Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer?

Journal of Translational Medicine

Carolin Lackner⁴  Peter Sedlmayr⁶  Berthold Huppertz⁶  Jochen Bernd Geigl¹  Rudolf Stauber²  Regina Riedl⁵  Hannelore Pock²  Richard Zigeuner³  Karin Kampel-Kettner³  Katja Fischereder³  Johannes Haybaeck⁴  Evelyn Zöhrer⁶  Thomas Kroneis⁶  Amin El-Heliebi⁶ 

[1] Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/VIII, Graz, 8010, Austria;Department of Internal Medicine, Division of Gastroenterology and Hepatology, Medical University of Graz, Auenbruggerplatz 15, Graz, 8036, Austria;Department of Urology, Medical University of Graz, Auenbruggerplatz 5/6, Graz, 8036, Austria;Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, Graz, 8036, Austria;Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz, Auenbruggerplatz 2, Graz, 8036, Austria;Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Harrachgasse 21/VII, Graz, 8010, Austria

关键词: Circulating endothelial cells;    Array comparative genomic hybridization (array-CGH);    ScreenCell®;    Renal cancer;    Circulating tumor microemboli;    Circulating tumor cells;   

Others  :  826543
DOI  :  10.1186/1479-5876-11-214

 received in 2013-05-24, accepted in 2013-09-13,  发布年份 2013

【 摘 要 】

Background

Single circulating tumor cells (CTCs) or circulating tumor microemboli (CTMs) are potential biomarkers of renal cell cancer (RCC), however studies of CTCs/CTMs in RCC are limited. In this pilot study we aimed to evaluate a novel blood filtration technique suited for cytomorphological classification, immunocytochemical and molecular characterization of filtered, so called circulating non-hematologic cells (CNHCs) - putative CTCs/CTMs - in patients with RCC.

Methods

Blood of 40 patients with renal tumors was subjected to ScreenCell® filtration. CNHCs were classified according to cytomorphological criteria. Immunocytochemical analysis was performed with antibodies against CD45, CD31 and carbonic anhydrase IX (CAIX, a RCC marker). DNA of selected CNHCs and respective primary tumors was analysed by array-CGH.

Results

CNHC-clusters with malignant or uncertain malignant cytomorphological features - putative CTMs - were negative for CD45, positive for CD31, while only 6% were CAIX positive. Array-CGH revealed that 83% of malignant and uncertain malignant cells did represent with a balanced genome whereas 17% presented genomic DNA imbalances which did not match the aberrations of the primary tumors. Putative single CTCs were negative for CD45, 33% were positive for CD31 and 56% were positive for CAIX.

Conclusions

The majority of CNHC-clusters, putative CTMs, retrieved by ScreenCell® filtration may be of endothelial origin. Morphological criteria seem to be insufficient to distinguish malignant from non-malignant cells in renal cancer.

