A Phase I Study of Quizartinib Combined with Chemotherapy in Relapsed Childhood Leukemia: a Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Study Todd M

Published OnlineFirst February 26, 2016; DOI: 10.1158/1078-0432.CCR-15-1998

Cancer Therapy: Clinical Clinical Cancer Research A Phase I Study of Quizartinib Combined with Chemotherapy in Relapsed Childhood Leukemia: A Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Study Todd M. Cooper1, Jeannette Cassar2, Elena Eckroth2, Jemily Malvar2, Richard Sposto2,3, Paul Gaynon2,3, Bill H. Chang4, Lia Gore5, Keith August6, Jessica A. Pollard7, Steven G. DuBois8, Lewis B. Silverman8, Javier Oesterheld9, Guy Gammon10, Daniel Magoon11, Colleen Annesley11, and Patrick A. Brown11

Abstract

Purpose: To determine a safe and biologically active dose of 60 mg/m2/day. Of 17 response evaluable patients, 2 had complete quizartinib (AC220), a potent and selective class III receptor response (CR), 1 complete response without platelet recovery tyrosine kinase (RTK) FLT3 inhibitor, in combination with salvage (CRp), 1 complete response with incomplete neutrophil and chemotherapy in children with relapsed acute leukemia. platelet recovery (CRi), 10 stable disease (SD), and 3 progressive Experimental Design: Quizartinib was administered orally to disease (PD). Of 7 FLT3-ITD patients, 1 achieved CR, 1 CRp, 1 Cri, children with relapsed AML or MLL-rearranged ALL following 5 and 4 SD. FLT3-ITD patients, but not FLT3 wild-type (WT) days of high-dose cytarabine and etoposide (AE). A 3þ3 dose patients, had signiﬁcantly lower blast counts post-quizartinib. escalation design was used to identify a safe and biologically FLT3 phosphorylation was completely inhibited in all patients. active dose. Plasma inhibitory assay (PIA) testing was performed Conclusions: Quizartinib plus intensive chemotherapy is well weekly to determine biologic activity. tolerated at 60 mg/m2/day with near complete inhibition of FLT3 Results: Toxicities were consistent with intensive relapsed phosphorylation in all patients. The favorable toxicity proﬁle, leukemia regimens. One of 6 patients experienced a dose-lim- pharmacodynamic activity, and encouraging response rates war- iting toxicity (DLT) at 40 mg/m2/day (elevated lipase) and 1 of rant further testing of quizartinib in children with FLT3-ITD AML. 9 had a DLT (hyperbilirubinemia) at the highest tested dose of Clin Cancer Res; 1–9. 2016 AACR.

Introduction is internal tandem duplication (ITD) on exon 14 of the FLT3 gene, resulting in constitutive activation and autophosphorylation of FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine FLT3 (1, 2). In both adults and children with AML, FLT3-ITD kinase that dimerizes and autophosphorylates when bound by mutations with high allelic burden (ratio > 0.4) confer a dismal FLT3 ligand, activating downstream pathways that induce cell prognosis (3–6). Therefore, the current standard of care for growth and inhibit apoptosis. The most common FLT3 mutation children with FLT3-ITD mutations is intensive induction chemotherapy followed by hematopoietic stem cell transplantation (HSCT). High levels of FLT3 wild-type receptor (FLT3-WT) also 1Cancer and Blood Disorders Center, Seattle Children's Hospital, Seat- tle, Washington. 2Children's Center for Cancer and Blood Diseases, promote constitutive activation of the FLT3 receptor and carry Children's Hospital of Los Angeles, Los Angeles, California. 3Keck poor prognosis similar to those with FLT3-ITD mutations (7, 8). School of Medicine, University of Southern California, Los Angeles, Among children with AML, about 30 percent have FLT3 disease: California. 4Doernbecher Children's Hospital,Oregon Health & Science University, Portland, Oregon. 5Children's Hospital of Colorado, Aurora, FLT3-ITD mutations (15%), kinase domain (KD) mutations Colorado. 6Children's Mercy Hospital and Clinics, Kansas City, Missouri. (5%), or overexpression of FLT3-WT (10%; refs. 6, 9). In addition, 7Maine Children's Cancer Program, Scarborough, Maine. 8Dana Farber c-KIT is a receptor tyrosine kinase that is commonly overexpressed Cancer Institute/Boston Children's Hospital, Boston, Massachusetts. 9Levine Children's Hospital, Charlotte, North Carolina. 10Clinical or mutated in childhood AML (19%) and serves as a potential Research and Development, Ambit Biosciences Corporation, San target for therapy (10). In children with acute lymphoblastic Diego, California. 11Johns Hopkins University School of Medicine, Bal- leukemia (ALL), the highest levels of FLT3-WT expression occur timore, Maryland. in MLL-rearranged (MLL-r) patients, which account for 80% of Note: Supplementary data for this article are available at Clinical Cancer infant and 5% of childhood ALL, and those with hyperdiploidy, Research Online (http://clincancerres.aacrjournals.org/). which account for 30% of childhood ALL (11–13). Corresponding Author: Todd M. Cooper, Cancer and Blood Disorders Center, First-generation FLT3 inhibitors were developed for the treat- Seattle Children's Hospital, 4800 Sand Point Way NE, M/S MB.8.501, Seattle, WA ment of solid tumors, demonstrating activity against a variety of 98105. Phone: 206-987-1533; Fax 206-987-3946; kinases including RAF kinase, PDGFR, VEGFR, c-KIT, and FLT3. [email protected] These less selective inhibitors demonstrated toxicity consistent doi: 10.1158/1078-0432.CCR-15-1998 with their off-target effects (14–16). In children, the ﬁrst-gener- 2016 American Association for Cancer Research. ation FLT3 inhibitors lestaurtinib, midostaurin, and sorafenib

