doi:10.1182/blood-2007-05-091215Prepublished online October 26, 2007;   

 Ulrike Bacher, Claudia Haferlach, Wolfgang Kern, Torsten Haferlach and Susanne Schnittger matters - an analysis of 3082 patientsPrognostic relevance of FLT3-TKD mutations in AML: the combination

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Prognostic relevance of FLT3-TKD mutations in AML:

the combination matters - an analysis of 3082 patients

Ulrike Bacher1, Claudia Haferlach2, Wolfgang Kern2, Torsten Haferlach2,

and Susanne Schnittger2*

1Bone Marrow Transplant Unit

University Hospital of Hamburg-Eppendorf

Martinistr. 52

20246 Hamburg, Germany 2MLL - Munich Leukemia Laboratory

Max-Lebsche-Platz 31

81377 Munich, Germany

Corresponding author:

*Susanne Schnittger

MLL, Munich Leukemia Laboratory

Max-Lebsche-Platz 31

81377 Munich

Phone.: 0049 89 990 17 300

Fax: 0049 89 990 17 309

Email: Susanne.Schnittger@mll-online.com

URL : www.mll-online.com

Blood First Edition Paper, prepublished online October 26, 2007; DOI 10.1182/blood-2007-05-091215

Copyright © 2007 American Society of Hematology

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Abstract

We characterized the mutation status of the FLT3 tyrosine kinase domain (FLT3-TLD)

in 3082 patients with newly diagnosed AML. FLT3-TKD mutations were detected in

147 of 3082 (4.8%) patients. Similar to the FLT3 juxtamembrane domain mutations

(FLT3-LM) there was a high correlation of FLT3-TKD mutations with normal karyotype

(88/1472; 6.0%). FLT3-TKD mutations were most frequent in the AML FAB subtypes

M5b (15/114; 13.2%), M3v (6/51; 11.8%), and M4 (39/484; 8.1%). Similar to FLT3-LM

the FLT3-TKD mutations show elevated peripheral leukocytes as compared to FLT3wt

AML. FLT3-TKD had a high incidence in cases with NPM1 mutations (23/262; 8.8%),

CEPBA mutations (6/76; 7.9%), and NRAS mutations (6/78; 7.7%). FLT3-TKD in

combination with FLT3-LM (17/594 patients; 2.9%) and KITD816 (1/44; 2.3%) was

rare. Unlike the FLT3-LM which are associated with inferior survival, prognosis was

not influenced by FLT3-TKD in the total cohort of 1720 cases, where follow-up data

were available (97 FLT3-TKD; 1623 FLT3-WT). In t(15;17)/PML-RARA with FLT3-

TKD mutations, in FLT3-LM/TKD double mutated, and in MLL-PTD/TKD double

mutated cases prognosis was unfavorably influenced by FLT3-TKD mutations. In

contrast, we found an additional favorable impact of FLT3-TKD on EFS in

prognostically favorable AML with NPM1- or CEPBA mutations.

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INTRODUCTION

Actual therapy concepts try to modify the intensitity of therapy of acute myeloid

leukemia (AML) in accordance to the individual risk of relapse. Karyotype represents

the strongest prognostic parameter in AML. However, 45% of all AML patients show a

normal karyotype and thus an intermediate prognosis.1-3 As AML with normal

karyotype is considered a heterogeneous disease with respect to molecular aspects,

the definition of suitable molecular markers for the determination of prognosis and for

minimal residual disease (MRD) detection is of increasing importance.

Mutations of the fms-tyrosine kinase (FLT3) were first described in 19974 and account

for the most frequent molecular mutations in AML.5;6 The FLT3 gene is a member of

the class III receptor tyrosine kinase family including c-kit, c-fms, and the platelet-

derived growth factor receptors.6-9 In normal bone marrow FLT3 expression is

restricted to immature hematopoietic progenitor cells and mediates stem cell

proliferation.6;8;10;11

FLT3 length mutations (FLT3-LM or FLT3-ITD for “internal tandem duplication”)

represent one of the most frequent genetic alterations in AML. They show a frequency

of 20%-27% in AML in adults9;12-16 and of 10-16% in childhood cases17;18 and are

associated with progression of myelodysplastic syndrome (MDS) to secondary AML

(s-AML).19 FLT3-LM mostly are represented by internal tandem duplications with/or

insertions or deletions in exons 11 and 12 of the human FLT3 gene on chromosome

13q12, which codes for the juxtamembrane domain of the FLT3 protein. The

mutations are heterogeneous and consist of internal tandem duplications of 6-30

amino acids resulting in an elongated FLT3 protein with constitutive PTK activity.20

These mutations lead to autophosphorylation of the receptor and result in increased

proliferation of AML cells in vitro and to a decrease of apoptosis. Constitutively active

FLT3 mutants have transforming potential in interleukin-3-dependent cells and

activate several signal transduction pathways, including STAT-5 and MAPK.21-24

FLT3-LM are highly associated with normal karyotype, t(15;17)/PML-RARA and

t(6;9)/DEK-CAN,15;16 and are prognostically unfavorable in adults12-16 and in pediatric

patients17;25;26.

