Leukemia (2012) 26, 2061–2068 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu

ORIGINAL ARTICLE Phase IB study of the FLT3 kinase inhibitor midostaurin with chemotherapy in younger newly diagnosed adult patients with

RM Stone1, T Fischer2, R Paquette3, G Schiller3, CA Schiffer4, G Ehninger5, J Cortes6, HM Kantarjian6, DJ DeAngelo1, A Huntsman-Labed7, C Dutreix7, A del Corral8 and F Giles9

This phase 1b trial investigated several doses and schedules of midostaurin in combination with daunorubicin and cytarabine induction and high-dose cytarabine post-remission therapy in newly diagnosed patients with acute myeloid leukemia (AML). The discontinuation rate on the 50-mg twice-daily dose schedule was lower than 100 mg twice daily, and no grade 3/4 nausea or vomiting was seen. The complete remission rate for the midostaurin 50-mg twice-daily dose schedule was 80% (FMS-like tyrosine kinase 3 receptor (FLT3)–wild-type: 20 of 27 (74%), FLT3-mutant: 12 of 13 (92%)). Overall survival (OS) probabilities of patients with FLT3-mutant AML at 1 and 2 years (0.85 and 0.62, respectively) were similar to the FLT3–wild-type population (0.78 and 0.52, respectively). Midostaurin in combination with standard chemotherapy demonstrated high complete response and OS rates in newly diagnosed younger adults with AML, and was generally well tolerated at 50 mg twice daily for 14 days. A phase III prospective trial is ongoing (CALGB 10603, NCT00651261).

Leukemia (2012) 26, 2061–2068; doi:10.1038/leu.2012.115 Keywords: FMS-like tyrosine kinase 3 receptor; acute myeloid leukemia; midostaurin; PKC412; newly diagnosed

INTRODUCTION dosing schedule; pretreatment with the inhibitor is antagonistic Mutations in the FMS-like tyrosine kinase 3 receptor (FLT3) occur with chemotherapy. FLT3 inhibition of cell cycle progression in in B25% of patients with acute myeloid leukemia (AML), cause AML cell lines renders such cells insensitive to S-phase-specific 10 constitutive activation, and are associated with poor prognosis.1–3 chemotherapeutic agents such as cytarabine. Cytogenetically normal patients with AML whose leukemia is We conducted a phase 1b trial in newly diagnosed patients with characterized by the presence of internal tandem duplication (ITD) wild-type and mutant FLT3 AML to examine the safety, efficacy mutations in FLT3 (FLT3-ITD) have a significantly shorter disease- and pharmacokinetics of combining midostaurin with an induc- free survival (DFS) and overall survival (OS) than patients without tion regimen of daunorubicin and cytarabine followed by high- the mutation.2,4 dose cytarabine consolidation. Several doses and schedules of The prognostic significance of the FLT3 mutation has led to the midostaurin were investigated. After discontinuing dose schedules pursuit of FLT3 inhibitors for the treatment of AML. Midostaurin involving 100 mg twice daily, we found schedules of midostaurin (PKC412) is a potent kinase inhibitor of FLT3, c-KIT, PDGFR-b, (50 mg twice daily taken sequentially or concomitantly for 14 days VEGFR-2 and protein kinase C, with demonstrated activity against per cycle) that could be combined tolerably with chemotherapy. cell lines containing mutant FLT3 and against FLT3-induced This analysis describes 29 patients who received midostaurin myeloproliferative disease in a mouse model.5 Midostaurin’s two 100 mg twice daily, and focuses on 40 patients who received major metabolites, CGP62221 and CGP52421, are also capable of midostaurin 50 mg twice daily. inhibiting FLT3 in vitro.6,7 In previous single-agent clinical studies with midostaurin at 75 mg three times daily8 or 50 or 100 mg PATIENTS AND METHODS twice daily,9 70% and 42% of patients with FLT3-mutant (n ¼ 55) and FLT3–wild-type (n ¼ 60) AML, respectively, had X50% Patients and objectives peripheral blood blast reduction. Duration of response was short, Previously untreated patients, aged 18–60 years, diagnosed with AML and complete remissions (CRs) were not observed. according to the World Health Organization criteria and with Karnofsky performance status X70 were eligible. Exclusion criteria included known Midostaurin’s activity, although limited, prompted a search for impaired gastrointestinal function or gastrointestinal disease that could optimization of outcomes with FLT3 inhibitors. Synergism significantly alter the absorption of midostaurin; receipt of any investiga- between FLT3 inhibitors and standard chemotherapeutic agents, tional agent within 30 days of day 1; any surgical procedure within 14 days including daunorubicin and cytarabine, has been demonstrated in of day 1; an ejection fraction of o50% as assessed by multigated preclinical studies. These studies highlight the importance of the acquisition scan or echocardiogram scan within 14 days of day 1; presence

1Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 2Department of Hematology/Oncology, Medical Center, Otto-von-Guericke-University, Magdeburg, Germany; 3UCLA Medical Center, Los Angeles, CA, USA; 4Karmanos Cancer Institute, Detroit, MI, USA; 5Technische Universita¨t, Dresden, Germany; 6MD Anderson Cancer Center, Houston, TX, USA; 7Novartis Pharma AG, Basel, Switzerland; 8Novartis Oncology, East Hanover, NJ, USA and and 9HRB Clinical Research Facility, National University of Ireland, Galway and Trinity College, Dublin, Ireland. Correspondence: Dr RM Stone, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. E-mail: [email protected] Previous presentations: Preliminary data from this study were presented at the 51st American Society of Hematology Annual Meeting in 2009. Received 22 February 2012; revised 30 March 2012; accepted 5 April 2012; accepted article preview online 27 April 2012; advance online publication, 25 May 2012 Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2062 of pulmonary infiltrates; history of or newly diagnosed myelodysplastic Maintenance therapy with midostaurin alone was allowed after syndrome; history of myeloproliferative disease or secondary AML; and completion of the planned chemotherapy (because of the potential prior chemotherapy (other than hydroxyurea) or radiation therapy. benefits of continuous inhibition of FLT3) and was administered for 14 The primary objectives were to evaluate the safety and tolerability of days in each 28-day cycle according to the patient’s original assignment. A twice-daily midostaurin (50 and 100 mg) administered either concomi- 50% dose reduction in midostaurin was allowed for grade 3/4 tantly with standard chemotherapy or sequentially after completion of nonhematological toxicity attributed to the drug. Midostaurin could be chemotherapy, and to determine the effect of midostaurin on the given at a 50% dose until toxicity was resolved, and then the drug could be pharmacokinetics of daunorubicin and cytarabine. The secondary objec- re-escalated. tives were to evaluate the efficacy of midostaurin in combination with standard chemotherapy by measuring the response rate, DFS and OS, and Study assessments to investigate the effect of FLT3 mutational status on the rate of patient response. The patient’s FLT3 mutation status was determined at baseline from bone marrow and/or blood samples. To determine the presence or absence of FLT3-ITD, FLT3 sequencing was carried out at a central laboratory Treatment plan (Transgenomic Labs, New Haven, CT, USA). If the central laboratory data Midostaurin 100 mg twice daily in combination with chemotherapy was were not available (for example, sample was not sent or was insufficient), administered on either a concomitant dose schedule starting on day 1 of a results from the local laboratory were used. The allelic ratio was not 28-day cycle or sequentially starting on day 8 (Figure 1, dose/schedule I). routinely determined. Cytogenetic abnormalities were characterized on After the first 14 patients, prolonged exposure was deemed too toxic, and the basis of the Cancer and Leukemia Group B criteria.11 All adverse events the study was amended to limit treatment to 14 days per chemotherapy (AEs) were recorded, regardless of causality. As the primary objective of the course (days 1–7 and 15–21 of the concomitant schedule; days 8–21 of the study was to determine safety and tolerability of the novel regimens, DFS sequential schedule) (Figure 1, dose/schedule II). Given intolerance of the duration (time from remission to relapse or death) was analyzed using 14-day-per-cycle exposure to midostaurin 100 mg twice daily, the study investigator-reported data. However, all patients were followed for survival was again amended to reduce the dose of midostaurin to 50 mg twice without censoring for alternative therapies such as stem cell transplant. daily in both the 14-day concomitant and sequential schedules (Figure 1, Follow-up occurred every 3 months after treatment discontinuation until dose/schedule III). death. The chemotherapy regimen consisted of a cycle of induction with daunorubicin 60 mg/m2 i.v. on days 1–3 and cytarabine 200 mg/m2 by continuous i.v. infusion on days 1–7. A bone marrow biopsy performed Pharmacokinetic analysis between days 21 and 28 was used to determine if a second cycle of Samples were collected to determine the concentration of midostaurin induction (daunorubicin 60 mg/m2 i.v. on days 1–2; cytarabine 200 mg/m2 and metabolites CGP52421 (the monohydroxy metabolite) and CGP62221 continuous i.v. infusion on days 1–5; and midostaurin given in the same (the desmethyl metabolite) during cycles 1–5. Concentrations were schedule used in the initial induction course) should be administered. determined by high-performance liquid chromatography/mass spectro- At the investigator’s discretion, the biopsy was delayed to insure that metry with a limit of quantification of 10 ng/ml. Plasma concentrations of re-induction did not occur during administration of midostaurin in patients daunorubicin and cytarabine (cycle 1, day 1) were analyzed separately by on the sequential arm. Patients who did not achieve CR at the end of a high-performance liquid chromatography with ultraviolet detection. The second cycle of induction (cycle 2) were discontinued from the study. limit of quantification was 5 ng/ml for daunorubicin and 10 ng/ml for Patients who achieved a CR at the end of cycle 1 or 2 received cytarabine. consolidation therapy for three cycles with high-dose cytarabine 3 g/m2 i.v. over 3 h every 12 h given every other day (days 1, 3 and 5) for six doses in Statistical analysis addition to midostaurin administered according to the schedule assigned during induction. Enrolled patients (n ¼ 69) who received at least one dose of midostaurin and/or standard chemotherapy were analyzed for both safety and efficacy. OS was considered as the time from first dose of any study drug to death; otherwise, patients were censored at the date last known to be alive. DFS Dose/schedule Schema n was considered as the time from first CR to relapse or death and was not daunorubicin censored for transplant. cytarabine Study conduct 7 I Continuous Concomitant This trial was registered with ClinicalTrials.gov as Novartis-CPKC412A2106. 100 mg BID 7 All patients signed informed consent forms approved by the relevant Sequential Institutional Review Boards. The study was performed at four centers in the United States and two centers in Germany. 8 II 14-day 100 mg BID Concomitant 7 Sequential RESULTS 20 Dose schedules I and II: 100 mg twice daily III 14-day Concomitant Twenty-nine patients received midostaurin 100 mg orally twice 50 mg BID 20 daily, with 14 patients receiving dose schedule I (continuous Sequential dosing beginning on day 1 (n ¼ 7) or day 8 (n ¼ 7)) and 15 patients receiving dose schedule II (days 1–7 and 15–21 (n ¼ 7) or days 1 7 14 21 28 8–21 (n ¼ 8)) (Figure 1). The discontinuation rate was high, with 23 28-day cycle of 29 patients (79%) failing to complete all planned therapy Figure 1. Schema of dose and schedule of midostaurin administra- (Figure 2). Gastrointestinal grade 3/4 AEs occurred at this dose: tion. Daunorubicin and cytarabine induction (3 þ 7) and high-dose 7 (24.1%) nausea, 7 (24.1%) vomiting and 4 (13.8%) diarrhea. cytarabine post-remission therapy was administered on a standard Intolerable gastrointestinal AEs led to discontinuation in two schedule. In addition, patients received midostaurin (indicated by patients (both grade 2). The frequency and grade of other AEs black bars) on one of three dose schedules: (I) midostaurin 100 mg occurring on dose schedules I and II were similar to that seen for twice daily (BID) for 21 or 28 days; (II) midostaurin 100 mg BID for 14 days; or (III) midostaurin 50 mg BID for 14 days. Within each dose patients treated with midostaurin 50 mg twice daily on dose schedule, patients were assigned to receive midostaurin on day 1 schedule III (data not shown). (concomitant with chemotherapy, days 1–7; 14–21 in dose CR was achieved by 13 of 29 patients (45%), including 8 of 23 schedules II and III) or day 8 (sequential with chemotherapy; days patients (35%) with FLT3–wild-type blasts and five of six patients 8–21 in dose schedules II and III). (83%) with FLT3-mutant blasts (three ITD and two tyrosine kinase

