The frequency of mutations in 

exons 11 and 17
of c-kit gene in patients with acute
myeloid leukemia​

Dr. Yusra Rashid

Haematology department
University of Health Sciences
 Introduction
• Leukaemia is a disorder in which there is a neoplastic
proliferation of hematopoietic cells causing  increased
infiltration of bone marrow.​
• Basic cause is the genetic damage, which is either
acquired from environmental agents like chemicals,
radiations/viruses or it may be inherited.
• But some mutations are spontaneous.
 Classification of leukaemia
• Acute lymphoblastic leukaemia (ALL) ​
• Chronic lymphocytic leukaemia (CLL) ​
• Chronic myeloid leukaemia(CML) ​
• Acute myeloid leukaemia (AML) 
 Acute myeloid leukemia
• AML is a clonal malignancy characterized by neoplastic
proliferation of blasts.​
• Blast cells proliferate in bone marrow and inhibit the
normal differentiation of haemopoietic cells resulting
in cytopenias.​
• These cells accumulate in bone marrow, go to blood and
sometimes spread to other areas of body like spleen, liver,
lymph nodes and central nervous system (CNS).​
• These abnormal cells interfere with normal hematopoietic
process and organs function.
• AML can occur in patients who are suffering from
another haematological disorder, or as a result of previous
chemotherapy or exposure to radiation ​
• But, mostly it occurs as a new malignancy in previously
healthy individuals.
 Gene mutations
• Mutations of Nucleophosmin 1 gene
• Mutations of DNA methyltansferase 3A gene
• Mutations of Fms-Like Tyrosine Kinase 3
• Mutations of Isocitrate Dehydrogenase
• Mutations of Ten–Eleven Translocation 2
• Mutations of Tumor Protein 53
• C-KIT GENE mutations
 C-kit gene
• Oncogene that encodes c-kit receptor (CD117), which has
tyrosine kinase activity
• Located on chromosome 4
• It is comprised of four domains an extra-cellular domain, a
transmembrane, an intracellular domain consists of a juxta-
membrane domain and a tyrosine kinase domain
• There is a wide distribution of c-kit receptor in hematopoietic
cells and other tissue cells.
• Bone marrow stem cells and CD34+ cells express c-kit
receptor.
• Mast cells, germ cells, melanoma cells, gastrointestinal cajal
cells express c-kit receptor.
 C-kit receptor activation mechanism
• The extracellular domain of c-kit has five Immunoglobulin-like
domains (D1-D5).
• D1-D2-D3 is a main unit which binds to SCF
• The second functional unit is composed of this SCF binding unit and
a D4 while the third one is D5.
• The binding affinity of D4-D4 and D5-D5 is low when SCF is not
present.
• When the SCF-mediated c-kit receptor acquires its certain level in
the local environment, the adjacent D4s join to D4-D4 complex by
overcoming the electrostatic repulsion among them.
• The combination of SCF ligand with c-kit receptor occurs in two
steps. In first step, SCF combines with the accompanying c-kit
domain with the help of electrostatic forces between SCF and the
functional unit D1-D2-D3. In second step, conformational changes
occur in SCF in order to give more stability to the SCF/c-kit
complex.
C-Kit dimer interactions. The figure on left shows the c-kit dimer model
having two free c-kit molecules whereas, the diagram on right shows that
the D4-D4 and D5-D5 domains are in close proximity by overcoming the
electrostatic repulsion forces between them, in actual SCF-induced c-kit
dimer.
• SCF binding to receptor activates receptor dimerization,
relieving auto-inhibitory interactions leading to trans-
phosphorylation within pairs of dimer.
• This causes recruitment and phosphorylation leading to
downstream signaling proteins activation.
• Various “hot spots” in KIT mutation related to intracellular
and extracellular juxta-membrane domains of exons 8, 9, 11
and 17 and leads to the disruption of auto-inhibitory
mechanism.
 C-KIT MUTATIONS IN AML
• Associated with bad prognosis in AML patients.
• The most commonly studied mutations are in exons 2, 8, 9, 11,
13 and 17.
 Objective
• To determine the frequency of mutations in exons 11 and 17 of
the c-kit gene in AML patients.
 Rationale
• This study will aid in the detection of c-kit gene mutations in
AML patients. This will help us to know the frequency of
mutations in exons 11 and 17 of c-kit gene, which may
contribute to the bad prognosis of AML as well as the idea of
association these mutations with AML.
 Methodology
• The present study was conducted on 80 samples taken from
INMOL hospital and Jinnah Hospital, Lahore after approval of
synopsis from ethical review committee of University of
Health Sciences, Lahore and the approval from ethical
committee of both hospitals.
• History and written consent was taken from the patients.
• STUDY DESIGN:
• It was a descriptive study.
• SETTINGS:
• The study conducted in Department of Haematology and the
Department of Human Genetics and Molecular Biology
University of Health Sciences, Lahore
• SAMPLE SIZE:
• The sample size was calculated by the following formula
keeping the confidence level equal to 95% and margin of error
equal to 10%.
• The sample size calculated for both exons was large enough to
carry out research within limited resources and budget.
Therefore, 80 diagnosed patients of AML were analyzed.
 Sample selection
• Inclusion criteria:
• Diagnosed cases of AML on the basis of clinical presentation,
>20% blasts in peripheral smear and bone marrow and
cytochemical stains.
• All age group either male or female
• Exclusion criteria:
• Patients on chemotherapy/radiotherapy
• Relapsed cases of AML
• For DNA extraction, both EDTA whole blood and bone
marrow smear slides were used. After DNA extraction,
polymerase chain reaction was performed. Using agarose gel
electrophoresis, the presence of PCR product was determined,
which was subsequently washed and sent for sequencing
analysis.
• The sequencing analysis was done to determine the mutations
in exons 11 and 17.
 Statistical analysis
• Statistical analysis was done using SPSS version 20.
• Qualitative variables like gender, clinical presentation, disease sub
classification and presence or absence of mutations were reported in form of
frequencies and percentages.
• Quantitative variables like patients’ age, CBC parameters, LDH and blasts
counts and duration of illness were reported in form of mean and standard
deviation.
• Normal distribution of the data was checked by applying the Shapiro Wilk
test and the p value > 0.05 was considered as significant. Mean ± SD was
reported for normal distributed parameters and the median (IQR) was
reported for not normal distributed parameters. Mann Whitney U was applied
on not normally distributed parameters while t-test was applied on normally
distributed parameters and the p value ≤0.05 considered as significant and
vice versa.
 Results
• Newly diagnosed 80 AML patients were included in the study.
• The female to male ratio was 1.8:1.
• These patients were categorized according to FAB
classification into M1 (11), M2 (30), M3 (20), M4 (16), M5
(2) and M6 (1).
 Clinical Features of AML Patients
100.00%
100%
85.00%
90%
77.50%
80%
67.00%
70%

