Hoch, Ute

1087 Background: EP is a unique topoisomerase 1 inhibitor that provides continuous exposure to SN38. EP demonstrated a 29% overall response rate in patients with mBC, leading to a phase III global BEACON study in patients with mBC. CTCs in patient blood samples provide a minimally invasive approach to detect PD markers of drug activity. We developed quantitative multiplex immunofluorescent assays to measure target-specific PD biomarkers for EP in CTCs isolated pre- and post-treatment. Methods: Assays for Top1, Top2, g-H2Ax, Rad51, ABCG2, and Ki-67 were developed using control (0.1% DMSO) and drug-treated (SN38, 10 uM) tumor cell lines (HCT116, MCF7, A549, SKBr3) and PBMCs from healthy donors. The optimal antibody for each biomarker was then multiplexed in a panel with antibodies against cytokeratin, CD45 and DAPI for phenotypic identification of CTCs. For analysis of BEACON pts, serial 7.5 mL whole blood samples were drawn and shipped ambient to Apocell (Houston, TX) for further processing. PBMCs were separated and CTCs were isolated using ApoStream technology. CTCs were stained for PD markers and analyzed using an iCys laser scanning cytometer equipped with image analysis software. Results: Antibodies bound to tumor cells showed staining confined to the nucleus (Top1, Top2, g-H2AX, Ki-67) or membrane (ABCG2), exhibited defined peak separation from their isotype controls, and signal strength correlated with cellular expression of high and low levels of markers. To date, ~ 80% of BEACON pts consent to participate in the CTC sub-study. As of 30 Oct 2012, data from 167 pre-dose blood samples from BEACON pts were available. 99% of blood samples were successfully processed. 93% had detectable CTCs, yielding a median of 216 CTCs (range: 7.5-14816). Staining was positive for Top1, Top2, g-H2Ax, Rad51, ABCG2, and Ki-67 in 82%, 89%, 16%, 53%, 31%, 52% of samples, respectively. Conclusions: EP target-specific pharmacodynamic biomarkers can be reliably measured in CTCs isolated from patients participating in BEACON. Sample collection and analysis of pre- and post-treatment samples is ongoing. Clinical trial information: NCT01492101.

26 Background: NKTR-262 is a novel intratumoral prodrug designed to promote tumor antigen release, an immune stimulatory environment, and antigen presentation. NKTR-214 increases CD8+ T cells and NK cells in the tumor microenvironment. The combination of NKTR-262 with NKTR-214 accesses innate and adaptive immunity to provide an abscopal response and systemic tumor immunity. The Phase 1b/2 REVEAL trial is enrolling. Methods: In a 3+3 Phase 1 design, pts who are relapsed/refractory to ≥1 CPI receive escalating doses of intratumoral (IT) NKTR-262 followed by combined q3w treatment of IT NKTR-262 and fixed-dose intravenous (IV) NKTR-214 (0.006 mg/kg). Phase 1 will evaluate PK, safety, efficacy, and determine recommended Phase 2 dose. Scans are conducted every 9 wks (± 1 wk). Peripheral blood (PB) and tumor tissue are collected to measure biomarkers of immune activation. In Phase 2 expansion, NKTR-262 + NKTR-214 ± IV nivolumab (360 mg) will be assessed in specific tumor cohorts of IO-naïve and experienced pts. Results: As of 06 Nov 2018, 11 pts had received at least one cycle of NKTR-262 + NKTR-214 therapy. 7/11 pts were efficacy evaluable with at least one on-treatment scan (4 MEL, 2 SARC, 1 CRC). 2/4 R/R melanoma pts had uPRs with 100% (scan 2) and 40% (scan 1) reductions in target lesions per RECIST 1.1, respectively. Both patients remain on treatment. Two SARC pts had stable disease at scan 1. No pts experienced G3 or higher TRAEs or immune-related adverse events (AEs). Most common G1-2 AEs were transient flu-like symptoms. Gene expression analysis of tumor and PB show dose-dependent induction (4 to 16-fold) of TLR target genes. Consistent with the mechanism of action for NKTR-214, PB flow cytometry shows an average 13-fold increase from baseline in Ki67+ CD8+T cells in 6 pts. Conclusions: NKTR-262 + NKTR-214 engage the immune activation cascade from local tumor antigen production to anti-tumor T cell response. Initial dose levels of NKTR-262 with fixed-dose NKTR-214 were well-tolerated with early evidence of clinical activity. These data support continued evaluation of the combination +/- nivolumab. Clinical trial information: NCT03435640.

