Birrer, Michael J.

Abstract Platinum-based DNA cross-linking agents are widely used anti-cancer drugs. Tumor resistance to platinum compounds is a major determinant of patient survival, including in high grade serous ovarian cancer (HGSOC). Remarkably, despite >30 years of literature on platinum responses in human cancer, none of these findings is used clinically as a predictive biomarker to stratify patients for platinum resistance, nor exploited therapeutically to treat platinum-resistant disease. Thus, understanding mechanisms of platinum resistance is an urgent goal, both to identify predictive biomarkers of platinum response (to spare patients with platinum-resistant tumors futile platinum therapy) and to develop efficacious therapies for platinum-resistant disease. To better understand mechanisms underlying platinum resistance in HGSOC, we performed comprehensive, dynamic (+/-carboplatin), multiomic profiling of DNA, RNA, protein and post-translational modifications (phosphorylation, ubiquitination, acetylation) to identify the cellular networks that respond to platinum treatment and associate with platinum resistance in three HGSOC intra-patient cell line pairs (PEA1S/PEA2R, PEO1S/PEO4R, PEO14S/PEO23R). The cell line pairs were derived from ascites or pleural effusions (Langdon et al., 1988) from three patients both before (PEA1S, PEO1S, PEO14S) and after (PEA2R, PEO4R, PEO23R, respectively) their tumors became clinically platinum resistant (i.e., in vivo development of resistance). The molecular profiles revealed extensive responses to carboplatin and differential responses between platinum-sensitive and platinum-resistant cells. Higher oxidative phosphorylation and fatty acid oxidation (FAO) pathway expression were observed in the platinum-resistant cells, which was further validated in patient-derived xenograft (PDX) models. We show that pharmacologic inhibition or CRISPR knockout of CPT1A, which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. Thus, FAO, and CPT1A in particular, represent a candidate therapeutic target to overcome platinum resistance in HGSOC. Citation Format: Hong Wang, Dongqing Hugang, Shrabanti Chowdhury, Sara Savage, Richard Ivey, Jacob Kennedy, Jeffrey Whiteaker, Chenwei Lin, Xiaonan Hou, Catherine Huntoon, Uliana Voytovich, Zahra Shire, Qing Yu, Steven Gygi, Andrew Hoofnagle, Zachary Herbert, Anna Calinawan, Larry Karnitz, S. John Weroha, Scott Kaufmann, Bing Zhang, Pei Wang, Michael Birrer, Amanda Paulovich. Multiomic analysis identifies CPT1A and fatty acid oxidation as a potential therapeutic target in platinum-refractory high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1426.

Abstract Background There is a critical need to identify patient characteristics associated with long-term ovarian cancer survival. Methods Quality of life (QOL), measured by the Functional Assessment of Cancer Therapy-Ovarian-Trial Outcome Index (FACT-O-TOI), including physical, functional, and ovarian-specific subscales, was compared between long-term survivors (LTS) (8+ years) and short-term survivors (STS) (<5 years) of GOG 218 at baseline; before cycles 4, 7, 13, 21; and 6 months post-treatment using linear and longitudinal mixed models adjusted for covariates. Adverse events (AEs) were compared between survivor groups at each assessment using generalized linear models. All P values are 2-sided. Results QOL differed statistically significantly between STS (N = 1115) and LTS (N = 260) (P < .001). Baseline FACT-O-TOI and FACT-O-TOI change were independently associated with long-term survival (odds ratio = 1.05, 95% confidence interval = 1.03 to 1.06 and odds ratio = 1.06, 95% confidence interval = 1.05 to 1.07, respectively). A 7-point increase in baseline QOL was associated with a 38.0% increase in probability of LTS, and a 9-point increase in QOL change was associated with a 67.0% increase in odds for LTS. QOL decreased statistically significantly with increasing AE quartiles (cycle 4 quartiles: 0-5 vs 6-8 vs 9-11 vs ≥12 AEs, P = .01; cycle 21 quartiles: 0-2 vs 3 vs 4-5 vs ≥6 AEs, P = .001). Further, LTS reported statistically significantly better QOL compared with STS (P = .03 and P = .01, cycles 4 and 21, respectively), with similar findings across higher AE grades. Conclusions Baseline and longitudinal QOL change scores distinguished LTS vs STS and are robust prognosticators for long-term survival. Results have trial design and supportive care implications, providing meaningful prognostic value in this understudied population.

