Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 28, 2017; 23(16): 2891-2898
Published online Apr 28, 2017. doi: 10.3748/wjg.v23.i16.2891
Droplet digital PCR for quantification of ITGA6 in a stool mRNA assay for the detection of colorectal cancers
Elizabeth Herring, Shigeru Kanaoka, Éric Tremblay, Jean-François Beaulieu
Elizabeth Herring, Éric Tremblay, Jean-François Beaulieu, Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC J1H5N4, Canada
Shigeru Kanaoka, Department of Gastroenterology, Hamamatsu Medical Center, Naka-ku, Hamamatsu 432-8580, Japan
Author contributions: Herring E and Kanaoka S collected, prepared and analyzed the material; Herring E, Tremblay É and Beaulieu JF processed the data; Tremblay É validated the biostatistics; Herring E and Beaulieu JF wrote the manuscript.
Supported by the Canadian Institutes of Health Research, No. PPP133373.
Institutional review board statement: The use of human material for this study was approved by the Institutional Research Ethics Committee of the Research Center of the Centre Hospitalier Universitaire de Sherbrooke.
Conflict-of-interest statement: Herring E and Beaulieu JF are among inventors of a patent application related to integrin α6 (Proliferation-associated modulation of the splicing of the integrin alpha 6 isoforms, #21150276743, US, Kind code: A1). The other authors have no conflict of interest to declare.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at jean-francois.beaulieu@usherbrooke.ca. Individual participant consent was not obtained for data sharing but the presented data are anonymized and there is no possibility of identification.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Jean-François Beaulieu, PhD, Professor, Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec J1H5N4, Canada. jean-francois.beaulieu@usherbrooke.ca
Telephone: +1-819-8218000 Fax: +1-819-5645320
Received: December 23, 2016
Peer-review started: December 28, 2016
First decision: January 19, 2017
Revised: February 14, 2017
Accepted: March 30, 2017
Article in press: March 30, 2017
Published online: April 28, 2017
Abstract
AIM

To investigate the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening.

METHODS

ddPCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6A transcripts in stool samples obtained from patients with various types of colorectal lesions (advanced adenomas and stage II-IV colorectal cancers) and control (patients displaying no pathological findings) using duplex TaqMan reactions for both methods. ITGA6 and ITGA6A were chosen for this proof-of-concept study based on their relative medium and low abundance in stool samples, respectively, as established in a previous study.

RESULTS

We found that the ddPCR and qPCR methods performed equally well in this TaqMan duplex assay for the detection of ITGA6 and ITGA6A transcripts in stools of patients with colorectal lesions. For ITGA6, receiver operating characteristic (ROC) curve analysis showed comparable areas under the curve of 0.91 (P < 0.0001) and 0.89-0.90 (P < 0.0001) for the prediction of advanced adenomas and colorectal cancers, respectively. ITGA6A, which was detected at very low levels in control patients, was found to be significantly elevated (over 40 times) in stage II and III colorectal cancers (P < 0.0002). Comparison of the two sets of data revealed a strong correlation of the copy numbers obtained by ddPCR and qPCR for both ITGA6 and ITGA6A.

CONCLUSION

We found that ITGA6 and ITGA6A detection in stools of patients with colorectal cancers with ddPCR is comparable to that of qPCR using TaqMan assays.

Keywords: Colorectal cancer, Advanced adenoma, Non-invasive screening, Biomarker, mRNA

Core tip: We investigated the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening using ITGA6 and ITGA6A as established medium and low abundance stool biomarkers. Our side by side comparison of ddPCR and qPCR on stool samples obtained from patients with advanced adenomas and colorectal cancers and controls using duplex TaqMan reactions revealed that both methods performed equally well.