Abstract
Purpose:
To evaluate the safety, activity, and emergence of FLT3-kinase domain (KD) mutations with combination therapy of crenolanib and sorafenib in acute myeloid leukemia (AML) with FLT3-internal tandem duplication (ITD).
Experimental Design:
After in vitro and xenograft efficacy studies using AML cell lines that have FLT3-ITD with/without FLT3-KD mutation, a pilot study was performed with crenolanib (67 mg/m2/dose, three times/day on days 1–28) and two dose-levels of sorafenib (150 mg/m2/day and 200 mg/m2/day on days 8–28) in 9 pediatric patients with refractory/relapsed FLT3-ITD-positive AML. Pharmacokinetic, pharmacodynamic, and FLT3-KD mutation analysis were done in both pre-clinical and clinical studies.
Results:
The combination of crenolanib and sorafenib in pre-clinical models showed synergy without affecting pharmacokinetics of each agent, inhibited p-STAT5 and p-ERK for up to 8 hours and led to significantly better leukemia response (P<0.005) and survival (P<0.05) compared with single agents. Fewer FLT3-KD mutations emerged with dose-intensive crenolanib (twice daily) and low-intensity sorafenib (three-times/week) compared to daily crenolanib or sorafenib (P<0.05). The crenolanib and sorafenib combination was tolerable without dose-limiting toxicities and 3 complete remissions (1 with incomplete count recovery) and 1 partial remission were observed in 8 evaluable patients. Median crenolanib apparent clearance showed a non-significant decrease during treatment (45.0 L/h/m2, 40.5 L/h/m2, and 20.3 L/h/m2 on days 1, 7, and 14, respectively) without drug-drug interaction. Only 1 patient developed a FLT3-KD mutation (FLT3 F691L).
Conclusions:
The combination of crenolanib and sorafenib was tolerable with antileukemic activities and rare emergence of FLT3-TKD mutations, which warrants further investigation.
Keywords: crenolanib, sorafenib, combination chemotherapy, pharmacokinetics, mutation, children
INTRODUCTION
Children with acute myeloid leukemia (AML) and activating FLT3 internal tandem duplication (ITD) mutations, which occur in ~15% of cases, have a poor event-free survival of 25–35% and are at high risk for disease relapse after conventional chemotherapy (1–3). Midostaurin and gilteritinib, tyrosine kinase inhibitors that target FLT3, have been approved for treatment of adult AML patients with FLT3 mutations and a variety of inhibitors are under development, although none are approved in children (1,4–7). FLT3 tyrosine kinase inhibitors (TKIs) are classified as type I and type II. Type I inhibitors (e.g., midostaurin, gilteritinib, and crenolanib) bind to the active conformation of the FLT3 receptor, while type II inhibitors (e.g., sorafenib and quizartinib) bind to the inactive conformation (8–12). Type I inhibitors inhibit FLT3 signaling in AML cells with ITD and/or kinase domain (KD) mutations but type II inhibitors exhibit greatly reduced activity in those with FLT3-KD mutations. Initial anti-leukemic activity has been observed with single-agent TKI therapy, but responses are often transient, and most patients develop resistance. A primary mechanism of clinical resistance to FLT3 inhibitors, particularly those with type II properties, is the development of secondary KD mutations in FLT3 (8–12).
We and others have proposed combination therapy with both a type I and type II FLT3 inhibitor as an effective strategy to target secondary KD resistance mutations and improve efficacy in FLT3-ITD-positive AML (13,14). Crenolanib, a second-generation inhibitor, showed high selectivity for FLT3 and preclinical activity against drug resistant FLT3-KD mutations (13–16). We hypothesized that using different combination strategies of sorafenib, a type II inhibitor, and crenolanib, a type I inhibitor, would alter resistance profiles in FLT3-ITD positive AML cells, and identify an effective schedule that could suppress the selection of clones with newly detected FLT3-KD mutations.
In the present study, we characterized the preclinical antileukemic activity of crenolanib and sorafenib administered in combination along with FLT3-KD mutational profiling in a FLT3-ITD-positive AML bone marrow xenograft mouse model. Then, we conducted a pilot study in pediatric patients with relapsed or refractory FLT3-ITD-positive AML with this combination to evaluate tolerability, response, pharmacokinetics and pharmacodynamics of both agents, and emerging mutation profiles.
MATERIALS AND METHODS
In vitro studies
Combinations of FLT3 inhibitors with type I (crenolanib, midostaurin) and type II (quizartinib, sorafenib) properties and representing more selective (crenolanib, quizartinib) and multikinase (midostaurin, sorafenib) inhibitors were evaluated in MOLM13 cells, containing a FLT3-ITD mutation, and MOLM13-RES cells, containing an additional FLT3 D835Y mutation, as described previously (14). Cells were exposed to increasing drug concentrations at a fixed concentration ratio for 72 h. Cell viability was assessed using the MTT cell proliferation reagent in 3 independent experiments (18 total replicates). Combination index (CI) values were determined using the method of Chao and Talalay using the computer software CalcuSyn (Biosoft) (17). CI values > 1.0, = 1.0, or < 1.0 represent antagonism, additive effect, or synergism, respectively.
FLT3-ITD positive MOLM13 xenograft mouse model
A FLT3-ITD positive MOLM13 bone marrow xenograft mouse model was used to model FLT3 inhibitor resistance in vivo. 1×106 MOLM13 cells were administered via tail vein injection to 8–12 weeks old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratories). Cell engraftment was assessed biweekly by noninvasive bioluminescence imaging, as previously described (14,18). In a pilot study (Supplementary Fig. 1), mice were treated with sorafenib 60 mg/kg orally once daily for 5 days per week until leukemic progression.
