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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Online
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1437-4331
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Volume 48, Issue 9 (Sep 2010)

Issues

Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer

Murugan Kalimutho
  • Department of Internal Medicine, University of Rome “Tor Vergata” Rome, Rome, Italy
/ Giovanna Del Vecchio Blanco
  • Gastroenterology Unit, Policlinic of Tor Vergata, University of Rome Tor Vergata, Rome, Italy
/ Paolo Gravina
  • Department of Laboratory Medicine,“U.O.C. Clinical Molecular Biology and Biochemistry”, University Hospital Tor Vergata, Rome, Italy
/ Micaela Cretella
  • Gastroenterology Unit, Policlinic of Tor Vergata, University of Rome Tor Vergata, Rome, Italy
/ Liliana Mannucci
  • Department of Laboratory Medicine,“U.O.C. Clinical Molecular Biology and Biochemistry”, University Hospital Tor Vergata, Rome, Italy
/ Elena Mannisi
  • Gastroenterology Unit, Policlinic of Tor Vergata, University of Rome Tor Vergata, Rome, Italy
/ Amanda Formosa
  • Department of Internal Medicine, University of Rome “Tor Vergata” Rome, Rome, Italy
/ Francesco Pallone
  • Gastroenterology Unit, Policlinic of Tor Vergata, University of Rome Tor Vergata, Rome, Italy
/ Giorgio Federici
  • Department of Internal Medicine, University of Rome “Tor Vergata” Rome, Rome, Italy
  • Department of Laboratory Medicine,“U.O.C. Clinical Molecular Biology and Biochemistry”, University Hospital Tor Vergata, Rome, Italy
/ Sergio Bernardini
  • Department of Internal Medicine, University of Rome “Tor Vergata” Rome, Rome, Italy
  • Department of Laboratory Medicine,“U.O.C. Clinical Molecular Biology and Biochemistry”, University Hospital Tor Vergata, Rome, Italy
Published Online: 2010-05-27 | DOI: https://doi.org/10.1515/CCLM.2010.245

Abstract

Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC).

Methods: Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis.

Results: We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/β-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of β-actin was 26.12±13.39 ng/mL for healthy, and 49.61±46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC.

Conclusions: This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results.

Clin Chem Lab Med 2010;48:1303–11.

Keywords: adenomatosis polyposis coli (APC); colorectal cancer; denaturing HPLC; faecal DNA; faeces; quantitative

About the article

Corresponding author: Dr. Murugan Kalimutho, PhD, Department of Laboratory Medicine,“U.O.C. Clinical Molecular Biology and Biochemistry”, University Hospital Tor Vergata, Viale Oxford 81, Rome 00133, Italy Phone: +3906-20902259, Fax: +3906-20902357,


Received: 2009-12-10

Accepted: 2010-03-15

Published Online: 2010-05-27

Published in Print: 2010-09-01


Citation Information: Clinical Chemistry and Laboratory Medicine, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2010.245. Export Citation

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