【 授权许可】

   
2013 El-Heliebi et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Paterlini-Brechot P, Benali NL: Circulating tumor cells (CTC) detection: clinical impact and future directions. Cancer Lett 2007, 253:180-204.
• [2]Zhao S, Liu Y, Zhang Q, Li H, Zhang M, Ma W, Zhao W, Wang J, Yang M: The prognostic role of circulating tumor cells (CTCs) detected by RT-PCR in breast cancer: a meta-analysis of published literature. Breast Cancer Res Treat 2011, 130:809-816.
• [3]Rahbari NN, Aigner M, Thorlund K, Mollberg N, Motschall E, Jensen K, Diener MK, Buchler MW, Koch M, Weitz J: Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer. Gastroenterology 2010, 138:1714-1726.
• [4]De Albuquerque A, Kubisch I, Stolzel U, Ernst D, Boese-Landgraf J, Breier G, Stamminger G, Fersis N, Kaul S: Prognostic and predictive value of circulating tumor cell analysis in colorectal cancer patients. J Transl Med 2012, 10:222-5876-10-222.
• [5]Pichler M, Hutterer GC, Chromecki TF, Jesche J, Kampel-Kettner K, Rehak P, Pummer K, Zigeuner R: External validation of the Leibovich prognosis score for nonmetastatic clear cell renal cell carcinoma at a single European center applying routine pathology. J Urol 2011, 186:1773-1777.
• [6]Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Staehler M, Negrier S, Chevreau C, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Anderson S, Hofilena G, Shan M, Pena C, Lathia C, Bukowski RM: Sorafenib for treatment of renal cell carcinoma: final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. J Clin Oncol 2009, 27:3312-3318.
• [7]Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Oudard S, Negrier S, Szczylik C, Pili R, Bjarnason GA, Garcia-del-Muro X, Sosman JA, Solska E, Wilding G, Thompson JA, Kim ST, Chen I, Huang X, Figlin RA: Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 2009, 27:3584-3590.
• [8]Small AC, Gong Y, Oh WK, Hall SJ, van Rijn CJ, Galsky MD: The emerging role of circulating tumor cell detection in genitourinary cancer. J Urol 2012, 188:21-26.
• [9]Parkinson DR, Dracopoli N, Petty BG, Compton C, Cristofanilli M, Deisseroth A, Hayes DF, Kapke G, Kumar P, Lee JS, Liu MC, McCormack R, Mikulski S, Nagahara L, Pantel K, Pearson-White S, Punnoose EA, Roadcap LT, Schade AE, Scher HI, Sigman CC, Kelloff GJ: Considerations in the development of circulating tumor cell technology for clinical use. J Transl Med 2012, 10:138-5876-10-138.
• [10]Christiansen JJ, Rajasekaran AK: Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res 2006, 66:8319-8326.
• [11]Krebs MG, Hou JM, Sloane R, Lancashire L, Priest L, Nonaka D, Ward TH, Backen A, Clack G, Hughes A, Ranson M, Blackhall FH, Dive C: Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches. J Thorac Oncol 2012, 7:306-315.
• [12]Raimondi C, Gradilone A, Naso G, Vincenzi B, Petracca A, Nicolazzo C, Palazzo A, Saltarelli R, Spremberg F, Cortesi E, Gazzaniga P: Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients. Breast Cancer Res Treat 2011, 130:449-455.
• [13]Joosse SA, Pantel K: Biologic challenges in the detection of circulating tumor cells. Cancer Res 2013, 73:8-11.
• [14]Zheng S, Lin HK, Lu B, Williams A, Datar R, Cote RJ, Tai YC: 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices 2011, 13:203-213.
• [15]Desitter I, Guerrouahen BS, Benali-Furet N, Wechsler J, Janne PA, Kuang Y, Yanagita M, Wang L, Berkowitz JA, Distel RJ, Cayre YE: A new device for rapid isolation by size and characterization of rare circulating tumor cells. Anticancer Res 2011, 31:427-441.
• [16]Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schutze K, Capron F, Franco D, Pazzagli M, Vekemans M, Lacour B, Brechot C, Paterlini-Brechot P: Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells. Am J Pathol 2000, 156:57-63.