Abstract Purpose: Preplanned exploratory analyses were performed to identify biomarkers in circulating tumor cells (CTC) predictive of response to the topoisomerase 1 inhibitor etirinotecan pegol (EP). Experimental Design: The BEACON trial treated patients with metastatic breast cancer (MBC) with EP or treatment of physician's choice (TPC). Blood from 656 of 852 patients (77%) was processed with ApoStream to enrich for CTCs. A multiplex immunofluorescence assay measured expression of candidate response biomarkers [topoisomerase 1 (Top1), topoisomerase 2 (Top2), Ki67, RAD51, ABCG2, γH2AX, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)] in CTCs. Patients were classified as Top1 low (Top1Lo) or Top1 high (Top1Hi) based on median CTC Top1 expression. Correlation of CTC biomarker expression at baseline, cycle 2 day 1 (C2D1), and cycle 4 day 1 with overall survival (OS) was investigated using Cox regression and Kaplan–Meier analyses. Results: Overall, 98% of samples were successfully processed, of which 97% had detectable CTCs (median, 47–63 CTCs/mL; range, 0–2,020 CTCs/mL). Top1, Top2, and TUNEL expression was detected in 52% to 90% of samples; no significant associations with OS were observed in pretreatment samples for either group. EP-treated patients with low C2D1Top1+ CTCs had improved OS compared with those with higher positivity (14.1 months vs. 11.0 months, respectively; HR, 0.7; P = 0.02); this difference was not seen in TPC-treated patients (HR, 1.12; P = 0.48). Patients whose CTCs decreased from Top1Hi to Top1Lo at C2D1 had the greatest OS benefit from EP (HR, 0.57; P = 0.01). Conclusions: CTC Top1 expression following EP treatment may identify patients with MBC most likely to have an OS benefit. Clin Cancer Res; 24(14); 3348–57. ©2018 AACR.

Abstract Background: At the time of ovarian cancer platinum resistance, standard therapies have limited activity. New anti-cancer drug combinations may lead to improved clinical outcomes. Activity of topoisomerase I (Top I) inhibitors or poly(ADP-ribose) polymerase (PARP) inhibitors have been reported in ovarian cancer and have demonstrated potentiation in other models. Objective: To investigate the efficacy of the Top I inhibitor etirinotecan pegol (NKTR-102) and the PARP inhibitor rucaparib (CO-338) as single agents and in combination in patient-derived xenograft (PDX) models of ovarian cancer. Methods: Five ovarian cancer PDX models in SCID mice were treated for 28 days with NKTR-102 (10 mg/kg/q7d IV), rucaparib (150 mg/kg/qd PO), or combination NKTR-102 plus rucaparib at the same doses and schedules. Of the five PDX models, four were platinum resistant and one was a platinum-sensitive positive control. Each treatment arm had a total of 10 mice and animals were observed daily for body weight and general condition. Tumor size was assessed weekly by ultrasound. The primary endpoint was change in tumor cross-sectional area (cm2) from pretreatment baseline to final tumor burden. Western blot was performed to investigate potential biomarkers of response. Results: Compared to vehicle-treated controls, statistically significant improvement (p<0.05) was observed in 3 of 5 models in single agent rucaparib treated tumors and in 5 of 5 models in single agent NKTR-102 treated tumors. The combination of NKTR-102 plus rucaparib also demonstrated statistically significant improvement compared to vehicle-treated controls in 5 of 5 models. The combination of NKTR-102 plus rucaparib induced maximal tumor regression in 2 of 5 tumors with a trend towards smaller tumors compared to single agent treatment arms. This was not found to be statistically significant in the 28 day study period under which the experiment was performed. Exploratory analysis to assess candidate drug response determinants (e.g.Top I, PAPR I, and PARP polymer formation levels) was investigated. Conclusion: NKTR-102 and rucaparib are well tolerated in mice and demonstrate activity in platinum-resistant and platinum-sensitive ovarian cancer PDX models. Combination therapy demonstrated a trend towards decreased tumor size when compared to single agent activity during treatment. Current efforts are focused on extending the time on study for the PDX models evaluated to determine if statistical superiority of the NKTR-102/rucaparib combination can be established. Citation Format: Caroline C. Nitschmann, Rachel M. Hurley, Marc A. Becker, Xiaonan Hou, Ute Hoch, Thomas C. Harding, Deborah H. Charych, Scott H. Kaufmann, Paul Haluska, Saravut J. Weroha. Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models. [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 B50.

