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Purpose Veliparib (V), an oral poly(ADP-ribose) polymerase (PARP) inhibitor, potentiates effects of alkylating agents and topoisomerase inhibitors in preclinical tumor models. We conducted a phase I trial of V with iv cyclophosphamide (C) and V plus iv doxorubicin (A) and C. Methods Objectives were to establish the maximum tolerated dose (MTD) of the combinations, characterize V pharmacokinetics (PK) in the presence and absence of C, measure PAR in peripheral blood mononuclear cells (PBMCs) and γH2AX in circulating tumor cells (CTCs). In Group 1, dose escalations of V from 10 to 50 mg every 12 h Days 1-4 plus C 450 to 750 mg/m2 Day 3 in 21-day cycles were evaluated. In Group 2, V doses ranged from 50 to 150 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 in 21-day cycles. In Group 3, patients received AC Day 1 plus V Days 1-7, and in Group 4, AC Day 1 plus V Days 1-14 was given in 21-day cycles to evaluate effects on γH2AX foci. Results Eighty patients were enrolled. MTD was not reached for V and C. MTD for V and AC was V 100 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 every 21 days. V PK appears to be dose-dependent and has no effect on the PK of C. Overall, neutropenia and anemia were the most common adverse events. Objective response in V and AC treated groups was 22% (11/49). Overall clinical benefit rate was 31% (25/80). PAR decreased in PBMCs. Percentage of γH2AX-positive CTCs increased after treatment with V and AC. Conclusion V and AC can be safely combined. Activity was observed in patients with metastatic breast cancer.

Background In preclinical Ewing sarcoma (ES) models, poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors were identified as a potential therapeutic strategy with synergy in combination with cytotoxic agents. This study evaluated the safety and dosing of the PARP1/2 inhibitor niraparib (NIR) with temozolomide (TMZ; arm 1) or irinotecan (IRN; arm 2) in patients with pretreated ES. Methods Eligible patients in arm 1 received continuous NIR daily and escalating TMZ (days 2‐6 [D2‐6]) in cohort A. Subsequent patients received intermittent NIR dosing (cohort B), with TMZ re‐escalation in cohort C. In arm 2, patients were assigned to NIR (days 1‐7 [D1‐7]) and escalating doses of IRN (D2‐6). Results From July 2014 to May 2018, 29 eligible patients (23 males and 6 females) were enrolled in arms 1 and 2, which had 7 dose levels combined. Five patients experienced at least 1 dose‐limiting toxicity (DLT) in arm 1 (grade 4 [G4] neutropenia for >7 days or G4 thrombocytopenia), and 3 patients experienced at least 1 DLT in arm 2 (grade 3 [G3] colitis, G3 anorexia, or G3 alanine aminotransferase elevation). The maximum tolerated dose was NIR at 200 mg every day on D1‐7 plus TMZ at 30 mg/m2 every day on D2‐6 (arm 1) or NIR at 100 mg every day on D1‐7 plus IRN at 20 mg/m2 every day on D2‐6 (arm 2). One confirmed partial response was observed in arm 2; the median progression‐free survival was 9.0 weeks (95% CI, 7.0‐10.1 weeks) and 16.3 weeks (95% CI, 5.1‐69.7 weeks) in arms 1 and 2, respectively. The median decrease in tumor poly(ADP‐ribose) activity was 89% (range, 83%‐98%). Conclusions The combination of NIR and TMZ or IRN was tolerable, but at lower doses in comparison with conventional cytotoxic combinations. A triple‐combination study of NIR, IRN, and TMZ has commenced., Preclinical evaluations have identified the EWS‐FLI1 translocation, pathognomonic of Ewing sarcoma, as a predictive factor of response to poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors with synergistic cell death in vivo with DNA damaging agents. This phase 1 study examines the dosing and safety of a combination of the PARP inhibitor niraparib with temozolomide or irinotecan.

Purpose: The Wee1 kinase inhibitor adavosertib abrogates cell-cycle arrest, leading to cell death. Prior testing of twice-daily adavosertib in patients with advanced solid tumors determined the recommended phase II dose (RPh2D). Here, we report results for once-daily adavosertib. Patients and Methods: A 3 + 3 dose-escalation design was used, with adavosertib given once daily on days 1 to 5 and 8 to 12 in 21-day cycles. Molecular biomarkers of Wee1 activity, including tyrosine 15–phosphorylated Cdk1/2 (pY15-Cdk), were assessed in paired tumor biopsies. Whole-exome sequencing and RNA sequencing of remaining tumor tissue identified potential predictive biomarkers. Results: Among the 42 patients enrolled, the most common toxicities were gastrointestinal and hematologic; dose-limiting toxicities were grade 4 hematologic toxicity and grade 3 fatigue. The once-daily RPh2D was 300 mg. Six patients (14%) had confirmed partial responses: four ovarian, two endometrial. Adavosertib plasma exposures were similar to those from twice-daily dosing. On cycle 1 day 8 (pre-dose), tumor pY15-Cdk levels were higher than baseline in four of eight patients, suggesting target rebound during the day 5 to 8 dosing break. One patient who progressed rapidly had a tumor WEE1 mutation and potentially compensatory PKMYT1 overexpression. Baseline CCNE1 overexpression occurred in both of two responding patients, only one of whom had CCNE1 amplification, and in zero of three nonresponding patients. Conclusions: We determined the once-daily adavosertib RPh2D and observed activity in patients with ovarian or endometrial carcinoma, including two with baseline CCNE1 mRNA overexpression. Future studies will determine whether CCNE1 overexpression is a predictive biomarker for adavosertib.

Purpose Our preclinical work identified depletion of ATR as a top candidate for topoisomerase 1 (TOP1) inhibitor synthetic lethality and showed that ATR inhibition sensitizes tumors to TOP1 inhibitors. We hypothesized that a combination of selective ATR inhibitor M6620 (previously VX-970) and topotecan, a selective TOP1 inhibitor, would be tolerable and active, particularly in tumors with high replicative stress. Patients and Methods This phase I study tested the combination of M6620 and topotecan in 3-week cycles using 3 + 3 dose escalation. The primary end point was the identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Results Between September 2016 and February 2017, 21 patients enrolled. The combination was well tolerated, which allowed for dose escalation to the highest planned dose level (topotecan 1.25 mg/m2, days 1 to 5; M6620 210 mg/m2, days 2 and 5). One of six patients at this dose level experienced grade 4 thrombocytopenia that required transfusion, a dose-limiting toxicity. Most common treatment-related grade 3 or 4 toxicities were anemia, leukopenia, and neutropenia (19% each); lymphopenia (14%); and thrombocytopenia (10%). Two partial responses (≥ 18 months, ≥ 7 months) and seven stable disease responses ≥ 3 months (median, 9 months; range, 3 to 12 months) were seen. Three of five patients with small-cell lung cancer, all of whom had platinum-refractory disease, had a partial response or prolonged stable disease (10, ≥ 6, and ≥ 7 months). Pharmacodynamic studies showed preliminary evidence of ATR inhibition and enhanced DNA double-stranded breaks in response to the combination. Conclusion To our knowledge, this report is the first of an ATR inhibitor-chemotherapy combination. The maximum dose of topotecan plus M6620 is tolerable. The combination seems particularly active in platinum-refractory small-cell lung cancer, which tends not to respond to topotecan alone. Phase II studies with biomarker evaluation are ongoing.

