臺灣博碩士論文加值系統

非小細胞肺癌為肺癌中較為常見的種類型態，而肺癌在台灣更是最為主要的癌症死亡原因 (17.5%)，因此發展血清中的腫瘤標記有助於對非小細胞肺癌的診斷，我們的團隊整合了兩種肺腺癌細胞株(CL1-0 ,CL1-5) 的分泌性蛋白體和來自肺腺癌病人的胸腔積液 (pleural effusion) 蛋白質體，來搜尋可能的非小細胞肺癌腫瘤標記。我們的研究團隊已經透過一維電泳和液相層析質譜儀的方法，建立兩種肺腺癌細胞株的分泌性蛋白質體資料庫和一個肺腺癌病人的肋肌膜液體蛋白質體資料庫，並交集出163個可能標的蛋白，再透過生物資訊學上的分析 (signal P、secretome P、TMHMM) 運算出在胺基酸序列上是否含有典型分泌性或非典型分泌性蛋白的胺基酸序列或者是穿膜蛋白的胺基酸序列，推測89個蛋白質，可能為分泌型蛋白的可能。經過文獻的搜尋，我們選擇了四個我們所感興趣的分子做為我們要研究的對象 angiogenin 、cystatin-C 、fetuin-A 和IGFBP2 ，並且在人類血清中或是胸腔積液中做驗證。我們的結果顯示出，在血清的蛋白質含量方面，angiogenin和IGFBP2 的含量在罹患非小細胞肺癌病人中有明顯升高，並且在統計上與正常人的血清相比具有顯著性的差異，然而 fetuin-A 的蛋白質含量在血清中則是低於正常人的，而計算area under curve (AUC) 數值，我們得到 angiogenin 為0.61 ，cystatin- C 為0.62，fetuin-A 為0.73，IGFBP2 為0.71 而傳統的腫瘤標記 CEA則為0.71，結合fetuin-A、IGFBP2、跟CEA 三個分子我們得到相較於個別使用下一個檢測能力更好的的組合 (AUC = 0.79)，而IGFBP2在檢測疾病分期 (早期: stage I, II ; 晚期: stage III, IV) 的應用上其AUC為 0.69在比較傳統分子CEA ( AUC = 0.57 ) 和fetuin-A ( AUC = 0.59 ) 都顯示出在區分疾病分期上的優勢 。更重要的是，IGFBP2在惡性腫瘤胸腔積液的蛋白質含量中，明顯高於肺炎、肺結核和非惡性腫瘤胸腔積液族群，並具有統計上的意義。最後，我們探尋了分泌性蛋白 fetuin-A在肺癌生成過程中所扮演的角色，並發現細胞外的fetuin-A可能在肺癌細胞中扮演一個移行抑制者的角色，而IGFBP2在CL1-0細胞株中初步可以發現具有促進移行的功能。總體來說，我們的結果顯示，藉由整合分泌性蛋白體資料庫和胸腔積液蛋白體資料庫的方式，可提供去尋找一個用於非小細胞肺癌新穎性的生物標記組合，
.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which is one of the most prominent causes of cancer-related mortality in Taiwan (17.5%). In this study, we integrate two adenocarcinoma cancer cell lines secretome, CL1-0, CL1-5 and human pleural effusion proteome from lung adenocarcinoma patients to discover serum/pleural effusion maker panel for NSCLC. Previously, we have generated two of lung adenocarcinoma cell lines secretome databases and one pleural effusion proteome database by one-dimensional gel electrophoresis in conjunction with nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS) platform. After integration of these three protein datasets, we found 163 proteins identified in all of these three datasets. The 163 identified proteins were further analyzed by bioinformatics software programs and 89 candidates are predicted as secreted protein. Based on literature search, we selected four potential markers, including angiogenin, cystatin-C, fetuin-A and IGFBP2 for further verification by using human sera and pleural effusions. Our results showed that the serum levels of angiogenin and IGFBP2 in NSCLC patients were significantly higher than those in healthy controls, whereas, serum level of fetuin-A was significantly decreased in NSCLC patients as compared to healthy controls. The area under curve (AUC) value for angiogenin, cystatin-C, fetuin-A, IGFBP2 and CEA was 0.61, 0.62, 0.73, 0.71 and 0.71, respectively. A combination of serum fetuin-A, IGFBP2 and CEA displayed higher diagnostic capacity (AUC 0.79) than either marker alone. The AUC value for IGFBP2, fetuin-A and CEA for prediction of disease stage (stage I/II vs. stage III/IV) was 0.69, 0.57 and 0.59, respectively. These results indicate IGFBP2 is a potential maker to distinguish disease stage. Importantly, protein level of IGFBP2 in malignant pleural effusion from cancer patients was significantly higher than those from patients without malignancy. We also characterized biological significance of secreted proteins in lung cancer progress. We found that extracellular fetuin-A inhibite cellular migration and extracellular IGFBP2 promote cellular migration of lung cancer cells. Collectively, our results suggest that integration of cancer cell secretome and pleural effusion proteome provide an efficient means of identifying novel marker panel for NSCLC.

