Tobias Goschzik

Supratentorial ependymomas of childhood carry C11orf95-RELA fusions leading to pathological activation of the NF-κB signaling pathway.

Methods). This fusion was not detectable in a large series of infratentorial and spinal ependymomas including myxopapillary ependymomas. It was also not detectable in ependymoblastomas, medulloblastomas or pineoblastomas. 14/19 samples from supratentorial ependymomas expressed this novel fusion transcript resulting in an N-terminal part of C11orf95 encoding 212aa of the hypothetical 678aa protein fused to relA that is thereby uncoupled from its normal upstream regulators. The c-terminal relA part of the putative fusion protein contains almost the full relA sequence; only the first three amino acids (encoded by exon 2) are deleted in-frame. The occurrence of the fusion did not seem to be correlated to a specific histology, although several but not all cases showed clear cell morphology, and the fusionpositive cases showed no predominant location within the supratentorium (supplementary Table 1). Interestingly, all five reLA fusion-negative samples were from female patients (two-tailed Fisher’s exact test, p = 0.03). The physiological function of C11orf95 is unclear. It has been described as a fusion partner of the gene coding the transcription factor Mkl2 in benign chondroid lipomas representing the molecular correlate of the t(11;16) (q13;p13) translocation found in these tumors [3, 5]. The breakpoint in chondroid lipomas is at exon 5, whereas it is at exon 2 in supratentorial ependymomas. RELA encodes relA (NF-κB3), a 65-kDa protein which interacts with IκB and p50 in the central signaling complex in the NF-κB pathway. After activation of cell surface receptors the signal is transmitted by the IKK complex that phosphorylates IκB and thereby controls the translocation of relA/p50 into the nucleus and transcription of specific target genes (reviewed in [4]). The key physiological function of NF-κB signaling is the orchestration of the inflammatory responses to both Clinical observations and studies on genetic alterations and gene expression indicated that supratentorial ependymomas differ from ependymomas of infratentorial or spinal location [6, 8]. To further elucidate the pathogenesis of supratentorial ependymomas, we performed paired-end rNA sequencing in 19 tumor samples from 18 patients (for clinical data, see supplementary Table 1). Mapping of the reads predicted a novel recurrent fusion mrNA between C11orf95, a gene with unknown function, and v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) encoding the relA p65 subunit of the central NF-κB complex. RELA is located approximately 1.9 Mbp telomeric from C11orf95 on the same chromosomal band 11q13. We confirmed the fusion by sanger sequencing of the cDNA (Fig. 1a; primer sequences, see supplementary

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

FGFR1 mutations in Rosette-forming glioneuronal tumors of the fourth ventricle.

Rosette-forming glioneuronal tumors (RGNTs) are rare glioneuronal tumors of the fourth ventricle region that preferentially affect young adults. Despite their histologic similarity with pilocytic astrocytomas (PAs), RGNTs do not harbor KIAA1549-BRAF fusions or BRAF mutations, which represent the most common genetic alteration in PAs. Recently, mutations affecting the hotspot codons Asn546 and Lys656 of fibroblast growth factor receptor 1 (FGFR1) have been described in PAs. They are considered to be the most frequent mechanism of mitogen-activated protein kinase activation, alternative to KIAA1549-BRAF fusion and BRAF mutations. To uncover possible molecular similarities between RGNTs and PAs, we performed a mutational study of FGFR1 in 8 RGNTs. An FGFR1 N546K mutation and an FGFR1 K656E mutation were found in the tumors of 2 patients. Notably, the patient with an FGFR1 K656E mutated RGNT had undergone a resection of a diencephalic pilocytic astrocytoma with pilomyxoid features 5 years before the discovery of the fourth ventricle tumor; the mutational analysis uncovered the presence of the same FGFR1 K656E mutation in the diencephalic tumor. These results indicate that, in addition to histologic similarities, at least a subgroup of RGNTs may show close molecular relationships with PAs. Whether FGFR1 mutated RGNTs represent a specific subset of this rare tumor entity remains to be determined.

PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway in papillary tumors of the pineal region.

