Introduction
Anti-tumor necrosis factor (anti-TNF) therapy can be safely withdrawn in a subset of patients with long-standing quiescent Crohn’s disease. However, accurately identifying which patients belong to this subset remains challenging. This study aimed to identify mRNA profiles associated with relapse risk and prolonged remission after anti-TNF withdrawal in CD patients in microscopic remission. Here, we leveraged the NanoString 360 Immuno Panel, which provides significant advantages over bulk sequencing by offering highly multiplexed, sensitive, and automated gene expression analysis of the tumor microenvironment without enzymatic amplification to contrast mucosal immune signatures in patients with and without three-year relapse following anti-TNF withdrawal.
Aims & Methods
Patients in the CEASE cohort with mucosal biopsies at anti-TNF withdrawal who later relapsed were matched to those in sustained remission based on biopsy location (ileum or colon).1 For patients with a relapse, only biopsies corresponding to the site of disease activity at relapse were selected for mRNA analysis. Microscopic remission at baseline was confirmed by a blinded expert pathologist. Relapse was defined as endoscopically confirmed inflammation, while sustained remission was defined as no relapse during follow-up. 785 mRNA’s of the nCounter Host Response Profiling panel were measured, and corrected to the expression of household genes (IO 360 panel). The 3 most up- and downregulated relapse-associated mRNAs were identified by first selecting on significance (p<0.05), followed by ranking based on log2 fold change. mRNA levels were normalized to the cohort mean, and the score was calculated as the sum of the most upregulated divided by the sum of the most downregulated mRNA’s. Hence, a Cox regression and ROC analysis were performed to examine the relationship between the score and relapse.
Results
Eleven patients who relapsed were matched to 11 in sustained remission based on biopsy location (7 colon, 4 ileum). Median age was 40 years, 45% male, with median anti-TNF exposure of 5.2 years. Baseline characteristics were comparable between groups; of note, 6/11 (55%) in the relapse group vs 4/11 (36%) in sustained remission received concomitant immunomodulators (p=0.67). Median follow-up was 3.3 years (IQR 2.6) and median time to relapse 15.8 months (IQR 9.5). The score, based on the 3 most up- and downregulated mRNAs (IFITM1, PIK3CA, RBPJ, IRF3, THOP1, NEU1), was higher in the relapse group (median 2.94 (IQR 1.2-6.0) vs 0.36 (IQR 0.24-0.56), p = 0.001) and associated with relapse risk (HR per one-point 1.25, 95% CI 1.08–1.45). The AUC was 0.89, with the best cut-off of 0.98 based on the Youden index, resulting in a sensitivity of 0.83 and specificity of 1.
| mRNA | Mean relapse group | Mean sustained remission group | Log 2 fold change | p-value | Immunological function |
| IFITM1-mRNA | 5.09E+14 | 1.88E+14 | 1.43 | 0.012 | Regulates CD4+ T helper cell differentiation |
| PIK3CA-mRNA | 3.36E+14 | 1.28E+14 | 1.39 | 0.004 | Enhancing CD8+ T-cell action via increased IL-2 |
| RBPJ-mRNA | 4.22E+14 | 1.73E+14 | 1.28 | 0.032 | Critical regulator of T cell differentiation |
| NEU1-mRNA | 1.59E+14 | 3.70E+14 | -1.22 | 0.046 | Cleaves sialic acids from monocyte glycoproteins |
| THOP1-mRNA | 2.31E+14 | 5.97E+14 | -1.37 | 0.010 | THOP1-/- mice exhibit better survival in sepsis |
| IRF3-mRNA | 5.08E+13 | 1.70E+14 | -1.74 | 0.014 | Canonical innate immunity transcription factor |
Conclusion
This study identifies a specific mucosal mRNA profile associated with relapse risk after anti-TNF withdrawal in Crohn disease patients in clinical, endoscopic and microscopic remission. This profile reliably distinguishes high-risk patients in this case-control study but requires further validation. Furthermore, it provides insights in mucosal immune mechanisms, suggesting that strong innate immunity may protect against CD relapse, whereas adaptive immunity is associated with risk for relapse.
References
1. Ten Bokkel Huinink S, de Jong DC, Nieboer D, et al. Validation and update of a prediction model for risk of relapse after cessation of anti-TNF treatment in Crohn's disease. Eur J Gastroenterol Hepatol 2022;34:983-992.
