FLT3 mutations in acute myeloid leukaemia

MYELOPROLIFERATIVE DISORDERS IN PRACTICE 2010; Vol 4 No 2

Clinical review

FLT3 mutations in acute myeloid leukaemia The last two decades have seen significant progress in understanding many of the underlying genetic and molecular abnormalities in acute myeloid leukaemia (AML). In parallel, there has been an evolution of therapeutic regimes with modest gains in survival. An array of mutations and deregulated gene expression have been identified which, even if not causative, contribute to the leukaemic phenotype. Here, we review mutations of Fmslike tyrosine kinase-3 (FLT3) and the associated diagnostic and therapeutic implications.

Risk stratification in AML Large studies in the 1990s demonstrated that the outcome of patients with AML could be predicted on the basis of diagnostic cytogenetics at presentation.1,2 Along with age,3,4 cytogenetics allowed patients to be divided into three risk strata: favourable, poor, and intermediate or standard. The largest group among intermediate-risk patients is those that are cytogenetically normal, and indeed 50% of all patients with AML fall into this category. In the UK Medical Research Council (MRC) AML10 study, these patients had a complete remission rate of 88%, a relapse risk at three years of 49% and a three-year overall survival of 45%.2 However, this category is not homogenous and survival rates vary between 24% and 42%.5 While cytogenetically normal, the leukaemic cells harbour one or more mutations, and a number of such genetic markers have been identified in AML, including mutations of FLT3, nucleophosmin-1 (NPM1), CCAATenhancer-binding protein-alpha (CEBPA), mixed lineage leukaemia (MLL), Wilms tumour-1 (WT1) and BAALC.6 These are broadly separated into class I and class II mutations. Class I (including FLT3 and RAS) mutations confer a proliferative advantage to the affected cells. Class II (including CEPBA, NPM1 and MLL) mutations alter the cells’ ability to differentiate.7 The importance of these studies is reflected in the most recent WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, which recognises AML with NPM1 or mutated CEBPA as distinct diagnostic entities; while AML with FLT3 mutations is not classified as a separate entity, it is recommended that FLT3 mutation analysis be carried out along with NPM1 and CEPBA in patients with AML.8

Mutations in the FLT3 gene FLT3 is a member of the type III receptor tyrosine kinase (RTK) subfamily; the FLT3 gene is located on chromosome 13. The FLT3 RTK consists of five immunoglobulin-like regions in the extracellular region, an intracellular juxtamembrane domain and two tyrosine kinase domains (TKDs).9,10 Paracrine or autocrine activation of this receptor by the FLT-3 ligand results in receptor dimerisation, autophosphorylation and subsequent downstream intracellular signalling. FLT3 and its ligand play an important role in proliferation, survival and differentiation of multipotent stem cells. In the mid-1990s, a Japanese group detected duplicated sequences in the FLT3 gene in the region coding for the juxtamembrane domain.11 More mutations have since been recognised: the two most common are internal tandem duplication (FLT3/ITD) and FLT3/TKD. The former map to the juxtamembrane region, while the latter are mainly point mutations.12,13 The mutations cause ligand-independent constitutive activation of the RTK with autophosphorylation and phosphorylation of downstream proteins involved in signal transduction and subsequent up-regulation of some genes and downregulation of others. They likely arise in a leukaemic stem cell, but may arise in a subclone. FLT3/ITD mutations occur in 23–27% of AML, and 32–34% of cytogenetically normal AML (CNAML) patients.12,14 TKD mutations occur in about 7% overall and 10% of CN-AML patients.13,15 FLT3 mutations are the most common genetic alteration in AML, present in 20–30% of de novo AML. Presence of these mutations varies between cytogenetic subgroups. They are more common in CN-AML (30–34%) and acute promyelocytic leukaemia with t(15;17) (37% ITD and 7.7% D835),15 but less common in core binding factor leukaemias.14,16,17 They also appear to be less common in AML with a complex karyotype. Many studies have shown the presence of FLT3 mutations to be more frequently associated with a higher white blood cell count and higher blast percentage in peripheral blood and marrow.18

FLT3 mutations and outcomes That presence of FLT3/ITD mutations in AML portends a poor prognosis was demonstrated by a number of clinical studies in which they were

S Osman Ahmed BSc MRCP Specialist Registrar in Haematology Panagiotis D Kottaridis MSc PhD FRCPath Consultant Haematologist, Royal Free Hospital, London 13

Clinical review


associated with an increased relapse risk and worse overall survival.12,14,19 Correlation of outcome with FLT3 mutation status, based on analysis of bioarchived samples of 854 patients enrolled in MRC trials AML10 and AML12, showed that the presence of an FLT3/ITD mutation increased the relapse risk to 64%, versus 44% in those without the mutation. Disease-free, event-free and overall survival were significantly inferior in the presence of the mutation (30% versus 46%, 23% versus 39%, 32% versus 44% respectively). As well as the qualitative presence of a mutation, there appeared to be quantitative influence of mutational burden on outcome.14 Further stratification of CN-AML could be carried out on the basis of a concurrent mutation of NPM1, the presence of which was found to be related to a favourable outcome.20 Thus, the worst outcomes were found in FLT3/ITD-positive/ NPM1-negative patients, and the best outcomes in those who were FLT3/ITD-negative/NPM1positive.21 There is some evidence that outcome in the presence of FLT3/TKD mutations may be superior to that in AML with FLT3/ITD mutations, although their impact is controversial.22

