Adamantinomatous and papillary craniopharyngiomas are characterized by distinct epigenomic as well as mutational and transcriptomic profiles

Abstract

Introduction: Craniopharyngiomas (CP) are rare epithelial tumors of the sellar region. Two subtypes, adamantinomatous (adaCP) and papillary CP (papCP), were previously identified based on histomorphological and epidemiological aspects. Recent data indicates that both variants are defined by specific genetic alterations, and influenced by distinct molecular pathways and particular origins. The fact that CP is an uncommon tumor entity renders studies on large cohorts difficult and exceptional. In order to achieve further insights distinguishing CP variants, we conducted whole genome methylation (450 k array) and microarray-based gene expression studies in addition to CTNNB1 and BRAF mutation analysis using a comprehensive cohort of 80 adaCP and 35 papCP.

Results: BRAF V600E mutations were solely found in the papCP subgroup and were not detectable in adaCP samples. In contrast, CTNNB1 mutations were exclusively detected in adaCP. The methylome fingerprints assigned DNA specimens to entity-specific groups (papCP (n = 18); adaCP (n = 25)) matching perfectly with histology-based diagnosis, suggesting that they represent truly distinct biological entities. However, we were not able to detect within the adaCP group (including 11 pediatric and 14 adult cases) a significant difference in methylation signature by age. Integrative comparison of the papCP with the adaCP group based on differential gene expression and methylation revealed a distinct upregulation of Wnt- and SHH signaling pathway genes in adaCP.

Conclusions: AdaCP and papCP thus represent distinct tumor subtypes that harbor mutually exclusive gene mutations and methylation patterns, further reflected in differences in gene expression. This study demonstrates that DNA methylation analyses are an additional method to classify CP into subtypes, and implicates a role of epigenetic mechanisms in the genesis of the respective CP variants. Detection of tumor-specific signaling pathway activation enables the possibility of target-oriented intervention.

Fig. 1

Detection of BRAF V600E mutation in papCP. SSCP analyses revealed shifted bands (▸) only in papCPs (a). DNA extraction and subsequent Sanger sequencing confirmed a BRAF V600E mutation (b). Pyrosequencing was utilized in cases with only slight SSCP bands or less available DNA concentrations. Case pap26 exhibited a low (9.2 %) frequency of BRAFV600E mutation (c). Immunohistochemical staining using a mutation specific BRAF V600E antibody (clone VE1) revealed a positive staining of pap23 rated with ++ (d). sc = staining control; wt = wild type; NTC = non template control

Fig. 2

Methylation profiling of CP subtypes. The scatterplot (a) shows samples projected onto the first two principle components derived by applying PCA to the most variable 10,000 probes already selected for the clustering. Pediatric CP are marked with white circles and adult CP with black circles. Unsupervised consensus clustering of 450 k methylation data revealed two distinct and stable clusters corresponding to papCP and adaCP, respectively. The upper part of the figure shows the consensus matrix (brown) that displays the stability of the clusters, i.e. in all of the 1, 000 resampling iterations of the consensus clustering the same samples were assigned to the same two clusters (b). The lower part of the figure shows a heatmap of the methylation pattern of the 10,000 most variable CpG sites used for clustering. Below the heatmap the two clusters resulting from the consensus clustering of gene expression data is shown. Furthermore, the distribution of age, BRAF and CTNNB1 mutations across samples was added. The sample marked with a red dot represents a case with histologically unsure subtype classification

Fig. 3

Comparison of adaCP and papCP methylation and gene expression profile. Volcano plot (a) showing the difference in median methylation of genes between adaCP (n = 17) and papCP (n = 8) samples on the x-axis and the –log10 transformed p-values of the corresponding t-test results on the y-axis. The volcano plot on the right hand side shows the difference in mean gene expression of genes between adaCP and papCP samples on the x-axis and corresponding –log10 transformed t-test p-values on the y-axis. Genes significantly differentially methylated and differentially expressed are marked red. AXIN2, PTCH1 and GLI2, indicated in blue, are significantly hypomethylated and show an increased gene expression in adaCP samples. (b) Plotting of tumor specific AXIN2, GLI2 and PTCH1 methylation values (beta scale, x-axis) and gene expression values (log2, y-axis) revealed a significant different clustering of adaCP and papCP subtypes. Corresponding box plots clearly emphasize that there is an inverse correlation between gene expression and methylation