To Study The Heterogeneity Within Acute Myeloid Leukemia With CEBPA Mutations

Part I The clinical relevance and prognostic significance of CEBPA mutations in cytogenetically normal acute myeloid leukemiaPurpose:To determine the clinical relevance and prognostic significance of mutations in the CCAAT/enhancer binding protein alpha (CEBPA) gene in cytogenetically normal acutemyeloid leukemia (CN-AML).Methods:The entire CEBPA coding region was screened in259patients with CN-AML by direct sequencing. We also examined the mutation status of NPM1, FLT3and DNMT3A. The clinical relevance and prognostic of CEBPA mutation were analyzed.Results:In a cohort of259CN-AML patients,79(30.5%) had CEBPA mutation.43cases (16.6%) were single-mutated (CEBPAsm) and36cases (13.9%) were double-mutated. Presence of CEBPA mutations was associated with French-American-British types Ml and M2(P=0.014). CEBPAdm patients had a low level of platelets (P=0.002) and were highly unlikely to have an NPM1mutation (P=0.003) compared with CEBPAsm cases. Concurrent recurrent molecular mutations were less frequently in CEBPAdm than in CEBPAsm cases (P=0.001). CEBPAdm was associated with a higher first complete remission rate (83.3%) compared with wild-type CEBPA (CEBPAwt)(52.8%) and CEBPAsm (55.8%), P=0.003. Only CEBPAdm patients had a significantly better overall survival (OS)(3-year OS:58.0±9.9%,35.5±4.2%, and30.5±7.9%for CEBPAdm, CEBPAwt, CEBPAsm, respectively, P=0.025). CEBPAdm patients also had a longer event-free survival (EFS) compared with CEBPAwt and CEBPAsm cases (3-year EFS:46.2±9.3%,25.8±3.5%, and23.8±7.1%, P=0.026). In multivariable analysis, CEBPAdm was a prognostic factor for favorable OS outcome (hazard ratio [HR]=0.52, P=0.045) and favorable EFS outcome (HR=0.52, P=0.018).Conclusion:We propose CEBPAdm should be clearly distinguished from CEBPAsm CN-AML. Only CEBPAdm patients were associated with favorable outcome. Part Ⅱ To explore recurrent mutations associated with mutated CEBPAPurpose:To explore potentially recurrent mutations associated with mutated CEBPA, study the biological activities of mutated protein, and then analyze its clinical relevance and prognostic significance.Methods:1. Whole gene sequencing of a familial acute myeloid leukemia (AML) with mutated CEBPA. Candidate recurrent mutations were verified by bidirectional Sanger sequencing of the corresponding gene after PCR amplification.2. The mutation region and expanded conserved region of candidate genes were screened in50AML cases with CEBPA mutations, and then50AML cases with wild-type CEBPA. The confirmed recurrent mutations were further screened in expanded AML samples.3. All detected mutations were analyzed by PolyPhen-2prediction. We constructed a plasmid with coding sequencing of mutated protein and along with a green fluorescent protein. The transcriptional activity of mutated protein was analyzed by Dual-Luciferase reporter assay system. The location of mutated protein was examined by fluorescence microscope.4. The clinical relevance and prognostic of recurrent mutations were analyzed in86AML patients with mutated CEBPA.Results:1. We found4candidate recurrent mutations associated with mutated CEBPA, namely CDC40, PRPS2, RAD21and GATA2mutations.2. In50AML cases with CEBPA mutations,4patients had GATA2mutations. In50AML cases with wild-type CEBPA, no mutations were found in4candidate genes. Overall, in expanded samples, we found8/86(9.3%)cases with mutated CEBPA had GATA2mutations.3. All detected GATA2mutations were identified by PolyPhen-2as probably damaging indicating influencing the function of GATA2protein. The main type of GATA2mutation (p.A318V/c.953C>T) had reduced transcriptional activity compared with wild type. However, mutated GATA2showed nuclear localization like the wild-type GATA2protein.4. There was no significant difference in clinical characteristics, molecular aberration and complete remission compared cases with GATA2mutations than with wild type. But patients with GATA2mut/CEBPAdm had a trend of longer OS compared than cases with GATA2mut/CEBPAsm, GATA2wt/CEBPAdm or GATA2wt/CEBPAsm (2-year OS:100%,66.7±27.2%,73.1±8.8%and44.5±7.8%, respectively, P=0.151). In addition, patients with GATA2mut/CEBPAdm had a significant better EFS than cases with GATA2mut/CEBPAsm, GATA2wt/CEBPAdm or GATA2wt/CEBPAsm (2-year EFS:100%,66.7±27.2%,56.6±10.0%and27.0±6.8%, P=0.015).Conclusion:GATA2mutations are frequent in AML cases with CEBPA mutations. CEBPAdm patients with GATA2mutation have even better outcomes; however,this should be further confirmed in a larger scale of sample.