In addition to the juxtamembrane domain mutations, mutations in the tyrosine kinase

domain (FLT3-TKD mutations) have been described in AML.7;9;16;27 FLT3-TKD

mutations are small mutations in the activation loop of FLT3 mostly representing point

mutations in codon D835 or deletions of codon I836. They induce constitutive tyrosine

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phosphorylation leading to activation of the receptor tyrosine kinase and are supposed

to represent gain-of-function mutations.7;22;28-32 Corresponding activation loop

mutations have been reported at position D816 of c-KIT and also in other receptor

tyrosine kinases, e.g. RET.21

Studies on the frequency of FLT3-TKD in AML were performed by Abu-Duhier et al.

(n=97), Yamamoto et al. (n=429), Thiede et al. (n=979), Moreno et al. (n=208), and by

Fröhling et al. in 224 patients with AML showing a normal karyotype.7;9;16;27;33

According to these studies FLT3-TKD show an incidence of 5.8%-7.7% in AML and

thus are less frequent than FLT3-LM.7;9;16;34

Due to the high frequency of FLT3 mutations in AML concentration focuses on the

development of FLT3 inhibitors for treatment of this AML subgroup. A number of

selective FLT3 tyrosine kinase inhibitors have demonstrated in vitro and in vivo

activity.35-39

Because of the low frequency the prognostic significance of FLT3-TKD mutations is

still unclear. In the presented study we analyzed the frequency of FLT3-TKD

mutations in 3082 patients with AML at diagnosis and did correlations to other

biological factors like karyotype, molecular mutations, cytomorphological subtypes,

and leukocyte count. The influence of FLT3-TKD on prognosis was evaluated in detail.

PATIENTS, MATERIAL, AND METHODS

Patients

Bone marrow samples or blood samples from 3082 consecutive patients with AML at

diagnosis were screened for FLT3-TKD mutations.

The range of age was 17.5 years to 91.8 years (median 63.1 years). Of the 3082

patients, 2546 (82.6%) had de novo AML, 334 (10.8%) had secondary AML following

myelodysplastic syndrome (MDS) (s-AML), and 202 (2.0%) had AML following

treatment of a previous malignant disease (t-AML). Patients were treated according to

protocols of the AMLCG study group (80%)40 or according to other intensive AML

therapy protocols. The study was approved by Ethics Committee of participating

institutions and informed consent was provided according to the Declaration of

Helsinki.

Samples were evaluated by cytomorphology, cytochemistry, multiparameter flow

cytometry, cytogenetics, fluorescence in situ hybridization (FISH), and molecular

genetics in parallel.41-44 The cytomorphological classification of AML was performed

according to the FAB classification.45;46 (table 1)

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Paired samples of 152 patients were compared with respect to the FLT-TKD mutation

status at diagnosis and at relapse.

Molecular analysis

Ficoll, mRNA extraction, cDNA synthesis, mutation analysis for FLT3-LM and control

PCR were performed as previously described (Schnittger Blood 2002). Analysis for

TKD mutations was performed by Lightcycler based melting curve analysis with

forward primer 17F: 5´-CCGCCAGGAACGTGCTTG-3´), reverse primer 17R: 5´-

ATGCCAGGGTAAGGATTCACACC-3´ and hybridization probes FLT3-sensor 5´-

ACTCATGATATCTCGAGCCAATCC-FL-3´ and FLT3-anchor 5´- LCred640-

AAGTCACATATCTTCACCACTTTCCCGT-3´-PH. The PCR reaction was carried out

in a 20 µl reaction volume with each 0.5 µM of forward and reverse primer, 0.75 µM

Hyb-Probes, 4 mM MgCl2 and 2 µl LightCycler-FastStart DNA Master Hybridization

Probes (Roche Diagnostics, Mannheim, Germany). LightCycler data were analyzed

using the LightCycler 3.0 software (Roche Diagnostics, Mannheim, Germany) and the

second derivative maximum method. Each 20 µl reaction contains 2 µl of cDNA, an

equivalent of about 3000 cells. Amplification was performed with 45 cycles using 60°C

annealing temperature. Final melting curve analysis was started at 40°C up to 95°C

with slop of 0.2°C/sec and continuous detection with channel F2/F1 (supplement,

figure 1).

Evaluation of this assay was performed in 100 samples by parallel assessment with

standard PCR, EcoRV restriction enzyme digestion and agarose gel electrophoresis

as was previously described.9

Screening for FLT3-LM, FLT3-TKD, NRAS mutations, and NPM1 mutations was

performed as described elsewhere.16;43;47;48 Mutations of the CEBPA gene were

analyzed by denaturing high performance liquid chromatography (DHPLC) (WAVE,

Transgenomics, Northumberland, USA) with subsequent direct sequencing of

aberrant fragments.

Sequencing analysis

Approximately 100 ng purified PCR products were directly sequenced with 3.3 pmol

primers as described above with BigDye Terminator Cycle Sequencing Kit (Applera,

Weiterstadt, Germany). After initial denaturation at 95 °C for 5 minutes, 25 cycles at

94°C for 15 seconds and 60°C for 4 minutes were performed. Sequence analysis was

performed on an ABI 310 or 3100 Sequence Detection system.

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Statistical analysis

Overall survival (OS) and event free survival (EFS) were performed according to

Kaplan-Meier. OS was calculated from time of diagnosis to death and EFS from time

of diagnosis to death, documentation of persistent leukemia, or relapse. The

correlation of OS and EFS with other parameters was assessed by Cox regression.

The comparison of survival curves was performed using double sided log rank test.

Comparisons of dichotomous variables between different groups were performed by

use of two-sided Fisher´s exact test. For statistical analysis SPSS (version 12.4)

software (SPSS, Chicago, IL) was used.

