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      Multidimensional Analysis of a Cell-Free DNA Whole Methylome Sequencing Assay for Early Detection of Gastric Cancer: Protocol for an Observational Case-Control Study


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          Commonly used noninvasive serological indicators serve as a step before endoscope diagnosis and help identify the high-risk gastric cancer (GC) population. However, they are associated with high false positives and high false negatives. Alternative noninvasive approaches, such as cancer-related features in cell-free DNA (cfDNA) fragments, have been gradually identified and play essential roles in early cancer detection. The integrated analysis of multiple cfDNA features has enhanced detection sensitivity compared to individual features.


          This study aimed to develop and validate an assay based on assessing genomic-scale methylation and fragmentation profiles of plasma cfDNA for early cancer detection, thereby facilitating the early diagnosis of GC. The primary objective is to evaluate the overall specificity and sensitivity of the assay in predicting GC within the entire cohort, and subsequently within each clinical stage of GC. The secondary objective involved investigating the specificity and sensitivity of the assay in combination with possible serological indicators.


          This is an observational case-control study. Blood samples will be prospectively collected before gastroscopy from 180 patients with GC and 180 nonmalignant control subjects (healthy or with benign gastric diseases). Cases and controls will be randomly divided into a training and a testing data set 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) will be constructed in the training data set. The model includes features such as the methylated fragment ratio, chromosomal aneuploidy of featured fragments, fragment size index, and fragment end motif. The performance of the model in distinguishing between patients with cancer and noncancer controls will then be evaluated in the testing data set. Furthermore, GC-related biomarkers, such as pepsinogen, gastrin-17, and Helicobacter pylori, will be measured for each patient, and their predictive accuracy will be assessed both independently and in combination with the THEMIS model


          Recruitment began in November 2022 and will be ended in April 2024. As of August 2022,250 patients have been enrolled. The final data analysis is anticipated to be completed by September 2024.


          This is the first registered case-control study designed to investigate a stacked ensemble model integrating several cfDNA features generated from a bisulfite-free whole methylome sequencing assay. These features include methylation patterns, fragmentation profiles, and chromosomal copy number changes, with the aim of identifying the GC population. This study will determine whether multidimensional analysis of cfDNA will prove to be an effective strategy for distinguishing patients with GC from nonmalignant individuals within the Chinese population. We anticipate the THEMIS model will complement the standard-of-care screening and aid in identifying high-risk patients for further diagnosis.

          Trial Registration

          ClinicalTrial.gov NCT05668910; https://www.clinicaltrials.gov/study/NCT05668910

          International Registered Report Identifier (IRRID)


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          Most cited references32

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              Detection of circulating tumor DNA in early- and late-stage human malignancies.

              The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

                Author and article information

                JMIR Res Protoc
                JMIR Res Protoc
                JMIR Research Protocols
                JMIR Publications (Toronto, Canada )
                20 September 2023
                : 12
                : e48247
                [1 ] Department of General Surgery First Hospital of Yulin Yulin China
                [2 ] Department of Gastrointestinal Surgery Xijing Hospital Air Force Military Medical University Xi'an China
                [3 ] Genecast Biotechnology Co, Ltd Wuxi China
                Author notes
                Corresponding Author: Xiaohua Li xjyylixiaohua@ 123456163.com
                Author information
                ©Yongjun Han, Jiangpeng Wei, Weidong Wang, Ruiqi Gao, Ning Shen, Xiaofeng Song, Yang Ni, Yulong Li, Li-Di Xu, Weizhi Chen, Xiaohua Li. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 20.09.2023.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included.

                : 17 April 2023
                : 11 August 2023
                : 17 August 2023
                : 18 August 2023

                gastric cancer,circulating cell-free dna,early detection,methylation,fragmentation,chromosomal instability,whole methylome sequencing,multidimensional model


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