Papillary tumors of the pineal region (PTPR) are recognized as a distinct entity in the World Health Organization classification of CNS tumors. Papillary tumors of the pineal region frequently show loss of chromosome 10, but no studies have investigated possible target genes on this chromosome. Chromosome 10 harbors the PTEN (phosphatase and tensin homolog) gene, the inactivation of which, by mutation or epigenetic silencing, has been observed in different brain tumors, including high-grade gliomas. In this study, we investigated copy number changes by molecular inversion probe (MIP) analysis and the mutational status of PTEN in 13 PTPR by direct sequencing. MIP analysis of 5 PTPR showed chromosome 10 loss in all cases. In addition, there were losses of chromosomes 3, 14, 22, and X, and gains of whole chromosomes 8, 9, and 12 in more than 1 case. One case had a homozygous PTEN deletion; and 2 point mutations in exon 7 of PTEN (G251D and Q261stop) were found. Immunohistochemistry revealed decrease or loss of the PTEN protein and increased expression of p-Akt and p-S6. These results indicated that PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway may play a role in the biology of PTPR. This evidence may lead to the possible use of PI3K/Akt/mTOR inhibitors in therapy for patients with PTPR.

Molecular stratification of medulloblastoma: comparison of histological and genetic methods to detect Wnt activated tumours

Wnt activation in medulloblastomas is associated with good outcome. Upfront testing and risk‐adapted stratification of patients will be done in future clinical studies. In a cohort of 186 paediatric medulloblastomas our aim was to identify the optimal methods in standard clinical practice to detect this subgroup.

Genomic Alterations of Adamantinomatous and Papillary Craniopharyngioma.

Craniopharyngiomas are rare histologically benign but clinically challenging neoplasms. To obtain further information on the molecular genetics and biology of craniopharyngiomas, we analyzed a cohort of 121 adamantinomatous and 16 papillary craniopharyngiomas (ACP, PCP). We extracted DNA from formalin-fixed paraffin-embedded tissue and determined mutational status of CTNNB1, BRAF, and DDX3X by Sanger sequencing, next generation panel sequencing, and pyrosequencing. Sixteen craniopharyngiomas were further analyzed by molecular inversion profiling (MIP); 76.1% of the ACP were mutated in exon 3 of CTNNB1 encoding for &bgr;-catenin and there was a trend towards a worse event-free survival in cases mutated at Thr41. Next generation panel sequencing of 26 ACP did not detect any recurrent mutations other than CTNNB1 mutations. BRAF V600E mutations were found in 94% of the PCP, but not in ACP. GISTIC analysis of MIP data showed no significant larger chromosomal aberrations but a fraction of ACP showed recurrent focal gains of chromosomal material, other cases showed loss in the chromosomal region Xq28, and a third group and the PCP had stable genomes. In conclusion, the crucial pathogenetic event appears to be WNT activation in ACP, whereas it appears to be activation of the Ras/Raf/MEK/ERK pathway by BRAF V600E mutations in PCP.

Biomarker-driven stratification of disease-risk in non-metastatic medulloblastoma: Results from the multi-center HIT-SIOP-PNET4 clinical trial

Purpose To improve stratification of risk-adapted treatment for non-metastatic (M0), standard-risk medulloblastoma patients by prospective evaluation of biomarkers of reported biological or prognostic significance, alongside clinico-pathological variables, within the multi-center HIT-SIOP-PNET4 trial. Methods Formalin-fixed paraffin-embedded tumor tissues were collected from 338 M0 patients (>4.0 years at diagnosis) for pathology review and assessment of the WNT subgroup (MBWNT) and genomic copy-number defects (chromosome 17, MYC/MYCN, 9q22 (PTCH1) and DNA ploidy). Clinical characteristics were reviewed centrally. Results The favorable prognosis of MBWNT was confirmed, however better outcomes were observed for non-MBWNT tumors in this clinical risk-defined cohort compared to previous disease-wide clinical trials. Chromosome 17p/q defects were heterogeneous when assessed at the cellular copy-number level, and predicted poor prognosis when they occurred against a diploid (ch17(im)/diploid(cen)), but not polyploid, genetic background. These factors, together with post-surgical tumor residuum (R+) and radiotherapy delay, were supported as independent prognostic markers in multivariate testing. Notably, MYC and MYCN amplification were not associated with adverse outcome. In cross-validated survival models derived for the clinical standard-risk (M0/R0) disease group, (ch17(im)/diploid(cen); 14% of patients) predicted high disease-risk, while the outcomes of patients without (ch17(im)/diploid(cen)) did not differ significantly from MBWNT, allowing re-classification of 86% as favorable-risk. Conclusion Biomarkers, established previously in disease-wide studies, behave differently in clinically-defined standard-risk disease. Distinct biomarkers are required to assess disease-risk in this group, and define improved risk-stratification models. Routine testing for specific patterns of chromosome 17 imbalance at the cellular level, and MBWNT, provides a strong basis for incorporation into future trials.

