Combined hepatocellular-cholangiocarcinoma (HCC-CCA) is a rare liver tumor comprising histologic features of both HCC and CCA. Due to its heterogeneous nature, treatment of combined HCC-CCA is a significant clinical challenge and prognosis remains poor. Therefore, further understanding of the tumor biology underlying the individual subtypes of this mixed tumor is required to improve treatment stratification and optimize treatment strategies. This study sought to identify epigenetic regulation underlying gene expression patterns in the individual components of combined HCC-CCA. Formalin fixed paraffin embedded tumor specimens from 10 patients diagnosed with combined HCC-CCA were utilized in this study. Hematoxylin and eosin staining was performed for each sample, and regions representative of the individual HCC and CCA components were delineated by a pathologist. Unstained slides were cut and dissected to separate HCC and CCA components. DNA and RNA extraction was performed for each sample for DNA methylation (n = 8 HCC and 7 CCA) and gene expression (n = 8 HCC and 8 CCA) profiling via reduced representation bisulfite sequencing and RNA-seq, respectively. As expected, DNA methylation levels at transcription start sites (TSS) were negatively correlated with gene expression in all samples (Spearman’s rho = -0.133 to -0.546; p < 2.2 x 10-16). Samples did not cluster by tumor subtype when comparing genome-wide DNA methylation and gene expression patterns. Interestingly, of the 5 patients with DNA methylation data available for both subtypes, 4 clustered by patient as opposed to cancer subtype, suggesting similar epigenetic regulatory patterns arising from development in the same microenvironment and genetic background. Differential gene expression analysis resulted in the identification of 58 differentially expressed genes (DEGs) between the HCC and CCA subtypes (q-value < 0.05). In addition, a total of 474 differentially methylated regions (DMRs) were identified between the HCC and CCA subtypes (minimum difference > 25%; q-value < 0.05). Of these, 422 DMRs overlapped with 324 known genes, 1 of which (STK38L) displayed increased expression (log2 fold change = 4.39; q-value = 0.01) associated with hypomethylation of 2 regions (-34.86% and -27.47%; q-value < 1 x 10-38) in the CCA compared to HCC group. STK38L encodes a serine/threonine kinase involved in Hippo signaling, a highly conserved signaling pathway that functions as a key coordinator of tissue growth and homeostasis. In all, these results provide insights into the epigenetic regulatory patterns associated with the two components of combined HCC-CCA. Future studies may aim to understand the effects of epigenetic regulation on treatment response for this deadly disease.
American Association for Cancer Research Annual Meeting, March 29 - April 3 2019, Atlanta, GA