The incidence of cancer-related morbidity and mortality is rapidly increasing globally, and accounted for nearly one fifth of all deaths in 2020 (Sung et al., 2021). High-cost treatment is a significant financial burden for cancer patients, with almost 286 billion dollars in 2021 and an increase of 8.2% - 581 billion dollars in 2030. In Vietnam, GLOBOCAN 2020 reported over 182,500 newly diagnosed cases and 122,690 cancer-related deaths (Sung et al., 2021). Among these, liver (14.5%), lung (14.4%), breast (11.8%), gastric (9.8%), and colorectal cancer (9%) are the five most common types. Up to 80% of cancer patients in Vietnam were diagnosed at stage III or stage IV, resulting in a high rate of 1 year mortality (25%) and a low 5-year survival rate compared to other countries (Pham et al., 2019). Diagnostic delays are associated with a lower chance of survival, greater treatment-associated problems, and higher costs (Hawkes, 2019). Cancer detection at earlier stages can improve the opportunity to control cancer progression, increase the patient survival rate, and lower medical expenses (Kakushadze et al., 2017).
Although currently guided screening tests have each been shown to provide better treatment outcomes and reduce cancer mortality, some of them are invasive, thus having low accessibility. Importantly, most of them are single cancer screening tests, which may result in high false positive rates when used sequentially (Sasieni et al., 2023). Multi-cancer early detection (MCED) tests can potentially overcome these challenges by simultaneously detecting multiple cancer types from a single test (Liu et al., 2020). Liquid biopsy, an emerging non-invasive approach for MCED, can capture a wide range of tumor features, including cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), exosomes, proteins, mRNA, and metabolites (Li et al., 2018; Nguyen et al., 2022b). Among them, ctDNA has become a promising biomarker for detecting early-stage cancers because it is a carrier of genetic and epigenetic modifications from cancer-derived DNA (Gao et al., 2022). Indeed, ctDNA detection has demonstrated several advantages in non-invasive diagnostic, prognostic, and monitoring of cancer patients during and after treatment (Pascual et al., 2022; Nguyen et al., 2020). Furthermore, ctDNA carrying tumor-specific alterations could be used to identify the corresponding unknown primary cancer and tumor localization.
In recent years, there has been considerable interest in exploring the potential of ctDNA alterations for early detection of cancer (Nguyen et al., 2020; Moser et al., 2023). One such approach is the PanSeer test, which uses 477 differentially methylated regions (DMRs) in ctDNA to detect five different types of cancer up to 4 years prior to conventional diagnosis (Im et al., 2021). The DELFI assay employs a genome-wide analysis of ctDNA fragment profiles to increase sensitivity in early detection (Zhou et al., 2022b). Recently, the Galleri test has emerged as a multi-cancer detection assay that analyzes more than 100,000 methylation regions in the genome to detect over 50 cancer types and localize the tumor site (Cohen et al., 2018).
Despite their great potential, there remain several challenges that these assays must solve to deliver accessible and reliable clinical adoption for the large population, including the low fraction of ctDNA in the blood of early-stage cancer patients, the heterogeneity of ctDNA signatures from diverse cancer types, subtypes and stages (Moser et al., 2023), and the high sequencing depth required. To address these challenges, recent studies have focused on multi-analyte approach - combining genomic and nongenomic features such as methylomics and fragmentomics to increase the detection of ctDNA and accuracy for tissue of origin (TOO) identification (Moser et al., 2023; Im et al., 2021; Zhou et al., 2022b). Advances in multimodal analysis approaches have led to the development of powerful screening tests that enable high sensitivity and cost-effectiveness. For example, CancerSEEK uses a combined approach of protein biomarkers and genetic alterations to detect and locate the presence of eight types of cancers (Cohen et al., 2018). In this assay, cancer-associated serum proteins play a complementary role in tumor localization as cfDNA mutations are not tissue specific. However, detecting both protein and genetic biomarkers are time-consuming and costly. Thus, the development of future MCED tests should endeavor to deliver a screening approach with high sensitivity, specificity, and TOO identification at cost-effective price to provide better clinical outcomes and treatment opportunities for all cancer patients.
In an effort to address the challenges of early cancer detection, we have developed a multimodal approach called SPOT-MAS (screening for the presence of tumor by DNA methylation and size). This assay was previously applied to cohorts of colorectal (Nguyen et al., 2022a) and breast cancer patients (Pham et al., 2023) and demonstrated ability for early detection of these cancers at high sensitivity across different cancer stages and patient age groups. In this study, we aimed to expand our multimodal approach, SPOT-MAS, to comprehensively analyze methylomics, fragmentomics, DNA copy number, and end motifs (EMs) of cfDNA and evaluate its utility to simultaneously detecting and locating cancer from a single screening test. As proof of concept, we used 2288 participants, including 738 non-metastatic patients and 1550 healthy controls, to train and fully validate this approach on five commonly diagnosed cancers, including breast, gastric, lung, colorectal, and liver cancer. Our findings demonstrate that the multimodal approach of SPOT-MAS enables profiling of multiple ctDNA signatures across the entire genome at low sequencing depth to detect five different cancer types in their early stages. Beyond detecting the presence of cancer signals, our assay was able to predict the tumor location, which is important for clinicians to fast-track the follow-up diagnostic and guide necessary treatment. Thus, SPOT-MAS has the potential to become a universal, simple, and cost-effective approach for early multi-cancer detection in a large population.