60%

50%

40%

30%

20%
6.20%
10%

0%
Fever Infections Gum Bleeding Bruises Lymph Nodes
 Mutational analysis
• All 80 patients of AML were analyzed for disease associated
mutations but none of them was found to have mutation in the
exon 11 and 17 of c-kit gene.
• However, two polymorphisms, an intronic variant upstream of
the coding sequence and a base pair deletion in the coding
region of exon 11 was found in six patients.
• Neither mutation nor polymorphism was found in exon 17.
 Discussion
• Total 80 diagnosed patients of AML were included. Out of
these, 67.5% were males and 37.5% were females. The male
to female ratio came out to be 1.8:1. Another study on
Pakistani population also showed the similar trend of male
predominance towards AML and the male to female ratio was
1.5:1 (Sultan et al., 2016).
• In current study, the median (IQR) age of presentation of 80
AML patients was 38.0 (24) years. This finding was nearly
consistent with an Indian study which showed median age of
40 years (Philip et al., 2015). This lower median age as
compared to other developed nations was, perhaps due to
different genetic makeup and late referring of patients to the
tertiary care hospitals.
• AML patients present with non-specific symptoms. Detailed
clinical history of AML patients in current study showed that,
the fever was the most common symptom (100%) followed by
bruises (85%), infections (77.5%), gum bleeding (67%) and
lymph nodes (6%). Similar findings were reported in another
AML study in India, in which fever occurred in most of the
patients followed by fatigue, infections, bleeding and bruises
(Preethi, 2014).
• Among haematological parameters, mean Hb (7.7 g/dl) was
low and mean WBC count (52.19 x103/ul) was high and mean
low Platelet count (58.7x103/l) was observed. Highest numbers
of blast percentages (35-95%) were also seen in under
discussion study. Current study findings were in concordance
with previous published data in Netherlands (de Jonge et al.,
2011).
• AML is actually a group of heterogeneous morphological
subtypes. Each subtype has its own morphological,
cytogenetic and phenotypic variation. For categorizing of
AML patients, the FAB classification is mostly used all over
the world. In present study, M2 was the most important
observed morphological subtype. This finding was consistent
with the previous study conducted in Karachi Pakistan
(Kulsoom et al., 2017)
• The survival statistics of AML patients in current study was
very bad as 100% included patients were died because of
disease. No patient could survive for more than 10 months.
However, in United States, the 5 years survival statistics of
AML patients was 27.4% and its percentage was high among
young age group.
• This lowest survival rate of under discussion AML patients
also suggested that old age had worst prognosis. It might be
possible; the old patients respond poorly to therapy and mostly
die during the induction therapy. Similar finding was also
observed in another study conducted in Bosnia (Jahic et al.,
2017).
• Incidentally, none of our collected samples showed any type of
mutation in both exons 11 and 17. Our findings were
remarkably similar to a one published Turkish data titled
"Molecular examination of mutations in acute myeloid
leukaemias patients from Turkey." They also did not find c-kit
gene mutations in any of their AML cases (Merdin et al.,
2021).
• Our findings were also consistent with those of a Brazilian
study published in 2012 on the detection of mutations in acute
myeloid leukaemias. They discovered mutations in exon 8 of
the c-kit gene, but none in exon 17 (Machado et al., 2012b).
Conclusion