Leukemia (2002) 16, 16991704 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.

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Prognostic signicance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy
N Boissel1, JM Cayuela2, C Preudhomme3, X Thomas4, N Grardel3, X Fund4, I Tigaud4, E Raffoux1, P Rousselot1, F Sigaux2, L Degos1, S Castaigne5, P Fenaux3 and H Dombret1
Departement dHematologie, Hopital Saint-Louis, Paris, France; 2INSERM U462, Hopital Saint-Louis, Paris, France; 3Departement dHematologie, Hopital Claude Huriez, Lille, France; 4Departement dHematologie, Hopital Edouard Herriot, Lyon, France; and 5Service dHematologie, CHG de Versailles, Versailles, France
1

FLT3 internal tandem duplications (FLT3-ITDs) are present in nearly 25% of patients with AML and have been associated with poor response to conventional therapy and poor outcome. We retrospectively evaluated the effect of reinforced courses of chemotherapy on the prognostic value of FLT3-ITDs in 159 AML patients prospectively enrolled in the ALFA-9000 trial, which randomly compared three reinforced induction regimens (standard 3+7 including high-dose daunorubicin, double induction, and timed-sequential therapy). FLT3-ITD was present in 40/159 (25%) blast samples and associated with high WBC (P = 0.002) and cytogenetics (P 0.001) with a higher incidence (35%) in patients with a normal karyotype. There was no difference in CR rate between FLT3-wt and FLT3-ITD patients (80% vs 78%). Relapse-free survival (RFS) was similar in both groups (5-year RFS, 33% vs 32%; P = 0.41), even after adjustment for age, sex, WBC, cytogenetics, and treatment arm. A trend to a worse survival was observed in the FLT3-ITD group (estimated 5-year OS, 23% vs 37%; P = 0.09), mainly in patients with a normal karyotype. This was associated with a dramatic outcome in relapsing FLT3-ITD patients (estimated 3-year postrelapse survival, 0% vs 27%; P = 0.04). These results suggest that the bad prognosis associated with FLT3-ITDs in AML might be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations might thus be useful to intensify induction as well as post-remission therapy in FLT3-ITD patients. Leukemia (2002) 16, 16991704. doi:10.1038/sj.leu.2402622 Keywords: FLT3; acute myeloid leukemia; prognosis

Introduction The fms-like tyrosine kinase 3 (FLT3) belongs to the class III receptor tyrosine kinase (RTK) family, which also includes KIT, FMS and PDGF receptor. FLT3 is expressed by early hematopoietic progenitors and its dimerization by FLT3 ligand (FL), expressed on bone marrow stroma, induces growth control signals in normal hematopoiesis.13 Mutations of the FLT3 gene have been reported in 25% of adult acute myeloid leukemia (AML), in 315% of myelodysplastic syndrome (MDS), and occasionally in chronic myeloid leukemia and lymphoproliferative disorders.47 Usually, the mutated gene presents an internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence, which frequently involves exon 14 and more rarely intron 14 or exon 15,5,8 according to the recently description of the FLT3 locus by Abu-Duhier et al.9 Duplicated sequences of the JM domains have variable lengths but can always be read inframe. The role of FLT3-ITDs in leukemogenesis is still unclear. However, FLT3-ITDs probably lead to a gain of proliferation consecutive to a spontaneous ligand-independent

