2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/650754摘要：曱狀腺結節的盛行率很高，其中曱狀腺癌約佔5%，以乳突癌(papillary thyroid cancer, PTC)與渡泡癌 (follicular thyroid cancer, FTC)居多。目前用來診斷曱狀腺結節良惡性的標準方法是細針穿刺細胞學檢查 (fine needle aspiration cytology, FNAC)，但約有15%的結節無法依此確定良惡性。因此，發展準確診斷 FNAC檢體的方法是急迫且重要的臨床問題。此外，探討分子生物學的變化，與結節的診斷和預後之關 係是相當有趣而且重要的。本計劃的主要目的為結合基因體、代謝物表現與細胞形態特徵等方法以瞭解其與PTC與FTC之診 斷及預後之關係。曱狀腺癌的臨床表現、代謝物和細胞型態都是重要的phenotype。為了促進曱狀腺癌的診斷與預後 評估的準確性，本計晝結合了基因體學和代謝體學來探討特定生物標記和曱狀腺癌致病機轉的關係。在 基因與臨床表現的研究中，BR4F、RAS等基因突變已明確被發現與曱狀腺癌相關，因為他們的突變會 造成MAPK signaling pathway活化，使細胞增生。根據外國文獻，當FNAC無法確診時，基因突變檢測 可協助區分良惡性，提升診斷正確性。在其他可能的致病基因清單中，sodium iodide symporter (NIS)是很 少被分析但值得進一步研究的基因，因為曱狀腺癌細胞攝取碘的能力與其分化程度及預後相關。本研究 除了將使用傳統的PCR-Sanger sequencing檢測分析NIS’ BRAF, RAS與其他可能之基因突變熱點、探討 國人基因變異和疾病診斷、預後相關性以外，同時會使用次世代定序更全面性地找尋其他仍未被發現的 單點基因突變或其他的基因突變方式。在代謝體方面的研究已經有文獻可以利用代謝物小分子來區別不同類別的曱狀腺結節。然而文獻中 大多是採用組織切片為樣品，無法作為術前曱狀腺結節診斷的方法。本計晝使用靈敏性佳的質譜儀來分 析樣品的代謝物，來找出和FNAC結果相對應的代謝生物性指標。首先，我們會先找出曱狀腺囊腫、良 性腫瘤、惡性腫瘤與正常曱狀腺組織的代謝物表現差異，並在利用FNAC取出的細胞上檢驗這些差異結 果。在細胞形態學方面，我們先前研究發現以電腦程式所分析的細胞影像特徵(computerized morphometry, CM)與PTC預後相關。本研究將進一步拓展此工具運用在FTC和其他FNAC無法區分良惡性的曱狀腺 結節，將與病理報告和預後進行對照。總結，本計劃將結合基因檢測、代謝體檢測、與CM來綜合分析曱狀腺FNAC檢體，以促進曱狀腺 癌的診斷與預後評估的準確性。預計納入120個檢體，包括（a) PTC和FTC、(b) FNAC無法確定良惡 性的曱狀腺瘤、（c)良性曱狀腺瘤。藉由phenotypes與genotype的對照比較，預期將增進FNAC的診斷 正確性和預後評估，並進一步對曱狀腺癌的致病機轉深入了解，以作為發展新治療的基礎。<br> Abstract: The prevalence of thyroid nodules is high and five to fifteen percent of these nodules are malignant; most of them are well-differentiated thyroid cancer, including papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). The current standard diagnostic tool is fine needle aspiration cytological (FNAC) examination of the nodules. The overall sensitivity and specificity is about 90%, which is far from perfect. Besides, 15% of the nodules evaluated by FNAC are indeterminate, especially in cases of follicular neoplasm. All of the obstacles become a thorny problem for both healthcare-givers and patients. In addition, study of the changes at molecular levels with the diagnosis and prognosis of thyroid cancer is very interesting and important.The goal of this study is to propose a reliable method for diagnosis and prognosis of papillary thyroid cancer and follicular thyroid cancer through analyzing genetic mutations, metabolites feature, and cellular morphologic characteristics. Clinical presentation, metabolites features, and cellular morphology are all important phenotypes of thyroid cancer. To improve the diagnostic and prognosis accuracy, this project integrates functional genomic approaches including genomics and metabolomics to establish a causal link between select biomarkers and disease pathogenesis.Among all the candidate genes, BRAFV600E and RAS mutation has been well-known detected in differentiated thyroid carcinoma. Mutation of BRAFV600E and RAS result in the activation of mitogen-activated protein kinase (MAPK) pathway and thus cell proliferation. They have also been associated with aggressive clinical behavior and poor prognosis. TERT promoter mutation was just discovered in thyroid cancer for one year, but has turned into the focus because of its risk in aggressive clinical behavior and higher prevalence in advanced thyroid cancer. Genetic mutations of several other genes were also reported. Sodium iodide symporter (NIS) is an important but under-explored target gene. BRAF mutation induces NIS regression and promotes epithelial to mesenchymal transition and invasion. We would like to examine the mutations of NIS, BRAFV600E, RAS, TERT, and other possible genes in FNAC specimens and explore the correlation of mutations, diagnosis, and prognosis. Sanger sequencing will be the method of choice for the known somatic mutation hotspots, but next-generation sequencing (NGS) will be used to more comprehensively search for disease-causing genes. NGS also has the potential to shed light on new pathophysiological pathways.Metabolomics has been demonstrated as a powerful tool to differentiate different types of thyroid nodules. However, previous studies mainly used tissue biopsy as study materials which limited its application in presurgical screening of thyroid nodules. This study uses a more sensitivity mass spectrometry based metabolomics platform to investigate tumor markers in FNAC specimens. We use a two steps approach by first identification of the metabolic differences between thyroid neoplasms, benign or malignant, and normal thyroid tissue in operative specimens and then test those differences on cells obtained through FNAC.According to our previous studies, cytological features of PTC, analyzed with computerized morphometry, are correlated with prognosis significantly. It helps to predict prognosis pre-operatively and to establish a risk-stratified treatment plan. In this current project, we plan to extend this tool to the diagnosis and/or prognosis of FTC and to nodules with uncertain FNAC reports to assist clinical management.In summary, this study applies a multi- and inter-disciplinary approach to improve the accuracy and sensitivity for diagnosis and prognosis of thyroid cancer using FNAC specimens. We will examine 120 samples, including groups of (a). PTC or FTC (40 samples), (b). Indeterminate (40 samples) and (c). Benign (40 samples). From our study with the comparison of phenotypes and genotypes, we anticipate to break the current diagnostic dilemma and limitation of poor prognostic prediction of thyroid nodules. Furthermore, we are longing to improve our understanding in thyroid cancer genesis which could disclosure novel diagnostic markers and treatment targets.曱狀腺結節的盛行率很高，其中曱狀腺癌約佔5%，以乳突癌(papillary thyroid cancer, PTC)與渡泡癌 (follicular thyroid cancer, FTC)居多。目前用來診斷曱狀腺結節良惡性的標準方法是細針穿刺細胞學檢查 (fine needle aspiration cytology, FNAC)，但約有15%的結節無法依此確定良惡性。因此，發展準確診斷 FNAC檢體的方法是急迫且重要的臨床問題。此外，探討分子生物學的變化，與結節的診斷和預後之關 係是相當有趣而且重要的。 本計劃的主要目的為結合基因體、代謝物表現與細胞形態特徵等方法以瞭解其與PTC與FTC之診 斷及預後之關係。 曱狀腺癌的臨床表現、代謝物和細胞型態都是重要的phenotype。為了促進曱狀腺癌的診斷與預後 評估的準確性，本計晝結合了基因體學和代謝體學來探討特定生物標記和曱狀腺癌致病機轉的關係。在 基因與臨床表現的研究中，BR4F、RAS等基因突變已明確被發現與曱狀腺癌相關，因為他們的突變會 造成MAPK signaling pathway活化，使細胞增生。根據外國文獻，當FNAC無法確診時，基因突變檢測 可協助區分良惡性，提升診斷正確性。在其他可能的致病基因清單中，sodium iodide symporter (NIS)是很 少被分析但值得進一步研究的基因，因為曱狀腺癌細胞攝取碘的能力與其分化程度及預後相關。本研究 除了將使用傳統的PCR-Sanger sequencing檢測分析NIS’ BRAF, RAS與其他可能之基因突變熱點、探討 國人基因變異和疾病診斷、預後相關性以外，同時會使用次世代定序更全面性地找尋其他仍未被發現的 單點基因突變或其他的基因突變方式。 在代謝體方面的研究已經有文獻可以利用代謝物小分子來區別不同類別的曱狀腺結節。然而文獻中 大多是採用組織切片為樣品，無法作為術前曱狀腺結節診斷的方法。本計晝使用靈敏性佳的質譜儀來分 析樣品的代謝物，來找出和FNAC結果相對應的代謝生物性指標。首先，我們會先找出曱狀腺囊腫、良 性腫瘤、惡性腫瘤與正常曱狀腺組織的代謝物表現差異，並在利用FNAC取出的細胞上檢驗這些差異結 果。在細胞形態學方面，我們先前研究發現以電腦程式所分析的細胞影像特徵(computerized morphometry, CM)與PTC預後相關。本研究將進一步拓展此工具運用在FTC和其他FNAC無法區分良惡性的曱狀腺 結節，將與病理報告和預後進行對照。 總結，本計劃將結合基因檢測、代謝體檢測、與CM來綜合分析曱狀腺FNAC檢體，以促進曱狀腺 癌的診斷與預後評估的準確性。預計納入120個檢體，包括（a) PTC和FTC、(b) FNAC無法確定良惡 性的曱狀腺瘤、（c)良性曱狀腺瘤。藉由phenotypes與genotype的對照比較，預期將增進FNAC的診斷 正確性和預後評估，並進一步對曱狀腺癌的致病機轉深入了解，以作為發展新治療的基礎。The prevalence of thyroid nodules is high and five to fifteen percent of these nodules are malignant; most of them are well-differentiated thyroid cancer, including papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). The current standard diagnostic tool is fine needle aspiration cytological (FNAC) examination of the nodules. The overall sensitivity and specificity is about 90%, which is far from perfect. Besides, 15% of the nodules evaluated by FNAC are indeterminate, especially in cases of follicular neoplasm. All of the obstacles become a thorny problem for both healthcare-givers and patients. In addition, study of the changes at molecular