ASP2215

FLT3 inhibitors for treating Acute Myeloid Leukemia

Mona Hassanein, MD, Muhamad H. Almahayni, MD, Sameh Gaballa, MD, Syed O. Ahmed, MD, Riad El Fakih, M.D

PII: S2152-2650(16)30107-0
DOI: 10.1016/j.clml.2016.06.002
Reference: CLML 802

To appear in: Clinical Lymphoma, Myeloma and Leukemia

Received Date: 4 March 2016
Revised Date: 11 May 2016
Accepted Date: 1 June 2016

Please cite this article as: Hassanein M, Almahayni MH, Gaballa S, Ahmed SO, Fakih, RE, FLT3 inhibitors for treating Acute Myeloid Leukemia, Clinical Lymphoma, Myeloma and Leukemia (2016), doi: 10.1016/j.clml.2016.06.002.

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Title: FLT3 inhibitors for treating Acute Myeloid Leukemia Article Type: Review
Corresponding Author: Dr. RIAD EL FAKIH, M.D Corresponding Author’s email: [email protected]
Corresponding Author’s Institution: KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTER First Author: MONA HASSANEIN, MD
Order of Authors: MONA HASSANEIN, MD; Muhamad H Almahayni, MD; SAMEH GABALLA, MD; SYED O AHMED, MD; RIAD EL FAKIH, M.D

Author affiliations:
RIAD EL FAKIH, KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTER RIADH, RIADH Saudi Arabia MONA HASSANEIN MD, KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTER
Muhamad H Almahayni, MD, KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTER SAMEH GABALLA, MD, MD ANDERSON CANCER CENTER
SYED O AHMED, MD, KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTER

Introduction:

Acute myelogenous leukemia (AML) is a clonal disorder of hematopoietic stem cells caused by acquired and occasionally inherited genetic alterations(1). FLT3 is a tyrosine kinase receptor that plays a role in proliferation and differentiation of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplication (ITD) mutation is a common molecular alteration in AML, occurring in approximately 20% to 30% of AML patients who have a comparatively poor clinical outcome and increased relapse rate(2). FLT3 mutations are divided into 2 categories: (1) FLT3/ITD mutations (internal tandem duplications) in or close to the juxtamembrane domain of the receptor and, (2) single amino acid substitutions (point mutations) in the activation loop of the tyrosine kinase domain (FLT3/TKD mutations)(3). FLT3 tyrosine kinase domain mutations are not common (͠ 7%) and have no characteristic clinical signature or prognostic impact (4), however they may be very significant clinically since they represent a mechanism of resistance to FLT3 tyrosine kinase inhibitors (TKIs)(5). FLT3-ITD modulates cell proliferation, survival and differentiation through constitutive activation of canonical pathways such as PI3K/AKT, MAPK/ERK, STAT5 and cooperates with other recurrent molecular abnormalities to induce acute leukemia(6)Figure 1. Clinically FLT3 mutated AML is associated with large disease burden (leukocytosis, packed bone marrow), and frequently seen in normal- cytogenetic AML; as far as FAB subtype, it’s clearly more common within the M3 subtype, and possibly M5 subtype, and has a fair distribution within the other FAB subtypes(7-10). A number of receptor tyrosine kinase inhibitors (TKIs) targeting FLT3 have been studied in AML patients as monotherapy or in combination with conventional chemotherapeutic regimes (11) Table 1. Early studies of FLT3 inhibitors(12, 13) proved that these drugs were able to inhibit FLT3 phosphorylation and induces apoptosis in vitro and in vivo and in both wild-type and constitutively activated mutant FLT3, however much higher drug concentrations were needed to inhibit FLT3-ITD phosphorylation in vivo. First generation FLT3 inhibitors (including sunitinib, midostaurin, and lestaurtinib) did not achieve sustained and robust FLT3 inhibition in early clinical trials, however second-generation inhibitors demonstrated better FLT3 specificity and better tolerance (14). Resistance to FLT3 inhibitors is challenging. Most patients treated with a single FLT3 inhibitor only had a transient and partial response because of resistance, which greatly limits the efficacy of FLT3-TKIs(15). Multiple mechanisms (point mutations, protective effect by bone marrow stromal cells, plasma inhibitory activity, high levels of FLT3 ligand) have been implicated in the development of drug resistance(5).

