Effects Of C/EBPα Mutations On Clinical Prognosis In Acute Myeloid Leukemia Patients

Background: Acute Myeloid Leukemia (AML) is usually considered to be induced by mutation leading to cell uncontrolled proliferation, differentiation and/or apoptosis pathway changes. As a member of C/EBP (CCAAT/enhancer binding protein, C/EBP) family, C/EBPαis an important transcription factor during the myeloid progenitor cell proliferation and differentiation. C/EBPαplays an important role in the process of AML cell proliferation and differentiation in hematopoietic stem system. The recently WHO classification of leukemia emphasizes on a routine mutational screen of C/EBPαmutations, associated FLT3 and NPM1 gene mutations within AML patients bone marrow for initial diagnosis. C/EBPαhas become an important prognostic reference indicator, and may be a new therapeutic target for AML patients. The destruction or losses of C/EBPαfunction are often associated with the occurrence of AML. The retrieve of C/EBPαexpression can be a treatment for inducing differentiation. A large number of studies show that, AML patients with C/EBPαmutations mainly include frameshift mutations in the N-terminal and disruption mutations in the C-terminal basic leucine zipper (bZIP) region. The presence of C/EBPαmutations have been described and demonstrated as significantly influencing the outcome of patients, such as Event-free survival (EFS), Overall survival (OS) and complete remission (RD). The patients with lower relapse rate and higher OS are closely related to the C/EBPαmutations. Therefore, the accuracy of the C/EBPαmutation detection has a great significancy for the treatment and prognosis with the risk classification for AML patients.Objective: Detect C/EBPαmutations in AML patients; compare the performance of direct sequencing method and multiplex-PCR fragment analysis for detecting C/EBPαmutations in patients with AML; analyze the relationship between the patients with C/EBPαmutations and clinical characteristics; and observe the clinical outcome of C/EBPα-WT and C/EBPαmutation patients who receivd remission induction therapy.Methods: Firstly, the polymerase chain reaction (PCR) direct sequencing was used to detect 60 samples of bone marrow and 10 samples of peripheral blood. Then, multiplex-PCR fragment analysis method was used to detect the AML patients with C/EBPαmutations again. The relationship between C/EBPαmutations and patient characteristics was analyzed using independent-samples t test. CR rate comparisons were performed using Chi-square test. The Kaplan-Meier method was used to estimate distributions of RD and OS of patients. Differences of RD and OS between two survival distributions were analyzed using the log-rank test.Results:①PCR direct sequencing showed 22 C/EBPαmutations in 8 (13.3%) of 60, including 5 (62.5%) cases with biallelic mutations and 3 (37.5%) cases with single mutations, of which there is only 1 case with N-terminal frameshift mutation;②1 case with N-terminal frameshift mutation was not detected with multiplex-PCR fragment analysis method. The method detects C/EBPαmutation percentages of 93% compared with the PCR direct sequencing method in this study;③there is no significant difference between the C/EBPα-WT and C/EBPαmutant patients in terms of sex, age, bone marrow blast cell count, platelet count and white blood cell (WBC) count. But C/EBPαmutant patients have more hemoglobin than the C/EBPα-WT patients;④the inverse correlation between expression of C/EBPα-WT and CD7 has not been detected with the samples in this study. There are other kinds patients besides C/EBPα-CD7+ and C/EBPα+CD7- patients with 60 samples. P value (P = 0.926) is not considered statistically significant. But CD7+ is not detected in C/EBPαmutant patients;⑤CR, OS, and RD of C/EBPαmutant patients are all higher than C/EBPα-WT patients, but the difference is not statistically significant (P values are 0.569,0.130 and 0.510, respectively);⑥CR (87.5%) and RD (9.25, 95% CI: 7.78-10.72) of C/EBPα+CD7- patients is significantly higher than those of C/EBPα-CD7+ (CR 25% and RD 5.188, 95% CI: 3.325-7.050), the differences of these two groups is statistically significant (P = 0.012 and P = 0.008, respectively).Conclusions:①These results show that mutations in the transcription factor C/EBPαare found in 13.3% AML patients, and biallelic mutations occur in 62.5% AML patients;②Although PCR direct sequencing is a high accuracy detection method to screen C/EBPαmutation, the combination of direct sequencing and multiplex-PCR fragment analysis may be an optimal approach for detecting the mutations in AML in future clinical application.③There is no significant difference between the C/EBPα-WT and C/EBPαmutant patients in terms of sex, age, bone marrow blast cell count, platelet count and white blood cell (WBC) count;④There only is a little difference between the C/EBPα-WT and C/EBPαmutant patients in terms of CR, OS, and RD in this study, which are should be further observed.⑤C/EBPα+CD7- could be used as a prognostic marker of relatively favorable outcome.