// Jung-Min Lee 1 , John L. Hays 2 , Victoria L. Chiou 1 , Christina M. Annunziata 1 , Elizabeth M. Swisher 3 , Maria I. Harrell 3 , Minshu Yu 1 , Nicolas Gordon 1 , Tristan M. Sissung 4 , Jiuping Ji 5 , William D. Figg 4 , Lori Minasian 1 , Stanley Lipkowitz 1 , Bradford J. Wood 6 , James Doroshow 7 and Elise C. Kohn 1 1 Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 2 Division of Medical Oncology, Department of Internal Medicine, The James Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH, USA 3 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Washington, Seattle, WA, USA 4 Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 5 National Clinical Target Validation Laboratory, Leidos Biomedical Research Inc, Frederick National Laboratory for Cancer Research, Frederick, MD, USA 6 Center for Interventional Oncology, Radiology, and Imaging Sciences, Clinical Center and National Cancer Institute, NIH, Bethesda, MD, USA 7 Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, MD, USA Correspondence to: Jung-Min Lee, email: // Keywords : triple negative breast cancer, olaparib, carboplatin Received : July 27, 2016 Accepted : March 13, 2017 Published : March 25, 2017 Abstract PURPOSE: To investigate the safety, activity, and potential biomarkers of response to olaparib and carboplatin combination in sporadic triple negative breast cancer (TNBC).EXPERIMENTAL DESIGN: Metastatic or recurrent TNBC patients with no germline BRCA mutation or with BRCAPro scores

Clinical pharmacodynamic assays need to meet higher criteria for sensitivity, precision, robustness, and reproducibility than those expected for research-grade assays because of the long duration of clinical trials and the potentially unpredictable number of laboratories running the assays. This report describes the process of making an immunoassay based on commercially available reagents "clinically ready". The assay was developed to quantify poly(ADP-ribose) (PAR) levels as a marker of PAR polymerase inhibitor activity for a proof-of-concept phase 0 clinical trial at the National Cancer Institute (NCI) and subsequent clinical trials. In this publication, we retrospectively examine the measures taken to validate the published PAR immunoassay and outline key lessons learned during the development and implementation of these procedures at both internal and external clinical trial sites; these measures included optimizing PAR measurements in tumor biopsies and peripheral blood mononuclear cells (PBMCs), reagent qualification, analytical validation and assay quality control, instrument qualification and method quality control, and support for external laboratories.

Purpose: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. Experimental Design: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. Results: The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. Conclusions: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

Purpose Wee1 tyrosine kinase phosphorylates and inactivates cyclin-dependent kinase (Cdk) 1/2 in response to DNA damage. AZD1775 is a first-in-class inhibitor of Wee1 kinase with single-agent antitumor activity in preclinical models. We conducted a phase I study of single-agent AZD1775 in adult patients with refractory solid tumors to determine its maximum-tolerated dose (MTD), pharmacokinetics, and modulation of phosphorylated Tyr15-Cdk (pY15-Cdk) and phosphorylated histone H2AX (γH2AX) levels in paired tumor biopsies. Patients and Methods AZD1775 was administered orally twice per day over 2.5 days per week for up to 2 weeks per 21-day cycle (3 + 3 design). At the MTD, paired tumor biopsies were obtained at baseline and after the fifth dose to determine pY15-Cdk and γH2AX levels. Six patients with BRCA-mutant solid tumors were also enrolled at the MTD. Results Twenty-five patients were enrolled. The MTD was established as 225 mg twice per day orally over 2.5 days per week for 2 weeks per 21-day cycle. Confirmed partial responses were observed in two patients carrying BRCA mutations: one with head and neck cancer and one with ovarian cancer. Common toxicities were myelosuppression and diarrhea. Dose-limiting toxicities were supraventricular tachyarrhythmia and myelosuppression. Accumulation of drug (t1/2 approximately 11 hours) was observed. Reduction in pY15-Cdk levels (two of five paired biopsies) and increases in γH2AX levels (three of five paired biopsies) were demonstrated. Conclusion This is the first report of AZD1775 single-agent activity in patients carrying BRCA mutations. Proof-of-mechanism was demonstrated by target modulation and DNA damage response in paired tumor biopsies.

Purpose: A phase I trial of veliparib in combination with topotecan plus carboplatin (T+C) demonstrated a 33% objective response rate in patients with hematological malignancies. The objective is to perform exposure–response analysis to inform the phase II trial design. Experimental Design: Pharmacokinetic, efficacy, and safety data from 95 patients, who were administered 10 to 100 mg b.i.d. doses of veliparib for either 8, 14, or 21 days with T+C, were utilized for exposure–efficacy (objective response and overall survival) and exposure–safety (≥grade 3 mucositis) analysis. Multivariate cox proportional hazards and logistic regression analyses were conducted. The covariates evaluated were disease status, duration of treatment, and number of prior therapies. Results: The odds of having objective response were 1.08-fold with 1,000 ng/hr/mL increase in AUC, 1.8-fold with >8 days treatment, 2.8-fold in patients with myeloproliferative neoplasms (MPN), and 0.5-fold with ≥2 prior therapies. Based on analysis of overall survival, hazard of death decreased by 1.5% for 1,000 ng/hr/mL increase in AUC, 39% with >8 days treatment, 44% in patients with MPN, while increased by 19% with ≥2 prior therapies. The odds of having ≥grade 3 mucositis increased by 29% with 1,000 ng.h/mL increase in AUC. Conclusions: Despite shallow exposure–efficacy relationship, doses lower than 80 mg do not exceed veliparib single agent preclinical IC50. Shallow exposure–mucositis relationship also supports the 80-mg dose. Based on benefit/risk assessment, veliparib at a dose of 80 mg b.i.d. for at least 14 days in combination with T+C is recommended to be studied in MPN patients. Clin Cancer Res; 23(21); 6421–9. ©2017 AACR.

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.