AML with FLT3 mutations:

FLT3-ITD mutations are associated with cytogenically normal (CN-AML) AML (65%-70%), high WBC (white blood cell) count, increased peripheral blood and bone marrow blasts, increased risk of relapse and poor OS. Presence of FLT3-ITD mutations is widely accepted as a poor prognostic factor in CN-AML(8). The early reports of the prognostic significance of FLT3/ITD mutation showed that it was an independent adverse factor for OS, prominently in subjects under 60 years of age(16). Outcomes of 854 patients, mostly 60 years of age or younger, who were included in the United Kingdom Medical Research Council (MRC) AML 10 and 12 trials, showed that testing for FLT3/ITD mutation should be part of routine work up because of its important prognostic value(17). The incidence and prognostic impact of FLT3 mutation in elderly are controversial. 140 patients over 55 years of age were enrolled in Southwest Oncology Group (SWOG) study 9031; FLT3 ITDs were detected in 34% of patients, and had no significant prognostic value with respect to OS, but rather with disease resistance(18). These results were comparable, to another study in which FLT3 mutation status was available in 388 of the patients, and was not associated with significantly poor clinical outcomes, but the incidence of FLT3-mutation was only 12%(19). Other retrospective studies showed on subgroup analysis, that old patients with FLT3 ITDs had a worse prognosis(20, 21). The allelic ratio (AR) of FLT3-ITD/WT has clinical implications, a high AR was associated with inferior DFS and OS in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML), treated on Cancer and Leukemia Group B frontline trials(22, 23). In another study, the impact of AR on the outcome of 323 patients with FLT3-ITD–positive AML, who received either intensive chemotherapy or autologous HSCT or allogeneic HSCT as post remission therapy, showed that patients with a high allelic ratio gained a major benefit after allogeneic HSCT performed in first CR(24).

NPM1 mutation is also frequent in CN – AML, as such a significant proportion of patients carry both FLT3-ITD and NPM1 mutations(25) . NPM1 is associated with relatively longer event-free survival, overall survival and a high rate of complete remission. These positive effects are lost in the presence of a coexisting FLT3- ITD(26).

First generation FLT3 Inhibitors: Sunitinib (SU11248)

Sunitinib is a TKI approved for RCC (renal cell carcinoma), GIST (gastrointestinal stromal tumor) and NET (neuroendocrine tumor). It also inhibits KIT, KDR and PDGFR kinases more sensitively than FLT3 kinase(27). In a phase one study Sunitinib as monotherapy led to complete or partial morphological remission in all 4 patients with FLT3-ITD, however the remission was short and patients developed significant toxicities(28). In a subsequent phase I/II trial, Sunitinib demonstrated a complete remission rate of 59% in elderly patients with FLT3 mutation (29).

Midostaurin (PKC412)

This compound inhibits both FLT3-ITD and FLT3-TKD kinase activity. In a phase I trial, 20 patients with FLT3 mutated relapsed/refractory AML or high-grade myelodysplastic syndrome, midostaurin at a dose of 75 mg 3 times daily by mouth decreased peripheral blast count by 50% in 14 patients (70%), seven patients (35%) had a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); it also reduced marrow blast counts by 50% in 6 patients. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology (30). In a combination therapy phase one trial, with daunorubicin and cytarabine induction and high-dose cytarabine post-remission therapy in newly diagnosed patients with AML, midostaurin 50 mg twice daily led to a complete remission rate of 80%. Overall survival of patients with FLT3-mutant AML at 1 and 2 years (0.85 and 0.62, respectively) was similar to the FLT3-wild-type patients (0.78 and 0.52, respectively)(30). Another 95 patients with AML or high risk MDS were treated on a phase II randomized trial with oral midostaurin at 50 or 100 mg twice daily. 71% of the patients with FLT3-mutant and 42% of the patients with FLT3 wild-type achieved significant reduction in circulating and bone marrow blasts ( >50%). The response rate and toxicity were not affected by the dose given(31). Midostaurin (25 or 50 mg orally twice daily) in combination with ATRA (all-trans retinoic acid) and CLAG chemotherapy in relapsed/refractory AML, phase one trial, showed twenty two percent complete remission rate and 11 % achieved complete remission with incomplete count recovery(32).

Results of the RATIFY study has been recently reported demonstrating the benefit of the addition of midostaurin to induction chemotherapy in younger patients with newly diagnosed AML. In this phase III study, 717 patients were randomized to receive standard induction and consolidation chemotherapy with or without midostaurin: patients in the midostaurin arm had 23% improvement in OS (HR
0.77 [p=0.0074]) and a significant prolongation in median survival, of note 57% of patients included in RATIFY trial received an allogeneic stem cell transplant at a Median time of 4.5 to 5.0 months. A significant improvement in EFS was observed after transplant in the midostaurin arm 8.2 versus 3.0 months with placebo (HR, 0.84; P = .025).(33). Midostaurin is currently being studied in the peritransplant setting and in combination with other targeted therapies.