levels with the diagnosis and prognosis of thyroid cancer is very interesting and important. The goal of this study is to propose a reliable method for diagnosis and prognosis of papillary thyroid cancer and follicular thyroid cancer through analyzing genetic mutations, metabolites feature, and cellular morphologic characteristics. Clinical presentation, metabolites features, and cellular morphology are all important phenotypes of thyroid cancer. To improve the diagnostic and prognosis accuracy, this project integrates functional genomic approaches including genomics and metabolomics to establish a causal link between select biomarkers and disease pathogenesis. Among all the candidate genes, BRAFV600E and RAS mutation has been well-known detected in differentiated thyroid carcinoma. Mutation of BRAFV600E and RAS result in the activation of mitogen-activated protein kinase (MAPK) pathway and thus cell proliferation. They have also been associated with aggressive clinical behavior and poor prognosis. TERT promoter mutation was just discovered in thyroid cancer for one year, but has turned into the focus because of its risk in aggressive clinical behavior and higher prevalence in advanced thyroid cancer. Genetic mutations of several other genes were also reported. Sodium iodide symporter (NIS) is an important but under-explored target gene. BRAF mutation induces NIS regression and promotes epithelial to mesenchymal transition and invasion. We would like to examine the mutations of NIS, BRAFV600E, RAS, TERT, and other possible genes in FNAC specimens and explore the correlation of mutations, diagnosis, and prognosis. Sanger sequencing will be the method of choice for the known somatic mutation hotspots, but next-generation sequencing (NGS) will be used to more comprehensively search for disease-causing genes. NGS also has the potential to shed light on new pathophysiological pathways. Metabolomics has been demonstrated as a powerful tool to differentiate different types of thyroid nodules. However, previous studies mainly used tissue biopsy as study materials which limited its application in presurgical screening of thyroid nodules. This study uses a more sensitivity mass spectrometry based metabolomics platform to investigate tumor markers in FNAC specimens. We use a two steps approach by first identification of the metabolic differences between thyroid neoplasms, benign or malignant, and normal thyroid tissue in operative specimens and then test those differences on cells obtained through FNAC. According to our previous studies, cytological features of PTC, analyzed with computerized morphometry, are correlated with prognosis significantly. It helps to predict prognosis pre-operatively and to establish a risk-stratified treatment plan. In this current project, we plan to extend this tool to the diagnosis and/or prognosis of FTC and to nodules with uncertain FNAC reports to assist clinical management. In summary, this study applies a multi- and inter-disciplinary approach to improve the accuracy and sensitivity for diagnosis and prognosis of thyroid cancer using FNAC specimens. We will examine 120 samples, including groups of (a). PTC or FTC (40 samples), (b). Indeterminate (40 samples) and (c). Benign (40 samples). From our study with the comparison of phenotypes and genotypes, we anticipate to break the current diagnostic dilemma and limitation of poor prognostic prediction of thyroid nodules. Furthermore, we are longing to improve our understanding in thyroid cancer genesis which could disclosure novel diagnostic markers and treatment targets.曱狀腺結節曱狀腺癌基因體代謝體曱狀腺細胞之電腦程式影像分析次世代定序thyroid nodulesthyroid carcinomagenomicscomputerized morphometrynext-generation sequencingmetabolomics