Abstract Background: The need remains for novel agents that prolong survival and/or improve QOL in women with aBC. EP is a long-acting topoisomerase 1 inhibitor engineered to produce sustained exposure to irinotecan and its active metabolite SN38. Given previous efficacy seen in an earlier phase II trial in MBC, EP 145 mg/m2 every 3 weeks was compared to TPC (one of 7 single-agent regimens) in the randomized phase 3 BEACON study (NCT01492101). As reported at ASCO 2015 (abstract 1001), EP prolonged median overall survival by 2.1 months, although this did not reach statistical significance (12.4 vs 10.3 months; HR 0.87, p=0.08). Grade ≥ 3 adverse events were significantly less common with EP (48% vs 63% with TPC, p<0.001). We now present results of the QOL analyses. Methods: Patients completed validated health-related QoL (HRQoL) questionnaires, EORTC QLQ-C30 (version 3.0) and breast cancer-specific QLQ-BR23, pretreatment and every 8 weeks until progression, death or withdrawal of consent. Questionnaires were scored according to the EORTC manual. For each scale, raw scores were standardized via a linear transformation to a range from 0 to 100. Absolute scores and changes from baseline were analyzed longitudinally and categorically using a 5-point difference calculated by treatment group. Comparisons between treatment groups were conducted to evaluate the differences in global health status, functional scores and symptoms over time. Results: The majority of patients who were randomized (total: 733/852 [86%], EP: 378/429 [88%], TPC: 355/423 [84%]) completed at least one post-baseline HRQoL questionnaire. In the EORTC QLQ-C30, grade ≥ 3 AEs significantly impacted HRQoL measured by global health status and 5 additional functional domains. Of the six domains, compared to TPC in a longitudinal analysis, EP was statistically superior in the mean treatment effect through Week 32 in global health status p=0.02 and physical functioning scale p=0.01. EP was also numerically superior in all other scales. In EORTC QLQ-C30 and BR-23, a total of 13 symptoms were measured and categorically analyzed. There were no treatment differences in 7 of 13 symptom scales. EP was associated with worsening of 3 symptom scales: appetite loss, nausea/vomiting, and diarrhea. TPC was associated with worsening of 2 symptom scales: dyspnea and systemic side effects. Conclusions: In the phase 3 BEACON trial comparing EP to TPC, the more favorable toxicity profile of EP resulted in an improvement in global health status and physical function (results of the symptom scales confirmed the different toxicities of the two arms). EP remains a promising investigational therapy for aBC. Citation Format: Cortes J, Awada A, Perez EA, Rugo HS, Twelves C, Im S-A, Zhao C, Hoch U, Ney J, Hannah AL, O'Shaughnessy J. Impact of treatment on quality of life (QOL) in the BEACON study, a randomized phase III trial of etirinotecan pegol (EP) versus treatment of physician's choice (TPC) in patients (pts) with advanced breast cancer (aBC) whose disease has progressed following anthracycline (A), taxane (T) and capecitabine (C). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-11-08.

