Published OnlineFirst November 10, 2016; DOI: 10.1158/1078-0432.CCR-16-1896 Cancer Therapy: Clinical Clinical Cancer Research Phase I Study of Epigenetic Priming with Azacitidine Prior to Standard Neoadjuvant Chemotherapy for Patients with Resectable Gastric and Esophageal Adenocarcinoma: Evidence of Tumor Hypomethylation as an Indicator of Major Histopathologic Response Bryan J. Schneider1, Manish A. Shah1, Kelsey Klute1, Allyson Ocean1, Elizabeta Popa1, Nasser Altorki2, Michael Lieberman3, Andrew Schreiner4, Rhonda Yantiss4, Paul J. Christos5, Romae Palmer6, Daoqi You7, Agnes Viale7, Pouneh Kermani1, and Joseph M. Scandura1,8 Abstract Purpose: Epigenetic silencing of tumor suppressor genes (TSG) by digital droplet, bisulfite qPCR in tumor samples collected at is an acquired abnormality observed in cancer and is prototyp- baseline and at resection. ically linked to DNA methylation. We postulated that pretreat- Results: All subjects underwent complete resection of residual ment (priming) with 5-azacitidine would increase the efficacy of tumor (R0). Three of the 12 patients (25%) achieved a surgical chemotherapy by reactivating TSGs. This study was conducted to complete response and 5 had partial responses. The overall identify a tolerable dose of 5-azacitidine prior to EOX (epirubicin, response rate was 67%. The most common toxicities were gas- oxaliplatin, capecitabine) neoadjuvant chemotherapy in patients trointestinal and hematologic. Hypomethylation of biomarker with locally advanced esophageal/gastric adenocarcinoma genes was observed at all dose levels and trended with therapeutic (EGC). response. Experimental Design: Eligible patients had untreated, locally Conclusions: Neoadjuvant VEOX was well-tolerated with advanced, resectable EGC, ECOG 0–2, and adequate organ func- significant clinical and epigenetic responses, with preliminary tion. 5-Azacitidine (V, 75 mg/m2) was given subcutaneously for 3 evidence that priming with V prior to chemotherapy may (dose level, DL 1) or 5 (DL 2) days prior to each 21-day cycle of augment chemotherapy efficacy. The recommended phase II EOX (E, 50 mg/m2; O, 130 mg/m2; X, 625 mg/m2 twice daily for trial schedule is 5-azacitidine 75 mg/m2 for 5 days followed by 21 days). Standard 3þ3 methodology guided V dose escalation. EOX chemotherapy every 21 days. Clin Cancer Res; 1–8. Ó2016 DNA methylation at control and biomarker regions was measured AACR. 1Division of Hematology/Oncology, Department of Internal Medicine, Weill Introduction 2 Cornell Medical College, New York, New York. Department of Thoracic Epigenetic silencing of tumor suppressor genes (TSG) is com- Surgery, Weill Cornell Medical College, New York, New York. 3Department monly observed in gastroesophageal cancer and is believed to play of Surgery, Weill Cornell Medical College, New York, New York. 4Department of Pathology, Weill Cornell Medical College, New York, New York. 5Division of a role in oncogenesis, metastasis, and chemotherapy resistance Biostatistics and Epidemiology, Department of Healthcare Policy and (1–7). 5-Azacitidine (V, Vidaza) is a cytosine analog that acts as Research, Weill Cornell Medical College, New York, New York. 6Clinical Trials a DNA hypomethylating agent (DHA) because it cannot accept a Office, Weill Cornell Medical College, New York, New York. 7Memorial Sloan- methyl donor in the 50-position of the pyrimidine ring and 8 Kettering Cancer Center, New York, New York. Division of Regenerative depletes cellular DNA methyltransferase I (DNMT1). Reactivated Medicine, Department of Internal Medicine; Weill Cornell Medical College, expression of TSGs is commonly implicated in the clinical activity New York, New York. of DHAs, as TSGs often regulate apoptosis, DNA repair, and Note: Supplementary data for this article are available at Clinical Cancer checkpoint control. In vitro, DHAs can sensitize resistant cancer Research Online (http://clincancerres.aacrjournals.org/). cells to a variety of cytotoxic agents (8–11) including the most Prior Presentation: Poster presentation at ASCO Annual Meeting, Chicago, IL; active chemotherapies for gastroesophageal cancer (platinum May 31, 2014. agents and epirubicin; refs. 10, 12–15). Yet this approach has Corresponding Author: Bryan J. Schneider, C411 Med Inn Building, 1500 East been sparsely studied in clinical trials and has never been tested in Medical Center Drive, SPC 5848, Ann Arbor, MI 48019. Phone: 734-647-8921; Fax: esophageal/gastric adenocarcinoma (EGC). 