Comprehensive validation of a cell-free DNA-based assay for multi-cancer detection: A Vietnamese longitudinal prospective cohort study of 9,024 participants.

102 Background: Blood-based multi-cancer early detection (MCED) has emerged as a promising method for enhancing early cancer detection efficiency and benefiting population health. However, the lack of evidence regarding their clinical utility across diverse populations has hindered their routine use. To address this, we conducted a comprehensive analytical and clinical validation of an MCED test, SPOT-MAS (Screening for the Presence Of Tumor by DNA Methylation And Size). Methods: Analytical validation was conducted on a retrospective cohort of 96 healthy and 169 cancer-confirmed individuals to determine the limit of detection, reproducibility, and potential interferents. Additionally, we launched a multi-center prospective study, named K-DETEK (ClinicalTrials.gov identifier: NCT05227261), to clinically validate the diagnostic performance. Our study recruited 10,027 asymptomatic individuals aged 40 years or older across 75 major hospitals in Vietnam, following them up for 12 months. Results: SPOT-MAS test demonstrated the capability to detect over 50% of cancer samples with 98% specificity at a tumor fraction of 0.049 (95% CI: 0.043-0.059). These results were consistent across intra- and inter-batch analyses. The test remained robust even with hemoglobin contamination up to 500 mg/dL and genomic DNA contamination of up to 100%. In the K-DETEK study, among 9,024 eligible participants, 43 (0.48%) showed a positive cancer signal. Of those, 25 were diagnosed with cancer through standard imaging and biopsy tests, with 21 matching tumor types with our predictions, resulting in a positive predictive value of 58.14% (95% CI: 43.33-71.62) and tumor-tissue-origin accuracy of 84%. 8,981 participants (99.52%) had a negative cancer signal, and 8,974 were confirmed cancer-free, indicating a negative predictive value of 99.92% (95% CI: 99.84-99.96). The test showed an overall sensitivity of 78.13% (95% CI: 61.25-88.98) and a specificity of 99.80% (95% CI: 99.68-99.87) for cases with tumors at all stages, with slightly lower sensitivity (74.05%, 95% CI: 56.64-87.32) for precancerous and early clinical stages. Conclusions: Our study represents the most comprehensive and largest validation study in Asia supporting the utility of SPOT-MAS as a multi-cancer blood test for early cancer detection. Its potential significance is underscored, particularly in low- and middle-income countries, emphasizing the need for accessible and effective screening tools. Clinical trial information: NCT05227261 .