臺灣博碩士論文加值系統

癌症是國人死亡的第一位原因，已經成為公眾健康的最重要議題。在我的博士論文研究中，我著重在乳癌和急性骨髓性白血病的研究。

I. 探討乳癌發生的遺傳因子與尋找早期乳癌復發的危險因子和解決方法
雖然乳癌的發生率在台灣相對於西方國家比較低，乳癌的病例在過去幾十年的快速增長。為了降低乳癌相關的死亡率，首先，我們研究乳癌的遺傳基因來找出乳癌發生的高危險族群，經過預防性治療或是定期篩檢，以防止晚期乳癌的發生，使乳癌可以早期診斷。其次，我們分析了早期（淋巴結陰性）乳癌患者，復發的臨床與病理因子和尋找可能的治療方法。在第一部分中，我們前瞻性招收早期發病或具有顯著家族史的乳癌患者，然後，我們以次世代定序方法，在這些患者，分析68個癌症風險相關基因。一共有133位患者接受基因篩檢，其中30為病患（22.6％）被發現攜帶致病性的突變，分別是9個 BRCA1、11個BRCA2、2個 RAD50、2個TP53和ATM、BRIP1、FANCI、MSH2、MUTYH和RAD51C各1個。三陰性乳癌（TNBC）有最高基因突變率（45.5％，p值= 0.025）。致病性基因變異的攜帶者被認為是發展惡性腫瘤的高風險族群，建議接受定期癌症篩檢。由於對於非BRCA基因的定期癌症篩檢方法目前沒有定論，我們基於個別基因生物學功能來制定策略;例如，攜帶RAD51C突變的病人，接受類似於對BRCA的癌症篩檢方式，是因為BRCA和RAD51C都參與同源重組的功能。我們本研究的初步結論認為多基因的次世代定序檢查，對於癌症風險評估，找出乳癌的高危險族群，具有臨床實用的價值。在第二部分，我們回溯性分析了2005年1月到2009年12月間的2216早期乳癌患者;其中，有716位患者經過乳房全部切除或是乳房保留手術，被診斷為pT1-2N0M0(淋巴結陰性)的早期乳癌。在經過為期47.0個月(中位數)的追蹤，有47位患者有乳癌復發。復發的顯著危險因素分別是淋巴血管侵犯（HR = 4.60，95％CI 2.32-9.10）和組織學惡性度3級（HR = 4.99，95％CI 1.06-23.48）;輔助放射線治療具有防止腫瘤復發的功效（HR = 0.35，95％CI，0.14-0.92）。此外，我們研究輔助化療和放療對患者的淋巴血管侵犯和組織學惡性度3級，是否具有治療效果。結果顯示，淋巴血管侵犯和組織學惡性度3級可以被輔助放射線治療減少其復發的影響(淋巴血管侵犯(+)/輔助放射線治療(+), 沒有復發; 組織學惡性度3級(+)/輔助放射線治(+), HR=0.82, 95% CI. 0.18-3.70)。然而，輔助化療沒有達到該效果。因此，我們認為，淋巴血管侵犯和組織學惡性度3級是預測淋巴結陰性早期乳癌患者的腫瘤復發危險因素。輔助放射線治療可對於具有這兩個危險因子的淋巴結陰性早期乳癌患者，提高無復發存活率。

