Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Abstract

Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

Keywords: ARID1A; DNA methylation; IDH; RNA sequencing; TCGA; cholangiocarcinoma; integrative genomics; lncRNAs; multi-omics; whole exome.

Figure 1. Somatic Alterations in Cholangiocarcinoma

(A and B) Significantly mutated genes identified using the MutSigCV algorithm, and additional genes with chromosomal alterations, hotspot mutations (red font), and possibly functional mutations, grouped by pathway. (A) TCGA analysis sample set (n = 38). (B) Additional sample set (n = 15). Left, mutation amount and percentage, plus epigenetic silencing for CDKN2A. Top, overall number of mutations per megabase. Bottom, mutation spectra signatures. Dashed yellow line in upper panel indicates median mutations/megabase.

Figure 2. mRNA Analysis of Cholangiocarcinoma Identified an IDH-Enriched Cluster

(A and B) Principal component analysis of RNA-seq expression data of 15,272 genes after exclusion of 541 normal liver genes. The heatmap in (A) shows the most strongly discriminant 973 genes (shared between the TCGA and the GSE26566 dataset) that define the three clusters. (B) Three-dimensional PCA plot of TCGA CCA samples. (C) Hierarchical clustering analysis of 40 samples from the CCA microarray dataset GSE26566, using the same 973 genes as in (A). Genes in heatmaps (A) and (C) are shown in the same order.

Figure 3. IDH Mutant Cancers Are Enriched for a High Mitochondrial Signature and Mitochondrial DNA Count and a Low Chromatin Modifier Signature

(A) GSEA analysis identified mitochondrial and chromatin modifier genes as significantly differentially expressed in the IDH-mutant-enriched mRNA cluster 1. Selected pathways are shown, omitting genesets that show a high degree of gene membership overlap with the displayed pathways. Full results are available in Table S6. NES, normalized enrichment score; size, geneset size. (B and C) Heatmaps of the most significant mitochondrial and chromatin-modifier genes for TCGA (B) and GSE26566 (C). TCGA samples are filtered for high purity (>0.65); unfiltered results are shown in Figure S9. (D and E) Quantification of mitochondrial signature (D) and mitochondrial DNA (E) count for different mutational subgroups, showing high-purity samples only. No subgroup by itself is significantly different from all other samples, indicating only enrichments and not exclusivity for high/low mitochondrial markers. Box and whisker plots show maximum and minimum bars. (F) Pan-cancer correlation analysis of mitochondrial and chromatin modifier genes. For each cancer, the genes on the x and y axes are the same and in the same order. Red signifies high positive Spearman correlation values; green denotes high negative values for each gene-gene comparison. C, chromatin modifiers; M, mitochondrial genes. Genes are listed in Table S8.

Figure 4. Cluster-of-Clusters Analysis of CCAs

(A) Unsupervised hierarchical clustering of copy number data. The clustering is performed on SCNAs that are determined by GISTIC analysis to be significantly altered, as shown in the lower heatmap. (B) Unsupervised hierarchical clustering of DNA methylation data. (C) The cluster-of-clusters analysis (COCA) was performed on the three platforms with the strongest degree of correlation (mRNA, copy number, and methyl-ation). Bottom, robustness scores indicating strength of cluster membership assignment for each sample. Selected information of interest is shown here; full clustering, genetic, clinical, and pathological data are available in Table S1.

Figure 5. ARID1A Is Hypermethylated and Has Low Expression in the IDH COCA

(A and B) TCGA methylation (A) and RNA-seq (B) data for 24 genes (36 probes) that show both IDH^mut-specific hypermethylation and downregulation in the IDH COCA subtype. (C and D) Methylation (C) and microarray (D) data for the same 24 genes and 36 methylation probes as in (A and B), in the publically available datasets GEO: GSE32079 and GSE26566. (E) Scatterplot of TCGA methylation and RNA expression values for ARID1A. (F) Location of the two ARID1A hypermethylated probes within the ARID1A promoter. ENCODE histone marks for H3K27Ac and H3K4me3 are shown.

Figure 6. Cross-Cancer Analysis Comparing TCGA Cholangiocarcinoma, HCC, and Pancreatic Adenocarcinoma

(A) Tumor map analysis incorporating mRNA, methylation, and copy number showing proximity of each sample. (B) COCA across miRNA, copy number, DNA methylation, RPPA, and Tumor Map for the three cancer types. Unsupervised clustering was performed within each data type across a cohort of 292 samples from CCA, HCC, and PDAC to derive cross-tumor subtypes (miRNA n = 4; copy number (CN) n = 5; DNA methylation (DNAmeth) n = 7; RPPA n = 6; tumor map n = 7; see Figure S6 for individual platform cluster solutions). Individual classification subtypes were then used as input for pan-tumor COCA analysis identifying three COCA classes (first bar; K1, light green; K2, dark blue; K3, light blue). Second annotation bar denotes histology type – CCA, HCCC, PDAC. Third bar indicates the CCA-specific subtype classification (ECC, METH2, Meth3, and IDH, cf. Figure 5). Fourth bar notes IDH1 mutation status (red, R172 mutations; blue, R132 mutations; black, other mutations). Fifth bar indicates samples with FGFR2 fusions. Sixth bar indicates CCA score, a median value of the 600 most-enriched genes in CCA (see C). The bottom heatmap indicates sample membership for each of the individual classification subtypes (black, subtype member; white, not a subtype member; gray, missing data). Each row is labeled by platform and subtype number. (C) Six hundred genes enriched in cholangiocarcinoma-like HCC.