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J Ayub Med Coll Abbottabad 2021;33(3)
http://www.jamc.ayubmed.edu.pk
399
ORIGINAL ARTICLE
PREVALENCE OF FLT-3 MUTATION IN ACUTE MYELOID
LEUKAEMIA
Kinza Sabir1, Nauman Ismat Butt2, Muhammad Akram1, Fahmina Ashfaq2, Jawayria
Sajid1, Kausar Bano1
1Department of Medical Oncology, Jinnah Hospital, Allama Iqbal Medical College, Lahore-Pakistan
2Department of Medicine, Chaudhary Muhammad Akram Research & Teaching Hospital, Azra Naheed Medical College, Lahore-
Pakistan
Background: FLT-3 mutation is a valuable prognostic marker in patients of AML being related
with bad prognosis and poor clinical response to conventional chemotherapeutic agents.
Frequency of FLT-3 mutation in AML varies from 25% to 35%. The objective of this study was to
determine prevalence of FLT-3 mutation in patients with Acute Myeloid Leukaemia. Methods:
This observational cross-sectional Study was conducted in Department of Oncology, Jinnah
Hospital Lahore from 1st October 2018 to 31st March 2019. Patients with acute myeloid
leukaemia, aged 15–60 years, of both genders were included. After taking consent, demographic
data was noted. Three ml of sample of blood was obtained from each patient and sent for detection
of FLT-3 mutation. Data was analysed using SPSS version 20.0. Chi square test was applied, p-
value <0.05 significant. Results: A total of 180 patients were enrolled in this study. The mean±SD
age of patients was 34.72±14.3 years, among which 38.3% were female and 61.7% male. The
mean±SD duration of disease was 3.39±2.8 months. The mean±SD haemoglobin level, TLC and
platelet counts were 7.2±2.3 g/dl, 30,913±63,573 per cm and 58.6±52.3×103 per cm. The blast cell
(%) count was 69.6±19.8. FLT-3 mutation was present in 18.9%. Conclusion: We conclude that
FLT-3 mutation to be present in only a minority of patients with Acute Myeloid Leukaemia
having no significant association with age, sex, haemoglobin, WBCs and blast counts.
Keywords: FLT-3 mutation; Acute Myeloid Leukaemia
Citation: Sabir K, Butt NI, Akram N, Ashfaq F, Sajid J, Bano K. Prevalence of FLT-3 mutation in acute myeloid
leukaemia. J Ayub Med Coll Abbottabad 2021;33(3):399–402.
INTRODUCTION
Acute Myeloid Leukaemia (AML) is a life-
threatening hematologic malignancy that is detonated
by accumulation of immature myelogenous cells in
the blood and bone marrow.1 It presents usually at a
later adulthood stage and has a substantial
proportionate mortality in all the cancer related
deaths throughout the world.2 AML is one of the most
common form of leukaemia in the Western world and
accounts for nearly 25% of all adult leukemia.3
Affected hematopoietic cells progressively
accumulate a variety of molecular alterations during
pre-leukemic evolution and disease propagation,
which include somatic mutations, epigenetic
alterations, cytogenetic abnormalities, and
transcriptomic changes.4 Cytogenetics, together with
mutation status, form the basis of risk classification
system and provide means for risk stratification in
AML patients.5
FMS-like tyrosine kinase 3 (FLT-3) is a
tyrosine-kinase receptor with vital roles in
hematopoietic stem/progenitor cell survival,
proliferation and differentiation. Mutations in FLT-3,
specifically internal tandem duplication in the juxta-
membrane domain (FLT3-ITD), are seen in
approximately 20% of AML patients.6 It is related
with bad prognosis of patients and a poor clinical
response to conventional chemotherapeutic agents.