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Translational Relevance Schwartz formula), cardiac (echocardiogram with shortening fraction 27%), and adequate performance status (Karnofsky Children with acute myeloid leukemia and internal tandem or Lansky >50%). Exclusion criteria included uncontrolled duplication mutations in the FLT3 receptor tyrosine kinase systemic infection, significant cardiovascular disease, and active have a poor prognosis despite intensive chemotherapy and central nervous system involvement (CNS3). Children with hematopoietic stem cell transplantation. FLT3 inhibition prolonged QT, using the Fridericia's formula to correct for represents a promising therapeutic strategy for improving heart rate (QTcF 450 ms) at study entry were not eligible. survival in this high-risk patient population. Clinical testing Institutional review boards at participating centers approved of first-generation FLT3 inhibitors in children has been limited the study and participating patients or their parents signed by off-target toxicity and an inability to achieve sustained FLT3 written informed consent. The original clinical trial was regis- inhibition. Comprehensive evaluation of FLT3 inhibitors tered at www.clinicaltrials.gov as NCT01411267. requires an agent that is sufficiently potent and selective and a pharmacodynamic assay that measures target inhibition. Study design Here we report the phase I clinical trial of quizartinib, a Treatment schedule. Eligible patients received cytarabine (1 g/m2/ second-generation FLT3 inhibitor, in combination with inten- dose i.v. every 12 hours) and etoposide (150 mg/m2/dose i.v. sive chemotherapy for children relapsed acute leukemia. We once daily) on days 1–5. Quizartinib was administered orally determined that quizartinib is safe, provides sustained phos- once daily on days 7–28. Patients were to receive up to 2 cycles of pho-FLT3 inhibition, and demonstrates promising activity in protocol therapy. Patients who achieved a complete response children with FLT3-ITD mutations. This trial may represent a (CR) or complete response without platelet recovery (CRp) after significant step in testing FLT3 inhibition as a therapeutic the second course of therapy could exit the study to pursue modality in children. alternative treatment or were eligible to continue single-agent quizartinib as continuation therapy.

Dose selection. Adult phase I studies determined the MTD to be have been studied most extensively. Quizartinib (AC220) is a 200 mg fixed daily dosing, with grade 3 QTcF prolongation second-generation FLT3 inhibitor demonstrating superior poten- proving dose limiting (19). However, PIA testing demonstrated cy and selectivity in preclinical models when compared with first- biologic activity via complete inhibition of phospho-FLT3 in a generation inhibitors. In addition to nanomolar potency against limited number of FLT3-ITD patients at 18 mg daily and in a larger FLT3-ITD mutations, quizartinib also demonstrates preclinical cohort of FLT3-ITD patients at 60 mg daily. Data from subsequent activity against FLT3-WT and c-KIT (17, 18). Clinical trials in phase II studies have indicated that the optimal dose in adults adults with relapsed or refractory AML demonstrate clinical activ- balancing activity and reduced QT prolongation is 60 mg; how- ity, manageable toxicity, and utility as a bridge to HSCT. The adult ever, adult phase I studies available during the development of phase I single-agent clinical trial demonstrated responses in non- this study demonstrated responses at 40 mg (20). In addition to FLT3-ITD AML, leading investigators to hypothesize that quizar- dosing data described above, this clinical trial was the first to tinib may have clinical activity against overexpression of FLT3-WT combine quizartinib with intensive chemotherapy despite not or c-KIT (19). having single-agent pediatric toxicity data. Therefore, we selected This is a first-in-child phase I study of quizartinib performed 25 mg/m2/daily as dose level one (DL1). This is roughly equiv- in the Therapeutic Advances in Childhood Leukemia/Lympho- alent to 40 mg fixed dosing using the average adult body surface ma Consortium (TACL). The primary objective of this study area of 1.73 m2. was to determine a safe and biologically active dose of quizartinib given in sequential combination with cytarabine and Statistical design. A3þ3 design (21) escalated doses of quizartinib etoposide (AE) in young patients with relapsed or refractory in approximately 50% dose increments beginning with 25 AML or MLL-r ALL. mg/m2/once daily. Blood from each patient was assayed for 2 Materials and Methods biologic activity using PIA testing. If DL1, DL2 (40 mg/m /day), and DL3 (60 mg/m2/day) were tolerated, escalation would stop Patients for assessment of quizartinib PIA. Additional patients would be The trial was open to accrual between September 2011 and accrued at DL3 to ensure that PIA was assessable in at least 9 March 2013. Data for all patients were current as of March patients; if 7 of 9 patients achieved PIA of >90% at 3 of 4 trough 2015. Patients were required to be between >1monthand21 time points, DL3 would be considered biologically active and years of age. Eligible patients were required to have ALL in escalation would stop. If not, dose escalation would proceed to first relapse (>25% bone marrow blasts) with MLL-r or hyper- DL4 (90 mg/m2/day) and DL5 (130 mg/m2/day). Secondary diploidy or to have AML in first relapse with 5% bone endpoints included the achievement of CR, determination of marrow blasts. Children with relapsed AML were included FLT3 PIA levels, and in vitro sensitivity testing. Two-way ANOVA regardless of FLT3-ITD status due to quizartinib activity against was utilized to investigate differences in marrow blasts inhibition overexpression of WT-FLT3, c-KIT overexpression and muta- among the patient groups as a result of increasing doses of tions, single-agent clinical activity in adult FLT3-WT patients, quizartinib. The Spearman rank correlation was used to assess and the combination of intensive AML reinduction therapy the relationship between the quizartinib in vitro sensitivity and used in this study. Other requirements included adequate liver change in bone marrow blasts on study. Change in bone marrow [serum bilirubin <1.5 times upper limit of normal (ULN) for blast count was evaluated using a paired t test. Unless otherwise age, ALT < 5 times ULN for age], renal (derived from the stated, P values refer to two-sided tests.