II. 探討微環境與EZH2基因在急性骨髓性白血病的影響
儘管化學治療可以促成急性骨髓性白血病病人達到緩解狀態，但是因為較高的復發率和緩解狀態維持具有困難，急性骨髓性白血病的患者預後目前仍然不佳， 這個現象暗示著化學治療無法根除白血病幹細胞。過去的基因體研究證明，白血病幹細胞具有大量的DNA甲基化和組蛋白修飾的特徵，這些特徵亦可以在復發或是難治的急性骨髓性白血病上頭發現，意味著急性骨髓性白血病復發和白血病幹細胞有關。Polycomb repressive complex 2（PRC2）及其核心蛋白EZH2，在真核細胞中是重要的表觀遺傳調控機制，因此我們探討EZH2在AML的影響。研究發現，高表現的EZH2預測急性骨髓性白血病患者的高復發率與較差的預後。EZH2的高表現在與白血病幹細胞相關聯，並通過抑制DAB2IP的表現，來活化ERK途徑。有趣的是，我們找到由骨髓基質細胞分泌的新細胞因子（稱為細胞因子X），來增加白血病細胞EZH2的表現，進而支持白血病幹細胞的存活。我們進一步證明不論是在體外或體內的研究，抑制細胞因子X / EZH2的路徑，可以減低白血病幹細胞以增強化療的效果。這些方法證明急性骨髓性白血病細胞可以通過依賴細胞因子X / EZH2，而達到化學治療耐藥性，以及提供針對細胞因子X / EZH2途徑的標靶治療和化療的治療依據。

Cancer is the leading cause of death in Taiwan and becomes the most important issue in public health. In my PhD thesis, I focused on breast cancer and acute myeloid leukemia.

I. Explore the genetic factor in breast cancer development and identification of risk factors on tumor recurrence of early breast cancer
Although the incidence of breast cancer is relative lower in Taiwan than that in western countries, the cases of breast cancer grows rapidly in the past decades. In order to reduce breast cancer-related mortality, first, we investigate the genetic factors to identify people with high-risk of breast cancer development so as to prevent advanced breast cancer by prophylactic intervention or help them to make an early diagnosis of breast cancer by regularly screening. Second, we analyzed poor clinic-pathologic factors related to recurrence in early stage (node-negative) breast cancer and search for resolution. In the first part, we prospectively enroll patients with early-onset or a significant familial history of breast cancer for genetic study. We then performed next-generation sequencing to analyze a sequencing panel containing 68 genes that had cancer risk association for these patients. A total of 133 patients were enrolled and 30 (22.6%) were found to carry germline deleterious mutations, 9 in BRCA1, 11 in BRCA2, 2 in RAD50, 2 in TP53 and one each in ATM, BRIP1, FANCI, MSH2, MUTYH, and RAD51C. Triple-negative breast cancer (TNBC) was associated with the highest mutation rate (45.5%, p=0.025). Mutation carriers were considered as high-risk to develop malignancy and advised to receive cancer screening. Screening protocols of non-BRCA genes were based on their biologic functions; for example, patients carrying RAD51C mutation received a screening protocol similar to that for BRCA, since BRCA and RAD51C are both involved in homologous recombination. We conclude that multiple gene sequencing in cancer risk assessment is clinically valuable to identify people with high risk of breast cancer. In the second part, we retrospective reviewed 2216 early breast cancer patients between January 2005 and December 2009; among them, 716 patients who received mastectomy or breast?駥onserving surgery and were proven to have pT1-2N0M0 breast cancer were enrolled in the study. Forty-seven of the 716 patients developed tumor recurrence during the 47.0 months of median follow-up. The significant risk factors of recurrence were LVI (HR = 4.60, 95% CI. 2.32-9.10) and Nottingham grade 3 (HR = 4.99, 95% CI. 1.06-23.48); adjuvant radiotherapy (HR = 0.35, 95% CI. 0.14-0.92) prevented tumor recurrence. Furthermore, we investigate the therapeutic impact of adjuvant chemotherapy and radiotherapy on patients with LVI and Nottingham grade 3. The adverse effect of LVI and grade 3 can be abrogated by adjuvant radiotherapy in RFS (LVI(+)/radiotherapy(+), no recurrence; grade 3(+)/radiotherapy(+), HR=0.82, 95% CI. 0.18-3.70). However, adjuvant chemotherapy did not. Therefore, we considered that LVI and Nottingham grade 3 were the independent risk factors predicting tumor recurrence for patients with NNBC. Adjuvant radiotherapy might be considered in NNBC patients with these unfavorable factors to improve the RFS.