Using previously published sorafenib and crenolanib pharmacokinetic data in NSG mice (14,18), simulations were performed to characterize crenolanib and sorafenib plasma exposure achieved at different doses and schedules administered to NSG mice and to identify clinically relevant schedules for combination therapy in the efficacy studies (Supplementary Fig. 2). Plasma concentration-time data were analyzed using population pharmacokinetic modeling via Monolix version 4.3.2 (19). Area under the concentration-time curve (AUC) over a 24-hour [AUC(0–24h)] or 48-hour [AUC(0–48h)] interval during steady-state dosing was estimated using the post-hoc model estimated curves for each individual mouse. Previously, crenolanib 15 mg/kg was identified as the maximum tolerated single-dose in NSG mice, and 15 mg/kg administered twice daily for 5 days per week was the maximum tolerated schedule (14).
For efficacy studies, mice were randomized to treatment groups based on signal intensity on day 10 after injection of MOLM13 cells and drug treatments were started. Sorafenib for oral administration and crenolanib for i.p. injection was formulated as previously described (14,18). Crenolanib was given twice daily. Sorafenib was administered once daily for 5 days per week, or every-other-day three-times a week on Monday, Wednesday, and Friday [MWF] alone or in combination with crenolanib given once or twice daily on the following schedules: sorafenib and crenolanib once daily (combination 1), sorafenib MWF with crenolanib twice daily (combination 2), and sorafenib once daily with crenolanib twice daily (combination 3). Combination 3, with full dose intensity of both drugs, was not tolerated after 1 week of treatment due to excessive weight loss and was not evaluated further. Leukemic burden was monitored with bi-weekly bioluminescence imaging, and mice were observed daily and euthanized humanely upon signs of terminal illness (e.g., lethargy, scruffy appearance, hunched, domed head, breathing heavy, or hind limb paralysis) or 20% decrease in body weight from baseline. Among 6 treatment groups involving a total of 52 mice, 6 mice were sacrificed before showing overt signs of terminal illness: (i) 2 mice in the vehicle-treated group were taken before signs of leukemia due to high imaging signaling; (ii) 3 mice were taken after the median survival was met in the respective treatment group (2 mice in the combination group 1, and 1 mouse in the combination group 2); and (iii) 1 mouse in the combination group 2 was taken without overt signs of leukemia symptoms but had lost 19% of body weight. Because the primary endpoint was time until sacrifice due to leukemia symptoms, survival analysis was conducted using Gray’s method (20) to include sacrifice for high imaging signal as a competing event; and mice taken for reasons other than leukemia symptoms were censored. Median survival times were estimated and pairwise comparisons of each treatment group were performed with Holm’s P-value (21). All animal studies were approved by the Animal Care and Use Committee at St. Jude Children’s Research Hospital.
FLT3 KD mutation analysis in mouse bone marrow MOLM13 cells
At the time of leukemic progression, mice were sacrificed, and bone marrow was harvested for assessment of leukemic cell infiltration and isolation of DNA or RNA. After bone marrow was collected, red blood cells were lysed (R7757, SIGMA), MOLM13 cells were stained using human CD45-PE antibody (130–091-230), labelled with PE-antibeads (130–048-801), and separated from mouse bone marrow cells using positive selection on AUTOMACS (all from MiltenyiBiotec). Cytospins were prepared of MOLM13 cells before and after MACS sorting, stained with Modified Wright-Giemsa Stain, and cells were counted under the microscope (40x) to determine percentage of leukemic infiltration. Further enrichment of MOLM13 cells was achieved after MACS sorting (median, 88% pre-sort; median 98% post-sort).
DNA was isolated from 0.5×106 MOLM13 cells using the Maxwell® 16 System (Promega). RNA was isolated from 1×106 cells using standard Trizol-Cholorform extraction. cDNA was generated from 0.5 μg of RNA using the SuperScript III First-Strand Synthesis System (ThermoFisher Scientific). FLT3-KD mutations were analyzed by deep amplicon sequencing using either 100 ng of DNA or 125 ng of cDNA. Libraries were prepared for either exon 17 or 20 using the Nextera XT kit and run on the Illumina HiSeq 2000. Sequences were aligned using CLC Genomics Workbench 6. Mutation burden in bone marrow MOLM13 cells obtained from mice across the different treatment groups was defined as the proportion of covering reads containing the mutation. The burden was set to zero when this proportion was less than 0.001 = 0.1% (set as lower limit of assay detection). For each bone marrow sample, the total mutation burden was defined as the sum of the burden of the individual mutations. The permutation t-test (10,000 permutations) was used to compare the total mutation across all treatments for all studies.
Patients
Children, adolescents, and adults aged 1 to 39 years with relapsed or refractory AML or mixed-phenotype acute leukemia with FLT3-ITD or -KD mutations, irrespective of the number of prior salvage regimens, were eligible. Inclusion and exclusion criteria for the clinical trial are described in the supplementary information. The protocol was approved by the institutional review board of St. Jude Children’s Research Hospital and was registered at ClinicalTrials.gov (NCT02270788). Written informed consent was obtained from all patients or their legal guardians with assent from the patients, as appropriate. The study was conducted in accordance with the Declaration of Helsinki.
Patient treatment plan
Based on the pediatric phase I study in diffuse intrinsic pontine glioma or recurrent high-grade glioma (NCT01393912), crenolanib was administered orally at a daily dose of 200 mg/m2 divided three times per day. In course 1, only one dose of crenolanib (67mg/m2) was given in the morning of day 1 to characterize the single-dose pharmacokinetic profile over a 24-hour period followed by three times daily dosing on days 2 to 28.
Sorafenib was given once daily based on a previous population pharmacokinetic model in children, indicating that estimated sorafenib steady-state trough concentrations at the 10th percentile were above a sorafenib concentration of 143 ng/mL that inhibited phospho-FLT3 of leukemia samples in a plasma inhibitory assay (22). For course 1, sorafenib was administered on days 8 to 28, in order to evaluate crenolanib single-agent pharmacokinetics during the first week of treatment. Sorafenib was administered at a dose of 150 mg/m2 (dose level 1, maximum dose 300 mg) or 200 mg/m2 (dose level 2, maximum dose 400 mg). Dose escalation followed a 3+3 design. Intrathecal (IT) chemotherapy was given on day 8. If central nervous system (CNS) disease was present, IT therapy was given weekly until the cerebrospinal fluid became clear of leukemia.
