Gemtuzumab ozogamicin plus midostaurin in combination with standard ‘7 + 3’ induction therapy in newly diagnosed AML: Results from the SAL‐MODULE phase I study

We conducted a phase I trial in newly diagnosed acute myeloid leukaemia (AML) to investigate the combination of two novel targeted agents, gemtuzumab ozogamicin (GO) and midostaurin, with intensive chemotherapy in FLT3‐mutated AML and CBF leukaemia. Three dose levels of midostaurin and one to three sequential doses of 3 mg/m2 GO in combination with ‘7 + 3’ induction were evaluated. Based on safety findings in 12 patients, our results show that 3 mg/m2 GO on Days 1 + 4 and 100 mg midostaurin on Days 8–21 can be safely combined with IC in newly diagnosed AML.


I N TRODUC T ION
Based on the results of randomized-controlled trials, the tyrosine kinase inhibitor midostaurin and the conjugated CD33 antibody gemtuzumab ozogamicin (GO) have been approved for combination treatment with standard intensive chemotherapy in acute myeloid leukaemia (AML). 1,2In newly diagnosed AML with FLT3 mutations (FLT3-mut), a combination of intensive chemotherapy with the tyrosine kinase inhibitor midostaurin is used as the standard of care, whereas GO is considered the standard of care in patients with core-binding factor (CBF) AML or NPM1 mutations. 3ubgroup analyses from the ALFA 0701 trial indicate that the addition of GO has a pronounced efficacy in the subgroup of patients with FLT3-ITD mutations, providing a rationale for the combined use of midostaurin plus GO with intensive chemotherapy for newly diagnosed FLT3-mut AML. 4 This could be due to the high expression of CD33 on FLT3-mut cells. 5,6On the other hand, there is evidence that the subgroup of CBF AML benefits from the inhibition of the tyrosine kinase KIT. 7Since midostaurin is a strong KIT inhibitor, the addition of both midostaurin and GO could be a promising approach to further improve treatment outcomes in the CBF-and FLT3-mutated AML subgroups.In order to prospectively evaluate this hypothesis, we set up the MOSAIC clinical trial consisting of a phase I dose-finding part to assess the feasibility of combining midostaurin plus GO with standard IC (MODULE), followed by a randomizedcontrolled phase II part evaluating the benefit of adding GO to the standard of care in FLT3-mut AML (MAGMA) and of adding midostaurin to the standard of care in CBF AML (MAGNOLIA).Here, we report the results of the phase I part (MODULE).

M ET HODS
MODULE was designed as an open-label, dose escalation, phase I trial following a 3 + 3 design.The primary aim was to determine the maximum tolerated dose (MTD) and recommended phase II dose for the combination of midostaurin and GO administered with 7 + 3 (cytarabine + daunorubicin) chemotherapy in the first induction cycle and to the assess tolerability and preliminary efficacy of this drug combination.Patients aged 18-75 years with newly diagnosed AML (WHO 2022) with evidence of CBF fusions and/or FLT3 mutations, and fit for intensive chemotherapy were eligible.In cohorts of three subjects, patients received chemotherapy with cytarabine 200 mg/m 2 continuous infusion over 7 days plus daunorubicin 60 mg/m 2 IV on 3 days (7 + 3) and additionally GO plus midostaurin in three predefined dose levels and one fall-back level (Table S1).Non-haematological DLTs were defined as ≥ grade 3 toxicity with a causal relationship to either midostaurin or GO.Haematological DLTs were defined as grade 4 neutropenia or thrombocytopenia beyond Day 42 after initiation of anti-leukaemic treatment in the absence of AML (<5% bone marrow blasts).Application of growth factors (G-CSF) was not permitted until the end of the DLT period.The trial was conducted in accordance with the principles of Good Clinical Practice (GCP) guidelines and the Declaration of Helsinki, and approved by the ethics committees of the participating sites.All patients gave written consent prior to trial participation.The study is registered at clini caltr ials.gov under NCT04385290.

