Colorectal cancer (CRC) is the third most common malignancy among men and women, with a relative 5-year survival rate of 63%. Survival strongly depends on the tumor stage at the time of initial diagnosis, ranging from 91% for localized disease to 13% for distantly metastasized tumors. Preventing CRC-related deaths requires effective screening strategies to detect tumors early, when curative treatment is still possible. Colonoscopy is the gold standard for CRC screening but adherence remains low in the general population. Therefore, alternative screening methods are urgently needed, particularly for individuals who decline colonoscopy.
Currently, immunological fecal occult blood tests (iFOBT) and circulating tumor DNA (ctDNA) assays are approved for CRC screening. However, their diagnostic accuracy—especially for early-stage cancers—remains limited. Available ctDNA blood tests are based either on DNA methylation biomarkers (e.g., SEPT9) or on panels of tumor-specific mutations. To date, no CRC ctDNA assay has combined both genetic and epigenetic biomarkers to maximize diagnostic performance.
CRC is characterized by several distinct molecular features: (1) aberrant DNA methylation, (2) frequent hotspot mutations in TP53, PIK3CA, APC, BRAF, and KRAS, and (3) amplification of chromosomal arms 8q, 13p/q, and 20q. Our goal is to develop, optimize, and evaluate the clinical performance of a ctDNA-based CRC test that integrates hotspot mutations, gene amplifications, and DNA methylation markers. To this end, we will identify DNA methylation sites located within commonly amplified genomic regions (8q, 13p/q, and 20q), exploiting the 'biological amplification' of methylated ctDNA fragments to enhance diagnostic sensitivity.
Next-generation sequencing (NGS) will be used to simultaneously assess mutations and DNA methylation in a single-tube assay using bisulfite-converted DNA. The diagnostic performance of this assay will be retrospectively evaluated in a case-control study comprising 64 CRC cases and 64 matched controls.