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

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.