II. Explore the role of microenvironment and EZH2 in acute myeloid leukemia
The prognosis of patients with acute myeloid leukemia (AML) remains poor because high relapse rate and failure to maintain durable remission despite poly-chemotherapy, indicating current treatments do not target leukemia stem cell (LSC). Previous genomic study showed that LSC population was characterized with extensive DNA methylation and an aberrant pattern of histone modifications and these significant epigenetic profiling was also identified in relapsed and refractory AML, indicating LSC associated with AML relapse. Polycomb repressive complex 2 (PRC2) and its core protein, EZH2, are important epigenetic machinery in eukaryotic cells, we investigate the impact of EZH2 in the AML. We found that high expression of EZH2 predicted high relapse rate and poor prognosis in AML patients. Up-regulated expression of EZH2 is associated with LSC and contributes to chemoresistance through silencing of DAB2IP to activate ERK pathway. Interestingly, we identify a novel cytokine (termed Cytokine X) secreted by bone marrow stromal cells, to upregulate EZH2 and support LSC survival. We further demonstrate that inhibition of Cytokine X/EZH2 pathway abrogate LSC properties to enhance chemotherapeutic effect in in vitro and in vivo studies. These approaches demonstrate how AML cells resistant to chemotherapy through Cytokine X /EZH2-dependent LSC properties and provide the basis for combinatorial targeting Cytokine X /EZH2 pathway and chemotherapy for AML.

目錄

中文摘要…………………………………………………………………..…..………i
英文摘要…………………………………………………………………..…………iv
誌謝辭………………………………………………………………………………viii
I. 探討乳癌發生的遺傳因子與尋找早期乳癌復發的危險因子和解決方法。
第一章 前言……………………………………………………………………..……1
(a) 探討乳癌發生的遺傳因子
第一節 研究背景………………………………………………………………..…3
第二節 研究目的…………………………………………………………………..7
第二章 研究方法
第一節 研究材料…………………………………………………………………..8
第二節 研究設計…………………………………………………………………..8
第三節 統計方法…………………………………………………………………14
第三章 研究結果
第一節 描述性統計分析…………………………………………………………14
第二節 推論性統計分析…………………………………………………………16
第四章 討論
第一節 結果討論…………………………………………………………………21
第二節 其他相關性討論…………………………………………………………22
第三節 研究限制…………………………………………………………………24
第五章 結論與建議
第一節 結論………………………………………………………………………25
(b) 尋找早期乳癌復發的危險因子和解決方法
第一節 研究背景………………………………………………………...………26
第二節 研究目的…………………………………………………………………27
第二章 研究方法
第一節 研究材料與設計…………………………………………………………28
第二節 統計方法…………………………………………………………………29
第三章 研究結果
第一節 描述性統計分析…………………………………………………………29
第二節 推論性統計分析…………………………………………………………31
第四章 討論
第一節 結果討論…………………………………………………………………34
第二節 其他相關性討論…………………………………………………………37
第三節 研究限制…………………………………………………………………38
第五章 結論與建議
第一節 結論………………………………………………………………………39
Reference…………………………………………………………………………..…40
表(table 1~ table 10) …………………………………………………………………48
圖(figure 1~ figure 11) ………………………………………………………………60

II. 探討微環境與EZH2基因在急性骨髓性白血病的影響
第一章 前言……………………………………………………………………..…71
第一節 研究背景………………………………………………………...………71
第二節 研究目的…………………………………………………………………75
第二章 研究方法
第一節 研究材料與設計…………………………………………………………75
第二節 統計方法…………………………………………………………………85
第三章 研究結果
第一節 實驗結果分析……………………………………………………………85
第四章 討論
第一節 結果討論………………………………………..………………………101
第五章 結論與建議
第一節 結論…………………………………………..…………………………107
Reference……………………………………………………………………………108
表(table 1~ table 5) …………………………………………………………………114
圖(figure 1~ figure 34) …………………………………..…………………………117
相關論文發表………………………………………………………………………152

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Part II
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