Analysis and Identification of Mutation in Chronic Myelogenous Leukemia (Cml) and Bcr_Abl in Leukemia Patients aged 40 to 50 Year

Rashmita Sahoo¹, Vidya Harini V², Fatemeh Esfandiari Ghalati¹ and Yaghoub Sadeghi²*

¹Head of Triesta Lab. HCG Hospital, Bangalore, India. ²Department of Biotechnology, Bangalore University, Bangalore India.

ABSTRACT: Chronic Myelogenous Leukemia (CML) is one of the few cancers that is known to caused by a single, specific genetic mutation in more than 90% of cases. The transformation to CML is caused by a reciprocal translocation of the BCR gene on chromosome 22 (at q11) and the ABL gene on chromosome 9 (at q34), resulting in a fused BCR-ABL gene dubbed the “Philadelphia chromosome”, approximately 44,000 people in the United States are diagnosed with some form of leukemia. The tyrosine kinase inhibitors (TKIs) Imatinib, Dasatinib, and Nilotinib inhibit activity of the BCR-ABL fusion protein, resulting in both hematologic responses. The aim of my study was to determine and confirm presence of Philadelphia chromosome and presence of BCR-ABL in patient with CML. Determination of the presence of the Ph chromosome by karyotyping at diagnosis is necessary as part of differential diagnosis and as prognosis. The standard methods of banding are the Q, G, R, and C banding techniques. In this study chromosomes have been treated to trypsin for taking different bands. karyotyping and FISH were equal, as shown in the present work, except for the fact that FISH was faster. In cases that did not present metaphases by karyotyping, FISH was extremely useful in providing a result. In daily practice, FISH can be very helpful since index of karyotyping, when the patient is receiving myelotoxic drugs, there may not be enough metaphases for analysis. RT-PCR is still more sensitive but its clinical meaning has to be clarified. Most of PCR include the detection of the translocation at the level of the RNA transcript. The method usually consists of two steps. The first reaction is that of obtaining cDNA from RNA by means of reverse transcription (RT). Subsequently, an aliquot of cDNA is transferred to another tube and subjected to the second reaction, that is, PCR amplification. Two different enzymes are used, namely, reverse transcriptase for the RT step and Taq polymerase for the PCR. Flow cytometry is a technique for analyzing large populations of single cells. For this method, the diagnostician has to analyze multiple bivariate dot plots from the cytometer. The flow cytometric immune bead assay is a fast and easy technique for specific detection of BCR-ABL proteins in leukemic cells. It is not dependent of the breakpoint position in the BCR gene, does not need special laboratory facilities other then a routine flow cytometer, provides results within several hours and it can be run in parallel to routine immunophenotyping.

KEYWORDS: Chronic Myelogenous Leukemia; Cancer; Mutatio; Mid-age

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