目 錄
指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
授權書…………………………………………………………………….
誌謝………………………………………………………………………….IV
中文摘要…………………………………………………………………..V
英文摘要……………………………………………………………….VII
目錄……………………………………………………………………..IX
圖表目錄………………………………………………………………..XI
第一章 序論 ………………………………………………………....1
第二章 材料與方法…………………………………………………………6
2.1 細胞培養………………………………………………………………6
2.2 收集癌細胞株的條件式培養…………………………………………6
2.3 病人族群與臨床樣品…………………………………………………7
2.4 胸腔積液樣品前處理…………………………………………………7
2.5 一維電泳分析及在膠體內蛋白質酶作用……………………………8
2.6 反式液相串聯式質譜分析……………………………………………8
2.7 蛋白質資料庫的比對與生物資訊學分析……………………………10
2.8 酵素連結免疫吸附分析………………………………………………11
2.9 生物統計方法分析……………………………………………………11
2.10 轉移盤細胞移行能力試驗……………………………………………12
2.11 細胞存活試驗…………………………………………………………12
第三章 實驗結果………………………………………………………………14
3.1 藉由整合肺癌細胞株和胸腔積液蛋白質體資料庫建立可能的腫瘤標記資料庫…………………………………………………………………………………14
3.2 肺癌細胞株和胸腔積液蛋白質體資料庫與過去文獻之比較……………18
3.3 腫瘤標記在病人血清中濃度的相關性……………………………………19
3.4 腫瘤標記在病人胸腔積液中濃度的相關性………………………………23
3.5 分泌性fetuin-A蛋白在CL1-0與CL1-5細胞株中所扮演的角色………25
3.6 分泌性IGFBP2蛋白在CL1-0細胞株中所扮演的角色……………………26
第四章 討論………………………………………………………………………27
第五章 未來研究方向……………………………………………………………39
附表………………………………………………………………………………40
附圖………………………………………………………………………………63
參考文獻…………………………………………………………………………83


