Leukemia Plasma Dna Integrity And Npm1 Mutations In The Gene Quantitative And Its Clinical Significance

Objective:To evaluate the clinical significance of detection of both circulating DNA concentration and integrity in patients with acute leukemia.Methods:Plasma DNA in patients with acute leukemia and control subjects were extracted and detected by fluorescent dye staining and fluorescent quantitative PCR(FQ-PCR). Agarose gel electrophoresis was performed to analyze the integrity of plasma DNA. FQ-PCR was employed to amplify large and short fragments ofβ-actin (ACTB) and DNA integrity index was calculated as the ratio of longer to shorter DNA fragments (ACTB 384/106). The dynamics of DNA integrity in 8 paired samples was analyzed during the progression of the disease.Results:The mean plasma DNA concentration of the 60 patients with acute leukemia was (152.4±242) ng/ml, which was 8 times that of the healthy control group (19.6±7.9) ng/ml, and also significantly higher than that of the lymphoma, benign disease of hematological system and solid tumor groups. The level of plasma DNA of leukemia was closely related to the age of the patients and the disease course. But no significant difference was found in FAB subtypes of acute myeloid leukemia. DNA concentrations were significantly increased in patients with acute leukemia (median 8.80 ng/ml) when compared with the healthy controls (median 3.42 ng/ml, P = 0.004). ROC curve analysis showed that the area under the curves (AUC) of circulating DNA levels for discrimination of patients with controls was 0.79.Agarose gel electrophoresis analysis showed a typical ladder pattern of DNA fragments from the plasma DNA of patients with acute leukemia. On the other hand, large and short products ofβ-actin were amplified by polymerase chain reaction, demonstrating increased integrity in plasma DNA of leukemia patients. ROC curve analysis showed that the AUC of circulating DNA integrity for discrimination of patients with controls was 0.88. The dynamics of DNA integrity in this 8 paired samples was analyzed during the progression of the disease. All patients at complete remission had a distinct reduction in plasma DNA integrity, while the plasma DNA integrity of these 8 patients at first relapse re-elevated.Conclusion:Both the concentration and integrity of plasma DNA are increased in leukemia patients, and plasma DNA may have important clinical value in both the diagnosis and monitoring of the leukemia patients. Objective:To construct quantitative plasmid standard pMDl8-T-NPM1A and develop a FQ-PCR method detecting plasma nucleophosmin mutation A (NPM1A) in leukemia patients, exploreing the clinical significance.Methods:NPM1A gene was amplified from plasma DNA and peripheral blood cell DNA which were extracted from AML patients. The standard quantitative plasmid pMDl8-T-NPM1A was constructed by T-A clone method and analyzed by PCR and DNA sequencing.Primers were designed according to the sequence of NPM1A gene,the FQ-PCR method for determination of NPM1A was established,the reaction condition was optimized and evaluated (linear range, sensitivity, specificity , reproducibility).The NPM1A level was detected by FQ-PCR in AML patientsResults:By PCR identification and DNA sequencing analysis confirmed that the construction of pMDl8-T-NPM1A was successful, and corresponded with request of quantitative standard.The better reaction condition was as follows:Each cycle was of 30 sec at 95℃,5sec at 95℃,and 30 sec at 60℃.The better reaction system was 25μl volumes containing SYBR Premix Ex Taq(2×): 12.5μl, forward primer (10pmol/u1): 0.5μl,reverse primer(10 pmol/u1): 0.5μl, sample(sclinic samples,positive standard sample,negative control and positive control): 2μl,ddH2O: 8μl . The developed PCR method showed high sensitivity (102copies/m1) and good specificity, the linear range was104～1012copies/m1, the coefficient variation (CV) was 3.21% in intra-assay and 4.87% in day to day.Changes of NPM1A lever in plasma coincided with it in peripheral blood cell. The level of plasma NPM1A DNA of AML patients was closely related to the clinical progress of disease.Conclusion:FQ-PCR is a sensitive and specific method to detect NPM1A in plasma, and could be recommended for monitoring of NPM1A lever of AML patients.This method can be used to detect the clinical progress of disease and evaluate prognosis of disease.