臺灣博碩士論文加值系統

目前受到美國食品藥物管理署認證的膀胱癌生物標誌分子並不足以應用於非侵入式的臨床診斷上。因此，如何快速的從大量的驗證分子中快速挑選出具新穎性的癌症生物標誌分子是一件重要的課題。在本論文中已成功建立了122個蛋白質的多重反應監測質譜測量法，其中包含了118個膀胱癌上升表現的候選標誌分子、兩個已知的膀胱癌生物標誌分子與兩個管家基因，並針對這122個蛋白質的胜肽進行質譜參數的優化。在122蛋白質於膀胱癌、疝氣、血尿、尿道感染等大量的臨床尿液樣品定量結果中，ANXA3與HSPE1在膀胱癌的樣品中有較高的偵測頻率。因此，進一步選擇這兩個分子進行驗證。其中HSPE1在西方墨點法與酵素免疫分析法中，於膀胱癌尿液的表現量較高於其他尿液，而免疫組織化學染色法中也進一步顯示HSPE1在膀胱癌組織有較高的表現量。綜合以上之結果，HSPE1具有應用於臨床診斷之潛力，並以本篇論文的研究結果作為未來膀胱癌診斷提供參考。

Bladder cancer biomarkers currently approved by the Food and Drug Administration are insufficiently reliable for use in non-invasive clinical diagnosis. Verification/validation of numerous biomarker candidates for BLCA detection is a crucial bottleneck for novel biomarker development. A multiplexed liquid chromatography multiple-reaction-monitoring mass spectrometry assay of 122 proteins, including 118 up-regulated tissue proteins, two known bladder cancer biomarkers and two housekeeping gene products, was successfully established for protein quantification in clinical urine specimens. Quantification of 122 proteins was performed on a large cohort of urine specimens representing a variety of conditions, including 142 hernia, 126 bladder cancer, 67 hematuria, and 59 urinary tract infection samples. ANXA3 (annexin A3) and HSPE1 (heat shock protein family E member 1), which showed the highest detection frequency in bladder cancer samples, were selected for further validation. Western blotting showed that urinary ANXA3 and HSPE1 protein levels were higher in bladder cancer samples than in hernia samples, and enzyme-linked immunosorbent assays confirmed a higher urinary concentration of HSPE1 in bladder cancer than in hernia, hematuria and urinary tract infection. Immunohistochemical analyses showed significantly elevated levels of HSPE1 in tumor cells, suggesting that HSPE1 could be a useful tumor tissue marker for the specific detection of bladder cancer. Collectively, our findings provide valuable information for future validation of potential biomarkers for bladder cancer diagnosis.

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致謝 iii
中文摘要 iv
Abstract v
Table of Contents vi
List of Tables viii
List of Figures ix
Abbreviations x
1. Introduction - 1 -
1.1 Bladder Carcinoma - 1 -
1.2 Urinary Biomarkers of Bladder Carcinoma - 1 -
1.3 Targeted Mass Spectrometry for Protein Biomarker Verification - 3 -
1.4 Specific Aims - 4 -
2. Methods - 5 -
2.1 Preparation of Clinical Urinary Specimens and BLCA Cell Extracts - 5 -
2.2 Tryptic Digestion of Urinary Proteins and Bladder Cancer Cell Extracts - 6 -
2.4 Development of MRM Methods for Quantifying Dimethylated Peptides - 7 -
2.5 LC−MRM/MS Analysis and Data Acquisition - 8 -
2.6 Processing of MRM-MS Data - 9 -
2.7 Western Blotting - 10 -
2.8 Immunohistochemical Staining - 10 -
2.9 Sandwich ELISA for HSPE1 Quantification in Urinary Proteins - 11 -
2.10 Possible Secretory Pathways of 130 Targeted Proteins - 12 -
2.11 Statistical Analysis - 12 -
3. Results - 14 -
3.1 Selection of protein biomarker candidates from the BLCA tissue proteome for MRM-MS assay development - 14 -
3.2 Establishment of the MRM assay: prescreening dimethyl-labeled signature peptide candidates in BLCA cell extracts - 14 -
