Development of a Novel Tissue-Specific Method to Detect Cytokeratin 20-Positive Circulating Tumor Cells in Metastatic Colorectal Cancer

Sze Chuen Cesar Wong, Charles Chan Ming Lok, Chu So Shan


Abstract: Introduction: Although many studies have shown the vast potential of circulating tumor cells (CTCs) detection in cancer diagnosis and prognosis, our understanding of their clinical significance is still far from complete. A major obstacle arises from the lack of well-established tumor or tissue-specific markers to detect CTCs by immunocytochemical staining after immunomagnetic enrichment (IE). Methods: We have established the utility of cytokeratin 20 (CK20), a gastrointestinal tract specific marker, for the specific detection and identification of colorectal cancer (CRC) CTCs. This breakthrough was successfully validated in spike-in experiments using CRC cell line models followed by a pilot study which recruited 32 metastatic CRC patients, 25 benign colorectal diseases patients and 27 normal subjects. Results: CK20-positive CTCs were detected in 90% metastatic CRC patients but not in benign colorectal diseases patients and normal subjects using this refined assay. Conclusions: These impressive results have laid the foundation for further development of CK20-positive CTCs as a promising marker in diagnosis, prognostication and treatment monitoring of metastatic CRC.


Tissue-specific; Cytokeratin 20-positive; Circulating tumor cells; Metastatic; Colorectal cancer

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Elshimali YI, Grody WW. The clinical significance of circulating tumor cells in the peripheral blood. Diagn Mol Pathol 2006;15:187-194.

Paterlini-Brechot P, Benali NL. Circulating tumor cells(CTC) detection: Clinical impact and future directions.Cancer Lett 2007;253:180-204.

Goeminne JC, Guillaume T, Symann M. Pitfalls in the detection of disseminated non-hematological tumor cells.Ann Oncol 2000:11:785-792.

Mocellin S, Keilholz U, Rossi CR, Nitti D. Circulating tumor cells: the ‘leukemic phase’ of solid cancers. Trends Mol Med 2006;12:130-139.

Pantel K, Hille C, Scher HI. Circulating tumor cells in prostate cancer. Clin Chem 2019;65:87-99.

Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J,Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781-791.

Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A,Reuben JM, Doyle GV, Matera J, Allard WJ, Miller MC,Fritsche HA, Hortobagyi GN, Terstappen LW. Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005;23:1420-1430.

Cristofanilli M, Mendelsohn J. Circulating tumor cells in breast cancer: Advanced tools for “tailored” therapy? Proc

Natl Acad Sci USA 2006;103:17073-17074.

UICC. TNM classification of malignant tumours, 5th edition. New York: Wiley and Sons, 1997.

Moll R, Lowe A, Laufer J, Franke WW. Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 1992; 140:427-447.

Schuster R, Max N, Mann B, Heufelder K, Thilo F, Grone J, Rokos F, Buhr HJ, Thiel E, Keilholz U. Quantitative real-time RT-PCR for detection of disseminated tumor cells in peripheral blood of patients with colorectal cancer using different mRNA markers. Int J Cancer 2004;108:219-227.

Vlems FA, Diepstra JH, Punt CJ, Ligtenberg MJ, Cornelissen IM, van Krieken JH, Wobbes T, van Muijen GN,Ruers TJ. Detection of disseminated tumor cells in blood and bone marrow samples of patients undergoing hepatic resection for metastasis of colorectal cancer. Br J Surg 2003;90:989-995.

Hardingham JE, Hewett PJ, Sage RE, Finch JL, Nuttall JD, Kotasek D, Dobrovic A. Molecular detection of bloodborne epithelial cells in colorectal cancer patients and in patients with benign bowel disease. Int J Cancer 2000;89:8-13.

Dandachi N, Balic M, Stanzer S, Halm M, Resel M, Hinterleitner TA, Samonigg H, Bauernhofer T. Critical evaluation of real-time reverse transcriptase-polymerase chain reaction for the quantitative detection of cytokeratin 20 mRNA in colorectal cancer patients. J Mol Diagn 2005;7:631-637.

