Background: Given the variable response to anti-TNF therapy in patients with Crohn’s disease (CD), we hypothesized there is a core biologic (inflammatory) signature associated with early response and endoscopic healing (EH). The primary objective was to identify plasma protein biomarkers associated with early biochemical remission (BioRem) and EH in children receiving anti-TNF therapy.

Methods: CD patients were enrolled (10/2019-12/2022) at four pediatric centers with longitudinal blood and stool specimens collected prior to the start of either infliximab (IFX) or adalimumab (ADAL) and throughout this observational study. At month3 (M3), fecal calprotectin remission (fCalRem) was defined as a fCal <250 µg/g and BioRem was defined as a fCal<250µg/g or CRP<.05g/dL. At month12 (M12), a research-only/mucosal healing colonoscopy was recommended and EH was defined as a Simple Endoscopic Score-CD (SES-CD)<3. Plasma protein abundance was determined by the aptamer based SOMAscan^TM (SomaLogic, Boulder, CO) at baseline (BL, n=80), M3 (n=78), and at M12 (n=79) from the CD patients and 30 healthy controls (HC).

Results: Eighty CD (63 IFX, 17 ADAL) were enrolled. During the one-year study, 58 patients had a colonoscopy, 10 declined endoscopy, 7 had surgery, and 5 stopped anti-TNF therapy. EH was achieved by 37/58 (64%, no difference by biologic). Aim1 was to identify a core CD proteome. Starting with 7312 protein analytes, we identified 2556 differently abundant proteins (1647, up, 909 down, Fig1a-b) between the 43 steroid-naïve CD patients (19 on prednisone, 18 on budesonide) and the 30 HC (FDR<0.05). In Fig1c-d, we highlight the identified biological processes and pathways. Aim2 was to identify a subset of BL and M3 CD core proteins (and pathways) that were differentially abundant between patients who achieved M3 fCalRem vs M3 fCalNonRem (Fig2a-d). Aim3 was to identify a predictive anti-TNF response signature from BL CD core proteins for M3 and M12 outcomes. As M3 BioRem was determined for all 80 patients (42/80, 52.5%), we assessed the change (delta) in protein abundance from BL to M3 in those who achieved BioRem. Of the 562 proteins that significantly changed between BL and M3, only 124 proteins changed in the BioRem cohort. Of these 124 proteins, we found 26 were statistically different between BioRem and BioNonRem at M3 and only 4/26 proteins (AGRN, SLC16A3, QSOX2, ADAMTS13) were differentially abundant at BL between BioRem and BioNonRem. Moreover, ADAMTS13^high abundance at BL was significantly associated with M3 fCalRem (OR 5.4 [1.4-27]) and M12 EH (OR 4.8 [1.2-21]).

Conclusions: We identified a core CD proteome and a BL protein signature that is associated with favorable outcomes. Additional analyses will aim to identify individual patient subsets who share a common proteome and achieve EH (controlling for a therapeutic exposure to the anti-TNF therapy).

Figure1. Plasma protein abundance was compared between 80 patients with Crohn’s disease (CD) and 30 healthy controls (HC) and in a subset of 43 prednisone/budesonide unexposed CD and the 30 HC to identify the pre-treatment CD core proteome of 2556 proteins (1647 upregulated, 909 downregulated). A). Hierarchical clustering of the core proteome was performed to produce the heatmap. B). The volcano plot demonstrates the proteins with the largest positive and negative fold-change between treatment-naïve CD and HC. C). The novel core proteome was submitted for ontological enrichment analysis (ToppCluster) with pathways (green) and biological processes (blue) presented. D). The table highlights key pathways and biological processes that were identified by the unbiased analysis of the treatment-naïve proteome.

Figure2. A). PCA plot was generated from Crohn’s disease (CD) patients who achieved end of induction (M3) fecal calprotectin remission (fCalRem, <250 µg/mL) vs. fCalNonRem using the patient’s baseline (pre-treatment) protein abundance. B). The proteome was submitted for ontological enrichment analysis (ToppCluster) with pathways (green) and biological processes (blue) presented for fCal remitters (baseline proteins). C). PCA plot was generated from CD patients who achieved M3 fCalRem vs. fCalNonRem using the patient’s M3 protein abundance. D). Enrichment analysis (ToppCluster) with pathways (green) and biological processes (blue) presented for fCal remitters (M3 proteins). Oval drawings highlight key groupings of patients in the PCA plots (A, C) and key biological processes/pathways (B, D).

Rebekah Karns

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