Background: Wee1 tyrosine kinase phosphorylates and inactivates cyclin dependent kinase (Cdk) 1 as part of DNA damage response (DDR) signaling, resulting in G2 cell cycle arrest to enable DNA repair. We are conducting a Phase I study of oral Wee1 inhibitor AZD1775 in patients (pts) with advanced solid tumors using twice-daily (Arm A) and once-daily (Arm B) dosing. Results for Arm A were published previously. Here, we report updated pharmacodynamic (PD), pharmacokinetic (PK), and clinical response results for Arm B. Methods: In Arm B, AZD1775 was administered once daily on days 1-5 and 8-12 of each 21-day cycle in a 3+3 design. Primary objectives were to establish safety, tolerability, PK, and MTD. Secondary objectives included determining PD effects on DDR markers in tumor tissue and circulating tumor cells (CTCs), and tumor response (RECIST 1.1). Post-treatment PD effects were established in Arm A. Given the importance of sustained molecular target control in maximizing targeted therapy efficacy, paired tumor biopsies in Arm B were obtained at baseline and day 8 (pre-dose) to assess durability of Wee1 inhibition and downstream PD effects. Immunofluorescence assays were used to measure target inhibition (pY15-Cdk1/2) and DDR markers (e.g., γH2AX). Results: Arm B included 42 pts; of the 34 evaluable for response, 6 (18%) had a partial response (PR; 4 ovarian, 2 endometrial), and 20 (59%) had stable disease (SD; 2-26 cycles, mean 7). Of 10 evaluable BRCA-deficient pts, 2 had a PR (20%) and 6 had SD (60%; mean: 7.17 cycles). AZD1775 was well-tolerated, as previously reported. Three of 11 paired biopsies had elevated baseline pY15-Cdk (8-23% nuclear area positive [NAP]) and showed persistence of target suppression following the dosing break during days 6-8. Of the 8 pts with low baseline pY15-Cdk (0-3% NAP), 5 had elevated pY15-Cdk levels at day 8 (range: 192-1094%), suggesting a target rebound effect. Of the 8 pts with elevated pY15-CdK at baseline or day 8, 4 (50%) achieved a PR. Day 8 γH2AX activation was detected for 3 pts, all who progressed within 2 cycles. Preliminary PK data revealed low plasma drug concentrations at day 8 pre-dose (55 ± 50 nM; n = 4) relative to peak levels at day 5 (1697 ± 1062 nM; n = 9). Conclusions: Daily AZD1775 exhibits antitumor activity in pts with both BRCA-proficient and -deficient ovarian and endometrial cancer. Responses amongst pts with elevated pY15-Cdk levels at baseline or day 8 provide additional clinical/PD evidence for the presumed mechanism of action of AZ1775 on the cell cycle and suggest that levels of pY15-Cdk may be an important correlate of response. In contrast, γH2AX activation on day 8 demonstrates the presence of drug-induced DNA double strand breaks that are not associated with clinical drug activity. PK data confirmed substantially reduced plasma AZD1775 concentrations at day 8. Ongoing genomic and CTC analyses may uncover additional molecular determinants of response. NCT01748825. Supported in part by NCI Contract HHSN261200800001E. Citation Format: Arjun Mittra, Geraldine O'Sullivan Coyne, Shivaani Kummar, Khanh Do, Ashley Bruns, Lamin Juwara, Richard Piekarz, Larry Rubinstein, Sheila Prindiville, Elad Sharon, Howard Streicher, Tiffaney Hsia, Ganesh Mugundu, Jiuping Ji, Deborah Wilsker, Angie B. Dull, Brandon Miller, Robert J. Kinders, Ralph Parchment, James H. Doroshow, Alice P. Chen, Naoko Takebe. DNA damage response and therapeutic activity following once-daily administration of the Wee 1 inhibitor AZD1775 (adavosertib) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT099.

Background In tumors carrying BRCA mutations, DNA damage caused by standard cytotoxic chemotherapy can be potentiated by poly [ADP-ribose] polymerase (PARP) inhibitors, leading to increased cell death through synthetic lethality. Individuals carrying mutations in BRCA have an increased incidence of triple negative breast cancer (TNBC). In order to assess the role of PARP inhibition in the treatment of TNBC, we conducted a randomized phase II trial of the combination of veliparib, a small molecule PARP inhibitor, with the cytotoxic agent cyclophosphamide versus cyclophosphamide alone in patients with refractory TNBC. Methods Adult patients with TNBC were randomized to receive oral cyclophosphamide 50 mg once daily with or without oral veliparib at 60 mg daily in 21-day cycles. Patients on the cyclophosphamide arm could crossover to the combination arm at disease progression. Results Forty-five patients were enrolled; 18 received cyclophosphamide alone and 21 received the combination as their initial treatment regimen. Lymphopenia was the most common grade 3/4 toxicity noted in both arms. One patient in the cyclophosphamide alone arm, and 2 in the combination arm had objective responses. Response rates and median progression free survival did not significantly differ between both treatment arms. Conclusion The addition of veliparib to cyclophosphamide, at the dose and schedule evaluated, did not improve the response rate over cyclophosphamide treatment alone in patients with heavily pre-treated triple-negative breast cancer.

Phase 0 trials are designed primarily to evaluate the pharmacodynamic and/or pharmacokinetic properties of selected investigational agents before initiating more traditional phase I testing. One of the major objectives of phase 0 trials is to interrogate and refine a target or biomarker assay for drug effect in human samples implementing procedures developed and validated in preclinical models. Thus, close collaboration between laboratory scientists and clinical investigators is essential to the design and conduct of phase 0 trials. Given the relatively small number of patients and tissue samples, showing a significant drug effect in phase 0 trials requires precise and reproducible assay procedures and innovative statistical methodology. Furthermore, phase 0 trials involving limited exposure of a study agent administered at low doses and/or for a short period allow them to be initiated under the Food and Drug Administration exploratory investigational new drug guidance with less preclinical toxicity data than usually required for traditional first-in-human studies. Because of the very limited drug exposure, phase 0 trials offer no chance of therapeutic benefit, which can impede patient enrollment, particularly if invasive tumor biopsies are required. The challenges to accrual are not insurmountable, however, and well-designed and executed phase 0 trials are feasible and have great potential for improving the efficiency and success of subsequent trials, particularly those evaluating molecularly targeted agents.

Purpose: PARP is essential for recognition and repair of DNA damage. In preclinical models, PARP inhibitors modulate topoisomerase I inhibitor–mediated DNA damage. This phase I study determined the MTD, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib, an orally bioavailable PARP1/2 inhibitor, in combination with irinotecan. Experimental Design: Patients with advanced solid tumors were treated with 100 mg/m2 irinotecan on days 1 and 8 of a 21-day cycle. Twice-daily oral dosing of veliparib (10–50 mg) occurred on days 3 to 14 (cycle 1) and days −1 to 14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Paired tumor biopsies were obtained after irinotecan alone and veliparib/irinotecan to evaluate PARP1/2 inhibition and explore DNA damage signals (nuclear γ-H2AX and pNBS1). Results: Thirty-five patients were treated. DLTs included fatigue, diarrhea, febrile neutropenia, and neutropenia. The MTD was 100 mg/m2 irinotecan (days 1 and 8) combined with veliparib 40 mg twice daily (days −1–14) on a 21-day cycle. Of 31 response-evaluable patients, there were six (19%) partial responses. Veliparib exhibited linear PK, and there were no apparent PK interactions between veliparib and irinotecan. At all dose levels, veliparib reduced tumor poly(ADP-ribose) (PAR) content in the presence of irinotecan. Several samples showed increases in γ-H2AX and pNBS1 after veliparib/irinotecan compared with irinotecan alone. Conclusions: Veliparib can be safely combined with irinotecan at doses that inhibit PARP catalytic activity. Preliminary antitumor activity justifies further evaluation of the combination. Clin Cancer Res; 22(13); 3227–37. ©2016 AACR.