圖 表 目 錄
表一、 收集自長庚紀念醫院的健康人和病人血清檢體資料………………40
表二、 收集自長庚紀念醫院的胸腔積液檢體資料…………………………41
表三、 89個candidate list………………………………………………42
表四、 4個candidate 在血清和胸腔積液中與臨床相關的文獻報導……49
表五、 89個分子在各種細胞株比對到的蛋白………………………………50
表六、 各個分子在比較正常人血清和病人血清的濃度變化………………59
表七、 比較過去文獻在血清中CEA的檢測效益……………………………60
表八、 比較過去文獻在血清中腫瘤標記組的檢測效益……………………61
表九、 比較過去文獻在胸腔積液中腫瘤標記組的檢測效益………………62
圖一 CL1-0 CL1-5和Pleural Effusion三個資料庫交集……………63
圖二 89個候選蛋白gene ontology分類圖………………………………64
圖三 過去的資料庫中與89個候選蛋白交疊圖……………………………65
圖四 比較各個分子在正常人與罹患非小細胞肺癌病人的血清含量………67
圖五 各個分子跟癌症分期相關的盒狀圖……………………………………69
圖六 IGFBP2 & FetuinA 整合ROC curve………………………………70
圖七 所有分子和整合的ROC curve…………………………………………71
圖八 IGFBP2 & fetuinA 和CEA比較癌細胞轉移ROC curve……………73
圖九 IGFBP2和fetuin-A的盒狀圖……………………………………………74
圖十 IGFBP2 、CEA和兩個分子整合ROC curve……………………………75
圖十一 IGFBP2 、CEA和兩個分子整合ROC curve…………………………76
圖十二 Fetuin-A蛋白在CL1-0與CL1-5細胞株中的功能測試……………77
圖十三 IGFBP2蛋白在CL1-0細胞株中的移行功能測試……………………78
圖十四 IGFBP2蛋白在CL1-0細胞株中的生長功能測試……………………79

參考文獻
Ahmedin Jemal, R.S., Elizabeth Ward, Yongping Hao, Jiaquan Xu and Michael J. Thun (2009). Cancer Statistics, 2009. CA Cancer J Clin 2009;59, 225-249.

Anderson, N.L., and Anderson, N.G. (2002). The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 1, 845-867.

Bach, P.B., Silvestri, G.A., Hanger, M., and Jett, J.R. (2007). Screening for lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 132, 69S-77S.

Barcellos-Hoff, M.H., and Ewan, K.B. (2000). Transforming growth factor-beta and breast cancer: Mammary gland development. Breast Cancer Res 2, 92-99.

Bendtsen, J.D., Jensen, L.J., Blom, N., Von Heijne, G., and Brunak, S. (2004a). Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Sel 17, 349-356.

Bendtsen, J.D., Nielsen, H., von Heijne, G., and Brunak, S. (2004b). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783-795.

Bitterlich, N., Muley, T., and Schneider, J. (2010). Centre-independent detection of non-small cell lung cancer (NSCLC) by means of classification with receiver operating characteristic (ROC)-based data transformation. Anticancer Res 30, 1661-1665.

Bitterlich, N., and Schneider, J. (2007). Decision guarantee in tumour marker analysis: a cut-off independent assessment. Anticancer Res 27, 1933-1939.

Borgia, J.A., Basu, S., Faber, L.P., Kim, A.W., Coon, J.S., Kaiser-Walters, K.A., Fhied, C., Thomas, S., Rouhi, O., Warren, W.H., et al. (2009). Establishment of a multi-analyte serum biomarker panel to identify lymph node metastases in non-small cell lung cancer. J Thorac Oncol 4, 338-347.

Chantapet, P., Riantawan, P., Lebnak, P., and Getngern, P. (2000). Utility of serum cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) as tumour markers for non-small cell lung cancer. J Med Assoc Thai 83, 383-391.

Chen, F., Li, W.M., Wang, D.M., Gao, S.S., Bao, Y., Chen, W.B., and Liu, D. (2008). [Clinical value of combined detection of serum tumor markers in lung cancer diagnosis]. Sichuan Da Xue Xue Bao Yi Xue Ban 39, 832-835.

Chu, Y.W., Yang, P.C., Yang, S.C., Shyu, Y.C., Hendrix, M.J., Wu, R., and Wu, C.W. (1997). Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 17, 353-360.

D'Amico, T.A., Brooks, K.R., Joshi, M.B., Conlon, D., Herndon, J., 2nd, Petersen, R.P., and Harpole, D.H., Jr. (2006). Serum protein expression predicts recurrence in patients with early-stage lung cancer after resection. Ann Thorac Surg 81, 1982-1987; discussion 1987.

Domej, W., Tilz, G.P., Foldes-Papp, Z., Demel, U., Rabold, T., and Holzer, H. (2002). Cystatin C of pleural effusion as a novel diagnostic aid in pleural diseases of different aetiologies. Clin Sci (Lond) 102, 373-380.