Leukemia (2012) 2061 – 2068 & 2012 Macmillan Publishers Limited Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2063 I. Continuous, 100 mg BID dosing

14 enrolled

7 assigned to sequential administration 7 assigned to concomitant administration

7 patients treated 7 patients treated

6 had 1 cycle of induction 7 had 1 cycle of induction 6 patients stopped early/discontinued 5 patients stopped early/discontinued therapy; 1 had 2 cycles therapy; 0 had 2 cycles 2 AEs 1 AE 2 treatment failures 3 treatment failures 0, 0, and 2 had 1, 2, and 3 cycles 1, 0, and 1 had 1, 2, and 3 cycles 1 protocol violation 1 withdrew consent of consolidation, respectively of consolidation, respectively 1 withdrew consent

II. 14-day, 100 mg BID dosing

15 enrolled

7 assigned to sequential administration 8 assigned to concomitant administration

7 patients treated 8 patients treated

7 had 1 cycle of induction 8 had 1 cycle of induction 7 patients stopped early/discontinued 5 patients stopped early/discontinued therapy; 0 had 2 cycles therapy; 0 had 2 cycles 1 AE 1 AE 2 treatment failures 1 treatment failure 0, 1, and 2 had 1, 2, or 3 cycles 1, 1, and 1 had 1, 2, or 3 cycles 1 transplant 3 withdrew consent of consolidation, respectively of consolidation, respectively 3 withdrew consent

III. 14-day, 50 mg BID dosing

40 enrolled

20 assigned to sequential administration 20 assigned to concomitant administration

11 patients stopped early/discontinueda 7 patients stopped early/discontinueda 16 had 1 cycle of induction 15 had 1 cycle of induction 2 AEs 1 AE therapy; 4 had 2 cycles therapy; 5 had 2 cycles 4 treatment failures 4 treatment failures 2 transplants 1 transplant 3, 0, and 11 had 1, 2, or 3 cycles 3, 1, and 8 had 1, 2, or 3 cycles 1 other therapy 1 other therapy of consolidation, respectively of consolidation, respectively 1 protocol violation 1 withdrew consent

aThree patients (n = 2 from the sequential arm; n = 1 from the concomitant arm) discontinued in complete remission before completing consolidation and were considered as completers by investigators. Figure 2. CONSORT flow diagram. It depicts patient numbers for enrollment, intervention allocation and follow-up. Abbreviations: AE, adverse event; BID, twice daily.