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Toxicity evaluation. Dose-limiting toxicity (DLT) was assessed in the absolute number of bone marrow or circulating leukemic the first course only. DLT was defined as a serious adverse event at blasts, development of extramedullary disease, or other labora- least possibly attributable to quizartinib. The DLT definition was tory or clinical evidence of progression. consistent with published principles adopted for Children's Oncology Group (COG) relapsed acute leukemia clinical trials PIA that combine molecularly targeted drugs with intensive chemo- Detailed procedures have been published previously (24). The therapy. In general, the guidelines incorporate tolerance of resolv- TF/ITD cell line was originally generated in the Small laboratory as ing grade 3 and some resolving grade 4 toxicities with stringent described (23). Low passage aliquots have been maintained in the safety monitoring (22). On the basis of prior COG studies Brown laboratory. The cells are authenticated every 6 months (last utilizing cytarabine/etoposide in AML, nonhematologic toxicities March 2015) using RT-PCR for FLT3 juxtamembrane domain and occurring in >10% of patients were either excluded (anorexia/ sequencing of the PCR products to confirm expression of the alopecia) or required resolution with supportive care within a original FLT3 ITD construct. Peripheral blood was drawn from specified period of time (nausea/vomiting/diarrhea/metabolic patients before chemotherapy, before and 2 hours after the first abnormalities). Attributions were made by individual investiga- dose of quizartinib on day 7 and at trough time points on days 14, tors and reviewed by the study committee. Hematologic DLT was 21, and 28. Plasma was isolated from peripheral blood by defined as failure to recover a peripheral ANC > 500/mL and centrifugation. For each time point, 5 106 TF/ITD cells were nontransfusion–dependent platelet count > 20,000/mL due to incubated with 500 mL of plasma for 1 hour. Clarified lysate was documented bone marrow aplasia/hypoplasia (as opposed to immunoprecipitated with anti-FLT3 antibody, electrophoresed malignant infiltration or other cause) for greater than or equal to with SDS-PAGE, and blotted with anti-phospho-tyrosine anti- 56 days, which is approximately 2 SDs greater than the average body, then stripped and reprobed with anti-FLT3 antibody. Pro- duration of neutropenia in the aforementioned cytarabine/etopo- teins were visualized by chemiluminescence and densitometric side regimens. analysis was performed.

Evaluable population for toxicity and DLT. Any patient who FLT3 genotyping received at least one dose of study drug was included in the Detailed procedures are published (25). RNA was isolated from toxicity tabulations. Any patient that experienced a DLT as defined baseline bone marrow or peripheral blood blasts enriched by in the protocol only needed to have received one dose to be Ficoll centrifugation. Contaminating germline DNA (gDNA) was evaluable for toxicity. Otherwise, patients must have received at removed from all samples by a DNase1 digestion, and 1 mg cDNA least 75% of the prescribed doses of quizartinib (i.e., at least 17 of was synthesized. The juxtamembrane domain was amplified and the prescribed 22 doses of quizartinib) on study to be evaluable gel electrophoresed, with the presence of an additional, higher for DLT. Patients who were not evaluable for DLT were replaced. molecular weight band indicating a FLT3/ITD. The kinase domain was amplified and digested with EcoRV. Pre- and postdigestion Evaluable population for response. A patient was considered evalu- samples were gel electrophoresed, with a double banded profile able for response if the patient received all or part of protocol indicating the presence of a point mutation. PCR products were therapy, and either (i) met the definition of progressive disease at sent to sequencing for confirmation. any time, or (ii) had bone marrow collected and was under follow-up for a sufficient period to evaluate their disease at the FLT3 expression by qPCR end of course 1. Deaths due to toxicity after receiving all or part of RNA was isolated using the RNeasy kit (Qiagen). All samples protocol therapy were considered as nonresponders. were treated with DNase1 to remove contaminating gDNA. One microgram of RNA was used to synthesize cDNA using the Verso Safety assessments cDNA synthesis kit (Thermo Scientific). Quantitative real-time Adverse events were graded using the NCI Common Toxicity PCR was performed using SYBR green and primers JMD-F (50- Criteria (CTCAE), version 4.0. Events were collected beginning TGTCGAGCAGTACTCTAAACA-30) and JMD-R (50-ATCCTAG- with the first dose of study therapy until 30 days following TACCTTCCCAAACTC-30) with GAPDH as a control. Samples removal from protocol therapy, and investigators evaluated each for severity and relationship to quizartinib and cytarabine/etopo- Table 1. Patient characteristics side. Electrocardiograms (ECG) were performed at screening, and Total evaluable patients ALL (N ¼ 4) AML (N ¼ 18) on days 7, 14, 21, 28 prior to quizartinib dosing and 2 and 4 hours Age (years) at enrollment postdose. Each ECG was performed in triplicate, reviewed locally, Median 2.8 yrs 13.1 yrs and transmitted for central review. Range 11 mo–19 yrs 1.8–21 yrs Gender Treatment response Male 1 7 Bone marrow aspirates and/or biopsies were performed at Female 3 11 baseline and on day 29 of each course. If the marrow was #Prior therapies hypoplastic and peripheral blood counts had not recovered to Median 2 3 m m Range 1–10 1–5 ANC 1,000/ L and platelets 100,000/ L in the absence of Prior HSCT leukemia, marrow testing was repeated not less than once every 14 No 4 7 days. Responses were defined per standard criteria and included Yes 0 11 CR, CRp, complete response with incomplete hematologic recov- FLT3/ITDþ ery (CRi), stable disease (SD), and progressive disease (PD; No – 9 – ref. 23). Of note, PD was defined as increase of at least 25% of Yes 9