Inhibition of poly (ADP-ribose) polymerase (PARP) sensitizes tumor cells to DNA damage that would normally be repaired through the base excision repair pathway. PARP inhibitors are active clinically against BRCA-deficient ovarian cancers. The PARP inhibitor talazoparib produces cytotoxicity in human cancer cell lines and animal models of tumors that harbor mutations that compromise DNA repair pathways. In this study, single agent talazoparib (1000 µg/day) was administered to patients with deleterious BRCA1 or BRCA2 mutations and advanced solid tumors in 28 day cycles. The primary objective of the trial was to examine pharmacodynamic (PD) effects of talazoparib; the secondary objective was to determine response rate in patients whose tumors carry BRCA mutations. Mandatory paired tumor biopsies were obtained pre-treatment and 3-6 hrs post-treatment on cycle 1 day 8. Optional biopsies were collected at the time of progression. One core from each time point was analyzed for PARP inhibition by a validated ELISA assay while the other core was used for IFA analysis of γH2AX. A total of 9 patients (pts) were enrolled and treated before this trial was closed due to lack of drug availability: [prostate (3), ovarian (2), breast (2), uterine sarcoma (1), pancreatic (1)]. Median age was 63 (range: 33-73 yrs); male-to-female ratio was 4:5; and the median number of prior treatments was 6 (range: 1-12). All 9 pts were evaluable for PD endpoints. One pt progressed during the first cycle of treatment; 8 pts were evaluable for clinical response. Mean time on study for evaluable pts was 8 cycles (range: 2-18); 5 of 8 (62%) pts experienced a documented partial responses [ovarian (2), prostate (2), breast (1)] lasting between 4 and 12 cycles (median: 6 cycles). Two pts had stable disease for 4 to 6 cycles, and one progressed after 2 cycles. The agent was well tolerated; the most frequent adverse events were hematologic including grade (gr) 4 anemia (1) and thrombocytopenia (1), and gr 3 anemia (2), neutropenia (1), lymphopenia (1). Decreases in PAR levels (>75%) were observed in all cycle 1 day 8 biopsy pairs, documenting a primary PD effect. Increased γH2AX expression was observed for 4/6 pts in post-dose biopsies; pre-treatment γH2AX levels, measured as %Nuclear Associated Protein (NAP), increased from a mean ± SD of 1.33 ± 1.08 to a post-treatment %NAP mean of 5.60 ± 0.78; (p=0.018), supporting a role for drug-enhanced DNA double strand breaks in the mechanism of action of talazoparib for BRCA mutant tumors. In summary, talazoparib demonstrated significant clinical activity as a single agent in patients with BRCA-deficient tumors and produced substantial reductions in tumor PAR levels in matched pre and post-treatment tumor biopsies. Citation Format: Robert S. Meehan, Alice P. Chen, Geraldine O’Sullivan Coyne, Shivaani Kummar, Jiuping Ji, Rasa Vilimas, Lamin Juwara, Robert J. Kinders, Katherine Ferry-Galow, Deborah Wilsker, Yiping Zhang, Angie B. Dull, Tony Navas, Lihua Wang, Ralph E. Parchment, James H. Doroshow. Pilot trial of talazoparib (BMN 673), an oral PARP inhibitor, in patients with advanced solid tumors carrying deleterious BRCA mutations [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 4678. doi:10.1158/1538-7445.AM2017-4678

Purpose: Veliparib, a PARP inhibitor, demonstrated clinical activity in combination with oral cyclophosphamide in patients with BRCA-mutant solid tumors in a phase I trial. To define the relative contribution of PARP inhibition to the observed clinical activity, we conducted a randomized phase II trial to determine the response rate of veliparib in combination with cyclophosphamide compared with cyclophosphamide alone in patients with pretreated BRCA-mutant ovarian cancer or in patients with pretreated primary peritoneal, fallopian tube, or high-grade serous ovarian cancers (HGSOC). Experimental Design: Adult patients were randomized to receive cyclophosphamide alone (50 mg orally once daily) or with veliparib (60 mg orally once daily) in 21-day cycles. Crossover to the combination was allowed at disease progression. Results: Seventy-five patients were enrolled and 72 were evaluable for response; 38 received cyclophosphamide alone and 37 the combination as their initial treatment regimen. Treatment was well tolerated. One complete response was observed in each arm, with three partial responses (PR) in the combination arm and six PRs in the cyclophosphamide alone arm. Genetic sequence and expression analyses were performed for 211 genes involved in DNA repair; none of the detected genetic alterations were significantly associated with treatment benefit. Conclusion: This is the first trial that evaluated single-agent, low-dose cyclophosphamide in HGSOC, peritoneal, fallopian tube, and BRCA-mutant ovarian cancers. It was well tolerated and clinical activity was observed; the addition of veliparib at 60 mg daily did not improve either the response rate or the median progression-free survival. Clin Cancer Res; 21(7); 1574–82. ©2015 AACR.

Purpose: To determine the safety and tolerability of olaparib with cisplatin and gemcitabine, establish the maximum tolerated dose (MTD), and evaluate the pharmacodynamic and pharmacokinetic profile of the combination. Experimental Design: We conducted a phase I study of olaparib with cisplatin and gemcitabine in patients with advanced solid tumors. Treatment at dose level 1 (DL1) consisted of olaparib 100 mg orally every 12 hours on days 1 to 4, gemcitabine 500 mg/m2 on days 3 and 10, and cisplatin 60 mg/m2 on day 3. PAR levels were measured in peripheral blood mononuclear cells (PBMC). Results: Dose-limiting toxicities (DLT) in two of three patients at DL1 included thrombocytopenia and febrile neutropenia. The protocol was amended to enroll patients treated with ≤2 prior severely myelosuppressive chemotherapy regimens and treated with olaparib 100 mg once daily on days 1 to 4 (DL−1). No DLTs were seen in six patients at DL−1. Because of persistent thrombocytopenia and neutropenia following a return to DL1, patients received 100 mg olaparib every 12 hours on day 1 only. No hematologic DLTs were observed; nonhematologic DLTs included gastrointestinal bleed, syncope, and hypoxia. Of 21 patients evaluable for response, two had partial response. Olaparib inhibited PARP in PBMCs and tumor tissue, although PAR levels were less effectively inhibited when olaparib was used for a short duration. Conclusions: Olaparib in combination with cisplatin and gemcitabine is associated with myelosuppression even at relatively low doses. Modified schedules of olaparib in chemotherapy naive patients will have to be explored with standard doses of chemotherapy. Clin Cancer Res; 18(8); 2344–51. ©2012 AACR.

