Expression And Clinical Significance Of PFKP In Adult Acute Myeloid Leukemia

Objective:Phosphofructokinase(PFK)is a key enzyme in the glycolytic pathway,which has different tissue distributions of the L(liver),M(muscle)and P(platelet)isoforms.The platelet isoform of phosphofructokinase(PFKP)can catalyzes the irreversible conversion of fructose-6-phosphate(F6P)to fructose-1,6-bisphosphate(F-1,6-BP).The gene transcription and translation level of PFKP can cause changes in glucose metabolism,but can also affect the expression level of multiple genes.Upregulated mRNA levels can be found in many solid tumors,such as neuroblastoma,human cervical cancer,liver cancer,kidney cancer,breast cancer,prostate cancer,leukemia,etc.Studies have shown that the level of PFKP expression is related to the survival of patients.However,there are relatively few reports on acute myeloid leukemia.The purpose of this study was to investigate the expression and clinical features of PFKP during the preliminary diagnosis and treatment of acute myeloid leukemia.Methods:Using the Oncomine Oncology Gene Chips Database(from https://www.oncomine.com/resource/login.html),TCGA Database(from http://ualcan.path.uab.edu/index.html),Data-mining the expression of PFKP gene in acute myeloid leukemia,and survival data were analyzed based on its expression level combined with clinical data.To collect the bone marrow fluid specimens of 55 patients of AML who were admitted in the First Affiliated Hospital of Guangzhou Medical University from November 2012 to July 2017(inclusion criteria: according to morphocytology and histochemistry of FAB classification,the percentage of patient?s peripheral blood or bone marrow blast cell exceeding 20% is diagnosed as AML.The items collected in the study included general data(age,sex),laboratory data(white blood cell count,red blood cell count,hemoglobin level,platelet count,WHO classification,bone marrow blast cell count).Application of reverse transcription real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)is used for the detecting relatively expression level of PFKP in AML patients in the initial diagnosis and regulated treatment achieved complete remission(bone marrow aspirate contains less than 5% of bone marrow blast cells).Using SPSS 19.0 and GraphPad Prism 5 to analyze: 1.Paired t-test was used to compare the mean of two groups of the relative expression levels of PFKP in bone marrow of AML patients in the initial diagnosis and achieved complete remission(CR)with standard induction therapy.And compare if there is a difference and whether the difference is statistically significant;2.Using the Mann-Whitney U test,the relative mRNA content of PFKP was statistically expressed as mean±standard deviation(X±s).The 55 patients with newly diagnosed acute myeloid leukemia were divided into low expression and high expression according to their relative expression of PFKP in bone marrow cells to compare whether there is any statistical difference between the two groups.The white blood cell count,red blood cell count,hemoglobin amount,platelet count,bone marrow fluid primordial cell count,CR rate,and 1 year recurrence rate are measurement data using two independent sample t-tests or two independent sample rank sum tests.The clinical features such as age,gender,and WHO classification were counted data using chi-square test.(If the number of cases is greater than or equal to 40 and all frequencies are greater than or equal to 5,use the ordinary chi-square test;if the number of cases is greater than or equal to 40,but the frequency is greater than or equal to 1 and less than 5,use the modified chi-square test;if the number of cases is less than 40 and the frequency is less than 1,use the Fisher exact method.Two independent sample t-tests are used for continuous variable comparisons,while two independent sample rank sum tests are used to compare continuous variables that do not follow the normal distribution or homogeneity test of variance.)3.The 47 patients with newly diagnosed acute myeloid leukemia were divided into low expression and high expression according to their relative expression of PFKP in bone marrow cells.(Of the 55 patients mentioned above,8 patients were remove out of the data because of follow-up loss,or the irregular treatment.)Log-rank analysis of Kaplan-Meier survival analysis was used for statistical analysis of the effect of PFKP expression in the survival rate of the two groups of patients,and whether it was statistically significant.