3.3 Establishment of the MRM assay: selection of the top three transitions and collision energy (CE) optimization - 17 -
3.4 Methodological verification of 122 protein candidates in clinical urine samples by MRM-MS - 18 -
3.5 Validation of prioritized proteins in clinical urine and tissue samples by Western blotting - 19 -
3.6 Validation of HSPE1 in clinical urine samples by sandwich ELISA - 20 -
3.7 Immunohistochemical detection of HSPE1 in clinical tissue samples - 22 -
4. Discussion - 24 -
5. References - 29 -
6. Publication List - 169 -

 
List of Tables
Table 1. The list of 130 protein biomarker candidates for MRM assay development and their secretory properties. - 35 -
Table 2. Criteria of peptide selection for MRM assay development. - 42 -
Table 3. The transition list of MRM assay of 122 bladder cancer associated tissue proteins. - 101 -
Table 4. Clinical information of the clinical specimens used for MRM-MS. - 142 -
Table 5. 29 detectable protein targets in cancer and non-cancer urine samples and their possible secretory pathways. - 143 -
Table 6. The CV values for quantifying 122 bladder cancer associated tissue proteins in clinical urine specimens. - 145 -
Table 7. Quantitative results of 12 detectable bladder cancer associated tissue proteins by MRM assay in clinical urine specimens. - 147 -
Table 8. The p-values, fold changes, sensitivity, specificity, and AUC values of HSPE1 ROC curves. - 149 -
Table 9. Correlation results between urinary HSPE1 expression and clinicopathological characteristics of the bladder cancer patients by a Chi-squared test. - 151 -
Table 10. The IHC scores of expressions of HSPE1 in clinical tissue slides of BLCA patient. - 152 -
Table 11. Correlations of HSPE1 protein expression (IHC scores) in tissue specimens with clinicopathological characteristic. - 156 -

 
List of Figures
Figure 1. Bladder cancer statistics of estimated new cancer cases and deaths from 2008 to 2018. - 157 -
Figure 2. Criteria for signature peptide selection in MRM prescreens of 122 BLCA tissue candidates. - 158 -
Figure 3. An example of MS/MS spectrum confirmation by Mascot Daemon and Analyst software. - 159 -
Figure 4. Selecting the best CE using the CE ramping mode in a QTRAP MS. - 160 -
Figure 5. Comparison of differences in CE between default and optimized value (∆CE) for all optimized Q1/Q3 transitions. - 161 -
Figure 6. Workflow for the methodological verification of protein biomarker candidates in clinical urine samples. - 162 -
Figure 7. Western blot pre-test of ANXA3, HSPE1, VPS29 and YWHAQ in bladder cancer and hernia pooling urinary specimens. - 163 -
Figure 8. Methodological verification of HSPE1 in individual urine specimens from BLCA and hernia patients by antibody-based analysis. - 164 -
Figure 9. Correlation analysis between urinary HSPEL levels with urine (A) RBC and (B) WBC numbers in BLCA patients (n = 119). - 166 -
Figure 10. Up-regulation of HSPE1 in individual bladder tumor tissue specimens, determined by IHC. - 167 -
Figure 11. Expression of HSPE1 in individual bladder tumor and adjacent normal tissue specimens, determined by IHC. - 168 -

1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015; 65: 87-108. doi: 10.3322/caac.21262.