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Table 2. Adverse event grade 3 attributable to quizartinib or cytarabine/etoposide, all doses combined for those evaluable for DLT (N ¼ 18) Attribution Quizartinib Regimen Category Name of toxicity Baseline only only Both Neither Total Blood and lymphatic system disorders Anemia 2 . 8 5 1 16 Febrile neutropenia . . 6 2 1 9 Eye disorders Eye disorders - Other, specify 1 . . . . 1 Vitreous hemorrhage 1 . . . . 1 Gastrointestinal disorders Constipation . 1 . . . 1 Diarrhea . 1 . . . 1 Enterocolitis . . . 1 . 1 Flatulence . . . . 1 1 Mucositis oral . . 2 . . 2 Nausea . . . . 1 1 Pancreatitis . . 1 . . 1 Rectal pain . . . . 2 2 Vomiting . 1 . . . 1 General disorders and administration Pain . . 1 . . 1 site conditions Immune system disorders Anaphylaxis . . . . 1 1 Infections and infestations Catheter related infection . . 1 1 . 2 Device related infection . . 1 . . 1 Infections and infestations - Other, specify . . 4 . . 4 Lung infection . . 2 1 . 3 Sepsis . . 1 . . 1 Skin infection . . 1 . . 1 Investigations Activated partial thromboplastin time prolonged . . . . 1 1 Alanine aminotransferase increased . . . 1 . 1 Alkaline phosphatase increased . . . . 1 1 Aspartate aminotransferase increased . . . 2 1 3 Blood bilirubin increased . 1 . . . 1 Creatinine increased . . . . 1 1 Electrocardiogram QT corrected interval prolonged . . . 1 . 1 Lipase increased . . . 1 . 1 Lymphocyte count decreased 3 . 7 4 1 15 Neutrophil count decreased 9 . 6 3 . 18 Platelet count decreased 4 . 8 5 1 18 White blood cell decreased 4 . 9 5 . 18 Metabolism and nutrition disorders Acidosis . . . . 1 1 Anorexia 3 . . 3 . 6 Hyperglycemia . . 2 . 3 5 Hypermagnesemia . . . . 1 1 Hypoalbuminemia . . 1 . . 1 Hypocalcemia . . 1 . 1 2 Hypokalemia 1 . 4 . 2 7 Hyponatremia . . 1 . . 1 Hypophosphatemia . 1 1 . 2 4 Tumor lysis syndrome . . 1 . . 1 Musculoskeletal and connective tissue disorders Pain in extremity . . . . 1 1 Nervous system disorders Headache . . . 1 1 2 Respiratory, thoracic and mediastinal disorders Apnea 1 . . . . 1 Hypoxia 1 . . . 1 2 Respiratory failure . . . . 1 1 Sore throat . . 1 . . 1 Skin and subcutaneous tissue disorders Rash maculo-papular . . 1 . 2 3 Vascular disorders Hypotension 1 . 1 . 1 3 conditions Fever . . 1 . . 1 31 5 73 36 31 176

were run in duplicate on a CFX96 Real-Time instrument (Bio- blood cells were lysed using a red blood cell lysis buffer (0.155 Rad) and the results were analyzed on the Bio-Rad CFX96 mol/L NH4Cl, 0.01 mol/L KHCO3, 0.1 mmol/L EDTA) and Manager. washed with PBS. Cells were plated with increasing doses of quizartinib (0, 5, 10, 20, and 50 nmol/L) with DMSO control Quizartinib in vitro sensitivity and maintained for 48 hours at 37 C þ 5% CO2 in AIM-V media Bone marrow or peripheral blood blasts were enriched using (Life Technologies) supplemented with 20% FBS (Gemini Bio- density centrifugation (Ficoll-Paque Plus; GE Healthcare) from sciences), 1% penicillin–streptomycin and 1% L-glutamine (Life baseline bone marrow aspirates or whole blood. Remaining red Technologies). WST-1 (Roche) was added to each well and

