A Novel Host Stool mRNA Extraction Method Improves Sensitivity of Detection for Colorectal Cancer and Precancerous Lesions

Abstract BACKGROUND Early detection of colorectal cancer (CRC) is essential for reducing disease-related mortality; however, the invasiveness and resource intensity of colonoscopy limit its widespread use in population screening. Although several non-invasive screening modalities are available, their sensitivity remains suboptimal, particularly for advanced precancerous lesions (APL). Stool-derived host RNA biomarkers represent a promising approach to improve screening performance. However, numerous technical challenges are associated with extracting and quantifying this RNA, including an abundance of PCR inhibitors, a high microbial background, and varying levels of RNA degradation. METHODS We developed an extraction method for the isolation of host RNA from stool with improved sensitivity of detection and better diagnostic performance than the conventional phenol-chloroform-based extraction. ECB-Extract ( E nrichment C apture By hybridization), uses hybridization capture of RNA under denaturing conditions by adding locked nucleic acid bases to the probes. The improved hybridization characteristics allow us to directly isolate host RNA from stool lysate. RESULTS ECB-Extract significantly improved PCR inhibitor removal, enabling scale-up of stool input and resulting in increased diagnostic sensitivity. We evaluated ECB-Extract against a conventional phenol-chloroform-based extraction method using the same panel of biomarkers in a pilot clinical cohort (N = 73). Using ECB-Extract, the sensitivity for detecting CRC and Advanced Precancerous Lesions (APL) was 96% and 50%, respectively, compared with 56% and 21% for the phenol-chloroform comparator at 96% specificity. In a larger cohort of 359 samples, ECB-Extract combined with a panel of the eight CRC-associated mRNA biomarkers achieved sensitivities of 95.7% for CRC and 51.2% for APL at a specificity greater than 90%. CONCLUSION These results demonstrate that ECB-Extract substantially improves stool host RNA extraction performance and, consequently, enhances diagnostic sensitivity for CRC and APL.