Additional courses were given for patients responding to therapy and both crenolanib and sorafenib were given on days 1 to 28.
Definition of dose-limiting toxicities in patients
The Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0 were used for toxicity evaluations. Dose-limiting toxicities (DLTs) were evaluated in the first course and included any grade 3 or 4 nonhematologic toxicities related to therapy except for grade 3 elevations of amylase, lipase, or total bilirubin or grade 3 or 4 elevations of ALT, AST, alkaline phosphatase, and gamma glutamyl transferase (GGT) that declined to grade 2 or lower within 14 days; grade 3 hypokalemia, hypocalcemia, hypophosphatemia, or hypomagnesemia that were correctable with oral supplements; grade 3 or 4 infection or fever; and grade 3 skin rash and hand-foot-skin reaction that returned to grade 2 or lower within 7 days. Dose-limiting hypertension was defined as an elevated diastolic blood pressure above the 95th percentile for age and gender that was not controlled by antihypertensive medications within 14 days or grade 4 or higher hypertension. Hematologic toxicities were only considered when bone marrow hypocellularity or aplasia (ANC < 0.5 × 109/L and platelet count < 20 × 109/L) persisted more than 42 days in the absence of leukemia or other causes.
Response criteria in patients
Response to single agent crenolanib was assessed by bone marrow aspiration on day 8 of course 1, and response evaluation to combination therapy was performed on day 28 of each cycle and thereafter if hypoplasia was observed. The bone marrow response to therapy was assessed by morphologic and flow cytometric studies. Complete remission (CR) was defined as bone marrow with <5% blasts, absolute neutrophil count (ANC) with ≥0.5 × 109/L, platelets with ≥75 × 109/L without transfusions, and no evidence of extramedullary disease. CR with incomplete blood count recovery (CRi) was defined as <5% blasts, no extramedullary disease, but ANC <500 cells/μL or platelet count <75,000 cells/μL. Partial response (PR) was defined as bone marrow with 5% to 25% blasts and decrease of at least 50% in blast percentage without extramedullary disease. Resistance disease with clinical benefit was defined as bone marrow with 5% to 25% blasts, decrease of at least 50% in blast percentage, or clearance of peripheral blasts with improvement of symptoms assessed by the investigator. MRD was defined as negative if the level was <0.1% (23).
Crenolanib and sorafenib pharmacokinetic studies in patients
For crenolanib serum pharmacokinetic studies, a blood sample was collected in red top tubes without anti-coagulant on days 1, 7, and 14 of course 1 before and at 0.5, 1, 2, 4, and 8 h after crenolanib administration; an additional 24 h sample was obtained after crenolanib treatment on day 1. For sorafenib plasma concentrations, a blood sample was collected in a green top heparinized tube on day 14 of course 1 before and at 1, 2, 4 and 8h after sorafenib administration. For both agents, one blood sample was collected before the morning dose on days 22 (± 2 days) and 28 (± 2 days) of course 1. Concentrations of crenolanib and sorafenib were measured by validated HPLC-based methods with tandem mass spectrometric detection in the Pharmacokinetics Shared Resource at St. Jude Children’s Research Hospital.
Pharmacokinetic data analysis in patients
The population pharmacokinetic and individual post-hoc estimates of crenolanib and sorafenib were determined by nonlinear mixed-effects modeling with Monolix (version 5.1.1, www.monolix.org), using the stochastic approximation expectation-maximization approach.
A linear one-compartment model with first-order absorption (crenolanib) or zero-order absorption (sorafenib), an absorption lag time, and first-order elimination was used to model the data. The parameters estimated included Tlag, the absorption lag time (in hours); ka, the first-order absorption constant (1/hours) or Tk0, the length of the zero-order absorption phase (hours); V/f, the apparent volume (L/m2); Cl/f, the apparent clearance (L/h/m2), where f is the unidentifiable bioavailability.
In addition, the individual post-hoc parameter values were used to estimate the area under the concentration curve (AUC), Cmax, and Cmin. The interindividual variability and inter-occasion variability of the parameters was assumed to be log-normally distributed. A proportional residual error model was used with assumed normal distribution of the residuals.
Plasma pharmacodynamic studies in patients
For assessment of FLT3 ligand concentrations, blood samples were collected in green top heparinized tubes at pre-treatment on days 7, 22 (±2 days) and 28 (±2 days) of course 1. A Milliplex Human Cytokine/Chemokine Magnet Bead Panel (Millipore Cat#HCYTOMAG-60K) was custom ordered to measure FLT3 ligand. Samples were thawed once and analyzed according to the manufacture’s protocol. Overnight incubations were used to improve assay sensitivity. Two controls were included: control 1 covered the concentration range of 106–221 pg/mL (measured 198.63 pg/mL), and control 2 covered the range of 521–1082 pg/mL (measured 591.27 pg/mL).
Next generation sequencing of FLT3-KD mutations and targeted gene panel in patient samples
DNA was isolated from leukemic blast samples using the QIAamp DNA Mini kit and the Blood and Body Fluid Spin Protocol (Qiagen). FLT3 KD mutations were analyzed by deep amplicon sequencing as previously described (24).
DNA samples were analyzed by a targeted 80-protein coding gene panel for mutation status using amplicon sequencing with the MiSeq platform (Illumina). DNA libraries were prepared and analyzed as previously described (24). Sequencing for FLT3 KD mutations and the targeted gene panel was performed in the Genomics Shared Resource at the Ohio State University.
Data availability
The data generated in this study are available upon request from the corresponding author. The genomic data were generated by the authors and included in the article.