Abstract Background: Etirinotecan pegol (EP) is a long-acting Top1 inhibitor providing sustained levels of active metabolite throughout the entire chemotherapy cycle. The phase 3 BEACON trial compared EP to treatment of physician's choice (TPC) in patients with advanced breast cancer, demonstrating a non-statistically significant 2.1 month difference in survival favoring EP in the intent to treat population. A novel aspect of the BEACON trial is to explore the utility of biomarkers measured in CTCs for predicting efficacy with EP. Pre- and post-treatment CTCs were isolated from blood of 77% of the 852 BEACON patients. Target-specific pharmacodynamic biomarkers for EP measured in CTCs were analyzed to identify patients most responsive to treatment with EP. Methods: Donation of blood samples for CTC analysis was voluntary. Participating BEACON patients had serial (baseline, Cycle 2 Day 1 [C2D1], Cycle 4 Day 1 [C4D1], End of Treatment) 7.5-mL whole blood samples drawn in EDTA tubes and shipped within 96 hours ambient to ApoCell (Houston, TX) for processing. PBMCs were separated by Ficoll® gradient, and CTCs were isolated using ApoStream® technology. Isolated cells were deposited on three slides and stained for DAPI, CD45, cytokeratin markers, as well as Top1, Top2, Ki67, γH2AX, Rad51, ABCG2, and TUNEL. Biomarkers were quantified by iCys® laser scanning cytometer equipped with image analysis software, and correlated with OS using Cox multiple regression and Kaplan-Meier analyses. Results: The CTC substudy yielded 611 pre-treatment, 519 C2D1, 268 C4D1, and 431 End of Treatment samples. Among the successfully processed blood samples, 98% had detectable CTCs, with a median of 63, 46, 51, and 57 CTCs/mL at baseline, C2D1, C4D1, and End of Treatment, respectively. Cox regression analyses of CTC number and percentage of Top1, Top2, Ki67, or TUNEL positive CTCs identified a correlation for post-treatment number of Top1-positive CTCs with OS in patients receiving EP. To assess the impact of Top1-positive CTCs, patients were classified as Top1-High (> median) or Top1-Low (≤ median) based on the percent of Top1-positive CTCs at baseline. Among the Top1-High patients at baseline, significantly improved OS (HR 0.54, p=0.007) was observed for those who converted to Top1-Low after their first treatment with EP (C2D1), but not TPC (HR 1.12, p=0.613). These results suggest that decreased number of Top1-positive CTCs may reflect EP target engagement with Top1, as these patients derived the most benefit from treatment. Conclusions: CTC collection and analysis was successfully incorporated into the phase 3 BEACON study, with 77% patient participation. CTC detection rate using ApoStream® was high, permitting evaluation of biomarkers at baseline and post-treatment. Significantly improved OS was observed in patients who had a decreased number of Top1-positive CTCs following cycle 1 of EP. Citation Format: Rugo HS, Cortes J, Awada A, O'Shaughnessy J, Twelves C, Im S-A, Hannah AL, Lu L, Sy S, Caygill K, Zajchowski D, Davis DW, Hoch U, Perez EA. Early change in topoisomerase 1 (Top1) positive circulating tumor cells (CTCs) is associated with overall survival (OS) in patients with advanced breast cancer after treatment with etirinotecan pegol. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-13-02.

