DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Amplification of the PLAG-family genes-PLAGL1 and PLAGL2-is a key feature of the novel tumor type CNS embryonal tumor with PLAGL amplification.

Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0-14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/ß-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.

Prognostic impact of copy number alterations and tumor mutational burden in carcinoma of unknown primary.

INTRODUCTION: Chromosomal aberrations are known to drive metastatic spread, but their profile is still elusive in carcinoma of unknown primary (CUP). Therefore, the aim of this study was to characterize the chromosomal aberration pattern in CUP depending on histological and clinical features and to assess its prognostic impact together with chromothripsis, tumor mutational burden (TMB), microsatellite instability (MSI), and mutational profiles as potential prognostic biomarkers. METHODS: Chromosomal aberrations and chromothripsis were detected by methylation-based copy number variation (CNV) analysis, whereas TMB and MSI were calculated based on large next-generation sequencing (NGS) panels. Putative primaries were assigned by consensus between two independent oncologists. RESULTS: CNV losses varied depending on putative primaries and were more abundant in patients harboring TP53 mutations and/or deletions 17p. CNV loss was prognostically adverse in localized CUP treated with surgery and/or radiotherapy, but not in disseminated poor-risk CUP treated with palliative chemotherapy. CNV loss also worsened the prognosis in squamous cell CUP. Chromothripsis was detected in 18/59 (30.5%) patients without prognostic effect. TMB was highest in cases with MSI, squamous cell histology, and with lung, anal or cervical putative primaries. CONCLUSION: Overall, CNV, chromothripsis, TMB, and MSI profiles in CUP are reminiscent of biological characteristics known from other cancer entities without a unifying CUP-specific signature. Markedly, high-level CNV loss is an adverse predictive biomarker in localized but not disseminated chemotherapy-treated CUP. This implies that chromosomal losses drive CUP progression, but also increase susceptibility to chemotherapy, with both effects apparently leveling out in disseminated CUP.

Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis.

OBJECTIVE: A detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options. DESIGN: We analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling. RESULTS: Patients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but ROBO2 mutations occurred exclusively in invasive CCA and CTNNB1 mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin. CONCLUSION: Integrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.

Anaplastic ganglioglioma-A diagnosis comprising several distinct tumour types.

AIMS: Anaplastic ganglioglioma is a rare tumour, and diagnosis has been based on histological criteria. The 5th edition of the World Health Organization Classification of Tumours of the Central Nervous System (CNS WHO) does not list anaplastic ganglioglioma as a distinct diagnosis due to lack of molecular data in previous publications. We retrospectively compiled a cohort of 54 histologically diagnosed anaplastic gangliogliomas to explore whether the molecular profiles of these tumours represent a separate type or resolve into other entities. METHODS: Samples were subjected to histological review, desoxyribonucleic acid (DNA) methylation profiling and next-generation sequencing. Morphological and molecular data were summarised to an integrated diagnosis. RESULTS: The majority of tumours designated as anaplastic gangliogliomas resolved into other CNS WHO diagnoses, most commonly pleomorphic xanthoastrocytoma (16/54), glioblastoma, isocitrate dehydrogenase protein (IDH) wild type and diffuse paediatric-type high-grade glioma, H3 wild type and IDH wild type (11 and 2/54), followed by low-grade glial or glioneuronal tumours including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumour and diffuse leptomeningeal glioneuronal tumour (5/54), IDH mutant astrocytoma (4/54) and others (6/54). A subset of tumours (10/54) was not assignable to a CNS WHO diagnosis, and common molecular profiles pointing to a separate entity were not evident. CONCLUSIONS: In summary, we show that tumours histologically diagnosed as anaplastic ganglioglioma comprise a wide spectrum of CNS WHO tumour types with different prognostic and therapeutic implications. We therefore suggest assigning this designation with caution and recommend comprehensive molecular workup.

Gene expression profiling of Group 3 medulloblastomas defines a clinically tractable stratification based on KIRREL2 expression.