R E SU LTS
Between September 2020 and April 2022 a total of 13 patients were enrolled.Of them, 12 patients were included in DLT evaluation.The median age of all enrolled 13 patients with at least one dose of GO was 49 years; favourable genetic risk per ELN 2022 was present in three patients (25%); intermediate and adverse in eight (67%) and one (8%) respectively (Table 1).
Four patients were treated at the first dose level.One patient discontinued study treatment on Day 6 of induction before commencement of midostaurin due to an infusionrelated reaction to GO (CTC grade 4).This patient did not qualify for DLT evaluation and was therefore replaced.
The other three patients in the first dose cohort completed the regular study period without DLTs.Seven patients were treated in the second dose level.One patient discontinued treatment on Day 15 due to nausea-related inability to swallow midostaurin.As the target dose of midostaurin for DLT evaluation was not met, the patient had to be replaced.One of three enrolled patients in the second dose cohort experienced neutropenic colitis CTCAE grade 3 on Day 14 of treatment, which was classified as DLT.The colitis had fully recovered by Day 27.Consequently, the second dose cohort was extended to six patients.Here, one 55-year-old female patient developed signs of sinusoidal obstruction syndrome (SOS) CTCAE grade 3 on Day 13 of treatment, classified as DLT.Apart from a pre-existing hepatic steatosis grade 1, this patient had no other relevant predisposing comorbidities and a body surface area below 2 m 2 (1.9 m 2 ).She was subsequently treated with defibrotide and supportive care until recovery on Day 28.In accordance with the protocol, the dose escalation was stopped on dose level 2 due to the occurrence of two DLTs in six evaluable patients, and the dose level 2 was defined as MTD.A total of five serious adverse events (infusion-related reaction, colitis, SOS, parvovirus-B19 infection and grade 4 neutropenia beyond Day 42) were observed among all enrolled patients.Two of the SAEs were observed in the same patient (colitis and parvovirus-B19 infection) at different time points, resulting in five SAEs in four patients.An unexpected increase in the frequency of common AML adverse events was not observed.Based on these safety data, the steering committee judged the MTD as safe and feasible.To substantiate the toxicological safety with respect to the recommended phase II dose, we extended the second dose cohort by another three patients, resulting in a total number of nine patients as described in the 3 + 3 + 3 design of dose escalation trials. 8,9o DLTs were observed in the additional three patients, underpinning this dose level as recommended as phase II dose for the subsequent randomized phase II part of the trial.No deaths occurred during the first 30 and 60 days after treatment start.
The overall response rate in the 12 patients of the full analysis set was 92%, with complete remission (CR) in nine patients (75%), CR with incomplete haematological recovery (CRi) in two patients (17%) and primary refractory disease in one patient (8%).The characteristics and safety data of all patients who qualified for DLT evaluation are presented in Table 1, Table S2 and Figure S1.
At the time of data cut, 11 of 12 patients were alive and in complete remission.Among those, 2/11 patients suffered a relapse but subsequently achieved an ongoing second CR.One patient died due to third AML relapse; eight patients received an allogeneic HCT.After a median follow-up time of 25 months, the 2-year RFS is 81% and the 2-year OS is 100%.The clinical course of individual patients is depicted in Figure 1.