& 2012 Macmillan Publishers Limited Leukemia (2012) 2061 – 2068 Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2064 domain (TKD) mutations). One patient from dose schedule I with Kaplan–Meier OS probabilities at 1 and 2 years, respectively, FLT3–wild-type blasts received two cycles of induction and did not were 0.85 (95% CI: 0.65–1.0) and 0.62 (95% CI: 0.35–0.88) in respond. Two patients (33%) with FLT3-mutant blasts and nine patients with FLT3-mutant AML, and 0.78 (95% CI: 0.62–0.93) and patients (39%) with FLT3–wild-type blasts survived more than 4 0.52 (96% CI: 0.33–0.71) in patients with FLT3–wild-type AML years. (Figure 3a). Kaplan–Meier DFS probabilities at 1 year for FLT3- mutant and FLT3–wild-type patients were 0.50 (95% CI: 0.22–0.78) Dose schedule III: 50 mg twice daily and 0.60 (95% CI: 0.39–0.81), respectively (Figure 3b). Patients on dose schedule III received midostaurin 50 mg twice daily Of the eight patients with FLT3-ITD mutations assessed for DFS, either concomitantly (days 1–7 and 15–21; n ¼ 20) with chemo- six relapsed, all within 1 year. Six of the nine patients with FLT3- therapy or sequentially (days 8–21; n ¼ 20) after chemotherapy ITD mutations assessed for OS have died. Of the four patients with (Figure 1). FLT3 mutations were noted in 13 of 40 patients: 9 with TKD mutations assessed for DFS and OS, one relapsed after 1 year FLT3-ITD mutations and 4 with TKD mutations. As expected, most and none have died. patients (77%) with FLT3-mutated blasts displayed normal cyto- Overall, three patients in the study received more than two genetics (Table 1).11 The percentage of patients with the FLT3 cycles of maintenance therapy (between 23 and 29 cycles). All mutation in the sequential and concomitant arms was similar. The three were in the midostaurin 50-mg twice-daily cohort. Of these discontinuation rate in the 14-day 50-mg twice-daily arm (45%) was three patients, two had FLT3-mutant AML and one had FLT3–wild- lower than the rate in the 14-day 100-mg twice-daily arm (80%). type AML. These patients tolerated the drug well (two patients with no midostaurin dose adjustments). All discontinued treat- ment while in CR and remained in CR at last follow-up. Two other Efficacy: 50 mg twice daily patients received less than 2 months of post-consolidation The CR rate (80% overall) was higher among patients with FLT3- maintenance therapy. Both patients discontinued treatment in mutant AML (12 of 13 patients (92%) compared with 20 of 27 CR; one (FLT3–wild-type) relapsed 1 month after discontinuation. patients (74%) with wild-type FLT3). The single patient with FLT3- By investigator report, post-treatment transplant was received by mutant AML who did not respond had an ITD mutation consisting 5 of 27 patients (19%) with FLT3–wild-type disease and 4 of 13 of a 43-bp insertion. The dosage schedule did not affect the rate of patients (31%) with FLT3-mutant disease. CR; 16 of 20 patients (80%) in both the sequential and concomitant groups achieved a CR. Of the patients who achieved Safety and tolerability: 50 mg twice daily CR, 9 of 12 patients (75%) with FLT3-mutant AML and 15 of 20 patients (75%) with FLT3–wild-type disease achieved CR after the Midostaurin was generally well tolerated in combination with first cycle of induction. chemotherapy at the 50-mg twice-daily dose. The amount of midostaurin and chemotherapy received during induction was similar on the two arms, with 14 of 20 patients (70%) in both the Table 1. Characteristics of patients treated on dose schedule III sequential and concomitant arms completing all planned induc- (n ¼ 40) by FLT3 mutation status tion therapy. All three cycles of consolidation treatment were received by 12 of 16 patients (75%) in the sequential arm and 8 of Characteristic, n (%) FLT3–wild-type FLT3-mutant Total 16 patients (50%) in the concomitant arm. A higher rate of n ¼ 27 n ¼ 13 N ¼ 40 discontinuation was noted in the concomitant (55%) compared Sex, male 17 (63) 7 (54) 24 (60) with the sequential (35%) schedule (Figure 2). Patients on the Agep60 years 27 (100) 12a (92) 39 (98) sequential arm were exposed to midostaurin for a median of 130 (median) (50) (46) (48.5) days (range, 7–975 days) and patients on the concomitant arm were exposed for a median of 89 days (range, 8–1016 days). Karnofsky performance score Exposure to midostaurin was similar between the FLT3-mutated 100 7 (26) 1 (8) 8 (20) group (median, 133 days; range, 21–975 days) and FLT3–wild-type 90 11 (41) 3 (23) 14 (35) group (median, 90 days; range, 7–1016 days). 80 4 (15) 3 (23) 7 (18) Nausea, diarrhea and vomiting were the most common 70 3 (11) 3 (23) 6 (15) Unknown 2 (7) 3 (23) 5 (13) nonhematological events in the induction and consolidation periods (Table 2). Overall, one episode (3%) of grade 3 diarrhea Cytogenetics11 occurred, lasted 1 day, and resolved without treatment. No grade Adverse 7 (26) 2 (15) 9 (23) 3/4 nausea or vomiting occurred. Grade 3/4 hepatic toxicity was Intermediate 7 (26) 1 (8) 8 (20) infrequent. No grade 3/4 peripheral edema was observed. Overall, (excluding normal) the toxicity reported was similar in the sequential and concomi- Favorable 5 (19) 0 5 (13) tant schedules. No deaths were recorded in either arm on Normal 5 (19) 10 (77) 15 (38) treatment or within 28 days of the last dose of study drug. Unknown 3 (11) 0 3 (8)

White blood cell count Pharmacokinetics: 50 mg twice daily p50 000 Â 109/l 21 (78) 12 (92) 33 (83) Plasma levels for midostaurin and its metabolites, on both the (median) (9000) (16 000) (13 000) sequential and concomitant dosing schedules, reached levels similar to those previously reported for patients with AML treated with Dosage schedule 9 Sequential 13 (48) 7 (54) 20 (50) midostaurin monotherapy (Figure 4; Table 3). During off-treatment Concomitant 14 (52) 6 (46) 20 (50) periods, trough concentrations of midostaurin and CGP62221 were similar, decreasing to low or undetectable levels. Differences were FLT3 mutation type observed in plasma concentrations of midostaurin and CGP62221 ITD NA 9 (69) NA between the two arms, which can be explained by the different Point mutation NA 4 (31) NA treatment schedules. For example, on day 15, the plasma concen- Abbreviations: FLT3, FMS-like tyrosine kinase 3 receptor; ITD, internal trations of drug in the concomitant arm are lower compared with the tandem duplication; NA, not applicable. aPatient aged 65 years at sequential arm, because patients in the concomitant arm had not enrollment was a protocol violation, but was included in all analyses. received midostaurin for 7 days, whereas those in the sequential arm were in the middle of the 14-day treatment block. In contrast,