Purpose: Oral administration of the alkylating agent cyclophosphamide at low doses, metronomic dosing, is well tolerated, with efficacy in multiple tumor types. PARP inhibition potentiates effects of cyclophosphamide in preclinical models. We conducted a phase I trial of the PARP inhibitor veliparib and metronomic cyclophosphamide in patients with refractory solid tumors and lymphoid malignancies. Experimental Design: Objectives were to establish the safety and maximum tolerated dose (MTD) of the combination; characterize veliparib pharmacokinetics (PK); measure poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies and peripheral blood mononuclear cells (PBMC); and measure the DNA-damage marker γH2AX in PBMCs and circulating tumor cells (CTC). Cyclophosphamide was administered once daily in 21-day cycles in combination with veliparib administered once daily for 7, 14, or 21 days. Results: Thirty-five patients were enrolled. The study treatment was well tolerated, and the MTD was established as veliparib 60 mg with cyclophosphamide 50 mg given once daily. Seven patients had partial responses; an additional six patients had disease stabilization for at least six cycles. PAR was significantly decreased in PBMCs (by at least 50%) and tumor biopsies (by at least 80%) across dose levels (DL); γH2AX levels were increased in CTCs from seven of nine patients evaluated after drug administration. Conclusions: The combination of veliparib with metronomic cyclophosphamide is well tolerated and shows promising activity in a subset of patients with BRCA mutations. A phase II trial of the combination compared with single-agent cyclophosphamide is ongoing in BRCA-positive ovarian cancer, triple-negative breast cancer, and low-grade lymphoma. Clin Cancer Res; 18(6); 1726–34. ©2012 AACR.

Background Poly(ADP-ribose) polymerase (PARP)facilitates DNA repair and PARP inhibitors may potentiate the effect of DNA-damaging chemotherapeutic agents in patients with cancer. Collection of peripheral blood mononuclear cells (PBMCs)as a surrogate tissue to monitor PARP inhibitor pharmacodynamic effects has several advantages over tumor biopsy collection, including minimally invasive sample collection and the ability to collect multiple samples for longitudinal assessment of drug effect. Methodology/Principal Findings Using our previously validated immunoassay for measuring poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies, we validated a method to quantify PAR levels in PBMCs to monitor the pharmacodynamic effects of the PARP inhibitor ABT-888 in clinical trials. The inter-individual variation in PAR levels was large. No significant difference (P = 0.67) was measured between median baseline PAR levels in 144 healthy volunteers (131.7 pg/1×107 PBMCs [interquartile range, 79.5–241.6]) and 49 patients with cancer (149.2 pg/1×107 PBMCs [interquartile range, 83.2–249.3]). In addition, PAR levels monitored in healthy volunteers over 3 weeks had considerable intra- and inter-individual variation (range, 44–1073 pg PAR/1×107 PBMCs). As a pharmacodynamic model, we quantified changes in PAR levels in human PBMCs treated ex vivo with clinically relevant concentrations of ABT-888. Of 40 healthy volunteer PBMC samples treated with ABT-888, 47.5% had greater than 50% PAR reduction compared to vehicle-treated controls. Considerable inter-sample heterogeneity in PAR levels was measured, and several ABT-888–insensitive samples were identified. Conclusions/Significance Our results emphasize the importance of using a validated method to measure PAR levels, and support further investigation into the role of PARP in PBMCs. To this end, the PAR immunoassay has been validated for use with PBMCs and incorporated into clinical trials to assess PBMCs as a potential pharmacodynamic surrogate for tumor biopsies in clinical trials of PARP inhibitors.

Purpose We conducted the first phase 0 clinical trial in oncology of a therapeutic agent under the Exploratory Investigational New Drug Guidance of the US Food and Drug Administration. It was a first-in-human study of the poly (ADP-ribose) polymerase (PARP) inhibitor ABT-888 in patients with advanced malignancies. Patients and Methods ABT-888 was administered as a single oral dose of 10, 25, or 50 mg to determine the dose range and time course over which ABT-888 inhibits PARP activity in tumor samples and peripheral blood mononuclear cells, and to evaluate ABT-888 pharmacokinetics. Blood samples and tumor biopsies were obtained pre- and postdrug administration for evaluation of PARP activity and pharmacokinetics. A novel statistical approach was developed and utilized to study pharmacodynamic modulation as the primary end point for trials of limited sample size. Results Thirteen patients with advanced malignancies received the study drug; nine patients underwent paired tumor biopsies. ABT-888 demonstrated good oral bioavailability and was well tolerated. Statistically significant inhibition of poly (ADP-ribose) levels was observed in tumor biopsies and peripheral blood mononuclear cells at the 25-mg and 50-mg dose levels. Conclusion Within 5 months of study activation, we obtained pivotal biochemical and pharmacokinetic data that have guided the design of subsequent phase I trials of ABT-888 in combination with DNA-damaging agents. In addition to accelerating the development of ABT-888, the rapid conclusion of this trial demonstrates the feasibility of conducting proof-of-principle phase 0 trials as part of an alternative paradigm for early drug development in oncology.

