This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giribaldi, G. Articles by Turrini, F. Search for Related Content PubMed PubMed Citation Articles by Giribaldi, G. Articles by Turrini, F.

JMD 2006, Vol. 8, No. 1
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

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

Giuliana Giribaldi^*, Simone Procida^*, Daniela Ulliers^*, Franca Mannu, Roberta Volpatto, Giorgia Mandili^*, Laura Fanchini, Oscar Bertetto, Gianruggero Fronda, Luigi Simula^¶, Elena Rimini^¶, Giovanni Cherchi^¶, Lisa Bonello^||, Milena Maria Maule^|| and Francesco Turrini^*

From the Università di Torino, Dipartimento di Genetica, * Biologia e Biochimica, Torino; Nurex S.r.l., Sassari; Centro Oncologico Ematologico Subalpino, Oncologia Medica, Torino; Ospedale San Giovanni Battista, Chirurgia Generale 7, Torino; Ospedale Santissima Annunziata, ^¶ Sassari; and the Center for Experimental Research and Medical Studies (CERMS), ^|| Torino, Italy

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.

This article has been cited by other articles:

				S. C. C. Wong, C. M. L. Chan, B. B. Y. Ma, E. P. Hui, S. S. M. Ng, P. B. S. Lai, M. T. Cheung, E. S. F. Lo, A. K. C. Chan, M. Y. Y. Lam, et al. Clinical Significance of Cytokeratin 20-Positive Circulating Tumor Cells Detected by a Refined Immunomagnetic Enrichment Assay in Colorectal Cancer Patients Clin. Cancer Res., February 1, 2009; 15(3): 1005 - 1012. [Abstract] [Full Text] [PDF]

				S. Hundt, U. Haug, and H. Brenner Blood Markers for Early Detection of Colorectal Cancer: A Systematic Review Cancer Epidemiol. Biomarkers Prev., October 1, 2007; 16(10): 1935 - 1953. [Abstract] [Full Text] [PDF]