Detection Of Myeloperoxidase Gene Expression By In Situ Hybridization In Patients With Acute Leukemia

Objective To investigate the difference and clinical significance of myeloperoxidase gene expression in acute leukemia.Methods (1) Preparation of MPO cDAN probe: In the study, human myeloperoxidase(MPO) gene cDNA segment was molecularly cloned with RT-PCR, PCR and other DNA recombinant techniques from an AML-M3 patient, which is considered always having highly expression of MPO gene. The size of cDNA cloned was 559bp. This cDNA segment was analysed by DNA sequencing and labeled with Digoxin. And then the a probe was used to detect MPO gene expression in bone marrow or blood cells through In Situ Hybridization(ISH) technique. (2) Patient samples: Bone marrow or blood cells were obtained from 60 patients with acute leukemia and 20 controls.Among these 60 patients, there were 30 new cases of acute myeloid leukemia (AML) ,which including 7 cases of AML-M1, 6 of AML-M2, 6 of AML-M3, 6 of AML-M4, 4 of AML-M5, 1 of AML-M7 . The others were 30 case of acute lymphoid leukemia, including 10 new or unremissioned cases and 20 completed remission cases after therapy. The blast percentage of all new cases weren't less then 60%. The control cases were patients without diseases of hematopoietic system and the results of their bone marrow inspection weren't found abnomily. (3) Criterion of positive samples : The positive cells showed blue and purpl granules in cytoplasm. Acoding the amount of granules in cytoplasm,the intensity of positive cells was divided into four degree.200 cells of every cases was countered and if the number of positive cells was beyond 5% ,the case was a positive one and considered having MPO gene expression. Then all data were dealed with statistics technique of rank sum , analysis of variance ,and Chi-squared test.Results (l)There didn't detect MPO mRNA using ISH in 30 cases of ALL,but all cases of AML, except AML-7, had high level of MPO gene expression.The positive rates of other cases of ALL and control cases were less than 5%, and had weak intensity. The difference between AML and ALL, control cases was significant in statistics(p<0.05).(2) In cases of AML, cases of AML-M3 showed the highest positive ratio(the mean ratio 84.7 + 17.4%) and the strongest positive intensity; then the flowing included cases of AML-M2 and M4(55.7+4.8% and 50.5+3.0% respectively). Cases of AML-M1 and M5(32.5+7.6% and 16.3+2.3% respectively) showed the more lower and weak level of positive ratio and intensity.(3) Compaering the of two methods, ISHanalysis of MPO gene expression (24/24)and cytochemical study of MPO(16/24),the result showed the ISH analysis of MPO gene expression more sensitive then that detected by cytochemical study of MPO(P<0.05).Conclusions The study shows that the ISH technique is a sensitive method in detecting the expression of MPO gene. The results shows that there is significantly different between AML and ALL in level of MPO gene expression. And among AML, AML-M3 has the highest level of MPO mRNA, the second is AML-M2 and M4, the last is AML-M1 and M5. ISH analysis of MPO gene expression might be serve an useful method for classification of acute leukemia.