Lestaurtinib (CEP701)

Lestaurtinib is a non-selective staurosporin analogue that inhibits both mutant and wild-type JAK2 with comparable potency in cellular assays for FLT3 inhibition. Lestaurtinib monotherapy showed reduction in disease burden in patients with relapsed/refractory AML, response was short-lived though (34). A phase II trial of oral lestaurtinib monotherapy for 8 weeks in old, unfit and therapy-naïve AML patients showed transient reductions in peripheral blood and marrow blasts and longer periods of transfusion independence in 3 of 5 patients with mutated FLT3 and in 5 of 22 evaluable patients with wild-type mutations(35). Levis and colleagues conducted a study of salvage chemotherapy followed by lestaurtinib for FLT3 mutant AML patients in first relapse. Lestaurtinib was generally well tolerated following chemotherapy, and in the intention-to treat population, there were 29 CRs/PRs in the lestaurtinib arm and 23 in the control arm, with no significant difference in overall survival between the arms(36). One of the therapeutic questions of the ongoing UK MRC AML17 trial is to assess the value of lestaurtinib in patients who are FLT/ITD-positive, following randomization to one of five induction arms, the protocol involves randomization of patients who are FLT3-positive to either receive or not receive lestaurtinib, with either three or four courses of consolidation chemotherapy. It is hoped that results of this and similar studies will elucidate the role and place of these agents in therapy.

Tandutinib

Tandutinib is a potent FLT3 inhibitor that also inhibits PDGFR and c-Kit. In vivo tandutinib was shown to inhibit FLT3-ITD-transformed hematopoietic cell lines and human AML cell lines expressing the mutant FLT3 receptor(37). 40 patients with relapsed/refractory AML or high risk MDS, were treated with tandutinib on a phase I study. 8 patients had FLT3-ITD mutations, 2 out of 5 evaluable patients with FLT3-ITD showed decrease in blast percentage in peripheral blood and bone marrow, the dose limiting toxicity was reversible muscle weakness. Other manageable toxicities were nausea, vomiting and less frequently diarrhea. Myelosuppression was not prominent(38).

Second generation FLT3 inhibitors: Quizartinib (AC220, Daiichi Sankyo)

Formerly known as AC220, Quizartinib was developed to treat FLT3 mutant AML as a “second-generation” inhibitor, It is a multikinase inhibitor also active against KIT and PDGFRA (39). A phase I study of Quizartinib in patients with relapsed/refractory AML, regardless of FLT3 mutation status, was completed using both intermittent (2 weeks on and 2 weeks off) and continuous dosing in sequential 28 day cycles. The maximum tolerated dose was 200 mg/day in the continuous dosing cohort and was not reached in the intermittent dosing cohort; grade III QTc prolongation is one of the main toxicity concerns (40). A variety of phase II studies have been conducted using Quizartinib in the relapsed and refractory setting. What is remarkable about all of these studies, whether patients were in first relapse or later, young or old, is the relatively remarkable composite complete remission rate and partial remission rate, which ranged ranged between 61% and 72%(41, 42). Despite the single-agent activity of Quizartinib, 50% of patients relapse within 3 months. The proposed mechanisms of resistance are discussed below.

Sorafenib

Sorafenib is a multikinase inhibitor active against VEGFR, PDGFR, RAF-1, KIT and FLT3. It is approved for the treatment of HCC (hepatocellular carcinoma) and RCC (renal cell cancer) (43). 16 patients with refractory/relapsed AML were treated on a phase one trial with oral Sorafenib 200-400 mg twice daily, no dose-limiting toxicity was observed. A significant decrease in the bone marrow and peripheral blood blasts was observed in all FLT3-ITD patients(44). A phase I/II trial of sorafenib in combination with idarubicin, high-dose cytarabine, in patients with relapsed AML was reported. The phase I included 10 relapsed patients, 7 of them had FLT3-ITD+ mutation, 4 of the 10 achieved CR. The phase II part had 45 patients, 14 were FLT3-ITD+. Thirty four (85%) achieved CR or PR, including 13 of 14 FLT3-mutated patients. This trial showed that sorafenib can be safely given with this chemotherapy, and also underlined the potent inhibitory effect on FLT3 mutant patients(45). In another phase II trial sorafenib was added to idarubicin, and cytarabine during induction and consolidation, then given alone for up to 1 year as maintenance therapy for patients who completed consolidation. 16 out of 18 patients went to complete remission (CR), the other 2 patients had incomplete platelet recovery but were in CR. 10 out 18 patients eradicated the FLT3-mutated clone, 6 out of 18 patients showed some residual FLT3-mutated cells, and 2 patients showed persistent FLT3-mutated clone. The elimination of FLT3-mutated population at the time of morphologic CR, however, was not predictive of relapse. After a median follow-up of 9 months (range, 1-16 months), 10 (55%) patients had relapsed, with a median CR duration of 8.8 months (range, 1-9.5 months)(46).
Another encouraging phase II trial of sorafenib in combination with azacytidine in patients with relapsed/refractory AML demonstrated an overall response rate of 46%, though remissions were short lived. The combination was relatively well tolerated and could serve as a bridge to transplant(47). A large German phase III study demonstrated the efficacy of sorafenib in the upfront setting of young patients with AML; most patients were FLT3 negative. The EFS/RFS/LFS (but not OS) were superior in the sorafenib arm, supporting the role of TKIs even in FLT3 negative AML(48).

