Novel transcriptomic panel identifies histologically active eosinophilic oesophagitis

Abstract

Background and aims: Eosinophilic oesophagitis (EoE) is characterised by symptoms of esophageal dysfunction and oesinophil tissue infiltration. The EoE Diagnostic Panel (EDP) can distinguish between active and non-active EoE using a set of 77 genes. Recently, the existence of distinct EoE variants featuring symptoms similar to EoE, such as oesophageal dysfunction but lacking eosinophil infiltration, had been determined.

Methods: We used oesophageal biopsies from patients with histologically active (n=10) and non-active EoE (n=9) as well as from healthy oesophageal controls (n=5) participating in the Swiss Eosinophilic Esophagitis Cohort Study (SEECS) and analysed the gene expression profile in these biopsies by total RNA-sequencing (RNA-seq). Moreover, we employed the publicly accessible RNA-seq dataset (series GSE148381) as reported by Greuter et al, encompassing a comprehensive genomic profile of patients presenting with EoE variants.

Results: A novel, diagnostic gene expression panel that can effectively distinguish patients with histologically active conventional EoE from patients with EoE in histological remission and control individuals, and from three newly discovered EoE variants was identified. Histologically Active EoE Diagnostic Panel (HAEDP) consists of 53 genes that were identified based on differential expression between histologically active EoE, histological remission and controls (p≤0.05). By combining the HAEDP with EDP, we expanded our knowledge about factors that may contribute to the inflammation in EoE and improved our understanding of the underlying mechanisms of the disease. Conversely, we suggested a compact group of genes common to both HAEDP and EDP to create a reliable diagnostic tool that might enhance the accuracy of EoE diagnosis.

Conclusion: We identified a novel set of 53 dysregulated genes that are closely associated with the histological inflammatory activity of EoE. In combination with EDP, our new panel might be a valuable tool for the accurate diagnosis of patients with EoE as well as for monitoring their disease course.

Keywords: oesophageal disease; oesophageal disorders; oesophagitis.

Figure 1

The Histologically Active EoE Diagnostic Panel (HAEDP) differentiates patients with histologically active eosinophilic oesophagitis (EoE) from patients with EoE in remission and control individuals. (A) Principal component analysis visualises the geometric distance between samples. Each dot corresponds to a patient sample. (B) Graphical representation of the differentially expressed genes in the analysis comparing (a) controls with EoE AF−, (b) EoE RF− with EoE AF− and (c) EoE RF+ with EoE AF+ (adjusted p≤0.05). (C) Venn diagrams of downregulated (log2FC ≤−2) and upregulated (log2FC ≥2) genes differentially expressed in histologically active EoE compared with controls and EoE in histological remission. Intersections of both Venn diagrams correspond to the HAEDP (controls n=5, EoE AF(+) n=5, EoE AF(−) n=5, EoE RF(+) n=4, EoE RF(−) n=5). FC, fold change.

Figure 2

The Histologically Active EoE Diagnostic Panel (HAEDP) assesses disease severity in patients with histologically active eosinophilic oesophagitis (EoE). (A) Principal component analysis visualises the geometric distance between samples after applying the HAEDP gene list. Each dot corresponds to a patient. (B) The overall disease activity score assessed by the physician was determined for the EoE1 and EoE2 subgroups (*p≤0.05 using non-parametric t-test analysis). Hierarchical heatmaps of 46 upregulated (C) and 7 downregulated (D) genes of HAEDP in patients with histologically active EoE samples. Each column represents a patient, and each row represents a gene. Hierarchical clustering was used to analyse the data, and distance methods were set as ‘Euclidean’ (controls n=5, EoE AF(+) n=5, EoE AF(−) n=5, EoE RF(+) n=4, EoE RF(−) n=5).

Figure 3

The Histologically Active EoE Diagnostic Panel (HAEDP) and EoE Diagnostic Panel (EDP) share a common gene list. (A) Graphical representations of the number of genes overlapping between the EDP identified by Wen et al and the HAEDP. Seventeen genes were common to both panels and visualised as significantly downregulated in histologically active eosinophilic oesophagitis (EoE) (B), significantly upregulated in histologically active EoE (C) and upregulated but significantly not different in histologically active EoE (D). In addition, 36 genes were uniquely found in HAEDP, and 60 genes were uniquely found in EDP. Data are represented as the normalised counts mean±SEM. *P≤0.05, **p≤0.01, ***p≤0.001 using one-way analysis of variance test followed by Holm-Šídák’s multiple comparisons test (controls n=5, EoE AF(+) n=5, EoE AF(−) n=5, EoE RF(+) n=4, EoE RF(−) n=5). IGFL1, IGF-like family member 1.

Figure 4

Combined EoE Diagnostic Panel (EDP) and Histologically Active EoE Diagnostic Panel (HAEDP) precisely differentiated histologically active eosinophilic oesophagitis (EoE). Hierarchical heat diagram of genes of the EDP and HAEDP that are upregulated (A) and downregulated (B) in patients with EoE. Each column in the diagram represents a patient, and each row represents a gene. The genes are clustered by panels: white represents genes exclusive to EDP only, yellow represents genes exclusive to HAEDP only and purple represents genes common to EDP and HAEDP (controls n=5, EoE AF(+) n=5, EoE AF(−) n=5, EoE RF(+) n=4, EoE RF(−) n=5).

Figure 5

Combined EoE Diagnostic Panel (EDP) and Histologically Active EoE Diagnostic Panel (HAEDP) detect conventional eosinophilic oesophagitis (EoE) among EoE variants. Hierarchical heatmap of genes of the EDP and HAEDP applied on the RNA-sequencing dataset from Greuter et al : (A) upregulated genes and (B) downregulated genes (C) principal component analysis after applying HAEP and EDP panels to visualise the distribution of the samples. (D) List of genes upregulated only in conventional EoE as compared with other EoE variants and control. Data are the normalised counts mean±SEM. *P≤0.001 using one-way analysis of variance test followed by Holm-Šídák’s multiple comparisons test (controls n=7, conventional EoE n=10, GERD n=6, EoE-like disease n=13, lymphocytic EoE n=5, non-specific EoE n=10).