Leukemia (2012) 2061 – 2068 & 2012 Macmillan Publishers Limited Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2065 a 1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3 Survival (probability) FLT3-mutant 0.2 FLT3–wild-type 0.1 FLT3-mutant censored FLT3–wild-type censored 0.0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 Time (months) Patients at risk 13 131212119988888665543100 27 27 24 22 21 20 17 14 14 14 14 12 12 11 11 9 5 3 2 1 0

b 1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2 FLT3-mutant FLT3–wild-type Disease-Free Survival (probability) 0.1 FLT3-mutant censored FLT3–wild-type censored 0.0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 Time (months) Patients at risk 12 129665555555444333000 20 19 15 14 12 11 10 10 10 9 9 9 9 8 7 4 3 3 2 2 0 Figure 3. (a) OS probability in patients with FLT3–wild-type and FLT3-mutant AML treated on dose schedule III. OS was assessed without censoring for alternative therapies such as stem cell transplant. (b) DFS probability in patients with FLT3–wild-type and FLT3-mutant AML treated on dose schedule III. the active metabolite CGP52421 displayed a long half-life and was tyrosine kinase inhibitors in chronic myeloid leukemia. However, maintained at stable levels between dosing treatment phases. AML is genetically more similar to blast-phase chronic myeloid Although the clearance of daunorubicin does not seem to be leukemia, with many more mutations required for the develop- affected by midostaurin (Table 4), the mean concentration of ment of the full disease phenotype. FLT3 may occur as a daunorubicin observed at 24 h (C24 h) after the first dose was secondary mutation rather than an initiator of the leukemic 17.4 ng/ml and undetectable (o5 ng/ml) with and without clone.12 The absence of primacy of FLT3 mutations, lack of concomitant administration of midostaurin, respectively. adequate pharmacokinetics and protection of the leukemic stem Therefore, a pharmacokinetic interaction between daunorubicin cells in the marrow niche have each been invoked to explain the and midostaurin could not be excluded. No interaction between disappointing clinical efficacy seen when potent FLT3 inhibitors cytarabine and midostaurin was observed (data not shown). were used as single agents in early-stage clinical trials in advanced FLT3-mutant AML.8,9,13–15 However, the biological activity (frequent reduction in peripheral blood blasts) coupled with DISCUSSION preclinical studies showing synergy between FLT3 inhibitors and It was originally hoped that single-agent FLT3 inhibitor therapy chemotherapy prompted an effort to address the feasibility of would have a profound effect on AML, similar to the benefit of combination therapy.

& 2012 Macmillan Publishers Limited Leukemia (2012) 2061 – 2068 Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2066

Table 2. Adverse events occurring during the induction/consolidation Midostaurin (concomitant) periods in greater than 20% of patients regardless of attribution for Midostaurin (sequential) Midostaurin patients treated on dose schedule III a 3500 Event, n (%) Concomitant Sequential Total n ¼ 20 n ¼ 20 N ¼ 40 3000

2500 Grade Grade Grade Grade Grade Grade 1/2 3/4 1/2 3/4 1/2 3/4 2000 Nausea 16 (80) 0 17 (85) 0 33 (83) 0 Neutropenia 2 (10) 16 (80) 0 13 (65) 2 (5) 29 (73) 1500 (including febrile) Thrombocytopenia 1 (5) 13 (65) 0 16 (80) 1 (3) 29 (73) 1000

Diarrhea 13 (65) 0 14 (70) 1 (5) 27 (68) 1 (3) Midostaurin (ng/ml) Vomiting 12 (60) 0 14 (70) 0 26 (65) 0 500 Hypokalemia 10 (50) 3 (15) 8 (40) 4 (20) 18 (45) 7 (18) Pyrexia 8 (40) 0 9 (45) 6 (30) 17 (43) 6 (15) 0 Headache 9 (45) 1 (5) 11 (55) 0 20 (50) 1 (3) Anemia 1 (5) 6 (30) 1 (5) 11 (55) 2 (5) 17 (43) 0 5 10 15 20 25 30 Insomnia 10 (50) 0 9 (45) 0 19 (48) 0 Induction cycle 1 (days) Constipation 9 (45) 0 9 (45) 0 18 (45) 0 Chills 8 (40) 0 9 (45) 0 17 (43) 0 Petechiae 5 (25) 2 (10) 10 (50) 0 15 (38) 2 (5) b CGP62221 CGP62221 (concomitant) Cough 6 (30) 0 9 (45) 0 15 (38) 0 CGP62221 (sequential) 3500 Hypomagnesemia 9 (45) 0 6 (30) 0 15 (38) 0 Rash 8 (40) 1 (5) 6 (30) 0 14 (35) 1 (3) Abdominal pain 3 (15) 0 10 (50) 1 (5) 13 (33) 1 (3) 3000 Peripheral edema 9 (45) 0 5 (25) 0 14 (35) 0 Epistaxis 5 (25) 0 4 (20) 3 (15) 9 (23) 3 (8) 2500 Hypotension 8 (40) 0 4 (20) 0 12 (30) 0 ALT increased 4 (20) 0 4 (20) 3 (15) 8 (20) 3 (8) 2000 Alopecia 7 (35) 0 4 (20) 0 11 (28) 0 Decreased 5 (25) 0 5 (25) 1 (5) 10 (25) 1 (3) 1500 appetite Hypocalcemia 7 (35) 1 (5) 2 (10) 1 (5) 9 (23) 2 (5) 1000 Pruritus 6 (30) 1 (5) 4 (20) 0 10 (25) 1 (3) CGP62221 (ng/ml) Anorexia 4 (20) 0 5 (25) 1 (5) 9 (23) 1 (3) 500 Anxiety 4 (20) 0 5 (25) 1 (5) 9 (23) 1 (3) AST increased 3 (15) 1 (5) 3 (15) 3 (15) 6 (15) 4 (10) 0 Depression 2 (10) 0 8 (40) 0 10 (25) 0 Fatigue 5 (25) 0 5 (25) 0 10 (25) 0 51015202530 Mucosal 4 (20) 1 (5) 5 (25) 0 9 (23) 1 (3) Induction cycle 1 (days) inflammation Blood bilirubin 4 (20) 1 (5) 3 (15) 1 (5) 7 (18) 2 (5) CGP52421 CGP52421 (concomitant) increased c 3500 CGP52421 (sequential) Transfusion 4 (20) 1 (5) 2 (10) 2 (10) 6 (15) 3 (8) reaction 3000 Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotrans- ferase. 2500