Gilteritinib (ASP2215 Astellas)

ASP2215 is a potent inhibitor of both FLT3-ITD and FLT3-TKD and also has activity against Axl. In a Phase I/II trial, 82 patients with relapsed/refractory FLT3-mutated AML, the ORR (Overall response rate) was 57%, and was 63% in the 68 patients treated with 80 mg dose or higher (49). A phase III study is ongoing comparing ASP2215 to other salvage chemotherapy.

Resistance to FLT3-TKIs:

As opposed to CML, where responses to TKI therapy are rapid and durable, responses to FLT3 inhibitors are transient, lasting for 3 to 6 months due to the emergence of resistance(50). The primary cause of resistance is the acquisition of point mutations in the ATP binding region of the FLT3-KD, thereby altering the conformational state and weakening the binding affinity to specific FLT3 inhibitors. Studies initially identified point mutations in the FLT3-ITD KD as a mediator of resistance to the FLT3 inhibitor midostaurin(11, 51). Zhang and colleges have shown that a mutation in either TKD1orTKD2 was sufficient to provide resistance to sorafenib-induced apoptosis and to upregulate phosphorylated FLT3(52). Nonmutational mechanisms of resistance include upregulation of parallel prosurvival pathways including the FOXO3A, MEK/ERK, PI3K/Akt/mammalian target of rapamycin (mTOR), and signal transducer and activator of transcription (STAT)5/PIM pathways, upregulation of the FLT3 ligand(36) or FLT3 receptor, mutations in other kinases (eg, CBL), activation of antiapoptotic proteins BCL2, MCL1, and BCL-x(L), and tumor microenvironment/stroma-mediated resistance(53). Secondary TKD mutation in FLT3-ITD patients treated with an inhibitor is another cause of resistance and poor outcomes as reported by Alvarado et al (54).

Trials to overcome resistance to FLT3-TKIs: Several agents (AMG 925, SAR302503, Ponatinib, G-749..) might overcome FLT3 Inhibitor resistance as shown in some preclinical studies(55-58).

Crenolanib

The tyrosine kinase inhibitor, Crenolanib, initially developed as an inhibitor of PDGFR, has activity against mutations in the activation loop of FLT3, the most common being a substitution at amino acid D835(59). As the primary mechanism of resistance to the FLT-3 inhibitor Quizartinib (AC220) is development of TKD mutations in the activation loop, it was hypothesized that Crenolanib would be a pan selective FLT-3 inhibitor that would overcome Quizartinib resistance. In a phase II trial in patients with relapsed/refractory AML, Crenolanib had better activity in FLT3-inhibitor naïve patients as compared to previously treated patients (complete remission with incomplete blood count recovery 23% vs 5% respectively)(60). Crenolanib was also tested against a panel of D835 mutant cell lines and showed superior cytotoxicity when compared with other available FLT3 TKIs such as quizartinib and sorafenib(61).NCT02283177 is investigating Crenolanib in combination with induction chemotherapy in patients with newly diagnosed AML with a FLT3-ITD or TKD mutation.

FLT3-TKI in combination with other small molecules

Simultaneous activation of several survival pathways is a frequent problem in neoplastic diseases. In AML redundant activation of the MAPK, JAK/STAT and PI3K/AKT pathway has been described (1). Targeting multiple pathways at the same time has been successful in oncology. In vitro experiments showed synergism by using midostaurin in combination with the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the dual pyruvate dehydrogenase (lipoamide) kinase isozyme 1/PI3K inhibitor BAG956 or sunitinib in combination with the mTOR-inhibitor RAD001 or the MAP kinase–ERK kinase ½ inhibitor AZD6244(62-65). Currently, mTOR-inhibitor RAD001 in combination with the tyrosine kinase inhibitor midostaurin (PKC412) phase I trial is under way (NCT00819546). Synergism has also been demonstrated in combination with the heat shock protein-90 inhibitor 17-AAG and the histone deacetylases inhibitor MS-275(66, 67). Toxicity, sequence and optimal combination of these small molecules needs to be defined in large trials.