Among mechanisms underlying cytotoxic drug resistance is activation of diverse DNA damage response (DDR) pathways. Poly(ADP-ribose) polymerases (PARP)-1/2 facilitate both single- and double-strand break (DSB) repair and play a key role in the base excision repair (BER) of chemotherapy-damaged DNA. The PARP inhibitor veliparib (V) potentiates the cytotoxicity of different chemotherapeutics, including temozolomide (TEM). TEM induces distinct alkylating events in neoplastic cells whose ultimate outcome depends on the interaction of BER, mismatch repair (MMR), O(6)-methylguanine-DNA methyltransferase (MGMT), and DSB repair. While clinical activity of TEM has been mainly observed in MGMT-deficient AML, V potentiated cytotoxicity of TEM in leukemia cells in vitro in the setting of MGMT overexpression or deficient MMR pathway (Mol Cancer Ther, 2009). Methods: We conducted a Phase I study to determine maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of V+TEM, using a 3+3 dose escalation. Patients (pts) ≥60 years (yrs) with newly diagnosed poor cytogenetic-risk AML/ALL who were not candidates for intensive therapy, or ≥18 yrs with relapsed/refractory AML/ALL, secondary AML (therapy-related or arising from MDS or MPN), and CMMoL-2 were eligible. Any number of prior regimens, including allogeneic transplant (alloSCT), were allowed. V was given orally day (d)1 once, then twice a day on d4-12 at one of 6 dose levels (DL) (DL1A-B 20mg; DL2-DL3-DL4-DL5-DL6: 40-80-120-150-200 mg). TEM was given orally once a day on d3-9 (DL1A 150 mg/m2/d; DL1B-DL6 200 mg/m2/d). 28-day cycles (cy) were repeated depending on response/tolerability (4-6 weeks delay allowed) with V on d1-8 and TEM d1-5. TEM was taken on empty stomach with antiemetics and V was taken irrespective of meals. Results: Forty-nine pts with median age 69 yrs (range, 22-88; 47% ≥70) were treated. Of 47 AML pts, 29 (62%) had secondary AML and 27 (57%) adverse karyotype. Median number of prior treatments for AML was 1 (range, 0-6): 18 (38%) had median 1 prior therapy (range, 1-3) for MDS; 30 (64%), 9 (18%), 34 (69%) received hypomethylating agents, alloSCT and intensive chemotherapy, respectively. Overall 42 (85%) pts were refractory to their last treatment. Pts received a median of 1 (range, 1-7) cy of therapy. Two did not complete cy 1, pt withdrawal d5 and progressive fungal pneumonia d9 with death d15 of progressive disease (PD). The MTD/RP2D was defined at V 150 mg and TEM 200 mg/m2; 2 of 4 pts treated at V 200 mg and TEM 200 mg/m2 developed dose-limiting toxicity of grade (gr) 3 oral mucositis/esophagitis. The most frequent drug-related toxicities (NCI CTC v4) were gr 1/2 nausea/vomiting (39%), fatigue (26%), oropharyngeal mucositis (26%), constipation (12%), and diarrhea (10%). Other common toxicities were infectious, including febrile neutropenia (29%), pneumonia (20%), bacteremia (18%). One (2%) pt died ≤d30 and 12 (24%) ≤d60 mainly of PD (1 pt fungal pneumonia before count recovery d31). Overall response rate was 33% (complete remission (CR), hematologic improvement (HI)/stable disease) with 8 (16%) pts achieving CR (1 CRi). Median overall survival was 5.03 months, for all responders 11.58 months, and for CR pts 19.89 months (Fig 1). Responses occurred at all DLs. Three CR pts underwent alloSCT; 2 remain in CR at ~3 yrs. Pharmacokinetics (PK): V or TEM PKs were not altered with co-administration. There was a correlation between the DLT of mucositis and V single (Cmax P=0.005; AUC P=0.009) and multiple dose exposure (Cmax P = 0.02; AUC P=0.03). Pharmacodynamics and pharmacoepigenetics: Four of 39 pts examined had MGMT methylation (3 CR; 75%) and 2 had BRCA-1 methylation (1 HI) in peripheral blood (PB) or bone marrow (BM) mononuclear cells (MC). Defective FancD2 pathway was observed in the BMMC of 19/19 pts using FancD2 ubiquitylation assays but did not correlate with response. V reduced PAR levels by >75% in PBMC of most pts and in the presence of TEM. Induction of γ-H2AX in CD34+ cells was seen upon V/V+TEM treatment. Conclusion: V plus TEM demonstrated safety and activity in this resistant and elderly leukemia population. Response rate was higher in MGMT methylated pts, but responses were also seen in pts who had no MGMT methylation, had failed multiple therapies, had secondary AML and/or adverse karyotype. Future clinical study should aim to identify pts with defective DDR pathways who are most likely to respond to this therapeutic approach. Figure 1. Figure 1. Disclosures Off Label Use: Temozolomide is not approved for AML. Beumer:Millenium: Other: Research support. Gore:Celgene: Consultancy, Honoraria, Research Funding.

Robust PD assay results are valuable for informing decisions about continued preclinical and clinical development of new agents and for identifying effective combinations of targeted agents. The National Cancer Institute's Division of Cancer Treatment and Diagnosis (DCTD) develops and validates PD assays to obtain accurate information about drug effect on intended molecular targets in first-in-human clinical trials. Our group utilizes quantitative immunofluorescence assays (qIFA) of PD biomarkers in FFPE slides prepared from pre- and post-dose tumor biopsies collected from patients on early phase clinical trials. Paired biopsies are fixed and paraffin embedded, in parallel with tissue controls, such that all tissues are sectioned onto the same slide. Stringent methods to maintain the biopsy orientation are utilized during fixation, blocking and microtomy. A large number of sections are generated to increase the likelihood of finding optimal tumor regions of interest for biomarker analyses. Flanking H&E slides are utilized to assess the quality of the tumor biopsies. Whole slide scans of the H&E slides are shared with a clinical pathologist who determines whether the sections are sufficient or insufficient for the intended analysis. The pathology review allows the assay operator to select the optimal range of slides to stain and quantitatively analyze for the biomarker(s) of interest. Pathology-guided regions allow the operator to focus on tumor regions of interest and avoid normal tissue and/or confounding regions compromised by handling and processing artifacts. For nuclear biomarkers such as γH2AX and related DNA damage response biomarkers the pathologist annotates areas of sufficient tumor content and viability. For biomarker changes in tissue architecture such as that seen in Epithelial Mesenchymal Transition biomarkers, including E-Cadherin, Vimentin and β-catenin, the pathology annotation denotes areas of normal tissue and necrosis to eliminate from the qIFA analysis. All other areas of sufficient tumor cellularity from the entire slide are evaluated for the EMT biomarkers. Use of the pathology-annotated whole slide image as a tool for guidance of the operator performing the quantitative evaluation of the PD biomarker helps to ensure a non-subjective analysis. Finally, steps are taken to ensure proper storage of the paraffin slides, including paraffin dipping after microtomy, which is critical for preservation of labile epitopes such as phosphorylated proteins. We will present details of these optimized methods as well as key lessons learned during the preclinical and clinical implementation of qIFA measurements of PD biomarkers for various molecular targeted agents. Funded by NCI Contract No HHSN261200800001E. Citation Format: Katherine V. Ferry-Galow, Scott M. Lawrence, Tony Navas, Hala R. Makhlouf, Donna O. Butcher, Brad A. Gouker, William H. Yutzy, Jiuping Ji, Robert Kinders, Ralph E. Parchment, Shivaani Kummar, Joseph E. Tomaszewski, James H. Doroshow. Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2015-5279

