Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 7, 2022; 28(25): 2920-2936
Published online Jul 7, 2022. doi: 10.3748/wjg.v28.i25.2920
Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis
Si-Yu He, Ying-Chun Li, Yong Wang, Hai-Lin Peng, Cheng-Lin Zhou, Chuan-Meng Zhang, Sheng-Lan Chen, Jian-Feng Yin, Mei Lin
Si-Yu He, Hai-Lin Peng, Cheng-Lin Zhou, Mei Lin, Department of Clinical Laboratory, Taizhou People's Hospital (Postgraduate Training Base of Dalian Medical University), Taizhou 225300, Jiangsu Province, China
Si-Yu He, Department of Clinical Laboratory, The First People's Hospital of Tianmen City, Tianmen 431700, Hubei Province, China
Ying-Chun Li, Yong Wang, Department of General Surgery, Taizhou People's Hospital (Postgraduate Training Base of Dalian Medical University), Taizhou 225300, Jiangsu Province, China
Chuan-Meng Zhang, Central Laboratory, Taizhou People's Hospital (Postgraduate training base of Dalian Medical University), Taizhou 225300, Jiangsu Province, China
Sheng-Lan Chen, Department of Laboratory, Taizhou Genewill Medical Laboratory Company Limited, Taizhou 225300, Jiangsu Province, China
Jian-Feng Yin, Department of Laboratory, Jiangsu CoWin Biotech Co., Ltd., Taizhou 225300, Jiangsu Province, China
Author contributions: He SY performed most of the experiments, analyzed some data, and drafted the manuscript; Li YC and Wang Y provided specimens for the study; Peng HL and Zhou CL gave constructive guidance on the study; Zhang CM contributed to the statistics; Chen SL and Yin JF performed some of the experiments; Lin M designed and supervised the study and edited the manuscript; all authors approved the final version of the article.
Supported by Taizhou Social Development Plan, No. TS202004; Natural Science Foundation of Nanjing University of Chinese Medicine China, No. XZR2020093; and Taizhou People's Hospital Medical Innovation Team Foundation, No. CXTDA201901.
Institutional review board statement: This study was reviewed and approved by the Ethics Committee of Taizhou people's Hospital in Jiangsu Province (No. KY201912501).
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (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: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Mei Lin, MD, PhD, Senior Scientist, Department of Clinical Laboratory, Taizhou People's Hospital (Postgraduate Training Base of Dalian Medical University), No. 366 Taihu Road, Taizhou 225300, Jiangsu Province, China. l_mei@163.com
Received: January 16, 2022
Peer-review started: January 16, 2022
First decision: March 8, 2022
Revised: March 18, 2022
Accepted: May 27, 2022
Article in press: May 27, 2022
Published online: July 7, 2022
Processing time: 168 Days and 21.5 Hours
Abstract
BACKGROUND

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Given its insidious onset, the condition often already progresses to advanced stage when symptoms occur. Thus, early diagnosis is of great significance for timely clinical intervention, efficacy enhancement, and prognostic improvement. Featuring high throughput, fastness, and rich information, next generation sequencing (NGS) can greatly shorten the detection time, which is a widely used detection technique at present.

AIM

To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC, and to establish a technological platform for CRC screening, diagnosis, and efficacy monitoring through fecal DNA detection.

METHODS

NGS was used to sequence the stool DNA of patients with CRC, which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease, as well as the tumor tissues of CRC patients. Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened, and their significances in diagnosing CRC and predicting patients' prognosis were comprehensively evaluated.

RESULTS

High mutation frequencies of TP53, APC, and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery. Contrastively, no pathogenic mutations of the above three genes were noted in the postoperative stools, the normal controls, or the benign intestinal disease group. This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients. The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent. Compared to the postoperative stools and the stools in the two control groups, the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools (χ2 = 7.328, P < 0.05; χ2 = 4.219, P < 0.05), suggesting that fecal TP53 and KRAS genes can be used for CRC screening, diagnosis, and prognostic prediction. No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups (χ2 = 0.878, P > 0.05), so further analysis with larger sample size is required. Among CRC patients, the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools, with a true positive rate of 59.26%, while the pathogenic mutation sites of KRAS occurred in 10 stools, with a true positive rate of 37.04%. The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67% (18/27) and 68.97%, respectively, both of which were higher than those of TP53 or KRAS mutation detection alone, suggesting that the combined genetic testing can improve the CRC detection rate. The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T (mutation start and stop positions were both 7579436 for the former, while 55249164 for the latter) were found in the preoperative stools and tumor tissues. These "undetected" mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression, which needs to be confirmed through further research. Some mutations of "unknown clinical significance" were found in such genes as TP53, PTEN, KRAS, BRAF, AKT1, and PIK3CA, whose clinical values is worthy of further exploration.

CONCLUSION

NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis. Fecal TP53 and KRAS can be used as specific genes for the screening, diagnosis, prognostic prediction, and recurrence monitoring of CRC. Moreover, the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Keywords: Colorectal cancer; Feces; Next generation sequencing; Diagnosis; Gene

Core Tip: Colorectal cancer (CRC) is characterized by high morbidity and mortality, as well as low early diagnosis rate. The development of current gold standard for clinical diagnosis of CRC is restricted due to its invasiveness. The purpose of this study is to explore the potential value of fecal gene detection based on next generation sequencing in the diagnosis of CRC, to screen specific genes or gene combinations suitable for CRC diagnosis and prognosis prediction in fecal DNA, and to establish a technical platform for fecal DNA detection for CRC screening, diagnosis, and efficacy monitoring.