Molecular screening of pointmutation [T3151] using allele specific oligonucleotides-polymerase chain reaction [ASO-PCR]

The development of the BCR-ABL targeted Imatinib Mesylate [IM] represent a paradigm shift in the treatment of chronic myeloid leukemia [CML]. However, a considerable number of CML patients have been reported to show resistance to IM, leading to relapses. The purpose of this paper is to screening for T3151 mutation in patients who are showing criteria of resistance or relapse, using Allele Specific Oligonucleotides Polymerase Chain Reaction [ASO-PCR]. A total of 24 DNA samples related to 18 CML patients of non-responder to IM were analyzed for T3151. They received daily dose of IM [300-400] mg for a mean duration 35 months. They were followed up hematologically, cytogenetically and molecularly every 3-6 months. They had not achieved complete hematological response [CHR], major cytogenetic response [MCyR] or major molecular response [MMR] along the follow up duration and until the end of this study. DNA was extracted from 100 micro l of venous blood [VB] using DNA isolation kit. Screening for the presence of T3151 mutation was done using [ASO-PCR]. PCR products were electrophoresed using 2.5% agarose gel electrophoresis. Agarose gel electrophoresis of ASO-PCR amplified products showed just one band of about [158 bp] in the lanes of wild-type alleles, indicating that non of those patients were harboring such mutation. There was no evidence that the initial lack of CHR, CyR or MMR in CML patients treated with IM due to the presence of T3151 mutation but those patients may have carried other types of mutations result in shift in sensitivity that may allow sufficient kinase activity to initiate disease progression in the presence of IM

Cytogenetic response of peripheral blood in chronic myeloid leukemia patients treated with Imatinb

Chronic myeloid leukemia [CML] is a stem cell disorder associated with an acquired chromosomal abnormality, Philadelphia chromosome [Ph], which arises from the reciprocal translocation of part of long arm of chromosome 9, in which proto-oncogene ABL gene [ablson] is located, to long arm of chromosome 22, in which BCR gene [break point cluster region] is located forming BCR-ABL fusion gene. The suppression of BCR-ABL is likely to be crucial for therapeutic success. The development of the BCR-ABL-targeted Imatinib mesylate represents a paradigm shift in the treatment of CML. This is a prospective study designed as a try to apply cytogenetic technique as a conformational diagnosis of Philadelphia chromosome [Ph] in CML patients and also, to follow up CML patients treated with imatinib mesylate [IM] for assessment of cytogenetic response of peripheral blood at different IM treatment duration. Prephral blood samples were collected from CML patients every 3-6ms. At first, [310] prephral blood [PB] samples related to 135 CML patients were cultured but only 181[58%] cultures related to [42] patients were successful [gave obvious metaphases]. The degree of cytogenetic response of peripheral blood was quantified according to the proportion of Philadelphia chromosome positive metaphases. The results showed that [64.28%] of CML achieved major peripheral blood cytogenetic response while [35.71%] achieved partial cytogenetic response. Conventional cytogenetic karyotyping is necessary for Ph-chromosome detection and also, as an assay for periodical assessment of cytogenetic response in CML patients treated with imatinib. Imatinib has resulted in cytogenetic responses in first line IM treated patients and in those who have failed previous IFN-a therapy and in CML patients at early and late chronic phase