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Table 3. Adverse event grade 3 attributable to quizartinib or cytarabine/etoposide, all doses combined for those not evaluable for DLT (N ¼ 4) Attribution Quizartinib Regimen Category Name of toxicity Baseline only only Neither Total Blood and lymphatic system disorders Anemia 1 . 2 . 3 Febrile neutropenia . . 1 1 2 General disorders and administration site conditions Pain . . . 1 1 Infections and infestations Infections and infestations - Other, specify . . 1 1 2 Skin infection . . 1 . 1 Soft tissue infection . . 1 . 1 Investigations Alanine aminotransferase increased . . . 1 1 Aspartate aminotransferase increased . . . 1 1 Blood bilirubin increased . . . 1 1 CPK increased . . . 1 1 Lymphocyte count decreased 2 . 1 . 3 Neutrophil count decreased 2 . 1 . 3 Platelet count decreased 2 . . 1 3 White blood cell decreased . . 2 . 2 Metabolism and nutrition disorders Anorexia . . . 1 1 Hyperglycemia . . . 1 1 Hypoalbuminemia . . . 1 1 Hypocalcemia . . . 2 2 Hypokalemia . . 1 3 4 Hypophosphatemia 1 . . 1 2 Musculoskeletal and connective tissue disorders Bone pain . . . 1 1 Pain in extremity . . . 1 1 Respiratory, thoracic and mediastinal disorders Dyspnea . . . 1 1 Hypoxia . . . 2 2 Pleural effusion . . . 1 1 Pulmonary edema . . . 1 1 Respiratory failure . . . 1 1 Vascular disorders Hypertension . . . 1 1 Hypotension . . . 1 1 Conditions Fever 1 . . 1 2 9 . 11 28 48 incubated for an additional 30 minutes and read within 4 hours to quizartinib and chemotherapy for evaluable and inevaluable on a Bio-Rad 680 Microplate Reader. Inhibition was plotted as a patients are listed in Tables 2 and 3, respectively. A dose-depen- percentage of the untreated control. dent increase in QTcF was observed (Supplementary Table S2). One patient experienced grade 3 QTcF prolongation, which Results occurred on the last day of quizartinib administration. This was not considered a DLT because it quickly resolved and did not limit Patient characteristics further doses of quizartinib. Twenty-two eligible patients were enrolled (Table 1). The median age was significantly lower in ALL patients, reflecting that Response 3 of 4 patients had relapsed MLL-r infant leukemia. Of 18 AML Seventeen patients were evaluable for response: 3 had ALL, 7 patients, 9 were FLT3-ITD and 9 were FLT3-WT. had FLT3-WT AML, and 7 had FLT3-ITD AML (Table 4). Five patients were not evaluable for response because they were Toxicity removed from protocol therapy prior to disease assessment with- Eighteen patients were evaluable for toxicity. Four patients were out meeting PD criteria (Supplementary Table S3). Of those not evaluable for toxicity according to protocol definition and evaluable for response, 2 achieved CR, 1 CRi, 1 CRp, 10 SD, and were replaced (Supplementary Table S1). Toxicities were consis- 3 had PD. Responses in the 7 evaluable FLT3-ITD AML patients tent with intensive relapsed leukemia regimens. None of 3 included 1 CR, 1 CRp, 1 CRi, and 4 SD. Patients with FLT3-ITD patients at DL1 experienced a DLT. One of 6 patients at DL2 AML demonstrated marked reduction in bone marrow blast experienced a DLT. This was a 19-year-old female with relapsed counts after protocol therapy. This was not true of FLT3-WT or MLL-r ALL and a history of prior asparaginase-related pancreatitis who experienced grade 3 lipase elevation on study. One of 9 Table 4. Response patients at DL3 experienced a DLT. This 19-year-old female with Evaluable (not evaluable) Overall ALL AML FLT3-WT AML FLT3-ITD FLT3-ITD AML experienced grade 4 hyperbilirubinemia on day for response 17 (5) 3 (1) 7 (2) 7 (2) 12, and quizartinib was stopped on day 15 due to investigator Overall response decision. Attribution by the treating physician was documented as CR 2 – 11 possibly related to quizartinib. The event did not resolve prior to CRi 1 –– 1 the patient's death, which was due to overwhelming aspergillosis CRp 1 1 and multi-organ failure occurring 12 days after the last dose of SD 10 1 5 4 PD 3 2 1 – quizartinib and 8 days after study removal. Toxicities attributable

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Figure 1. A, bone marrow blasts before and after treatment in FLT3/ITD AML patients. B, bone marrow blasts before and after treatment in FLT3 wild-type (WT) AML (solid lines) and ALL (dotted lines) patients. P values from paired t test.