2. Saad A, Hanbury DC, McNicholas TA, Boustead GB, Morgan S, Woodman AC. A study comparing various noninvasive methods of detecting bladder cancer in urine. BJU Int. 2002; 89: 369-73. doi:
3. Jordan AM, Weingarten J, Murphy WM. Transitional cell neoplasms of the urinary bladder. Can biologic potential be predicted from histologic grading? Cancer. 1987; 60: 2766-74. doi:
4. Gontero P, Oderda M, Altieri V, Bartoletti R, Cai T, Colombo R, Curotto A, Di Stasi S, Maffezzini M, Tamagno S, Serretta V, Sogni F, Terrone C, et al. Are referral centers for non-muscle-invasive bladder cancer compliant to EAU guidelines? A report from the vesical antiblastic therapy Italian study. Urol Int. 2011; 86: 19-24. doi: 10.1159/000321926.
5. Lotan Y, Roehrborn CG. Sensitivity and specificity of commonly available bladder tumor markers versus cytology: results of a comprehensive literature review and meta-analyses. Urology. 2003; 61: 109-18; discussion 18. doi:
6. Carpinito GA, Stadler WM, Briggman JV, Chodak GW, Church PA, Lamm DL, Lange PH, Messing EM, Pasciak RM, Reservitz GB, Ross RN, Rukstalis DB, Sarosdy MF, et al. Urinary nuclear matrix protein as a marker for transitional cell carcinoma of the urinary tract. J Urol. 1996; 156: 1280-5. doi:
7. Jamshidian H, Kor K, Djalali M. Urine concentration of nuclear matrix protein 22 for diagnosis of transitional cell carcinoma of bladder. Urol J. 2008; 5: 243-7. doi:
8. Grossman HB, Messing E, Soloway M, Tomera K, Katz G, Berger Y, Shen Y. Detection of bladder cancer using a point-of-care proteomic assay. JAMA. 2005; 293: 810-6. doi: 10.1001/jama.293.7.810.
9. Villicana P, Whiting B, Goodison S, Rosser CJ. Urine-based assays for the detection of bladder cancer. Biomark Med. 2009; 3: 265. doi: 10.2217/bmm.09.23.
10. Huber S, Schwentner C, Taeger D, Pesch B, Nasterlack M, Leng G, Mayer T, Gawrych K, Bonberg N, Pelster M, Johnen G, Bontrup H, Wellhausser H, et al. Nuclear matrix protein-22: a prospective evaluation in a population at risk for bladder cancer. Results from the UroScreen study. BJU Int. 2012; 110: 699-708. doi: 10.1111/j.1464-410X.2011.10883.x.
11. Kinders R, Jones T, Root R, Bruce C, Murchison H, Corey M, Williams L, Enfield D, Hass GM. Complement factor H or a related protein is a marker for transitional cell cancer of the bladder. Clin Cancer Res. 1998; 4: 2511-20. doi:
12. Quek ML, Sanderson K, Daneshmand S, Stein JP. New molecular markers for bladder cancer detection. Curr Opin Urol. 2004; 14: 259-64. doi:
13. Leyh H, Marberger M, Conort P, Sternberg C, Pansadoro V, Pagano F, Bassi P, Boccon-Gibod L, Ravery V, Treiber U, Ishak L. Comparison of the BTA stat test with voided urine cytology and bladder wash cytology in the diagnosis and monitoring of bladder cancer. Eur Urol. 1999; 35: 52-6. doi: 19819.
14. Oge O, Kozaci D, Gemalmaz H. The BTA stat test is nonspecific for hematuria: an experimental hematuria model. J Urol. 2002; 167: 1318-9; discussion 9-20. doi:
15. Thomas L, Leyh H, Marberger M, Bombardieri E, Bassi P, Pagano F, Pansadoro V, Sternberg CN, Boccon-Gibod L, Ravery V, Le Guludec D, Meulemans A, Conort P, et al. Multicenter trial of the quantitative BTA TRAK assay in the detection of bladder cancer. Clin Chem. 1999; 45: 472-7. doi:
16. Chen CL, Chung T, Wu CC, Ng KF, Yu JS, Tsai CH, Chang YS, Liang Y, Tsui KH, Chen YT. Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer. Mol Cell Proteomics. 2015; 14: 2466-78. doi: 10.1074/mcp.M115.051524.