DISCUS SION
Clinical development and approval of various novel agents for AML treatment in the last few years has led to a relevant clinical improvement for certain subgroups of the disease.Even though targeted approaches potentially cause less unspecific toxicity, undesired off-target effects or augmented haematological toxicities still restrict their clinical use and applicability, in particular when combined with conventional chemotherapy backbones such as '7 + 3'.On the other hand, we still observe unsatisfactory responses and long-term remissions even with tolerable and established combinations of novel agents with chemotherapy, indicating medical need and suggesting the option to attack cancer cells with various modalities by addressing multiple targets.Therefore, new-generation trials testing the possibility of a combination of more than one novel agent, and identifying the optimal dose combination balancing risks and benefits represent the logical next step in the clinical development of AML treatment.
In line with this approach, the prospective dose-finding phase I trial presented here aimed to explore the tolerability and optimal dosing for the combination of a standard 7 + 3 backbone with the two targeted agents midostaurin and GO, based on findings from previous clinical trials suggesting an additive beneficial effect of combining the two drugs in two important genetically defined AML subgroups, CBF and FLT3-mut AML.In the dose-finding part of the trial presented here, the combination of both novel agents and intensive induction resulted in an overall response rate of 92%.
Our results show that the approved single dose of GO 3 mg/m 2 on Days 1 + 4 and the approved dose of midostaurin mg/day on Days 8-21 can be safely combined with intensive chemotherapy in newly diagnosed AML.We draw this conclusion based on the fact that the majority of patients in the dosing cohorts did not experience DLTs.These findings are supported by data from the British NCRI AML19 v2 trial, in which patients were randomized between one or two doses of GO (3 mg/m 2 capped at 5 mg on Days 1 and 4) in combination with cytarabine and daunorubicin.Patients with FLT3 mutations could enter the MIDOTARG pilot and receive 500 mg midostaurin twice daily for 14 days following the completion of chemotherapy.Combinations of GO and midostaurin were well tolerated. 10A retrospective case series from the Czech Republic reports the tolerability of the combination even with three doses of GO. 11 Although the combination of two GO doses was tolerable in our trial, our experience still suggests an increase in AEs compared to chemotherapy with one of the agents alone, indicating an upper dose limit for their double combination.Currently, we are conducting two RCTs evaluating GO plus midostaurin plus 7 + 3 versus GO plus 7 + 3 in CBF AML and versus midostaurin plus 7 + 3 in FLT3-mutated AML.The results of these comparisons will allow a more detailed and robust assessment of the amount of excess toxicity caused by the double combination and, most importantly, measure the additive or synergistic antileukaemic efficacy of adding one or both targeted agents.

AU T HOR C ON T R I BU T ION S
CR and CDB designed the study.CR wrote the study protocol.MR, FF, MW, CDB and CS provided input to the study protocol.CR, CS, LR, LF, BNH, MH, RN, SS, KSE, KW, JHM, JMM, TK, FS, CDB and MB treated patients and provided data.CR, CS, LR, LF, BNH, MH, RN, SS, KSE, KW, JHM, JMM, TK, FS, CDB, MB, FF and MR continuously reviewed safety information.CR, MR, SZ and LR performed statistical analysis and created visualizations.CR and MR developed the first draft.All authors interpreted the data, revised the manuscript and approved its final version.

F U N DI NG I N FOR M AT ION
This study was supported in part by research funding from Novartis and Pfizer.

AC K NOW L E D GE M E N T
Open Access funding enabled and organized by Projekt DEAL.

DATA AVA I L A BI L I T Y S TAT E M E N T
The protocol will be available upon request.During the ongoing part 2 of the study, we are not able to share individual patient-level data outside the participating institutions.

E T H IC S S TAT E M E N T
The trial was conducted in accordance with the principles of Good Clinical Practice (GCP) guidelines and the Declaration of Helsinki, and approved by the ethics committees of the participating sites.

PAT I E N T C ONSE N T S TAT E M E N T
All patients gave written consent prior to trial participation.

C L I N IC A L T R I A L R E GIS T R AT ION (I NC LU DI NG T R I A L N U M BE R)
The study is registered at clini caltr ials.gov under NCT04385290.

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I G U R E 1 Clinical and molecular features and outcome in 12 patients evaluable for efficacy.Clinical and molecular features and outcome in patients treated within the SAL MODULE trial.Left panel, mutational landscape.Each row represents a patient, each column represents a gene/ alteration; in addition, clinical features (left) and ELN22 risk classification (right) are shown.Right panel: swimmer plot displaying individual patient outcomes.ADV, ELN22 adverse risk; AR, allelic ratio; CR, complete remission; ELN22, European LeukemiaNet 2022 risk satisfaction; FAV, ELN22 favorable risk; N, no/not dected.ND, no data/not assessed/not applicable; Y, yes/present.
Patient characteristics of all 12 patients eligible for DLT evaluation.