MLL-r ALL patients (Fig. 1). Of the 10 patients with SD, 4 were and 3 ALL). To be considered sufficient, samples were required to FLT3-ITD AML, 5 were FLT3-WT AML and 1 was MLL-r ALL. The contain at least 1 106 viable leukemic blasts by Trypan blue relative percent change in bone marrow blast percentage was exclusion and morphologic examination. As shown in Fig. 3A, a strikingly different in the 4 FLT3-ITD AML patients with SD (mean striking difference in IVS was demonstrable, with FLT3-ITD sam- reduction 86%) compared with the 5 others with SD (mean ples demonstrating a clear dose-dependent cytotoxic effect at the increase of 5%, P ¼ 0.002). Three FLT3-ITD patients (1 CR, 1 0—20 nmol/L concentration levels at which quizartinib is pri- CRp, 1 SD) and 1 FLT3-WT (CR) received HSCT after protocol marily active against FLT3, and below the concentrations at which therapy. Because of small sample size, and in the context of the more nonspecific kinase inhibition or other off-target effects phase I study, we are not able to draw statistically significant would be expected. The FLT3-WT AML samples showed a more conclusions regarding overall survival. Supplementary Table S4 modest cytotoxic effect and the MLL-r ALL samples showed no illustrates a comparison of 1-year survival between evaluable cytotoxic effect (Fig. 3; Supplementary Table S5; P < 0.005 FLT3-WT and FLT3-ITD AML patients. comparing the 3 groups). Of the 15 patients with IVS data, 10 also had bone marrow blast counts available before and after PIA therapy. As shown in Fig. 3 B, there was a statistically significant PIA FLT3 inhibition was nearly complete at all patients at all positive correlation (Spearman rank correlation coefficient r ¼ dose levels (Fig. 2). In 19 patients with PIA assessment, 9 had 0.7, P ¼ 0.04) between IVS and change in bone marrow blast – 100% inhibition, 9 had 97% 99% inhibition, and 1 had 94% percentage, suggesting that the IVS assay has the potential to serve 2 inhibition (Supplementary Table S5). As DL3 (60 mg/m ) had 1 as a biomarker of clinical response to quizartinib. of 9 patients with DLT and 9 of 9 patients with >90% inhibition by PIA, this dose level was defined as the recommended phase II dose. Discussion This clinical trial demonstrated that quizartinib in combination FLT3 genotyping and quantitative expression of FLT3 mRNA with intensive AML chemotherapy is safe and biologically active at FLT3 genotyping was confirmed in 20 of 22 patients for whom a dose of 60 mg/m2 given once daily. The unique design selected a marrow at study entry was submitted (Supplementary Fig. S1). RP2 dose (RP2D)-based on both safety and pharmacodynamic The FLT3 genotype of 2 patients was based on reports from endpoints. There was no dose escalation past the 60 mg/m2 dose outside clinical laboratories. The size of the FLT3-ITD insertions based upon adult phase I/II data demonstrating complete ranged from 18 to 105 base pairs, and the mutant:wild-type allelic responses at doses as low as 40 mg, potent inhibition of FLT3 ratio ranged from 0.62 to 2.82. One patient had no detectable phosphorylation at doses as low as 12 mg, and dose-dependent wild-type FLT3, indicating loss of heterozygosity (LOH) for FLT3- prolongation of QTcF (19). The current adult phase III study of ITD (Patient 10, Supplementary Table S5). No patients had quizartinib for FLT3/ITD AML starts all patients at 30 mg fixed detectable D835 point mutations. Quantitative expression of dosing, escalating to 60 mg fixed dosing based on QTcF evaluation FLT3 transcript was measured using qPCR (Supplementary Table (www.clinicaltrials.gov identifier NCT02039726). Despite inten- S5; Supplementary Fig. S2), demonstrating a high degree of sive ECG monitoring, our study only demonstrated only one interpatient variability in the AML patients, particularly in the transient grade 3 elevation of QTcF that was not dose limiting. FLT3 WT cohort. Although there was a dose-dependent increase in QTcF from baseline (Supplementary Table S2), 60 mg/m2 daily dosing did In vitro sensitivity not restrict study drug administration in any patients. Our results Of the 22 eligible patients enrolled, sufficient material to support further study of quizartinib in children with FLT3/ITD perform 48-hour in vitro sensitivity (IVS) testing was available mutations at 60 mg/m2 once daily dosing. However, our PD for 15 patients at study entry (7 FLT3-ITD AML, 5 FLT3-WT AML, testing demonstrates FLT3 inhibition at lower doses and it is

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DL1 (25 mg/m2/day) 1 2 3 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28

pFLT3

tFLT3

DL2 (40 mg/m2/day) 4 5 7 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28

pFLT3

tFLT3

8 9 10 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28

pFLT3

tFLT3

DL3 (60 mg/m2/day) 11 12 13 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28 Pre D7H0 D7H2 D14 D21 D28

pFLT3

tFLT3

Figure 2. PIA representing 12 patients treated during the dose-finding phase of the study. Pre, before chemotherapy; D7H0 ¼ before first dose of quizartinib; D7H2 ¼ 2 hours after first dose of quizartinib; D14, D21, and D28 ¼ trough on indicated day.

possible that future pediatric clinical trials may mimic adult removed because of infection without having received 75% of quizartinib development and study its efficacy at lower doses. prescribed quizartinib therapy. We recognize that those with This trial was the first in adults or children to give quizartinib in detectable peripheral blasts clearly did not respond to quizartinib, combination with intensive chemotherapy. This strategy but were not evaluable because they did not meet our protocol addressed the inherent difficulty in enrolling children with definition of progressive disease. Rather than retrospectively relapsed leukemia onto single-agent phase I studies while also change our protocol definitions for evaluability, we detail the providing the vital combination data required to incorporate circumstances surrounding all patients not evaluable for toxicity quizartinib into de novo AML therapy. Importantly, we demon- and response in Supplementary Tables S1 and S3. Of interest, of strated that adding quizartinib to intensive chemotherapy does those that were removed from protocol therapy for persistence of not result in detectable additive toxicity. Infection, myelosuppres- peripheral blasts on therapy, none harbored FLT3-ITD mutations. sion, and febrile neutropenia were common and consistent with Not only did FLT3-ITD patients have a higher overall response rate intensive AML regimens, but there were no hematologic DLTs and (ORR; ORR¼CRþCRpþCRi) compared with non-ITD patients no indication that the combination increased the severity of these (43% vs. 14%) but FLT3-ITD AML patients with SD clearly toxicities. demonstrated improvement in blast count compared with FLT3-ITD patients clearly demonstrated superior bone marrow FLT3-WT AML and ALL patients with SD. There was one CR responses compared with those with FLT3-WT AML and MLL-r among the FLT3 WT patients (patient 9 in Supplementary Table ALL (Fig. 1). Of the 5 patients not evaluable for response, 3 were S5). Interestingly, this patient had the highest FLT3 expression by removed from protocol therapy due to persistence of leukemia qPCR of the evaluable patients, and demonstrated a high degree detected in the peripheral blood. The other 2 patients were of in vitro sensitivity to quizartinib. Unfortunately, there did not

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Cooper et al.