Specific targeted therapy with tyrosine kinase
inhibitors such as ibrutinib, midostaurin, sorafenib
and sunitinib along with bone marrow transplantation
is needed to improve the outcome in these patients.6
Literature has shown that the frequency of FLT-3
mutation in AML varies and was reported to range
from 25–35%.7 However the emergence of drug
resistance8 to FLT3-inhibitors has posed a crucial
challenge in optimal management of patients,
warranting the desire for further comprehending the
biology and complexity of FLT-3 in AML. FLT-3
mutation is a valuable prognostic marker in patients
of AML. Very limited published data is available
regarding presence and prognosis of FLT-3 mutation
in AML patients in the Pakistani population. It is not
known whether ethnic differences play a role in the
progression and prognosis of disease. Relapse of
leukaemia and poor response to conventional
treatment in our population leads to increased disease
burden, in-hospital stay and mortality. It is highly
important to have information regarding the
magnitude of the disease in Pakistani population, so
that early targeted therapy can be given to these
patients thereby reducing disease morbidity and
J Ayub Med Coll Abbottabad 2021;33(3)
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400
mortality in our country that has limited resources
and a significant financial strain.
The objective of this study was to determine
frequency of FLT-3 mutation in patients with Acute
Myeloid Leukaemia presenting to Department of
Oncology, Jinnah Hospital Lahore.
MATERIAL AND METHODS
This observational cross-sectional study was
conducted for duration of six months from 1st
October 2018 to 31st March 2019 in the
Department of Oncology, Jinnah Hospital Lahore.
Keeping margin of error of 6% and confidence
level of 95%, the sample size was 180 patients
(Allahyari et al.9 showed frequency of 21%) using
non-probability consecutive sampling technique.
Patients of both sexes, aged 15–60 years,
diagnosed with Acute Myeloid Leukaemia
(presence of at least 20% myeloblast cells in bone
marrow biopsy of the patient by microscopic
examination) were included. Patients with AML
secondary to myelodysplastic syndrome and patients
of Chronic Myeloid Leukaemia in acute blast crisis
were excluded. Written informed consent was taken.
Information regarding demographic data was noted
after which 3 ml of blood sample was obtained from
each patient using aseptic technique and sent to the
pathology laboratory for detection of FLT3 mutation
using Polymerase Chain Reaction (PCR) -
Qualitative. Presence of FLT3 mutation was labelled
and noted. Confidentiality of the data was ensured.
Data was entered and analysed using SPSS version
20.0. Stratification of outcome using chi-square test
was done, keeping p-value <0.05 as statistically
significant.
RESULTS
A total of 180 patients fulfilling the inclusion and
exclusion criteria were enrolled in the study after
taking informed consent from the patient’s attendant.
The mean±SD age of patients was 34.72±14.3 years,
among which 38.3% (69 patients) were female and
61.7% (111 patients) were male. In this study, 8.3%
(15 patients) belonged to high socioeconomic status,
31.7% (57 patients) belonged to middle
socioeconomic status, where as 60.0% (108 patients)
belonged to low socioeconomic status. The mean±SD
duration of disease was 3.39±2.8 months. The
mean±SD duration haemoglobin level, TLC and
platelet counts were 7.2±2.3 g/dl, 30,913±63,573 per
cm and 58.6±52.3 x103 per cm. The blast cell (% in
the bone marrow) count was 69.6±19.8. FLT3
mutation was present in 34 patients (18.9%) where as
it was absent in 146 patients (81.1%). Stratification
for outcome was done as shown in table-1.
Table-1: Stratification of outcome
Variables Pearson Chi-square
coefficient p-value
Age 3.647 .177
(non-significant)
Sex 2.495 .114
(non-significant)
Socioeconomic Status 0.992 .609
(non-significant)
Duration of Disease 2.479 .290
(non-significant)
Haemoglobin level 2.516 .284
(non-significant)
TLC 5.072 .079
(non-significant)
Platelet Count 2.181 .336
(non-significant)
Blast Cell Count 3.132 .209
(non-significant)
DISCUSSION
The presence of FLT-3 aberrancies in high-risk
leukaemia patients was identified 2 decades ago and
comprehension about the biology, clinical
implications, and specific targeting therapies is still
on-going. Whether FLT3-ITD is an initiating event in
leukaemia genesis or not still remains controversial,
but the conspicuous development of intrinsic
resistance to FLT-3 inhibitor therapy reinforces its
position as one of the crucial co-operating events in
the generation of human AML. The FLT-3 mutation
is considered a poor prognostic factor as it is
associated with higher rates of early disease relapse
and overall shorter survival despite chemotherapy.
