Stratified Prognostic Analysis Of C-KIT Gene Mutation In Adult AML Patients With RUNX1-RUNX1T1 Fusion Gene

Objective:The incidence of C-KIT gene in AML patients with RUNX1-RUNX1T1 fusion gene positive is 30-50%,and the prognosis is poor,which is often related to lower CR duration and overall survival rate.According to the "second hit" theory of leukemia,the RUNX1-RUNX1T1 fusion gene alone is not enough to cause the occurrence of leukemia.It needs to be combined with the first type of mutation to cause the disease,and C-KIT is An important mutation in the "second hit" theory.Although C-KIT is recognized as a poor prognostic factor for AML patients with RUNX1-RUNX1T1 fusion gene positive,the prognosis of clinical patients still has a large heterogeneity.Here,we collected clinical data of 54 AML patients with RUNX1-RUNX1T1 fusion gene positive with C-KIT mutations,Prognostic analysis was conducted through three levels of C-KIT mutation domain,C-KIT mutation burden,and other concomitant mutations except C-KIT.to analyze the prognostic impact of concomitant mutations.Methods:1.Case dataFrom July 2009 to December 2019,54 cases of AML patients with RUNX1-RUNX1T1 fusion gene positive and C-KIT gene mutation were newly treated in the Department of Hematology of our hospital.Data is collected from medical records,including basic information such as age,gender,concomitant mutations,C-KIT gene mutation types,etc.,through the C-KIT mutation functional domain,C-KIT mutation load,and other concomitant mutations other than C-KIT Level for prognostic analysis.2.TreatmentAll patients follow adult AML guidelines.Accept cytarabine plus anthracycline("37" regimen)-based standard induction chemotherapy regimen,and large doses of cytarabine(3 g/m2,per 12 h 1 time,6 doses)3～4 courses after induction chemotherapy and Some patients were treated with dexamethasone.Selective hematopoietic stem cell transplantation was performed in combination with patient’s wishes and economic status.3.Assessment indicatorsOverall survival(OS)was defined from the date of diagnosis to the date of death or last follow-up.The time of disease-free survival(DFS)is defined as the CR achieved after chemotherapy to disease recurrence、 death or last follow-up date.Recurrence(RP)is defined as the proportion of primordial cells in blood or bone marrow > again0.05 or recurrence at any extramedullary site after reaching CR.Criteria for complete remission(CR)refer to the Diagnostic and Therapeutic Criteria for Hematology(4th Edition),after treatment,the symptoms and signs of leukemia disappeared completely,and Peripheral neutrophil >1.5 × 10 L,platelet count >100 × 109/ L,leukemic cells were not found in leukocyte classification;Bone marrow myeloblasts type I+ type II/primitive monocyte + naive monocyte≦5%,red blood cells and megakaryocyte cell lines are normal and have no extramedullary leukemia.4.Second-generation sequencing and Sanger first-generation sequencing to determine accompanying mutations and C-KIT mutation typesSecond generation sequencing and Sanger first generation sequencing :DNA,of bone marrow samples Amplification by ordinary PCR,response procedure :95℃ pret denaturation 3 min,95℃ denaturation 30 s,60℃ annealing 30 s,72℃ extension 45 s,35 cycles,72℃ re-extension by 10 min,4℃HOLD.Amplified target gene products were sent to Beijing Huada Gene Technology Service Co.,Ltd for Second generation sequencing and Sanger first generation sequencing.Comparison of first generation sequencing results with NCBI gene bank sequences of National Biotechnology Information Center;5.Quantitative detection of mutation level by dd-PCRFrom C-KIT sequencing,a total of 43 patients with C-KIT D816V/Y and D822 K mutations were selected,and the C-KIT gene mutation level of 43 patients was absolutely quantified at the DNA level using dd-PCR technology based on MGB probes.And use statistical methods to analyze its prognosis.6.The AML of 54 patients with RUNX1-RUNX1T1 fusion gene positive with C-KIT mutation were divided into two groups according to functional domain C-KIT gene protein domain is divided into three parts: extracellular region,transmembrane region and cytosolic region,the 1-519 amino acid residues form the extracellular domain,which is encoded by the 5′end of the E1(containing the 5′-terminal untranslated region starting point),E2-E9 and E10,and contains five Ig,and functionality is SCF combination;520-543 amino acid residues form a short transmembrane region,which is encoded by E10 and functions as a signal transduction;the 544-976 amino acid residues form a cytosolic region encoded by the E10 3’terminal,E11-E20,the region also includes inserted fragments of the inserted kinase region: the E14 、 E15 3’terminal and the E16 5’ terminal.E12 contains a ATP binding site(Gly-X-Gly-X-X-Gly).E21 contains the translated terminator and 3" UTR,this region has tyrosine kinase activity.E17 corresponds to Tyrosine kinase 2 domain,and E8 and E9 correspond to Extracellular domain.7.Classify other concomitant mutations except C-KIT gene mutations according to their functionsThe other concomitant mutations of 54 patients were classified into three groups:proliferation,differentiation and epigenetic genes.8.dd-PCR quantitative detection of mutation levelsThe threshold value of C-KIT gene mutation load was determined according to the ROC curve Jordan index,and 43 patients were divided into C-KIT gene mutation high level group and low level group.Results:1.The 54 AML patients were divided into Tyrosine kinase 2domain and Extracellular domain groups according to the different functional domains of C-KIT mutation at the first diagnosis.The results were not statistically different between the two groups: five-year survival(OS),five-year disease-free survival(DFS),first recurrence(RP1)and first remission(CR1);2.Classification of other concomitant mutations by function,shows that the epigenetic genome is statistically significant for its prognosis(OS : P=0.040;DFS :P=0.010).It shows that clinical attention should be paid to the occurrence of epigenetic genome mutations at the first diagnosis;3.Statistical analysis of concomitant mutations other than C-KIT in 54 patients at the time of first diagnosis showed that the concomitant ASXL1 mutations were statistically significant for their prognosis(OS:P<0.001;DFS:P<0.001).It shows that the ASXL1 mutation at the first diagnosis is a negative factor for the poor prognosis and short survival of AML patients with C-KIT gene mutation and RUNX1-RUNX1T1 fusion gene positive;4.dd-PCR,of bone marrow DNA samples from 43 patients at first diagnosis,and the C-KIT gene mutation results were divided into two groups according to the ROC curve Jordan index,Comparison of two groups of five-year OS and five-year DFS(OS: P=0.005;DFS:P=0.006)and(RP1: P =0.023),the results were statistically significant.It shows that the C-KIT gene mutation burden at the first diagnosis is an adverse factor affecting the prognosis of AML patients with RUNX1-RUNX1T1 fusion gene positive.High-level patients have a short survival time and are prone to recurrence。5.The three C-KIT stratifications were analyzed for clinical characteristics between groups,and the results showed that the ratio of Epigenetic modification gene and proliferation group is lower than the differentiation group in bone marrow blasts(P(epigenetic modification gene)VS(differentiation)=0.023;P(proliferation)VS(differentiation)=0.008).Conclusion:1.For newly diagnosed acute myeloid leukemia patients with RUNX1-RUNX1T1 fusion gene positive with C-KIT gene mutation,C-KIT gene mutation burden is of great significance for clinical prediction of relapse,assessment of prognosis,and timely adjustment of treatment plan.When accompanied by ASXL1 mutations or when accompanied by epigenetic mutations,clinical attention should be paid and timely prevention.2.The ratio of epigenetic modification gene and proliferation group to differentiated group of bone marrow blasts is low.