Dong, F., Wu, H.B., Hong, J., and Rechler, M.M. (2002). Insulin-like growth factor binding protein-2 mediates the inhibition of DNA synthesis by transforming growth factor-beta in mink lung epithelial cells. J Cell Physiol 190, 63-73.

Dowling, P., O'Driscoll, L., Meleady, P., Henry, M., Roy, S., Ballot, J., Moriarty, M., Crown, J., and Clynes, M. (2007). 2-D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins. Electrophoresis 28, 4302-4310.

El-Badry OM, R.J., Helman LJ, Cooper MJ, Rechler MM, Israel MA. (1989). Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II. J Clin Invest 84, 829-839.

Ferrer, J., Villarino, M.A., Encabo, G., Felip, E., Bermejo, B., Vila, S., and Orriols, R. (1999). Diagnostic utility of CYFRA 21-1, carcinoembryonic antigen, CA 125, neuron specific enolase, and squamous cell antigen level determinations in the serum and pleural fluid of patients with pleural effusions. Cancer 86, 1488-1495.
Ferrigno, D., Buccheri, G., and Biggi, A. (1994). Serum tumour markers in lung cancer: history, biology and clinical applications. Eur Respir J 7, 186-197.

Gao, X., and Xu, Z. (2008). Mechanisms of action of angiogenin. Acta Biochim Biophys Sin (Shanghai) 40, 619-624.

Good, D.M., Thongboonkerd, V., Novak, J., Bascands, J.L., Schanstra, J.P., Coon, J.J., Dominiczak, A., and Mischak, H. (2007). Body fluid proteomics for biomarker discovery: lessons from the past hold the key to success in the future. J Proteome Res 6, 4549-4555.

Heffner, J.E. (2008). Diagnosis and management of malignant pleural effusions. Respirology 13, 5-20.

Henskens YMC, V.E.a.A.A. (1996). Cystatinsin health and diseases. Biol Chem Hoppe-Seyler 377, 71-86.

Holly JM, W.J. (1989). Insulin-like growth factors; autocrine, paracrine or endocrine? New perspectives of the somatomedin hypothesis in the light of recent developments. J Endocrinol 122, 611-618.

Huang, L.J., Chen, S.X., Huang, Y., Luo, W.J., Jiang, H.H., Hu, Q.H., Zhang, P.F., and Yi, H. (2006). Proteomics-based identification of secreted protein dihydrodiol dehydrogenase as a novel serum markers of non-small cell lung cancer. Lung Cancer 54, 87-94.

Huang, W.W., Tsao, S.M., Lai, C.L., Su, C.C., and Tseng, C.E. (2010). Diagnostic value of Her-2/neu, Cyfra 21-1, and carcinoembryonic antigen levels in malignant pleural effusions of lung adenocarcinoma. Pathology 42, 224-228.

I, P.P.a.M. (1998). Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. cell 93, 1135-1145.

Janssen-Heijnen, M.L., and Coebergh, J.W. (2003). The changing epidemiology of lung cancer in Europe. Lung Cancer 41, 245-258.

Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E.L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305, 567-580.
Kulasingam, V., and Diamandis, E.P. (2007). Proteomics analysis of conditioned media from three breast cancer cell lines: a mine for biomarkers and therapeutic targets. Mol Cell Proteomics 6, 1997-2011.

Kundranda, M.N., Henderson, M., Carter, K.J., Gorden, L., Binhazim, A., Ray, S., Baptiste, T., Shokrani, M., Leite-Browning, M.L., Jahnen-Dechent, W., et al. (2005). The serum glycoprotein fetuin-A promotes Lewis lung carcinoma tumorigenesis via adhesive-dependent and adhesive-independent mechanisms. Cancer Res 65, 499-506.

Kuralay, F., Tokgoz, Z., and Comlekci, A. (2000). Diagnostic usefulness of tumour marker levels in pleural effusions of malignant and benign origin. Clin Chim Acta 300, 43-55.

