Herein, we report the validation results of a previously established methylation-based signature for SVZ contact of GBM [12]. To our knowledge, this is the first attempt to validate the reported SVZ methylation signature. The first step to verify the molecular classifier was the imaging assessment of SVZ contact. We chose a multi-reader, interdisciplinary approach to reduce misclassification biases. However, eleven cases (16.9%) had no agreement on SVZ contact. This is partly due to the definition of the SVZ, which, while formally standardized, still holds the potential for differing interpretation. Precise measurement is crucial as only a few millimeters differences can determine SVZ contact. Adeberg et al. also experienced a variability of 38% in the validation process of their findings [12]. However, only one rater assessed the imaging at three different time points, indicating an intraobserver variability in contrast to our interobserver variability. Such differences in MRI-based GBM SVZ contact assessment have been previously described, but the clinical implications are still ambiguous and need further clarification [13, 17].

In the neuroradiological assessment of high-grade gliomas usually two- or even three neuroradiologists assess the available imaging and find a consensus on tumor location and its spatial extension. This helps to improve the accuracy, consistency, and reliability of radiological assessment of gliomas. In addition, advanced imaging techniques such as diffusion tensor imaging, perfusion-weighted imaging, magnetic resonance spectroscopy, and positron emission tomography-computed tomography are becoming more regularly used. They can provide additional information about the spatial configuration of tumors [18]. Nevertheless, more precise and reliable methods are necessary to determine SVZ contact, given its potential role for risk stratification and future individualization of patient care [13, 19].

Next, we validated a previously described methylation-based signature to determine the tumors’ SVZ involvement and its role for OS [12]. The association between the MRI T1ce imaging rater assessment and SVZM was solid, with the signature showing a considerable performance concerning sensitivity and NPV. Moreover, the OS was reduced for T1-ce SVZ + and SVZM + compared to T1ce SVZ- and SVZM-. For methylation-based classification, we observed a prognostic separation, especially within the first year following diagnosis. Median OS was comparable between SVZ + (12.6 months) and SVZM + (13.0 months), hinting towards comparable prognostic separation. Consensus SVZ classification was time-independent – in contrast to SVZM – and thus conferred a more robust readout for this cohort. One can speculate that the close connection to the SVZ is indeed what enables these GBM to present such aggressive phenotypes. Besides, the SVZM signature yielded a high sensitivity and NPV, underlining the potential to utilize it as a screening tool for SVZ contact and to confirm the absence of it in case of corresponding imaging findings.

In our cohort, SVZM classified 81% of tumors as SVZ-associated, whereas in the original study, SVZM positive and negative GBM were equally distributed [12]. In most published studies that concentrate on MRI-based SVZ definition, the proportions of participants belonging to the SVZ-positive and SVZ-negative groups were relatively comparable, ranging from 54 to 70% [20, 21]. Barami et al. mentioned that 93 out of 100 of low- to high-grade glioma patients (53 GBM) had shown contact with the lateral ventricular wall based on MRI [22]. It is worth noting that in these studies, the criterion for defining SVZ contact was based on lateral contact with the lateral ventricles rather than contact with a 5 mm margin adjacent to the lateral ventricles. However, our findings suggest that a substantial proportion of GBM might be associated with the SVZ, with the limitation of potential sampling biases.

Adeberg et al. described an “epigenetic and transcriptional silencing of MAB21L2/LRBA in SVZM + tumors “, possibly resulting in transforming growth factor β activity and a dysregulated immune response. We were able to confirm the LRBA/BA21L2 locus as an important component of the SVZM signature. Adeberg et al. also noted a hypomethylation resulting in increased gene expression for 90% of the CpG that defines SVZM + [12]. It must be noted that some authors have reported that SVZ contact is not associated with any molecular signature, including methylation, while other authors have suggested specific changes in gene expression levels of GBMs with SVZ contact [23, 24]. Furthermore, a relationship may exist between driver mutations in both the healthy SVZ tissue and tumor tissue, suggesting that SVZ NSCs might be the source of GBM [6, 8]. Another area of interest is the role of cerebrospinal fluid (CSF) in GBM formation. As the SVZ is located close to the cerebrospinal fluid compartment, tumors contacting the SVZ could experience changes in their microenvironment and hence develop an altered methylation status. CSF has been shown to promote tumorigenic capacities in vitro and tumor growth in vivo and to cause transcriptomic changes in human GBM, leading to higher malignancy [25]. The altered DNA methylation signature in SVZ + tumors could possibly be connected to CSF exposure.

In our patient cohort, we noted that SVZ involvement was accompanied by a trend of larger tumor volume, worse KPS, and a higher incidence of multifocal tumor spread, which may all have negative implications for the observed OS (Suppl. Fig. 5). A plausible explanation for this observation is that larger tumors have a higher likelihood of contacting a specific area, in our case, the SVZ. Furthermore, the feasibility of surgical removal is hampered in large tumors where critical structures are more likely to be involved, which in turn results in worse performance status. Finally, infiltration of the ventricles is another factor that may occur in cases with SVZ contact, potentially exerting a negative influence on survival.

In our cohort, tumors with MRI-based SVZ involvement had a higher likelihood of presenting as multifocal at the date of diagnosis. This observation was also made by Lim et al., where 40% of SVZ-associated GBM presented as multifocal compared to 14% in the SVZ- group. Ahmadipour et al. showed similar results in their cohort in which 49% of SVZ + GBM were multifocal compared to 18% in the SVZ- group [26, 27]. Various studies have assessed the role of SVZ contact as a prognostic factor, with many of them confirming its negative association with survival [13, 28, 29].

DNA methylation analysis and the use of large-scale methylation arrays have enabled the classification of CNS tumors into different subtypes [10]. There is an ongoing search for genetic and epigenetic signatures that can help us to further understand the complex mechanisms involved in GBM formation. The described DNA methylome-based classifier of SVZ contact may be a valuable asset to improve diagnostic accuracy and patient stratification. This validation analysis provides further evidence on the potential utility of methylation signatures to improve patient and tumor stratification. Nevertheless, further evaluation with a higher number of patients who clearly show no signs of radiological SVZ involvement is needed to further validate its prognostic role.

This work has some limitations that should be considered when interpreting the results. The low number of cases that show no signs of SVZ involvement limits the generalizability of our findings. Multivariable analysis was limited due to the small number of patients and events. A much larger and more homogeneous patient population with balanced baseline characteristics and prospective study design is warranted to ultimately determine the validity of the DNA methylation-assisted classification of SVZ involvement in GBM. Finally, future research should also investigate ways to improve and tweak the SVZ signature given the potential for further refinement.