Dysembryoplastic Neuroepithelial Tumor of the Septum Pellucidum and the Supratentorial Midline: Histopathologic, Neuroradiologic, and Molecular Features of 7 Cases.

Dysembryoplastic neuroepithelial tumors (DNTs) are one of the most common epilepsy-associated low-grade glioneuronal tumors of the central nervous system. Although most DNTs occur in the cerebral cortex, DNT-like tumors with unusual intraventricular or periventricular localizations have been reported. Most of them involve the septum pellucidum and the foramen of Monro. In this study, we have described the neuroradiologic, histopathologic, and molecular features of 7 cases (4 female and 3 male; patient age range, 3 to 34 y; mean age, 16.7 y). The tumors, all localized near the supratentorial midline structures in proximity to the foramen of Monro and septum pellucidum, appeared in magnetic resonance imaging as well-delimited cystic lesions with cerebrospinal fluid-like signal on T1-weighted and T2-weighted images, some of them with typical fluid-attenuated inversion recovery ring sign. Histologically, they shared features with classic cortical DNTs but did not display aspects of multinodularity. From a molecular point of view the cases investigated did not show KIAA1549-BRAF fusions or FGFR1 mutations, alterations otherwise observed in pilocytic astrocytomas, or MYB and MYBL1 alterations that have been identified in a large group of pediatric low-grade gliomas. Moreover, BRAFV600E mutations, which so far represent the most common molecular alteration found in cortical DNTs, were absent in this group of rare periventricular tumors.

High-Resolution Genomic Analysis of Cribriform Neuroepithelial Tumors of the Central Nervous System

Abstract Cribriform neuroepithelial tumors (CRINET) are one of several recently characterized entities in the broad spectrum of solid tumors with SMARCB1-INI1 loss. This neoplasm seems to be exceedingly rare and displays unique neuropathologic and clinical features. To date, only a few cases of CRINET have been characterized from a molecular point of view. In this study, we investigated the molecular features of 3 cases of CRINET using multiplex ligation-dependent probe amplification and molecular inversion profiling approaches. Along with mutations and deletions of SMARCB1-INI1, molecular inversion profiling analysis revealed a stable genomic profile without significant large chromosomal changes. Focal alterations (gains) were observed in individual cases at chromosomes 4q12 (PDGFRA), 12q15 (MDM2), 7p15.1 (NPY), and 18q11.2 (CDH2). Genomic Identification of Significant Targets in Cancer analysis highlighted focal alterations, including gains at chromosomes 16q23.2 (MAF), 17q23 (AXIN2), and 8p12 (ADAM3A). No cases showed BRAFV600E or CTNNB1 mutations. These data indicate that CRINET present stable genetic features and lack alterations commonly identified in other pediatric brain tumors. Further studies are required to determine whether specific alterations and specific signaling pathways, in addition to SMARCB1-INI1, may be implicated in the biology of this rare tumor and whether there are additional molecular similarities between CRINET and atypical teratoid/rhabdoid tumors.

Medulloblastoma with extensive nodularity: a tumour exclusively of infancy?

