Evaluation and Validation of Plasma Proteins Using Two Different Protein Detection Methods for Early Detection of Colorectal Cancer




Bhardwaj, Megha
Gies, Anton
Weigl, Korbinian
Tikk, Kaja
Benner, Axel
Schrotz-King, Petra
Borchers, Christoph H.
Brenner, Hermann

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Objective: Plasma protein biomarkers could be an efficient alternative for population-based screening for early detection of colorectal cancer (CRC). The objective of this study was to evaluate and validate plasma proteins individually and as a signature for early detection of CRC. Methods: In a three-stage design, proteins were measured firstly by liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and later by proximity extension assay (PEA) in a discovery set consisting of 96 newly diagnosed CRC cases and 94 controls free of neoplasms at screening colonoscopy. Two algorithms (one for each measurement method) were derived by Lasso regression and .632+ bootstrap based on 11 proteins that were included in both the LC/MRM-MS and PEA measurements. Additionally, another algorithm was constructed from the same eleven biomarkers plus amphireglin, the most promising protein marker in the PEA measurements that had not been available from the LC/MRM-MS measurements. Lastly the three prediction signatures were validated with PEA in independent samples of participants of screening colonoscopy (CRC (n = 56), advanced adenoma (n = 101), and participants free of neoplasm (n = 102)). Results: The same four proteins were included in all three prediction signatures; mannan binding lectin serine protease 1, osteopontin, serum paraoxonase lactonase 3 and transferrin receptor protein 1, and the third prediction signature additionally included amphiregulin. In the independent validation set from a true screening setting, the five-marker blood-based signature including AREG presented areas under the curves of 0.82 (95% CI, 0.74–0.89), 0.86 (95% CI, 0.77–0.92) and 0.76 (95% CI, 0.64–0.86) for all, early and late stages CRC, respectively. Conclusion: Two different measurement methods consistently identified four protein markers and an algorithm additionally including amphiregulin, a marker measured by PEA only, showed promising performance for detecting early stage CRC in an independent validation in a true screening setting. These proteins may be potential candidates for blood-based tests for early detection of CRC.


The authors acknowledge the cooperation of gastroenterology practices and clinics in patient recruitment and of Labor Limbach in sample collection. They also thank Katja Butterbach, Katarina Cuk, Ulrike Schlesselmann, Sabine Eichenherr, Romana Kimmel and Rosa Orihuela Vicente for their excellent work in laboratory preparation of blood samples and Isabel Lerch, Susanne Köhler, Utz Benscheid, Jason Hochhaus, and Maria Kuschel for their contribution in data collection, monitoring, and documentation. C.H.B. and the University of Victoria-Genome British Columbia Proteomics Centre are grateful to Genome Canada and Genome British Columbia for financial support through the Genomics Innovation Network (project codes 204PRO for operations and 214PRO for technology development) and the Genomics Technology Platform (264PRO). C.H.B. is also grateful for support from the Leading Edge Endowment Fund, and the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, QC, Canada). C.H.B. is also grateful for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, QC, Canada).


colorectal cancer, early detection, screening, biomarkers, prevention, proximity extension assay, LC/MRM-MS, proteomics


Bhardwaj, M., Gies, A., Weigl, K., Tikk, K., Benner, A., Schrotz-King, P., Borchers, C. H., & Brenner, H. (2019). Evaluation and validation of plasma proteins using two different protein detection methods for early detection of colorectal cancer. Cancers, 11(10). https://doi.org/10.3390/cancers11101426