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Professional article/Strokovni prispevek
FMS-LIKE TYROSINE KINASE (FLT3) GENE ITD
MUTATION IN ACUTE MYELOID LEUKEMIA
MUTACIJA GENA FLT3 PRI AKUTNI MIELOBLASTNI LEVKEMIJI
Rajko Kusec1, 2, Maruska Marusic-Vrsalovic1, Tanja Vranic-Bobetic1, Slobodanka Ostojic2,
Hrvoje Mingo2, Branimir Jaksic2
1 Institute of Clinical Chemistry, Merkur University Hospital, Zagreb, Croatia
2 Department of Medicine, Hematology, Merkur University Hospital, Zagreb, Croatia
Arrived 2004-03-01, accepted 2004-03-11; ZDRAV VESTN 2004; Supl. I: 5–7
Ključne besede: gen FLT3; levkemija; notranja tandemska
podvojitev; prognoza; citogenetika
Izvleček – Izhodišča. FLT3 je recepTOR razreda III za tirozin
kinazo. Nahaja se v normalnih matičnih celicah in levke-
mičnih celicah pri aktuni mieloblastni levkemiji (AML). No-
tranja tandemska podvojitev (ITD) gena FLT3 na eksonih 14
in 15 vodi do nastanka neodvisnih dimerov in aktivacije s
posledično proliferacijo in inhibicijo apoptoze, kar v končni
fazi vodi do leVkemogeneze. Pregledali smo vzorce serije bol-
nikov z AML za prisotnost mutacije FLT3/ITD in ugotavljali
morebitno povezavo med prisotnostjo mutacije s klinično
sliko in potekom bolezni.
Metode. Z metodo verižne reakcije s polimerazo v realnem
času (RT-PCR) smo preiskali 67 vzorcev kostnega mozga bol-
nikov z AML za prisotnost ITD na eksonih 14 in 15 gena FLT3.
Rezultati. V skupini pregledanih vzorcev je bila incidenca
FLT3/ITD mutacije 16,4%. Mutacijo smo ugotovili pri 2 do 6
bolnikIH s translokacijo t (15;17), pri 1 od 4 s translokacijo
kariotip. Večina je imela fenotip AML M2 in M4 po FAB raz-
delitvi. Pri bolnikih z mutacijo je bolezen kazala slabši potek
in preživetje, ni pa bilo nobenih razlik v osnovnih hemato-
loških parametrih med skupinami z mutacijo in brez.
Zaključki. Znano je, da je mutacija FLT3/ITD najpogostejša
samostojna mutacija pri bolnikih z AML in predstavlja ne-
ugoden dejavnik napovedi poteka bolezni. Zaradi tega bi mo-
rali to preiskavo vključiti v redno molekularno diagnostično
preiskovanje bolnikov z AML.
Key words: FLT3 gene; leukemia; internal tandem duplica-
tion; prognostics; cytogenetics
Abstract – Background. FLT3 is a class III receptor tyrosine
kinase expressed in normal stem cells and blasts of myeloid
leukemia. Internal tandem duplication (ITD) of the FLT3 ge-
ne affecting the exons 14 and 15 leads to ligand-independent
FLT3 dimerization and constitutive activation. This stimula-
tes proliferation and induces inhibition of apoptosis which
contributes to leukemogenesis. We have screened a panel of
acute myeloid leukemia (AML) patients for the occurrence of
FLT3/ITD mutation and correlated this mutation to patients’
survival and basic hematological parameters.
Methods. RT-PCR for ITD in exons 14 and 15 of FLT3 gene
was done on bone marrow samples of 67 AML patients at
diagnosis.
Results. There was a 16.4% incidence of FLT3/ITD mutation
in the cohort of examined patients. By cytognetic subgroups
there were 2/6 t(15;17) and 1 of 4 t(8;21) positive patients.
The rest had normal and 2 had complex karyotype. Majority
were of FAB M2 or M4 phenotype. For a subset of patients
taken into comparative survival analysis there was a clear
disadvantage for FLT3/ITD patients. No difference was
found for basic hematological parameters between two
groups.
