台灣地區甲狀腺癌之基因研究

Abstract

甲狀腺結節有很高的盛行率，其中甲狀腺癌約5%，以乳突癌(papillary thyroid cancer, PTC)與濾泡癌(follicular thyroid cancer, FTC)居多。目前用來診斷甲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。 甲狀腺癌的臨床表現和細胞型態都是重要的phenotype，為了促進甲狀腺癌的診斷與預後評估的準確性，本計畫將透過基因體學來研究特定生物標記和甲狀腺癌致病機轉的關係。在基因與臨床表現的研究中，BRAF、RAS等基因突變已明確被發現與甲狀腺癌相關，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，因為甲狀腺癌細胞攝取碘的能力與其分化程度及預後相關。本研究使用次世代定序來分析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，同時會更全面性地找尋其它仍未被發現的單點基因突變或其他的基因突變方式。 本計劃的主要目的為結合基因體與細胞形態特徵等方法來分析甲狀腺FNAC檢體，以促進甲狀腺癌的診斷與預後評估的準確性，進一步探討分子生物學的變化，以瞭解其與PTC與FTC之診斷及預後之關係。本研究共收案37位病患，其中包含Group 1— 穿刺細胞學報告為malignant的病人 15位，Group 2— 穿刺細胞學報告為suspicious or indeterminate的病人20位，及Group 3— 穿刺細胞學報告為benign的病人2位。基因檢測結果顯示15個Group1的檢體中全部15個檢體皆檢測出帶有致病的基因變異，陽性率100%。20位Group2的病人中，其中三位開刀的結果是良性增生性結節，這三個穿刺細胞學檢體基因檢測皆未檢出致病的基因變異；其餘17位開刀結果為甲狀腺癌，15位檢測出有致病的基因變異，陽性率88%。而2個Group3的檢體中，一位開刀是良性結節，其檢體並無基因變異；另一位則開刀結果為poorly-differentiated carcinoma，基因檢測出PTEN基因變異。 利用本研究所提出之方法，透過甲狀腺穿刺殘餘之檢體，藉由whole genome amplification可獲得適當量之DNA進行次世代定序，減少病人必須額外接受穿刺之風險。此外本實驗設計次世代定序平台可大規模的檢測26個與甲狀腺癌相關的基因之變異及23種fusion gene，透過一次性的次世代定序結果，能夠同時判讀點突變、小片段缺失、及rearrangement之變異，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，並進一步對甲狀腺癌的致病機轉深入了解，以作為將來發展新治療的基礎。

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 only about 90%, and 15% of the nodules evaluated by FNAC are indeterminate. All of the obstacles become a thorny problem for both healthcare-givers and patients. 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 and cellular morphologic characteristics. Clinical presentation and cellular morphology are all important phenotypes of thyroid cancer. To improve the diagnostic and prognosis prediction, this project integrates genomic approaches to establish a causal link between selected biomarkers and disease pathogenesis. We enrolled the patients aged 20 to 85 years old who received FNAC in NTUH. According to cytology results, we classified subjects into three groups, including Group1 - malignant, Group2 – Indeterminate, and Group3 – Benign. The remaining material in the two FNA needles was collected and whole genome amplified afterward. We studied genetic variants through next-generation sequencing of a tailored panel consisting of 26 genes and 23 genetic fusions. 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 have analyzed 37 samples including 15 subjects in Group1, 20 subjects in Group2, and 2 subjects in Group3. We found pathogenic variants in all Group1 samples (positive rate 100%) and 15 pathology-proved malignant Group2 samples (positive rate 88%). The most common variant is BRAFV600E, 7 in Group1 and 10 in Group2. Genetic fusion of NCOA4/RET, RBPMS/NTRK3, and CCDC6/RET can be detected via our DNA-based method.