{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T12:30:20Z","timestamp":1790080220092,"version":"4.0.1"},"reference-count":63,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,7,5]],"date-time":"2023-07-05T00:00:00Z","timestamp":1688515200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Oncol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Colorectal cancer (CRC) can develop through several dysregulated molecular pathways, including the serrated pathway, characterized by CpG island methylator (CIMP) phenotype. Although the tumor tissue is a commonly tested material, sample types such as stool or plasma, bring a new, non-invasive approach. Several cancer-related methylated genes have been identified in CRC patients, including gene<jats:italic>GRIA4<\/jats:italic>, showing promising diagnostic potential. The aim of our study was to develop a sensitive droplet digital PCR (ddPCR) assay to examine<jats:italic>GRIA4<\/jats:italic>hypermethylation status in CRC patients and evaluate its diagnostic potential in tissue and liquid biopsy samples.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In total, 23 patients participated in this study, 7 patients with primary CRC and 16 patients with liver metastasis of clinically known CRC. We obtained tumor and non-tumor tissues (N=17), blood samples pre- and post-surgery (N=22), and blood of five volunteers without a personal cancer history. We have developed and optimized a ddPCR assay for<jats:italic>GRIA4<\/jats:italic>hypermethylation detection, from tissue and plasma samples.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>We detected significantly increased<jats:italic>GRIA4<\/jats:italic>methylation in tumor tissues compared to their adjacent non-tumor tissue, p&amp;lt;0.0001. Receiver operating characteristic (ROC) analysis defined cutoff values to separate primary tumors and metastases from non-tumor colon\/rectum, specifically 36.85% for primary tumors and 34.81% for metastases. All primary tumors were above this threshold. When comparing the methylation levels of metastatic vs. non-tumor tissue, a smaller increase was observed in liver metastasis versus colon tissue (3.6\u00d7 gain; p=0.001), then in liver metastasis versus adjacent liver tissue (17.4\u00d7 gain; p&amp;lt;0.0001). On average,<jats:italic>GRIA4<\/jats:italic>hypermethylation in primary tumor plasma was 2.8-fold higher (p=0.39), and in metastatic plasma, 16.4-fold higher (p=0.0011) compared to healthy individuals. Hypermethylation in metastatic plasma was on average 5.9 times higher (p=0.051) than in primary tumor plasma. After tumor removal surgery, average hypermethylation decrease in plasma was 1.6\u00d7 for primary (p=0.037) and 4.5\u00d7 for metastatic patients (p=0.023).<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Based on our data, it can be inferred that<jats:italic>GRIA4<\/jats:italic>serves as a tissue specific biomarker for the colon\/rectum tissue, thus is suitable for cancer classification. This biomarker showed the potential to be an attractive target for early non-invasive detection of metastases of clinically known CRC, although additional analysis has to be performed.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fonc.2023.1205791","type":"journal-article","created":{"date-parts":[[2023,7,5]],"date-time":"2023-07-05T17:54:31Z","timestamp":1688579671000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":11,"title":["Hypermethylated GRIA4, a potential biomarker for an early non-invasive detection of metastasis of clinically known colorectal 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