Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma

Abstract

Background and aims Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings. Methods Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from ten patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or x-ray contrast solution on RNA quality. Results RNA was of variable quantity (100–1500 ng) and poor quality (Agilent RNA Integrity Number (RIN) <5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130bp. Differential gene expression by microarray analysis identified 1,140 up-regulated genes and 1,001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 upregulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3 and PNMA2 protein expression in cancer samples. Conclusions RNA isolated from biliary brushings, is suitable for molecular analysis of biliary diseases using qPCR and microarray.

Cholangiocarcinoma and dominant strictures in patients with primary sclerosing cholangitis

“…The incidence thereafter was approximately 1% per year in those with dominant strictures. The high rate of CC (particularly at presentation) highlights the difficult clinical problem of differentiating malignant from benign dominant biliary strictures and demonstrates the need for improved diagnostic tests such as genomic studies and fluorescence in situ hybridisation (FISH) analysis which has been shown to improve the diagnostic accuracy of biliary cytology sampling [21–22]. Those with new presentations of PSC with evidence of a dominant stricture require thorough evaluation in order to exclude the presence of super-imposed CC, with consideration of a full range of diagnostic tests at the time of ERCP, including exfoliative cytology, intraductal biopsy and cholangioscopy…”

Oncofetal long noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2

“…Mouse Pvt1 is an oncogene that was originally identified as a common retroviral integration site in murine leukemia virus (MLV)‐induced T lymphomas . The human PVT1 oncogene, which is located at 8q24.21, was found to be up‐regulated in a series of human tumors . However, the precise mechanism of lncRNA‐hPVT1 and the downstream molecules associated with its action have not been described.…”

Analysis of differentially expressed genes in malignant biliary strictures

“…Our analysis of the chip data from cholangiocarcinoma tissues (Chapman et al, 2012) identified several differentially expressed genes, including GSTA1 and GSTA3. According to the network analysis, these two genes interacted with CYP450 members and thus participate in the CYP450 exogenous chemical metabolism.…”

“…The data set GSE34166 (Chapman et al, 2012), including raw files and probe annotations, was downloaded from the Gene Expression Omnibus (GEO) database, which contains 10 biliary stricture samples, representing 4 benign and 6 malignant tissues (cholangiocarcinoma). Chip data were acquired by using the GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array.…”