| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 102 === Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders with great variability in the pathogenesis, clinical course and response to therapy. Advances in molecular research have greatly improved our understanding of the leukemogenesis in AML. The aim of the present doctoral thesis is to first investigate the detailed molecular genetic alterations in patients afflicted with AML. We plan to elucidate the role of RUNX1, WT1 and DNMT3A mutations, three molecular alterations not well studied before, and single or double CEBPA mutation (CEBPAsingle-mut or CEBPAdouble-mut) in the leukemogenesis of AML. Second, it is of utmost significance to understand the clinic-biologic features, associated cytogenetic and molecular abnormalities and clinical relevance of these molecular aberrations in AML patients. Further, we can depict the comprehensive picture of total 17 frequent molecular genetic alterations in AML patients from the project and other researches from our team. In addition to the importance of these genetic alterations in the development of AML, we also perform sequential studies to explore the role of these events in the disease progression. As we aware, the integrated prognostic system is more power than a single biomarker to risk-stratify the heterogeneous AML patients into different groups with distinct outcome. Therefore, the integration of cytogenetic and molecular changes we design will improve the prognostic stratification of AML patients, especially those with intermediate-risk cytogenetics, and may lead to better decision on therapeutic strategy.
In the subproject one (RUNX1 mutation in AML), we found that RUNX1 mutations could be detected in a substantial proportion (13.2%) of patients with de novo non-M3 AML. RUNX1 mutations were closely associated with male gender, older age, immature FAB subtypes (M0 and M1) and trisomy 8. Most runt homology domain (RHD) mutations were missense mutations (64.5%), while most transcription activation domain (TAD) mutations were of frameshift mutations (76.5%). Among the 62 patients with RUNX1 mutations, 31 (50%) showed additional molecular abnormalities at diagnosis. They were mutually exclusive with CEBPA and NPM1 mutations, but were closely associated with MLL/PTD. Furthermore, the RUNX1 mutation predicted lower complete remission (CR) rate and was an independent poor-risk factor for overall survival (OS) and disease-free survival (DFS).
In the subproject two (CEBPA mutation in AML), about two third of patients with CEBPA mutations had CEBPAdouble-mut both at TAD1 and bZIP domains. CEBPAdouble-mut patients had a higher CR rate and a significant longer DFS and OS than those with CEBPAwild or CEBPAsingle-mut. CEBPAsingle-mut was closely associated with CD56 expression but inversely correlated with HLA-DR, CD7 and CD15 expression. Compared to the patients with CEBPAdouble-mut, those with CEBPAsingle-mut had a higher incidence of concurrent FLT3/ITD, FLT3/TKD, MLL/PTD or RUNX1 mutation and had a poorer prognosis. This study provides evidences independently from previous ones, stressing the differences in biologic characteristics between CEBPAsingle-mut and CEBPAdouble-mut AML and their possible prognostic implication.
In the subproject three (WT1 mutation in AML), 29 different kinds of WT1 mutations were detected in 32 (6.8%) patients. We showed that WT1 mutations occurred with similar frequencies in patients with normal cytogenetics (CN-AML, 7.0%) and those with abnormal cytogenetics (6.3%). The mutation was closely associated with younger age, FAB M6 subtype and t(7;11)(p15;15), but inversely related to M0 subtype. Among the 32 patients with WT1 mutations, 23 (72%) showed additional molecular abnormalities at diagnosis; sixteen (69.6%) of them had at least one concurrent Class II mutation and 13 (56.5%), Class I mutation. Furthermore, the WT1 mutation predicted higher relapse rate and was an independent poor-risk factor for OS and relapse-free survival (RFS) among total cohort and CN-AML patients. Incorporation of the these gene mutations, including NPM1/FLT3-ITD, CEBPAdouble-mut, WT1 mutation and age at diagnosis that are closely associated with prognosis, into survival analyses can better stratify patients into different risk groups.
