Distinct blood protein profiles associated with ileal and colonic ulcers in Crohn’s disease

Introduction
Ileal and colonic Crohn’s disease (CD) are nowadays considered as separate entities. Studies are needed to better characterise the biological specificities of these subphenotypes. In fine, this research should offer opportunities for the development of personalised medicine.

Aim
We aim to: 1) characterise the blood protein profiles associated with ileal and colonic ulcers; 2) find blood biomarker candidates more specifically associated with ileal or colonic ulcers and comparing their performance to CRP.

Methods
By combining different technologies (proximity extension assay, selected reaction monitoring and high-sensitivity turbidimetric immunoassay (hsCRP)), 207 serum proteins were measured in CD patients presenting no endoscopic lesions (endoscopic remission) (n=23), isolated ileal ulcers (n=17) or isolated colonic ulcers (n=16). To compare the endoscopic activity between the ileum and colon, and in order to obtain an assessment of the overall lesion burden, we used the Crohn’s disease endoscopic index (CDEIS) without dividing its total score (sum of each gut segment) by the number of inspected segments. This score was retrospectively calculated and called “total CDEIS”. The Wilcoxon-Mann-Whitney test was used for the differential analyses (isolated ileal ulcers vs endoscopic remission; isolated colonic ulcers vs endoscopic remission). From this analysis, proteins showing a differential abundance were selected and their abundance values were re-scaled to be comprised between 0 and 1. Then, those proteins were systematically combined by two using the product or the sum of their re-scaled values (n=30 combinations for the detection of ileal ulcers; n=306 combinations for the detection of colonic ulcers). The classification performance (isolated ileal ulcers vs endoscopic remission; isolated colonic ulcers vs endoscopic remission) of individual markers or their combinations were given by the area under the receiver operating characteristics curve (AUROC). The AUROC values were statistically compared to a random classifier (AUROC=0.5) and the AUROC of CRP using the bootstrap test (2000 replications).

Results
The median of the total CDEIS was higher in CD patients with isolated colonic ulcers than those with isolated ileal ulcers (23.1 vs 8.0). When compared to endoscopic remission, the presence of isolated ileal ulcers and isolated colonic ulcers were specifically associated with the level of 6 and 18 serum proteins, respectively: (high level: JUN, CNTNAP2; low level: FCRL6, LTA, CLEC4A, NTF4); (high level: hsCRP, IL6, APCS, CFB, MBL2, IL7, IL17A, CCL19, CXCL10, CSF3, IL10, CLEC4G, MMP12, VEGFA; low level: CLEC3B, GSN, TNFSF12, TPSAB1). No protein was associated with both isolated ileal ulcers and isolated colonic ulcers. All CD patients in endoscopic remission showed a normal level of hsCRP (<5 mg/L). Compared to CD patients in endoscopic remission, CD patients with isolated colonic ulcers showed an increased median level of hsCRP (1.3 vs 5.9 mg/L, p-value=0.0045) and this was not the case for CD patients presenting isolated ileal ulcers (1.3 vs 2.2 mg/L, p-value=0.13). hsCRP detected ileal and colonic ulcers with an AUROC of 0.64 (p-value=0.07) and 0.77 (p-value=0.001), respectively. To discriminate ileal ulcers from endoscopic remission, 20 proteins showed an AUROC significantly different than 0.5 and numerically higher than hsCRP (0.65-0.75 vs 0.64, non-significant): FCRL6, LTA, JUN, CLEC4A, CNTNAP2, NTF4, ITGB6, ITM2A, TNFSF12, APCS, IL17C, SERPINA3, CLEC4G, MASP1, CD83, PIK3AP1, LY75, C8G, CFB, TREM1. To discriminate colonic ulcers from endoscopic remission, 2 proteins showed an AUROC significantly different than 0.5 and numerically higher than hsCRP: CSF3 (0.85 vs 0.77, non-significant), IL7 (0.79 vs 0.77, non-significant). To discriminate ileal ulcers from endoscopic remission (AUROC), CLEC4A × LTA was the best combination when compared with hsCRP (0.82 vs 0.64, p-value=0.057). To discriminate colonic ulcers from endoscopic remission (AUROC), hsCRP × CSF3 was the best combination when compared with hsCRP (0.85 vs 0.77, p-value=0.020).

Conclusions
In CD patients, ileal and colonic ulcers were associated with distinct systemic responses. hsCRP showed a better capacity to detect patients with colonic than ileal ulcers. Some markers might help to detect ileal ulcers or to improve the ability of CRP for detecting colonic ulcers.