Complete remission was defined by <5% blasts in a normocellular bone marrow with

normal levels of peripheral neutrophil and platelet counts.

RESULTS

Frequency of the FLT3-TKD mutation

In total 3082 samples were analysed at diagnosis. Of these 147 were positive for a

FLT3-TKD mutation (4.8%). As estimated from the LightCycler curves and sequence

data 137 had a mutation/wt ratio corresponding to 20-100% of mutated cells. Ten

cases had a TKD/wt ratio of approximately 10%. Lower ratios were not observed. On

the other hand 2935 cases (95.2%) tested negative for a FLT3-TKD mutation.

Sequencing analysis

To further characterize the detected mutations PCR products of cases with low

melting temperatures profiles were directly sequenced. In 10 cases sequencing with a

sensitivity of approximately 10% did not reveal a mutation as the mutated clone as

derived from the melting peak was below 10% of the whole cell population. Of 147

mutated cases 137 (93.2%) could be further characterized. Changes of D835 were

most frequent (122/137; 89.1%). In detail, a change from asparagine to tyrosine

(D835Y) was found in 68/147 cases (49.6%). In 118/122 cases with FLT3-TKD

mutations (96.7%) only one allele was mutated. Compound heterozygotes were found

in 4/122 cases with FLT3-TKD mutations (3.3%) (1. asp > ala / asp > tyr; 2. asp > tyr /

asp > glu; 3. asp > tyr / asp > his; 4. asp > tyr / asp > glu). In one case we found

mutations of both D835 and a ∆I836 (1/137; 0.7%). ∆I836 was observed in 14 cases

(10.2%). (table 2)

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Biological parameters of patients with FLT3-TKDmut compared to FLT3-TKDwt

The 147 FLT3-TKD mutated cases included 72 males (49.0%) and 75 females

(51.0%), thus revealing no sex difference. Median age of the FLT3-TKD mutated

patients was 59.5 years (range 18.7 - 84.6 years) compared to 63.2 years (range 17.5

- 91.8 years)in the unmutated group (n.s.). Thus, FLT3-TKD mutations were not

significantly correlated with gender or age. The median white blood count (WBC) was

40.0 x 109/L (range: 1.2 x109/L - 332.0 x109/L) in the cohort with FLT3-TKD mutations

compared to 50 x 109/L (range: 1.0 - 840.0 x109/L) in the cohort with FLT3-LM vs. 7.6

x 109/L (range: 115.0 - 462.0 x109/L) in the patients with FLT3- wildtype. Thus, WBC

was significantly higher in FLT3-TKD-mutated than in FLT3wt AML (p<0.001), and

lower than in FLT3-LM (p=0.04).

Correlation of the FLT3-TKD mutation status with AML history

The distribution of FLT3-TKD mutations in de novo AML, s-AML, and t-AML revealed

the highest incidence in de novo AML (135/2546; 5.3%) compared to s-AML (8/334;

2.4%) and t-AML (4/202; 2.0%). Thus, FLT3-TKD mutations were significantly more

frequent in de novo than in s-AML after MDS or precedent chemotherapy (p=0.0101).

Correlation of the FLT3-TKD mutation status to cytomorphology

The FAB subtype was available in 2762/3082 patients (89.6%) and was correlated to

the FLT3-TKD status. Overall FLT3-TKD mutations were detected in all morphologic

subgroups. They were significantly overrepresented in AML M3v (6/51; 11.8%;

p=0.0204), M4 (39/484; 8.1%; p=0.0003), and M5b (15/114; 13.2%; p<0.0001). In

contrast, they were significantly underrepresented in the subtypes M2 (25/1054) and

M6 (M6: 0/112; 0%; p=0.0147). (table 3)

Correlation of the FLT3-TKD mutation status with cytogenetics

Karyotype was available in 3018/3082 cases (97.9%). In 64/3082 cases (2.1%)

karyotype was not available due to a low number of analyzed metaphases. Cases

were grouped into 12 categories: normal karyotype, t(15;17)/PML-RARA,

t(8;21)/AML1-ETO, inv(3)/t(3;3), inv(16)/t(16;16)/CBFB-MYH11, 11q23/MLL, complex

aberrant karyotype (defined by ≥3 chromosomal abnormalities), trisomy 8, 5q- or loss

of chromosome 5, 7q- or loss of chromosome 7, and all others, the so called “other

aberrations”.

FLT3-TKD mutations were observed in all cytogenetic subgroups. However, some

specific associations with distinct cytogenetic subgroups were observed. Compared to

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the total cohort they were significantly overrepresented in normal karyotype (88/1384;

6.0%; p=0.0033) when compared to the overall cohort. They showed an elevated

frequency in t(15;17)/PML-RARA (10/130; 7.7%); but this association did not reach

significance. In complex aberrant karyotype they were significantly underrepresented

(4/430; 0.9%; p<0.0001). (table 4)

Correlation of the FLT3-TKD mutation status with other molecular markers

The correlation of FLT3-TKD with other molecular markers was analyzed for FLT3-

LM, MLL-PTD, and mutations in NPM1, NRAS, KITD816, and CEPBA. FLT3-TKD

mutations were significantly overrepresented in the patients with NPM1 mutations

(23/262; 8.8%) (p<0.0001). In patients with FLT3-LM they were significantly

underrepresented (17/594; 2.9%; p=0.0066). (table 5)

Stability of the FLT3-TKD in paired samples from diagnosis and relapse

The stability of FLT3-TKD was analyzed by evaluation of 152 paired samples at

diagnosis and relapse. The mutation status was TKDwt/TKDwt at both time points in

139 cases. Four cases revealed an FLT3-TKD mutation at diagnosis as well as at

relapse. Thus, the TKD status was unchanged at diagnosis and relapse in 143 cases

(94.1%). In contrast, nine cases (5.9%) that were FLT3-TKD mutated at diagnosis did

not reveal this mutation at relapse. This meant that of 13 cases that were positive at

diagnosis, 9 (69%) lost the mutation at relapse. Although the limited number of cases

has to be taken into account, this may suggest that FLT3-TKD is a rather unstable

mutation not valid for follow-up studies.