Lee, D.Y., Kim, S.J., and Lee, Y.C. (1999). Serum insulin-like growth factor (IGF)-I and IGF-binding proteins in lung cancer patients. J Korean Med Sci 14, 401-404.

Lee, J.H., and Chang, J.H. (2005). Diagnostic utility of serum and pleural fluid carcinoembryonic antigen, neuron-specific enolase, and cytokeratin 19 fragments in patients with effusions from primary lung cancer. Chest 128, 2298-2303.

Li, W., Yang, X., Wang, K., Tan, W., Li, H., and Ma, C. (2008). FRET-based aptamer probe for rapid angiogenin detection. Talanta 75, 770-774.

Li, Y.P., Hu, C.P., and Yang, H.Z. (2003). [Clinical value of tumor supplied group of factor combined with CEA in diagnosing tuberculosis pleural effusion and malignant pleural effusion]. Hunan Yi Ke Da Xue Xue Bao 28, 608-610.

Liang, Q.L., Shi, H.Z., Qin, X.J., Liang, X.D., Jiang, J., and Yang, H.B. (2008). Diagnostic accuracy of tumour markers for malignant pleural effusion: a meta-analysis. Thorax 63, 35-41.

Light, R.W. (2006). The undiagnosed pleural effusion. Clin Chest Med 27, 309-319.

Lombardi, C., Tassi, G.F., Pizzocolo, G., and Donato, F. (1990). Clinical significance of a multiple biomarker assay in patients with lung cancer. A study with logistic regression analysis. Chest 97, 639-644.
Lou, X., Xiao, T., Zhao, K., Wang, H., Zheng, H., Lin, D., Lu, Y., Gao, Y., Cheng, S., Liu, S., et al. (2007). Cathepsin D is secreted from M-BE cells: its potential role as a biomarker of lung cancer. J Proteome Res 6, 1083-1092.

Maskell NA, G.F., Davies RJ. (2003). Standard pleural biopsy versus CTguided cutting-needle biopsy for diagnosis of malignant disease in pleural effusions: a randomized controlled trial. Lancet 361, 1326-1330.

Mathews, S.T., Chellam, N., Srinivas, P.R., Cintron, V.J., Leon, M.A., Goustin, A.S., and Grunberger, G. (2000). Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor. Mol Cell Endocrinol 164, 87-98.

Melamed, M.R., Flehinger, B.J., and Zaman, M.B. (1987). Impact of early detection on the clinical course of lung cancer. Surg Clin North Am 67, 909-924.

Miedouge, M., Rouzaud, P., Salama, G., Pujazon, M.C., Vincent, C., Mauduyt, M.A., Reyre, J., Carles, P., and Serre, G. (1999). Evaluation of seven tumour markers in pleural fluid for the diagnosis of malignant effusions. Br J Cancer 81, 1059-1065.

Mishina, T., Dosaka-Akita, H., Hommura, F., Nishi, M., Kojima, T., Ogura, S., Shimizu, M., Katoh, H., and Kawakami, Y. (2000). Cyclin E expression, a potential prognostic marker for non-small cell lung cancers. Clin Cancer Res 6, 11-16.

Moller, S., Croning, M.D., and Apweiler, R. (2001). Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17, 646-653.

Naumnik, W., Niklinska, W., Ossolinska, M., and Chyczewska, E. (2009). Serum cathepsin K and cystatin C concentration in patients with advanced non-small-cell lung cancer during chemotherapy. Folia Histochem Cytobiol 47, 207-213.

Nielsen, H., and Krogh, A. (1998). Prediction of signal peptides and signal anchors by a hidden Markov model. Proc Int Conf Intell Syst Mol Biol 6, 122-130.

Olchovsky, D., Shimon, I., Goldberg, I., Shulimzon, T., Lubetsky, A., Yellin, A., Pariente, C., Karasik, A., and Kanety, H. (2002). Elevated insulin-like growth factor-1 and insulin-like growth factor binding protein-2 in malignant pleural effusion. Acta Oncol 41, 182-187.