Conclusions. As it is evident today that FLT3/ITD is the single
most common genetic abnormality in AML that also presents
unfavorable clinical prognostic marker, it should be included
in molecular diagnostic testing of acute myeloid leukemia.
Introduction
Deregulated tyrosine kinase activity has been implicated in
the pathogenesis of myeloid malignancies. Recently a FMS-
like tyrosine kinase-3 (FLT3) gene, a member of the PDGF-R
subfamily has been recognized as an important molecule in
acute myeloid leukemia. FLT3 is primarily expressed in hema-
topoietic stem cells, where it plays a role in hematopoiesis. It
is activated by ligand binding that causes its dimerisation and
activation and receptor phorphorylation (1). Activation of FLT3
leads to cell proliferation and activation. An activating somatic
mutation in the form of internal tandem duplication in the
juxtamembrane region of the gene has been described (2).
Since the identification of this mutation, and another, D835
activation loop domain mutation, a wealth of reports has emer-
ged suggesting that FLT3 mutation is the single most common
molecular of genetic abnormality in acute myeloid leukemia
with direct clinical impact on the disease outcome. An average
frequency of approximately 20% for FLT3/ITD and 7% for
D835 has been reported in the literature, associated more
frequently with standard risk cytogenetics, PML/RARα  rear-
rangement, less frequent with core binding factor leukemia,
ZDRAV VESTN 2004; 73: I-5–7
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secondary or pediatric AML. Except of elderly AML presence
of FLT3/ITD is associated with shorter survival.
In the present study we have examined the panel of 67 AML
patients for FLT3/ITD mutation and have determined its pro-
gnostic significance and correlation to basic hematological da-
ta in a subset of them.
Materials and methods
Patient samples
Bone marrow at diagnosis was obtained from 67 AML patients
treated at the Department of hematology, Merkur University
Hospital in Zagreb, Croatia. All patient related procedures were
in accordance with the Helsinki declaration and accepted ethi-
cal standards. Prognostic impact of FLT3/ITD mutation was
tested on a subset of 28 patients, median age 53, range 19–85
years. Mutation positive and negative patients in analysis did
not differ by age or sex distribution.
PCR amplification of FLT3/ITD
Exons 14 and 15 were amplified using primers FLT3F 5'-caatt-
taggtatgaaagcc-3' FLT3R 5'-caaactctaaattttctct-3', as designed
by the molecular genetics working group of European Orga-
nization for Research and Treatment of Cancer (EORTC). PCR
reaction was performed in 25 ml reaction buffer, containing
1ml of cDNA, 10pmol of each primer, 0,1 mM dNTP, 5mM
MgCl
2
 and 1U of Taq DNA polymerase (Applied Biosystems).
PCR was performed with cycling conditions of initial denatura-
tion step at 94 0C for 5 min, 36 cycles of 94 0C for 30 sec., 55 0C
for 1 min and 72 0C for 1 min, with a final extension step of 72
0C for 7 minutes. Unmutated PCR products were seen as 130
bps products on 3,5% agarose gel. Samples showing additio-
nal longer PCR products were considered FLT3/ITD+. All mu-
tation suspected samples were repeatedly tested for confir-
mation.
Statistical analysis
Curves for overall survival for ITD+, and FLT3/WT patients
were calculated by the Kaplan-Meier, and Wilcoxon test for
difference between groups was calculated. All statistical analy-
ses were performed by using MedCalc statistical package
(MedCalc software, Mariakerke, Belgium).
Results
Eleven of 67 AML patients (16.4%) were shown to have FLT3/
ITD mutation in the form of additional larger gene amplicon
by PCR. The sizes of ITD varied between 30 and approxima-
tely 100 bp (Figure 1). All positive patients also expressed the
unmutated allele.
Among the mutated patients there were 2 PML patients, 1
with core binding factor leukemia and others had either nor-
mal standard cytogenetics or complex abnormalities (2 pati-
ents). Morphological classification and hematological data of
positive patients are given in Table 1. For the subset of 28
tested patients, 11 mutated and 17 non-mutated we calcula-
ted overall survival that showed trend for worse outcome for
FLT3/ITD+ patients but with the given sample size the diffe-
rence was not significant (Figure 2). However, median survi-
val for FLT3/ITD+ patients was 3 months vs. 16 months for
non-mutated patients (p = 0.0431). Two groups did not differ
in the grade of leukocytosis, hemoglobin or platelet counts at
diagnosis.
