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  • 學位論文

臨床代謝體樣品收集變異研究與建立偵測乳癌定量模型

Variations Arising from Sample Collection in Clinical Metabolomics and Quantitative Model Building for the Diagnosis of Breast Cancer

指導教授 : 郭錦樺

摘要


代謝體學是一個近年來受到許多關注的新研究方法,具有應用於個人化醫療的潛力,但目前此類研究方法仍有一些挑戰。本論文研究臨床代謝體學可能之誤差來源,論文包含兩部分:第一部分針對收集臨床檢體的採血管、採樣體積以及在健康受試者中自體代謝波動等三種變因進行探討。本論文第二部分則為開發一個利用定量代謝體方法針對乳癌進行診斷的分析平台。 在本論文第一部分,我們針對三種常用的血漿(plasma)採樣管,其管內各含不同的抗凝血劑,如肝素(Heparin)、乙二胺四乙酸(EDTA)或是檸檬酸(Citrate),在4mL和8mL的採樣體積下進行分析,並和血清樣品(serum)進行比較。研究發現使用三種採樣管具有不同的結果,並且其中以Heparin管和血清樣品具有較相近的結果;此外,不同的採樣體積對於使用Citrate採樣管以及EDTA採樣管的樣品,有顯著的代謝物濃度差異。對於健康受試者中自體代謝波動分析結果發現,在一個月內四次的不同時間的採樣下,進行健康人的代謝體自體差異分析,結果發現由食物或藥物而導致的代謝物變化有可能高達20倍以上,但內生性的代謝物則在2倍差異以內。 在本論文的第二部分,我們開發了一個利用定量代謝體方法,對於乳癌病患進行血液分析。首先開發了兩個平行的液相層析結合質譜儀 (LC-MS) 的分析平台,並針對52個可能與乳癌相關的代謝物進行分析,比較乳癌患者與健康受試者血液中,此52個代謝物之濃度差異,結果顯示17個代謝物具有偵測乳癌之潛力。因此,本研究後續利用柱後注入內標法 (post-column infused internal standard method) 結合LC-MS方法針對上述17個代謝物進行絕對定量,進而針對停經前後婦女分別建構乳癌預測模型。此模型對於停經前婦女的AUROC為0.940,其專一性及靈敏度分別為94.2%和88.4%;而對於停經後婦女的AUROC為0.828,專一性及靈敏度則分別為85.1%和73.5%。 本論文的結果顯示,使用代謝體研究方法須有合適的實驗設計,才能夠得到正確的結果。本論文同時也建構了一個具有前瞻性診斷乳癌的分析平台。

並列摘要


Metabolomics is a field of science that is gaining much popularity in recent years. Metabolomics shows high potential in the area of personalized medicine, yet there remain many challenges within this study approach. This thesis evaluated several challenges in clinical metabolomics. The first part of this study aimed to examine three possible sources of variation seen in metabolomic type studies, including variations that arise from sampling tube types, sampling volumes and also from natural fluctuations within a healthy individual. The second part of this thesis worked on building a quantitative model to diagnose breast cancer using the metabolomic approach. In the first part, three plasma collection tube types containing the anticoagulants Heparin, EDTA and Citrate were compared with each other and against Serum collection tubes at two different collection volumes (4mL and 8mL). Considerable difference was seen among all tube types, with Heparin tubes displaying the closest results to that of serum tubes. Collection volume greatly influenced the metabolome observed from citrate tubes, and moderately for that of EDTA tubes. Plasma was collected from three healthy individuals four times over the period of a month to see the natural fluctuations of the metabolome under normal conditions. Compounds originating from exogenous sources such as food and drugs fluctuated at extensive folds (>20) whereas the majority of compounds of endogenous origin were regulated within 2 folds. In the second part of the thesis, a quantitative metabolomic method to diagnose breast cancer was developed. Two Liquid Chromatography coupled to Mass Spectrometry (LC-MS) methods were developed to examine 52 selected metabolites in a targeted approach. Breast cancer patients were compared against controls and 17 compounds showed potential to differentiate the two groups. Two post-column infused internal standard LC-MS methods were developed to accurately quantify the select compounds, and applied to a different cohort to build the diagnostic model. The AUROC of the prediction models were 0.940 for premenopausal women, with a specificity of 94.2% and a sensitivity of 88.4%, and 0.828 for postmenopausal women, with a specificity of 85.1% and a sensitivity of 73.5%. Careful consideration for study design must be in place when investigating and interpreting data under metabolomic approach. We present a prospective model for the prediction of breast cancer using a panel of metabolites.

參考文獻


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