FLT3 inhibitors Post-transplant:

FLT3 tyrosine kinase inhibitors are also under active investigation as maintenance therapy after HSCT. Patients bearing the ITD mutation have a higher likelihood of relapse, and it is possible that maintenance therapy with FLT-3 inhibitors will reduce recurrence rates. A multicenter phase I study examined quizartinib maintenance therapy in FLT3 positive AML patients in CR after allogeneic HSCT. Two dose levels were tested: 40 mg (n = 7) and 60 mg (n = 6) given daily in 28 day cycles. Only 1 patient relapsed, suggesting a lower than expected relapse rate. Although the study did not investigate higher doses, 60 mg daily is the recommended dose given also emerging data outside the HSCT realm(68). A phase I study led by the Massachusetts General Hospital team investigated the oral inhibitor tyrosine kinase inhibitor sorafenib. FLT3+ AML patients (n = 22) received the drug after myeloablative (n= 12) or reduced intensity (n= 10) HSCT. The drug was started between HSCT days 45 and 120, and administered continuously for twelve 28-day cycles. Disease status at HSCT was first CR (n =16), second CR (n = 3), or refractory (n= 3). The maximum tolerated dose was 400 mg twice daily. Common toxicities included skin rash and gastrointestinal symptoms. There were no indications of effects on GVHD rates (incidence of chronic GVHD was 42%). After a median follow-up of 14.5 months, 1 year progression-free and overall survival was 84% and 95%(69). These very promising results clearly deserve further evaluation, hopefully in a randomized controlled fashion.

Conclusion:

FLT3 mutations are a frequent finding in AML, and associated with a short RFS (~7 months with FLT3/ITD versus ~ 11 months with other AML subtypes). FLT3/ITD positive AML at relapse is a very aggressive disease with a dire prognosis(70); it is characterized by the shortest survival. Having said that, targeting activated FLT3- kinase represents an attractive option in AML. Several FLT3-tyrosine kinase inhibitors (TKIs) have been tried as monotherapy or in combination with other chemotherapeutic agents. The role and place of these inhibitors in the sequence of AML therapy (induction, consolidation, maintenance), or in combination with conventional chemotherapy still unclear. However with the emerging data from phase II and phase III trials (RATIFY), these agents are gaining grounds and the landscape treatment of FLT3 mutated AML is changing. The guidelines of various major organizations, such as NCCN and ELN, now recommend FLT3 mutation testing upon diagnosis, especially in patients presenting with high disease burden and normal cytogenetics; the same guidelines also recommend treatment with FLT3 inhibitors (in the presence of FLT3 mutation) or enrollment on clinical trials whenever possible. The majority of patients enrolled on FLT3 inhibitors trials so far are advanced, relapsed/refractory cases. In vitro, FLT3 mutated AML cells from newly diagnosed patients do not seem to be “addicted” to FLT3 signaling, a fact probably related to the polyclonal nature of the disease when it first presents and that FLT3 is not a founder mutation but a later event that grant proliferative advantage, unlike in CML where TKI target the driver mutation. Therefore, FLT3 inhibitors are probably targeting a subset of the leukemia cells and thus would have little use as a single agent in this setting. In combination, however, these agents may help a high-allelic-ratio patient achieve remission, and after achieving remission they may help to suppress the relapse of the FLT3/ITD clone. At present, AC220 appears to completely suppress FLT3-ITD autophosphorylation, but in trials the response was transient, and resistance has become evident after relatively short periods of treatment. Multiple mechanisms of resistance are proposed and include ligand interference, poorly understood relationships between mutation and wild-type signals, inadequate dosing, presence of residual dormant, noncycling cells that harbor FLT3-ITD mutations, among other as-yet unknown mechanisms. Several newer agents may overcome resistance and provide sustained inhibition of FLT3 phosphorylation and downstream effectors in FLT3-ITD–expressing cell lines. Next generation FLT3 inhibitors, such as crenolanib, a pan selective FLT-3 inhibitor, showed very promising results in phase II studies. Several FLT3 inhibitors are in phase III clinical trials, and multiple phase I/II trials are exploring a role for these novel compounds in conjunction with conventional chemotherapy or hematopoietic stem cell transplantation are ongoing. These developments and recent studies are grounds for optimism in a disease subset that otherwise runs an aggressive course.

