# High Accuracy Stool Biomarkers of Precancerous Colorectal Cancer Identified Using a 2000-Plex Immunoproteomic Screen

## TL;DR

This study identifies promising stool proteins that can detect early signs of colorectal cancer and precancerous lesions with high accuracy.

## Contribution

The largest immunoproteomic screen of stool proteins in colorectal cancer, identifying novel biomarkers with improved diagnostic accuracy.

## Key findings

- Stool proteins like fibrinogen and MMP-9 showed high accuracy in distinguishing CRC from healthy controls.
- Stool fibrinogen achieved a diagnostic accuracy of 86% for detecting advanced adenomas.
- Functional analysis linked CRC-related proteins to pathways like coagulation and inflammation.

## Abstract

Given the morbidity and mortality associated with colorectal cancer (CRC), novel biomarkers are clearly warranted, especially for early detection. An antibody-based screen of 2000 proteins was utilized to identify stool proteins that were elevated in patients with CRC, compared with healthy controls (HCs). Thirty-seven lead candidates were selected for ELISA validation in three independent cohorts comprised of CRC patients, advanced adenoma patients, and HCs, drawn from two different ethnicities. Of the 2000 proteins interrogated, 116 were differentially expressed in CRC stool, with 45 being elevated at twofold or higher; 37 of these proteins were selected for ELISA validation in three independent patient cohorts. Stool matrix metalloproteinase (MMP)-8, MMP-9, hemoglobin, Peptidoglycan Recognition Protein-S (PGRP-S), haptoglobin, and fibrinogen emerged as being most discriminatory for distinguishing CRC from HCs (area under the curve, 0.91–0.95), across cohorts and ethnicities, with several of these being significantly higher in more advanced stages of CRC. Stool fibrinogen, MMP-9, hemoglobin, MMP-8, and PGRP-S were the top 5 stool proteins with the highest accuracy for distinguishing advanced adenoma from HC, with stool fibrinogen topping the list with a receiver operating characteristic area under the curve value of 0.86. Functional pathway analysis revealed a significant over-representation of pathways related to antioxidant activity, integrin/receptor binding, cytokines, blood coagulation, and lipoprotein biosynthesis in patients with CRC compared with HC. Nuclear factor IC and IKZF2 were identified as key regulators of the molecular cascades over-represented in CRC. Stool fibrinogen, MMP-8, MMP-9, PGRP-S, haptoglobin, and myeloperoxidase emerge as promising biomarkers for distinguishing CRC/advanced adenomas/healthy stools, meeting or outperforming current yardsticks.

•First unbiased immunoproteomic screen of 2000 stool proteins in colorectal cancer (CRC), representing the largest such proteomic screen in this disease.•Thirty-seven lead candidates were selected for ELISA validation in three independent cohorts, representing the largest ELISA validation program involving CRC biosamples.•Report several stool biomarkers that detect advanced adenomas with significantly improved accuracy, with stool fibrinogen topping the list with a diagnostic accuracy of 86%, easily eclipsing the diagnostic performance of current tests.•Uncovered several stool proteins with improved diagnostic accuracy for CRC as well as stool proteins that discriminate CRC from advanced adenomas.

First unbiased immunoproteomic screen of 2000 stool proteins in colorectal cancer (CRC), representing the largest such proteomic screen in this disease.

Thirty-seven lead candidates were selected for ELISA validation in three independent cohorts, representing the largest ELISA validation program involving CRC biosamples.

Report several stool biomarkers that detect advanced adenomas with significantly improved accuracy, with stool fibrinogen topping the list with a diagnostic accuracy of 86%, easily eclipsing the diagnostic performance of current tests.

Uncovered several stool proteins with improved diagnostic accuracy for CRC as well as stool proteins that discriminate CRC from advanced adenomas.

The holy grail in colon cancer biomarker research is to identify biomarkers that can accurately detect early precancerous lesions, namely advanced adenomas. Here, we report several stool biomarkers that detect advanced adenomas with significantly improved accuracy, with stool fibrinogen topping the list with a diagnostic accuracy of 86%, easily eclipsing the diagnostic performance of current test. Further validation of these novel biomarkers in additional patient cohorts as well as in randomized clinical trials could significantly aid in early detection of CRC and better treatment for this leading cancer.