Abstract Background: The influence of sex hormone and insulin/insulin-like growth factor (IGF) axis signaling on endometrial cancer recurrence is unknown. We evaluated these pathways in a prospective cohort of Gynecologic Oncology Group (GOG)0210 trial endometrial adenocarcinoma patients. Methods: Stage II–IV patients (N = 816) were included in this study. Pretreatment specimens were tested for tumor mRNA and protein expression of IGF1, IGF2, IGF-binding proteins (IGFBP)-1 and -3, insulin (IR) and IGF-I receptors (IGF1R), phosphorylated IR/IGF1R (pIGF1R/pIR), and estrogen (ER) and progesterone receptors (PR) using qPCR and IHC. Serum concentrations of insulin, IGF-I, IGFBP-3, estradiol, estrone, and sex hormone binding globulin were measured. HRs and 95% confidence intervals (CI) for progression-free survival were calculated from Cox models adjusting for age, stage, and grade. Results: Recurrence occurred in 280 (34%) cases during a median of 4.6 years of follow-up. ER positivity (HR, 0.67; 95% CI, 0.47–0.95), IR positivity (HR, 0.53; 95% CI, 0.29–0.98), and circulating IGF-I (highest vs. lowest quartile: HR, 0.66; 95% CI, 0.47–0.92) were inversely associated with recurrence risk. Circulating estradiol (highest vs. lowest tertile: HR, 1.55; 95% CI, 1.02–2.36) and pIGF1R/pIR positivity (HR, 1.40; 95% CI, 1.02–1.92) were associated with increased recurrence risk. Conclusions: Circulating estradiol and tumor tissue phosphorylated (activated) IGR1R/IR were independently associated with higher risk of recurrence in patients with endometrial cancer. Impact: This study may inform future clinical trials of endocrine-targeted adjuvant therapies in patients with endometrial cancer that could include baseline assessment of serum and tissue biomarkers of estradiol and insulin signaling pathways.

Abstract Although high-grade serous ovarian cancers (HGSOC) are highly chemosensitive with an 85% initial response rate to platinum-based chemotherapy, 15% of patients are “exceptional nonresponders,” with platinum-refractory tumors that remain stable or progress during treatment. Unfortunately, we have no predictive biomarkers to identify refractory patients up front, and they receive futile chemotherapy through which most patients become too ill to be eligible for clinical trials. Hence, no progress has been made in treating these deadly tumors. The goal of this study is to identify mechanisms of platinum refractoriness to: i) predict refractory HGSOCs up front and ii) identify potential new drug targets in refractory disease to point to desperately needed new therapeutic approaches. Of note, 80-90% of patients who are initially platinum responsive will relapse and develop platinum-resistant disease, and it is possible that findings in platinum-refractory tumors might also provide insights into platinum-resistant tumors. Our NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC)-funded approach combines genomic and proteomic (“proteogenomic”) analyses of both preclinical models (0, 8, and 24 hours post-platinum exposure) and treatment-naïve human tumors. For preclinical models, we studied a well-characterized collection of patient-derived xenograft (PDX) models (10 sensitive, 10 refractory), as well as intrapatient HGSOC cell line pairs derived from patients before and after the development of platinum resistance. For the PDX models, proteogenomic profiling included RNASeq, WES, global proteomics, and phosphoproteomics at all 3 time points (0, 8, 24 hours). For cell line models (3 sensitive, 3 resistant), proteogenomic profiling was performed at all 3 timepoints (0, 8, 24 hours), and experiments were performed in complete biologic triplicate. Analyses included RNASeq, WES, global proteomics, phosphoproteomics, ubiquitin proteome, acetylated proteome, and pTyr. A large collection of 275 human HGSOCs (an approximate equal balance of platinum sensitive and refractory tumors) is currently undergoing proteomic profiling, and genomic profiles (WGS, RNASeq) will be performed on a subset. In parallel, we have performed a comprehensive review of 31 years of published work on platinum responses of human cancers, identifying ~700 genes implicated in the response and scoring each gene with respect to strength of the published evidence. Using a Bayesian approach, we are integrating the curated candidates from the literature with our empirical proteogenomic datasets to identify a candidate signature for detecting platinum-refractory disease prior to chemotherapy. We are also performing gene-regulatory network analysis to identify potential drivers of chemo response. NextGen, targeted, multiplex, multiple reaction monitoring mass spectrometry-based assays are being developed to quantify proteins in the signature for validation studies, using independent patient cohorts. This abstract is also being presented as Poster A62. Citation Format: Jacob J. Kennedy, Shrabanti Chowdhury, Sara R. Savage, Xiaonan Hou, Catherine J. Huntoon, Richard G. Ivey, Qing Yu, Chenwei Lin, Dongqing Huang, Lei Zhao, Uliana J. Voytovich, Regine M. Schoenherr, Zahra Shire, Steven J. Skates, Jeffrey R. Whiteaker, Andrew N. Hoofnagle, Samuel C. Mok, Bing Zhang, Larry M. Karnitz, S. John Weroha, Steven P. Gygi, Scott H. Kaufmann, Pei Wang, Michael J. Birrer, Amanda G. Paulovich. Proteogenomic approach to identify mechanisms of platinum refractoriness in high-grade serous ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR02.