Olsen, J.V., de Godoy, L.M., Li, G., Macek, B., Mortensen, P., Pesch, R., Makarov, A., Lange, O., Horning, S., and Mann, M. (2005). Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4, 2010-2021.

Parkin, D.M., Pisani, P., and Ferlay, J. (1993). Estimates of the worldwide incidence of eighteen major cancers in 1985. Int J Cancer 54, 594-606.

Pavesi, F., Lotzniker, M., Cremaschi, P., Marbello, L., Acquistapace, L., and Moratti, R. (1988). Detection of malignant pleural effusions by tumor marker evaluation. Eur J Cancer Clin Oncol 24, 1005-1011.

Pepe, M.W.M.M.S. (2002). Combining Several Screening Tests: Optimality of the Risk Score. Biometrics 58, 657.

Pfister, D.G., Johnson, D.H., Azzoli, C.G., Sause, W., Smith, T.J., Baker, S., Jr., Olak, J., Stover, D., Strawn, J.R., Turrisi, A.T., et al. (2004). American Society of Clinical Oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol 22, 330-353.

Planque, C., Kulasingam, V., Smith, C.R., Reckamp, K., Goodglick, L., and Diamandis, E.P. (2009). Identification of five candidate lung cancer biomarkers by proteomics analysis of conditioned media of four lung cancer cell lines. Mol Cell Proteomics 8, 2746-2758.

Porcel, J.M., Vives, M., Esquerda, A., Salud, A., Perez, B., and Rodriguez-Panadero, F. (2004). Use of a panel of tumor markers (carcinoembryonic antigen, cancer antigen 125, carbohydrate antigen 15-3, and cytokeratin 19 fragments) in pleural fluid for the differential diagnosis of benign and malignant effusions. Chest 126, 1757-1763.

Prakash UB, R.H. (1985). Comparison of needle biopsy with cytologic analysis for the evaluation of pleural effusion: analysis of 414 cases. Mayo

Clin Proc 60, 158-164.

R., L. (2003). Medical thoracoscopy. In: Light RW, Gary Lee YC, editors. Textbook of pleural diseases. London: Arnold, 498-512.

Sanchez de Cos Escuin, J., Lopez Parra, S., Disdier Vicente, C., Martin Vicente, M.J., Masa Jimenez, J.F., and Dominguez Retortillo, C. (1996). [Diagnostic utility of carcinoembryonic antigen, neuron-specific enolase and squamous cell carcinoma antigen in malignant pleural effusion]. An Med Interna 13, 369-373.

Sato, H., Yazawa, T., Suzuki, T., Shimoyamada, H., Okudela, K., Ikeda, M., Hamada, K., Yamada-Okabe, H., Yao, M., Kubota, Y., et al. (2006). Growth regulation via insulin-like growth factor binding protein-4 and -2 in association with mutant K-ras in lung epithelia. Am J Pathol 169, 1550-1566.

Schneider, J. (2006). Tumor markers in detection of lung cancer. Adv Clin Chem 42, 1-41.

Schneider, J., Bitterlich, N., Velcovsky, H.G., Morr, H., Katz, N., and Eigenbrodt, E. (2002). Fuzzy logic-based tumor-marker profiles improved sensitivity in the diagnosis of lung cancer. Int J Clin Oncol 7, 145-151.

Shitrit D, Z.B., Shitrit AB, Shlomi D, Kramer MR. (2005). Diagnostic value of Cyfra 21-1, CEA, Ca 19-9, Ca 15-3, and Ca 125 assays in pleural effusions: analysis of 116 cases and review of literature. Oncologist 10, 501-507.

Silvestri, G.A., Gould, M.K., Margolis, M.L., Tanoue, L.T., McCrory, D., Toloza, E., and Detterbeck, F. (2007). Noninvasive staging of non-small cell lung cancer: ACCP evidenced-based clinical practice guidelines (2nd edition). Chest 132, 178S-201S.

Sonnhammer, E.L., von Heijne, G., and Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6, 175-182.

