Comprehensive aptamer-based screen of 1317 proteins uncovers improved stool protein markers of colorectal cancer

Background: To screen and validate novel stool protein biomarkers of colorectal cancer (CRC). Methods: A novel aptamer-based screen of 1317 proteins was used to uncover elevated proteins in the stool of patients with CRC, as compared to healthy controls (HCs) in a discovery cohort. Selected biomarker candidates from the discovery cohort were ELISA validated in three independent cross-sectional cohorts comprises 76 CRC patients, 15 adenoma patients, and 63 healthy controls, from two different ethnicities. The expression of the potential stool biomarkers within CRC tissue was evaluated using single-cell RNA-seq datasets. Results: A total of 92 proteins were significantly elevated in CRC samples as compared to HCs in the discovery cohort. Among Caucasians, the 5 most discriminatory proteins among the 16 selected proteins, ordered by their ability to distinguish CRC from adenoma and healthy controls, were MMP9, haptoglobin, myeloperoxidase, fibrinogen, and adiponectin. Except myeloperoxidase, the others were significantly associated with depth of tumor invasion. The 8 stool proteins with the highest AUC values were also discriminatory in a second cohort of Indian CRC patients. Several of the stool biomarkers elevated in CRC were also expressed within CRC tissue, based on the single-cell RNA-seq analysis. Conclusions: Stool MMP9, fibrinogen, myeloperoxidase, and haptoglobin emerged as promising CRC stool biomarkers, outperforming stool Hemoglobin. Longitudinal studies are warranted to assess the clinical utility of these novel biomarkers in early diagnosis of CRC.