Abstract Purpose: GOG-0218, a double-blind placebo-controlled phase III trial, compared carboplatin and paclitaxel with placebo, bevacizumab followed by placebo, or bevacizumab followed by bevacizumab in advanced epithelial ovarian cancer (EOC). Results demonstrated significantly improved progression-free survival (PFS), but no overall survival (OS) benefit with bevacizumab. Blood samples were collected for biomarker analyses. Experimental Design: Plasma samples were analyzed via multiplex ELISA technology for seven prespecified biomarkers [IL6, Ang-2, osteopontin (OPN), stromal cell–derived factor-1 (SDF-1), VEGF-D, IL6 receptor (IL6R), and GP130]. The predictive value of each biomarker with respect to PFS and OS was assessed using a protein marker by treatment interaction term within the framework of a Cox proportional hazards model. Prognostic markers were identified using Cox models adjusted for baseline covariates. Results: Baseline samples were available from 751 patients. According to our prespecified analysis plan, IL6 was predictive of a therapeutic advantage with bevacizumab for PFS (P = 0.007) and OS (P = 0.003). IL6 and OPN were found to be negative prognostic markers for both PFS and OS (P < 0.001). Patients with high median IL6 levels (dichotomized at the median) treated with bevacizumab had longer PFS (14.2 vs. 8.7 months) and OS (39.6 vs. 33.1 months) compared with placebo. Conclusions: The inflammatory cytokine IL6 may be predictive of therapeutic benefit from bevacizumab when combined with carboplatin and paclitaxel. Aligning with results observed in patients with renal cancer treated with antiangiogenic therapies, it appears plasma IL6 may also define those patients with EOC more or less likely to benefit from the addition of bevacizumab to standard chemotherapy.

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 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 Purpose: Somatic mosaic mutation in the PPM1D gene have been associated with risk of breast and ovarian cancer. Somatic mosaic mutations in blood cells may be associated with older age and chemotherapy exposure. We wanted to determine if somatic mosaic mutations in PPM1D and in other genes are associated with older age and repeated exposure to chemotherapy, rather than a contributor to risk of disease. Experimental design: We used targeted capture and massively parallel genomic sequencing to sequence DNA from PBMCs from affected patients for PPM1D as well as 60 other DNA repair genes. For a number of samples with PPM1D mutations, we re-sequenced at greater depth on patients where PBMCs that were collected at diagnosis and recurrence. Results: We identified somatic mosaic mutations in exon 6 of PPM1D in 4 out of 474 (0.8%) individuals that were newly diagnosed and had no previous chemotherapy, in 3 out of 29 (10%) individuals newly diagnosed but receiving chemotherapy and in 58 of 274 (21%) individuals with relapsed platinum resistant ovarian cancer (p<0.0001). The presence of PPM1D mutations in exon 6 was strongly associated with more advanced age (p=0.011, Wilcoxon test) and more than one chemotherapy regimen (p=0.026, Fisher's exact test). In paired blood samples from 13 patients, PPM1D mutations present at relapse were either not detected or detected at a lower percentage compared to blood obtained soon after diagnosis. Somatic mosaic mutations were also identified in TP53 in 7 of 274 (2.5%) women with relapsed platinum resistant cancer. In one of these cases, we had an earlier blood sample from diagnosis in which the mutation was not present at time near diagnosis. Conclusion: We found that somatic mosaic PPM1D mutations in PMBCs are associated with advanced age and repeated exposure to chemotherapy. Mosaic mutations in other genes were not confirmed with the exception of TP53, for which mutations are also detectable after extensive treatment. It seems likely that mosaic PPM1D mutations in blood from cancer patients may be a result of selection by chemotherapy of age-induced somatic mutations during exposure to chemotherapy. Further studies are needed to determine the clinical significance of these mutations. Citation Format: Maria I. Harrell, Barbara M. Norquist, Tom Walsh, Mark F. Brady, Ming K. Lee, Robert Hershberg, Kimberly R. Kalli, Heather Lankes, Eric Q. Konnick, Colin C. Pritchard, Bradley J. Monk, John K. Chan, Robert Burger, Scott H. Kaufmann, Michael J. Birrer, Elizabeth M. Swisher. Old age and chemotherapy contribute to the selection of PPM1D somatic mosaic mutations in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B71.