2000

This study was designed to determine a safe and tolerable dose 1500 of midostaurin that could be administered with standard induction and post-remission chemotherapy. Achieving such a regimen was 1000

more challenging than initially expected. We had previously shown CGP52421 (ng/ml) that 75 mg three times daily8 and 50 and 100 mg each twice daily9 500 were well tolerated and reasonably efficacious when given conti- nuously as single agents to patients with AML. However, 100-mg 0 twice-daily midostaurin given either concomitantly with induction 51015202530 chemotherapy (beginning on the first day of chemotherapy) or Induction cycle 1 (days) sequentially (on the eighth day after the start of chemotherapy) led to grade 3/4 nausea and vomiting and a high rate of Figure 4. (a) Median trough concentration-time profile of midos- discontinuation. Tolerability improved for patients who received taurin during the first cycle of induction therapy (midostaurin 50-mg midostaurin 50 mg twice daily for 14 days per cycle in both the twice-daily cohort). (b) Median trough concentration-time profile of concomitant and sequential arms. Given the slightly higher degree CGP62221 during the first cycle of induction therapy (midostaurin 50-mg twice-daily cohort). (c) Median trough concentration-time of tolerability in the sequential arm, the fact that a pharmacokinetic profile of CGP52421 during the first cycle of induction therapy interaction between midostaurin and daunorubicin could not be (midostaurin 50-mg twice-daily cohort). Open boxes represent excluded in our study, and concerns in other studies about possible values from patients on the concomitant arm (day 1–7 and day antagonism if a FLT3 inhibitor was given before chemotherapy,10 15–22 dosing); closed circles represent values from patients on the the sequential schedule was chosen for further evaluation. sequential arm (day 8–22 dosing).

Leukemia (2012) 2061 – 2068 & 2012 Macmillan Publishers Limited Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2067 Table 3. Concentration of midostaurin and its two metabolites with 50-mg twice-daily midostaurin administration during daunorubicin/cytarabine induction

Concomitant administration of midostaurin with chemotherapy (days 1–7 and 15–21 per cycle)

Cycle Days Midostaurin (ng/ml) CGP62221 (ng/ml) CGP52421 (ng/ml)

n Median Min Max n Median Min Max n Median Min Max

Induction cycle 1 1 14 LLOQ LLOQ LLOQ 14 LLOQ LLOQ LLOQ 6 LLOQ LLOQ LLOQ Day 1 13 1170 355 2300 13 189 64.8 593 5 170 52.9 812 ( þ 2h) Day 1 13 1010 630 3380 13 385 219 626 5 137 115 209 ( þ 4h) 2 13 1490 LLOQ 4980 13 871 LLOQ 1710 6 361 228 460 4 12 2355 1680 8710 12 2315 919 3310 5 832 570 1090 6 14 2525 1200 11 300 14 2730 1970 4850 6 1360 770 1550 15 11 87.6 11.7 7100 11 528 184 3950 6 1235 882 1680 15 ( þ 2 h) 11 1870 87.1 7300 11 630 195 3400 6 1170 814 1920 15 ( þ 4 h) 11 1420 96.3 7120 11 845 297 3510 6 1465 893 1860 16 11 1820 60 7520 11 1030 283 4460 6 1665 880 2240 22.5 11 1550 LLOQ 9470 11 2630 66.7 4870 5 2850 2390 3840 29.5 11 63.05 LLOQ 1810 8 425.9 LLOQ 2980 5 1900 1570 2930 First consolidation 1.5 9 LLOQ LLOQ LLOQ 9 LLOQ LLOQ 16.4 1 1170 cycle 15.5 9 387 LLOQ 1040 9 210 14.1 986 1 810 29.5 9 LLOQ LLOQ 83.8 9 65.8 LLOQ 1090 2 809; 2990

Sequential administration of midostaurin with chemotherapy (days 8–21 per cycle)

Induction cycle 1 8 18 LLOQ LLOQ LLOQ 18 LLOQ LLOQ LLOQ 11 LLOQ LLOQ LLOQ 8(þ 2 h) 18 1495 460 2880 18 130 LLOQ 612 11 70.8 35.9 194 8(þ 4 h) 18 1585 635 3660 18 244.5 42.2 810 11 113 58.2 207 9 17 1680 922 3290 17 732 136 1420 11 293 145 414 15 17 3660 1290 17 100 17 2690 1280 5500 10 1460 903 1670 22.5 16 2755 904 19 200 16 3050 1570 8200 9 2800 1630 3540 29.5 12 57.2 0 4970 10 409.5 38.9 2590 6 1955 1350 3330 First consolidation 1.5 14 LLOQ LLOQ 91.2 14 LLOQ LLOQ 217 7 1740 835 3110 cycle 15.5 14 1330 635 3790 14 1435 513 3560 8 2685 1490 4210 29.5 12 6.75 LLOQ 4000 12 157.5 LLOQ 1980 7 2230 1440 5320 Abbreviation: LLOQ, lower limit of quantification. Shaded rows indicate the active dosing period. Induction cycle 1 refers to the daunorubicin/cytarabine induction. First consolidation cycle refers to the first of three high-dose cytarabine consolidation cycles. The LLOQ is 10 ng/ml.

