| Part 1: Detection of 5-m C levels of genome in patients withacute myeloid leukemiaObjective: To explore the changes of 5-m C levels of whole genome and the change methylation levels after treatment with decitabine in patients with acute myeloid leukemia(AML),and analyse the relationship betwewn the clinical features and the prognosis. Methods: Total of 42 cases of AML patients(22 males,20 females, median age 63 years) were enrolled in this study from Jan, 2013 to Dec, 2014. The patients were treated with decitabine(20mg/(m2·d)×3~5d) based chemotherapy. All the AML patients were divided into three groups according to their clinical status which were de novo group(20 cases), relapsed/ refractory group(10 cases), remission group(12 cases) which was also used as a positive control. t The peripheral blood were collected at-1, 1 and 7 day of combination chemotherapy from all the patients. The 5- m C levels was detected by using the of quantitutive assay. The other 13 patients with iron deficiency anemia as the no rmal control. Result: 1)The 5- m C levels in de novo AML, relapsed/refrctory AML, postivie and normal controls were 0.160 ± 0.089 ng, 0.270 ± 0.136ng0.043 ± 0.018 ng and 0.052 ± 0.022 ng respectively. The 5- m C level of newly diagnosed and relapsed patients was higher than those in normal and positive controls(p<0.05), the relapsed/refractory group is the highest in all groups(p <0.05). 2) After teratment with decitabine, positive control group post-treatment one day and 7 days 5- m C content was 0.050 ± 0.016 ng and 0.046 ± 0.009 ng, similar with per- treatment(p> 0.05);In de novo patients post-treatment 1 day and 7 days, 5-m C content was 0.084 ± 0.030 ng,and 0.055 ± 0.028 ng, significant differences(p <0.05); relapsed/ refractory patients after treatment(one day and 7 days) 5- m C content was 0.190±0.127 ng and 0.142±0.112 ng,of which post-treatment 1day and 7 days than per-treatment significantly decreased(p <0.05). 3) After treatment, patients who were CR and NR, the 5- m C content was 0.063±0.037 and 0.127±0.105, respectively. Compared with the control group, CR group close to normal, NR was significantly higher than normal(p> 0.05). 4) Per-treatment 5- m C levers no significant correlation with gender, age, FAB type, original cell, white blood cell count,hemoglobin, a nd platelet. 5) Per-treatment 5-m C levels has an effect on OS and PFS of AML, whose 5- m C levles below 0.113 have a longer time of OS and DFS. Conclusion: The genome methylation in the AML patients of de nove and relapsed/refractory was increase,post-treatment with decitabin the methylation of genome was reduced and close to normal after remission; the OS and DFS is shorter in the patients who has a highter level of global methylation.Part 2: Detection of methylation status on gene promoter of stem cellrelated transcription factor Sox2, Oct4, Klf4, C-Myc, and Nanog inpatients with acute myeloid leukemiaObjective : To investigate the methylation status of transcription factor such as SOX2, OCT4, KLF4, C-MYC and NANOG genes in patients with acute myeloid leukemia(AML) and the change methylation status after treatment with decitabine. Methods: Methylation-specific PC R assay was used to detect the promoter methylation status of SOX2, OCT4, KLF4, CMYC, and N ANOG gene in peripheral blood samples(per-treatment, post-treatment 1 and 7 days from 42 AML patients(22 males, 20 females, median age 63 years) who were treated by decitabine(20mg/m2×3~5d) based chemotherapy. 42 cases of AML patients were divided into de novo group(20 cases),relapsed/refractory group(10 cases), remission group(12 cases) which was also severed as a positive control, and the other 13 patients with iron deficiency anemia as the normal control. Result: 1) In normal controls,2cases(15.4%) partially methylated of Sox2 and 4 patients(30%) detected partially methylated of Nanog, while Oct4, C-Myc, Klf4 gene methylation was not detected. In positive controls, Sox2, Oct4, Klf4, C myc and Nanog gene methylation rate was 50, 50, 41.7, 33.3 and 100% respectively. Sox2, Oct4, Klf4, C myc and Nanog gene methylation were analysed among the 30 cases AML patients of de nove and relapsed/refractory disease. they were 90%,76.7%,70%,86.7% and 100%respectively. 2) Before demethylation treatment,the Sox2, Oct4, K lf4, C-Myc and Nanog gene methylation positive rates were 85%, 75%, 70%, 80%,and 100% of de nove patients,and relapsed patients Sox2, Oct4, K lf4, C-Myc and Nanog gene methylation positive rates were 100%, 80%, 70%, 100%, 100%; the methylation rate approaching in two groups(p> 0.05),butthey were higher than the normal controls(p <0.05).Sox2 and C-Myc gene in de novo and relapsed groups the methylation was higher than the positive controls(p <0.05). 3) Post-treatment with decitabine, the rate of gene methylation was no significant change of positive control(p> 0.05); the gene of Sox2, Oct4, K lf4 and C-Myc methylation positive rate was 33.3, 40, 21.4, and 45% respectively, which were much lower than those of pre-treatment of de nevo group, the difference was statistically significant(p <0.05). In relapsed group Sox2, K lf4 and C-Myc gene methylation positive rate was lower than that in pre-treatment(33.3, 0% and 50% respectively), the difference was statistically significant(p <0.05). 4) The levels of Sox2 or Oct4 gene methylation have an impact on overal survival(OS)and disease free survival(DFS) in comparison with patients with unmethylated, former is shorter. The OS of patients with combined Sox2 and Oct4 gene methylation is poor in comparison with those who only have one such gene methylationed. Conclusion: The manner fo stem cell related transcription factor Sox2, Oct4, Klf4, C-Myc, and Nanog gene promoter methylation can be seen in AML patients,and the methylation positive rate decline after decitabine treatment; Sox2 and Oct4 gene methylation has an important influence on the prognosis.Part 3: Impacts of TET2, DNMT3 A, and ASXL1 gene mutations onthe prognosis in patients with myelodysplastic syndromeObjective: To detect TET2, DNMT3 A and ASXL1 mutation in patients with myelodysplastic syndrome(MDS), and analyse the relationship between gene mutation and clinical characteristics and impact on prognosis. Methods: The genome amplification and direct sequencing technology were employed to detect ten-eleven translocation 2(TET2, exon3 and exon11), DNA methyltransferase3A(DNMT3A, exon23), additional sex combs- like protein 1(ASXL1, exon12) gene mutations 20 cases of MDS patients and 6 cases of healthy donors’ periphery blood samples as controls. Result: 1) TET2, ASXL1, and DNMT3 A mutations was not detected in the controls. 20 cases of MDS patients, 3 cases(15%) was detected TET2 gene mutation, one case of homozygous mutation, two cases of heterozygous mutations; 4 cases(20%) was detected ASXL1 gene mutation, homozygous mutation and miscellaneous mutation in two cases,respectively.DNMT3 A was not detected exon23 mutation.One case of TET2 and ASXL1 mutations,one case exist two position mutations in ASXL1 gene. 2) TET2 or ASXL1 mutation rate was no difference among WHO subtype, karyotype, risk stratification. Mutated and with wild-type patients showed no significant difference between sex, age, white blood cell count, hemoglobin, platelet count(p> 0.05). TET2 or ASXL1 mutation had no significant effect on overal survival(OS)(p> 0.05).Conclusion:MDS with TET2, ASXL1 mutations is common, but the effect on survival time is uncertain. |
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