Ning Y, Hanna DL, Zhang W, Mendez A, Yang D,El-Khoueiry R, Matsusaka S, Sunakawa Y, Stremitzer S, Parekh A, Okazaki S, Berger MD, Barzi A, Lenz HJ.Cytokeratin-20 and survivin-expressing circulating tumor cells predict survival in metastatic colorectal cancer patients by a combined immunomagnetic qRT-PCR approach. Mol Cancer Ther 2015;14:2401-2408.

Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Tangen C. An evaluation of the carcinoembryonic antigen (CEA) test for monitoring patients with resected colon cancer. JAMA 1993;270:943-947.

Allegra CJ, Paik S, Colangelo LH, Parr AL, Kirsch I,Kim G, Klein P, Johnston PG, Wolmark N, Wieand HS.Prognostic value of thymidylate synthase, Ki-67, and p53 in patients with Dukes’B and C colon cancer: a national cancer institute-national surgical adjuvant breast and bowel project collaborative study. J Clin Oncol 2003:21:241-250.

Cascinu S, Staccioli MP, Gasparini G, Giordani P, Catalano V, Ghiselli R, Rossi C, Baldelli AM, Graziano F, Saba V,Muretto P, Catalano G. Expression of vascular endothelial growth factor can predict event-free survival in stage II colon cancer. Clin Cancer Res 2000;6:2803-2807.

Watanabe T, Wu TT, Catalano PJ, Ueki T, Satriano R,Haller DG, Benson AB 3rd, Hamilton SR. Molecular predictors of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 2001;344:1196-1206.

Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, Hamilton SR, Laurent-Puig P, Gryfe R,Shepherd LE, Tu D, Redston M, Gallinger S. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer.N Engl J Med 2003;349:247-257.

Katsumata K, Sumi T, Mori Y, Hisada M, Tsuchida A, AokiT. Detection and evaluation of epithelial cells in the blood of colon cancer patients using RT-PCR. Int J Clin Oncol 2006;11:385-389.

Giribaldi G, Procida S, Ulliers D, Mannu F, Volpatto R,Mandili G, Fanchini L, Bertetto O, Fronda G, Simula L,Rimini E, Cherchi G, Bonello L, Maule MM, Turrini F.Specific detection of cytokeratin 20-positive cells in blood of colorectal and breast cancer patients by a high sensitivity real-time reverse transcriptase-polymerase chain reaction method. J Mol Diagn 2006;8:105-112.

Riethdorf S, Fritsche H, Muller V, Rau T, Schindlbeck C,Rack B, Janni W, Coith C, Beck K, Janicke F, Jackson S,Gornet T, Cristofanilli M, Pantel K. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res 2007;13:920-928.

Wong CS, Cheung MT, Ma BB, Hui EP, Chan AC, Chan CK, Lee KC, Cheuk W, Lam MY, Wong MC, Chan CM,Chan JK, Chan AT. Presence of cytokeratin 20 positive cells in pN0 lymph nodes and circulating cytokeratin 20 mRNA in pN0 colorectal cancer patients. Int J Surg Pathol


Wong SC, Ng SS, Cheung MT, Luk LY, Chan CM, Lai PB, Ma BB, Hui EP, Lam MY, Au TC, Chan AT. Clinical significance of CDX2-positive circulating tumour cells in colorectal cancer patients. Brit J of Cancer 2011;104:1000-1006.

Wong SC, Chan CM, Ma BB, Hui EP, Ng SS, Lai PB,Cheung MT, Lo ES, Chan AK, Au TC, Lam MY, Chan AT.Clinical significance of cytokeratin 20-positive circulating tumor cells detected by a refined immunomagnetic enrichment assay in colorectal cancer patients. Clin Cancer Res 2009;15:1005-1012.

Wong SC, Lo ES, Lee KC, Chan JK, Hsiao WL. Prognostic and diagnostic significance of β-catenin nuclear immunostaining in colorectal cancer. Clin Cancer Res 2004;10:1401-1408.



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