Refrences

1. Kornblau SM, Womble M, Qiu YH et al. Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia. Blood 2006;108(7): 2358-2365.
2. Frohling S, Scholl C, Levine RL et al. Identification of driver and passenger mutations of FLT3 by high-throughput DNA sequence analysis and functional assessment of candidate alleles. Cancer Cell 2007;12(6): 501-13.
3. Dicker F, Haferlach C, Sundermann J et al. Mutation analysis for RUNX1, MLL-PTD, FLT3-ITD, NPM1 and NRAS in 269 patients with MDS or secondary AML. Leukemia;24(8): 1528-32.
4. Abu-Duhier FM, Goodeve AC, Wilson GA et al. Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol 2001;113(4): 983-8.
5. Smith CC, Wang Q, Chin CS et al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature;485(7397): 260-3.
6. Kim HG, Kojima K, Swindle CS et al. FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia. Blood 2008;111(3): 1567-74.
7. Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia;17(9): 1738-1752.
8. Thiede C, Steudel C, Mohr B et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002;99(12): 4326-35.
9. Schnittger S, Schoch C, Dugas M et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 2002;100(1): 59-66.
10. Haferlach T, Schoch C, Schnittger S et al. Distinct genetic patterns can be identified in acute monoblastic and acute monocytic leukaemia (FAB AML M5a and M5b): a study of 124 patients. British journal of haematology 2002;118(2): 426-431.
11. Heidel F, Solem FK, Breitenbuecher F et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood 2006;107(1): 293-300.
12. Levis M, Allebach J, Tse K-F et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 2002;99(11): 3885-3891.
13. O’Farrell A-M, Abrams TJ, Yuen HA et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 2003;101(9): 3597-3605.
14. Wander SA, Levis MJ, Fathi AT. The evolving role of FLT3 inhibitors in acute myeloid leukemia: quizartinib and beyond. Ther Adv Hematol;5(3): 65-77.
15. Kancha RK, Grundler R, Peschel C, Duyster J. Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations. Exp Hematol 2007;35(10): 1522-6.
16. Kiyoi H, Naoe T, Nakano Y et al. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood 1999;93(9): 3074-3080.
17. Kottaridis PD, Gale RE, Frew ME et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001;98(6): 1752-1759.

