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標題: | CEBPA基因甲基化在急性骨髓性白血病中的應用 Application of CEBPA Methylation in Acute Myeloid Leukemia |
作者: | Tsung-Chin Lin 林宗縉 |
指導教授: | 林亮音(Liang-In Lin) |
關鍵字: | CCAAT/加強子結合蛋白α,基因甲基化,急性骨髓性白血病,預後,高解析度解離分析, CEBPA,DNA methylation,AML,prognosis, HRM, |
出版年 : | 2011 |
學位: | 博士 |
摘要: | 表觀基因修飾作用所造成的抑癌基因去活化是細胞走向癌化過程中的重要機制之一,其中經常可見到啟動子區域的CpG島有高度甲基化的現象。在某些癌症中,CEBPA遠端啟動子的高度甲基化會造成CEBPA的表現下降,然而CEBPA甲基化對於AML的臨床意義仍未釐清。為了調查CEBPA甲基化對於AML臨床特徵的相關性,我們使用MassARRAY對於3株血癌細胞株及193位原發性AML病患的CEBPA甲基化狀態進行偵測,並以雙向階層式分群法將病患分成CEBPA高度甲基化及CEBPA低度甲基化兩群進行分析。結果顯示CEBPA甲基化程度與CEBPA表現量呈現負相關。在125位接受標準前導性治療的病患中,CEBPA高度甲基化的病患相較於CEBPA低度甲基化的病患具有較高的完全緩解率(93.3% vs. 73.6%, P = 0.116)。在正常染色體核型的病患中,排除了帶有CEBPA或NPM1突變的病患後,存活分析顯示CEBPA高度甲基化的病患具有較長的整體存活期(P = 0.028)。多變項Cox比例風險回歸分析顯示CEBPA高度甲基化可作為整體存活期(相對風險為0.406, 95%信賴區間自0.166至0.996, P = 0.050)及無疾病存活期(相對風險為0.416, 95%信賴區間自0.223至0.777, P = 0.006)之低風險的獨立預後因子。因此CEBPA高度甲基化對於AML病患而言是屬於預後較佳的因子之一。
由於CEBPA高度甲基化是屬於AML預後較佳的因子之一,而且和大多數在癌症中發現的基因異常甲基化一樣,都是屬於異質性甲基化的型態,因此需要建立一套針對異質性甲基化的快速篩檢方法。在接下來的研究中,我們建立了以高解析度解離檢測的解離溫度(Tm)和解離波寬(Width)計算而得的甲基化指數,來評估異質性甲基化的程度。甲基化指數 = [AML病患樣本的波寬 / 正常對照組的波寬] + [AML病患樣本的Tm值 - 正常對照組的Tm值]。我們發現甲基化指數和MassARRAY測得的甲基化百分比之間有高度相關性(R2 = 0.80, P < 0.001)。甲基化指數的同次再現性(within-run)變異係數為0.9%,異次再現性(between-run)變異係數為2.6%。以雙向階層式分群法將病患分成CEBPA高度甲基化及CEBPA低度甲基化兩群後,以接受器操作特性曲線(ROC curve)分析甲基化指數應用於CEBPA甲基化篩檢的實用性,結果顯示當甲基化指數為1.412時,敏感度為97.14%,特異性為95.89%,ROC curve下的面積為0.983(95%信賴區間 = 0.952-0.996, P < 0.001)。此外CEBPA甲基化指數於完全緩解時下降、復發時上升,也反映了AML的疾病狀態。因此我們所發展的HRM檢測搭配甲基化指數的方法,具有和MassARRAY相近的甲基化程度評估能力,可望作為一套簡單且快速的CEBPA甲基化與否的篩檢方法,用以評估AML的預後。 Epigenetic inactivation of tumor suppressor genes, often in association with aberrant DNA methylation of CpG islands in the promoter region, is a key fact of tumorigenesis. Hypermethylation of the distal CEBPA promoter region has been reported to result in down-regulation of CEBPA expression in several malignancies. However, the clinical implication of CEBPA hypermethylation in AML remains unclear. To investigate the correlation between CEBPA hypermethylation and clinical features in AML, quantitative MassARRAY analyses for CEBPA methylation status were performed on 3 leukemic cell lines and a cohort of 193 patients. High CEBPA methylation group (CEBPAhigh-meth, n = 28) and low methylation group (CEBPAlow-meth,n = 165) were defined by using two-way hierarchical clustering. CEBPA methylation was inversely correlated with CEBPA expression. With a median follow-up of 48 months, among the 125 patients receiving standard induction therapy, CEBPAhigh-meth was associated with better treatment response (complete remission rate 93.3% versus 73.6%, P = 0.116). In patients with normal karyotype and without CEBPA and NPM1 mutations, the CEBPAhigh-meth had longer overall survival (OS) than the CEBPAlow-meth (P = 0.028). Multivariate analysis further supported that the CEBPA methylation was an independent prognostic factor for disease free-survival (hazard ratio [HR] = 0.416; 95% confidence interval [CI], 0.223-0.777, P = 0.006), and OS (HR = 0.406; 95% CI, 0.166-0.996, P = 0.050). We conclude that CEBPA hypermethylation is a favorable prognostic biomarker for AML patients. Given that CEBPA hypermethylation could be a favorable prognosis factor in AML and that CEBPA methylation demonstrated heterogeneous pattern which is a common finding in various malignancies, a rapid and efficient screening method deserved to be developed for heterogeneous methylation pattern. Therefore, in the second part of our study is to set up an algorithm called methylation index deduced from high resolution melting (HRM) profile, including Tm shifting and Tm distribution (width). Methylation Index= [patient width/reference width] + [patient Tm – reference Tm]. The methylation index was highly correlated with the exact methylation levels obtained from MassARRAY method (R2 = 0.80, P < 0.001). The coefficient of variation of within-run and between-run reproducibility for methylation index was 0.9% and 2.6%, respectively. With the cut-off methylation index of 1.412, corresponding to an area under the ROC curve of 0.983 (95% CI, 0.952-0.996; P < 0.001), the best sensitivity and specificity of 97.14% and 95.89%, respectively, was obtained to dichotomize into two groups with low and high CEBPA methylation status. In addition, CEBPA methylation index decreased at complete remission and increased again at relapse, indicating the disease statuses. Taken together, the methylation index from HRM profiles could be a simple and efficient screening method to determine CEBPA methylation status in AML. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38484 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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