Prognostic significance of the FLT3-TKD mutation

When all patients were taken into account, OS and EFS were not significantly

influenced by the presence of FLT3-TKD mutations (supplement, figure 2 a-2b). This

pattern remained unchanged if all cases with FLT3-LM were excluded (supplement,

figure 2 c-d). We then compared OS and EFS with respect to FLT3-TKD mutations in

different cytogenetic subgroups. In t(8;21), inv16/t(16;16), 11q23/MLL

rearrangements, inv(3)/t(3;3), +8, and in complex aberrant karyotype the influence of

FLT3-TKD could not be determined due to the rare occurrence in these cytogenetic

subgroups. In normal karyotype OS and EFS did not differ significantly in dependence

on FLT3-TKD mutations (supplement, figure 3 a-e). In patients with karyotypes

associated with an intermediate prognosis FLT3-TKD had no prognostic impact as

well (supplement, figures 4a-d). Also when this analysis was restricted to the de novo

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AML case no prognostic significance could be worked out (supplement, figures 2 e-f

and 3e-f).

In the subgroup with t(15;17) EFS was significantly deteriorated by FLT3-TKD

mutations (p=0.051) (figure 1 b). This became even more evident, when the analysis

was restricted to the cases without FLT3-LM (figure 1 c-d).

Furtheron, we analzyed the prognostic impact of FLT3-TKD mutations in dependence

on FLT3-LM, MLL-PTD, NPM1, and CEPBA. In the NRAS mutated patients the

influence of FLT3-TKD mutations could not be analyzed due to the low incidence in

this subgroup. Kaplan Meier plots showed an unfavorable impact of FLT3-TKD in

prognostically unfavorable groups like FLT3-LM (figure 2) and MLL-PTD (figure 3a-b).

This cooperative adverse effect of FLT3-TKD and MLL-PTD was even stronger, after

the FLT3-LM positive cases had been excluded (figure 3c-d). In contrast there seems

to be a favorable impact in the two subgroups with the favorable markers NPM1

(figure 4a-4b) and CEBPA (figure 5). For NPM1 this cooperative positive effect was

even stronger, when the FLT3-LM mutated cases were excluded. If this analysis was

further broken down to de novo AML, normal karyotype and exclusion of FLT3-LM

mutated cases, FLT3-TKD/NPM1 double mutated cases had a highly significantly

better EFS than sole FLT3-TKD mutated (p=0.010) or FLT3wt/NPM1wt cases

(p=0.028) (figure 4e-f). Because of the limited sample numbers in subgroups a

comparable detailed analysis was not possible for all other mutations.

DISCUSSION

Molecular mutations especially in normal karyotype AML are of increasing importance

for the definition of prognosis in AML. Some mutations have been shown to be

prognostically favorable like NPM148;49 and CEBPA50;51. Others like the MLL-PTD are

unfavorable.52;53 Whereas for the FLT3-LM an association with an unfavorable

prognosis within normal karyotype was clearly established, the prognostic impact of

FLT3-TKD remained unclear, so we did a detailed analysis on 3082 patients with

AML. Although we performed a very specific and sensitive assay which was

potentially able to detect not only the codon 835/836 mutations but covered a total of

17 codons in the TKD domain, the frequency of FLT3-TKD in our study was only

4.8%. This slightly lower frequence when compared to previous studies.9;12;16;27;54

(table 6) might be explained by differences of age as the median age of patients in this

study was 63 years in comparison to (<50 years in comparative studies. Likewise to

the FLT3-LM the FLT3-TKD seem to be more frequently associated with de novo AML

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rather than with s-AML or t-AML as suggested here and in some previous reports.15;33

Only one analysis found an equal distribution of FLT3-TKD in de novo and in s-AML.27

Like previous studies9;33;54 we found the D835Y mutation being the most frequent

amino acid exchange followed by the D835V and the D835E mutations. Thus, the

mutations are not randomly distributed within the TK domain. The occurrence of two

different TKD mutations in combination was rare in only 3% of all cases at all. This

suggests that a compound heterozygous mutation status does not provide a further

advantage for the leukemic clone.

Similar to AML with FLT3-LM, the FLT3-TKD is clearly associated with higher WBC

when compared to FLT3-wt AML. However, median WBC in FLT3-TKD was lower

than in FLT3-LM which is in accordance to some previous studies,33;54 although others

were not able to show any influence of the FLT3-TKD on peripheral leukocytes.9;27;54

A preliminary analysis of paired samples from diagnosis and relapse suggests low

stability of this mutation. Loss of the FLT3-TKD mutation at relapse was found in 69%

of cases which were positive at diagnosis (6% of all cases). This was in the range of

two previous studies that observed loss in 3%55 and in 8% of the cases, respectively13.