FLT3 mutations and transplantation As FLT3 is a poor prognostic marker, it may be reasonable to suggest that patients with FLT3 mutations be considered for transplantation in first remission in the presence of a suitable donor. Analysis of data from the AML10 and AML12 trials showed that, on the basis of a donor versus no-donor analysis, evidence to recommend an allogeneic transplant in the presence of an FLT3 mutation was lacking.23 On the other hand, data published by the German-Austrian Acute Myeloid Leukaemia Study Group suggested that patients with CN-AML who were FLT3/ITD-positive and had a donor had superior relapse-free survival compared with those who did not. The analysis included mutation information for NPM1, CEBPA, MLL and NRAS in addition to FLT3 status.21 Whether FLT3/ITD per se should be an indication for transplantation needs to be further elucidated based on the results of prospective trials incorporating information about the presence or absence of these other mutations.

Targeted therapy for FLT3 The presence of an activating mutation in a tyrosine kinase suggested that targeted inhibition of the constitutively active RTK might be of therapeutic value in a fashion similar to that of imatinib in chronic myeloid leukaemia (CML). A number of small molecules were developed with selective in vitro cytotoxicity against leukaemic 14

blasts harbouring the FLT3 mutations. These include CEP-701 (lestaurtinib), SU11248 (sunitinib) and PKC412 (midostaurin).24 However, these agents may not be comparable in efficacy to the imatinib model: acute leukaemias require multiple hits and FLT3 inhibition might target one mutation while leaving others intact; FLT3 mutations may also occur at a later stage and downstream from the leukaemic stem cell, which would not be targeted by specific inhibitors; finally, the development of resistance is possible. Nevertheless, preclinical and clinical trials with these agents have demonstrated inhibition of FLT3 phosphorylation in a sustained fashion with clearance of peripheral and, to a lesser degree, marrow blasts.24–27 Administration of lowdose cytarabine was shown in vitro to have a synergistic effect on cytotoxicity.26,28 CEP-701 has been shown in early clinical trials as monotherapy in patients with refractory or relapsed AML to be associated with a short-lived reduction in disease burden.27 In Phase II studies of untreated elderly patients, CEP-701 led to responses in three of five patients with FLT3/ITD.25 In a randomised study of relapsed patients receiving chemotherapy with or without CEP-701, ten of 17 patients showed a response versus four of 17 receiving chemotherapy alone. In vitro sensitivity of leukaemic cells and adequate drug levels were required for a response to be observed.28 Of note, it appears that in vitro, cells with FLT3/TKD mutations show less susceptibility to CEP-701-induced cytotoxicity than do blasts expressing FLT3/ ITD.29 One aim of the ongoing AML17 trial is to assess the value of CEP-701 in FLT/ITD-positive patients.30 It is hoped that results of this and similar trials will further elucidate the place of these agents in therapy. Sorafenib, an agent approved for advanced renal and hepatocellular carcinoma, but which also inhibits FLT3/ITD, has been shown to elicit responses in relapsed AML, although numbers in these studies were small.31 Numerous Phase I and II trials are currently investigating the role of these agents as monotherapy or in combination with chemotherapy in the de novo, relapsed and transformed settings.

Future prospects and issues Recent progress in understanding the molecular pathology of AML has helped further refine the system of risk-stratifying patients, with the hope that this will lead to the right patient getting the right risk-adapted therapy in a way that will improve overall outcomes. However, the expansion in clinical study data and potential diagnostics in AML also poses a number of issues. The most important with regard to FLT3/ITD is the


