Page 80 - Molecular features of low-grade developmental brain tumours

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Previous studies on SEGA methylation have shown that SEGAs are a unique entity among CNS tumours 17,27,29. However, the molecular mechanisms targeted by methylation changes in SEGA have not been well studied yet. In the present study, we identified substantial methylation changes in SEGAs compared to periventricular control tissue that appeared to be independent of the TSC1/TSC2 mutation or other clinical information available. GO analysis showed an enrichment of the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. Previous gene expression studies on SEGA found differential expression of similar pathways, indicating that these pathways are already affected on DNA level and might therefore be important drivers in SEGA pathogenesis 16-18. Differential expression of genes related to the immune system and the ECM organization has also been seen in cortical tubers 17,40. Furthermore, several studies have documented dysregulation of inflammation and ECM organization related pathways in cortical tubers, suggesting that these processes might be conserved across TSC pathology 41-44. Therefore, it would be of interest to investigate if DNA methylation changes related to these processes are also present in cortical tubers and other TSC lesions.
The role of mTOR pathway activation due to loss of function mutations in TSC1/ TSC2 in TSC is well established. Furthermore, loss of heterozygosity (LOH) of TSC1 or TSC2 has been reported in approximately 80% of SEGAs and has also been found in other TSC hamartomas 15,17,30. In vitro experiments show that mTOR inhibitors can reduce cell size and cell proliferation of SEGA cells. Currently, mTOR inhibitors are amongst the treatment options for SEGA associated with TSC 45. Therefore, we aimed to investigate methylation changes on genes related to the mTOR signaling pathway. GO term analysis on the differentially methylated CpGs between SEGA and control tissue did not reveal the mTOR pathway as a principal target. Moreover, by directly investigating mTOR pathway related genes, we found only a small number of differentially methylated CpGs on 6/35 mTOR related genes, indicating that DNA methylation changes most likely do not contribute to the mTOR activation in SEGA.
Figure 6. Detection of SEGA subgroups based on histological markers and clinical data. a-e. ROC curves and scatterplots for detection of SEGA1 or SEGA2 based on CD3 (a), HLA-DP/DQ/DR (b), GFAP (c), MAP2 (d) and pS6 (e) positivity. The point in the ROC curve indicates the most optimal % of positivity to separate the SEGA1 and SEGA2 group, followed by the proportion of SEGA1 and SEGA2 that is correctly detected. Scatterplots show the spread between samples for the % of positive area for SEGA1 (blue) and SEGA2 (SEGA2a light red and SEGA2b dark red). The red line indicates the most optimal % of positivity to separate the SEGA1 and SEGA2 group based on the ROC curve. *p-value<0.05, **p-value<0.01, ***p-value<0.001, Mann-Whitney U test. f. Variable Importance plots obtained from Random Forest in R for detection of SEGA1 and SEGA2 based on the clinical data (Table 1). Each point represents the mean decrease Gini value, indicative of the importance of each variable. Variables are listed from most important to least important. Antiepileptic drugs were also categorized in 3 groups: GABA blockers (treatment GABA), valproic acid (treatment valproic acid) and sodium channel blockers (treatment sodium channels). g. Variable Importance plots obtained from Random Forest in R for detection of SEGA1, SEGA2a and SEGA2b based on the clinical data (Table 1). Each point represents the mean decrease Gini value, indicative of the importance of each variable. Variables are listed from most important to least important. Antiepileptic drugs were also categorized in 3 groups: GABA blockers (treatment GABA), valproic acid (treatment valproic acid) and sodium channel blockers (treatment sodium channels). h. Scatterplot showing no difference in tumour size (mm) in SEGA1 (blue) compared to SEGA2 (SEGA2a light red and SEGA2b dark red).