BRCA 1/2 dysfunction sensitizes cells to inhibition of PARP enzymatic activity, due to the persistence of DNA breaks normally repaired by homologous recombination (HR), thus cancer pts carriers of germ line BRCA deleterious mutations have been targeted for treatment with PARP inhibitors. An example of HR repair is the FA pathway where BRCA 1/2 collaborate with several other genes resulting in FancI and FancD2 activation and nucleus repair foci formation. We have developed a triple stain immunofluorescence based method to evaluate FA pathway functionality (foci formation) (FancD2/DAPI/Ki67) (FATSI) in paraffin embedded tumor tissues, and hypothesized that among patients with sporadic tumors, a significant proportion with tumoral dysfunctional FA pathway can be identified. A two steps clinical trial (NCT01017640) was designed to 1- screen solid tumor patients for absence of FA tumor foci formation, and 2- evaluate safety and recommended doses in these pts of the PARPi veliparib, either alone or in combination with chemotherapy (MMC). 607 pts were consented, 532 had their tumor tissue screened, 142 (27%) had absent foci. All major solid tumors were represented in FA dysfunction, notably 36 of 105 (34%) colorectal and 40 of 135 (30%) breast cancers. 52 pts received 192 cycles of veliparib, 29 pts as monotherapy in escalating dose cohorts (starting at 50 mg PO BID continuously), and 23 following MMC 10 mg-sqm every 4 weeks (40mg-sqm cumulative cap) at 50 mg BID at increased duration cohorts (1, 2 and 3 wks) every 4 wks (100 and 200 mg BID for 3 wks in subsequent cohorts). Recommended dose levels are: veliparib 300 mg BID as monotherapy (2DLT/3 pts at 400 BID:G3 fatigue) and MMC/veliparib 10mg-sqm/200 mg BID three weeks duration. Analysis for germ line mutations (BRCA1 and 2 sequencing and for the 5 most common BRCA1 large rearrangements) in PBMC among 50 pts showed 7 pts (3 breast, 1 ampullary, 2 ovarian) with BRCA abnormalities: 6 known deleterious mutations and 1 polymorphism. Five RECIST criteria responses occurred (1CR, 4 PR) (1 lung, 2 breast, 1 ovarian, 1 endometrial), all in the combination arm. Two breast cancer pts (39 and 13 cycles) and 1 ovarian (6 cycles) on monotherapy, plus 1 breast (15 cycles), 1 colon (7 cycles) on the combination, had prolonged stability. rH2AX (n=49) and PAR (n=17) analysis in PBMC suggests veliparib dose dependency (blunting of rH2AX induction, lower PAR level and slower recovery for higher doses). The trial was amended to enroll an expanded cohort of 10 FATSI negative colorectal patients who will undergo fresh biopsy for whole exome sequencing (RNAseq) and FA/DNA repair gene panel analysis prior to treatment at the recommended dose in the combination arm, with the goal of identifying the specific genetic defect mediating the FA dysfunction, as well as partner mutations to be targeted in subsequent trials. Supported by R01CA152101 & N01CM62207 Citation Format: Miguel A. Villalona-Calero, Wenrui Duan, Weiqiang Zhao, Konstantin Shilo, Jiuping Ji, Jennifer Thurmond, Adam Norris, Jeffrey Rose, Rachel Layman, John Marshall, Tanios Bekaii-Saab, Alice Chen. Phase I trial of veliparib or mitomycin (MMC) + veliparib in patients with sporadic solid tumors screened for somatic deficiency in the Fanconi Anemia (FA) pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2013-LB-139

2520 Background: The PARPi O has single-agent activity against BRCA1/2 mutation carriers (Br/Ov mut+) Br/OvCa. We hypothesized addition of C to O to stress the DNA repair machinery would further im...

3000 Background: The nuclear enzyme PARP is essential in recognition and repair of DNA damage. In preclinical models, PARP inhibitors work as sensitizing agents for DNA-damaging agents such as Ir. V is an orally bioavailable PARP 1 and 2 inhibitor. This phase I study determined the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), PK and PD of V together with Ir. Methods: Eligibility included patients with performance status 0-2; ≥ age 18; adequate bone marrow, hepatic and renal function. Cycles were 21 days. Ir was given i.v. 100 mg/m2 over 90 min on Days 1 and 8. Twice daily (BID) oral dosing of V (10-50mg) occurred Days 3-14 (Cycle 1) and Days -1-14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Tumor biopsies and matched peripheral blood mononuclear cells (PBMCs) were collected for PD evaluation of PAR, γ-H2AX, PARP1, TOPO1, and ERCC1 after Ir alone and after the combination. Results: 32 pts were treated (2 lung, 14 breas...

Abstract 3634 PARP is activated in response to DNA single strand (SS) breaks and is pivotal to the base excisional repair pathway for chemotherapy-damaged DNA. The orally bioavailable PARP inhibitor V delays DNA repair and potentiates the cytotoxicity of multiple classes of chemotherapy drugs including topoisomerase I inhibitors and platinating agents in leukemia cell lines. A previous clinical trial of T+C yielded promising results in adults with refractory acute leukemia at the maximum tolerated dose (MTD) of T 1.6 mg/m2/d + C 150 mg/m2/d by intravenous continuous infusion (IVCI) × 120 hrs. We are conducting a Phase I dose-escalation trial of V given orally twice daily Days 1–8 with T+C given by 120 hr IVCI Days 3–7. Deaths due to myocardial infarct and splenic infarcts with ascites occurred in one pt each when C 150 mg/m2 was added to V 10 mg BID and T 1.3 mg/m2, resulting in adjustment of the dose escalation schema for C and T.Dose levelVeliparib Oral Daily Days 1–8Topotecan Continuous IV Days 3–7Carboplatin Continuous IV Days 3–7Clinical Responses/#Pts1A10 mg p.o. BID1.0 mg/m2/day—1/3(CRi)1B10 mg p.o. BID1.3 mg/m2/day—1/3(CR)2A10 mg p.o. BID1.3 mg/m2/day150 mg/m2 IV0/42C10 mg p.o. BID1.0 mg/m2 IV120 mg/m2 IV2/6(CRi, PR)2D10 mg p.o BID1.2 mg/m2 IV120 mg/m2 IV1/6(PR)2E10 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CRi)3C20 mg p.o. BID1.0 mg/m2 IV120 mg/m2 IV0/63D20 mg p.o BID1.2 mg/m2 IV120 mg/m2 IV2/6(CR, PR)3E20 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CR)440 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CR)5§80 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/4(CR, CRi) Of 56 pts treated at doses of V 10–80 mg orally BID Days1–8 and T 1.0–1.2 mg/m2/d + C 120–150 mg/m2/d for 120 hrs IVCI Days 3–7, 25 (45%) have experienced > grade 3 non-hematologic toxicity, 6(11%) have had grade 4 toxicity and 1 (4%) died from sepsis with multi-organ failure. Overall response rate is 27% (8CR, 4 CRi, 3 PR), with 7 in relapsed AML, 7 in refractory AML and 1 refractory ALL. Pharmacokinetic (PK) studies in pt plasma, marrow supernatant, and marrow blasts demonstrated that V is detectable in marrow supernatant and cells, does not accumulate with multiple dose administrations in plasma but does in marrow, and is not altered by T or C administration. Nuclear staining for phosphorylated histone H2AX (γH2AX) demonstrated that V 10 mg BID increased DNA damage by > 2-fold in 11/22 (50%) on Day 1 and 13/18 (72 %) on Day 4 after 24 hrs of T+C. A PAR ELISA demonstrated that V suppresses PAR levels to Disclosures: No relevant conflicts of interest to declare.

