| Objective1. To identify predictive variables in patients with refractory/relapsed acute myeloid leukemia(r AML) receiving allogeneic hematopoietic stem cell transplantation(allo-HSCT).2. To investigate gene mutations and clone evolution in r AML patients undergoing allo-HSCT at diagnosis and at relapse after transplantation with next-generation sequencing3. To explore the expression and potential leukemogenesis of Notch2 gene in AML patients and cell lines.Methods1. We enrolled 101 consecutive r AML patients treated with allogeneic HSCT at the First Affiliated Hospital of Soochow University between October 2003 and December 2012. The patients were retrospectively assessed to evaluate the effect of clinical characteristicson outcomes, such as age, FAB classification, stratification of cytogenetic and molecular risk, disease status at transplantation, performance status at transplantation, number of chemotherapy sessions and the type of transplantation, and to identify predictive variables.2. The study group consisted of samples from 23 r AML patients at diagnosis and 20 AML patients(including 8 r AML) at diagnosis and at relapse following allo-HSCT. We used a DNA+RNA specific gene panel, which targeted 390 tumor-related genes(full coding exons and related introns) for DNA sequencing and the transcriptome for RNA sequencing(RNA-seq), to analyze genetic aberrations of r AML patients at diagnosis and the clone evolution after the relapse following allo-HSCT. The mean count of sequencing reads obtained per sample was 50 million and themean sequencing depth was over 300×.3. By real-time fluorescent quantitative PCR, Notch2 gene m RNA expression level of bone marrow mononuclear cells for 52 adult de novo AML patients was detected, as well as 10 cases of acute lymphoblastic leukemia(ALL) and 10 cases of normal control. Notch2 gene m RNA expression level was compared in AML patients and ALL patients/normal controls, and among AML different subgroups. The correlation between Notch2 gene m RNA expression level and disease-free survival(DFS) and overall survival(OS) were analyzed. Notch2 overexpression plasmid and Notch2 inhibition sh RNA were transfected in myeloid leukemia cell lines THP-1, respectively, its effects on cell proliferation was analyzed.Results1. Impact of clinical characteristics on the outcome of allo-HSCT in r AML patientsOne hundred and one r AML patients underwent allo-HSCT were included in this study. The median age was 34 years(range, 14–58 years). All patients received myeloablative conditioning. The majority of transplants were HLA-matched, including 38 HLA-identical siblings and 33 matched but unrelated donors. Thirty patients received mismatched transplants from related donors.Of all patients, 53 achieved complete morphological remission(CR-AML) before transplantation, while 48 failed to do so(NR-AML). NR patients most likely carried the FLT3-ITD mutation(P=0.006), had a poor performance status(P=0.001), received transplants from unrelated donors(P=0.027) and were subjected to remission induction therapy less often(P=0.002). The 5 year estimate of overall survival in the CR-AML and NR-AML groups was found to be 46% and 18%, respectively, while the 5 year estimate of the incidence of relapse was determined to be 45% and 81%, respectively. In contrast, the 5 year estimate of NRM in both groups was similar(28% versus 30%).Amongst the CR patients, 32 patients displayed minimal residual disease(MRDpos) based on a multiparameter flow cytometric assay, while 21 showed no evidence of residual disease(MRDneg). The distribution of characteristics such as age, gender, FAB classification, risk stratification and performance status, was similar in both groups of patients. The 5 year OS estimate in MRDneg and MRDpos patients as 51% and 41%, respectively(P=0.37); the DFS estimate was calculated as 55% and 36%, respectively(P=0.20); the RI estimate was calculated as 21% and 29%, respectively(P=0.43).Using univariate analysis, we determined that within the entire cohort of patients, excluding those diagnosed with M5/M6 de novo AML, the presence of favorable cytogenetic and molecular risk, CR status at the time of transplantation and the performance status are significantly associated with survival. From the multivariate analyses, the prognostic factors associated with improved DFS and OS are CR status(hazard ratio [HR]=0.31, 95% CI 0.12-0.81, P=0.017) and Zubrod-ECOG-WHO score<3(HR=0.25, 95%CI 0.14-0.45, P=0.001) at the time of transplantation. From the univariate analyses, we determined that the type of AML, cytogenetic and molecular risk, status of FLT3-ITD mutation, performance status and the number of chemotherapy sessions, have a statistically