Development and validation of a blood-based assay for gastric cancer early detection: A multidimensional analysis of cell-free DNA whole methylome sequencing—protocol for an observational, case‒control study

Background: Cancer-related features in cell-free DNA (cfDNA) fragments have gradually been identified and play essential roles in noninvasive early cancer detection. Integrated analysis of several cfDNA features has enhanced detection sensitivity compared to single features. To facilitate early diagnosis of gastric cancer, an assay based on assessing genomic-scale methylation and fragmentation profiles of plasma cfDNA will be developed and validated for early cancer detection. Methods This is an observational case‒control study. Blood samples will be prospectively collected before gastroscopy from 180 gastric cancer patients and 180 nonmalignant control subjects (healthy or with benign gastric diseases). Cases and controls will be randomly divided into a training and a testing dataset at a ratio of 2:1. Plasma cfDNA will be isolated and extracted, followed by bisulfite-free low-depth whole methylome sequencing. A multidimensional model named THorough Epigenetic Marker Integration Solution (THEMIS) including methylation, fragmentation, and chromosomal copy number alternation will be constructed in the training dataset. The performance of the model in differentiating cancer patients from noncancer controls will then be evaluated in the testing dataset. Discussion This is the first registered case‒control study designed to investigate a stacked ensemble model integrating several cfDNA features, including methylation pattern, fragmentation profile, and chromosomal copy number alternation, in identifying the GC population. This study will reveal whether multidimensional analysis of cfDNA will be an effective strategy for differentiating GC patients from nonmalignant individuals in a Chinese population. Trial registration This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of the Air Force Medical University (Xi’an, China; approval No. KY20222222-F-1) and registered on the Chinese Clinical Trial Registry with registration number ChiCTR2200065623. The study was registered on clinicalTrial.gov with a registration number: NCT05668910.