In the subproject four (DNMT3A mutation in AML), DNMT3A mutations at 30 different positions, most commonly in the MTase domain, were demonstrated. All the nonsense, frameshift, and in-frame mutations generated truncated peptide with complete or partial deletion of the MTase domain and were suggested to abolish the catalytic activity of this enzyme. The missense R882 mutations, the most common DNMT3A mutations, resulted in impaired enzyme activity. DNMT3A mutations could be detected in a substantial proportion of patients with de novo AML (14% of total patients; 15.2% of non-M3 AML; 19.5% of intermediate-risk cytogenetics and 22.9% of CN-AML). DNMT3A mutations were closely associated with older age, FAB M4/M5 subtypes and intermediate-risk cytogenetics and CN-AML. Among the 70 patients with DNMT3A mutations, 68 (97.1%) showed additional molecular abnormalities at diagnosis. They were mutually exclusive with CEBPA mutation, but were closely associated with FLT3/ITD, NPM1, PTPN11 and IDH2 mutations. Furthermore, the DNMT3A mutation was an independent poor-risk factor for OS and RFS among total cohort and CN-AML patients. Incorporation of DNMT3A mutation with eight other prognostic factors, including age, WBC counts, cytogenetics, NPM1/FLT3-ITD, CEBPA, RUNX1, WT1, and IDH2 mutations, into survival analyses can better stratify AML patients into different risk groups.
In the subproject five (integrated prognostic system in AML patients), early assessment of cytogenetics and mutational profiling of eight relevant genes, including CEBPA, NPM1, FLT3, RUNX1, WT1, IDH2, ASXL1 and DNMT3A, may provide a framework for risk stratification in 318 non-M3 AML patients. Using the proposed classification by integrating cytogenetic and mutational profiles, we reduced the proportion of patients in intermediate-risk group, as defined by cytogenetics alone, from 72% to 24.5%, defined by both cytogenetic and molecular changes. Three fourth of intermediate-risk patients defined by cytogenetics alone can be reliably reclassified into favorable- (35.2%) or unfavorable-risk (40.2%) group according to the molecular genotype. The patients with intermediate-risk cytogenetics but favorable molecular genotype (mutation of NPM1, IDH2 or CEBPAdouble-mut in the absence of FLT3/ITD) had similar treatment response to those with favorable-risk cytogenetics. Similarly, the prognosis of the patients with intermediate-risk cytogenetics but unfavorable molecular genotype (mutation of RUNX1, WT1, ASXL1, or DNMT3A) was as poor as those with unfavorable-risk cytogenetics. The integrated cytogenetic and molecular classification further refines the prognostic prediction models and may guide the therapeutic decision.
In the subproject six (comparative analyses of genetic alterations in paired samples), we found that mutational shifts, including mutational loses and gains, occurred in 61 (52.1%) of 117 patients at relapse. Class I mutations, which activate signal transduction, were lost more frequently (50.8%) than Class II mutations (15.6%), which cause transcriptional deregulation. Genetic evolution with acquisition of novel mutations at relapse were identified in 15 individuals (12.8%), all involving Class I, WT1 or ASXL1 mutations, but not Class II or NPM1 mutations. Sequential study showed that mutations of CEBPA, RUNX1, IDH1/2 or DNMT3A remained quitely stable during the clinical course. In contrast, the instability of WT1 and TET2 mutations was noticed during disease progression. Our findings distinctly help judge which genetic alteration can be used as a biomarker for minimal residual disease (MRD) monitoring.
In conclusions, the present doctoral thesis combined genetic association studies, molecular results and clinical data to demonstrate how the genetic alterations, especially RUNX1, WT1 and DNMT3A mutations are involved in the leukemogenesis of AML. Incorporation of cytogenetic changes and molecular alterations as an integrated prognostic system can better risk-stratify the heterogeneous AML patients, especially intermediate-risk cytogenetics into different prognostic groups with distinct outcome. Finally, the gene mutations which are stable during treatment courses can also be used as biomarkers to monitor MRD.
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