Screening of BCR-ABL Kinase domain Mutations from

Chronic Myeloid Leukemia patients: A preliminary study

Nazneen Islam, Toheed Kausar, Zahra Hasan, Tariq Moatter

Molecular Pathology,Department of Pathology and Microbiology,
Aga Khan University Hospital Karachi.
Introduction
Chronic myelogenous Leukemia (CML
Chromosomal translocation t(9;22) (q34;q11); yields
Philadelphia chromosome.

Fusion gene BCR-ABL encodes constitutively active
protein tyrosine kinase that promotes;
Cell survival
Cell proliferation
Inhibition of apoptosis.


Chromosomal translocation t(9;22)
Mechanism of action of BCR-ABL
tyrosine kinase
Mechanism of action of BCR-ABL
tyrosine kinase inhibitor (Imatinib)
Imatinib (STI571;Glevic )

Selective inhibitor of Tyrosine kinase

CHR 95% of patients
CCyR 76% of patients
3 log in BCR-ABL.. 57% of patients
CMR 4% of patients

Resistance to Imatinib
50% to 90% of CML patients acquired resistance to Imatinib due
BCR ABL kinase domain point Mutations.
Impair Imatinib Binding
4 region in Kinase domain where mutation can occur
P loop
Activation Loop
Binding domain
Catalytic Domain






Frequency of BCR ABL kinase Domain
Mutation
100 different mutations have been identified
16 common mutations ---85% of all mutations
F
r
e
q
u
e
n
c
y

Method for Mutation Analysis
RNA Extraction
Complimentary
DNA formation
RT-PCR
DNA
Sequencing
DNA sequencing of ABL kinase Domain
P loop
Drug Binding
loop
Catalytic
Domain
Activation
Loop
5TTAGGATCCTAGATTCGGAATCGTTGATCCTTATCACAGTCAGATATACTGGTAATCCATTCGATACGAGTAGATACAGA
TCGTTAGGATCCTTTCGTTGATCCACTGGTAATCCATTCGATTAGATACAGATCCAGTCAGATATACTGGTAATCCAAGTCA
GATATACTGCTGGTAATCCAAACCACAGTCAGATATACTGGTAATCCATTCGCTCACAGTCAGAGGGTACTGGTAATCCAT
TCGATACGAGTAGATACAGATCGTTAGGATCCTTTCGTTGATCCTTCGTTGATCGTTGATCCACTGGTAATCATATGAATA3
716 bp
BCR ABL
M31T
E355G
E255K
V3791

Results
S
#
Patient Age Sex Mutation
Detected
Type of Mutation

1 Patient#1 40 M No
2 Patient#2 59 M Yes M351T
3 Patient#3 55 F No
4 Patient#4 30 M No
5 Patient#5 54 M No
6 Patient#6 31 M
Yes
E355G
7 Patient#7 60 F Yes V379I
8 Patient#8 55 F No
9 Patient#9 21 M No
10 Patient#10 97 F Yes E255K
11 Patient#11 66 M No
Conclusion

BCR-ABL kinase domain mutations are associated with
imatinib Resistance in CML patients.
Timely identification of Imatinib resistance in CML
patients help to set therapeutic strategies with 2
nd

generation TKIs.
Direct DNA sequencing is a method of Choice in
detection of ABL kinase Domain mutation.
References
Tensuz Tauchi ,Kanzuna OhyasShilei,Molecular mechanism of
resistance of leukemia to Imatinib; Leukemia research
28SI(2004)S39-S45.
C.Cameron Yin;Detection and Molecular monitoring of minimal
residual disease in chronic myelogenous leukemia.
Dragana Milojknovic; Mechanism of resistance to imatinib ad
second generation tyrosine kinase in chronic myeloid leukemia
;Clin-Cancer-Res 2009;15(24);7519-27.
Micheal J.,mauro;Defining and managing Imatinib resistance
;American Society of Hematology.
Acknowledgment
Faculty Members:
Dr. Tariq Moatter
Dr. Zahra Hasan

Lab Fellows:
Toheed Kausar
Samina Ghani
Sheeba Parveen
Azra Samreen