significant correlation with survival. Using the multivariate analyses, three independent prognostic factors are significantly associated with higher OS rates: Zubrod-ECOG-WHO score < 3(HR=0.13, 95%CI 0.045-0.391, P<0.001), favorable/intermediate risk group(HR=0.30, 95%CI 0.103-0.853, P=0.026) and absence of FLT3/ITD mutation(HR=0.22, 95% CI 0.058-0.831, P=0.024).2. Clone evolution of r AML patients following allo-HSCT(1) Gene mutations of r AML patients at diagnosisThe median number of gene mutations of each sample in r AML patients at diagnosis was 4(range, 2-15). The most frequent gene mutations were FLT3-ITD, TET2 mutation and MLL fusion gene. Patients with FLT3-ITD tended to have a poor outcome, and FLT3 inhibitor could improve the overall remission rate and outcomes(2 year-OS 33% vs. 75%).(2) Gene mutations in AML patients at diagnosis and relapse following allo-HSCT6 mutations(range, 2-11) were detected per sample in 20 AML patients at diagnosis. The four most frequently mutated genes in our patient cohort were FLT3, DNMT3 A, CEBPA and NPM1. Genes involved in signal transduction and epigenetic regulation were the most frequent mutated groups. The most frequently mutated genes in r AML patietents were FLT3 and TET2.The median number of additional gene mutations after first relapse following HSCT in each sample was 11(range, 3-18). We identified 5 genes containing mutations in at least 4 independent samples(MSH3, KMT2 C, TSC2, WT1 and NOTCH2). Chromatin modification genes and cancer suppressor genes were the most frequent mutated genes. The most frequently mutated genes in r AML were TSC2, TNFAIP3, IGF1 R and WT1. Notch2 mutations were only found at the time of relapse following HSCT which could not be detected at diagnosis, furthermore, 3 out of 4 patients with Notch2 mutations were r AML.(3) Clone evolution patterns of relapse following allo-HSCTThree patterns related to clone evolution of relapse following allo-HSCT: 1. Gene mutations are exactly the same at diagnosis and relapse following allo-HSCT, which means a new clone is predominant at relapse; 2. Additional mutations occur at the relapse of transplantation at the base of original clone; 3. There is a clone loses some original gene mutations but gains additional mutations.3. Expression and primary leukemogenesis of Notch2 gene in adult patients with AML(1) Detection of Notch2 gene m RNA expression levels in de novo AML patientsNotch2 gene m RNA expression level in AML group was higher than that in normal control group(P<0.05) and in ALL group(P=0.180). According to FAB classification, the median Notch2 gene m RNA expression level in FAB-M3 subgroup patients was 0.07494, while that of other subgroups was 0.04637(P=0.174). According to chromosome karyotypic classification, Notch2 gene m RNA expression level in t(15,17) group and 11q23/MLL group were 0.07494 and 0.06200, respectively, while the expression of other karyotypic groups was 0.04762(P=0.22).(2) Correction between Notch2 expression and prognosisThrough Kaplan-Meier analysis in normal karyotypic AML patients, EFS was significantly superior in high Notch2 gene m RNA expression(≥0.04) group, compared with that in the group of patients with low expression(<0.04)(DFS of 2 years were 51% and 17%, respectively)(P = 0.047). 2 year-OS in two groups were 63% and 38%, respectively(P=0.528). There was no relationship between Notch2 gene m RNA expression with prognosis in patients with other chromosome abnormalities(P > 0.05).(3) The overexpression of Notch2 in myeloid cell lines THP-1 made the ability of growth and colony formation in THP-1 cells decreased. Although Notch2 was effectively inhibited by sh RNA in THP-1 cell lines, it was not found to have obvious influence on cell proliferation.Conclusion1. Our study showed that r AML patients in remission prior to transplantation displayed better outcomes than those who were not. In addition, r AML patients in remission before transplantation who had better physical performance or favorable/intermediate risk stratification may have better outcomes.2. Three clonal evolution patterns might occur in the diagnosis and relapse after allo-HSCT. Gene mutations involved in signal transduction and epigenetic regulation were frequent at diagnosis, while chromatin modification genes and cancer suppressor genes were the most frequent mutated genes at the time of relapse following HSCT.3. The Notch2 transcripts level was up-regulated in AML and high expression of Notch2 c DNA was associated with inferior 2 years’ DFS in patients with normal karyotype. Overexpression of Notch2 likely inhibited the proliferative and clonogenic abilities of AML cell line THP-1. |
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