• [17]Hofman VJ, Ilie MI, Bonnetaud C, Selva E, Long E, Molina T, Vignaud JM, Flejou JF, Lantuejoul S, Piaton E, Butori C, Mourad N, Poudenx M, Bahadoran P, Sibon S, Guevara N, Santini J, Venissac N, Mouroux J, Vielh P, Hofman PM: Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls. Am J Clin Pathol 2011, 135:146-156.
• [18]Hofman V, Bonnetaud C, Ilie MI, Vielh P, Vignaud JM, Flejou JF, Lantuejoul S, Piaton E, Mourad N, Butori C, Selva E, Poudenx M, Sibon S, Kelhef S, Venissac N, Jais JP, Mouroux J, Molina TJ, Hofman P: Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker. Clin Cancer Res 2011, 17:827-835.
• [19]Hou JM, Krebs MG, Lancashire L, Sloane R, Backen A, Swain RK, Priest LJ, Greystoke A, Zhou C, Morris K, Ward T, Blackhall FH, Dive C: Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J Clin Oncol 2012, 30:525-532.
• [20]Khoja L, Backen A, Sloane R, Menasce L, Ryder D, Krebs M, Board R, Clack G, Hughes A, Blackhall F, Valle JW, Dive C: A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker. Br J Cancer 2012, 106:508-516.
• [21]Pinzani P, Salvadori B, Simi L, Bianchi S, Distante V, Cataliotti L, Pazzagli M, Orlando C: Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection. Hum Pathol 2006, 37:711-718.
• [22]Vona G, Estepa L, Beroud C, Damotte D, Capron F, Nalpas B, Mineur A, Franco D, Lacour B, Pol S, Brechot C, Paterlini-Brechot P: Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer. Hepatology 2004, 39:792-797.
• [23]Pailler E, Adam J, Barthelemy A, Oulhen M, Auger N, Valent A, Borget I, Planchard D, Taylor M, Andre F, Soria JC, Vielh P, Besse B, Farace F: Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive Non-small-cell lung cancer. J Clin Oncol 2013, 31:2273-2281.
• [24]Sobin LH, Gospodarowicz MK, Wittekind C: TNM Classification of Malignant Tumours (Uicc International Union Against Cancer). 7th edition. Hoboken, NJ, USA: John Wiley & Sons; 2009.
• [25]van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FB, Verhoef S, Nederlof PM: A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer 2006, 94:333-337.
• [26]Liu L, Qian J, Singh H, Meiers I, Zhou X, Bostwick DG: Immunohistochemical analysis of chromophobe renal cell carcinoma, renal oncocytoma, and clear cell carcinoma: an optimal and practical panel for differential diagnosis. Arch Pathol Lab Med 2007, 131:1290-1297.
• [27]Gupta R, Balzer B, Picken M, Osunkoya AO, Shet T, Alsabeh R, Luthringer D, Paner GP, Amin MB: Diagnostic implications of transcription factor Pax 2 protein and transmembrane enzyme complex carbonic anhydrase IX immunoreactivity in adult renal epithelial neoplasms. Am J Surg Pathol 2009, 33:241-247.
• [28]Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, Merrill MJ, Proescholdt MA, Oldfield EH, Lee J, Zavada J, Waheed A, Sly W, Lerman MI, Stanbridge EJ: Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol 2001, 158:905-919.
• [29]Geigl JB, Speicher MR: Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis. Nat Protoc 2007, 2:3173-3184.
• [30]Fiegler H, Carr P, Douglas EJ, Burford DC, Hunt S, Scott CE, Smith J, Vetrie D, Gorman P, Tomlinson IP, Carter NP: DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 2003, 36:361-374.
• [31]Klatte T, Rao PN, de Martino M, LaRochelle J, Shuch B, Zomorodian N, Said J, Kabbinavar FF, Belldegrun AS, Pantuck AJ: Cytogenetic profile predicts prognosis of patients with clear cell renal cell carcinoma. J Clin Oncol 2009, 27:746-753.
• [32]Brunelli M, Eble JN, Zhang S, Martignoni G, Cheng L: Gains of chromosomes 7, 17, 12, 16, and 20 and loss of Y occur early in the evolution of papillary renal cell neoplasia: a fluorescent in situ hybridization study. Mod Pathol 2003, 16:1053-1059.
• [33]Presti JC Jr, Moch H, Reuter VE, Huynh D, Waldman FM: Comparative genomic hybridization for genetic analysis of renal oncocytomas. Genes Chromosomes Cancer 1996, 17:199-204.
• [34]Baudis M, Cleary ML: Progenetix.net: an online repository for molecular cytogenetic aberration data. Bioinformatics 2001, 17:1228-1229.