Therefore, it the FLT3-TKD mutations might be less suitable as MRD markers when

compared to other markers. However, this is a matter of future studies. Efforts were

already made to establish a real-time PCR assay for the follow-up of FLT3-TKD

mutations.56

We found a relatively high frequency of FLT3-TKD in t(15;17)/PML-RARA when

compared with the total cohort. This association between the TKD mutations and

acute promyelocytic leukemia (APL) confirmed the results of our previous analysis

which included part of the patients of this study.57 Likewise to the FLT3-LM57 we found

the FLT3-TKD significantly associated with the FAB subtype M3v (11.8%), whereas in

M3 the frequency (4.7%) was comparable to the overall frequency of FLT3-TKD

mutations in AML. In conclusion, this association of the FLT3-LM and of the TKD

mutations with PML-RARA in parallel with the low frequency of other molecular

markers in APL suggests a cooperation of both subtypes of FLT3 mutations with PML-

RARA to cause AML with preference for the cytomorphological M3v subtype.58 Such

cooperation is further emphasized by the previous observation of a higher PML-RARA

expression in FLT3-LM positive patients in comparison to FLT3-LM negative

patients.57

In contrast to the FLT3-LM which are significantly underrepresented in the subgroup

with inv(16)/CBFB-MYH11 (Schnittger et al.: 0%; Thiede et al. 0.6%),15;16 the

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frequency of FLT3-TKD in inv(16)/CBFB-MYH11 was only slightly lower than the

overall frequency in this study and in previous analyses (this study: 5.0%; Care et al.:

4.8%; Thiede et al.: 5.0%).15;16;59 A recent large study found the TKD mutations even

significantly associated with inv(16).54 Thus, the relationship between the FLT3-TKD

mutations and CBFB-MYH11 is yet not clear. In contrast to FLT3-LM which were

observed in 90% of t(6;9)/DEK-CAN cases,16 the FLT3-TKD mutations were not

detected in this rare entity. In addition, the FLT3-TKD mutations were significantly

underrepresented in AML with complex aberrant karyotype in our study which was in

correspondance to the FLT3-LM being also rare in this cytogenetic subgroup

according to our precedent analysis.15 In conclusion, the FLT3-LM and the TKD

mutations show many parallels with respect to their distribution within the different

cytogenetic AML subgroups – as demonstrated by their association to normal

karyotype, to t(15;17)/PML-RARA preferentially in AML M3v, and by their low

occurrence in complex aberrant karyotype.

Another focus of this analysis was the distribution of the FLT3-TKD mutations within in

AML with different molecular mutations. According to the two hit hypothesis at least

two different mutation types are needed to induce AML. Type I mutations encode

tyrosine kinases and increase proliferation, whereas type II mutations encode

transcription factors and block differentiation.21 The overrepresentation of FLT3-TKD –

being interpreted as type I mutations - in cases with NPM1, CEPBA, and NRAS

mutations (representing type II mutations) in this analysis might support this model.

FLT3-TKD and FLT3-LM seem to differ with respect to their molecular cooperation

partners, as the FLT3-LM were found underrepresented in NRAS mutated cases in

contrast to the overrepresentation of TKD mutations in this subgroup.12 Secondly, the

FLT3-TKD did not differ from the overall frequency in patients with MLL-PTD, whereas

the FLT3-LM were overrepresented in MLL-PTD positive cases.60

Our data support previous suggestions that FLT3-TKD and FLT3-LM show no61 or

rare coincidence9;16;33;62. It was suggested that the FLT3-LM and FLT3-TKD are

caused by totally different underlying mutational mechanisms and in addition do not

cooperate well to amplify any leukemogenic effect. The same seems to be true for

FLT3-TKD and KIT mutations as also these two targets were very rare in this study

The correlation of the FLT3-TKD mutations with cytomorphology allowed further

speculations with respect to their role in leukemogenesis. The association with the

monocytic subtype FAB M5 in this and in previous studies9;16 like the FLT3-LM which

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show the a similar distribution9;15;16 is indicative for a participation of FLT3 mutations in

monocyte differentiation in AML16;63. It had previously been shown that FLT3 was

continuously expressed during monocyte differentiation, and that FLT3 was needed

for optimal differentiation of monocytes from CD34 positive stem cells.64 In addition,

the underrepresentation of FLT3-TKD mutations in the FAB subtype M6 (own study)16

might be due to the low rate of FLT3-TKD mutations in complex aberrant karyotype,

as AML M6 is correlated with this cytogenetic subgroup.65

With respect to prognosis in the presented study which included 3082 AML cases

there was no influence on OS or EFS in the overall AML population, in de novo AML,

nor in the normal or intermediate karyotype subgroups in accordance with most

previous studies in adults,33;54 in elderly patients,62 and in children26. However, there

are as well reports of a worsened outcome,27 and the metaanalysis of Yanada et al.

on 1160 patients resulted in a significantly worse outcome.66 This is in contrast to a

large recent study which found a significantly improved survival in the patients with

FLT3-TKD mutations.54 Another study showed an inferior outcome in the intermediate

cytogenetic subgroup, whereas there was no impact of the FLT3-TKD mutations on

survival in the poor cytogenetic subgroup.16 These different findings may result from

selection within the cohorts. As we could show the prognostic effect of the FLT3-TKD

is dependend on additional mutations in single patients which came out by detailed

subgroup analyses that have not been performed previously.

