Background: A multi-cancer early detection (MCED) test that uses targeted methylation-based cfDNA technology to detect cancer and predict cancer signal origin (CSO) has potential to efficiently identify malignancies for which effective screening modalities do not exist. A previous version of a blood-based MCED test demonstrated favorable classification and test characteristics. Samples from the ongoing PATHFINDER study were reanalyzed in a prespecified interim analysis to evaluate performance of a more recent version of the test with an updated classifier (eg, updated CSO localization, hematological signal threshold) that is planned for clinical implementation as a general multi-cancer screening tool. Methods: PATHFINDER (NCT04241796) is an interventional, prospective study in which results (cancer signal detected/not detected and predicted CSO) using a previous version of the MCED test are returned to investigators, and those with a signal detected undergo further diagnostic testing. In this prespecified interim analysis, samples from those enrolled as of October 6, 2020 were reanalyzed with the more recent version of the MCED test (these results were not returned to investigators). The positive predictive value (PPV) for cancer detection, overall CSO accuracy, and concordance between the two test versions were assessed. Results: A total of 4011/4047 (99%) participants (pts) were analyzable (mean [SD] age 63.9 [8.7] years, 62% female, 92% white, 24% with prior cancer history, 39% ever smoker [4% current], 6% with genetic cancer predisposition). Cancer signal was detected in 0.95% (38/4011). A total of 27/38 also had signal detected by the previous version of the MCED test, including 19 who reached diagnostic resolution (13 with cancer diagnosis and 6 without); 11/38 were discordant positives. Nine different cancer types were detected in the 13 pts (2 stage I, 3 stage II, 2 stage III, and 3 stage IV); 1 had no AJCC stage expected, 1 metastatic recurrence and 1 stage evaluation underway. A conservative minimal PPV assuming all discordant positives are false positives, was 43.3% (13/30, 95% CI 27.4-60.8%) based on 19 pts with diagnostic resolution and 11 discordant positives. High negative percent agreement (PA) 99.7% (99.5-99.8%) between the two test versions was observed. Positive PA of 43.5% (95% CI, 31.9-55.9%) was consistent with the more stringent threshold for hematologic signal in the recent MCED version, as most discrepant cases had hematologic CSO with the previous MCED test. Among 13 detected cancers, accuracy of the top CSO prediction was 92.3% (12/13, 95% CI 66.7-99.6%). Conclusions: In this prespecified interim analysis, the more recent version of the MCED test detected cancers with high PPV and high accuracy of CSO prediction, supporting readiness for use in clinical practice. Full enrollment cohort data will be available at the meeting. Clinical trial information: NCT04241796