Both paediatric and adult clinical trials are underway
to investigate the role of various FLT-3 inhibitors in
conjunction with chemotherapy in FLT-3 positive
AML patients. In the present study, we analysed 180
patients of AML for presence of FLT-3 mutation. In
addition, we studied the clinicopathological features
to determine their correlation with FLT-3 mutation as
independent prognostic factors. According to the
result of this study, FLT-3 mutation was found in
18.9% AML patients and it showed no statistically
significant association with age, sex, socioeconomic
status, duration of disease, haemoglobin, TLC,
platelets and blast cell count. Stratification of this
data with FLT-3 mutation showed insignificant
association with WBC and blast percentages as in
this study 49.4% patients presented with high WBC
count >11000 per cm and 84.4% presented with blast
counts ≥50%. In this study, 130 patients were aged
between 15 to 45 years and among them FLT-3 was
positive in 21.8% while 47 patients were aged
between 46–60 years and among them 10.6% were
positive for FLT-3 mutation.
In the cross-sectional study conducted in
Iran on 100 AML patients by Allahyari et al9 PCR for
FLT-3 mutation was done. Mean age at diagnosis
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was 28.5 years and 52% patients were male. FLT-3
mutation was present in 21% but there were no
significant differences between sex, age or
visceromegaly and these results were consistent with
the findings of our study. Frequency of FLT-3
mutation in our study was also consistent with the
study conducted by Ishfaq et al10 in which 55 patients
were enrolled. Twenty-five patients had acute
lymphoblastic leukaemia (ALL) and 30 had AML.
The polymerase chain reaction demonstrated FLT3/
ITD mutations in 4% ALL patients while in AML it
was present in 13.3%. In AML, a statistically
significant association was found between higher
WBC count and FLT-3, and the mutation was more
common in elderly patients aged 31–40 years. In
ALL, no statistically significant association was
found with the clinical features however the mutation
was more common in age groups aged 21–30 years.
Yunus et al11 in Malaysia studied mutational analysis
of exons 14–15 and 20 of the FLT-3 gene in 54
patients utilizing PCR-CSGE (conformational
sensitive gel electrophoresis) to characterise FLT-3
mutations in adult AML cases. Mutations in FLT-3
exon 14–15 were seen in 13% and no mutation
identified in FLT-3 exon 20. FLT3-ITD mutations
were significantly associated with a high blast cell
percentage and high white blood cell count however
no significant difference was noted in overall median
survival within 2 years. Seventy newly diagnosed
chemotherapy-naive AML patients were enrolled by
Raezei et al12 in Iran for detecting the FLT-3 and
NPM-1 gene mutations by PCR followed by direct
sequencing. The frequencies of mutations in FLT3-
ITD, FLT3-TKD and NPM-1 were 25.9%, 5.9% and
20.8% respectively. The FLT3-ITD mutation was
more common in non-M3 subset of AML (FAB
classification). No mutation was identified in either
FLT3-TKD or NPM-1 genes in patients with M3
AML. No significant correlation was seen between
the presence of FLT3-ITD and NPM-1 mutations.
Considering the high stability of NPM-1 gene, it may
be utilized in conjunction with FLT-3 to monitor
patients, especially for detection of minimal residual
disease.
A total of 113 AML patients were evaluated
at diagnosis based on routine morphology and
cytochemistry and classified according to the WHO
criteria by Sazawal et al13 in India. The distribution
of AML subtypes was M1 (1 patient), M2 (32
patients), M3 (57 patients), M4 (14 patients), M5 (1
patient), M6 (1 patient) and 7 patients where
morphological subtype could not be classified. RT-
PCR was performed to identify PML/RARalpha,
AML-1/ETO, CBFbeta/MYH-11 and FLT3/ITD. Of
the 57 patients with M3 subtype, 55 had the PML-
RARalpha fusion transcript. The prevalence of bcr-3
(short isoform) was 62% and bcr-1 (long isoform)
38% with no correlation with age, sex or white blood
cell count. FLT3/ITD mutation was more frequent in
APL subtype (17.5%), the frequency greater in
patients with bcr3 isoform (70%) than in those with
in bcr1 isoform (30%). Patients with FLT3/ITD
mutation had a significantly higher median white cell
count, short median overall survival. AML1-ETO
fusion transcript was detected in 16 of 56 patients
with no correlation with clinical or haematological
parameters. Patients with APL who have FLT-3
mutation had a poorer prognosis. Therefore, it was
concluded that rapid identification of specific
translocations at diagnosis is important for prognostic
purposes and their detection should be incorporated
into routine assessment. In India, Chauhan et al14
showed FLT3-ITD mutation to be present in 23%
with association to young age less than 15 years and
high WBC count. However, no significant difference
was reported in the response rates to conventional
chemotherapy in patients with or without FLT3/ITD
mutation.
