Abstract 3176: Improved colorectal cancer screening by new stool-based protein markers
Authors: Bosch Linda J. , De Wit Meike , Hiemstra Annemieke C. , Piersma Sander R. , Pham Thang V. , Oudgenoeg Gideon , Coupe Veerle , Scheffer George , Mongera Sandra , Terhaar Sive droste Jochim , Oort Frank , van Turenhout Sietze , Ben Larbi Ilhame , Mulder Chris , Carvalho Beatriz , Fijneman Remond JA , Jimenez Connie R. , Meijer Gerrit A. 
Published: July 2016

Abstract
Background
The fecal immunochemical test (FIT) is used in many countries for non-invasive screening for colorectal cancer (CRC), but its characteristics leave room for improvement. We aimed to identify novel stool-based protein markers in stool that outperform or complement hemoglobin in detecting CRC.
Materials and methods
A series of large scale proteomic discovery and validation was conducted on a total of 313 human stool specimens. A discovery set consisted of 22 stool specimens (12 CRC and 10 controls), and isolated proteins were analyzed by an in-dept mass spectrometry GeLC-MS/MS workflow. An external validation set consisted of 291 independent stool specimens (79 CRC, 40 advanced adenoma (AA), 43 nonadvanced adenoma (A) and 129 controls), and isolated proteins were analyzed by single-shot mass spectrometry (Q-Exactive). As proof of concept, commercially available meso scale discovery (MSD) assays were performed on an independent validation set of 72 FIT fluid samples set including 14 CRC, 16 AA, 18 A and 24 controls.
Results
Of the 468 human proteins quantified in the discovery set, 93 were significantly enriched in CRC vs controls (p&lt;0.05). Of these, 29 were significantly found enriched in CRC vs controls in the validation set (q&lt;0.05). CART analysis and exhaustive search revealed a combination of 4 proteins as the most optimal combination to differentiate CRC from controls. A logistic regression model of this combination of proteins detected CRC with a sensitivity of 73% as compared to 43% for hemoglobin (HBA1) alone at a fixed specificity of 95% (p = 0.00003). Another combination of 4 proteins was the most optimal combination to differentiate AA from controls, which showed a sensitivity of 48% vs 8% for hemoglobin (p = 0.0002) at fixed specificity of 95%. Evaluation of one of the markers on FIT fluid samples provided proof of concept that these proteins can be detected in FIT fluid and can significantly discriminate CRC from healthy controls (p &lt; 0.001).
Conclusion
Proteome profiling on stool samples revealed 29 validated proteins significantly enriched in CRC samples compared to controls. A panel of 4 complementary protein markers outperformed hemoglobin for detection of CRC as well as AA. Proof of concept for detecting the proteins in FIT fluids confirmed the high potential of these markers for screening purposes in a non-invasive and cost-effective manner.
Citation Format: Linda J. Bosch, Meike De Wit, Annemieke C. Hiemstra, Sander R. Piersma, Thang V. Pham, Gideon Oudgenoeg, Veerle Coupe, George Scheffer, Sandra Mongera, Jochim Terhaar Sive droste, Frank Oort, Sietze van Turenhout, Ilhame Ben Larbi, Chris Mulder, Beatriz Carvalho, Remond JA Fijneman, Connie R. Jimenez, Gerrit A. Meijer. Improved colorectal cancer screening by new stool-based protein markers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3176.