DNA methylation, combined with RNA sequencing, provide novel insight into molecular classification of chordomas and their microenvironment

ABSTRACT Chordomas are rare tumors of notochord remnants, occurring mainly in the sacrum and skull base. In spite of slow growth, they are highly invasive what makes the treatment challenging. Because of low incidence the molecular background of chordomas is poorly recognized. Our study aims to determine role of DNA methylation abnormalities in skull base chordomas including its role in deregulation of gene expression. We subjected 32 tumor and 4 normal nucleus pulposus (NP) samples to profiling of DNA methylation with EPIC microarrays and gene expression with RNAseq. Genome-wide DNA methylation analysis showed two distinct chordoma clusters (subtypes C and I) with different patterns of aberrant DNA methylation. C Chordomas are characterized by general hypomethylation with hypermethylation of CpG islands, while I chordomas are generally hypermethylated. These differences were reflected by distinct distribution of differentially methylated probes (DMPs). Differentially methylated regions were determined in each chordoma subtype indicating aberrant methylation in known tumor-related genes and regions encoding small RNAs in C chordomas. Correlation between methylation and expression was observed in minority of these genes. Upregulation of TBXT in chordomas appeared related to lower methylation at tumor-specific DMR in gene promoter. Gene expression-based clusters of tumor samples did not overlap with DNA methylation subtypes. Nevertheless, the subtypes substantially differ in transcriptomic profile that shows immune activation in I chordomas and enhanced proliferation in C chordomas. Immune enrichment in chordomas I was confirmed with deconvolution methods (cohesively based on methylation and transcriptomic data). Copy number analysis showed higher chromosomal instability in C chordomas. All but one have 9p deletion ( CDKN2A / B ) and downregulation of genes encoded in related chromosomal band. No significant difference in patients’ survival was observed between tumor subtypes, however, shorter survival was observed in patients with higher number of copy number alterations.