dimerization and phosphorylation of the FLT3 receptor.10 This constitutive activation of the FLT3 receptor abolishes IL3 dependency in the transfected IL3-dependent cell line 32D,11 and is associated with higher engraftment rates of AML cells in NOD/SCID mouse.12 More recently, punctual mutations of D835 within the A-loop of FLT3 have also been reported in AML and other hematological malignancies, leading to similar constitutive FLT3 activation.13,14 Several retrospective studies have evaluated the prognostic signicance of FLT3-ITDs in childhood and adult AML.12,1521 Internal tandem duplications of the FLT3 gene are observed in all AML subtypes according to the FrenchAmericanBritish (FAB) classication, usually associated with a high white blood cell count (WBC). Higher frequencies of mutations are observed in t(15;17) and normal karyotypes. Among the three favorable cytogenetic translocations, t(8;21) translocation and inversion of the chromosome 16 are rarely associated with FLT3-ITD, as the frequency of FLT3-ITD is increased in AML with t(15;17). In contrast, lower frequencies are found in adverse risk cytogenetics. The inuence of FLT3 status on the achievement of complete remission is unclear. However, the presence of FLT3-ITD was associated with a signicant reduction in relapse-free and overall survival in all young adult studies. The presence of D835 mutation did not seem to be associated with similar clinical features and particularly not with high WBC, but also tended to worsen relapse-free survival.13,14 The aim of the present study was to analyze if a reinforced strategy for remission induction, as used by our group for several years, is able to negate, at least in part, the bad prognosis associated with FLT3-ITD in AML adults. We thus evaluated the clinical features and the prognostic signicance associated with FLT3-ITDs in 159 adults with AML, all prospectively enrolled in a randomized trial comparing three strategies of reinforced induction (standard 3+7 induction with high-dose daunorubicin, double induction, and timed-sequential induction). We conrmed the prevalence of these mutations in an adult population and the prole of associated clinical features. However, we did not observe the strong negative impact of FLT3-ITD on patient outcome, as reported until now. Methods

Patients and treatments

A total of 159 peripheral blood or bone marrow samples from 159 AML adult patients from three institutions were retrospectively analyzed for the presence of FLT3-ITD by PCR on genomic DNA. All these patients (aged 1565 years) had been diagnosed with de novo AML (ie without any antecedent myelodysplastic or myeloproliferative syndrome or prior history

Correspondence: H Dombret, Service Clinique des Maladies du Sang, Hopital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France; Fax: 331 4249 9345 Received 31 October 2001; accepted 23 April 2002

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of exposure to radiation, chemotherapy, or carcinogens) and randomized in the ALFA-9000 prospective trial. Patients with AML-M3 were not considered for inclusion in this trial. Patients were randomized at diagnosis to receive one of the three following induction regimens: arm 1 (high-dose daunorubicin 3+7 induction) consisted of 80 mg/m2/day daunorubicin for 3 days (days 13) and 200 mg/m2/day cytarabine as continuous infusion for 7 days (days 17); arm 2 (double induction) consisted of the same regimen, followed on day 20 by a second induction course comprising 12 mg/m2/day mitoxantrone for 2 days (days 20 and 21) and 500 mg/m2/12 h cytarabine as 3 h i.v. bolus infusion for 3 days (days 2022); arm 3 (timed-sequential induction) consisted of 80 mg/m2/day daunorubicin for 3 days (days 13) and 500 mg/m2/day cytarabine as continuous infusion for 3 days (days 13), followed on day 8 by 12 mg/m2/day mitoxantrone for two days (days 8 and 9) and 500 mg/m2/12 h cytarabine as 3 h i.v. bolus infusion for 3 days (days 810). Salvage therapy consisted of 3000 mg/m2/12 h cytarabine as 3 h i.v. bolus infusion for 6 days (days 16) and 100 mg/m2/day amsacrine for 3 days (days 79). With the exception of younger patients with an HLA-identical sibling and considered by center investigators as eligible for allogeneic bone marrow transplantation in rst complete remission (CR), all other CR patients then received two courses of consolidation. The rst consolidation course was administered in out-patients and consisted of 90 mg/m2/day amsacrine for 1 days (day 1) and 60 mg/m2/12 h s.c. cytarabine for 5 days (days 15). The second consolidation course was an intensive timed-sequential EMA course administered in hospitalized patients comprising 12 mg/m2/day mitoxantrone for 3 days (days 13) with 500 mg/m2/day cytarabine as continuous infusion for 3 days (days 13), followed on day 8 by 200 mg/m2/day etoposide for 3 days (days 810) with 500 mg/m2/12 h cytarabine as continuous infusion for 3 days (days 810). Patients aged more than 50 years received an attenuated-dose EMA course with half doses of cytarabine and etoposide. All patients with AML-M4 and AML-M5 subtypes or with a WBC more than 100 109/l were also randomized at diagnosis to receive central nervous system prophylaxis (six intrathecal injections of 15 mg methotrexate, 30 mg/m2 cytarabine, and 4 mg dexamethasone, followed at the end of the consolidation phase by a 18 Gy cranial irradiation) or not. Preliminary results of the ALFA 9000 trial have been presented at the 38th annual meeting of the American Society of Hematology.22 Final long-term analysis will be published shortly.

