Fecal HBA mRNA as an Alternative to the Protein-based FIT Assay for Colorectal Cancer Screening: A Computational and Clinical Validation Study

Abstract Background: Current US FDA cleared stool based colorectal cancer (CRC) molecular screening tests include fecal immunochemical tests (FIT) for occult blood detection, but integration with nucleic acid-based biomarkers requires separate workflows, increasing complexity and cost. Objective To evaluate fecal hemoglobin alpha (HBA) mRNA as an RNA workflow compatible alternative to FIT, enabling streamlined multi-target molecular assays. Methods: Computational analysis of GTEx RNA-seq data identified blood-specific genes. Clinical validation used RT-qPCR on stool samples from 246 participants (CRC, advanced precancerous lesions [APL], non-APL, negatives). FIT analysis was performed in parallel using standard methods. Diagnostic performance was compared to FIT using random forest models and cross-validation. Results: HBA1/HBA2 mRNA was identified as the best mRNA biomarker for the detection of occult blood in stool because of their high blood specificity and abundance. In clinical samples, HBA mRNA Ct values were inversely correlated with FIT hemoglobin concentration (r =-0.44, p < 0.001) and achieved comparable CRC sensitivity (87.0% vs. 84.4%) at matched specificities (95.2%). Combining HBA mRNA with CRC-associated mRNA markers yielded superior APL sensitivity compared to FIT combining with CRC-associated mRNA markers (52.6% vs. 48.8%). Conclusion: Fecal HBA mRNA offers diagnostic accuracy comparable to FIT while simplifying workflows for nucleic acids-based CRC and APL screening.