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Background: Non-invasive multi-cancer early detection (MCED) tests have shown promise in enhancing early cancer detection. However, their clinical utility across diverse populations remains underexplored, limiting their routine implementation. This study aims to validate the clinical utility of a multimodal non-invasive circulating tumor DNA (ctDNA)-based MCED test, SPOT-MAS (Screening for the Presence Of Tumor by DNA Methylation And Size). Methods: We conducted a multicenter prospective study, K-DETEK (ClinicalTrials.gov identifier: NCT05227261), involving 9057 asymptomatic individuals aged 40 years or older across 75 major hospitals and one research institute in Vietnam. Participants were followed for 12 months. Results: Of the 9024 eligible participants, 43 (0.48%) tested positive for ctDNA. Among these, 17 were confirmed with malignant lesions in various primary organs through standard-of-care (SOC) imaging and biopsy, with 9 cases matching our tissue of origin (TOO) predictions. This resulted in a positive predictive value of 39.53% (95%CI 26.37–54.42) and a TOO accuracy of 52.94% (95%CI 30.96–73.83). Among the 8981 participants (99.52%) who tested negative, 8974 were confirmed cancer-free during a 12-month period after testing, yielding a negative predictive value of 99.92% (95% CI 99.84–99.96). The test demonstrated an overall sensitivity of 70.83% (95%CI 50.83–85.09) and a specificity of 99.71% (95% CI 99.58–99.80) for detecting various cancer types, including those without SOC screening options. Conclusions: This study presents a prospective validation of a multi-cancer early detection (MCED) test conducted in a lower middle-income country, demonstrating the potential of SPOT-MAS for early cancer detection. Our findings indicate that MCED tests could be valuable additions to national cancer screening programs, particularly in regions where such initiatives are currently limited. Trial registration: ClinicalTrials.gov ID: NCT05227261. Date of registration: 07/02/2022. [ABSTRACT FROM AUTHOR]

Aim: Cancers lacking standard screening (LSS) options account for approximately 70% of cancer-related deaths due to late-stage diagnosis. Circulating tumor DNA (ctDNA) is a promising biomarker for multi-cancer early detection. We previously developed SPOT-MAS, a multimodal ctDNA-based assay analyzing methylation and fragmentomic profiles, effective in detecting common cancers (breast, colorectal, liver, lung and gastric). This study extends the analysis to five LSS cancers: endometrial, esophageal, head and neck, ovarian and pancreatic. Methods: SPOT-MAS was applied to profile cfDNA methylation and fragmentomic patterns in 739 healthy individuals and 135 LSS cancer patients. Results: We identified 347 differentially methylated regions and observed genome-wide hypomethylation across all five LSS cancers. Esophageal and head and neck cancers showed an enrichment of short cfDNA fragments (<150 bp). Eleven 4-mer end motifs were consistently altered in cfDNA fragments across all LSS cancers. Many significant signatures were consistent with previous observations in common cancers. Notably, SPOT-MAS achieved 96.2% specificity and 74.8% overall sensitivity, with a lower sensitivity of 60.7% in early-stage cancers. Conclusion: This proof-of-concept study demonstrates that SPOT-MAS a non-invasive test trained on five common cancer types, could detect a number of LSS cancer cases, potentially complementing existing screening programs. Plain Language Summary Many cancers do not have standard tests, so they are often found too late, which leads to about 70% of cancer deaths. We've created a blood test that can help find cancer early. This test has already worked well for common cancers like breast and lung cancer, and now we're testing it on five harder-to-detect cancers: endometrial, esophageal, head and neck, ovarian and pancreatic cancers. In our study, we tested our blood test on 739 healthy people and 135 patients with these difficult cancers. Our method correctly identified healthy people 96.2% of the time and found cancer cases 74.8% of the time. This new test could help with screening for types of cancer that do not have good tests right now. Article highlights Approximately 70% of cancer deaths result from late-stage diagnoses in cancers lacking standard-of-care screening (LSS). The SPOT-MAS test is a ctDNA-based assay designed to detect five common cancers (breast, lung, colorectal, gastric and liver) and has been extended to five additional LSS cancers: endometrial, esophageal, head and neck, ovarian and pancreatic. The assay analyzed cfDNA methylation and fragmentomic patterns in 135 LSS cancer patients and 739 healthy individuals. It identified 347 differentially methylated regions and specific fragmentomic alterations. SPOT-MAS achieved 96.2% specificity and 74.8% overall sensitivity, with 60.7% sensitivity for early-stage detection. This study suggests that SPOT-MAS could serve as a non-invasive method for detecting LSS cancers. [ABSTRACT FROM AUTHOR]

Background: Multi-cancer early detection (MCED) through a single blood test significantly advances cancer diagnosis. However, most MCED tests rely on a single type of biomarkers, leading to limited sensitivity, particularly for early-stage cancers. We previously developed SPOT-MAS, a multimodal ctDNA-based assay analyzing methylation and fragmentomic profiles to detect five common cancers. Despite its potential, SPOT-MAS exhibited moderate sensitivities for early-stage cancers. This study investigated whether integrating hotspot mutations into SPOT-MAS could enhance its detection rates., Method: A targeted amplicon sequencing approach was developed to profile 700 hotspot mutations in cell-free DNA and integrated into the SPOT-MAS assay, creating a single-blood draw workflow. This workflow, namely SPOT-MAS Plus was retrospectively validated in a cohort of 255 non-metastatic cancer patients (breast, colorectal, gastric, liver, and lung) and 304 healthy individuals., Results: Hotspot mutations were detected in 131 of 255 (51.4%) cancer patients, with the highest rates in liver cancer (96.5%), followed by colorectal (59.3%) and lung cancer (53.7%). Lower detection rates were found for cancers with low tumor mutational burden, such as breast (31.3%) and gastric (41.9%) cancers. In contrast, SPOT-MAS demonstrated higher sensitivities for these cancers (51.6% for breast and 62.9% for gastric). The combination of hotspot mutations with SPOT-MAS predictions improved early-stage cancer detection, achieving an overall sensitivity of 78.5% at a specificity of 97.7%. Enhanced sensitivities were observed for colorectal (81.36%) and lung cancer (82.9%)., Conclusion: The integration of genetic and epigenetic alterations into a multimodal assay significantly enhances the early detection of various cancers. Further validation in larger cohorts is necessary to support broader clinical applications., (© 2025 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)