Discussion
Since the discovery of the existence of FLT3/ITD and D835
mutations in myeloid leukemia some 7 years ago there is an
increasing body of research results accumulating that is sugge-
sting an important role of aberrantly expressed FLT3 gene in
the biology and clinical behavior of the disease. Our results fit
into so far reported frequencies of this molecular event in
AML. For adult AML frequencies from 17.3% (3) to higher
26.2% (4). Recent review by Levis et al. (5) summarizes the
results of multiple studies and confirms some of the impressi-
ons had from individual reports. For instance, in pediatric AML
incidence of FLT3/ITD is lower than in adults (from 6–15%)
(6). Normal cytogenetic or t(15;17) AML have higher inciden-
ce than core-binding factors leukemia (t(8;21) and inv(16)).
Secondary AML has less of this mutation than primary tumors
and poor risk cytogenEtics is not associated frequently with
FLT3 mutation (5).
Figure 1. Detection of FLT3/ITDs by PCR. Lane 1: Molecular
weight marker 100 bp lader; Lane 2: wild type FLT3 amplicon;
Lanes 3-6: FLT4/ITD positive cases. Note that additional bands
varie in size (arrows). Lane 8: blank, negative control with no
DNA.
Figure 2. Kaplan-Meier survival curve for FLT3/ITD+ (full
line, 1) and LFT3/wt patients (dashed line, 2).
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Prognostic impact of FLT3 mutation today is also believed to
be clear: it does adversely affect survival. Adult and pediatric
AML patients with FLT3 mutation live worse than non-FLT3
mutated patients (5, 7). Our results are in accordance with this
understanding. Overall incidence of FLT3/ITD mutation in a
panel of 72 consecutive leukemia we screened was 16.4%.
We had 2 FLT3/ITD+ cases of 6 t(15;17) promyelocytic leuke-
mia (30%). There was one t(8;21) AML with mutated FLT3 of
4 with the same karyotype. Interestingly, among our positive
cases there were 2 with complex karyotypes both of which
were including an extra copy of chromosome 13, to which
FLT3 maps. Both patients had relapsing and resistant disease.
In our assay we could only register somewhat longer ITDs in
Table 1. Clinical data of FLT3/ITD-positive patients.
Razpr. 1. Klinični podatki o FLT3/ITD pozitivnih bolnikih.
Age/Sex Cytogenetics FLT3/ITD WBC Outcome
Starost/Spol FAB Citogenetika size (bp) × 109/L Izid
30, M M2 45, x,–y, t(8;21) 70 76.3 A,
Allo BMT
40, F M3 46, xx, t(15;17) 70 34.0 D
49, M M3 46, xy, t(15;17) 70 5.4 A
57, M M2 normal 70 35.0 A
58, M M2 49, xy, del(9) (q13; q22)
+11, +13, +14 80 7.4 D
43, M M2 normal 80 485.0 D
52, F M1+CLL normal 30 127.0 D
65, F M4 normal 30 26.11 D
60, F M4 46, xx, der(19), t(17; 19)/
48, xx, +8, +13 100 18.6 D
85, F M1 normal 30 1.2 D
69, M M2 normal 30 48.11 A
A = alive, D = dead
those patients but it was not possible to relate to the gene
expression level itself.
In conclusion, our results confirmed the significant presence
of FLT3/ITD mutation in primary adult AML that was also asso-
ciated with poorer clinical performance of the disease. Based
on the numerous studies with similar experience one can ex-
pect the molecular testing for FLT3/ITD and possibly D835
mutation to be introduced into the routine molecular diagno-
stic workup of acute myelogenous leukemia. An additional
argument for this will certainly be targeting of FLT3 receptor
by the new generation of kinase inhibitors that will as drugs
enter clinical practice in the near future.
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KUSEC R, MARUSIC-VRSALOVIC M, VRANIC-BOBETIC T ET AL. FMS-LIKE TYROSINE KINASE (FLT3) GENE ITD MUTATION IN ACUTE MYELOID LEUKEMIA