Kuchenbauer et al15 correlated FLT-3
expression with clinical parameters, FAB types,
cytogenetics and flowcytometry in 207 AML
patients. FLT-3 levels correlated with high
percentages of bone marrow blast cells, high
leucocyte counts and M5 FAB subtype. In patients
with normal cytogenetics no impact on overall
survival was detected regardless of FLT-3 mutation,
while in the patients with normal cytogenetics and
wild-type FLT3, worse overall survival was noted.
Likewise, a study conducted in China by Lee et al16
on 52 AML patients showed FLT3-ITD positivity in
28.8% of AML patients which was associated
significantly with absolute leucocyte counts and bone
marrow blasts counts. Patients with t (15;17) showed
higher prevalence of FLT3-ITD and significantly
associated with worse overall survival.
None of WHO FAB subtype is directly
associated with FLT-3 mutation in AML as results
are variable in different countries according to
different citied studies. Further studies are required to
validate this to determine any geographic
significance. Most of the studies performed proved
the association between high WBCs count and blasts
percentage with FLT-3 mutation, which was not seen
in our study. Limitations of our study include a
relatively small sample size and the inability to see
association of various FAB subtypes with FLT-3
mutation. FLT3/ITD positive AML patients are often
treated by Allogenic Stem Cell Transplant (SCT)17
and there is an increasing interest to target the FLT-3
receptor tyrosine kinase with specific
inhibitors.18,19 FLT-3 inhibitors such as quizartinib,
sorafenib and midostaurin are undergoing clinical
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402
trials, and on 28th April 2017 the FDA approved
midostaurin for treating FLT-3 positive adult AML
patients.20
CONCLUSION
We conclude that this study showed FLT-3 mutation
to be present in only a minority of patients with
Acute Myeloid Leukaemia with no specific
association with age, sex, haemoglobin, WBCs and
blast counts. However, further studies with a larger
number of patients are necessary to determine the
prevalence of FLT-3 mutation in patients with AML
and its correlation with severity of disease and
prognosis so that appropriate therapeutic
advancements can be considered depending upon
magnitude and prognosis of the disease.
AUTHORS' CONTRIBUTION
This study was conceived and designed by KS, MA
and KB. MA, NIB and JS did the initial literature
research and designed the proforma for data
collection. KS and JS did the data collection,
assembly and patient assessment. Data analysis and
interpretation was performed by NIB and FA. KS,
NIB and FA were involved in manuscript writing.
MA and KB did the final critical review and
corrections. NIB is the corresponding author on
behalf of all other authors.
REFERENCES
1. Checkoway H, Dell LD, Boffetta P, Gallagher AE, Crawford
L, Lees PS, et al. Formaldehyde exposure and mortality risks
from acute myelogenous leukemia and other
Lymphohematopoietic Malignancies in the US National
Cancer Institute cohort study of workers in Formaldehyde
Industries. J Occup Environ Med 2015;57(7):785–94.
2. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myelogenous
leukemia. N Engl J Med 2015;373(12):1136–52.
3. Cancer Genome Atlas Research Network, Ley TJ, Miller C,
Ding L, Raphael BJ, Mungall AJ, et al. Genomic and
epigenomic landscapes of adult de novo acute myeloid
leukemia. N Engl J Med 2013;368(2):2059–74.
4. Marcucci G, Haferlach T, Dohner H. Molecular genetics of
adult acute myeloid leukemia: prognostic and therapeutic
implications. J Clin Oncol 2011;29(5):475–86.
5. Granfeldt Østgård LS, Medeiros BC, Sengeløv H, Nørgaard
M, Andersen MK, Dufva IH, et al. Epidemiology and clinical
significance of secondary and therapy-related acute
myelogenous leukemia: a national population-based cohort
study. J Clin Oncol 2014;33(31):3641–9.