RESULTS
Combinations of crenolanib, a type I inhibitor, and sorafenib, a type II inhibitor, are more effective than single-agent FLT3 inhibitors in vitro and in a mouse model
We evaluated different combinations of type I and type II FLT3 inhibitors in MOLM13 and MOLM13-RES AML cell lines. The combination of crenolanib and sorafenib showed the greatest level of synergism in both cell lines by assessment of combination index values (Fig. 1A). We then investigated the efficacy of prolonged drug treatments in a MOLM13 xenograft model. Starting on day 10 after MOLM13 cell injection, different schedules of sorafenib and crenolanib, alone and in combination, were evaluated with the goal to identify a tolerable and effective TKI combination regimen. In comparison to single-agent cohorts, combination treatment groups induced initial regression in leukemic burden by day 20 and suppressed further outgrowth, as assessed by bioluminescence imaging (Fig. 1B); and reduced leukemic cell infiltration in the bone marrow (P<0.005) (Supplementary Fig. 3A). Survival analysis demonstrated that combination regimens significantly prolonged median survival (Fig. 1C) compared to single-agent treatment groups (crenolanib twice daily with sorafenib thrice weekly versus crenolanib twice daily [P = 0.0021] or sorafenib thrice weekly [P = 0.0033]; sorafenib once daily with crenolanib once daily versus sorafenib once daily [P = 0.028]).
Figure 1. Combinations of crenolanib (CRE), a type I inhibitor, and sorafenib (SOR), a type II inhibitor, are more effective than single-agent FLT3 inhibitors.
(A) Type I and type II FLT3 inhibitors exert differential combinatorial antileukemic effects in MOLM13 and MOLM13 TKI resistant cells. Combination index values > 1.0, = 1.0, or < 1.0 represent antagonism, additive effect, or synergism, respectively. (B) Mean (± SD) whole body bioluminescence signal over the duration of treatment is shown for each treatment group; Vehicle (n=9), CRE BID (n=9), SOR MWF (n=9), SOR QD (n=10), CRE BID + SOR MWF (n=7), and SOR QD + CRE QD (n=8). Mice were treated until signs of leukemic progression. (C) Survival curves of mice in the different treatment groups as shown in Figure 1B. (D) Heatmap of total KD mutation burden across treatment groups (data provided in Supplementary Table 1). Each row represents a treated mouse and each column represents a mutation locus. Mutations in FLT3 exons 17 and 20 were assessed by deep amplicon sequencing. Treatment groups are separated by alternating backgrounds of gray and light blue. The intensity of the red color increases with mutation burden according to the color scale shown at the bottom.
Abbreviations: CRE, crenolanib; SOR, sorafenib; BID, twice daily; MWF, three-times per week on Monday, Wednesday, and Friday; and QD, once daily.
A pharmacodynamic study was conducted following administration of sorafenib, crenolanib, or the combination to assess the effect of drug treatments on FLT3 downstream signaling. For these studies, crenolanib was given twice daily with sorafenib once daily for 2 days. At 1 h after drug administration on day 2, the drug combination inhibited p-STAT5 to 7.5% and p-ERK to 35% of vehicle-treated mice. No inhibition of p-STAT5 or p-ERK was observed with single-agent TKIs (Supplementary Fig. 3B). Recovery of p-ERK and partial recovery of p-STAT5 was observed at 8 h with combination treatment, indicating that at least twice daily dosing of crenolanib may be required for optimal FLT3 inhibition (14,18). To assess the potential for a drug-drug interaction, pharmacokinetic studies were performed in mice throughout the course of the pharmacodynamic study and efficacy studies. Sorafenib and crenolanib plasma exposure were similar when drugs were administered alone or in combination acutely or chronically (Supplementary Fig. 3C).
Crenolanib suppresses the emergence of KD mutations in a MOLM13 xenograft model
At leukemic progression on TKI therapy, MOLM13 cells were isolated from the bone marrow and assessed for FLT3-KD mutations. Mutations were not observed in mice treated with vehicle, whereas KD mutation pattern and burden varied according to treatment group (single agent versus combination) (Supplementary Table 1). KD mutations were observed at more diverse residues with sorafenib administered once daily alone (D835H/Y, N841/K, V843/A, and G846/C) or with low-intensity (once daily) crenolanib (D835/Y/N/A, N841/Y/K), compared to treatment regimens with more-intensive crenolanib administered twice daily alone (D835/H/Y, V843/I), low-intensity (3 days per week) sorafenib alone (V843/A), or the combination (F691L, D835H/Y); in the last combination group, the F691L mutation was observed in only one mouse. In addition, the mutation burden of sorafenib given once daily alone or with crenolanib once daily was significantly greater than in the group treated with vehicle (P = 0.031 and 0.018, respectively); whereas mutation burden in the treatment groups receiving crenolanib twice daily, sorafenib three-times per week, or crenolanib twice daily with sorafenib three-times per week did not differ significantly from the group treated with vehicle (P = 0.23, 1.0, and 0.094, respectively) (Fig. 1D and Supplementary Fig. 4).
Patient characteristics
Based on encouraging preclinical data, we conducted a pilot study of crenolanib in combination with sorafenib in patients with refractory or relapsed hematologic malignancies with FLT3 mutations. All 9 patients enrolled on the trial had AML (median age, 9.7 years; range, 5.6–17.8 years) and their clinical features are summarized in Table 1. Patients had received a median of 3 previous regimens (range, 1–6); 4 had received HCT and 6 had previously been treated with sorafenib. Three patients had active CNS leukemia. All patients had a FLT3-ITD including the one (Patient 8) who developed extramedullary disease (right inguinal nodule) after HCT. This patient developed graft-versus-host disease after initiation of protocol therapy with near complete resolution of lymphadenopathy and, therefore, the chemotherapy was held on day 14 of course 1. Patients received a median of 3 courses (range, 1–4).
Table 1.