or stem cell transplant. Consequently, we supported moving Table 4. Effect of 50-mg twice-daily midostaurin administration on ahead with this schedule, although further dose optimization may daunorubicin pharmacokinetic parameters, cycle 1, day 1 be possible. Daunorubicin Concomitant Sequential Concomitant/ This study demonstrated an encouragingly high CR rate of 92% parameter, midostaurin midostaurin sequential in 13 patients with FLT3-mutant AML exposed to midostaurin median n ¼ 13 n ¼ 13 midostaurin 50 mg for 14 days of each 28-day cycle. plus induction (mean±s.d.) chemotherapy also led to a high CR rate (93%) in 15 newly diagnosed patients with FLT3-mutant AML.16 While the poor ± ± Cmax (ng/ml) 89 (98 38) 76 (229 267) 1.17 prognosis noted in patients with FLT3-ITD mutations is thought to ± ± Tmax (h) 0.5 (0.5 0.2) 0.5 (0.6 0.4) 1.0 be becaus of a high relapse rate,1–4,17–21 it is possible that a higher ± ± C24 h (ng/ml) 17.4 (26 32) LLOQ (0 0) 17.4/LLOQ likelihood of CR or ‘deeper’ CR could be beneficial in reducing AUC (0–24 h) 189 (373±327) 159 (267±254) 1.19 (ng/ml) relapse rates. Although no firm conclusions can be drawn on the CL (l/h/m2) 283 (269±130) 270 (330±226) 1.05 basis of 13 patients, four of whom had TKD mutations, it is 2 Vz (l/m ) 694 (717±266) 775 (946±696) 0.90 interesting to note that Kaplan–Meier DFS and OS probabilities in this group of patients were similar to those in the 27 patients with Abbreviations: AUC0–24 h, area under the plasma concentration-time curve FLT3–wild-type disease. The extent to which post-protocol from time 0 to 24 h; CL, apparent oral clearance; Cmax, maximum plasma therapy (for example, stem cell transplant) influenced this concentration; C , plasma concentration at 24 h; LLOQ, lower limit of 24 h relatively favorable survival outcome in the FLT3-mutant cohort quantification; Tmax, time to Cmax; Vz, apparent volume of distribution. The LLOQ is 5 ng/ml. is unclear. The results of our study, showing high CR and OS rates in patients with FLT3-mutant AML with acceptable tolerability, Although there was a 45% discontinuation rate in the cohort of suggest that the addition of midostaurin to chemotherapy may 40 patients receiving midostaurin 50 mg twice daily in combina- improve outcomes for newly diagnosed younger patients with tion with chemotherapy, most of these patients stopped for FLT3-mutant AML. However, AML is a heterogeneous disease and reasons other than toxicity, such as relapse, failure to achieve CR the current study was small and did not account for the influence