6. Medinger M, Lengerke C, Passweg J. Novel Prognostic and
Therapeutic Mutations in Acute Myeloid Leukemia. Cancer
Genomics Proteomics 2016;13(5):317–29.
7. Elyamany G, Awad M, Fadalla K, Albalawi M, Al Shahrani
M, Al Abdulaaly A. Frequency and prognostic relevance of
FLT3 mutations in saudi acute myelogenous leukemia
patients. Adv Hematol 2014;2014:141360.
8. Smith CC, Wang Q, Chin CS, Salerno S, Damon LE, Levis
MJ, et al. Validation of ITD mutations in FLT3 as a
therapeutic target in human acute myeloid
leukaemia. Nature 2012;485(7397):260–3.
9. Allahyari A, Sadeghi M, Ayatollahi H, Yazdi HN, Tavakol
M. Frequency of FLT3 (ITD, D835) Gene Mutations in
Acute Myelogenous Leukemia: a Report from Northeastern
Iran. Asian Pac J Cancer Prev 2016;17(9):4319–22.
10. Ishfaq M, Malik A, Faiz M, Sheikh I, Asif M, Khan MN, et
al. Molecular characterization of FLT3 mutations in acute
leukemia patients in Pakistan. Asian Pac J Cancer
Prev 2012;13(9):4581–5.
11. Yunus NM, Johan MF, Ali Nagi Al-Jamal H, Husin
A, Hussein AR, Hassan R. Characterisation and Clinical
Significance of FLT3-ITD and non-ITD in Acute Myeloid
Leukaemia Patients in Kelantan, Northeast Peninsular
Malaysia. Asian Pac J Cancer Prev 2015;16(12):4869–72.
12. Rezaei N, Arandi N, Valibeigi B, Haghpanah S, Khansalar
M, Ramzi M. FMS-Like Tyrosine Kinase 3 (FLT3) and
Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid
Leukemia Patients with Normal Karyotypes: Mutation Status
and Clinical and Laboratory Characteristics. Turk J
Haematol 2017;34(4):300–6.
13. Sazawal S, Kumar B, Hasan SK, Dutta P, Kumar R, Chaubey
R, et al. Haematological & molecular profile of acute
myelogenous leukaemia in India. Indian J Med
Res 2009;129(3):256–61.
14. Chauhan PS, Bhushan B, Mishra AK, Singh LC, Saluja
S, Verma S, et al. Mutation of FLT3 gene in acute myeloid
leukemia with normal cytogenetics and its association with
clinical and immunophenotypic features. Med
Oncol 2011;28(2):544–51.
15. Kuchenbauer F, Kern W, Schoch C, Kohlmann
A, Hiddemann W, Haferlach T, et al. Detailed analysis of
FLT3 expression levels in acute myeloid leukemia.
Haematologica 2005;90(12):1617–25.
16. Lee JN, Kim HR, Shin JH, Joo YD. Prevalence of FLT3
internal tandem duplication in adult acute myelogenous
leukemia. Korean J Lab Med 2007;27(4):237–43.
17. Pratz KW, Sato T, Murphy KM, Stine A, Rajkhowa T, Levis
M. FLT3-mutant allelic burden and clinical status are
predictive of response to FLT3 inhibitors in
AML. Blood 2010;115(7):1425–32.
18. Stone RM, Mandrekar SJ, Sanford BL, Stine A, Rajkhowa
T, Levis M. Midostaurin plus Chemotherapy for Acute
Myeloid Leukemia with a FLT3 Mutation. N Engl J
Med 2017;377(5):454–64.
19. Patnaik MM. The importance of FLT3 mutational analysis in
acute myeloid leukemia. Leuk Lymphoma 2018;59:2273–86.
20. Larrosa‐Garcia M, Baer MR. FLT3 Inhibitors in Acute
Myeloid Leukemia: Current Status and Future
Directions. Mol Cancer Ther 2017;16(6):991–1001.
Submitted: February 2, 2020 Revised: -- Accepted: November 8, 2020
Address for Correspondence:
Nauman Ismat Butt, Department of Medicine, Chaudhary Muhammad Akram Research & Teaching Hospital,
Azra Naheed Medical College, Lahore-Pakistan
Cell: +92 345 465 1049
Email: nauman_ib@yahoo.com
