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However, traditional bisulfite\u2010based methods often encounter sensitivity limitations in detecting early\u2010stage malignancies or certain cancer types. In the INSPECTOR study, we developed a MCED and cancer signal origin (CSO) system specifically designed for early\u2010stage or hard\u2010to\u2010detect cancers, including those of the lung, breast, colorectum, liver, esophagus, stomach, pancreas, and ovary.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We established a comprehensive methylation marker discovery database (\n                      <jats:italic>n<\/jats:italic>\n                      = 6,342) by integrating public datasets (\n                      <jats:italic>n<\/jats:italic>\n                      = 4,699) and in\u2010house samples (\n                      <jats:italic>n<\/jats:italic>\n                      = 1,643), all processed using human TET (hTET) enzyme\u2010assisted whole\u2010methylome sequencing (GM\u2010seq). This enabled the design of a targeted panel encompassing 155,362 methylated CpG sites. Leveraging hTET\u2010assisted high\u2010depth next\u2010generation sequencing (NGS), our blood test achieved a median unique depth of 1,093\u00d7. Multicenter case\u2010control cohorts, including various pathological subtypes, were used for training, validation, and independent validation of MCED and CSO models, and to verify the clinical feasibility.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Clinical validation was conducted across multi\u2010center case\u2010control cohorts, including 1,071 participants in the training set, 581 in the validation set, and 824 in the independent validation set. The MCED assay demonstrated robust performance with a specificity of 99.1% and sensitivity of 83.2% in the training set, 99.0% and 81.8% in the validation set, and comparable results in the independent validation set (99.0% specificity, 81.9% sensitivity). Notably, sensitivity reached 65.5% for stage I cancers, 79.7% for stage II, and 71.3% for stages I\u2010II combined. The sensitivities for different cancer types were as follows: esophageal (79.2%), gastric (76.1%), colorectal (86.2%), pancreatic (66.7%), liver (100.0%), lung (72.9%), breast (88.9%), and ovarian (87.9%). The CSO model exhibited strong accuracy, with top\u20101 cancer origin prediction rates of 87.9% (validation) and 87.4% (independent validation), rising to 95.1% and 94.5% for top\u20102 predictions, respectively. For stage I cancers specifically, the top\u20101 accuracy was 85.5%.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>These findings underscore the efficacy of the hTET\u2010assisted cfDNA methylation sequencing system across diverse cancer types, particularly in early stages. Enzyme\u2010assisted NGS test of methylated cfDNA thus enhances the clinical utility of non\u2010invasive blood\u2010based screening.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/cac2.70071","type":"journal-article","created":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T10:22:23Z","timestamp":1761819743000},"page":"1645-1665","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["The INSPECTOR study: enhanced feasibility for clinical translation of a multi\u2010cancer early detection method based on enzyme\u2010assisted high signal\u2010to\u2010noise ratio sequencing of methylated circulating tumor DNA"],"prefix":"10.34133","volume":"45","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6312-9299","authenticated-orcid":false,"given":"Hui\u2010Yan","family":"Luo","sequence":"first","affiliation":[{"name":"Department of Medical Oncology Sun Yat\u2010sen University Cancer Center  Guangzhou Guangdong P. 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