Patient characteristics and response.
| Patient | Dose level | Age (year) | Sex | Disease status (All AML) | No. previous regimens | Previous HCT | CNS leukemia | No. Courses Received | PB blast clearance | Response by flow cytometry (% AML cells in marrow) | Best Response | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pre-treatment | Day 8 | Day 28 | Best response‡ | |||||||||||
| 1* | 1 | 14.3 | M | 1st relapse | 1 | Y | N | 3 | Y** | 26 | 8.5 | 1.04 | 0.07 | CR |
| 2* | 1 | 10.6 | M | Primary refractory | 3 | N | N | 3 | Y | 72 | 15.6 | 56.4 | 15.6 | RDCB |
| 3* | 1 | 5.6 | M | Relapsed/refractory | 6 | Y | N | 4 | N | 56 | 48.41 | 20.01 | 8.8 | PR |
| 4 | 2 | 9.7 | F | Primary refractory | 1 | N | N | 2 | Y | 71 | 58.37 | 2.302 | 2.302 | CRi |
| 5 | 2 | 5.7 | M | Primary refractory | 3 | N | N | 1 | Y | 24 | 58.63 | 33.85 | 33.85 | RDCB |
| 6* | 2 | 12.5 | M | Relapsed/refractory | 3 | N | Y | 4 | Y | 98 | 42.38 | 0 | 0 | NR† |
| 7 | 2 | 5.7 | F | Relapsed/refractory | 2 | N | Y | 2 | Y | 92 | 73.05 | 83.67 | 73.05 | NR |
| 8* | 2 | 17.8 | F | Relapsed/refractory | 3 | Y | N | 1 | Y** | 0 | 0 | 0 | 0 | NE‡ |
| 9* | 2 | 6.2 | F | 1st relapse | 1 | Y | Y | 3 | Y | 92 | 22 | 0 | 0 | CR |
Patients who received prior sorafenib therapy.
No peripheral blood at initiation of treatment.
Best response during course 1 or subsequent courses.
Had persistent CNS disease although minimal residual disease was negative in the bone marrow.
This patient did not have PB or bone marrow involvement but presented with right inguinal nodule with positive FLT3-ITD. Response and toxicities were not evaluable because the patient developed graft-versus-host disease and chemotherapy was held but had near complete disappearance of inguinal node adenopathy.
Abbreviations: AML, acute myeloid leukemia; No., number; HCT, hematopoietic cell transplant; CNS, central nervous system; PB, peripheral blood; M, male; F, female; Y, yes; N, No; CR, complete remission; RDCB, Resistant disease with clinical benefit; PR, Partial response; CRi, Complete remission with incomplete blood count recovery; NR, No response; NE, not evaluable; FLT3-ITD, FLT3-internal tandem duplication
Toxicity profile in pediatric patients with relapsed or refractory FLT3-ITD-positive AML
Toxicities in 8 evaluable patients are presented in Table 2. Crenolanib 200 mg/m2 administered in 3 divided doses was well-tolerated and no patients required a crenolanib dose reduction. Sorafenib was administered to the first three patients at 150 mg/m2 from day 8 onward. As no DLTs were observed, sorafenib was dose-escalated to 200 mg/m2 for the next six patients without DLTs. The combination of crenolanib and sorafenib was well-tolerated with most adverse events being grades 1 and 2. Grade 1 rash was seen in 4 patients following the addition of sorafenib, and one of 2 patients with rash treated at dose level 2 concomitantly developed grade 2 hand-foot skin reaction which resolved within 5 days. No grade 4 or 5 toxicities were seen. Grade 3 non-hematologic or non-infectious toxicities were hypokalemia (n=3), increased alanine aminotransferase (n=2), and weight loss (n=1).
Table 2.
Toxicity profile of crenolanib in combination with sorafenib.
| Toxicity | Crenolanib 200 mg/m2 divided three times daily | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||||
| Sorafenib 150 mg/m2 (n=3) | Sorafenib 200mg/m2 (n=5) | All patients (n=8) | ||||||||||
|
| ||||||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
|
| ||||||||||||
| Blood and lymphatic system disorders | ||||||||||||
| Febrile neutropenia | 1 | 1 | ||||||||||
|
| ||||||||||||
| Cardiac disorders | ||||||||||||
| Hypertension | 1 | 1 | 1 | 1 | ||||||||
| Sinus bradycardia | 1 | 1 | ||||||||||
|
| ||||||||||||
| Ear and labyrinth disorders | ||||||||||||
| Ear pain | 1 | 1 | ||||||||||
|
| ||||||||||||
| Eye disorders | ||||||||||||
| Blurred vision | 1 | 1 | ||||||||||
|
| ||||||||||||
| Gastrointestinal disorders | ||||||||||||
| Nausea | 2 | 4 | 6 | |||||||||
| Vomiting | 2 | 4 | 6 | |||||||||
| Diarrhea | 1 | 1 | 2 | |||||||||
| Mucositis oral | 2 | 2 | ||||||||||
| Constipation | 2 | 2 | ||||||||||
| Pancreatitis | 1 | 1 | ||||||||||
| Abdominal pain | 1 | |||||||||||
| Ascites | 1 | 1 | ||||||||||
|
| ||||||||||||
| General disorders and administration site conditions | ||||||||||||
| Fever | 1 | 1 | ||||||||||
| Edema face | 1 | 1 | ||||||||||
| Edema limbs | 1 | 1 | ||||||||||
| Malaise | 1 | 1 | ||||||||||
| Fatigue | 2 | 2 | ||||||||||
|
| ||||||||||||
| Infections and infestations | ||||||||||||
| Enterocolitis infectious | 1 | 2 | ||||||||||
| Otitis media | 1 | 1 | ||||||||||
