Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 21, 2017; 23(47): 8291-8299
Published online Dec 21, 2017. doi: 10.3748/wjg.v23.i47.8291
Gene mutations in stool from gastric and colorectal neoplasia patients by next-generation sequencing
Omar Youssef, Virinder Sarhadi, Homa Ehsan, Tom Böhling, Monika Carpelan-Holmström, Selja Koskensalo, Pauli Puolakkainen, Arto Kokkola, Sakari Knuutila
Omar Youssef, Virinder Sarhadi, Homa Ehsan, Sakari Knuutila, Department of Pathology, Faculty of Medicine, University of Helsinki, Helsinki 00014, Finland
Tom Böhling, Department of Pathology, University of Helsinki and HUSLAB, Helsinki University Hospital, Helsinki 00014, Finland
Monika Carpelan-Holmström, Selja Koskensalo, Pauli Puolakkainen, Arto Kokkola, The HUCH Gastrointestinal Clinic, University Central Hospital of Helsinki, Helsinki 00290, Finland
Author contributions: Youssef O conducted sequencing of the samples and prepared the manuscript; Ehsan H prepared the samples for sequencing; Kokkola A, Carpelan-Holmström M, Koskensalo S and Puolakkainen P contributed clinical support for the participating subjects and arranged for stool sample collection; Sarhadi V, Böhling T and Knuutila S contributed to the study design and writing of the manuscript.
Institutional review board statement: The research study protocol was approved by the Hospital District of Helsinki and Uusimaa (HUS) Review Board (Ethical permission number 351/13/03/02/2014).
Conflict-of-interest statement: The authors declare no conflicts of interest.
Data sharing statement: Dataset available from sakari.knuutila@helsinki.fi
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: Sakari Knuutila, PhD, Professor, Department of Pathology, Faculty of Medicine, University of Helsinki, Haartmaninkatu 4, Helsinki 00014, Finland. sakari.knuutila@helsinki.fi
Telephone: +358-504482797 Fax: +358-294126700
Received: October 3, 2017
Peer-review started: October 5, 2017
First decision: October 18, 2017
Revised: November 1, 2017
Accepted: November 14, 2017
Article in press: November 14, 2017
Published online: December 21, 2017
Processing time: 77 Days and 16.8 Hours
ARTICLE HIGHLIGHTS
Research background

Stool DNA sample is a simple, noninvasive source for studying genetic markers of diagnostic/prognostic or predictive significance in colorectal cancer. The significance of stool DNA testing is, however, not well known for stomach cancers and for benign tumors. Current assays screen individual or few mutations only, and do not cover all important cancer mutations. Amplicon-based NGS could, thus, provide a sensitive method for DNA testing from stool samples in gastrointestinal (GIT) malignancies.

Research motivation

The main challenge in stool DNA-based genetic testing is that only a small proportion of stool DNA is of human origin, thus requiring a very sensitive test. We, therefore, hypothesized that diagnostic value of stool-based DNA testing could be enhanced by applying sensitive amplicon–based next-generation sequencing (NGS) to stool DNA. With the application of NGS we could screen all important mutations in 50 genes from a small amount of input DNA in a single test.

Research objectives

The objective of the study was to apply NGS for screening hotspot mutations in commonly mutated genes in GIT malignancies from stool DNA. The aim was also to see if mutations could be detected in patients with gastric cancer and in patients with early neoplasms, in addition to those with colorectal cancer.

Research methods

Mutation detection was performed by amplicon-based NGS using the Ion AmpliSeq Cancer Hotspot Panel v2 and Ion AmpliSeq Colon and Lung Cancer panel v2. Template preparation was done using the Ion OneTouch™ 2 System and sequencing was performed on Ion PGM (Thermo-Fisher Scientific). Sequencing data analysis and variant calling was performed by using the Torrent Suite Software v.5.2.2 with variant caller plugin. All single nucleotide variants were analyzed for previously reported hotspot mutations (reported in the COSMIC database) and novel variations, i.e. not reported in either COSMIC or dbSNP databases.

Research results

Hotspot mutations in stool DNA were found in APC, CDKN2A and EGFR in patients with stomach neoplasms and in AKT1, APC, ERBB2, FBXW7, KIT, KRAS, NRAS, SMARCB1, SMO, STK11 and TP53 in patients with colorectal neoplasia. APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions.

Research conclusions

This study demonstrates that NGS-based mutation screening can be successfully applied to stool DNA from patients with GIT neoplasms. In addition to mutation detection in stool DNA from colorectal cancer patients, mutations can also be detected from gastric cancer patients, as well as from patients with premalignant or benign neoplasms.

Mutation testing from stool DNA is mainly carried out for individual gene mutations by PCR-based methods for colorectal cancer screening. Since the amount of DNA of human origin is very low in stool, it was hypothesized that amplicon-based NGS could be highly sensitive and suitable for studying a large number of mutations, which could greatly enhance the diagnostic value of stool DNA testing. The methods used in this study require low input of DNA, can amplify around 200 targeted regions of important cancer genes, and together with the high sensitivity of NGS provide a great advantage over prevailing methods for mutation detection from stool DNA.

This study showed that mutations can also be detected in stool DNA from patients with stomach neoplasms. Detection of mutations in stool DNA of patients with premalignant neoplasm, and also in patients with stage I and II of tumor, demonstrates its application for early detection of GIT neoplasms.

The results of this study could have implication in future NGS-based stool DNA diagnostic tests that could be useful for screening of GIT malignancies and for detection at the premalignant stage. It could also act as a guide in a targeted therapy regimen, and to ease follow up of the treatment.

Research perspectives

Genetic mutations can be detected by amplicon–based NGS in stool DNA from patients with GIT tumors other than colorectal cancer also. Moreover, early neoplastic changes in GIT can also be detected in stool DNA. These results open up possibilities of development of NGS-based stool DNA test. Further testing of this method on a larger number of samples from patients with different GIT malignancies, premalignant lesions and healthy individuals is needed to fully assess it applicability in cancer diagnostics.