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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.

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

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

Poly (ADP-ribose) (PAR) polymerases (PARP1 and PARP2) are activated in response to DNA single-strand breaks and are pivotal to the base excision repair pathway for homologous recombination-defective cells or for the chemotherapy-damaged genome. ABT-888, AZD2281, and BSI201 are three presumed PARP inhibitors currently being evaluated in phase 1-3 clinical trials; each has demonstrated therapeutic activity either as a single agent for patients with BRCA-deficient malignancies or in combination with DNA-damaging chemotherapeutic agents. To compare the pharmacodynamics of these agents, we employed the BRCA1-defective breast cancer cell line MX-1 as a model. By monitoring γH2AX foci formation with a validated slide-based immunofluorescence assay, we found that γH2AX foci increased in a dose- and time-dependent fashion during treatment with each of the three drugs. However, utilizing our validated quantitative chemiluminescence ELISA for PAR (Clin. Cancer Res. 14: 6877, 2008), we unexpectedly found that PARP1/2 inhibition was dose and time dependent only for tumor cells treated with ABT-888 or AZD2281, but not BSI201. This observation was confirmed using ex vivo treatment of PBMCs from human whole blood. To investigate the mechanism of action of these drugs further, we used next-generation sequencing to identify genes that were induced or suppressed by exposure to these drugs. We found that a 4-hour exposure of MX-1 cells to a clinically achievable concentration of either ABT-888 or BSI201 produced expression changes in dramatically different sets of genes. Gene ontology and pathway analyses indicated that BSI201 specifically suppressed genes in the telomere pathway, suggesting PARP5/6 as potential targets and a different mechanism of γH2AX foci formation, whereas ABT-888 induced genes in the chromosome organization network, consistent with PARP1/2 as primary targets. These experiments strongly suggest that the major therapeutic mechanism of action of BSI201, unlike ABT-888 and AZD2281, is not mediated by inhibition of PARP1 or PARP2. Funded, in part, by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4527. doi:10.1158/1538-7445.AM2011-4527