DNA methylation analysis has become a powerful tool in neuropathology. Although DNA methylation-based classification usually shows high accuracy, certain samples cannot be classified and remain clinically challenging. We aimed to gain insight into these cases from a clinical perspective. To address, central nervous system (CNS) tumors were subjected to DNA methylation profiling and classified according to their calibrated score using the DKFZ brain tumor classifier (V11.4) as "≥ 0.84" (score ≥ 0.84), "0.3-0.84" (score 0.3-0.84), or "< 0.3" (score < 0.3). Histopathology, patient characteristics, DNA input amount, and tumor purity were correlated. Clinical outcome parameters were time to treatment decision, progression-free, and overall survival. In 1481 patients, the classifier identified 69 (4.6%) tumors with an unreliable score as "< 0.3". Younger age (P < 0.01) and lower tumor purity (P < 0.01) compromised accurate classification. A clinical impact was demonstrated as unclassifiable cases ("< 0.3") had a longer time to treatment decision (P < 0.0001). In a subset of glioblastomas, these cases experienced an increased time to adjuvant treatment start (P < 0.001) and unfavorable survival (P < 0.025). Although DNA methylation profiling adds an important contribution to CNS tumor diagnostics, clinicians should be aware of a potentially longer time to treatment initiation, especially in malignant brain tumors.

Peripheral metastases of glioblastoma (GBM) are very rare despite the ability of GBM cells to pass through the blood-brain barrier and be disseminated through the peripheral blood. Here, we describe a detailed genetic and immunological characterization of a GBM metastasis in the skeleton, which occurred during anti-PD-1 immune checkpoint therapy. We performed whole genome sequencing (WGS) and 850 K methylation profiling of the primary and recurrent intracranial GBM as well as one of the bone metastases. Copy number alterations (CNA) and mutational profiles were compared to known genomic alterations in the TCGA data base. In addition, immunophenotyping of the peripheral blood was performed. The patient who was primarily diagnosed with IDH-wildtype GBM. After the resection of the first recurrence, progressive intracranial re-growth was again detected, and chemotherapy was replaced by PD-1 checkpoint inhibition, which led to a complete intracranial remission. Two months later MR-imaging revealed multiple osseous lesions. Biopsy confirmed the GBM origin of the skeleton metastases. Immunophenotyping reflected the effective activation of a peripheral T-cell response, with, however, increase of regulatory T cells during disease progression. WGS sequencing demonstrated distinct genomic alterations of the GBM metastasis, with gains along chromosomes 3 and 9 and losses along chromosome 4, 10, and 11. Mutational analysis showed mutations in potentially immunologically relevant regions. Additionally, we correlated tumour-infiltrating lymphocyte and microglia presence to the occurrence of circulating tumour cells (CTCs) in a larger cohort and found a decreased infiltration of cytotoxic T cells in patients positive for CTCs. This study exemplifies that the tumour microenvironment may dictate the response to immune checkpoint therapy. In addition, our study highlights the fact that despite an effective control of intracranial GBM, certain tumour clones have the ability to evade the tumour-specific T-cell response and cause progression even outside of the CNS.

Group 3 medulloblastoma is one of the most aggressive types of childhood brain tumors. Roughly 30% of cases carry genetic alterations in MYC, SMARCA4, or both genes combined. While overexpression of MYC has previously been shown to drive medulloblastoma formation in mice, the functional significance of SMARCA4 mutations and their suitability as a therapeutic target remain largely unclear. To address this issue, we combined overexpression of MYC with a loss of SMARCA4 in granule cell precursors. Both alterations did not increase proliferation of granule cell precursors in vitro. However, combined MYC overexpression and SMARCA4 loss successfully induced tumor formation in vivo after orthotopic transplantation in recipient mice. Resulting tumors displayed anaplastic histology and exclusively consisted of SMARCA4-negative cells although a mixture of recombined and non-recombined cells was injected. These observations provide first evidence for a tumor-promoting role of a SMARCA4 deficiency in the development of medulloblastoma. In comparing the transcriptome of tumors to the cells of origin and an established Sonic Hedgehog medulloblastoma model, we gathered first hints on deregulated gene expression that could be specifically involved in SMARCA4/MYC driven tumorigenesis. Finally, an integration of RNA sequencing and DNA methylation data of murine tumors with human samples revealed a high resemblance to human Group 3 medulloblastoma on the molecular level. Altogether, the development of SMARCA4-deficient medulloblastomas in mice paves the way to deciphering the role of frequently occurring SMARCA4 alterations in Group 3 medulloblastoma with the perspective to explore targeted therapeutic options.

Risk stratification of children with ependymomas of the posterior fossa in current therapeutic protocols is mainly based on clinical criteria. We aimed to identify independent outcome predictors for this disease entity by a systematic integrated analysis of clinical, histological and genetic information in a defined cohort of patients treated according to the German HIT protocols.

TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6%±8.7%, respectively (p<0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89%±2% vs. 57.4%±1.8% (p<0.01)). In contrast, β-catenin mutation sensitized TP53 mutant cells to radiation (p<0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5%±1.5% in lithium treated cells vs. 56.6±3% (p<0.01)) accompanied by increased number of γH2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33%±8% for lithium treated cells vs. 27%±3% for untreated controls (p=0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.

CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients' quality of life.