Medulloblastomas (MB) molecularly designated as Group 3 (Grp 3) MB represent a more clinically aggressive tumor variant which, as a group, displays heterogeneous molecular characteristics and disease outcomes. Reliable risk stratification of Grp 3 MB would allow for appropriate assignment of patients to aggressive treatment protocols and, vice versa, for sparing adverse effects of high-dose radio-chemotherapy in patients with standard or low-risk tumors. Here we performed RNA-based analysis on an international cohort of 179 molecularly designated Grp 3 MB treated with HIT protocols. We analyzed the clinical significance of differentially expressed genes, thereby developing optimal prognostic subdivision of this MB molecular group. We compared the transcriptome profiles of two Grp 3 MB subsets with various outcomes (76 died within the first 60 months vs. 103 survived this period) and identified 224 differentially expressed genes (DEG) between these two clinical groups (Limma R algorithm, adjusted p-value < 0.05). We selected the top six DEG overexpressed in the unfavorable cohort for further survival analysis and found that expression of all six genes strongly correlated with poor outcomes. However, only high expression of KIRREL2 was identified as an independent molecular prognostic indicator of poor patients' survival. Based on clinical and molecular patterns, four risk categories were outlined for Grp 3 MB patients: i. low-risk: M0-1/MYC non-amplified/KIRREL2 low (n = 48; 5-year OS-95%); ii. standard-risk: M0-1/MYC non-amplified/KIRREL2 high or M2-3/MYC non-amplified/KIRREL2 low (n = 65; 5-year OS-70%); iii. high-risk: M2-3/MYC non-amplified/KIRREL2 high (n = 36; 5-year OS-30%); iv. very high risk-all MYC amplified tumors (n = 30; 5-year OS-0%). Cross-validated survival models incorporating KIRREL2 expression with clinical features allowed for the reclassification of up to 50% of Grp 3 MB patients into a more appropriate risk category. Finally, KIRREL2 immunopositivity was also identified as a predictive indicator of Grp 3 MB poor survival, thus suggesting its application as a possible prognostic marker in routine clinical settings. Our results indicate that integration of KIRREL2 expression in risk stratification models may improve Grp 3 MB outcome prediction. Therefore, simple gene and/or protein expression analyses for this molecular marker could be easily adopted for Grp 3 MB prognostication and may help in assigning patients to optimal therapeutic approaches in prospective clinical trials.

Oligosarcomas, IDH-mutant are distinct and aggressive.

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Clinicopathologic and molecular analysis of embryonal rhabdomyosarcoma of the genitourinary tract: evidence for a distinct DICER1-associated subgroup.

Embryonal rhabdomyosarcoma (ERMS) of the uterus has recently been shown to frequently harbor DICER1 mutations. Interestingly, only rare cases of extrauterine DICER1-associated ERMS, mostly located in the genitourinary tract, have been reported to date. Our goal was to study clinicopathologic and molecular profiles of DICER1-mutant (DICER1-mut) and DICER1-wild type (DICER1-wt) ERMS in a cohort of genitourinary tumors. We collected a cohort of 17 ERMS including nine uterine (four uterine corpus and five cervix), one vaginal, and seven urinary tract tumors. DNA sequencing revealed mutations of DICER1 in 9/9 uterine ERMS. All other ERMS of our cohort were DICER1-wt. The median age at diagnosis of patients with DICER1-mut and DICER1-wt ERMS was 36 years and 5 years, respectively. Limited follow-up data (available for 15/17 patients) suggested that DICER1-mut ERMS might show a less aggressive clinical course than DICER1-wt ERMS. Histological features only observed in DICER1-mut ERMS were cartilaginous nodules (6/9 DICER1-mut ERMS), in one case accompanied by foci of ossification. Recurrent mutations identified in both DICER1-mut and DICER1-wt ERMS affected KRAS, NRAS, and TP53. Copy number analysis revealed similar structural variations with frequent gains on chromosomes 2, 3, and 8, independent of DICER1 mutation status. Unsupervised hierarchical clustering of array-based whole-genome DNA methylation data of our study cohort together with an extended methylation data set including different RMS subtypes from genitourinary and extra-genitourinary locations (n = 102), revealed a distinct cluster for DICER1-mut ERMS. Such tumors clearly segregated from the clusters of DICER1-wt ERMS, alveolar RMS, and MYOD1-mutant spindle cell and sclerosing RMS. Only one tumor, previously diagnosed as ERMS arising in the maxilla of a 6-year-old boy clustered with DICER1-mut ERMS of the uterus. Subsequent sequencing analysis identified two DICER1 mutations in the latter case. Our results suggest that DICER1-mut ERMS might qualify as a distinct subtype in future classifications of RMS.

