Spatial heterogeneity in DNA methylation and chromosomal alterations in diffuse gliomas and meningiomas

Abstract

Adult-type diffuse gliomas and meningiomas are the most common primary intracranial tumors of the central nervous system. DNA methylation profiling is a novel diagnostic technique increasingly used also in the clinic. Although molecular heterogeneity is well described in these tumors, DNA methylation heterogeneity is less studied. We therefore investigated the intratumor genetic and epigenetic heterogeneity in diffuse gliomas and meningiomas, with focus on potential clinical implications. We further investigated tumor purity as a source for heterogeneity in the tumors. We analyzed genome-wide DNA methylation profiles generated from 126 spatially separated tumor biopsies from 39 diffuse gliomas and meningiomas. Moreover, we evaluated five methods for measurement of tumor purity and investigated intratumor heterogeneity by assessing DNA methylation-based classification, chromosomal copy number alterations and molecular markers. Our results demonstrated homogeneous methylation-based classification of IDH-mutant gliomas and further corroborates subtype heterogeneity in glioblastoma IDH-wildtype and high-grade meningioma patients after excluding samples with low tumor purity. We detected a large number of differentially methylated CpG sites within diffuse gliomas and meningiomas, particularly in tumors of higher grades. The presence of CDKN2A/B homozygous deletion differed in one out of two patients with IDH-mutant astrocytomas, CNS WHO grade 4. We conclude that diffuse gliomas and high-grade meningiomas are characterized by intratumor heterogeneity, which should be considered in clinical diagnostics and in the assessment of methylation-based and molecular markers.

Fig. 1. Overview of tumor purity estimations assessed by pathological evaluation based on histology, and four methylation-based methods; InfiniumPurify^,, PAMES, RF_absolute and RF_estimate.

Left; IDH-mutant glioma, middle; glioblastoma IDH-wildtype and right; meningioma. Each bar represents one biopsy and the biopsies for each patient are grouped together. Not analyzed tumor biopsies are marked with an X. The dashed line represents tumor purity of 70%.

Fig. 2. Evaluation of tumor purity across the methylation classes.

All samples were classified by the MNP methylation-based brain classifier (version 12.5) and (A) shows the distribution of tumor biopsies by the methylation class families and class calibrated scores. B Tumor purity estimations among methylation classes as estimated by histology, InfiniumPurify^,, PAMES, RF_absolute, and RF_estimate. Histopathology resulted in a more profound discrimination between the tumor methylation classes and control tissue class.

Fig. 3. Intratumor DNA methylation heterogeneity in methylation subtyping and differentially methylated positions (DMP) between biopsy pairs.

A Top panel: Tumor purity estimations based on histopathological evaluation. Each bar represents one biopsy and biopsies from the same patient are grouped together. X denotes a biopsy which could not be analyzed by histology. Middle panel: Biopsy grade based on 2021 WHO criteria. Bottom two panels: Methylation-based class family and subtype/subclass calibrated scores respectively from the MNP methylation-based brain classifier (version 12.5). B Intratumor DMPs found in the brain tumors before and after correcting for methylation artifacts of copy number alterations with SeSAMe. Intratumor DMPs in glioblastoma IDH-wildtype from some samples and the low-grade meningioma (GU-lgMNG) are from Wenger et al. GU-LGG lower-grade glioma. GU-HGG high-grade glioma. GU-hgMNG high-grade meningioma. Recurrent tumors are denoted with an R. Patients with subtype/subclass heterogeneity are marked with an asterisk (*) symbol.

Fig. 4. Inter- and intratumor heterogeneity in methylation-based biomarkers.

A Methylation age varies within IDH-mutant glioma, glioblastoma IDH-wildtype and high-grade meningioma. The dashed line represents y = x as a reference. B Stemness index differs more in glioblastoma IDH-wildtype and high-grade meningioma compared to IDH-mutant glioma. Methylation age in glioblastoma IDH-wildtype from some samples and low-grade meningioma (GU-lgMNG) are from Wenger et al. GU-LGG lower-grade glioma. GU-HGG high-grade glioma. GU-hgMNG high-grade meningioma. Recurrent tumors are denoted with an R. Each biopsy is represented by a circle and color coded according to patient identity.

Fig. 5. Copy number alterations inferred from methylation array data differed intratumorally in IDH-mutant glioma.

A Heterogeneous status of CDKN2A/B homozygous deletion was detected in one out of two patients with IDH-mutant astrocytoma, CNS WHO grade 4 (GU-LGG-90). B Left panel: Hematoxylin and eosin stain and Right panel: MRI scans of the tumors for localization of the biopsies (red dot). Scale bars: 50 µm.

Fig. 6. Copy number alterations inferred from methylation array data differed intratumorally in meningioma.

A Left panels: Heterogeneous status of MDM2 amplification was identified in the primary tumor of one out of seven high-grade meningioma patients (GU-hgMNG-14). Right panels: MDM2 status was confirmed by immunohistochemical staining of MDM2. B Left panels: Amplification of MDM2 was identified in all biopsies of the recurrent tumor (GU-hgMNG-14R). Right panels: immunohistochemical staining of MDM2. Recurrent tumors are denoted with an R. Scale bars: 50 µm.