Figure 3. A, quizartinib in vitro sensitivity (IVS) by diagnosis and FLT3 genotype. Data points represent mean SE for the indicated group. B, Spearman rank correlation of IVS and change in bone marrow blasts before versus after treatment. OD, optical density; IVS, in vitro sensitivity.

appear to be a response signal in the small number (n ¼ 3) of results provide strong support for further clinical testing of qui- evaluable MLL-r ALL patients. While this subset of patients was zartinib in children with FLT3-ITD–mutated AML. included in the trial due to expected overexpression of WT FLT3 and sensitivity to FLT3 TKI, these 3 individual patients actually Disclosure of Potential Conflicts of Interest expressed relatively low levels of FLT3 by qPCR (Supplementary P. Gaynon reports receiving speakers bureau honoraria from JAZZ and Sigma Fig. S2), a finding associated with low level dependence on FLT3 Tau, and is a consultant/advisory board member for JAZZ. G. Gammon is an fl signaling and modest FLT3 TKI-induced cytotoxicity in prior employee of Daiichi Sankyo. No potential con icts of interest were disclosed by the other authors. preclinical studies (12, 13). Importantly, testing of these 3 sam- fi in vitro ples con rmed a lack of sensitivity to quizartinib (Fig. 3). Authors' Contributions Full assessment of efficacy of FLT3 TKI in MLL-r ALL and the Conception and design: T.M. Cooper, R. Sposto, P. Gaynon, L. Gore, potential of FLT3 expression level to serve as a biomarker of G. Gammon, P.A. Brown response will require larger studies. Development of methodology: T.M. Cooper, P. Gaynon, P.A. Brown Comprehensive evaluation of FLT3 inhibition as a promising Acquisition of data (provided animals, acquired and managed patients, therapeutic modality requires interrogation of an agent that is provided facilities, etc.): T.M. Cooper, J. Cassar, E. Eckroth, P. Gaynon, sufficiently potent and selective. The PIA assay has been validated L. Gore, K. August, S.G. Dubois, L.B. Silverman, J. Oesterheld, D. Magoon, as a surrogate measure of pharmacodynamic activity in response P.A. Brown fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, to targeted therapies (24). More speci cally, the ability to achieve computational analysis): T.M. Cooper, J. Cassar, J. Malvar, R. Sposto, fi sustained phospho-FLT3 inhibition using the PIA assay has de n- P. Gaynon, B.H. Chang, J.A. Pollard, S.G. Dubois, P.A. Brown itively been linked to response (26). To date, the clinical efficacy of Writing, review, and/or revision of the manuscript: T.M. Cooper, J. Malvar, FLT3 inhibitors has been limited by their off-target toxicities and R. Sposto, P. Gaynon, B.H. Chang, L. Gore, K. August, J.A. Pollard, S.G. Dubois, their inability to consistently inhibit phospho-FLT3 (26). The PIA L.B. Silverman, J. Oesterheld, G. Gammon, P.A. Brown assay in this trial demonstrated near total phospho-FLT3 inhibi- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Cassar, E. Eckroth, R. Sposto, P.A. Brown tion in every patient across all dose levels, showing that quizarti- Study supervision: T.M. Cooper, P.A. Brown nib is pharmacodynamically superior to any FLT3 inhibitor that Other (performed correlative laboratory studies): C. Annesley has been published in pediatric trials to date. Given the near complete inhibition in all patients, correlation of PIA with Grant Support response was not possible. In addition, the potency of quizartinib This work was financially supported by grants from the Phase One Foun- makes selection of a RP2D based on the PIA assay not feasible. dation to Children's Hospital of Los Angeles, TACL Consortium. Quizartinib Interestingly, our IVS assays of quizartinib-induced in vitro cyto- was provided by Ambit Biosciences Corporation. The correlative laboratory toxicity strongly correlate with response in this clinical trial. assays were funded by grants (to P. Brown) from the Leukemia and Lymphoma Society (LLS Scholar in Clinical Research Grant #2365-12) and the American Further study in a larger trial will be necessary to determine Cancer Society (ACS Research Scholar Grant #120237). whether this biologic correlate may provide an additional tool The costs of publication of this article were defrayed in part by the payment of by which to predict likelihood of clinical response to FLT3 page charges. This article must therefore be hereby marked advertisement in inhibition. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This clinical trial demonstrates that quizartinib added to intensive chemotherapy is a safe and active therapy that provides Received August 20, 2015; revised January 12, 2016; accepted February 9, continuous suppression of FLT3 phosphorylation in vivo. Our 2016; published OnlineFirst February 26, 2016.

