A Phase 1 Study of Azacitidine (AZA) in Combination with Fludarabine and Cytarabine in Relapse/Refractory Childhood Leukemia: A Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Study
Authors: Sun Weili , Gaynon Paul S , Sposto Richard , Bittencourt Henrique , Place Andrew E. , Messinger Yoav H , Fraser Chris , Dalla-Pozza Luciano , van der Giessen Jeannette , Eckroth Elena , Yang Xiaojing , Liang Gangning , Jones Peter A. , Wayne Alan S. , Cooper Todd 
Published: October 2019

Abstract
Background: Growing evidence indicates that aberrant DNA hypermethylation is associated with leukemogenesis, drug resistance, and relapse. It has been shown that pre-treating leukemia cells with AZA or decitabine could partially reverse the aberrant DNA methylation, restore the expression of tumor suppressor genes, and sensitize cells to chemotherapeutic agents.
Study design: TACL2011-002 was a phase I study with an expansion cohort, examining the use of AZA in sequence with chemotherapy in pediatric leukemia. The objectives were 1) to evaluate the feasibility and toxicity profile of this combination; 2) to obtain preliminary response data in patients with acute myeloblastic leukemia (AML); 3) to study the extent of hypomethylation pre- and post-AZA therapy. Children 1-21 years of age with relapsed/refractory (R/R) leukemia were eligible for the phase I portion. A rolling six design was employed followed by an expansion to 12 response evaluable AML patients at the selected dose. Patients received AZA at a dose of 75mg/m2/day (dose level 1, DL1) subcutaneously on days 1-5, followed by intravenous (IV) fludarabine (30mg/m2/dose daily) and IV cytarabine (2gm/m2/dose daily) on days 6-10. If < 2 patients experienced dose-limiting toxicity (DLT), then DL1 would be the expansion cohort dose. If ≥ 2 patients experienced DLT, the dose of AZA was to be reduced to 50mg/m2/day (DL0). Patients were to receive up to 2 courses of therapy. DNA methylation was evaluated at days 0, 5 and the end of course 1 using PCR and the Infinium HumanMethylation450 assay.
Results: Fifteen patients were enrolled, 13 with AML and 2 with ALL. Fourteen patients were evaluable for toxicity and response. One AML patient was removed from study after day 3 due to central nervous system treatment needs and was replaced. The median number of prior treatment regimens was 2 (range 1-5). 5/12 patients with AML had prior hematopoietic stem cell transplant (HSCT) while none with ALL had prior HSCT. Toxicities were typical of intensive chemotherapy regimens. Non-hematologic toxicities ≥ grade 3 attributed to AZA included: febrile neutropenia (n=6), infections (n=3), AST elevation (n=1), oral hemorrhage (n=1), hypokalemia (n=1). No patients experienced DLT. 7/12 AML patients had complete response (CR) or CRi (CR with incomplete count recovery), including 3/5 that had received prior HSCT. 1/2 ALL patients had partial response (PR). DNA methylation analysis was performed in 4 patients to date. Compared to baseline, all patients had DNA demethylation by G-LINE analysis on day 5. At the end of course 1, DNA demethylation persisted in 2 patients who achieved CR, wheras no demethylation were detected in the other 2 patients who were non-responders.
Conclusions: AZA at 75mg/m2/day plus fludarabine and cytarabine is well tolerated in pediatric patients with relapsed leukemia. The favorable toxicity profile and encouraging response rates in AML warrants further testing of AZA in this patient population. Methylation analysis is ongoing to assess the possible correlation between DNA demethylation and response.
Table Response AML ALL Overall Patient evaluable for response 12 2 14 CR 6 6 CRi 1 1 PR 1 1 2 SD* 1 1 2 PD# 3 3
*SD = Stable Disease; #PD = Progression of Disease
ClinicalTrials.gov Identifier: NCT01861002
This study was supported by Gateway for Cancer Research

Disclosures
Off Label Use: Azacitidine is not approved by FDA to treat childhood leukemia.