18. Stirewalt DL, Kopecky KJ, Meshinchi S et al. FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia. Blood 2001;97(11): 3589-3595.
19. Daver N, Dumlao TL, Ravandi F et al. Effect of NPM1 and FLT3 mutations on the outcomes of elderly patients with acute myeloid leukemia receiving standard chemotherapy. Clinical Lymphoma Myeloma and Leukemia;13(4): 435-440.
20. Nakano Y, Kiyoi H, Miyawaki S et al. Molecular evolution of acute myeloid leukaemia in relapse: unstable N†ras and FLT3 genes compared with p53 gene. British journal of haematology 1999;104(4): 659-664.
21. Rombouts WJ, Blokland I, Lowenberg B, Ploemacher RE. Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the Flt3 gene. Leukemia 2000;14(4): 675-683.
22. Whitman SP, Archer KJ, Feng L et al. Absence of the Wild-Type Allele Predicts Poor Prognosis in Adult de Novo Acute Myeloid Leukemia with Normal Cytogenetics and the Internal Tandem Duplication of FLT3 A Cancer and Leukemia Group B Study. Cancer Research 2001;61(19): 7233-7239.
23. Whitman SP, Maharry K, Radmacher MD et al. FLT3 internal tandem duplication associates with adverse outcome and gene-and microRNA-expression signatures in patients 60 years of age or older with primary cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. Blood;116(18): 3622-3626.
24. Schlenk RF, Kayser S, Bullinger L et al. Differential impact of allelic ratio and insertion site in FLT3-ITD–positive AML with respect to allogeneic transplantation. Blood;124(23): 3441-3449.
25. Falini B, Mecucci C, Tiacci E et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005;352(3): 254-66.
26. Schlenk RF, Dohner K, Krauter J et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008;358(18): 1909-18.
27. Demetri GD, van Oosterom AT, Garrett CR et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet 2006;368(9544): 1329-38.
28. Fiedler W, Serve H, Dohner H et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005;105(3): 986-93.
29. Fiedler W, Kayser S, Kebenko M et al. Sunitinib and Intensive Chemotherapy in Patients with Acute Myeloid Leukemia and Activating FLT3 Mutations: Results of the AMLSG 10-07 Study (ClinicalTrials.gov No. NCT00783653). Blood;120(21): 1483-1483.
30. Stone RM, DeAngelo DJ, Klimek V et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005;105(1): 54-60.
31. Fischer T, Stone RM, Deangelo DJ et al. Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3. J Clin Oncol;28(28): 4339-45.
32. Ramsingh G, Westervelt P, McBride A et al. Phase I study of cladribine, cytarabine, granulocyte colony stimulating factor (CLAG regimen) and midostaurin and all-trans retinoic acid in relapsed/refractory AML. Int J Hematol;99(3): 272-8.
33. Stone RM, Mandrekar S, Sanford BL et al. The Multi-Kinase Inhibitor Midostaurin (M) Prolongs Survival Compared with Placebo (P) in Combination with Daunorubicin (D)/Cytarabine (C) Induction (ind), High-Dose C Consolidation (consol), and As Maintenance (maint) Therapy in Newly Diagnosed Acute My…. Blood;126(23): 6-6.
34. Smith BD, Levis M, Beran M et al. Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. Blood 2004;103(10): 3669-76.
35. Knapper S, Burnett AK, Littlewood T et al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood 2006;108(10): 3262-70.
36. Levis M, Ravandi F, Wang ES et al. Results From a Randomized Trial of Salvage Chemotherapy Followed by Lestaurtinib for FLT3 Mutant AML Patients in First Relapse. Blood;114(22): 788-788.
37. Kelly LM, Yu JC, Boulton CL et al. CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML). Cancer Cell 2002;1(5): 421-32.
38. DeAngelo DJ, Stone RM, Heaney ML et al. Phase 1 clinical results with tandutinib (MLN518), a novel FLT3 antagonist, in patients with acute myelogenous leukemia or high-risk myelodysplastic syndrome: safety, pharmacokinetics, and pharmacodynamics. Blood 2006;108(12): 3674-81.
39. Zarrinkar PP, Gunawardane RN, Cramer MD et al. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood 2009;114(14): 2984-92.
40. Cortes JE, Kantarjian H, Foran JM et al. Phase I study of quizartinib administered daily to patients with relapsed or refractory acute myeloid leukemia irrespective of FMS-like tyrosine kinase 3-internal tandem duplication status. J Clin Oncol;31(29): 3681-7.
41. Levis MJ, Perl AE, Dombret H et al. Final Results of a Phase 2 Open-Label, Monotherapy Efficacy and Safety Study of Quizartinib (AC220) in Patients with FLT3-ITD Positive or Negative Relapsed/Refractory Acute Myeloid Leukemia After Second-Line Chemotherapy or Hematopoietic Stem Cell Transp…. Blood;120(21): 673-673.
42. Cortes JE, Perl AE, Dombret H et al. Final Results of a Phase 2 Open-Label, Monotherapy Efficacy and Safety Study of Quizartinib (AC220) in Patients ≥ 60 Years of Age with FLT3 ITD Positive or Negative Relapsed/Refractory Acute Myeloid Leukemia. Blood;120(21): 48-48.
43. Wilhelm SM, Carter C, Tang L et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64(19): 7099-109.
44. Zhang W, Konopleva M, Shi YX et al. Mutant FLT3: a direct target of sorafenib in acute myelogenous leukemia. J Natl Cancer Inst 2008;100(3): 184-98.