2605 Background: Inhibition of poly (ADP-ribose) (PAR) polymerase (PARP) is expected to enhance the DNA-damaging effects of chemotherapy. PARP has also been implicated in endothelial cell migration. A is an orally available, potent inhibitor of PARP-1 and PARP-2. C is an alkylator that has effects on thrombospondin 1, endothelial cells and T reg cells. Methods: Objectives were to establish the safety, tolerability, and MTD of the combination of A with metronomic C; determine the PK of A; and evaluate PARP inhibition in PBMCs and tumor samples. Patients (pts) had histologically documented solid tumors and lymphoid malignancies that had progressed following standard therapy; ≥ 18 years old; KPS > 70%; adequate bone marrow, hepatic and renal function. Cycle length was 21 d. DLT was defined as drug-related gr ≥ 3 nonhematologic or gr 4 hematologic toxicities during cycle 1. PBMCs and optional tumor biopsies were collected, and PAR levels were determined using a validated ELISA. Results: Eighteen pts have been...

3027 Background: Poly (ADP-ribose) polymerase (PARP) group of NAD+-dependent enzymes participate in multiple DNA- related functions. Increased PARP activity can lead to drug resistance. Olaparib (O), a potent oral inhibitor of PARP-1 and PARP-2 may partially overcome resistance to C and G when used in combination. Methods: Eligibility: histologically confirmed metastatic or unresectable solid tumors, PS 0–2, normal organ functions. Treatment at dose level 1 (DL1): O 100 mg orally bid on days (D) −2 to 2, G 500 mg/m2/h on D1 and D8, C 60 mg/m2 on D1 after G; q21 days ×6. Doses (same schedule) at DL −1: O 100 mg once daily, G 400 mg/m2/h, C 50 mg/m2. Peripheral blood mononuclear cells (PBMCs) were collected and processed within 2 hours and poly (ADP-ribose) (PAR) concentration determined by immunoassay. Results: From 5/2008 to 11/2009, 23 patients (pts) enrolled; 14 males, median age 52 yrs (25- 88). Tumor types: 8 NSCLC; 3 ovarian; 3 pancreatic; 2 esophageal; 2 thymic ca; 1 mesothelioma; 1 adrenocortical c...

14024 Background: Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a molecular sensor of DNA breaks that facilitates DNA repair and controls genomic stability. Treatment with single doses of ABT-888, a novel potent PARP-1 inhibitor, reduced PAR levels in peripheral blood mononuclear cells (PBMCs) and tumor biopsies in an on-going Phase-0 trial at the NIH Clinical Center. As a corollary to this study, we investigated whether ABT-888 can act as a sensitizer for radiation therapy and chemotherapy in human cancer cell culture, xenograft tumors and PBMCs to support future combination clinical trials. Methods: Inhibition of PARP-1 by ABT-888 was determined by a quantitative PAR chemiluminescence immunoassay validated for the Phase 0 trial. Since gamma-H2AX (?-H2AX) is a marker of DNA damage, we also developed and validated ?H2AX assays to monitor the effects of PARP-1 inhibition during treatment with Topo I inhibitors and radiation. Human monocytic leukemia (THP-1) and breast carcinoma (MCF-7) cell lines were treated with Topo I inhibitors including indenoisoquinoline, camptothecin and topotecan or irradiated with 0.5 to 10 Gy in the presence of ABT 888. We further evaluated these effects in human blood ex vivo to confirm the observations made in cell culture. Results: We found that ABT-888 inhibited PAR, but did not significantly increase DNA damage. Combination of ABT-888 with a Topo I inhibitor produced over 275% increase of DNA damage in THP-1 leukemia cells compared to indenoisoquinoline alone. ?H2AX foci per cell were 9.5 ± 0.8 in MCF-7 treated with 0.5 Gy/50 nM ABT-888 in comparison to 4.0 ± 0.6 with radiation alone. When whole blood was treated in the presence of ABT-888, camptothecin- induced DNA damage in PBMCs was also increased 2–3 fold, with maximum ?H2AX expression at 2 hours post treatment. Conclusions: We conclude that ABT-888 is a highly potent PARP-1 inhibitor that can enhance the DNA damaging effects of chemotherapy and radiation therapy of human cancer. Funded by NCI Contract N01-CO-12400. No significant financial relationships to disclose.

10565 Background: DNA double-strand breaks (DSBs) caused by exposure to DNA damaging agents initiate phosphorylation of histone H2AX to form gamma-H2AX, which is considered a surrogate marker of DSBs. However, it is a challenge to quantitatively measure gamma-H2AX in clinical samples such as tumor biopsies. The aim of this study was to develop an immunohistochemical detection method for gamma-H2AX and to quantitatively evaluate the levels of gamma-H2AX in paraffin-embedded tumor samples. Methods: Human breast cancer MCF-7 cells were treated with the topoisomerase I inhibitor irinotecan at 1 μM or vehicle for 1 h, and fixed in 10% neutral buffered formalin and embedded in paraffin. Staining with gamma-H2AX antibody was performed on sections of treated MCF-7 cells, tumor specimens, and biopsies at baseline and after doxorubicin-containing chemotherapy from cancer patients. Numbers of foci and level of gamma-H2AX expression per tumor nucleus were determined by manual counting under a light microscope and an Automated Cellular Imaging System. Results: There was a rise in the mean numbers of nuclear foci and intensity of gamma-H2AX in MCF-7 cells treated with irinotecan versus vehicle (19.9 ± 2.7 vs. 9.95 ± 3.6; P < 0.0001 and 61.2 ± 8.5 vs. 16.2 ± 13.6; P < 0.0001 by Wilcoxon rank sum test). The level of gamma-H2AX foci in human tumor samples was 18.8 ± 13.1, 44.8 ± 14.5, 51.2 ± 20.8, or 69.7 ± 21.2 in carcinomas of the breast, colon, ovary, or prostate. In a patient with stable disease, levels of gamma-H2AX foci were 62.7 ± 26.9 at baseline and 67.2 ± 25.3 after doxorubicin-containing regimen chemotherapy. Conclusions: Our data suggest that the quantitative immunohistochemical detection of gamma-H2AX levels is facilitated by a digital imaging system, and is a reliable method to measure the effects of DNA damaging agents in cells and paraffin-embedded human tumor samples. Its application may help evaluate tumor response to various DNA damaging agents currently in the clinic and those presently undergoing clinical development. No significant financial relationships to disclose.