| Skin infection | 1 | 1 | ||||||||||
| Other (Enterobacter cloacae bacteremia) | 1 | 1 | ||||||||||
| Other (Enterococcus faecium bacteremia) | 1 | 1 | ||||||||||
| Other (Parvovirus) | 1 | 1 | ||||||||||
|
| ||||||||||||
| Injury, poisoning and procedural complications | ||||||||||||
| Postoperative hemorrhage | 1 | 1 | ||||||||||
|
| ||||||||||||
| Investigations | ||||||||||||
| Hypoalbuminemia | 2 | 3 | 2 | 5 | 2 | |||||||
| Hyperglycemia | 2 | 1 | 3 | 3 | 3 | |||||||
| Hypokalemia | 1 | 3 | 2 | 3 | ||||||||
| Hypophosphatemia | 3 | 2 | 3 | 2 | ||||||||
| Hyponatremia | 4 | 4 | ||||||||||
| Hypocalcemia | 1 | 1 | 1 | 2 | ||||||||
| Hypomagnesemia | 2 | 1 | 2 | 1 | ||||||||
| Hyperkalemia | 3 | 3 | ||||||||||
| Hypermagnesemia | 1 | |||||||||||
|
| ||||||||||||
| Metabolism and nutrition disorders | ||||||||||||
| Alanine aminotransferase increased | 3 | 3 | 2 | 3 | 3 | 2 | ||||||
| Aspartate aminotransferase increased | 3 | 3 | 2 | 6 | 2 | |||||||
| Blood bilirubin increased | 1 | 1 | 2 | 1 | ||||||||
| Weight loss | 1 | 1 | ||||||||||
| Serum amylase increased | 1 | 1 | ||||||||||
|
| ||||||||||||
| Musculoskeletal and connective tissue disorders | ||||||||||||
| Pain in extremity | 1 | 1 | 1 | |||||||||
| Back pain | 1 | 1 | ||||||||||
|
| ||||||||||||
| Nervous system disorders | ||||||||||||
| Headache | 2 | 1 | 2 | 1 | ||||||||
|
| ||||||||||||
| Psychiatric disorders | ||||||||||||
| Agitation | 1 | 1 | ||||||||||
|
| ||||||||||||
| Renal and urinary disorders | ||||||||||||
| Cystitis noninfective | 1 | 1 | ||||||||||
| Urinary tract pain | 1 | |||||||||||
|
| ||||||||||||
| Skin and subcutaneous tissue disorders | ||||||||||||
| Rash maculo-papular | 2 | 2 | 4 | |||||||||
| Hand-foot skin reaction | 1 | 1 | ||||||||||
| Urticaria | 1 | 1 | ||||||||||
| Pruritis | 1 | 1 | ||||||||||
Crenolanib and sorafenib pharmacokinetics and pharmacodynamics in pediatric patients with relapsed or refractory FLT3-ITD-positive AML
During week 1 of single agent crenolanib administration, median (range) crenolanib apparent oral clearance (Cl/f) values on days 1 and 7 were 45.0 L/h/m2 (5.4–1112 L/h/m2) and 40.5 L/h/m2 (2.4–162 L/h/m2), respectively, indicating a modest (10%), though not significant decrease in Cl/f over 7 days of treatment. The crenolanib Cl/f values observed in this study were in the range of those observed in adults on days 1 (33 L/h/m2) and 15 (34 L/h/m2) (25). On day 14, after 1 week of combination treatment with sorafenib, median crenolanib Cl/f (20.3 L/h/m2; range, 2.6–162 L/h/m2) decreased, though not significantly, by 55% and 50%, compared to days 1 and 7, respectively; given the extremely wide range of values for crenolanib Cl/f on all pharmacokinetic study days and the small number of subjects, definitive conclusions cannot be made regarding intra-occasion variability in crenolanib Cl/f.
When given in combination with crenolanib, the median sorafenib Cl/f on day 14 was 2.9 L/h/m2 (range, 1.0–3.9 L/h/m2), a result similar to those reported previously in children receiving single-agent sorafenib (22). Crenolanib and sorafenib exposure parameters are summarized in Table 3.
Table 3.
Crenolanib and sorafenib pharmacokinetic parameters.
| Crenolanib | Sorafenib | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|||||||||||
| Crenolanib (mg/m2 divided tid) | Sorafenib (mg/m2 qd) | N | Day | Cmax (ng/mL) | AUC(0–8h) (ng*h/mL) | Cl/f (L/h/m2) | Cmin (ng/mL) | Cmax (mg/L) | AUC(0–24h) (mg*h/L) | Cl/f (L/h/m2) | Cmin (mg/L) |
|
| |||||||||||
| 200 | 150 | 3 | 1 | 135 (29–179) | 710 (164–970) | 51.7 (45.0–184.7) | --- | --- | --- | --- | --- |
| 7 | 190 (101–226) | 1001 (486–1284) | 63.3 (40.5–153.9) | --- | --- | --- | --- | --- | |||
| 14 | 233 (79–393) | 1088 (418–2887) | 62.4 (20.3–162.3) | 64.5 (17.5–158.5) | 4.2 (2.4–5.0) | 89 (43–112) | 1.6 (1.0–3.8) | 3.1 (1.3–4.1) | |||
| 200 | 6 | 1 | 198 (6–384) | 972 (30–1884) | 42.9 (5.4–1112.0) | --- | --- | --- | --- | --- | |
| 7 | 397 (49–1398) | 2479 (330–9142) | 22.9 (2.4–120.4) | --- | --- | --- | --- | --- | |||
| 14 | 465 (91–2571) | 3160 (650–19201) | 16.6 (2.6–111.4) | 229.9 (27.5–1179.0) | 1.8 (1.0–3.4) | 32 (6–61) | 3.0 (2.6–3.9) | 1.1 (0.8–2.2) | |||
|
|
|||||||||||
| All Dose Levels | 1 | 45.0 (5.4–1112.0) | --- | ||||||||
| 7 | 40.5 (2.4–153.9) | --- | |||||||||
| 14 | 20.3 (2.6–162.3) | 2.9 (1.0–3.9) | |||||||||
Abbreviations: tid, three times daily; qd, once daily; Cmax, maximum plasma concentration; AUC, area under the concentration-time curve from time 0 to 8 hours (0–8h) or from 0 to 24 hours (0–24h); Cl/f, apparent oral clearance; Cmin, minimum plasma concentration at the time end of the dosing interval.