Accordingly, if subgroups with defined genetic profiles are regarded a significant

prognostic impact could be worked out. The only cytogenetic subgroup with an

obvious prognosis effect of the FLT3-TKD – a negative one in this case - was

t(15;17)/PML-RARA. Other negative effects were detected in AML with FLT3-LM or

MLL-PTD which are themselves are prognostically unfavorable. Paradoxically in

NPM1 and CEPBA mutated cases, which per se have a favorable prognosis, FLT3-

TKD mutations had a further favorable prognostic impact. These effects were even

stronger when the analysis was restricted to de novo AML or AML without FLT3-LM.

As the case numbers in some of the double mutated groups were rather small -

especially in the FLT3-TKD/CEBPA double mutated group - these finding have to be

taken with caution and should serve mainly as working hypothesis for further studies

within clinical trials.

Different signalling properties of both mutation subtypes were suggested by

Choudhary et al. who showed that FLT3-LM gain a function over ligand-activated

FLT3-wildtype which is not gained by FLT3-TKD mutations.29 Gene expression

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profiling with microarray analysis showed different gene expression patterns for FLT3-

LM and for FLT3-TKD positive AML.67 The differences between FLT3-TKD and FLT3-

LM regarding the distribution within distinct cytogenetic subgroups, the coincidence

with other molecular markers, and the differences with respect to the prognostic

impact in this and in the precedent studies emphasize the diversity within both

molecular subtypes. In conclusion, FLT3-LM mutations and FLT3-TKD mutations

should be regarded as two biologically and prognostically different mutations within a

single gene.

ACKNOWLEDGEMENT

This investigation was performed in part in the Laboratory for Leukemia Diagnostics;

Medical Department III, (Head: Prof. Dr. W. Hiddemann), Ludwig-Maximilians-

University Munich. We greatly appreciate the excellent technical help of Gudrun

Mellert, Claudia Tschulik, Madlen Fuchs, Theresa Förster, and Nina Leopold. We

would like to thank all participants of the AMLCG study group for sending bone

marrow or blood samples to our laboratory for reference diagnosis and for submitting

clinical data, as part of the patients were treated within the AMLCG study group.

Author Contribution: S.S. was principal investigator. U.B. and S.S. analyzed data

and wrote the manuscript. WK was responsible for immunophenotyping and

contributed in statistical evalualion. TH was responsible for cytomorphology. C.H. was

responsible for cytogenetics. S.S. did the molecular genetic analysis.

Conflict of Interest Disclosure:

CH, WK, TH, and SS in part own the MLL Munich Leukemia Laboratory which is

offering comprehensive leukaemia diagnostics.

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Tables and figures

Table 1: Characterization of the patient cohort (n=3082)

number of patients (n) percentage (%)

history of AML

de novo AML 2546 82.6

secondary AML (s-AML) 334 10.8

t-AML 202 2.0

total 3082 100.0

cytogenetic subgroup

normal 1472 47.8

t(15;17)* 130 4.2

t(8;21) 88 2.9

trisomy 8 129 4.2

11q23/MLL 109 3.5

5q-/-5 31 1.0

7q-/-7 60 1.9

inv(16)/t(16;16) ** 119 3.9

inv(3)/t(3;3) 44 1.4

t(6;9)(p23;q34) 6 0.2

complex aberrant 430 14.0

other aberrations 400 15.0

total 3018 100.0

FAB subtype

M0 132 4.8

M1 491 17.8

M2 1054 38.2

M3 85 3.1

M3v 51 1.8

M4 484 17.5

M4eo 118 4.3

M5a 107 3.9

M5b 114 4.1

M6 112 4.1

M7 14 0.5

total 2762 100.0

(*136 cases of FAB M3/M3v morphology were associated to 130 cases of t(15;17) in chromosome banding analyses due to additional 6 cryptic PML-RARA rearrangements; **118 cases of FAB M4eo morphology were associated to 119 cases of inv(16)/t(16;16) due to atypical morphology in one case)

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Table 2: Distribution of point mutations in 137 cases with FLT3-TKD mutations

amino acid exchange number of patients (n)

percentage (%)

D835Y 68 49.6 D835H 20 14.6 D835V 14 10.2 D835E 11 8.0 D835A 3 2.2 D835S 1 0.7 D835N 1 0.7 D835 compound heterozygous: D835Y+D835E D835Y+D835A D835Y+D835H

2 1 1

1.4 0.7 0.7

D835 – total 122 89.1 �I836 14 10.2 D835V and �I836 compound heterozygous

1 0.7

total 137 100.0

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Table 3: Correlation of FLT3-TKD mutations with the FAB subtype in 2762 patients with AML at diagnosis FAB n total n FLT TKDmut % FLT TKDmut p M0 132 3 2.3 0.158 M1 491 31 6.3 0.096 M2 1054 25 2.4 <0.001 M3 85 4 4.7 0.949 M3v 51 6 11.8 0.020 M4 484 39 8.1 <0.001 M4eo 118 5 4.2 0.751 M5a 107 6 5.6 0.711 M5b 114 15 13.2 <0.001 M6 112 0 0.0 0.015 M7 14 0 0.0 0.397 total 2762 134 4.9

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Table 4: Correlation of FLT3-TKD mutations with cytogenetics in 3018 patients

