肝內膽管癌 (Intrahepatic cholangiocarcinoma, ICC) 在臨床上並不常見，佔全世界原發性肝癌的5-15%。在東南亞因環境地理因素，發病率偏高。相關的致病因子包含慢性發炎性膽道疾病、肝內膽管結石、膽道寄生蟲感染、膽道畸形、或病毒感染等危險因子。ICC的轉移侵略性及死亡率皆高，且預後不佳。
在人類固態腫瘤發現到，基因的拷貝數變異有助於腫瘤的發生，其改變的染色體區域可能影響相關腫瘤基因的表現。文獻報告中，肝癌約64.5%有染色體1q區域拷貝數增加的情形。KIF14與TPM3兩個基因位於染色體1q，在肝細胞癌的研究上發現其RNA高度表現和染色體1q拷貝數放大增加有相關。TPM3和KIF14有相似的細胞激素功能，過度的表現會可能會影響細胞分化。這兩種分子可能和肝癌等致癌機轉有關。
在我們先前的研究結果發現，位於染色體1q21.3區域的ADAMTSL4，在合併肝細胞及膽道癌 (combined hepatocellular carcinoma and cholangiocarcinoma) 有經常性的拷貝放大變異現象。ADAMTSL4有多種生物功能包括細胞粘附、血管生成和神經系統發育。擴增的 ADAMTSL4可能與致癌機轉有著相關聯。染色體1q拷貝數變化在ICC尚未被探討。推測染色體1q拷貝數的變化在ICC也扮演著重要的角色。
本研究共收集86個 ICC 檢體，萃取DNA並利用定量PCR分析基因拷貝數的變異。結果顯示，拷貝數變異增加 (Gain) 的比例在3個基因ADAMTSL4、TPM3和KIF14分別為81.4% (70位)、60.5% (52位)、50% (43位)。單變數分析顯示該3個基因拷貝數變異增加會顯著影響病人預後 (progression free survival，p=0.022, 0.015, 0.029)。經過癌症分期 (stage) 校正後，多變數分析依然呈顯著意義影響病人預後，ADAMTSL4 (HR=2.423, p=0.021)、TPM3 (HR=2.186, p=0.007)、KIF14 (HR=1.931 , p=0.014)，因此肝內膽管癌病人ADAMTSL4、TPM3、KIF14基因拷貝數變異是重要的致癌機轉和預後指標，更進一步研究其致癌機轉可望作為日後發展基因標靶治療之參考，給肝內膽管癌病人的治療帶來新的曙光。

Intrahepatic cholangiocarcinoma (ICC) is relatively infrequent, accounting for 5-15% of primary liver cancer worldwide. The risk factors include chronic inflammatory biliary disease, hepatolithiasis, parasitic biliary infestation, biliary malformations, and viral infection. ICC is an aggressive cancer with a high rate of metastasis and fatality, and poor prognosis.
The copy number (CN) alterations have been observed in human solid tumors and known to contribute to the tumorigenesis. The altered chromosomal regions may affect the activities of cancer-related genes. Recurrent copy number gains of 1q has been reported in 64.5 % of hepatocellular carcinoma. RNA expression levels of KIF14 (kinesin family member 14) and TPM3 (tropomyosin 3) are most highly expressed and correlated with copy number status in the amplification of chromosomal 1q. TPM3 and KIF14 have similar function of cytokines, and are associated with cell differentiation. These two molecules may be involved in carcinogenesis, including liver cancer. In our previous study, we found ADAMTSL4 (ADAMTS-like 4), located on 1q21.3, was frequently amplified in combined hepatocellular carcinoma and cholangiocarcinoma. The biological functions of ADAMTSL4 include cellular adhesion, angiogenesis, and patterning of the developing nervous system. Amplification of ADAMTSL4 may be associated with carcinogenesis. Copy number changes of chromosome 1q have not been explored in the ICC. We hypothesize that copy number alterations of chromosome 1q also play a role in ICC.
In this study, DNA extraction and quantitative polymerase chain reaction were used to explore CN alterations and expression of target genes and associated mutation in 86 cases of ICC. Our result showed that the CN variations of gain for these three genes were ADAMTSL4 (81.4%, 70 cases), TPM3 (60.5%, 52 cases), and KIF14 (50%, 43 cases), respectively. The CN alterations of ADAMTSL4, TPM3, and KIF14 gene were showed significantly associated with cancer stage and progression free survival of ICC patients. The progression free survival of these 3 genes were p=0.022, p=0.015, and p=0.029 respectively. The genes CN alterations associated with stage were ADAMTSL4 (HR=2.423, p=0.021), TPM3 (HR=2.186, p=0.007), and KIF14 (HR=1.931, p=0.014). We were confident that this project was feasible, and ADAMTSL4, TPM3, and KIF14 gene CN alterations study were very likely shed lights on the molecular pathogenesis of ICC.

第一章 介紹 1
1、 肝內膽管癌流行病學 1
2、 肝內膽管癌與肝細胞癌之相似性 2
3、 肝內膽管癌臨床診斷 4
4、 基因的拷貝數變異 5
5、 基因表達 6
第二章 研究動機 10
第三章 材料與方法 11
材料 11
1、 檢體來源 11
2、 DNA萃取試劑 11
3、 定量即時聚合酶鏈鎖反應 11
4、 免疫組織化學染色分析(Immunohistochemical analysis) 12
方法 12
1、 檢體組織中的DNA萃取 12
2、 定量聚合酶反應評估拷貝數改變 (Quantitative polymerase chain reaction for evaluation of CN changes) 13
3、 免疫組織化學染色法(Immunohistochemical analysis) 14
4、 統計分析 (Statistical analysis ) 15
第四章 結果 16
1、 病患的臨床病理特質 16
2、 基因拷貝數(Gene Copy Numbers) 16
3、 基因拷貝數與存活期有相關聯 17
4、 免疫組織化學染色表現 18
第五章 討論 19
第六章 結論和展望 23
參考文獻 24
表1.整體存活率和無病存活率預後因素的單一分析結果 29
表2. 三個標靶的基因拷貝數表現情形 30
表3. 癌症腫瘤分期調整後無病存活率分析 31
表4. 目標基因拷貝數增加與疾病發展風險係數相關分析 32
圖1. 肝內膽管癌組織學分型 33
圖2. 合併肝細胞及膽管癌之ADAMTSL4表現 34
圖3 . 肝內膽管癌ADAMTSL4拷貝數變異增加與病人預後表現 35
圖4 . 肝內膽管癌TPM3拷貝數變異增加與病人預後表現 36
圖5. 肝內膽管癌KIF14拷貝數變異增加與之病人預後表現 37
圖6. 肝內膽管癌病患之不同種基因拷貝數變異增加與病人預後表現 38
圖7. KIF14於肝內膽管癌的免疫組織化學染色表現 39
附件一、人體試驗倫理委員會同意書 40
附件二、海報發表 42

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