dened according to the NCI criteria.23 The duration of CR was calculated from the date of rst CR until the date of rst relapse. Relapse-free survival (RFS) was calculated as survival without relapse or death from the date of rst CR. Post-relapse survival (PRS) was calculated from the date of rst relapse until death. Cytogenetic features were classied according to the CALGB classication: (1) favorable: inv(16) and t(8;21); (2) intermediate: normal karyotype; (3) unfavorable: all other abnormalities.24 Patient characteristics and CR rate comparisons were performed using Fishers exact test for binary variables and the MannWhitney test for continuous variables. Data on treatment failure were estimated by the KaplanMeier method25 and compared using the log rank test.26 Survival comparisons were adjusted for age, sex, initial degree of leukocytosis, randomization arm, and cytogenetics with the Cox model27 and tested by the likelihood-ratio test. A P value less than 0.05 was considered to indicate statistical signicance. All calculations were performed using the STATA software, version 7.0.(Stata Corporation, College Station, TX, USA). Results

Incidence of FLT3-ITDs
A longer genomic PCR product corresponding to an ITD of the FLT3 gene was detected in 40 of the 159 diagnosis samples (25%). The specicity of these products was conrmed by internal probe hybridization (Figure 1). One sample displayed a loss of the wild-type allele, as recently described by Whitman et al28 (Figure 1, lanes 6 and 7). Two samples displayed a longer PCR product of low intensity, suggesting the presence of the FLT3 mutation in a subset of AML cells only (Figure 1, lanes 4 and 9). Patient characteristics according to the presence or absence of FLT3-ITD are presented in Table 1. The presence of FLT3-

FLT3-ITD detection
Genomic DNA extracted from tumor cells harvested at diagnosis was amplied as described elsewhere,15 using primer 11F and 12R located on FLT3 exon 14 and 15, respectively. PCR products were run on a 6% polyacrylamide gel and visualized after bromide ethidium staining. FLT3-ITDs were detected as abnormal longer products. Cases displaying faint abnormal sized bands were conrmed after hybridization with an internal oligonucleotide probe 5 -TGAGACTCC TGTTTTGCTAAT- 3 , located on exon 15.

Statistical analysis
Response to initial therapy was evaluated after induction or after induction and salvage. Complete remission (CR) was
Leukemia

Figure 1 FLT3 PCR products. (Top) Reverse view of ethidium bromide stained 6% polyacrylamide gel. (Bottom) Autoradiography after hybridization with an internal 32P-labeled internal oligonucleotide probe. Lanes 1 to 5 and 8 to 11 show PCR results in nine patients, including three patients with FLT3-ITD (lanes 2, 4 and 9). The two patients with mutated PCR products of low intensity are represented (lanes 4 and 9). Lanes 6 and 7 show PCR results obtained in the patients displaying a loss of the wild-type FLT3 allele (lane 6: peripheral blood; lane 7: bone marrow). These results indicate that control hybridization with an internal probe may be performed in the absence of systematic sequencing of PCR products. MWM : molecular weight marker VI (Boehringer, Ingelheim, Germany). Arrow, position of a wild-type FLT3 allele (329 bp).

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Table 1

Patient characteristics (n = 159)

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Total
No. (patients) Median age (range) WBC (109/l) (range) Sex (M/F) FAB subtypes MO M1 M2 M4 M5 M6 M7 Unclassied Cytogenetics Favorable t(8,21) inv(16) Unfavorable Intermediate Unknown Induction 3+7 Double Timed-sequential 159 44y (1565) 21 (0.6400) 79/80 1 38 39 28 36 5 4 8 23 10 13 41 79 16 52 57 50

FLT3/ITD
40 43y (1964) 59 (0.6400) 14/26 0 16 6 4 11 1 0 2 0 0 0 7 28 5 9 15 16

wt FLT3
119 44y (1565) 13 (1.4329) 65/54 1 22 33 24 25 4 4 6 23 10 13 34 51 11 43 42 34

P values

0.002 * 0.04**

0.001**

0.0008**

WBC, white blood cells; FAB, FrenchAmericanBritish. *, by the MannWhitney test; **, by the Fisher test.