• [35]Thomas ML: The leukocyte common antigen family. Annu Rev Immunol 1989, 7:339-369.
• [36]Dorai T, Sawczuk IS, Pastorek J, Wiernik PH, Dutcher JP: The role of carbonic anhydrase IX overexpression in kidney cancer. Eur J Cancer 2005, 41:2935-2947.
• [37]Grabmaier K, A de Weijert MC, Verhaegh GW, Schalken JA, Oosterwijk E: Strict regulation of CAIX(G250/MN) by HIF-1alpha in clear cell renal cell carcinoma. Oncogene 2004, 23:5624-5631.
• [38]Parums DV, Cordell JL, Micklem K, Heryet AR, Gatter KC, Mason DY: JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol 1990, 43:752-757.
• [39]Mathiesen RR, Fjelldal R, Liestol K, Due EU, Geigl JB, Riethdorf S, Borgen E, Rye IH, Schneider IJ, Obenauf AC, Mauermann O, Nilsen G, Christian Lingjaerde O, Borresen-Dale AL, Pantel K, Speicher MR, Naume B, Baumbusch LO: High-resolution analyses of copy number changes in disseminated tumor cells of patients with breast cancer. Int J Cancer 2012, 131:E405-E415.
• [40]Hou Y, Song L, Zhu P, Zhang B, Tao Y, Xu X, Li F, Wu K, Liang J, Shao D, Wu H, Ye X, Ye C, Wu R, Jian M, Chen Y, Xie W, Zhang R, Chen L, Liu X, Yao X, Zheng H, Yu C, Li Q, Gong Z, Mao M, Yang X, Yang L, Li J, Wang W, et al.: Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell 2012, 148:873-885.
• [41]Hu DG, Webb G, Hussey N: Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol Hum Reprod 2004, 10:283-289.
• [42]Fuhrmann C, Schmidt-Kittler O, Stoecklein NH, Petat-Dutter K, Vay C, Bockler K, Reinhardt R, Ragg T, Klein CA: High-resolution array comparative genomic hybridization of single micrometastatic tumor cells. Nucleic Acids Res 2008, 36:e39.
• [43]Geigl JB, Obenauf AC, Waldispuehl-Geigl J, Hoffmann EM, Auer M, Hormann M, Fischer M, Trajanoski Z, Schenk MA, Baumbusch LO, Speicher MR: Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays. Nucleic Acids Res 2009, 37:e105.
• [44]Fiegler H, Geigl JB, Langer S, Rigler D, Porter K, Unger K, Carter NP, Speicher MR: High resolution array-CGH analysis of single cells. Nucleic Acids Res 2007, 35:e15.
• [45]Pugh TJ, Delaney AD, Farnoud N, Flibotte S, Griffith M, Li HI, Qian H, Farinha P, Gascoyne RD, Marra MA: Impact of whole genome amplification on analysis of copy number variants. Nucleic Acids Res 2008, 36:e80.
• [46]Molnar B, Ladanyi A, Tanko L, Sreter L, Tulassay Z: Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients. Clin Cancer Res 2001, 7:4080-4085.
• [47]Stott SL, Hsu CH, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, Rothenberg SM, Shah AM, Smas ME, Korir GK, Floyd FP Jr, Gilman AJ, Lord JB, Winokur D, Springer S, Irimia D, Nagrath S, Sequist LV, Lee RJ, Isselbacher KJ, Maheswaran S, Haber DA, Toner M: Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A 2010, 107:18392-18397.
• [48]Farace F, Massard C, Vimond N, Drusch F, Jacques N, Billiot F, Laplanche A, Chauchereau A, Lacroix L, Planchard D, Le Moulec S, Andre F, Fizazi K, Soria JC, Vielh P: A direct comparison of Cell Search and ISET for circulating tumour-cell detection in patients with metastatic carcinomas. Br J Cancer 2011, 105:847-853.
• [49]De Giorgi V, Pinzani P, Salvianti F, Panelos J, Paglierani M, Janowska A, Grazzini M, Wechsler J, Orlando C, Santucci M, Lotti T, Pazzagli M, Massi D: Application of a filtration- and isolation-by-size technique for the detection of circulating tumor cells in cutaneous melanoma. J Invest Dermatol 2010, 130:2440-2447.
• [50]Pinzani P, Scatena C, Salvianti F, Corsini E, Canu L, Poli G, Paglierani M, Piccini V, Pazzagli M, Nesi G, Mannelli M, Luconi M: Detection of circulating tumor cells in patients with adrenocortical carcinoma: a monocentric preliminary study. J Clin Endocrinol Metab 2013, 98:3731-3738.
• [51]Adebayo Awe J, Xu MC, Wechsler J, Benali-Furet N, Cayre YE, Saranchuk J, Drachenberg D, Mai S: Three-dimensional telomeric analysis of isolated circulating tumor cells (CTCs) defines CTC subpopulations. Transl Oncol 2013, 6:51-65.