17. Theodorescu D, Wittke S, Ross MM, Walden M, Conaway M, Just I, Mischak H, Frierson HF. Discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis. Lancet Oncol. 2006; 7: 230-40. doi: 10.1016/S1470-2045(06)70584-8.
18. Frantzi M, van Kessel KE, Zwarthoff EC, Marquez M, Rava M, Malats N, Merseburger AS, Katafigiotis I, Stravodimos K, Mullen W, Zoidakis J, Makridakis M, Pejchinovski M, et al. Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study. Clin Cancer Res. 2016; 22: 4077-86. doi: 10.1158/1078-0432.CCR-15-2715.
19. Chen YT, Chen CL, Chen HW, Chung T, Wu CC, Chen CD, Hsu CW, Chen MC, Tsui KH, Chang PL, Chang YS, Yu JS. Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology. J Proteome Res. 2010; 9: 5803-15. doi: 10.1021/pr100576x.
20. Lei T, Zhao X, Jin S, Meng Q, Zhou H, Zhang M. Discovery of potential bladder cancer biomarkers by comparative urine proteomics and analysis. Clin Genitourin Cancer. 2013; 11: 56-62. doi: 10.1016/j.clgc.2012.06.003.
21. Anderson NL. The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum. Clin Chem. 2010; 56: 177-85. doi: 10.1373/clinchem.2009.126706.
22. Rifai N, Gillette MA, Carr SA. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol. 2006; 24: 971-83. doi: 10.1038/nbt1235.
23. Issaq HJ, Veenstra TD. Would you prefer multiple reaction monitoring or antibodies with your biomarker validation? Expert Rev Proteomics. 2008; 5: 761-3. doi: 10.1586/14789450.5.6.761.
24. Makawita S, Diamandis EP. The bottleneck in the cancer biomarker pipeline and protein quantification through mass spectrometry-based approaches: current strategies for candidate verification. Clin Chem. 2010; 56: 212-22. doi: 10.1373/clinchem.2009.127019.
25. Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics. 2006; 5: 573-88. doi: 10.1074/mcp.M500331-MCP200.
26. Chen CL, Chung T, Wu CC, Ng KF, Yu JS, Tsai CH, Chang YS, Liang Y, Tsui KH, Chen YT. Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer. Mol Cell Proteomics. 2015. doi: 10.1074/mcp.M115.051524.
27. MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, Kern R, Tabb DL, Liebler DC, MacCoss MJ. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010; 26: 966-8. doi: 10.1093/bioinformatics/btq054.
28. Kessner D, Chambers M, Burke R, Agus D, Mallick P. ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics. 2008; 24: 2534-6. doi: 10.1093/bioinformatics/btn323.
29. Chang KP, Yu JS, Chien KY, Lee CW, Liang Y, Liao CT, Yen TC, Lee LY, Huang LL, Liu SC, Chang YS, Chi LM. Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology. J Proteome Res. 2011; 10: 4935-47. doi: 10.1021/pr200311p.
30. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011; 8: 785-6. doi: 10.1038/nmeth.1701.
31. Bendtsen JD, Jensen LJ, Blom N, Von Heijne G, Brunak S. Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Sel. 2004; 17: 349-56. doi: 10.1093/protein/gzh037.
32. Bendtsen JD, Kiemer L, Fausboll A, Brunak S. Non-classical protein secretion in bacteria. BMC Microbiol. 2005; 5: 58. doi: 10.1186/1471-2180-5-58.
33. Chen CL, Lai YF, Tang P, Chien KY, Yu JS, Tsai CH, Chen HW, Wu CC, Chung T, Hsu CW, Chen CD, Chang YS, Chang PL, et al. Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients. J Proteome Res. 2012; 11: 5611-29. doi: 10.1021/pr3008732.