ITD was not related to patient age, but was more frequently observed in females when compared to males (32% vs 19%; P = 0.04). As previously reported, FLT3-ITDs was strongly associated with high WBC (P = 0.002, by the MannWhitney test) and normal karyotype (P 0.001) with a higher 35% incidence in the 79 patients from the intermediate cytogenetic subgroup. Conversely, FLT3-ITD was never detected in the 23 patients from the favorable cytogenetic subgroup with t(8;21) translocation (10 patients) or inversion of the chromosome 16 (13 patients).

Outcome of patients with FLT3/ITD

One hundred and twenty-six of the 159 patients (79%) achieved a complete remission (CR). In univariate analysis, CR rate was similar in patients with or without FLT3-ITD (78% vs 80%; P = 0.82). The need of a salvage course to reach the CR was also comparable in patients with or without FLT3-ITD (one out of 31 vs 11 out of 95; P = 0.29). As indicated in Figure 2, relapse-free survival (RFS) was similar in patients with or without FLT3-ITD (estimated 5-year RFS, 32% vs 33%; P = 0.41 by the log-rank test). This result remained unchanged after censoring at transplant time the nine patients from the wild-type FLT3 group and the ve patients from the FLT3-ITD group who received an allogeneic transplantation in rst CR (not shown). As indicated in Figure 3, overall survival was slightly shortened in patients with FLT3-ITD, but this difference did not reach statistical signicance (estimated 5-year survival, 23% vs 37%; P = 0.09 by the log-rank test). A similar trend was observed in the 79 patients from the intermediate cytogenetic subgroup (P = 0.07 by the log-rank test), while overall survival was strictly identical in the 41 patients from the unfavorable

Figure 2 Relapse-free survival according to the FLT3 status. Patients with FLT3-ITD (n = 31) or not (n = 95) have similar relapsefree survival (P = 0.41 by the log-rank test).

cytogenetic subgroup (P = 0.71 by the log-rank test). Here again, results remained roughly unchanged after censoring allografted patients at transplant time. In six variable multivariate analyses including age (as a continuous variable), sex, randomization arm, WBC (as a continuous variable), cytogenetics (as a three subgroup variable), and FLT3 status, the only prognostic factors identied were cytogenetics for RFS P = 0.05) and overall survival (P 0.001) and age for overall survival (P = 0.02). In this multivariate model, the presence of FLT3-ITD had no prognostic value for RFS (relative risk in the FLT3-ITD group, 0.97; 95% CI 0.54
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Discussion Since the rst clinical study on FLT3-ITD in AML patients reported in 1999 by Kiyoi et al,15 three others studies have conrmed the poor prognosis associated with FLT3-ITD in young adults with AML (Table 2).12,18,21 It appears that FLT3ITDs are associated with a signicant increase in relapse rate rather than a decrease in the rate of patients reaching complete remission. Furthermore, in the recent large study from the Medical Research Council, the presence of FLT3-ITD was the worst prognosis factor for CR duration and RFS when tested in multivariate analyses.21 In the ALFA-9000 trial, our group raised the question whether RFS could be prolonged in young adults with AML using intensied induction strategies.22 In the present study, we thus evaluated retrospectively the prognosis signicance of FLT3-ITD in 159 young adults treated in this protocol. Patients were randomized to receive higher than conventional doses of chemotherapy for induction. One induction arm comprised higher doses of daunorubicin than standard 3+7 induction regimens (240 mg/m2 vs 135150 mg/m2). The two other induction arms were based on double chemotherapy strategies (double induction and timed-sequential chemotherapy). In addition, post-remission therapy also included one course of timed-sequential therapy in all patients. The incidence of FLT3-ITD observed in our study (25%) was similar to the 1327% reported before (Table 2). As internal duplications of the FLT3 gene were always found in-frame, we did not sequence them as sequencing of FLT3 mutants is now considered as useless in FLT3-ITD studies dealing with large patient populations.21,29 We conrmed the association of FLT3 mutations with higher WBC, and also observed lower frequencies of FLT3-ITD in t(8;21), inv(16) and high risk cytogenetics, as normal karyotypes were associated with a higher frequency of FLT3-ITD. We were, however, unable to observe similar conclusions as displayed in Table 2. In the context of intensied induction, the presence of FLT3-ITD was not associated with a lower CR rate or RFS. We only observed a trend for a worse overall survival associated with a very short outcome in the FLT3-ITD population after relapse. This observation may be related to the results reported by the German AMLCG Group. In the AMLCG study, the loss of the normal wild-type allele, which is a relatively rare event at AML diagnosis, was more frequently detected at AML relapse (S Schnittger et al, personal communication).30 Moreover, a recent study pointed out the very bad outcome associated with the loss of FLT3 wild-type allele in a group of young adults with primary AML and normal cytogenetics.28 It is thus very likely that increasing rates of mutant receptors, as reported at relapse time, induce increasing rates of constitutively active homodimers, leading to a worse prognosis after relapse. Unfortunately, FLT3 status was not evaluated at relapse time in the present study, because of the lack of available material. Overall, the dramatic outcome observed in these patients after relapse may legitimate intensive post-remission treatment including allogeneic bone marrow transplantation in rst complete remission. Given the relatively low number of patients tested in the present study, the lack of prospective comparison with a standard-dose induction regimen, and the lack of randomization stratication for FLT3-ITD, we can not strongly conclude that intensifying induction regimen interacts with the poor prognosis previously reported in FLT3-ITD patients. However, as