FLT3 ligand concentrations over 1 course of treatment are illustrated in Supplementary Fig. 5. Minimal changes in FLT3 ligand concentrations were observed from baseline to day 7 during single agent crenolanib. However, concentrations increased in seven of nine patients between days 7 and 28, during combination treatment. As FLT3 ligand may increase in response to FLT3 inhibition as an acute resistance mechanism, these data suggest target engagement over a cycle of treatment.
Leukemia response in pediatric patients with relapsed or refractory FLT3-ITD-positive AML
Clinical responses in the 9 patients enrolled on the trial are listed in Table 1. Two patients (patients 1 and 8) did not have detectable peripheral blasts at enrollment. Among 7 patients, 5 had decreases (1% or less) in peripheral blast percentages on day 8, and 6 patients had disappearance in peripheral blasts on day 29 (except patient 3) (Figure 2A). For bone marrow evaluation, 7 of 8 evaluable patients had decrease in blasts percentages after 7 days of crenolanib treatment. After crenolanib and sorafenib combination, 2 patients had CR and 1 patient had CRi. For the best response, 2 patients had CR (1 MRD negative), 1 had a CRi, 1 had a PR, and 2 had resistant disease with clinical benefit (Table 1 and Figure 2B). Patient 6 had clearance of bone marrow blasts (MRD negative) but blasts in cerebrospinal fluid persisted. Response was not evaluable in patient 8 because the patient developed graft-versus-host disease and chemotherapy was held on day 14 but near complete disappearance of inguinal node adenopathy was seen. All 3 patients with CR or CRi received HCT and 2 are currently alive (1 died from transplant-related toxicities) (Figure 2C).
Figure 2. Changes in peripheral blood and bone marrow blasts and overall survival.
Changes in blast percentages in (A) peripheral blood and (B) bone marrow are shown during cycle 1. (C) Overall survival is shown for 9 patients treated in this regimen. Patients 4 and 9 (asterisks) are alive without evidence of disease at 54 months and 76 months, respectively. For peripheral blood and bone marrow, patients 1 and 8 and patient 8 did not have detectable blasts at enrollment, respectively.
Abbreviation: Pt, patient
Next generation sequencing of samples in pediatric patients with relapsed or refractory FLT3-ITD-positive AML
FLT3-KD mutations were analyzed before and during treatment with crenolanib and sorafenib. Despite 6 patients receiving prior sorafenib treatment, no baseline KD mutations were observed (Supplementary Table 2). During the course of treatment, the emergence of a low frequency clone with FLT3 F691L (VAF [variant allele frequency] 0.0025) was observed in one patient at the time of disease progression during course 3 after a transient response; similar to what was observed in the MOLM13 xenograft model. All other patients remained clear of any FLT3-KD mutations during treatment.
A targeted 80-gene panel was used to categorize other mutations that co-occurred with FLT3-ITD. We observed baseline mutations in CEBPα, KMT2A, KMT2C, MAPK1, TP53, WT1 as well as other mutations (Figure 3A, Supplementary Table 3) with VAF ranging 0.1 to ~1.0. In two patients who achieved a decline in bone marrow blasts during course 1, no significant change in mutation VAF was observed at course 3 or 4 of treatment compared to baseline (Figure 3B, C). No CR was observed in 4 patients with baseline WT1 mutations while and 3 CR or CRi were seen in 6 patients without mutation.
Figure 3. OncoPrint of individual mutations detected by an 80-gene targeted panel.
Mutations detected (A) before TKI treatment and (B) over the course of TKI treatment. Mutations characterized as: missense (green), in frame (red), truncating (black). Samples identified as R (relapsed/refractory pre-TKI) and T (during TKI treatment). (C) Mutation variant allele frequency (VAF) shown for pre-TKI and during treatment for two patients. Detected FLT3-KD mutation and its frequency are indicated below VAF graph.
Abbreviation: TKI, tyrosine kinase inhibitor
DISCUSSION
Despite significant advances with the use of FLT3 inhibitors in adults with FLT3-ITD-positive AML, and the approval of midostaurin in 2017 and gilteritinib in 2018 (6,7), none are approved for use in children (1–3). Currently, several trials are evaluating FLT3 inhibitors in children. These include midostaurin in combination with chemotherapy in newly diagnosed FLT3 mutated AML (NCT03591510) and gilteritinib for frontline treatment or relapsed/refractory disease (NCT04293562 and NCT04240002). Recently, a small case series of pediatric patients with AML and FLT3-mutations receiving gilteritinib/chemotherapy combination regimens was reported (26). With the paucity of drug development efforts, there remains a continued need to identify effective and tolerable treatment regimens for pediatric AML with FLT3 mutations. In this present study, we demonstrated that crenolanib and sorafenib combinations were effective and prolonged survival compared to either drug alone in a MOLM13 murine xenograft model, as well in comparison to our recent study of gilteritinib using the same preclinical model (median survival, 55 or 58 days for crenolanib and sorafenib combinations versus 31 days for gilteritinib alone) (27). We also demonstrated that the combination regimen using dose-intensive crenolanib in combination with low-intensity sorafenib suppressed the emerging FLT3 KD mutation burden compared to single-agent dose-intensive sorafenib. Based on these promising results, we conducted this pilot study showing that the combination of crenolanib and sorafenib was well-tolerated with no significant pharmacokinetic drug-drug interactions between the two agents. Importantly, this study showed preliminary evidence of antileukemic activity in patients with relapsed or refractory FLT3-ITD-positive AML, with only one case with an emergence of FLT3-KD mutation during treatment.