734-647-8792; E-mail: [email protected] We hypothesized that pretreatment (i.e., priming) with a hypo- doi: 10.1158/1078-0432.CCR-16-1896 methylating agent will sensitize the adenocarcinoma to cytotoxic Ó2016 American Association for Cancer Research. therapy and increase the efficacy of neoadjuvant chemotherapy by www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 10, 2016; DOI: 10.1158/1078-0432.CCR-16-1896 Schneider et al. 1,500/mm3, platelet count 100,000/mm3), and adequate organ Translational Relevance function [serum creatinine 1.5Â the institutional upper limit of This article describes an open-label, phase I study that we normal (ULN), total bilirubin 1.5Â ULN, and aspartate ami- performed to explore the feasibility, safety, and biologic activ- notransferase (AST)/alkaline aminotransferase (ALT) 2.0Â ity of epigenetic priming with 5-azacitidine prior to neoadju- ULN]. Patients were ineligible if they had squamous cell carci- vant epirubicin, oxaliplatin, and capecitabine chemotherapy noma or cervical esophageal cancer, congestive heart failure (New in patients with potentially resectable esophageal/gastric can- York Heart Association Class II or greater), active angina pectoris, cer. Complete response is a requisite for the cure of esophageal or a myocardial infarction within 6 months or were unable to take and gastric adenocarcinoma, and this study demonstrates oral medication. The trial was approved by the Weill Cornell that it is safe to combine the epigenetic modifier azacitidine Institutional Review Board and informed consent was obtained with full-dose, neoadjuvant, cytotoxic chemotherapy as an from all patients. approach to improve complete response rates and improve survival. These findings provide the foundation for a subse- Drug and dosage administration quent phase II study to more clearly characterize the efficacy of EOX chemotherapy included epirubicin 50 mg/m2 and oxali- epigenetic priming during neoadjuvant chemotherapy. We platin 130 mg/m2 on day 1 plus capecitabine 625 mg/m2 (round- expect that this study defining the feasibility of epigenetic ed to the nearest 500 mg) orally twice daily for a 21-day cycle. priming in gastric and esophageal adenocarcinoma will trans- Patients received azacitidine (V) 75 mg/m2 by subcutaneous late to therapeutic advances in more common forms of cancer, injection daily for either 3 (DL 1) or 5 days (DL 2) and received as transcriptional silencing of TSGs by DNA hypermethylation EOX on the last day of azacitidine (Table 1). VEOX chemotherapy is seen in most, if not all, forms of cancer. was given for 3 cycles prior to resection. Intrapatient dose esca- lation/decrease of azacitidine was not allowed. Subsequent EOX doses were reduced if clinically significant day 12 (Æ2 days) neutropenia or thrombocytopenia were observed. Additional dose reductions of EOX agents were allowed for hand–foot reactivating expression of chemotherapy-sensitizing TSGs during syndrome, mucositis, diarrhea, or neurotoxicity (Supplementary the window of exposure to cytotoxic agents. We previously found Table S1). that "epigenetic priming" using a DHA prior to intensive chemo- Dose-limiting toxicities (DLT) during the cycle 1 or 2 halted therapy for acute myelogenous leukemia (AML) showed favorable neoadjuvant chemotherapy for that patient. All toxicities must activity without additive toxicity, and this approach is currently have returned to grade 1 prior to beginning the next cycle of being tested in a large, multicenter, randomized phase II study neoadjuvant chemotherapy and neutrophils and platelets much sponsored by NCI (clinicaltrials.gov NCT00538876 and have recovered to 1,500/mL and 100,000/mL, respectively. All NCT01627041; refs. 16, 17). Others have found that DHAs can patients underwent surgical resection 2 to 4 weeks from the last be safely combined with chemotherapy for solid tumor malig- cycle of neoadjuvant VEOX. Postsurgical therapy was left to the nancies, although experience is quite limited (18–22). We con- discretion of the treating clinician. Azacitidine (V) was supplied ducted an open-label phase I evaluation of the feasibility, safety, by Celgene & Co., Inc. in 100-mg vials. and biologic activity of epigenetic priming using the hypomethy- lating agent 5-azacitidine prior to full-dose, neoadjuvant epiru- Trial design bicin, oxaliplatin, and capecitabine (EOX) chemotherapy in Astandard3þ3 phase I strategy was used to escalate the patients with surgically resectable EGC (clinicaltrials.gov azacitidine dose level if the clinical toxicity was acceptable. NCT01386346). Tumor DNA hypomethylation was analyzed in Toxicities were graded according to the NCI Common Toxicity samples obtained before and following azacitidine treatment as a Criteria version 4.0 (23, 24). DLTs during the first cycle were pharmacodynamic biomarker of epigenetic response. defined as grade 3 nonhematologic toxicity (exceptions noted below), grade 4 neutropenia lasting