Noninvasive Multitarget Stool DNA Methylation Assay in the Early Detection of Multiple Gastrointestinal Cancers

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With an estimated 4.8 million new cases and 3.4 million deaths worldwide recorded in 2018, cancers of the gastrointestinal tract account for over one-quarter (26%) of global cancer incidence and over one-third (35%) of all cancer-related deaths.

Although the U.S. Food and Drug Administration (FDA) has approved several types of stool-based tests and the SEPT9 gene methylation blood-based assay for the detection of colorectal cancer, there are no FDA-approved early detection tests for multiple gastrointestinal cancers.

A prospective study in China investigating a noninvasive, multitarget stool DNA methylation assay for the early detection and localization of multiple gastrointestinal cancers has found the test allowed for the early detection and identification of the tissue of origin of gastrointestinal cancers. The study is being presented by Sun et al during the 2023 ASCO Breakthrough meeting, taking place August 3 to 5 in Yokohama, Japan (Abstract 41).

Study Methodology

Researchers prospectively enrolled 124 patients diagnosed with gastrointestinal cancers who had not undergone treatment and 92 healthy controls who underwent gastrointestinal cancer–related examinations every 3 years. The types of gastrointestinal malignancies included in the study were colorectal, gastric, esophageal, pancreatic, and ampullary cancers.

Stool DNA was extracted from the supernatant of a mixture containing 5 g of fresh stool and 25 ml of preservative solution using the stool DNA extraction kit. The researchers then subjected the samples to bisulfite conversion using the EZ DNA Methylation Kit, and then treated them with a stool DNA multitarget methylation assay kit to detect the methylation levels of three target regions of stool DNA—including the genes PDX1, COL4A2-1, and FGF5—with quantitative polymerase chain reaction.

Based on the guidelines of the study protocol, a positive result was defined as ΔCt value (ΔCt = Ct [target gene] – Ct [GAPDH]) < 10 for any of the three genes (where Ct = cycle threshold in gene expression measurement and GAPDH = glyceraldehyde-3-phosphate dehydrogenase). Multiple logistic regression models for single gastrointestinal cancers were constructed, including the ∆Ct value of each gene to adjust for tissue of origin of the gastrointestinal cancers.


Overall, 98 of 124 patients (79%) tested positive for cancer, and 88 of 92 healthy controls (96%) tested negative. By cancer type, the positivity rates were 71%, 83%, 75%, 81%, and 91% for colorectal cancer (n = 24/34), gastric cancer (n = 19/23), esophageal cancer (n = 18/24), pancreatic cancer (n = 17/21), and ampullary cancer (n = 20/22), respectively.

The researchers also found that among four positive healthy controls, three were diagnosed with advanced adenomas and one was a pregnant woman. For the multiple logistic regression model, the sensitivity of colorectal cancer was 88% (n = 30/34), gastric cancer was 91% (n = 21/23), esophageal cancer was 88% (n = 21/24), pancreatic cancer was 90% (n = 19/21), and ampullary cancer was 95% (n = 21/22).

“The multitarget stool DNA methylation assay allowed [the] early and accurate detection and identification of tissue-of-origin gastrointestinal cancers,” concluded the study authors.

Disclosure: Funding for this study was provided by Guangdong Second Provincial General Hospital. For full disclosures of the study authors, visit

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