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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.

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Introduction

Chronic myelogenous Leukemia (CML

Chromosomal translocation t(9;22) (q34;q11); yieldsPhiladelphia chromosome.

Fusion gene BCR-ABL encodes constitutively activeprotein tyrosine kinase that promotes;

Cell survival

Cell proliferation

Inhibition of apoptosis.

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Chromosomal translocation t(9;22)

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Mechanism of action of BCR-ABLtyrosine kinase

Mechanism of action of BCR-ABLtyrosine 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

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Resistance to Imatinib

50% to 90% of CML patients acquired resistance to Imatinib dueBCR 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

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Frequency of BCR ABL kinase DomainMutation

100 different mutations have been identified

16 common mutations ---85% of all mutations

Frequency

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Method for Mutation Analysis

RNA ExtractionComplimentaryDNA formation

RT-PCRDNA

Sequencing

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DNA sequencing of ABL kinase Domain

P loopDrug Binding

loop

Catalytic

Domain

Activation

Loop

5TTAGGATCCTAGATTCGGAATCGTTGATCCTTATCACAGTCAGATATACTGGTAATCCATTCGATACGAGTAGATACAGA

TCGTTAGGATCCTTTCGTTGATCCACTGGTAATCCATTCGATTAGATACAGATCCAGTCAGATATACTGGTAATCCAAGTCA

GATATACTGCTGGTAATCCAAACCACAGTCAGATATACTGGTAATCCATTCGCTCACAGTCAGAGGGTACTGGTAATCCAT

TCGATACGAGTAGATACAGATCGTTAGGATCCTTTCGTTGATCCTTCGTTGATCGTTGATCCACTGGTAATCATATGAATA3

716 bp

BCR ABL

M31TE355G

E255KV3791

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Results

S#

Patient Age Sex MutationDetected

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

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Conclusion

BCR-ABL kinase domain mutations are associated withimatinib Resistance in CML patients.

Timely identification of Imatinib resistance in CMLpatients help to set therapeutic strategies with 2ndgeneration TKIs.

Direct DNA sequencing is a method of Choice indetection of ABL kinase Domain mutation.

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References

Tensuz Tauchi ,Kanzuna OhyasShilei,Molecular mechanism ofresistance of leukemia to Imatinib; Leukemia research28SI(2004)S39-S45.

C.Cameron Yin;Detection and Molecular monitoring of minimalresidual disease in chronic myelogenous leukemia.

Dragana Milojknovic; Mechanism of resistance to imatinib adsecond 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.

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Acknowledgment

Faculty Members:Dr. Tariq Moatter

Dr. Zahra Hasan

Lab Fellows:Toheed Kausar

Samina Ghani

Sheeba Parveen

Azra Samreen

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