Figure 3 Overall survival according to the FLT3 status. A trend for a longer survival was observed in patients without FLT3-ITD (n = 119) when compared to those with FLT3-ITD (n = 40) (P = 0.09 by the logrank test).

to 1.74; P = 0.91) or survival (relative risk in the FLT3-ITD group, 1.25; 95% CI 0.77 to 2.04; P = 0.37). To further analyze the trend for a worse overall survival (observed in univariate analysis only) in the context of similar CR rates and RFS, we compared the post-relapse survival (PRS) in patients with or without FLT3-ITD. Overall, 68 of the 126 CR patients relapsed (51 in the wild-type FLT3 group and 17 in the FLT3-ITD group). Complete remission duration was similar in both FLT3 groups (estimated 5-year relapse rate, 67% in the wild-type group vs 68% in the ITD group; P = 0.44 by the log-rank test). As indicated in Figure 4, PRS was, however, signicantly shortened in patients with FLT3-ITD (estimated 3-year PRS, 0% vs 27%; P = 0.04 by the log-rank test), even after adjustment on cytogenetics using the Cox model (P = 0.05).

Figure 4 Post-relapse survival according to the FLT3 status. Postrelapse survival was signicantly longer in patients without FLT3-ITD (n = 51) than in those with FLT3-ITD (n 17) (P = 0.04 by the logrank test).
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Table 2

Previous FLT3-ITD prognostic signicance in young adults with AML

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References

No. (patients)

Age (range)

% FLT3/ITD

CR rates (P value)

Survival rates OS (P) DFS (P)

NC 0.006 0.001 0.001

Protocols

Abu-Duhier et al18 Kiyoi et al15 Kottaridis et al21 Rombouts et al12

106 201 854 81

41y 49y 41y 51y

(1574) (NC) ( 1563) (1688)

13 23 27 22

NC NS 0.05 0.03

0.0002 0.004 0.001 0.002

UKMRC AML-10, -11, -12 AML-87, -89, -92 UKMRC AML10, AML12 HOVON-29, -4, -31, AML11

OS, overall survival; DFS, disease-free survival; NS, non signicant; NC, non communicated.

recently reported by the German Multicenter Treatment Trial,31 our study suggests that the bad prognosis associated with FLT3-ITD may be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations in newly diagnosed AML patients might thus be useful to intensify induction as well as post-remission therapy in those with FLT3-ITD. References
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