Cytogenetic subgroup n total n FLT TKD mut % FLT TKD mut p normal 1472 88 6.0 0.003 t(15;17)/PML-RARA 130 10 7.7 0.116 t(8;21) 88 2 2.3 0.260 trisomy 8 129 6 4.7 0.934 11q23/MLL 109 7 6.4 0.421 5q-/-5 31 0 0.0 0.209 7q-/-7 60 1 1.7 0.251 inv(16)/t(16;16) 119 6 5.0 0.902 inv(3)/t(3;3) 44 2 4.5 0.936 t(6;9)(p23;q34) 6 0 0.0 0.582 complex aberrant 430 4 0.9 <0.001 other aberrations 406 19 4.7 0.900 total 3018 145 4.8

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Table 5: Correlation of FLT3TKD mutations with other molecular mutations (+: positive mutation status; -: negative mutation status).

molecular mutation

N mutation status

nr. FLT3-TKD mut +

% p

+ 338 12 3.6 FLT3-LM

1051 - 713 57 8.0

0,007

+ 84 6 7.1 MLL-PTD

1044 - 960 64 6.7

0,867

+ 221 20 9.0 NPM1

428 - 207 50 24.2

<0.001

+ 45 4 8.9 CEPBA

363 - 318 25 7.9

0,812

+ 78 3 3.8 NRAS

888 - 810 52 6.4

0,368

+ 44 1 2.3 KITD816

2532 - 2488 118 4.7

0,443

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Table 6: Frequency of FLT3-TKD mutations in this study and in previous studies

Reference total (n) positive FLT-TKD mutation

status (n)

percentage FLT-TKD mutations

this study 3082 147 4.8% Abu-Duhier et al., 2001 97 7 7.2% Yamamoto et al., 2001 429 30 7.0% Thiede et al., 2002 979 75 7.7% Moreno et al., 2003 208 20 7.7% Andersson et al., 2004 109 (>60 years) 11 10.1% Aurewarakul et al., 2005 256 15 5.8% Wang et al., 2005 143 92 6.3% Mead et al., 2007 1107 127 11% overall 5303 397 7.5%

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Legends for figures

Figure 1: a) Overall survival and b) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in t(15/17)/PML-RARA positive AML. c) Overall survival and b) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in t(15/17)/PML-RARA positive AML exclusive all cases that are FLT3-LM mutated. Figure 2: a) Overall survival and b) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in dependance on FLT3-LM.

Figure 3: a) Overall survival and b) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in dependance on MLL PTD. c) Overall survival and d) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in dependance on MLL PTD exclusive all cases that are FLT3-LM mutated. Figure 4: a) Overall survival and b) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FL3T-TKDmut) in dependance on NPM1 mutations. c) Overall survival and d) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FL3T-TKDmut) in dependance on NPM1 mutations all cases that are FLT3-LM mutated. e) Overall survival and f) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FL3T-TKDmut) in dependance on NPM1 mutations exclusive all cases that are FLT3-LM mutated in de novo normal karyotype AML. Figure 5: a) Overall survival and b) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in dependance on CEPBA mutations. c) Overall survival and d) event free survival of FLT3-TKD-wiltdtpye (FLT3-wt) versus FLT3-TKD mutated cases (FLT3-TKDmut) in dependance on CEPBA mutations exclusive all cases that are FLT3-LM mutated.

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Figure 1

300025002000150010005000

days

1,0

0,8

0,6

0,4

0,2

0,0

Surv

ival

OS in t(15;17)

FLT3 TKD wt: n=100

FLT3 TKD mut: n=10

FLT3 TKD wt: n=100

FLT3 TKD mut: n=10

p=0.132 p=0.051

a b

300025002000150010005000

days

1,0

0,8

0,6

0,4

0,2

0,0

Surv

ival

EFS in t(15;17)

F

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bloodjournal.hematologylibrary.org

From

Figure 1

FLT3 wt: n=49

FLT3 TKD mut: n=6

FLT3 wt: n=49

FLT3 TKD mut: n=6

p=0.065 p=0.017

c dOS in t(15;17)

FLT3-LM mutated excludedEFS in t(15;17)

FLT3-LM mutated excluded

F

or personal use only. by guest on June 6, 2013.

