Copyright
©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR
Susana Olmedillas-López, Dennis César Lévano-Linares, Carmen Laura Aúz Alexandre, Luz Vega-Clemente, Edurne León Sánchez, Alejandro Villagrasa, Jaime Ruíz-Tovar, Mariano García-Arranz, Damián García-Olmo
Susana Olmedillas-López, Luz Vega-Clemente, Alejandro Villagrasa, Mariano García-Arranz, Damián García-Olmo, Foundation Health Research Institute-Fundación Jiménez Díaz University Hospital, Madrid 28040, Spain
Dennis César Lévano-Linares, Mariano García-Arranz, Damián García-Olmo, Department of Surgery, School of Medicine, Universidad Autónoma de Madrid, Madrid 28029, Spain
Dennis César Lévano-Linares, Jaime Ruíz-Tovar, Department of Surgery, Rey Juan Carlos University Hospital, Madrid 28933, Spain
Carmen Laura Aúz Alexandre, Department of Pathology, Fundación Jiménez Díaz University Hospital, Madrid 28040, Spain
Edurne León Sánchez, Department of Biomedicine and Biotechnology, Universidad de Alcalá, Madrid 28805, Spain
Damián García-Olmo, Department of Surgery, Fundación Jiménez Díaz University Hospital, Madrid 28040, Spain
Author contributions: Olmedillas-López S and Lévano-Linares DC contributed equally to this work; Olmedillas-López S, Lévano-Linares DC, García-Arranz M and García-Olmo D conceived and designed the experiments; Olmedillas-López S, Aúz Alexandre CL, Vega-Clemente L, León Sánchez E and Villagrasa A performed the experiments; Olmedillas-López S, Lévano-Linares DC, Ruíz-Tovar J, García-Arranz M and García-Olmo D analyzed the data; Olmedillas-López S and Lévano-Linares DC wrote the paper.
Supported by “Fondo de Investigaciones Sanitarias (FIS)-FEDER”, Ministry of Health, Spain, No. PI13/01924 to García-Olmo D; and RETIC Program of ISCIII-FEDER, No. RD12/0019/0035 to Olmedillas-López S.
Institutional review board statement: This study was reviewed and approved by the Institutional Ethics Committee for Clinical Research of the Fundación Jiménez Díaz University Hospital (FJD) (PIC 63/2016_FJD).
Conflict-of-interest statement: The authors have no conflict of interest to declare.
Data sharing statement: Individual participant consent was not obtained for data sharing but the presented data are anonymized and there is no possibility of identification. No additional data are available.
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: Susana Olmedillas-López, PhD, Postdoc Researcher, Foundation Health Research Institute-Fundación Jiménez Díaz University Hospital, Avda. Reyes Católicos 2, Madrid 28040, Spain.
susana.olmedillas@fjd.es
Telephone: +34-91-5504800-2781 Fax: +34-91-5505353
Received: June 28, 2017
Peer-review started: June 28, 2017
First decision: August 15, 2017
Revised: September 15, 2017
Accepted: September 26, 2017
Article in press: September 26, 2017
Published online: October 21, 2017
Processing time: 115 Days and 21.2 Hours
AIM
To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients.
METHODS
In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.
RESULTS
Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation.
CONCLUSION
ddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.
Core tip: The potential of droplet digital PCR (ddPCR) to detect KRAS G12D mutation in stool DNA from colorectal cancer (CRC) patients was examined as a proof-of-concept for the applicability of this technology to study DNA biomarkers in stool-derived DNA. It was shown that KRAS G12D detection in stool-derived DNA from CRC patients by ddPCR is feasible and provides comparable results to the analysis of formalin-fixed paraffin-embedded tissue by pyrosequencing. These results suggest that analysis of KRAS mutations and other molecular biomarkers in stool by ddPCR could represent a complementary non-invasive approach to standard screening tests for CRC.