Sthaneshwar, P., Yap, S.F., and Jayaram, G. (2002). The diagnostic usefulness of tumour markers CEA and CA-125 in pleural effusion. Malays J Pathol 24, 53-58.

Swallow, C.J., Partridge, E.A., Macmillan, J.C., Tajirian, T., DiGuglielmo, G.M., Hay, K., Szweras, M., Jahnen-Dechent, W., Wrana, J.L., Redston, M., et al. (2004). alpha2HS-glycoprotein, an antagonist of transforming growth factor beta in vivo, inhibits intestinal tumor progression. Cancer Res 64, 6402-6409.

Tarn AC, L.R. (2001). Biochemical analysis of pleural fluid: what should we measure? . Ann Clin Biochem 38, 311-322.

Tomida, M., Mikami, I., Takeuchi, S., Nishimura, H., and Akiyama, H. (2009). Serum levels of nicotinamide N-methyltransferase in patients with lung cancer. J Cancer Res Clin Oncol 135, 1223-1229.

Tyan, Y.C., Wu, H.Y., Lai, W.W., Su, W.C., and Liao, P.C. (2005). Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry. J Proteome Res 4, 1274-1286.

Uzbeck, M.H., Almeida, F.A., Sarkiss, M.G., Morice, R.C., Jimenez, C.A., Eapen, G.A., and Kennedy, M.P. (2010). Management of malignant pleural effusions. Adv Ther 27, 334-347.

Villena, V., Lopez-Encuentra, A., Echave-Sustaeta, J., Martin-Escribano, P., Ortuno-de-Solo, B., and Estenoz-Alfaro, J. (1996). Diagnostic value of CA 72-4, carcinoembryonic antigen, CA 15-3, and CA 19-9 assay in pleural fluid. A study of 207 patients. Cancer 78, 736-740.

Villena, V., Lopez-Encuentra, A., Echave-Sustaeta, J., Martin-Escribano, P., Ortuno-de-Solo, B., and Estenoz-Alfaro, J. (2003). Diagnostic value of CA 549 in pleural fluid. Comparison with CEA, CA 15.3 and CA 72.4. Lung Cancer 40, 289-294.

Wagner, I.C., Guimaraes, M.J., da Silva, L.K., de Melo, F.M., and Muniz, M.T. (2007). Evaluation of serum and pleural levels of the tumor markers CEA, CYFRA21-1 and CA 15-3 in patients with pleural effusion. J Bras Pneumol 33, 185-191.

Wegiel, B., Jiborn, T., Abrahamson, M., Helczynski, L., Otterbein, L., Persson, J.L., and Bjartell, A. (2009). Cystatin C is downregulated in prostate cancer and modulates invasion of prostate cancer cells via MAPK/Erk and androgen receptor pathways. PLoS One 4, e7953.

Werle, B., Schanzenbacher, U., Lah, T.T., Ebert, E., Julke, B., Ebert, W., Fiehn, W., Kayser, K., Spiess, E., Abrahamson, M., et al. (2006). Cystatins in non-small cell lung cancer: tissue levels, localization and relation to prognosis. Oncol Rep 16, 647-655.

WM., E. (1990). Investigation of pleural effusion: comparison between fibreoptic thoracoscopy, needle biopsy and cytology. Resp Med 84, 23-26.
Wu, C.C., Chien, K.Y., Tsang, N.M., Chang, K.P., Hao, S.P., Tsao, C.H., Chang, Y.S., and Yu, J.S. (2005). Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model. Proteomics 5, 3173-3182.

Yuan, Y., Wang, F., Liu, X.H., Gong, D.J., Cheng, H.Z., and Huang, S.D. (2009). Angiogenin is involved in lung adenocarcinoma cell proliferation and angiogenesis. Lung Cancer 66, 28-36.

Zebrowski, B.K., Yano, S., Liu, W., Shaheen, R.M., Hicklin, D.J., Putnam, J.B., Jr., and Ellis, L.M. (1999). Vascular endothelial growth factor levels and induction of permeability in malignant pleural effusions. Clin Cancer Res 5, 3364-3368.