Integrative Discovery and Validation of Stool and Plasma Small RNA Biomarkers for Early Colorectal Cancer Detection
A.R. SANTOS D. 1,2,3,4, EIRAS M. 1,2,3, GONZALEZ-SANTOS M. 1,2, SOARES J. 1,2, CALDEVILLA R. 1,2,3, LARA-SANTOS L. 1,2,3,5,6, DINIS-RIBEIRO M. 2,4,7,8, BRANDÃO A. 2,9, LIMA L. 1,2
1 Experimental Pathology and Therapeutics Group; Portuguese Oncology Institute of Porto (IPO Porto), 4200-072, Porto, Portugal; 2 RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO-Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), Porto, Portugal; 3 Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313, Porto, Portugal; 4 Faculty of Medicine (FMUP), University of Porto, 4200-072, Porto, Portugal; 5 FF-I3ID, University Fernando Pessoa, 4249-004, Porto, Portugal; 6 Department of Surgical Oncology, Portuguese Oncology Institute of Porto (IPO-Porto), 4200-072, Porto, Portugal; 7 Precancerous Lesions and Early Cancer Management Group, Research Centre of IPO Porto (CI-IPOP), 4200-072, Porto, Portugal; 8 Department of Gastroenterology, Portuguese Oncology Institute of Porto, 4200-072 , Porto, Portugal; 9 Cancer Genetics Group, Research Center of IPO Porto (CI-IPOP), Portuguese Oncology Institute of Porto, 4200-072, Porto, Portugal
Background
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with prognosis strongly dependent on early detection. Current non-invasive screening tests, such as the fecal immunochemical test (FIT), demonstrate limited sensitivity for precancerous lesions, particularly high-grade dysplasia (HGD), and generate substantial false positives. Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), are detectable in stool and plasma and reflect tumorigenic processes, offering potential as non-invasive biomarkers. Both knowledge-driven candidate approaches and unbiased high-throughput discovery strategies may enhance early detection, yet their complementary roles have not been systematically explored.
Objectives
This study aimed to (i) evaluate literature-derived candidate miRNAs in stool and plasma for early detection of CRC and HGD, (ii) assess their potential as complementary tools to FIT-based screening, and (iii) identify novel stool- and plasma-derived sncRNA candidates using small RNA sequencing with integrative feature selection.
Methods
A two-phase approach was undertaken. In the candidate-based phase, four miRNAs (miR-21-5p, miR-92a-3p, miR-451a, miR-199a-5p), selected through a systematic literature review, were measured in plasma samples from 80 individuals and stool samples from 96 individuals undergoing colonoscopy, classified as no lesion (NL), low-grade dysplasia (LGD), high-grade dysplasia (HGD), or CRC. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves, sensitivity, specificity, and predictive values.
In the discovery phase, smallRNA sequencing was performed on stool and plasma samples from 36 individuals (equally divided across NL, LGD, HGD, and CRC groups). Candidate biomarker selection integrated differential expression analysis (DESeq2), gene-level area under the curve (AUC), Wilcoxon testing, and LASSO regression to prioritize sncRNAs with diagnostic potential.
Results
Candidate-based analyses demonstrated strong early detection performance. Stool-derived miRNA panels achieved sensitivities of 88% for CRC and 91% for HGD, rising to 96% for HGD when combined panels were applied. Circulating miRNAs outperformed FIT for HGD detection, with individual sensitivities exceeding 85%, and miR-21-5p demonstrating excellent CRC detection (AUC = 0.90). Negative miRNA results accurately excluded disease, with negative predictive values up to 98%, supporting their use in refining colonoscopy referrals after positive FIT. RNA-seq discovery identified 39 differentially expressed stool sncRNAs associated with colorectal lesions, with nine candidates prioritized through integrative selection. Plasma and stool analyses revealed distinct biomarker profiles, and 43 additional markers would have been missed using differential expression alone, highlighting the value of the integrative approach for early detection biomarker discovery.
Conclusions / Implications for practice or policy
These findings demonstrate that candidate-based miRNA evaluation and unbiased sncRNA discovery are complementary strategies for advancing early detection of colorectal cancer and precancerous lesions. Stool and plasma small RNA biomarkers offer strong non-invasive diagnostic potential, outperform FIT for HGD detection, and may refine screening pathways to reduce unnecessary colonoscopies. Future validation of RNA-seq–identified candidates may further improve early detection accuracy and facilitate translation into population-based CRC screening programs.