Correlation between DNA Methylation and Cell Proliferation Identifies New Candidate Predictive Markers in Meningioma

Abstract

Meningiomas are the most common primary tumors of the central nervous system. Based on the 2021 WHO classification, they are classified into three grades reflecting recurrence risk and aggressiveness. However, the WHO's histopathological criteria defining these grades are somewhat subjective. Together with reliable immunohistochemical proliferation indices, other molecular markers such as those studied with genome-wide epigenetics promise to revamp the current prognostic classification. In this study, 48 meningiomas of various grades were randomly included and explored for DNA methylation with the Infinium MethylationEPIC microarray over 850k CpG sites. We conducted differential and correlative analyses on grade and several proliferation indices and markers, such as mitotic index and Ki-67 or MCM6 immunohistochemistry. We also set up Cox proportional hazard models for extensive associations between CpG methylation and survival. We identified loci highly correlated with cell growth and a targeted methylation signature of regulatory regions persistently associated with proliferation, grade, and survival. Candidate genes under the control of these regions include SMC4, ESRRG, PAX6, DOK7, VAV2, OTX1, and PCDHA-PCDHB-PCDHG, i.e., the protocadherin gene clusters. This study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation and provides potential biomarkers bearing prognostic and therapeutic value for the clinical management of meningioma.

Keywords: Ki-67; MCM6; biomarkers; genome-wide DNA methylation; meningioma; methylome; proliferation signature; survival.

Figure 1

CNVs identified by DNAm in the cohort of 48 meningiomas. Each column represents individual patients ordered from left to right by increasing frequency of genetic alterations. Genetic alterations are ordered on the y axis from top to bottom by decreasing frequency of genetic alterations. CNV: copy-number variation, DNAm: DNA methylation.

Figure 2

Manhattan plot representing the association between DNAm at the CpG level and high-grade (>1) vs. low-grade meningiomas. The dashed and solid lines indicate FDR thresholds after p-value correction for genome-wide multitesting (Benjamini–Hochberg). FDR: false discovery rate.

Figure 3

UpSet plot summarizing the overlap between CpGs highly correlated with proliferation indices and grades in meningiomas. Every possible intersection is displayed (30 in total, including the 8 original hit lists shown on the bottom-left side). All intersections are inclusive. CpG regulatory contexts (gene and CGI annotations) are represented as proportions of barplot heights. Proportions of open-sea CpGs are displayed on CGI context bar plots. Average methylation levels are represented as boxplots of means (beta value) for each CpG of the intersected lists. Methylation differentials represent the range of each CpG beta value (max–min) and the densities of changes. DNAm: DNA methylation; methyl: methylation; CGI: CpG island; pos. correl: positive correlation; neg. correl: negative correlation; hypo: hypomethylation; hyper: hypermethylation; LI: labeling index.

Figure 4

DNAm proliferation signature in meningiomas. Hierarchical clustering heat map depicting 310 CpGs highly correlated between DNAm and three proliferative markers: mitotic index, Ki-67, and MCM6 expressions (metrics: Euclidean distance and complete linkage). CGI: CpG island; DNAm: DNA methylation; LI: labeling index; WHO: World Health Organization.

Figure 5

Manhattan plots representing the association between DNAm at the CpG level and survival. Upper panel: associations with progression-free survival (PFS). Lower panel: associations with overall survival (OS). Univariate Cox statistics (Wald test). p-value cutoffs running multivariate analyses on selected CpGs are indicated by the topmost dashed lines in each panel. For the PFS panel, the second dashed line represents the threshold for computing hit enrichments over chromosomic regions.