(P < 0.05 was considered statistically significant and all tests were two-sided.)Result:1.Oncomine tumor gene chip database,TCGA database mining analysisOncomine tumor gene microarray database can be used to excavate data from five groups of research institutes.combined and meta-analyzed the results,compared the relative mRNA expression level of PFKP in bone marrow fluid of acute myeloid leukemia and normal people,which can be found that the expression of PFKP in bone marrow fluid of patients with acute myeloid leukemia was significantly upregulated(P<0.05).Selected one of the institutional data for statistics,including 6 cases of normal people,23 cases of AML patients,using GraphPad Prism 5 software to plot and analyze,calculated P <0.05,the difference is statistically significant.The TCGA database was used to explore the effect of PFKP gene expression in patients with AML on the Survival prognosis.Compared with the high expression of 38 patients and the low expression of 125 patients,the survival curve of PFKP patients with low expression was found to be more stable and have a longer survival period,while the survival curve of the expression patient was steeper and have a shorter survival period(p=0.0092<0.05,the difference was statistically significant).From the chart,it can also be seen intuitively that the median survival period is longer in the low-expression group than in the high-expression group.2.Compare of the relatively expression level of PFKP in AML patients in initial diagnosis and regulated treatment achieved complete remissionThe qRT-PCR technique was used to detect the relative expression levels of PFKP in 6 patients in the initial diagnosis and regulated treatment achieved complete remission.The two-sample paired t-test of SPSS 19.0 software was used to compare the mean of the two groups,t=2.425,P=0.0358 <0.05,the difference was statistically significant.The relative expression level of PFKP was higher in the initial diagnosis of AML than CR.3.Analysis of the clinical features of AML of PFKP expression level in the initial diagnosisThe qRT-PCR technique was used to detect the relative expression of PFKP in patients with AML in the initial diagnosis.Mann-Whitney U test of SPSS 19.0 software was used to calculate the relative mRNA levels of PFKP using the mean±standard deviation(X±s)statistics.The 47 newly diagnosed AML patients,according to the median expression of the relative expression level of PFKP,was divided into two groups: low expression and high expression.There were 28 cases in low expression group and 27 cases in the other group.The white blood cell count,red blood cell count,hemoglobin amount,platelet count,bone marrow fluid primordial cell count,CR rate,and 1 year recurrence rate are measurement data using Student's t-tests.The clinical features such as age,gender,and WHO classification were counted data using chi-square test.It can be concluded that the P value is greater than 0.05,and there is no statistically significant difference in grouping.4.Survival analysis of PKFP gene expression levels in newly diagnosed AML patientsThe relative expression level of PFKP in patients of AML in initial diagnosis were detected by qRT-PCR.47 patients of newly diagnosed acute myeloid leukemia were grouped according to the relative expression level of PFKP in bone marrow fluids.The onset time was the starting point of observation,and the endpoint event was death,the follow-up deadline was July 12,2017.The overall survival(OS)was measured from the date of diagnosis to death or the final follow-up.Log-rank analysis ogKaplan-Meier survival analysis was used for statistical analysis,according to the relative expression level of PFKP in the bone marrow fluid,patients were divided into high and low expression groups.P = 0.043,P value Less than 0.05,the result was statistically significant.The observational starting point was the time of standard treatment achieved complete remission,and the recurrence or death event was the end point.The follow-up deadline was July 12,2017.The disease free survival(DFS)started from the complete remission after standard treatment to the time of recurrence or death(for any reason).Log-rank analysis ogKaplan-Meier survival analysis was used for statistical analysis,according to the relative expression level of PFKP in the bone marrow fluid,patients were divided into high and low expression groups.P = 0.039,P value Less than 0.05,the result was statistically significant.This indicates that AML patients with low expression of PFKP gene have longer OS and PFS than high expression.Conclusion:1.Comparing the relative mRNA expression level of PFKP in myeloid fluid of acute myeloid leukemia and normal human,the expression level of PFKP in bone marrow fluid of acute myeloid leukemia patients was significantly increased(P<0.05).AML patients with low expression of PFKP gene have longer OS and PFS than high expression.2.Comparing the relative expression levels of AML patients in initial diagnosis and standard treatment achieved complete remission,the relative expression level of PFKP at the time of initial diagnosis of AML was higher than that of CR.3.Clinical features of newly diagnosed AML patients such as age,sex,WHO classification,white blood cell count,red blood cell count,hemoglobin amount,platelet count,bone marrow fluid initial cell count,CR rate,1-year recurrence rate,analyzed and compared with PFKP gene expression level of newly diagnosed AML,were no statistically significant difference.