Medulloblastoma with extensive nodularity (MBEN) represents a rare medulloblastoma (MB) variant usually occurring in the first 3 years of life and associated with a good outcome [1]. It was previously designated as ‘cerebellar neuroblastoma’ and differs from the more common nodular/desmoplastic medulloblastoma by expanded lobular architecture and advanced neurocytic differentiation. MBENs show SHH pathway activation and can occur in patients with germline mutations of Patched1 (PTCH1) or Suppressor of fused (SUFU) [1–3]. It is believed that these tumours are derived from external granule cell progenitor cells, similar to desmoplastic/ nodular medulloblastomas [4]. Although adult medulloblastoma may show frequent SHH activation, MBEN has never been reported in adult patients. A 39-year-old male patient developed ataxia. The MRI showed a 2.8 cm contrast-enhancing cerebellar midline lesion (Figure 1a). After incomplete resection, the neuropathological examination revealed a tumour with a predominant (about 80% of the tumour tissue) grape-like lobular architecture and presence of reticulin-free islands with a neuropil-like matrix (Figure 1b–c), positive for synaptophysin (Dako, Hamburg, Germany). These nodules contained small cells with neurocytic cytology (Figure 1d) strongly positive for NeuN (Millipore, Darmstadt, Germany) (Figure 1e). Focally, the tumour cells displayed a streaming pattern in the nodular areas. The internodular regions with increased cellularity displayed a dense reticulin meshwork. Here, the tumour cells showed hyperchromatic nuclei and scant cytoplasm. The tumour cells of these internodular areas expressed the low affinity neurotrophin receptor p75-NTR (Thermo-Scientific, Waltham, MA, USA) (Figure 1f), a known SHH target [5] and Yap-1 (Cell Signaling, Danvers, MA, USA) indicative of SHH pathway activation. Neither nuclear positivity for ß-catenin (Roche-Ventana, Darmstadt, Germany) nor nuclear accumulation of p53 (Dako) was found. No expression of cytokeratins (Bachem, Weil am Rhein, Germany) and neurofilament protein (Dako) was observed. The proliferation marker MIB-1 (Ki67) (Dako) stained more than 20% of tumour cells in the internodular areas but was almost negative within the nodular areas. After the histological diagnosis, the patient received combined radio-chemotherapy. Radiotherapy was administered to the entire neuraxis (single dose 1.6 Gy, 5 fractions/week, total dose 35.2 Gy) with a boost to the posterior fossa (single dose 1.8 Gy, 5 fractions/ week, total dose 19.8 Gy). The total cumulative dose was 55.0 Gy. After irradiation, the residual tumour was no longer detectable in MRI. After radiotherapy, the patient received chemotherapy (CCNU/cisplatin, since the 5th cycle CCNU/carboplatin). Actually, 11 months after diagnosis, the patient completed the six of the eight planned cycles of chemotherapy. Clinically, the patient showed a treatment-related polyneuropathy and high-frequency hearing loss in one ear. For further molecular characterization, genomic DNA was extracted from FFPE tissue using standard methods (DNeasy Kit, Qiagen, D€ usseldorf, Germany). A molecular inversion probe (MIP) analysis (Oncoscan v3, Affymetrix, Santa Clara, CA, USA) revealed, besides chr. 7q gain, no significant cytogenetic alterations or allelic imbalances (Figure 1g). In particular, there was no allelic loss of 9q (PTCH1) or 10q (SUFU). MYC, MYCN and GLI2 were not amplified. The mutational probes included in the MIP assay revealed the presence of a PIK3CA E545K (c.1633 g>a) mutation, which was further confirmed by direct sequencing (Eurofins MWG Operon, Ebersberg, Germany) (not shown). Conversely, no TP53 allelic losses or mutations were evident in the MIP assay. The mutational analysis of exon 6 of Smoothened (SMOH) showed presence of an activating L412F mutation (Figure 1h). Medulloblastoma is the most common malignant intracranial tumour in childhood but can also be observed, more rarely, in adults [1]. Like MB in infants and small children, adult MBs show frequent activation of the SHH signaling pathway, usually caused by somatic mutations in PTCH1 or SMOH, [6]. Despite