34. Wu CC, Hsu CW, Chen CD, Yu CJ, Chang KP, Tai DI, Liu HP, Su WH, Chang YS, Yu JS. Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas. Mol Cell Proteomics. 2010; 9: 1100-17. doi: 10.1074/mcp.M900398-MCP200.
35. Getzenberg RH, Konety BR, Oeler TA, Quigley MM, Hakam A, Becich MJ, Bahnson RR. Bladder cancer-associated nuclear matrix proteins. Cancer Res. 1996; 56: 1690-4. doi:
36. Akaza H, Miyanaga N, Tsukamoto T, Ishikawa S, Noguchi R, Ohtani M, Kawabe K, Kubota Y, Fujita K, Obata K, Hirao Y, Kotake T, Ohmori H, et al. [Evaluation of urinary NMP22 (nuclear matrix protein 22) as a diagnostic marker for urothelial cancer--screening for urothelial cancer in patients with microscopic hematuria. NMP Study Group]. Gan To Kagaku Ryoho. 1997; 24: 837-42. doi:
37. Raitanen MP, Marttila T, Nurmi M, Ala-Opas M, Nieminen P, Aine R, Tammela TL, Finnbladder G. Human complement factor H related protein test for monitoring bladder cancer. J Urol. 2001; 165: 374-7. doi: 10.1097/00005392-200102000-00005.
38. Sprung RW, Martinez MA, Carpenter KL, Ham AJ, Washington MK, Arteaga CL, Sanders ME, Liebler DC. Precision of multiple reaction monitoring mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue. J Proteome Res. 2012; 11: 3498-505. doi: 10.1021/pr300130t.
39. Unwin RD, Griffiths JR, Leverentz MK, Grallert A, Hagan IM, Whetton AD. Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol Cell Proteomics. 2005; 4: 1134-44. doi: 10.1074/mcp.M500113-MCP200.
40. Lu CM, Lin JJ, Huang HH, Ko YC, Hsu JL, Chen JC, Din ZH, Wu YJ. A panel of tumor markers, calreticulin, annexin A2, and annexin A3 in upper tract urothelial carcinoma identified by proteomic and immunological analysis. BMC Cancer. 2014; 14: 363. doi: 10.1186/1471-2407-14-363.
41. Surinova S, Schiess R, Huttenhain R, Cerciello F, Wollscheid B, Aebersold R. On the development of plasma protein biomarkers. J Proteome Res. 2011; 10: 5-16. doi: 10.1021/pr1008515.
42. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc. 2009; 4: 484-94. doi: 10.1038/nprot.2009.21.
43. Hsu JL, Huang SY, Chow NH, Chen SH. Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem. 2003; 75: 6843-52. doi: 10.1021/ac0348625.
44. Yao Z, Li W, Lin Y, Wu Q, Yu F, Lin W, Lin X. Proteomic Analysis Reveals That Metabolic Flows Affect the Susceptibility of Aeromonas hydrophila to Antibiotics. Sci Rep. 2016; 6: 39413. doi: 10.1038/srep39413.
45. Hartl FU, Hayer-Hartl M. Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol. 2009; 16: 574-81. doi: 10.1038/nsmb.1591.
46. McClellan AJ, Tam S, Kaganovich D, Frydman J. Protein quality control: chaperones culling corrupt conformations. Nat Cell Biol. 2005; 7: 736-41. doi: 10.1038/ncb0805-736.
47. Doyle SM, Shorter J, Zolkiewski M, Hoskins JR, Lindquist S, Wickner S. Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat Struct Mol Biol. 2007; 14: 114-22. doi: 10.1038/nsmb1198.
