Hypoxia Associated Integration of Epigenetic, Metabolic, and Immune Biomarkers in Blood and Urine for Early...

Background: Detection of colorectal cancer (CRC) currently relies mainly on invasive procedures such as colonoscopy. In experimental models, tumor hypoxia induces epigenetic, metabolic, and immune changes via hypoxia-inducible factor (HIF) signaling. Building on these published insights, this study evaluated whether a panel of biomarkers previously associated with hypoxia-related processes, plasma methylated SEPT9 (mSEPT9), urinary N¹, N¹²-diacetylspermine (DiAcSpm), and systemic inflammatory indices (NLR, PLR, and LMR)-could be combined into a non-invasive diagnostic panel and compared with standard serum tumor markers. This study focused solely on diagnostic performance; it did not directly assess tumor hypoxia or HIF expression in patients. This study was conducted in a clinical diagnostic setting (patients with confirmed CRC, polyps, or benign surgical conditions) and does not represent a population-based screening cohort. Limitations include the lack of an external validation cohort; all analyses were performed on a single dataset, and the reported performance metrics may be optimistic. Independent validation is required before clinical implementation. Methods: This prospective single-center study enrolled 382 participants: 142 with CRC, 62 with colorectal polyps, and 178 non-malignant controls. Plasma mSEPT9 was quantified by real-time PCR, urinary DiAcSpm by ELISA, and inflammatory indices from blood counts. Serum tumor markers (CEA, CA19-9, CA125, and AFP) were measured by immunoassay. Diagnostic accuracy was assessed using ROC analysis and multivariable logistic regression. Results: mSEPT9 (AUC 0.843) and DiAcSpm (AUC 0.831) demonstrated significantly higher diagnostic accuracy than CEA (AUC 0.660) and CA19-9 (AUC 0.649). A combined panel including mSEPT9, DiAcSpm, NLR, PLR, and LMR achieved an AUC of 0.947, with 85.9% sensitivity and 92.9% specificity. This panel also showed strong performance for early-stage CRC (AUC 0.905). Conclusions: A multimarker panel of biomarkers (mSEPT9, DiAcSpm, NLR, PLR, and LMR) provides a non-invasive diagnostic approach for CRC detection in a clinical case-control setting. Validation in asymptomatic screening populations is required before any screening claim can be made. These findings are associative; direct evidence that tumor hypoxia drives these biomarker changes was not obtained and requires future investigation.