A Role for CXCR3 Ligands as Biomarkers of Post-Operative Crohn's Disease Recurrence

Abstract

Background and aims: Crohn's disease [CD] recurrence following ileocolic resection [ICR] is common. We sought to identify blood-based biomarkers associated with CD recurrence.

Methods: CD patients undergoing ICR were recruited across six centres. Serum samples were obtained at post-operative colonoscopy. A multiplex immunoassay was used to analyse 92 inflammation-related proteins [Olink Proteomics]. Bayesian analysis was used to identify proteins associated with increasing Rutgeerts score. Identified proteins were used in receiver operating characteristic [ROC] analysis to examine the ability to identify CD recurrence [Rutgeerts score ≥i2]. Existing single cell data were interrogated to further elucidate the role of the identified proteins.

Results: Data from 276 colonoscopies in 213 patients were available. Median time from surgery to first and second colonoscopy was 7 (interquartile range [IQR] 6-9) and 19 [IQR 16-23] months, respectively. Disease recurrence was evident at 60 [30%] first and 36 [49%] second colonoscopies. Of 14 proteins significantly associated with Rutgeerts score, the strongest signal was seen for CXCL9 and MMP1. Among patients on anti-tumour necrosis factor drugs, CXCL9 and CXCL11 were most strongly associated with Rutgeerts score. Both are CXCR3 ligands. Incorporation of identified proteins into ROC analysis improved the ability to identify disease recurrence as compared to C-reactive protein alone: area under the curve [AUC] 0.75 (95% confidence interval [CI]: 0.66-0.82] vs 0.64 [95% CI 0.56-0.72], p = 0.012. Single cell transcriptomic data provide evidence that innate immune cells are the primary source of the identified proteins.

Conclusions: CXCR3 ligands are associated with CD recurrence following ICR. Incorporation of novel blood-based candidate biomarkers may aid in identification of CD recurrence.

Keywords: CXCL9; Rutgeerts score; biomarker.

Figure 1.

Change in Rutgeerts score from first colonoscopy [n = 202] to second colonoscopy [n = 74]. Rutgeerts scores i0 and i1 [non-recurrence] are shown in blue and green, respectively. Rutgeerts scores ≥i2 [recurrence] are shown in red.

Figure 2.

Association of inflammatory Olink panel protein analytes with Rutgeerts score. Posterior mean of protein-specific effects of Rutgeerts score [solid dots] and corresponding 95% credible intervals [error bars] are shown. Effect estimates represent average changes in per cent of mean protein value for each 1 unit increase in Rutgeerts score [e.g. moving from i1 to i2]. The proteins on the x-axis are arranged by effect estimate. Proteins significantly associated with increased Rutgeerts score [i.e. those with 95% credible intervals not overlapping zero] are highlighted in red.

Figure 3.

Interaction effect between current anti-TNF use and Rutgeerts score. [A] For each protein, coefficients for the interaction term between current anti-TNF use and Rutgeerts score are plotted [solid dots] along with corresponding 95% credible intervals [error bars]. Proteins with credible intervals not overlapping zero for the interaction effect, i.e. for which the effect of Rutgeerts score on protein values are statistically different between those who are currently on anti-TNF and those who are not on anti-TNF, are highlighted in blue on the x-axis. [B] The interaction effect of Rutgeerts score on protein values, with patients stratified according to current anti-TNF use. Only proteins for which an association with Rutgeerts score differs significantly when comparing patients currently on anti-TNF to those not on anti-TNF are presented. Effect estimates represent average changes in per cent of mean protein value for each 1 unit increase in Rutgeerts score [e.g. moving from i1 to i2]. Values from patients currently on anti-TNF are represented in red, and those from patients not currently on anti-TNF are represented in green. Only proteins with non-overlapping credible intervals in A are shown.

Figure 4.

Receiver operating characteristic [ROC] curve of [i] CRP, [ii] CXCL9 and [iii] CRP/CXCL9/MMP1/IL5/ST1A1 in identifying post-operative Crohn’s disease recurrence.

Figure 5.

Single cell RNA sequencing expression of resected ileal tissues summarizing cellular sources in the ileum of blood protein analytes. Heatmap showing single-cell expression of selected genes, derived from analysis of blood protein analytes, per cluster [rows] in each cluster [columns] in cells from 11 ileal CD tissue resection. Expression is scaled normalized RNA expression.