Analytical and clinical validation of a circulating tumor DNA-based assay for multi-cancer early detection

The development of multi-cancer early detection (MCED) through a single blood test has emerged as a promising method for improving the efficiency of early cancer detection and benefiting population health. However, the lack of analytical validation and clinical evidence for their utility in diverse populations have prevented their use in clinical practice. To address these challenges, we conducted a comprehensive analytical and clinical validation for an MCED test, SPOT-MAS (Screening for the Presence Of Tumor by DNA Methylation And Size). The analytical validation was to establish the limit of detection, reproducibility of test results and assess the impact of potential interferents on test performance. Specifically, SPOT-MAS could detect at least 50% of cancer samples at a specificity of 98% if the samples have tumor fraction more than 0.049 (95% CI: 0.043-0.059). The results were consistently reproduced for both intra- and inter-batch analysis. Moreover, our test remained robust at hemoglobin contamination of 500 mg/dl and genomic DNA contamination of up to 100%. To validate the performance of SPOT-MAS test in clinical settings, we launched a multi-center prospective trial, named K-DETEK, of 10,027 asymptomatic participants in Vietnam. Our assay achieved a positive predictive value of 58.14% (95%CI: 43.33-71.62) with 84.00% (95%CI: 65.35-93.60) accuracy in predicting tumor location and a negative predictive value of 99.92% (95%CI: 99.83-99.96). To our knowledge, this is the first and largest prospective validation study in Asia supporting the utility of SPOT-MAS as a multi-cancer blood test for early detection in a low- and middle-income country, where a nationwide cancer screening program is urgently needed but currently not available. ### Competing Interest Statement LHDN, THHN, NMP, BLT, THGN, DHV, THT, TDN, VTCN, YTL, THN, VUT, MPL, TMTT, MNN, TTVV, ANN, TTN, NNTD, HTN, PLD, LAKH, TAN, HTPN, CTTC, TVP, HST are employees of Gene Solutions. All other authors declare no competing interests. ### Clinical Trial NCT05227261 ### Funding Statement The study was funded by Gene Solutions ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of the University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam, gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The analytical data are available upon reasonable request by email to the corresponding authors (LST, MDP). Raw sequencing data are not publicly available due to ethical and regulatory restrictions.