identification of the most appropriate induction and post-remission therapy, combining standard chemotherapy and FLT3 inhibitors. With regard to transplantation, reduction in non-relapse mortality as a result of advances in the treatment of complications may tip the balance in favour of transplantation for patients with FLT3 mutations. With regard to targeted therapies, results of ongoing trials are awaited, and the search continues for a ‘silver bullet’ for unfavourable mutations. An key consideration underpinning the search for better outcomes is whether FLT3 is the causative mutation in AML patients that harbour the mutation, or a feature of the genomic instability and one of many genetic aberrations occurring in the leukaemic clone. The answer will have implications for the efficacy of targeted therapies, which may not then have the same efficacy as imatinib has in CML, where BCR-ABL is sufficient and necessary to cause the phenotype. A further practical consideration is that most hospitals and laboratories will not routinely carry out analysis for FLT3/ITD and other mutations. Cost, logistics and the lack of technical expertise may not make it feasible for all AML patients in a given region to have mutational analysis, not least outside the Western world. In the meantime, enrolment of patients with AML into clinical trials allows physicians to circumvent local logistical limitations and obtain essential information in risk-stratifying patients, and allows more patients to be tested for mutations. Developments in gene-expression profiling may further aid in refining patient risk groups and allow prognostication with greater accuracy5 ■ References 1. Byrd JC, Mrozek K, Dodge RK et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002; 100: 4325–4336. 2. Grimwade D, Walker H, Oliver F et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties. Blood 1998; 92: 2322–2233. 3. Juliusson G, Antunovic P, Derolf A et al. Age and acute myeloid leukemia: real world data on decision to treat and outcomes from the Swedish Acute Leukemia Registry. Blood 2009; 113: 4179–4187. 4. Stone RM, O'Donnell MR, Sekeres MA. Acute myeloid leukemia. Hematology Am Soc Hematol Educ Program 2004: 98–117. 5. Mrozek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD. Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification? Blood 2007; 109: 431–448. 6. Mrozek K, Bloomfield CD. Chromosome aberrations, gene mutations and expression changes, and prognosis in adult acute myeloid leukemia. Hematology Am Soc Hematol Educ Program 2006: 169–177. 7. Gilliland DG, Jordan CT, Felix CA. The molecular basis of leukemia. Hematology Am Soc Hematol Educ Program 2004: 80–97. 8. Swerdlow SH, Campo E, Harris NL et al (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th edn. Lyon, France: IARC Press, 2008. 9. Abu-Duhier FM, Goodeve AC, Wilson GA et al. Genomic structure of human FLT3: implications for mutational analysis. Br J Haematol 2001; 113: 1076–1077. 10. Parcells BW, Ikeda AK, Simms-Waldrip T, Moore TB, Sakamoto KM. FMS-like tyrosine kinase 3 in normal hematopoiesis and acute myeloid leukemia. Stem Cells 2006; 24: 1174–1184. 11. Nakao M, Yokota S, Iwai T et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996; 10: 1911–1918. 12. Frohling S, Schlenk RF, Breitruck J et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute

Clinical review

myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood 2002; 100: 4372–4380. 13. Yamamoto Y, Kiyoi H, Nakano Y et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97: 2434–2439. 14. 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: 1752–1759. 15. 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: 983–988. 16. Care RS, Valk PJ, Goodeve AC et al. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol 2003; 121: 775–777. 17. Noguera NI, Breccia M, Divona M et al. Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol. Leukemia 2002; 16: 2185–2189. 18. 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: 59–66. 19. Kottaridis PD, Gale RE, Linch DC. Flt3 mutations and leukaemia. Br J Haematol 2003; 122: 523–538. 20. Schnittger S, Schoch C, Kern W et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 2005; 106: 3733–3739. 21. Schlenk RF, Dohner K, Krauter J et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008; 358: 1909–1918. 22. Mead AJ, Linch DC, Hills RK et al. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood 2007; 110: 1262–1270. 23. Gale RE, Hills R, Kottaridis PD et al. No evidence that FLT3 status should be considered as an indicator for transplantation in acute myeloid leukemia (AML): an analysis of 1135 patients, excluding acute promyelocytic leukemia, from the UK MRC AML10 and 12 trials. Blood 2005; 106: 3658–3665. 24. Small D. FLT3 mutations: biology and treatment. Hematology Am Soc Hematol Educ Program 2006: 178–184. 25. 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: 3262–3270. 26. Knapper S, Mills KI, Gilkes AF et al. The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of cytotoxicity varies with dependence on FLT3 signaling in both FLT3-mutated and wild-type cases. Blood 2006; 108: 3494–3503. 27. 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: 3669–3676. 28. Levis M, Smith BD, Beran M et al. A Randomized, Open-Label Study of Lestaurtinib (CEP-701), an Oral FLT3 Inhibitor, Administered in Sequence with Chemotherapy in Patients with Relapsed AML Harboring FLT3 Activating Mutations: Clinical Response Correlates with Successful FLT3 Inhibition. ASH Annual Meeting Abstracts 2005; 106: 403. 29. Mead AJ, Gale RE, Kottaridis PD et al. Acute myeloid leukaemia blast cells with a tyrosine kinase domain mutation of FLT3 are less sensitive to lestaurtinib than those with a FLT3 internal tandem duplication. Br J Haematol 2008; 141: 454–460. 30. http://aml17.cardiff.ac.uk/default.html (last accessed 27 July 2010) 31. Metzelder S, Wang Y, Wollmer E et al. Compassionate use of sorafenib in FLT3-ITD-positive acute myeloid leukemia: sustained regression before and after allogeneic stem cell transplantation. Blood 2009; 113: 6567–6571.

Key points ● Karyotype analysis at diagnosis of acute myeloid leukaemia (AML) is fundamental for prognostic stratification. ● FLT3/internal tandem duplication (ITD) mutations can be detected in about 25% of all AML patients. ● FLT3/tyrosine kinase domain (TKD) mutations represent the second most frequent (7–11%) mutation of FLT3. ● The prognostic role of FLT3/TKD remains controversial. ● Prospective trials are needed to elucidate the role of allogeneic stem cell transplantation in patients with FLT3/ITD mutations. ● A number of small molecule tyrosine kinase inhibitors with activity against FLT3 have entered Phase I and II clinical trials.

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