& 2012 Macmillan Publishers Limited Leukemia (2012) 2061 – 2068 Midostaurin in combination with chemotherapy in FLT3-mutant AML RM Stone et al 2068 of parameters such as stem cell transplantation or gene mutations cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a other than FLT3. Nevertheless, these promising safety results marker for the detection of minimal residual disease. Blood 2002; 100: 59–66. enabled initiation of the ongoing, international, prospective, 5 Weisberg E, Boulton C, Kelly LM, Manley P, Fabbro D, Meyer T et al. Inhibition of randomized double-blind phase III study (CALGB 10603, mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase NCT00651261) of standard induction and post-remission che- inhibitor PKC412. Cancer Cell 2002; 1: 433–443. 6 Manley PW, Boulton C, Caravatti G, Gilliland DG, Griffin J, Kung A et al. Preclinical motherapy with placebo or midostaurin at 50 mg twice daily on profile of PKC412 (midostaurin) as an FLT3 inhibitor for the therapy of AML. 94th days 8 through 21 of each chemotherapy cycle and as annual meeting of the American Association for Cancer Research on July 11–14, maintenance therapy in newly diagnosed patients with AML 2003 in Washington, DC. AACR 2003; Poster 1004. younger than 60 years of age. 7 Levis M, Brown P, Smith BD, Stine A, Pham R, Stone R et al. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cyto- toxic response to FLT3 inhibitors. Blood 2006; 108: 3477–3483. CONFLICT OF INTEREST 8 Stone RM, DeAngelo DJ, Klimek V, Galinsky I, Estey E, Nimer SD et al. Patients with Employment or Leadership Position: Alice Huntsman-Labed, Novartis Pharma AG (C); acute myeloid leukemia and an activating mutation in FLT3 respond to a small- Catherine Dutreix, Novartis Pharma AG (C); Adam del Corral, Novartis Pharmaceuticals molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105: 54–60. Corporation (C); Consultant or Advisory Role: Richard M Stone, Genzyme (C), Celgene 9 Fischer T, Stone RM, DeAngelo DJ, Galinsky I, Estey E, Lanza C et al. Phase IIB trial (C), Ariad (C); Ronald Paquette, Novartis (C); Gary Schiller, Genzme (C); Charles A of oral midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and Schiffer, Pfizer (C), Micromet (C), Celgene (C), Ambit (C), Ariad (C); Jorge Cortes, multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high- Novartis (C), Ariad (C), Ambit (U); Hagop M. Kantarjian, Novartis (C); Daniel J risk with either wild-type or mutated FLT3. J Clin Oncol DeAngelo, Novartis (C); Francis Giles, Novartis (C); Stock Ownership: Gerhard 2010; 28: 4339–4345. Ehninger, Novartis; Alice Huntsman-Labed, Novartis; Honoraria: Thomas Fischer, 10 Levis M, Pham R, Smith BD, Small D. In vitro studies of a FLT3 inhibitor combined Novartis; Ronald Paquette, Novartis; Gerhard Ehninger, Novartis; Research Funding: with chemotherapy: sequence of administration is important to achieve syner- Richard M Stone, Novartis; Thomas Fischer, Novartis; Gary Schiller, Genzyme; Charles gistic cytotoxic effects. Blood 2004; 104: 1145–1150. Schiffer, Pfizer, Ariad, Novartis, Celgene, Bristol-Myers Squibb, Ambit; Jorge Cortes, 11 Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC et al. Pretreat- Novartis, Ariad, Ambit; Hagop M Kantarjian, Novartis, Pfizer, Bristol-Myers Squibb; ment cytogenetic abnormalities are predictive of induction success, cumulative Francis Giles, Novartis; Expert Testimony: None; Other Remuneration: None. incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002; 100: 4325–4336. ACKNOWLEDGEMENTS 12 Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K et al. DNA sequencing of The authors acknowledge the study coordinators, nurses and physicians who a cytogenetically normal acute myeloid leukaemia genome. Nature 2008; 456: contributed to this study and the patients and their families for their participation. 66–72. Susie Crowley provided secretarial assistance. Sandra Harris and Erinn Goldman 13 Smith BD, Levis M, Beran M, Giles F, Kantarjian H, Berg K et al. Single-agent of Articulate Science, LLC, provided medical writing assistance. This study was CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients sponsored by Novartis Pharmaceuticals Corporation. with relapsed or refractory acute myeloid leukemia. Blood 2004; 103: 3669–3676. 14 DeAngelo DJ, Stone RM, Heaney ML, Nimer SD, Paquette RL, Klisovic RB et al. Phase 1 clinical results with tandutinib (MLN518), a novel FLT3 antagonist, in AUTHOR CONTRIBUTIONS patients with acute myelogenous leukemia or high-risk myelodysplastic syn- drome: safety, pharmacokinetics, and pharmacodynamics. Blood 2006; 108: Conception and design: Richard M Stone, Thomas Fischer, Charles A Schiffer, 3674–3681. Gerhard Ehninger, Jorge Cortes, Hagop M Kantarjian, Daniel J DeAngelo and 15 Zhang W, Konopleva M, Shi YX, McQueen T, Harris D, Ling X et al. Mutant FLT3: a Francis Giles. Collection and assembly of data: Richard M Stone, Ronald Paquette, direct target of sorafenib in acute myelogenous leukemia. J Natl Cancer Inst 2008; Charles A Schiffer, Gerhard Ehninger, Jorge Cortes, Hagop M Kantarjian, Daniel J 100: 184–198. DeAngelo, Alice Huntsman-Labed, Catherine Dutreix, Adam del Corral and Francis 16 Ravandi F, Cortes JE, Jones D, Faderl S, Garcia-Manero G, Konopleva MY et al. Giles. Data analysis and interpretation: Richard M Stone, Thomas Fischer, Gary Phase I/II study of combination therapy with sorafenib, idarubicin, and cytarabine Schiller, Charles A Schiffer, Jorge Cortes, Hagop M Kantarjian, Daniel J DeAngelo, in younger patients with acute myeloid leukemia. J Clin Oncol 2010; 28: Alice Huntsman-Labed, Catherine Dutreix, Adam del Corral and Francis Giles. 1856–1862. 17 Kayser S, Schlenk RF, Londono MC, Breitenbuecher F, Wittke K, Du J et al. Manuscript writing: Richard M Stone wrote the first draft of the manuscript and Insertion of FLT3 internal tandem duplication in the tyrosine kinase domain-1 is all authors edited and commented on subsequent drafts. Final approval of associated with resistance to chemotherapy and inferior outcome. Blood 2009; manuscript: All authors gave final approval of the manuscript. Provision of study 114: 2386–2392. materials or patients: Richard M Stone, Thomas Fischer, Gary Schiller, Charles A 18 Breitenbuecher F, Markova B, Kasper S, Carius B, Stauder T, Bo¨ hmer FD et al. A Schiffer, Jorge Cortes, Daniel J DeAngelo and Francis Giles. novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in AML. Blood 2009; 113: 4063–4073. 19 Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA et al. The REFERENCES presence of a FLT3 internal tandem duplication in patients with acute myeloid 1 Thiede C, Steudel C, Mohr B, Schaich M, Scha¨kel U, Platzbecker U et al. Analysis of leukemia (AML) adds important prognostic information to cytogenetic risk group FLT3-activating mutations in 979 patients with acute myelogenous leukemia: and response to the first cycle of chemotherapy: analysis of 854 patients from the association with FAB subtypes and identification of subgroups with poor prog- United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001; 98: nosis. Blood 2002; 99: 4326–4335. 1752–1759. 2 Whitman SP, Archer KJ, Feng L, Baldus C, Becknell B, Carlson BD et al. Absence of 20 Whitman SP, Ruppert AS, Radmacher MD, Mrozek K, Paschka P, Langer C et al. the wild-type allele predicts poor prognosis in adult de novo acute myeloid leu- FLT3 D835/I836 mutations are associated with poor disease-free survival and a kemia with normal cytogenetics and the internal tandem duplication of FLT3: a distinct gene-expression signature among younger adults with de novo cyto- cancer and leukemia group B study. Cancer Res 2001; 61: 7233–7239. genetically normal acute myeloid leukemia lacking FLT3 internal tandem dupli- 3 Weisberg E, Barrett R, Liu Q, Stone R, Gray N, Griffin JD. FLT3 inhibition and cations. Blood 2008; 111: 1552–1559. mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat 21 Mead AJ, Linch DC, Hills RK, Wheatley K, Burnett AK, Gale RE. FLT3 tyrosine kinase 2009; 12: 81–89. domain mutations are biologically distinct from and have a significantly more 4 Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C et al. Analysis of FLT3 favorable prognosis than FLT3 internal tandem duplications in patients with acute length mutations in 1003 patients with acute myeloid leukemia: correlation to myeloid leukemia. Blood 2007; 110: 1262–1270.

Leukemia (2012) 2061 – 2068 & 2012 Macmillan Publishers Limited