title: 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.
authors.affiliation: Department of Internal Medicine, University of Rome "Tor Vergata" Rome, Rome, Italy. mceric_rugan@hotmail.com
authors.forename: Murugan
authors.initials: M
authors.lastname: Kalimutho
authors.forename: Giovanna Del Vecchio
authors.initials: Gdel V
authors.lastname: Blanco
authors.forename: Paolo
authors.initials: P
authors.lastname: Gravina
authors.forename: Micaela
authors.initials: M
authors.lastname: Cretella
authors.forename: Liliana
authors.initials: L
authors.lastname: Mannucci
authors.forename: Elena
authors.initials: E
authors.lastname: Mannisi
authors.forename: Amanda
authors.initials: A
authors.lastname: Formosa
authors.forename: Francesco
authors.initials: F
authors.lastname: Pallone
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
authors.forename: Sergio
authors.initials: S
authors.lastname: Bernardini
mesh_terms: D000199:Actins / Q000235:genetics; D011125:Adenomatous Polyposis Coli / Q000150:complications* / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000150:complications*; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005240:Feasibility Studies; D005243:Feces; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D009691:Nucleic Acid Denaturation; D010865:Pilot Projects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D000199:Actins; D004247:DNA