48. Legname G, Fossati G, Gromo G, Monzini N, Marcucci F, Modena D. Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10. FEBS Lett. 1995; 361: 211-4. doi:
49. Monzini N, Legname G, Marcucci F, Gromo G, Modena D. Identification and cloning of human chaperonin 10 homologue. Biochim Biophys Acta. 1994; 1218: 478-80. doi:
50. Czarnecka AM, Campanella C, Zummo G, Cappello F. Heat shock protein 10 and signal transduction: a "capsula eburnea" of carcinogenesis? Cell Stress Chaperones. 2006; 11: 287-94. doi:
51. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrial chaperones in cancer: from molecular biology to clinical diagnostics. Cancer Biol Ther. 2006; 5: 714-20. doi:
52. Cappello F, David S, Rappa F, Bucchieri F, Marasa L, Bartolotta TE, Farina F, Zummo G. The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase. BMC Cancer. 2005; 5: 139. doi: 10.1186/1471-2407-5-139.
53. Shan YX, Liu TJ, Su HF, Samsamshariat A, Mestril R, Wang PH. Hsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells. J Mol Cell Cardiol. 2003; 35: 1135-43. doi:
54. Lin KM, Hollander JM, Kao VY, Lin B, Macpherson L, Dillmann WH. Myocyte protection by 10 kD heat shock protein (Hsp10) involves the mobile loop and attenuation of the Ras GTP-ase pathway. FASEB J. 2004; 18: 1004-6. doi: 10.1096/fj.03-0348fje.
55. Akyol S, Gercel-Taylor C, Reynolds LC, Taylor DD. HSP-10 in ovarian cancer: expression and suppression of T-cell signaling. Gynecol Oncol. 2006; 101: 481-6. doi: 10.1016/j.ygyno.2005.11.014.
56. Cappello F. HSP60 and HSP10 as diagnostic and prognostic tools in the management of exocervical carcinoma. Gynecol Oncol. 2003; 91: 661. doi:
57. Cappello F, Bellafiore M, David S, Anzalone R, Zummo G. Ten kilodalton heat shock protein (HSP10) is overexpressed during carcinogenesis of large bowel and uterine exocervix. Cancer Lett. 2003; 196: 35-41. doi:
58. Tetu B, Popa I, Bairati I, L'Esperance S, Bachvarova M, Plante M, Harel F, Bachvarov D. Immunohistochemical analysis of possible chemoresistance markers identified by micro-arrays on serous ovarian carcinomas. Mod Pathol. 2008; 21: 1002-10. doi: 10.1038/modpathol.2008.80.
59. Ghobrial IM, McCormick DJ, Kaufmann SH, Leontovich AA, Loegering DA, Dai NT, Krajnik KL, Stenson MJ, Melhem MF, Novak AJ, Ansell SM, Witzig TE. Proteomic analysis of mantle-cell lymphoma by protein microarray. Blood. 2005; 105: 3722-30. doi: 10.1182/blood-2004-10-3999.
60. Cavanagh AC, Morton H. The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10. Eur J Biochem. 1994; 222: 551-60. doi:
61. Takagi M, Nishimura K, Oguri N, Ohnuma K, Ito K, Takahashi J, Yasuda Y, Miyazawa K, Sato K. Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo. Theriogenology. 1998; 50: 255-62. doi:
62. Ghaffari Laleh V, Ghaffari Laleh R, Pirany N, Moghadaszadeh Ahrabi M. Measurement of EPF for detection of cow pregnancy using rosette inhibition test. Theriogenology. 2008; 70: 105-7. doi: 10.1016/j.theriogenology.2008.02.003.
63. Nancarrow CD, Evison BM, Scaramuzzi RJ, Turnbull KE. Detection of induced death of embryos in sheep by the rosette inhibition test. J Reprod Fertil. 1979; 57: 385-9. doi:
64. Anraku I, Rajasuriar R, Dobbin C, Brown R, Lewin SR, Suhrbier A. Circulating heat shock protein 60 levels are elevated in HIV patients and are reduced by anti-retroviral therapy. PLoS One. 2012; 7: e45291. doi: 10.1371/journal.pone.0045291.
