Frequency And Clinical Features Of ASXL2 Gene Mutation In Patients With AML1-ETO Positive Acute Myeloid Leukemia

Objective:To investigate frequency of additional sex combs-like 2(ASXL2) gene mutation in acute myeloid leukemia patients with AML1-ETO fusion gene. To analyze the clinical indicators of patients with AML1-ETO fusion gene positive and confirm the clinical features of patients with mutant ASXL2. Detect “Collaborative molecμles ”(C-Kit, FLT3,ASXL1, N/KRAS gene mutations) in patients with AML1-ETO fusion gene and confirm relationship with ASXL2 gene mutations.Methods:1.The mutation occurrence of ASXL2.Primers were designed according to the 11 and 12 exons of ASXL2. Mutation analysis of ASXL2 gene of DNA in 180 de novo AML patients( Including 59 patients with AML1-ETO fusion gene positive and 121 patients with AML1-ETO fusion gene negative)was performed by using polymerase chain reaction(PCR). The PCR products are derived from different primers, that were designed on the different position of the ASXL2 gene,and conduct eletrophoresis in agarose gel. The resμlts were analysised by gel imager.2.Clinical features of ASXL2 gene mutation in acute myeloid leukemia patients with AML1-ETO fusion gene.Make a collection of clinical indicators of patients with AML1-ETO fusion gene positive, for example, white blood cell counts, the hemoglobin levels, platelet counts, the proportion of acidophilic cell, the proportion of primitive cell, infiltrate degree of liver,spleen, central nervous system metastases and lymph nodes, immunophenotype, response to treatment and survival analysis. Make using of statistics and analytical method, carryingon the comparision of the clinical indicators of patients with mutant ASXL2 and ones without mutant ASXL2.3. Analysis of coexistence of “Collaborative molecμles ” and ASXL2 gene mutation.To investigate the mutation occurrence of ASXL1, C-Kit, FLT3, N/KRAS gene mutations in 59 patients with AML1-ETO fusion gene. Mutation analysis of ASXL1,C-Kit,FLT3-TKD, N/KRAS was performed by using polymerase chain reaction(PCR) followed by sequence analysis. The PCR products of FLT3-ITD was conducted eletrophoresis in polyacrylamide gel and the resμlts were analysised by gel imager.Results:1. In a total of 59 AML patients with AML1-ETO fusion gene positive, 11.9%(7/59)patients harboured ASXL2 gene mutations and 121 patients with AML1-ETO fusion gene negative, 3.3%(4/121)patients harboured ASXL2 gene mutations. Differences were not observed in frequency of ASXL2 gene mutations between patients with AML1-ETO fusion gene positive and ones with AML1-ETO fusion gene negative(P=0.055).2.In patients with AML1-ETO fusion gene positive: The hemoglobin levels of patients with mutated ASXL2 gene[56.2(38.0~72.0)g/L] were significantly lower than those with wild type ASXL2[69.0(37.2 ~ 154.0) g/L](P=0.038). Differences were not observed in white blood cell counts, platelet counts, the proportion of acidophilic cell, and the proportion of primitive cell in the marrow between patients with mutant ASXL2 and ones without mutant ASXL2(P>0.05). None of all 59 patients suffered from liver, spleen,central nervous system metastases. Moreover, enlarged lymph nodes was similar between patients with mutant ASXL2 and ones without mutant ASXL2( P=0.859).Immunophenotype: patients with ASXL2 mutations showed a lower surface expression level of CD33 compared to ones without mutant ASXL2(P=0.033). cyCD3 was not expressed in the both groups. Expression levels of CD117, cMPO, HLA-DR, CD34, CD38,CD13, CD44, CD15, CD64, CD11 b, CD56, CD19, cyCD79 a, CD7 were similar between patients with mutant ASXL2 and ones without mutant ASXL2. All of 59 patients were in remission(P=0.577). The median survival time for patients with mutant ASXL2 were 8months and ones without mutant ASXL2 were 22 months, but overall survival was similarbetween them(P=0.631).3. In 58 AML patients with AML1-ETO fusion gene positive: ASXL2 gene mutations exist alone in two patients. ASXL2 mutations are mutually exclusive with ASXL1 mutations. Differences were not observed in C-Kit, FLT3, RAS gene mutations between patients with mutant ASXL2 or ASXL1 and ones without mutant ASXL2 or ASXL1(P>0.05). C-Kit gene mutations are not coexist with the RAS gene mutation.Conclusions:1. ASXL2 mutation may be a new event that can cooperate with AML1-ETO to induce leukemia. The mutation frequency of ASXL2 in AML1-ETO positive AML patients in China is lower than those in Westen. ASXL2 gene mutations are also found in patients with AML1-ETO fusion gene negative, which means that ASXL2 mutations are not specifically recurrent in AML patients with AML1-ETO fusion gene positive.2. Patients in AML1-ETO positive AML with ASXL2 mutation show specific clinical characteristics of hemoglobin levels and expression level of CD33.3. ASXL2 and ASXL1 are homologous gene, which belong to a family of ASXL,suggests only one of the mutations are needed because of the similar biological functions.One of the proliferation-promoting gene(ASXL2 or ASXL1, C-Kit, FLT3, N/KRAS)mutations can cooperate with AML1-ETO to induce leukemia.