• [52]Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012, 366:883-892.
• [53]Duda DG, Duyverman AM, Kohno M, Snuderl M, Steller EJ, Fukumura D, Jain RK: Malignant cells facilitate lung metastasis by bringing their own soil. Proc Natl Acad Sci U S A 2010, 107:21677-21682.
• [54]Ballif BC, Rorem EA, Sundin K, Lincicum M, Gaskin S, Coppinger J, Kashork CD, Shaffer LG, Bejjani BA: Detection of low-level mosaicism by array CGH in routine diagnostic specimens. Am J Med Genet A 2006, 140:2757-2767.
• [55]Patel A, Kang SH, Lennon PA, Li YF, Rao PN, Abruzzo L, Shaw C, Chinault AC, Cheung SW: Validation of a targeted DNA microarray for the clinical evaluation of recurrent abnormalities in chronic lymphocytic leukemia. Am J Hematol 2008, 83:540-546.
• [56]Park Y, Kitahara T, Takagi R, Kato R: Does surgery for breast cancer induce angiogenesis and thus promote metastasis? Oncology 2011, 81:199-205.
• [57]Chang YS, di Tomaso E, McDonald DM, Jones R, Jain RK, Munn LL: Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc Natl Acad Sci U S A 2000, 97:14608-14613.
• [58]Mancuso P, Calleri A, Bertolini F: Circulating endothelial cells and circulating endothelial progenitors. Recent Results Cancer Res 2012, 195:163-170.
• [59]Ribatti D, Ranieri G, Basile A, Azzariti A, Paradiso A, Vacca A: Tumor endothelial markers as a target in cancer. Expert Opin Ther Targets 2012, 16:1215-1225.
• [60]Strijbos MH: Circulating tumour cells and circulating endothelial cells as biomarkers in oncology. Acta Clin Belg 2011, 66:332-336.
• [61]Shaked Y, Ciarrocchi A, Franco M, Lee CR, Man S, Cheung AM, Hicklin DJ, Chaplin D, Foster FS, Benezra R, Kerbel RS: Therapy-induced acute recruitment of circulating endothelial progenitor cells to tumors. Science 2006, 313:1785-1787.
• [62]Shaked Y, Henke E, Roodhart JM, Mancuso P, Langenberg MH, Colleoni M, Daenen LG, Man S, Xu P, Emmenegger U, Tang T, Zhu Z, Witte L, Strieter RM, Bertolini F, Voest EE, Benezra R, Kerbel RS: Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 2008, 14:263-273.
• [63]Strijbos MH, Gratama JW, Schmitz PI, Rao C, Onstenk W, Doyle GV, Miller MC, de Wit R, Terstappen LW, Sleijfer S: Circulating endothelial cells, circulating tumour cells, tissue factor, endothelin-1 and overall survival in prostate cancer patients treated with docetaxel. Eur J Cancer 2010, 46:2027-2035.
• [64]Matsusaka S, Suenaga M, Mishima Y, Takagi K, Terui Y, Mizunuma N, Hatake K: Circulating endothelial cells predict for response to bevacizumab-based chemotherapy in metastatic colorectal cancer. Cancer Chemother Pharmacol 2011, 68:763-768.
• [65]Gruenwald V, Beutel G, Schuch-Jantsch S, Reuter C, Ivanyi P, Ganser A, Haubitz M: Circulating endothelial cells are an early predictor in renal cell carcinoma for tumor response to sunitinib. BMC Cancer 2010, 10:695. BioMed Central Full Text
• [66]Mancuso A, Di Paola ED, Leone A, Catalano A, Calabro F, Cerbone L, Zivi A, Messina C, Alonso S, Vigna L, Caristo R, Sternberg CN: Phase II escalation study of sorafenib in patients with metastatic renal cell carcinoma who have been previously treated with anti-angiogenic treatment. BJU Int 2012, 109:200-206.
• [67]Vroling L, van der Veldt AA, de Haas RR, Haanen JB, Schuurhuis GJ, Kuik DJ, van Cruijsen H, Verheul HM, van den Eertwegh AJ, Hoekman K, Boven E, van Hinsbergh VW, Broxterman HJ: Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib. Angiogenesis 2009, 12:69-79.
• [68]Hernandez-Yanez M, Heymach JV, Zurita AJ: Circulating biomarkers in advanced renal cell carcinoma: clinical applications. Curr Oncol Rep 2012, 14:221-229.
• [69]Pantel K, Deneve E, Nocca D, Coffy A, Vendrell JP, Maudelonde T, Riethdorf S, Alix-Panabieres C: Circulating epithelial cells in patients with benign colon diseases. Clin Chem 2012, 58:936-940.