Identification of cancer-related genes FGFR2 and CEBPB in choledochal cyst via RNA sequencing of patient-derived liver organoids

by Yongqin Ye, Vincent Chi Hang Lui, Rosana Ottakandathil Babu, Zhongluan Wu, Weifang Wu, Patrick Ho Yu Chung, Kenneth Kak Yuen Wong, Bin Wang, Paul Kwong Hang Tam

Background

Choledochal cysts (CC) are congenital bile duct anomalies with 6–30% risk for developing bile duct cancer. However, the molecular mechanisms underlying cancer risk of CC are unknown. We sought to identify the gene expression changes underlying the cancer risk of CC patients.

Methods

Liver organoids (n = 51) were generated from liver/bile duct biopsies of CC (n = 7; type I) and hepatoblastoma (n = 5; HB: non-tumor & tumor) for RNA sequencing. Bioinformatics analysis was conducted to identify differentially expressed cancer-related genes in CC and controls. We compared CC with non-cancerous and cancerous controls, normal adjacent non-tumor region of hepatoblastoma (HB) liver as non-cancerous control and tumor region as non-CC cancer control (HB-tumor). Reverse transcription real-time quantitative PCR (RT-qPCR) verification and immunohistochemistry of selected genes was conducted in additional CC and HB liver biopsies.

Findings

HB non-tumor and HB tumor organoids displayed distinct gene expression profiles. Expression profiling separated CC organoids into two clusters, one overlapping with HB non-tumor and the other one with HB tumor organoids. Genes selected based on their log2FoldChange values for RT-qPCR verification in 31 CC and 11 HB non-tumor liver tissues revealed significantly elevated expression of FGFR2 in 7 and CEBPB in 2 CC liver tissues (CC vs HB: 4.082 vs. 0.7671, p<0.01; 2.506 vs. 1.210, p<0.01). Distinctive positive staining in bile ducts were seen in CC, HB tumor and non-tumor liver tissues for FGFR2 and CEBPB. Percentages of CEBPB-immuno-positive or FGFR2-immuno-positive bile duct cells in CC and HB-tumor liver were higher than that in HB non-tumor liver.

Interpretation

The study identified dysregulated genes related to cancer pathways in CC patients suggesting cancer risk. The findings suggest that the elevated expression of FGFR2 and CEBPB in liver may contribute to cancer development in CC patients.