OF8 Clin Cancer Res; 2016 Clinical Cancer Research

Quizartinib for Children with Relapsed Leukemia

References 1. Rosnet O, Buhring HJ, deLapeyriere O, Beslu N, Lavagna C, Marchetto S, disease in a patient with FLT3-ITDþ acute myeloid leukemia. Leuk Res et al. Expression and signal transduction of the FLT3 tyrosine kinase 2009;33:348–50. receptor. Acta Haematol 1996;95:218–23. 16. Smith BD, Levis M, Beran M, Giles F, Kantarjian H, Berg K, et al. Single-agent 2. Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, et al. Internal CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in tandem duplication of the flt3 gene found in acute myeloid leukemia. patients with relapsed or refractory acute myeloid leukemia. Blood Leukemia 1996;10:1911–8. 2004;103:3669–76. 3. Iwai T, Yokota S, Nakao M, Okamoto T, Taniwaki M, Onodera N, et al. 17. Kampa-Schittenhelm KM, Heinrich MC, Akmut F, Dohner H, Dohner K, Internal tandem duplication of the FLT3 gene and clinical evaluation in Schittenhelm MM. Quizartinib (AC220) is a potent second generation childhood acute myeloid leukemia. The Children's Cancer and Leukemia class III tyrosine kinase inhibitor that displays a distinct inhibition profile Study Group, Japan. Leukemia 1999;13:38–43. against mutant-FLT3, -PDGFRA and -KIT isoforms. Mol Cancer 2013; 4. Meshinchi S, Woods WG, Stirewalt DL, Sweetser DA, Buckley JD, Tjoa 12:19. TK, et al. Prevalence and prognostic significance of Flt3 internal 18. Zarrinkar PP, Gunawardane RN, Cramer MD, Gardner MF, Brigham D, tandem duplication in pediatric acute myeloid leukemia. Blood Belli B, et al. AC220 is a uniquely potent and selective inhibitor of FLT3 2001;97:89–94. for the treatment of acute myeloid leukemia (AML). Blood 2009; 5. Frohling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis K, et al. 114:2984–92. Prognostic significance of activating FLT3 mutations in younger adults (16 19. Cortes JE, Kantarjian H, Foran JM, Ghirdaladze D, Zodelava M, Borthakur to 60 years) with acute myeloid leukemia and normal cytogenetics: a study G, et al. Phase I study of quizartinib administered daily to patients with of the AML Study Group Ulm. Blood 2002;100:4372–80. relapsed or refractory acute myeloid leukemia irrespective of FMS-like 6. Brown P, Small D. FLT3 inhibitors: a paradigm for the development of tyrosine kinase 3-internal tandem duplication status. J Clin Oncol targeted therapeutics for paediatric cancer. Eur J Cancer 2004;40:707–21. 2013;31:3681–7. 7. Chillon MC, Gomez-Casares MT, Lopez-Jorge CE, Rodriguez-Medina C, 20. Cortes J, Tallman MS, Schiller G, Trone D, Gammon G, Goldberg S, et al. Molines A, Sarasquete ME, et al. Prognostic significance of FLT3 mutational Results of a phase 2 randomized, open-label, study of lower doses of status and expression levels in MLL-AF4þ and MLL-germline acute lym- quizartinib (AC220; ASP2689) in subjects with FLT3-ITD positive relapsed phoblastic leukemia. Leukemia 2012;26:2360–6. or refractory acute myeloid leukemia (AML). Oral Session 615. American 8. Stam RW, Schneider P, de Lorenzo P, Valsecchi MG, den Boer ML, Pieters R. Society of Hematology. Annual Meeting 2013;494. Prognostic significance of high-level FLT3 expression in MLL-rearranged 21. Le Tourneau C, Lee JJ, Siu LL. Dose escalation methods in phase I cancer infant acute lymphoblastic leukemia. Blood 2007;110:2774–5. clinical trials. J Natl Cancer Inst 2009;101:708–20. 9. Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia 2003; 22. Horton TM, Sposto R, Brown P, Reynolds CP, Hunger SP, Winick NJ, et al. 17:1738–52. Toxicity assessment of molecularly targeted drugs incorporated into mul- 10. Pollard JA, Alonzo TA, Gerbing RB, Ho PA, Zeng R, Ravindranath Y, Dahl G, tiagent chemotherapy regimens for pediatric acute lymphocytic leukemia et al. Prevalence and prognostic significance of KIT mutations in pediatric (ALL): review from an international consensus conference. Pediatr Blood patients with core binding factor AML enrolled on serial pediatric coop- Cancer 2010;54:872–8. erative trials for denovo AML. Blood 2010;115:2372–9. 23. Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH, 11. Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA, et al. et al. Revised recommendations of the International Working Group for FLT3 mutations in childhood acute lymphoblastic leukemia. Blood Diagnosis, Standardization of Response Criteria, Treatment Outcomes, 2004;103:3544–6. and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. 12. Brown P, Levis M, Shurtleff S, Campana D, Downing J, Small D. FLT3 J Clin Oncol 2003;21:4642–9. inhibition selectively kills childhood acute lymphoblastic leukemia cells 24.LevisM,BrownP,SmithBD,StineA,PhamR,StoneR,etal.Plasma with high levels of FLT3 expression. Blood 2005;105:812–20. inhibitory activity (PIA): a pharmacodynamic assay reveals insights into 13. Armstrong SA, Kung AL, Mabon ME, Silverman LB, Stam RW, Den Boer ML, the basis for cytotoxic response to FLT3 inhibitors. Blood 2006;108: et al. Inhibition of FLT3 in MLL. Validation of a therapeutic target identified 3477–83. by gene expression based classification. Cancer Cell 2003;3:173–83. 25. Levis M, Allebach J, Tse KF, Zheng R, Baldwin BR, Smith BD, et al. A FLT3- 14. Fiedler W, Serve H, Dohner H, Schwittay M, Ottmann OG, O'Farrell AM, targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells invitro and in et al. A phase 1 study of SU11248 in the treatment of patients with vivo. Blood 2002;99:3885–91. refractory or resistant acute myeloid leukemia (AML) or not amenable to 26. Levis M, Ravandi F, Wang ES, Baer MR, Perl A, Coutre S, et al. Results conventional therapy for the disease. Blood 2005;105:986–93. from a randomized trial of salvage chemotherapy followed by lestaur- 15. Safaian NN, Czibere A, Bruns I, Fenk R, Reinecke P, Dienst A, et al. Sorafenib tinib for patients with FLT3 mutant AML in first relapse. Blood 2011; (Nexavar) induces molecular remission and regression of extramedullary 117:3294–301.

www.aacrjournals.org Clin Cancer Res; 2016 OF9

A Phase I Study of Quizartinib Combined with Chemotherapy in Relapsed Childhood Leukemia: A Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Study

Todd M. Cooper, Jeannette Cassar, Elena Eckroth, et al.

Clin Cancer Res Published OnlineFirst February 26, 2016.

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