhibitor olaparib in recurrent ovarian and breast cancer: Analysis of the dose escalation and ovarian cancer expansion cohort [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 CT008. doi:10.1158/1538-7445.AM2017-CT008

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 Poly(ADP-ribose) polymerase (PARP) inhibitors have shown promising single-agent activity in ovarian cancers with BRCA1 and BRCA2 mutations (C. Scott, JCO 33:1397, 2015). Ongoing studies are testing the efficacy of PARP inhibitors in ovarian cancers with other genetic or epigenetic alterations that impair the homologous recombination (HR) pathway of DNA repair. It is less clear whether PARP inhibitor clinical activity can be enhanced in HR deficient cancers or even extended to HR proficient ovarian cancers through judicious combinations. Our preclinical studies have demonstrated that PARP inhibitors enhance the cytotoxic effects of selected therapeutic agents through a variety of mechanisms: By trapping PARP at sites of DNA damage, PARP inhibitors enhance the cytotoxicity of topotecan (A. Patel, J. Biol. Chem. 287: 4198, 2012).By inhibiting base excision repair, PARP inhibitors enhance the cytotoxicity of floxuridine (A. Huehls, Cancer Res. 71: 4944, 2011), an agent previously shown to have clinical activity in ovarian cancer. Building on the first of these observations, we conducted a phase 1 clinical trial of topotecan weekly x 3 in combination with veliparib (NCT01012817). Using a standard 3+3 design, patients were treated with veliparib PO twice daily on days 1-3, 8-10 and 15-17 and topotecan IV on days 2, 9 and 16 every 28 days. The dose-limiting toxicity was neutropenia. The MTD was veliparib 300 mg PO twice daily and topotecan 3 mg/m2/dose. Among 52 evaluable patients, objective responses (1 CR, 3 PRs) and stable disease (22 patients, 6 lasting ≥ 8 cycles) were observed. While there was no clear correlation between germline BRCA1 or BRCA2 mutations and clinical benefit, a patient with a germline RAD51D mutation had stable disease for 14 cycles. A phase 2 cohort in platinum-resistant ovarian cancer has just opened. Building on the second set of preclinical observations, we are also conducting a phase 1 trial of veliparib and IP floxuridine in patients with recurrent ovarian cancer confined to the peritoneal cavity (NCT01749397). Using a rolling 6 design, patients are treated with veliparib PO twice daily on days 1-10 and floxuridine IP on days 3-5 every 21 days. The trial is currently enrolling at dose level 5 (veliparib 300 mg PO twice daily on days 1-10 and floxuridine 3 gm/dose IP on days 3-5). Grade 3 toxicities in any cycle have included nausea (1), fatigue (1) and neutropenia (3). Among 20 evaluable patients, treatment duration has ranged from 2-33 cycles. These studies, which build on two different aspects of PARP1 function, illustrate two different approaches to potentially enhancing the activity of PARP inhibitors in relapsed ovarian cancer. These studies were funded in part by NIH grants P50 CA136393, R01 CA190423, and UM1 CA186686 as well as SU2C-AACR DT16-15. Citation Format: Scott H. Kaufmann, Andrea E. Wahner Hendrickson, Maria I Harrell, Michael E. Menefee, Edward J. Tanner, David G, Mutch, Elizabeth M. Swisher, and Larry M. Karnitz. PARP INHIBITOR COMBINATIONS FOR THE TREATMENT OF OVARIAN CANCER [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 IS04.

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.