Early Molecular Stratification of High-risk Primary Biliary Cholangitis

Abstract

High-risk primary biliary cholangitis (PBC), defined by inadequate response at one year to Ursodeoxycholic acid (UDCA), is associated with disease progression and liver transplantation. Stratifying high-risk patients early would facilitate improved approaches to care. Using long-term follow-up data to define risk at presentation, 6 high-risk PBC patients and 8 low-risk patients were identified from biopsy, transplant and biochemical archival records. Formalin-fixed paraffin-embedded (FFPE) liver biopsies taken at presentation were graded (Scheuer and Nakanuma scoring) and gene expression analysed using the NanoString® nCounter PanCancer Immunity 770-gene panel. Principle component analysis (PCA) demonstrated discrete gene expression clustering between controls and high- and low-risk PBC. High-risk PBC was characterised by up-regulation of genes linked to T-cell activation and apoptosis, INF-γ signalling and leukocyte migration and down-regulation of those linked to the complement pathway. CDKN1a, up-regulated in high-risk PBC, correlated with significantly increased expression of its gene product, the senescence marker p21^WAF1/Cip, by biliary epithelial cells. Our findings suggest high- and low-risk PBC are biologically different from disease outset and senescence an early feature in high-risk disease. Identification of a high-risk 'signal' early from standard FFPE tissue sections has clear clinical utility allowing for patient stratification and second-line therapeutic intervention.

Keywords: NanoString® nCounter PanCancer Immunity Panel; PBC; Prognosis; Stratification; UDCA.

Fig. 1

Principle component analysis (PCA) for transcriptional signatures in low-risk and high-risk early PBC. PCA displays sample clustering (low-risk PBC, high-risk PBC and controls) following identification of the top 57 differentially expressed genes by two-tailed t-test.

Fig. 2

Dendrogram of genes identified by PCA to most separate high- and low-risk PBC groups. Dendrogram generated from the 34 genes identified by t-test to most separate high- and low-risk PBC. Colours indicate scale of gene expression (red: up-regulation; blue: down-regulation).

Fig. 3

Pathway analysis and heatmap for high- and low-risk PBC.Pathway analysis of the top 8 pathways of differentially expressed genes for high- and low-risk PBC (red: up-regulation; blue: down-regulation).

Fig. 4

GeneMANIA pathway analysis of up-regulated genes in high- and low-risk PBC. Diagrams show GeneMANIA linked up-regulated gene-products that participate within the same biological pathway in (a) high-risk PBC and (b) low-risk PBC.

Fig. 5

p21^WAF1/Clp expression by biliary epithelial cells from time-zero, low-risk PBC, high-risk PBC and explanted liver biopsies. (a) Representative images generated by immunohistochemistry showing p21^WAF1/Clp expression by biliary epithelial cells from time-zero (T0), high-risk PBC, low-risk PBC and explanted liver biopsies. (b) Cumulative data of p21^WAF1/Clp expression by biliary epithelial cells from T0, high-risk PBC, low-risk PBC and explanted liver biopsies. One-way ANOVA (*p < 0.05, **p < 0.001).