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From

Figure 2

OS in FLT3 TKD and FLT3 LM EFS in FLT3 TKD and FLT3 LM

1) FLT3 TKD- / FLT3 LM-: n=656

2) FLT3 TKD+ / FLT3 LM+: n=12

3) FLT3 TKD+ / FLT3 LM-: n=57

4) FLT3 TKD- / FLT3 LM+: n=326

1) FLT3 TKD- / FLT3 LM-: n=656

2) FLT3 TKD+ / FLT3 LM+: n=12

3) FLT3 TKD+ / FLT3 LM-: n=57

4) FLT3 TKD- / FLT3 LM+: n=326

2) vs. 1) p=n.s.2) vs. 3) p=n.s2) vs. 4) p=n.s

2) vs. 1) p=0.0992) vs. 3) p=0.2112) vs. 4) p=0.701

a b

F

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From

Figure 3

1) FLT3 TKD- / MLL PTD-: n=896

2) FLT3 TKD+ / MLL PTD+: n=6

3) FLT3 TKD+ / MLL PTD-: n=64

4) FLT3 TKD- / MLL PTD+: n=78

1) FLT3 TKD- / MLL PTD-: n=896

2) FLT3 TKD+ / MLL PTD+: n=6

3) FLT3 TKD+ / MLL PTD-: n=64

4) FLT3 TKD- / MLL PTD+: n=78

2) vs. 1) p=0.0302) vs. 3) p=0.0202) vs. 4) p=0.152

2) vs. 1) p=0.0202) vs. 3) p=0.0162) vs. 4) p=0.117

a bOS in FLT3-TKD and MLL-PTD OS in FLT3-TKD and MLL-PTD

F

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From

Figure 3

1) FLT3 TKD- / MLL PTD-: n=609

2) FLT3 TKD+ / MLL PTD+: n=53) FLT3 TKD+ / MLL PTD-: n=52

4) FLT3 TKD- / MLL PTD+: n=49

1) FLT3 TKD- / MLL PTD-: n=609

2) FLT3 TKD+ / MLL PTD+: n=5

3) FLT3 TKD+ / MLL PTD-: n=52

4) FLT3 TKD- / MLL PTD+: n=49

2) vs. 1) p=0.0202) vs. 3) p=0.0152) vs. 4) p=0.046

1) vs. 3) p=0.0231) vs. 4) p=0.0223) vs. 4) p=0.071

c dOS in FLT3-TKD and MLL-PTD

FLT3-LM mutated excludedEFS in FLT3-TKD and MLL-PTD

FLT3-LM mutated excluded

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From

Figure 4

a b

2) FLT3 TKD+ / NPM1+: n= 201) FLT3 TKD- / NPM1-: n=292

3) FLT3 TKD+ / NPM1-: n= 774) FLT3 TKD- / NPM1+: n=202

2) vs. 1) p=0.1452) vs. 3) p=0.0202) vs. 4) p=0.098

2) FLT3 TKD+ / NPM1+: n= 201) FLT3 TKD- / NPM1-: n=292

3) FLT3 TKD+ / NPM1-: n= 774) FLT3 TKD- / NPM1+: n=202

2) vs. 1) p=0.4192) vs. 3) p=0.1192) vs. 4) p=0.205

EFS in FLT3-TKD and NPM1(all cases)

OS in FLT3-TKD and NPM1(all cases)

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Figure 4

2) FLT3 TKD+ / NPM1+: n= 161) FLT3 TKD- / NPM1-: n=238

3) FLT3 TKD+ / NPM1-: n= 634) FLT3 TKD- / NPM1+: n=112

2) vs. 1) p=0.2902) vs. 3) p=0.0752) vs. 4) p=0.250

c d EFS in FLT3-TKD and NPM1(excluded all FLT3-LM+ cases)

OS in FLT3-TKD and NPM1(excluded all FLT3+ cases)

2) FLT3 TKD+ / NPM1+: n= 161) FLT3 TKD- / NPM1-: n=238

3) FLT3 TKD+ / NPM1-: n= 634) FLT3 TKD- / NPM1+: n=112

2) vs. 1) p=0.0692) vs. 3) p=0.0082) vs. 4) p=0.011

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From

Figure 4

e f

2) vs. 1) p=0.1542) vs. 3) p=0.0842) vs. 4) p=0.308

2) FLT3 TKD+ / NPM1+: n= 161) FLT3 TKD- / NPM1-: n=101

3) FLT3 TKD+ / NPM1-: n= 284) FLT3 TKD- / NPM1+: n=106

OS in FLT3-TKD and NPM1 (excluded all FLT3+ cases)

Only de novo, NK

OS in FLT3-TKD and NPM1 (excluded all FLT3+ cases)

Only de novo, NK

2) FLT3 TKD+ / NPM1+: n= 161) FLT3 TKD- / NPM1-: n=101

3) FLT3 TKD+ / NPM1-: n= 284) FLT3 TKD- / NPM1+: n=106

2) vs. 1) p=0.0282) vs. 3) p=0.0102) vs. 4) p=0.092

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From

Figure 5

2) FLT3 TKD+ / CEBPA+:n=4

1) FLT3 TKD- / CEBPA-: n=293

3) FLT3 TKD+ / CEBPA-: n=25

4) FLT3 TKD- / CEBPA+: n=41

2) FLT3 TKD+ / CEBPA+:n=4

1) FLT3 TKD- / CEBPA-: n=293

3) FLT3 TKD+ / CEBPA-: n=25

4) FLT3 TKD- / CEBPA+: n=41

2) vs. 1) p=0.0932) vs. 3) p=0.0372) vs. 4) p=0.124

2) vs. 1) p=0.1202) vs. 3) p=0.0742) vs. 4) p=0.204

a b

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From

Figure 5

2) FLT3 TKD+ / CEBPA+:n=4

1) FLT3 TKD- / CEBPA-: n=172

3) FLT3 TKD+ / CEBPA-: n=22

4) FLT3 TKD- / CEBPA+: n=21

2) FLT3 TKD+ / CEBPA+:n=4

1) FLT3 TKD- / CEBPA-: n=172

3) FLT3 TKD+ / CEBPA-: n=22

4) FLT3 TKD- / CEBPA+: n=21

c dOS in FLT3-TKD and CEBPAFLT3-LM mutated excluded

EFS in FLT3-TKD and CEBPAFLT3-LM mutated excluded

2) vs. 1) p=0.1982) vs. 3) p=0.1712) vs. 4) p=0.239

2) vs. 1) p=0.1442) vs. 3) p=0.0492) vs. 4) p=0.131

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From