Assessment The Frequency of E255k and F359V Mutations by ASO-PCR in Non-Responders Chronic Myeloid Leukemia Patients Using Imatinib mesylate Therapy

Chronic myeloid leukaemia (CML) is a clonal disorder of a pluripotent stem cell. In the last decade Imatinib Mesylate (IM) which is first generation tyrosine kinase inhibitor, was introduced into the clinical management of CML , however many patients experience treatment failure because of many mutations within the fusion gene which is known as break point cluster region-Ablson gene (BCR-ABL). These mutations account for about 30-50% of cases of IM resistance, thus the molecular diagnosis of these mutated genes is highly recommended to be included in the standard procedure for follow-up of CML patients who failed to get benefit from Imatinib . Identifying the mutations, F359V and E255K during Imatinib treatment, it provides to the clinician relevant information for choosing the second generation of tyrosine kinase inhibitor (Dasatinib) . In this cross-section study screening for the two mutations were done. Seventy patients were recruited from Baghdad Medical City /Teaching Hospital /Hematology Department, during the period between September 2011 to June 2012. They were diagnosed clinically and hematologically as Chronic Myeloid Leukemia (CML) and they were on IM in different doses (400-800 mg/day) for at least one and a half year. Along with 10 healthy age match subjects who served as technical negative control . Those patients were classified according to their responsiveness to IM into four groups:
1- Response failure group which included 22 patients whose Florescence in situ hybridization (FISH) for BCR-ABL gene was more than 35% despite normal WBCs count while using IM (600-800mg/day) for at least one and a half year . 2- Suboptimal responder group which included 16 patients whose FISH for BCR-ABL gene was 1-35% despite normal WBCs count, while using IM (600mg/day )for at least one and a half year . 3- Advanced disease group which included 14 patients who were in accelerated and blastic phases regardless of their FISH results ,those patients have high WBCs count and blast cells more than 10% in peripheral blood/or bone marrow while using IM (800mg/day ) for at least one and a half year .
4- Optimal responder group which included 18 patients receiving 400mg/day Imatinib and whose FISH result was less than 1% with normal WBCs count and they were included as disease control group.
Peripheral blood (PB) sample was taken from each patient and control. DNA was extracted from PB samples using commercial available DNA extraction kit. Molecular screening for the presence of E255K and F359V mutations in DNA of CML patients and optimal responder was done using Allele Specific Oligonucleotide-Polymerase Chain Reaction (ASO-PCR). Amplified products were electrophoresed in 2.5% agarose gel. The mean age for CML patients at diagnosis was 40.45 ± 2.56 years, ranging from 25 -75 years . Male to female ratio in CML patients was 1.12:1 . The mean duration of the disease was longest in the optimal group with mean of 7.75± 0.28 years ,followed by that of the advanced group (5.78± 0.96 years) whereas the disease duration of both the failure and the suboptimal groups were comparable ( 4.97±0.47 , 4.68 ±0.33 years respectively ) . The mean of WBC count was highest in the advanced group (61.06±10.47)×109/L , whereas it was normal in the other groups . The mean of FISH reading for BCR- ABL gene was highest in the advanced group of CML patients( 74±3.20%) , followed by the response failure group (67.40 ± 3.24 % ) then the suboptimal group (18.87± 2.66 % ) while it was undetectable in the optimal responder group. Furthermore there was a positive correlation between the WBC count and the FISH % in the advance and suboptimal groups but this correlation did not reach the level of significance. Finally the result of molecular screening by ASO – PCR technique for the mutation E255K and F359V showed that all CML patients were negative for both mutations. In conclusion, there may be another variants for these mutations such as E255V and/or F359I/C, another types of point mutations in BCR-ABL or another mechanisms of resistance such as over expression ,drug influx and efflux could be the causes of IM treatment failure or suboptimal response in the studied CML patients.