This study was designed as a try to apply molecular, conventional cytogenetic, and immunological techniques as conformational diagnosis of BCR-ABL gene and protein, and also to follow up chronic myeloid leukemia patients (CML) treated with imatinb mesylate (IM) for assessment of response at different IM treatment durations. The study recruited 165 patients, of them,135 CML previously or newly diagnosed, 17 Acute Lymphocytic Leukemia(ALL), 3 Acute Myelocytic leukemia (AML), 9 Chronic Lymphocytic Leukemia(CLL), 1 Chronic Myelomonocytic Leukemia(CMML) at the National Center of Hematology (NCH)-AL- Mustensseria University in the period between February 2006 to August 2008. Also, 15 healthy individuals (as a negative control) were included. Peripheral blood (PB) samples were taken from each subject and divided into two parts: (1) the heparinized was blood used for blood culturing for cytogenetic analysis, as well as, lymphocyte separation for immunological study and (2) EDTA-treated PB was used for RNA and DNA extraction for molecular studies. At the end of this study, the results obtained from the 42 CML patients were only analyzed because other CML patients were either lost for regular follow up, had long gap in IM treatment or their samples were not sufficient for testing. The overall mean value for age of CML patients at diagnosis and at sampling were (32.83±0.67) Ys and (34.71±1.02) Ys, respectively. Most frequent age group was between (20-29)Ys. Male to female ratio was 1:1.62. The overall mean value for age of ALL, AML CLL CMML and healthy individuals were (20.05, 44.33, 57, 55 and 38.2)years, respectively.The male to female ratio were (1.8:1, 2:1, 1:2, one male and 1:1), respectively. From a total of 42 CML patients, 66.66% were previously treated (pretreated with hydroxyuria or interferon-alfa) and (33.33%) were already treated with IM as a first line treatment. At sampling, 95.23% of CML patients were at chronic phase, from them, 32.5% were at early- chronic phase (less than 12ms from diagnosis) and 67.5% were at late- chronic phase(whom had a median time from diagnosis of 36ms), while (4.76%) at accelerated phase. During the follow up period and until the end of this study, (83.33%) of CML patients were at chronic phase and (16.66%) were at accelerated phase. The mean value of CML disease duration (from diagnosis until the end of this study) was (54.31, ±3.61) ms. The mean value of time from diagnosis to starting IM treatment in CML patients treated with IM as a first line treatment and in newly diagnosed were (1.78±0.87)ms and (18.21±5.25)ms, respectively. There was a significant difference between these two groups (p=0.034, LSD=15.17). Duration of gap in IM treatment was studied. It was shown that (9.52%) of CML patients had no gap in IM treatment while (90.48%) had an overall mean of gap duration of (10.30±1.02) ms. Hematological follow up of those patients was done using complete blood picture and differential count every 3-6 ms. Results were shown that (52.38%) of patients had achieved complete hematological response, while (47.16%) had only partial hematological response. CML patients were followed up for assessment of cytogenetic response to IM using blood culturing every 3-6ms. At first, (310) 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 was quantified according to the proportion of Philadelphia chromosome positive metaphases. The results showed that (64.28%) of CML achieved major cytogenetic response while (35.71%) achieved partial cytogenetic response. PB samples of ALL, AML, CLL, CMML and healthy individuals were cultured for one time, in order to screen for the presence of Philadelphia chromosome. Successful cultures from those leukemia patients and healthy individuals were negative for Philadelphia chromosome. Molecular screening for the presence of bcr-abl in (34) CML patients, (12)ALL patients, (1)AML patients,(1)CMML patient and (2) healthy individuals, were done using Multiplex-Single Step-Reverse Transcriptase- Polymerase Chain Reaction (M-SS-RT-PCR). At first, RNA was extracted from PB samples, then amplified using M-SS-RT-PCR. Amplified products were electrophoresid in 1.5% agarose gel. The results showed that all CML patients were positive for bcr-abl while all the others were negative for this gene. For assessment of molecular response to IM, quantification of bcr-abl in CML patients were done using Real Time-Reverse Transcriptase-Polymerase Chain Reaction (RT-RT-PCR). The first analysis point was after overall mean of IM treatment duration (23.1) ms and the second analysis point were after overall mean of IM treatment duration (35.95) ms.The results of RT-RT-PCR reaction were recorded as ratio between transcripts of bcr-abl /abl ×100 at these two time
point of analysis. The mean values of ratio at first and second time points of analysis were (0.879) and (0.471), respectively. There was a statically significant difference (p=0.05, LSD=0.0324). Also, RT-RT-PCR results were recorded as log reduction of bcr-abl transcripts. There is no significant differences in the percentage of patients who achieved complete molecular response(CMR) (≥4 log reduction) and major molecular response(MMR) (3 log reduction), but there is a significant differences in the percentage of patients who achieved only Minor-MR(2 log reduction) (p=0.05,LSD=13.789) and patients who not achieved MR(< 2log reduction) (p=0.05,LSD=5.779). DNA samples from (18) CML patients, who show signs of hematological, cytogenetic and/or molecular non-response or relapse, were screened for T315I mutation using Allele Specific oligonucleotides (ASO). The results showed that this mutation dose not detected in any of those patients. Immunological screening for the presence of BCR-ABL protein was done using Immunocytochemistry staining technique. A total of (42) CML patients, (10) ALL patients, (2) AML patients, (1) CMML patient and (8) healthy individuals were screened. Lymphocytes were separated from heparinized PB samples, smeared and fixed on positive charged slides. Monoclonal antibody specific for BCR-ABL protein was used as primary antibody. The results showed that all CML patients were positive for BCR-ABL protein and all the others were negative. In conclusion, the results of CML diagnosis obtained from hematological, cytogenetic, molecular and immunologic analysis were consistent with each other. Obligatory complementary tests that should be performed for any CML patient who start to receive IM, in order to assess the full response to therapy.