Cholangiocarcinogenesis is a developmental process driven by distinct sequential alterations of the cellular transcriptome

Background and aims:

As in other organs, carcinogenesis of bile duct tumors today is considered a multistep process, morphologically reflected in the existence of two histologically defined precursor lesions, biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasia of the bile duct (IPNB). However, current knowledge on the molecular alterations in cholangiocarcinogenesis is scarce and no information is available regarding the chronological sequence of the changes involved. By deciphering the course of transcriptional alterations in cholangiocarcinogenesis, this study aimed to identify early, potentially driving genetic events.

Methods:

Of 12 patients with distal cholangiocarcinoma (dCCA), non-neoplastic biliary epithelium (NB), high-grade BilIN (BilIN-3) or IPNB, and invasive tumor was isolated by laser capture microdissection of FFPE tissue sections resulting in three matched sample sets for both BilIN and IPNB (n = 36, respectively). Total RNA was extracted using the RNeasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and gene expression of a panel of 770 cancer-relevant genes was assessed quantitatively using the nCounter® System (NanoString Technologies). Significantly dysregulated mRNAs were validated by quantitative RT-PCR. To correct for multiple testing, the false discovery rate (FDR) was controlled at 0.1 using the Benjamini-Hochberg procedure.

Results:

RNA was obtained in sufficient quantity and adequate quality from the BilIN matched sample set. We identified 180 genes with significantly altered expression from non-neoplastic tissue to invasive dCCA, of which WNT10A, COL5A1 and COL1A2 ranked highest (each p < 0.0001). Of these, a subset of 126 genes demonstrated gradual up- or downregulation. Notably, the three Wnt-signaling pathway proteins WNT5B (fold change: NB vs. BilIN 2.4, BilIN vs. dCCA 2.2), WNT10A (NB vs. BilIN 3.6, BilIN vs. dCCA 7.9) and AXIN2 (NB vs. BilIN 3.1, BilIN vs. dCCA 1.9) were among the proportionally strongest, sequentially upregulated genes. The three genes with most prominent gradual downregulation were FGF19 (dCCA vs. BilIN 47.2, BilIN vs. NB 2.5), BNIP3 (dCCA vs. BilIN 5.8, BilIN vs. NB 2.8) and PCK1 (dCCA vs. BilIN 3.8, BilIN vs. NB 2.4). Strikingly, gene expression of several collagen encoding genes was markedly augmented in the sequence from BilIN to dCCA (e.g. COL1A1 and COL1A2, p < 0.0001, fold change > 30). First in vitro experiments confirmed robust protein expression of WNT5B and WNT10A in different cholangiocarcinoma cell lines by Western-Blot analysis.

Conclusion:

Here, we show that multistep cholangiocarcinogenesis is accompanied by distinct sequential changes in the cellular transcriptome. In particular, we identified WNT5B, WNT10A and AXIN2 to be increasingly expressed during this process, indicating dysregulation of the Wnt-signaling pathway. Secondly, late carcinogenesis seems to be associated with a significant upregulation of collagen genes, possibly creating a tumor-promoting microenvironment. Next, we will analyze the IPNB matched sample set to evaluate the observed molecular alterations in comparison to BilIN. Building on this, in vitro and in vivo experiments will be performed with the ultimate aim to establish new options for targeted therapies in cholangiocarcinoma.