65. Shamaei-Tousi A, D'Aiuto F, Nibali L, Steptoe A, Coates AR, Parkar M, Donos N, Henderson B. Differential regulation of circulating levels of molecular chaperones in patients undergoing treatment for periodontal disease. PLoS One. 2007; 2: e1198. doi: 10.1371/journal.pone.0001198.
66. Taipale M, Jarosz DF, Lindquist S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat Rev Mol Cell Biol. 2010; 11: 515-28. doi: 10.1038/nrm2918.
67. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer. 2005; 5: 761-72. doi: 10.1038/nrc1716.
68. Karapanagiotou EM, Syrigos K, Saif MW. Heat shock protein inhibitors and vaccines as new agents in cancer treatment. Expert Opin Investig Drugs. 2009; 18: 161-74. doi: 10.1517/13543780802715792.
69. Ciocca DR, Calderwood SK. Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones. 2005; 10: 86-103. doi:
70. Janik S, Schiefer AI, Bekos C, Hacker P, Haider T, Moser J, Klepetko W, Mullauer L, Ankersmit HJ, Moser B. HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications. Sci Rep. 2016; 6: 24267. doi: 10.1038/srep24267.
71. Behnsawy HM, Miyake H, Kusuda Y, Fujisawa M. Small interfering RNA targeting heat shock protein 70 enhances chemosensitivity in human bladder cancer cells. Urol Oncol. 2013; 31: 843-8. doi: 10.1016/j.urolonc.2011.07.007.
72. He LF, Guan KP, Ye HY, Ren L, Yan Z, Wang SW, Hou SK. Heat shock protein 70 expression in relation to apoptosis in primary bladder transitional cell carcinoma. Chin Med J (Engl). 2005; 118: 2093-6. doi:
73. Syrigos KN, Harrington KJ, Karayiannakis AJ, Sekara E, Chatziyianni E, Syrigou EI, Waxman J. Clinical significance of heat shock protein-70 expression in bladder cancer. Urology. 2003; 61: 677-80. doi:
74. Sheng KH, Yao YC, Chuang SS, Wu H, Wu TF. Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis. Proteomics. 2006; 6: 1058-65. doi: 10.1002/pmic.200402015.
75. Lee H, Choi SK, Ro JY. Overexpression of DJ-1 and HSP90alpha, and loss of PTEN associated with invasive urothelial carcinoma of urinary bladder: Possible prognostic markers. Oncol Lett. 2012; 3: 507-12. doi: 10.3892/ol.2011.522.
76. Cardillo MR, Sale P, Di Silverio F. Heat shock protein-90, IL-6 and IL-10 in bladder cancer. Anticancer Res. 2000; 20: 4579-83. doi:
77. Margel D, Pevsner-Fischer M, Baniel J, Yossepowitch O, Cohen IR. Stress proteins and cytokines are urinary biomarkers for diagnosis and staging of bladder cancer. Eur Urol. 2011; 59: 113-9. doi: 10.1016/j.eururo.2010.10.008.
78. Atsu N, Ekici S, Oge OO, Ergen A, Hascelik G, Ozen H. False-positive results of the NMP22 test due to hematuria. J Urol. 2002; 167: 555-8. doi:
79. Miyake M, Goodison S, Rizwani W, Ross S, Bart Grossman H, Rosser CJ. Urinary BTA: indicator of bladder cancer or of hematuria. World J Urol. 2012; 30: 869-73. doi: 10.1007/s00345-012-0935-9.
80. Todenhofer T, Hennenlotter J, Tews V, Gakis G, Aufderklamm S, Kuehs U, Stenzl A, Schwentner C. Impact of different grades of microscopic hematuria on the performance of urine-based markers for the detection of urothelial carcinoma. Urol Oncol. 2013; 31: 1148-54. doi: 10.1016/j.urolonc.2011.10.011.