Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer.
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.
Kalimutho
Murugan
M
Department of Internal Medicine, University of Rome "Tor Vergata" Rome, Rome, Italy. mceric_rugan@hotmail.com
Blanco
Giovanna Del Vecchio
Gdel V
Gravina
Paolo
P
Cretella
Micaela
M
Mannucci
Liliana
L
Mannisi
Elena
E
Formosa
Amanda
A
Pallone
Francesco
F
Federici
Giorgio
G
Bernardini
Sergio
S
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
D000199:Actins; D004247:DNA