Array-based DNA methylation profiling of primary lymphomas of the central nervous system

Abstract

Background: Although primary lymphomas of the central nervous system (PCNSL) and extracerebral diffuse large B-cell lymphoma (DLBCL) cannot be distinguished histologically, it is still a matter of debate whether PCNSL differ from systemic DLBCL with respect to their molecular features and pathogenesis. Analysis of the DNA methylation pattern might provide further data distinguishing these entities at a molecular level.

Methods: Using an array-based technology we have assessed the DNA methylation status of 1,505 individual CpG loci in five PCNSL and compared the results to DNA methylation profiles of 49 DLBCL and ten hematopoietic controls.

Results: We identified 194 genes differentially methylated between PCNSL and normal controls. Interestingly, Polycomb target genes and genes with promoters showing a high CpG content were significantly enriched in the group of genes hypermethylated in PCNSL. However, PCNSL and systemic DLBCL did not differ in their methylation pattern.

Conclusions: Based on the data presented here, PCNSL and DLBCL do not differ in their DNA methylation pattern. Thus, DNA methylation analysis does not support a separation of PCNSL and DLBCL into individual entities. However, PCNSL and DLBCL differ in their DNA methylation pattern from non- malignant controls.

Figure 1

DNA methylation analyses in PCNSL as compared to hematopoietic controls and systemic DLBCL. (A) Principal Components Analysis (PCA) significantly separates control samples (blue circles) from PCNSL (orange circles) and systemic DLBCL (yellow circles) (p = 1.2 × 10^-12; q = 3.9157 × 10^-11) [40,41]. (B) Heatmap generated by applying the same conditions as in (A) red: high methylation level, green: low methylation level; blue squares: hematopoietic controls, orange squares: PCNSL; Hematopoieteic controls have been analysed in replicates and are shown as individual samples demonstrating good reproducibility (C) Hierachical cluster analysis of DNA methylation data obtained from the 296 CpG loci (corresponding to 194 genes) differentially methylated between five PCNSL (orange) and 10 hematopoietic controls (blue) including 49 systemic DLBCL (yellow boxes). CpGs differentially methylated in PCNSL and normal controls did not distinguish between PCNSL and systemic DLBCL. (D) Unsupervised hierarchical cluster analysis of methylation values of all 1,284 CpGs from 10 hematopoietic controls (blue boxes), five PCNSL (orange boxes) and 49 systemic DLBCL (yellow boxes). While normal and malignant samples were delineated according to their DNA methylation pattern, PCNSL and systemic DLBCL did not segregate.

Figure 2

Enrichment of PcG marks and HCP promotors in the group of genes methylated in PCNSL but unmethylated in non-neoplastic hematopoietic tissues. Bar plot of the different DNA methylation subsets showing percentage and absolute numbers (numbers below each diagram) of (A) genes containing PcG marks in ESCs and (B) genes with one, two or three PcG marks, respectively. The meP/umC group was significantly enriched for PcG target genes (p < 0.0001; Fisher's exact test) and genes containing all three PcG marks (p < 0.0001). "array" reflects the distribution of the respective genes on the GoldenGate Cancer Panel I array. (C) Comparison of the content of EED, SUZ12 and 3 mK27-H3 target genes in the different groups and the GoldenGate array. The content of genes containing the individual marks is comparable in PCNSL. (D) Bar plot showing the percentage of promoter subtypes according to their CpG content in the different DNA methylation subsets. Genes de novo methylated in PCNSL (meP/umC) predominantly had promoters with high CpG content (p < 0.0001). In contrast, genes having promoters with low CpG content were enriched in the group showing high methylation in PCNSL and normal controls (meL/meC; p < 0.0001).