Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

Abstract

With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed nextgeneration sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrencefree survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While »70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, »70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.

A genomic case study of mixed fibrolamellar hepatocellular carcinoma

“…Conventional HCC is characterized by recurrent point mutations (including in TP53 , CTNNB1 , and TERT ), HBV integration sites, structural variants, and copy number variants (CNVs) affecting chromatin regulators and the TERT promoter, among others [ 6 – 8 ]. Overall, FL-HCCs show fewer large-scale chromosomal alterations and less frequent methylation of tumor suppressor promoters with methylation changes primarily near liver developmental genes when compared with conventional HCC [ 9 – 11 ]. Most notably, a novel DNAJB1:PRKACA fusion has been identified in association with pure FL-HCC but not in conventional HCC [ 9 , 11 – 13 ].…”

Fibrolamellar Carcinoma: Recent Advances and Unresolved Questions on the Molecular Mechanisms

“…52 However, a slightly lower methylation in FLC has also been reported by others with a third of these tumors hypomethylated and more aggressive as judged by size, the presence of microsatellites, vascular invasion, number of tumors, and overall survival. 32 Other tumors show what is called "methylator phenotype" an increased methylation on CpGrich domains. 53 An examination of the methylation status of tumor suppressor gene promoters in FLC compared with HCC and paired nontumor liver of each did not detect a methylator phenotype.…”

“…Finally, two tumor suppressor genes (DLUE7 and ZNF709) and eight putative tumor suppressor genes showed increased methylation. 32 A seventh unresolved question is why doesn't the immune system attack FLC? On the one hand, FLC is at the extreme low end of tumor mutational load.…”

“…A histological sample was not available in this specific case. proper enzymatic activity, the changes in the transcriptome, the segregation between FLC and HCC have since been reproduced in several papers, 10,[29][30][31][32][33] although in one study 33 the chimera was found in only 79%. In some studies, RNA-Seq analysis showed that in proposed cases of FLC that did not contain the DNAJB1-PRKACA fusion, the transcriptome clustered with cases of HCC rather than FLC, and these cases where eventually adjudicated as HCCs.…”

“…In some studies, RNA-Seq analysis showed that in proposed cases of FLC that did not contain the DNAJB1-PRKACA fusion, the transcriptome clustered with cases of HCC rather than FLC, and these cases where eventually adjudicated as HCCs. 10,32 The genomic data on the formation of the chimeric protein and the highly consistent changes in the transcriptome and proteome of the FLC tumor, relative to the adjacent normal tissue, offered several important insights into the disease. One important insight was that FLC was not a genetically inherited disease.…”