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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

Editorial Board Member: Gillery, Philippe / Kazmierczak, Steven / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Whitfield, John B.

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

Murugan Kalimutho1 / Giovanna Del Vecchio Blanco3 / Paolo Gravina2 / Micaela Cretella3 / Liliana Mannucci2 / Elena Mannisi3 / Amanda Formosa1 / Francesco Pallone3 / Giorgio Federici1, 2 / Sergio Bernardini1, 2

1Department of Internal Medicine, University of Rome “Tor Vergata” Rome, Rome, Italy

2Department of Laboratory Medicine,“U.O.C. Clinical Molecular Biology and Biochemistry”, University Hospital Tor Vergata, Rome, Italy

3Gastroenterology Unit, Policlinic of Tor Vergata, University of Rome Tor Vergata, Rome, Italy

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,

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 9, Pages 1303–1311, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.245, May 2010

Publication History

Received:
2009-12-10
Accepted:
2010-03-15
Published Online:
2010-05-27

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

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