45. Ravandi F, Cortes JE, Jones D et al. Phase I/II study of combination therapy with sorafenib, idarubicin, and cytarabine in younger patients with acute myeloid leukemia. Journal of Clinical Oncology;28(11): 1856-1862.
46. Al-Kali A, Cortes J, Faderl S et al. Patterns of molecular response to and relapse after combination of sorafenib, idarubicin, and cytarabine in patients with FLT3 mutant acute myeloid leukemia. Clinical Lymphoma Myeloma and Leukemia;11(4): 361-366.
47. Rollig C, Serve H, Huttmann A et al. Addition of sorafenib versus placebo to standard therapy in patients aged 60 years or younger with newly diagnosed acute myeloid leukaemia (SORAML): a multicentre, phase 2, randomised controlled trial. Lancet Oncol;16(16): 1691-9.
48. Röllig C, Müller-Tidow C, Hüttmann A et al. Sorafenib versus placebo in addition to standard therapy in younger patients with newly diagnosed acute myeloid leukemia: results from 267 patients treated in the randomized placebo-controlled SAL-Soraml trial. Blood;124(21): 6-6.
49. Levis MJ, Perl AE, Altman JK et al. Results of a first-in-human, phase I/II trial of ASP2215, a selective, potent inhibitor of FLT3/Axl in patients with relapsed or refractory (R/R) acute myeloid leukemia (AML). In: ASCO Annual Meeting Proceedings. p. 7003.
50. Weisberg E, Sattler M, Ray A, Griffin JD. Drug resistance in mutant FLT3-positive AML.
Oncogene;29(37): 5120-34.
51. Cools J, Mentens N, Furet P et al. Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia. Cancer Res 2004;64(18): 6385-9.
52. Zhang W, Gao C, Konopleva M et al. Reversal of acquired drug resistance in FLT3-mutated acute myeloid leukemia cells via distinct drug combination strategies. Clin Cancer Res;20(9): 2363-74.
53. Zeng Z, Shi YX, Samudio IJ et al. Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML. Blood 2009;113(24): 6215-24.
54. Alvarado Y, Kantarjian HM, Luthra R et al. Treatment with FLT3 inhibitor in patients with FLT3†mutated acute myeloid leukemia is associated with development of secondary FLT3–tyrosine kinase domain mutations. Cancer;120(14): 2142-2149.
55. Li C, Liu L, Liang L et al. AMG 925 is a dual FLT3/CDK4 inhibitor with the potential to overcome FLT3 inhibitor resistance in acute myeloid leukemia. Molecular cancer therapeutics;14(2): 375-383.
56. Kesarwani M, Huber E, Azam M. Overcoming AC220 resistance of FLT3-ITD by SAR302503. Blood Cancer J;3: e138.
57. Zirm E, Spies†Weisshart Br, Heidel F et al. Ponatinib may overcome resistance of FLT3†ITD harbouring additional point mutations, notably the previously refractory F691I mutation. British journal of haematology;157(4): 483-492.
58. Lee HK, Kim HW, Lee IY et al. G-749, a novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of acute myeloid leukemia. Blood;123(14): 2209-2219.
59. Smith CC, Lasater EA, Lin KC et al. Crenolanib is a selective type I pan-FLT3 inhibitor. Proceedings of the National Academy of Sciences;111(14): 5319-5324.
60. Randhawa JK, Kantarjian HM, Borthakur G et al. Results of a phase II study of crenolanib in relapsed/refractory acute myeloid leukemia patients (pts) with activating FLT3 mutations. Blood;124(21): 389-389.
61. Galanis A, Ma H, Rajkhowa T et al. Crenolanib is a potent inhibitor of FLT3 with activity against resistance-conferring point mutants. Blood;123(1): 94-100.
62. Mohi MG, Boulton C, Gu T-L et al. Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs. Proceedings of the National Academy of Sciences of the United States of America 2004;101(9): 3130-3135.
63. Weisberg E, Banerji L, Wright RD et al. Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL–and mutant FLT3-expressing cells. Blood 2008;111(7): 3723-3734.
64. Ikezoe T, Nishioka C, Tasaka T et al. The antitumor effects of sunitinib (formerly SU11248) against a variety of human hematologic malignancies: enhancement of growth inhibition via inhibition of mammalian target of rapamycin signaling. Molecular Cancer Therapeutics 2006;5(10): 2522-2530.
65. Nishioka C, Ikezoe T, Yang J et al. Blockade of MEK/ERK signaling enhances sunitinib-induced growth inhibition and apoptosis of leukemia cells possessing activating mutations of the FLT3 gene. Leukemia research 2008;32(6): 865-872.
66. Al Shaer L, Walsby E, Gilkes A et al. Heat shock protein 90 inhibition is cytotoxic to primary AML cells expressing mutant FLT3 and results in altered downstream signalling. British journal of haematology 2008;141(4): 483-493.
67. Nishioka C, Ikezoe T, Yang J et al. MS-275, a novel histone deacetylase inhibitor with selectivity against HDAC1, induces degradation of FLT3 via inhibition of chaperone function of heat shock protein 90 in AML cells. Leukemia research 2008;32(9): 1382-1392.
68. Sandmaier BM, Khaled SK, Oran B et al. Results of a Phase 1 Study of Quizartinib (AC220) As Maintenance Therapy in Subjects with Acute Myeloid Leukemia in Remission Following Allogeneic Hematopoietic Cell Transplantation. Blood;124(21): 428-428.
69. Chen Y-B, Shuli L, Andrew LA et al. Phase I Trial of Maintenance Sorafenib after Allogeneic Hematopoietic Stem Cell Transplantation for Patients with FLT3-ITD AML. Blood;124(21): 671-671.
70. Ravandi F, Kantarjian H, Faderl S et al. Outcome of patients with FLT3-mutated acute myeloid leukemia in first relapse. Leukemia research;34(6): 752-756.