Abstract Background: Etirinotecan pegol (EP) is the first long-acting topoisomerase 1-inhibitor providing sustained level of active metabolite that concentrate in the tumor for an entire chemotherapy cycle. In a Phase 2 study (Lancet Oncology 2013), EP demonstrated a 29% overall response rate in patients with metastatic breast cancer, leading to the Phase 3 global BEACON study in this population. Detection of circulating tumor cells (CTCs) using immunomagnetic EpCAM-based methods have been conceptually accepted as a "liquid tumor biopsy". To circumvent the limited recovery of CTCs these methods provide for molecular profiling applications, we isolated CTCs based on an antibody-independent, continuous flow dielectrophoresis (DEP) technology (ApoStream®). We developed quantitative multiplex immunofluorescent assays for target-specific pharmacodynamics (PD) biomarkers for EP in CTCs isolated pre- and post-treatment. Here we present the distribution of the PD biomarkers in CTC samples collected from BEACON patients. Methods: Donation of blood samples for CTC analysis was voluntary. BEACON patients participating in the substudy had serial (pre-dose, Cycle 2 Day 1, Cycle 4 Day 1, End of Treatment) 7.5-mL whole blood samples drawn in EDTA tubes and shipped ambient to ApoCell (Houston, TX) within 96 hrs for processing. PBMCs were separated by Ficoll® gradient, and CTCs were isolated using ApoStream® technology. Isolated cells were deposited on three slides and stained for DAPI, CD45, and cytokeratin markers, as well as 2-3 of the seven PD markers, and analyzed by iCys® laser scanning cytometer equipped with image analysis software. CTC count, mean fluorescence intensity (MFI) of PD biomarkers, and % marker-positive cells were analyzed for their distribution. Results: 75% of the 852 BEACON patients participated in the CTC substudy, yielding 649 pre-treatment CTC samples. As of Mar14, 538 Cycle 2 Day 1, 281 Cycle 4 Day 1, and 394 End of Treatment samples were collected. 98% of pre-treatment blood samples were successfully processed. 97% had detectable CTCs, with a median of 504 CTCs (interquartile range: 128-1432). PD markers could be analyzed in 83-92% of samples after implementing a minimum requirement of 10 CTCs analyzed. Staining was positive for Top1, Top2, μH2Ax, Rad51, ABCG2, Ki67, and TUNEL in 82%, 71%, 14%, 30%, 20%, 56%, and 94% of samples, respectively. Percent of marker-positive cells varied from 0.2-69%, with 6- to 37-fold interquartile ranges. Median MFI in these cells varied between 25x103 and 100x103, with 1.2- to 1.6-fold interquartile ranges. Conclusions: CTC collection was successfully incorporated into the BEACON study, with 75% patient participation. CTC detection rate using ApoStream® was high, permitting evaluation of biomarkers at baseline and over time. EP target-specific PD biomarkers can be measured in CTCs isolated from patients participating in BEACON and will be assessed for their potential to predict clinical response. Final BEACON efficacy and safety results are expected in early 2015, which will allow further analysis of CTCs with patient outcome (response, PFS, OS) and change in CTCs at time of progression. Citation Format: Edith A Perez, Katie Caygill, Alison L Hannah, Javier Cortes, Ahmad Awada, Joyce O'Shaughnessy, Christopher Twelves, Hope Rugo, Seock-Ah Im, Kenna Anderes, Darren W Davis, Ute Hoch. Etirinotecan pegol target-specific pharmacodynamic biomarkers in circulating tumor cells from patients with metastatic breast cancer in the phase 3 BEACON study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-10-03.

Abstract Purpose: This study was designed to establish the maximum tolerated dose (MTD) and to evaluate tolerability, pharmacokinetics, and antitumor activity of etirinotecan pegol. Experimental Design: Patients with refractory solid malignancies were enrolled and assigned to escalating-dose cohorts. Patients received 1 infusion of etirinotecan pegol weekly 3 times every 4 weeks (w × 3q4w), or every 14 days (q14d), or every 21 days (q21d), with MTD as the primary end point using a standard 3 + 3 design. Results: Seventy-six patients were entered onto 3 dosing schedules (58–245 mg/m2). The MTD was 115 mg/m2 for the w × 3q4w schedule and 145 mg/m2 for both the q14d and q21d schedules. Most adverse events related to study drug were gastrointestinal disorders and were more frequent at higher doses of etirinotecan pegol. Late onset diarrhea was observed in some patients, the frequency of which generally correlated with dose density. Cholinergic diarrhea commonly seen with irinotecan treatment did not occur in patients treated with etirinotecan pegol. Etirinotecan pegol administration resulted in sustained and controlled systemic exposure to SN-38, which had a mean half-life of approximately 50 days. Overall, the pharmacokinetics of etirinotecan pegol are predictable and do not require complex dosing adjustments. Confirmed partial responses were observed in 8 patients with breast, colon, lung (small and squamous cell), bladder, cervical, and neuroendocrine cancer. Conclusion: Etirinotecan pegol showed substantial antitumor activity in patients with various solid tumors and a somewhat different safety profile compared with the irinotecan historical profile. The MTD recommended for phase II clinical trials is 145 mg/m2 q14d or q21d. Clin Cancer Res; 19(1); 268–78. ©2012 AACR.