In this study, we treated nine pediatric patients with FLT3-ITD positive AML. These patients were heavily pre-treated including four patients who had received HCT previously. The combination therapy of crenolanib and sorafenib without cytotoxic chemotherapy was tolerable with no DLTs, and only grade 3 non-hematologic or non-infectious toxicities were observed. Based on data from our prior sorafenib population pharmacokinetic model in children (22), sorafenib was given once daily with the aim to maintain adequate exposure to inhibit FLT3 while reducing the severity of skin toxicities. Indeed, the incidences and severity of skin rash (none for grade 2 or higher) and hand-foot skin reaction (only 1 with grade 2; 12.5%) in this study were quite low as compared to those observed in our previous study of sorafenib in combination with clofarabine and cytarabine in pediatric AML (66.7% and 19.0% for skin rash in concurrent and sequential regiments, respectively, and 66.7% and 28.6% for hand-foot skin reaction) (22,28). In addition, preliminary antileukemic activity was observed. Six of seven evaluable patients had disappearance of peripheral blasts, and CR, CRi, or PR were seen in four of eight evaluable patients. Of the three CR or CRi patients who received subsequent HCT, two are alive in remission. Notably, among three patients who had previous HCT, two had CR and one had PR with counts recovery. Although patient 3 had refractory disease to 5 previous salvage regimens after relapsing from HCT, his disease was controlled at PR for 3 courses. Therefore, the less intensive therapy with combination of type I and type II inhibitors can be an option for patients with FLT3-ITD positive AML, even for those relapsing after HCT.
One potential advantage of developing type I FLT3 inhibitors is their ability to suppress the outgrowth of AML clones carrying newly detected FLT3-KD mutations, which occurs during treatment with type II inhibitors, such as sorafenib and quizartinib, and confers resistance to these agents (8–12). In our patients treated with crenolanib and sorafenib, we did not observe the emergence of FLT3 D835 mutations but only a low frequency clone with FLT3 F691L mutation in one patient. This is consistent with what has been reported in adult patients receiving single agent crenolanib (29). In addition, several co-occurring mutations were observed in pre-treatment samples, primarily in genes associated with epigenetic regulation and transcription factors. In two patients with post-treatment samples, we did not observe post-crenolanib expansion of mutations, but neither were they cleared during treatment. We also observed no CR in patients with baseline WT1 mutations. The worse prognosis of patients with co-occurring FLT3-ITD and WT1 mutations is well documented in pediatric AML (30). While clinical benefit was observed in several patients, existing co-occurring mutations were not cleared, highlighting the need for continued development efforts to improve on existing therapies through combinatorial treatment strategies.
This study is the first to report crenolanib pharmacokinetic data in children. Crenolanib clearance values when administered alone during the first week of treatment were similar to those reported in adults with AML (25). As crenolanib is metabolized by CYP3A4 and sorafenib is metabolized by both CYP3A4 and UGT1A9, we performed pharmacokinetic studies of both agents after co-administration for one week to assess for a potential drug-drug interaction. Although there was a trend for lower crenolanib clearance values on day 14 compared to days 1 and 7, it was not statistically significant. Sorafenib clearance values on day 14 were similar to values we reported previously in children with AML (22). Although there was drug-drug interaction potential between crenolanib and sorafenib through CYP3A4, our previous preclinical pharmacokinetic studies in mice lacking the liver uptake transporter OATP1B, which is a major transporter of the UGT1A9-mediated sorafenib-glucuronide metabolite, indicated that glucuronidation may be the more prominent metabolic pathway for sorafenib (31,32).
In conclusion, we demonstrated that the combination of a type I with a type II FLT3 inhibitor was effective in a preclinical murine model of FLT3-ITD-positive AML in both antileukemic activity and suppression of the emergence of drug-resistant secondary FLT3-KD mutations. Clinically, we determined the drug combination was tolerable, the use of less dose-intensive sorafenib resulted in a lower incidence of severe skin toxicities, preliminary antileukemic activity was observed, and the occurrence of secondary FLT3-KD mutations was uncommon. This regimen provides an option for less-intensive therapy in children with FLT3-ITD-positive AML and warrants further investigation.
Supplementary Material
Translational relevance.
Acute myeloid leukemia (AML) with FLT3-internal tandem duplication (ITD) mutation is associated with poor outcome and can develop resistance to single-agent tyrosine kinase inhibitor therapy by developing FLT3-kinase domain (KD) mutations. We evaluated whether combination therapy with type I (crenolanib) and type II (sorafenib) inhibitors can improve the anti-AML effects of single-agent therapy. In pre-clinical models, the combination showed synergy, inhibited p-STAT5 and p-ERK, and led to significantly better leukemia response and survival of the xenografted mice with fewer FLT3-KD mutations when compared with single agents. A feasibility study in 9 children with refractory/relapsed FLT3-ITD-positive AML showed that crenolanib combined with less intensive once daily sorafenib was tolerable without dose-limiting toxicities or drug-drug interaction. Additionally, preliminary anti-AML activity was observed. Only 1 patient had the emergence of a FLT3-KD mutation (FLT3 F691L). This combination regimen warrants further investigation.
ACKNOWLEDGEMENTS
We thank the clinical, research and pharmacokinetic nurses at St. Jude Children’s Research Hospital who participated in this study. The research reported in this paper was supported by the National Cancer Institute of the National Institutes of Health Cancer Center Support Grants P30 CA021765 and P30 CA016058, R01CA138744 [to SDB], by ALSAC, and by AROG Pharmaceuticals.
Acknowledgment of research support:
This study was supported by National Institutes of Health (NIH) Cancer Center Support Grants P30 CA021765 and P30 CA016058, by NIH R01 CA138744 (to SDB), by ALSAC, and by AROG pharmaceuticals.
Footnotes
Conflicts of interest: Dr. Inaba received research support from AROG pharmaceuticals. The other authors declare no potential conflicts of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data generated in this study are available upon request from the corresponding author. The genomic data were generated by the authors and included in the article.