Published online Jun 28, 2022. doi: 10.3748/wjg.v28.i24.2705
Peer-review started: December 4, 2021
First decision: January 8, 2022
Revised: January 14, 2022
Accepted: May 13, 2022
Article in press: May 13, 2022
Published online: June 28, 2022
Colorectal cancer (CRC) is one of the most prevalent malignancies in China with an increasing ratio of colon to rectal cancer over the decades. Early screening is the key to reducing this burden. The traditional screening methods, including colonoscopy, fecal immunochemical test (FIT) and serum carcinoembryonic antigen (CEA), have reached a bottleneck, especially for early-stage colon cancer (ECC) due to its occult onset. Thus, precision and effective non-invasive biomarkers are highly desirable.
Detection of aberrant methylated DNA in stool has been proven to be a promising noninvasive method for the early diagnosis of CRC. However, the reported screening efficacy of the same biomarker varied across different studies. Moreover, effective biomarkers for detecting ECC are scarce, especially in the Chinese population.
The objective of our study was to identify a novel assay based on stool DNA (sDNA) methylation biomarkers which could improve the effectiveness of ECC screening. We also investigated the influence of clinicopathologic covariates on test performance.
We performed a blinded, single-center, case-control study using archived stool samples from 125 ECC patients and 125 individuals with normal colonoscopy (controls); the subjects were randomly assigned to a training or test set at a 1.5:1 ratio. Targeted bisulfite sequencing (TBSeq) was performed on five pairs of preoperative and postoperative sDNA samples from ECC patients to identify DNA methylation biomarkers. Pyrosequencing (PSQ) was used for validation of the candidate biomarkers in large samples. A stepwise logistic regression analysis was applied to the data of the training set to develop a multiplex stool-based assay. The detection performance was further evaluated in the test set and combined set. In addition, the association of detection sensitivity with clinicopathologic covariates were analyzed by χ2 test.
Through TBSeq, the three top hypermethylated genes, paired box 8, Ras-association domain family 1 and secreted frizzled-related protein 2, that were involved in the important cancer pathways were selected as biomarkers. Based on the PSQ results, an sDNA panel containing the three biomarkers was developed by logistic regression modelling. Receiver operating characteristic (ROC) analysis showed that this panel offered an advantage over any single biomarker, FIT or serum CEA in the detection of ECC. Further analysis revealed that the inclusion of FIT could effectively improve the detection accuracy. In the combined set, the sensitivity, specificity and area under the ROC curve for the sDNA panel combining FIT reached 80.0%, 93.6% and 0.918, respectively. Moreover, this multiplex assay maintained excellent performance in the subgroup by tumor stage. Additionally, we found that the detection sensitivity of the multiplex assay was significantly higher in T4 than in T1-3 stage tumors (P = 0.041), but was not affected by tumor site, tumor stage, histological differentiation, age or sex.
The present study identified a novel multiplex stool-based assay, including three sDNA methylation biomarkers and FIT, capable of detecting ECC with high sensitivity and specificity. Importantly, the detection rate by this assay was related to T stage but not tumor site, tumor stage, histological differentiation, etc.
Our study provided a new and promising approach for improvement of ECC screening in the Chinese population. Further demonstrations on a large-scale, prospective multicenter study are needed to conclusively evaluate its value.