Intimal sarcomas and undifferentiated cardiac sarcomas carry mutually exclusive MDM2, MDM4, and CDK6 amplifications and share a common DNA methylation signature.

Undifferentiated mesenchymal tumors arising from the inner lining (intima) of large arteries are classified as intimal sarcomas (ISA) with MDM2 amplification as their molecular hallmark. Interestingly, undifferentiated pleomorphic sarcomas (UPS) of the heart have recently been suggested to represent the cardiac analog of ISA due to morphological overlap and high prevalence of MDM2 amplifications in both neoplasms. However, little is known about ISAs and cardiac UPS without MDM2 amplifications and molecular data supporting their common classification is sparse. Here, we report a series of 35 cases comprising 25 ISAs of the pulmonary artery, one ISA of the renal artery and 9 UPS of the left atrium. Tumors were analyzed utilizing the Illumina Infinium MethylationEPIC BeadChip array, enabling copy number profile generation and unsupervised DNA methylation analysis. DNA methylation patterns were investigated using t-distributed stochastic neighbor embedding (t-SNE) analysis. Histologically, all ISAs and UPS of the left atrium resembled extra-cardiac UPS. All cases exhibited highly complex karyotypes with overlapping patterns between ISA and UPS. 29/35 cases showed mutually exclusive amplifications in the cell-cycle associated oncogenes MDM2 (25/35), MDM4 (2/35), and CDK6 (2/35). We further observed recurrent co-amplifications in PDGFRA (21/35), CDK4 (15/35), TERT (11/35), HDAC9 (9/35), and CCND1 (4/35). Sporadic co-amplifications occurred in MYC, MYCN, and MET (each 1/35). The tumor suppressor CDKN2A/B was frequently deleted (10/35). Interestingly, DNA methylation profiling (t-SNE) revealed an overlap of ISA and cardiac UPS. This "ISA" methylation signature was distinct from potential histologic and molecular mimics. In conclusion, our data reveal MDM4 and CDK6 amplifications in ISAs and UPS of the left atrium, lacking MDM2 amplification. We further report novel co-amplifications of various oncogenes, which may have therapeutic implications. Finally, the genetic and epigenetic concordance of ISAs and UPS of the left atrium further supports a shared pathogenesis and common classification.

Accurate calling of KIAA1549-BRAF fusions from DNA of human brain tumours using methylation array-based copy number and gene panel sequencing data.

AIMS: KIAA1549-BRAF fusions occur in certain brain tumours and provide druggable targets due to a constitutive activation of the MAP-kinase pathway. We introduce workflows for calling the KIAA1549-BRAF fusion from DNA methylation array-derived copy number as well as DNA panel sequencing data. METHODS: Copy number profiles were analysed by automated screening and visual verification of a tandem duplication on chromosome 7q34, indicative of the KIAA1549-BRAF fusion. Pilocytic astrocytomas of the ICGC cohort with known fusion status were used for validation. KIAA1549-BRAF fusions were called from DNA panel sequencing data using the fusion callers Manta, Arriba with modified filtering criteria and deFuse. We screened DNA methylation and panel sequencing data of 7790 specimens from brain tumour and sarcoma entities. RESULTS: We identified the fusion in 337 brain tumours with both DNA methylation and panel sequencing data. Among these, we detected the fusion from copy number data in 84% and from DNA panel sequencing data in more than 90% using Arriba with modified filters. While in 74% the KIAA1549-BRAF fusion was detected from both methylation array-derived copy number and panel sequencing data, in 9% it was detected from copy number data only and in 16% from panel data only. The fusion was almost exclusively found in pilocytic astrocytomas, diffuse leptomeningeal glioneuronal tumours and high-grade astrocytomas with piloid features. CONCLUSIONS: The KIAA1549-BRAF fusion can be reliably detected from either DNA methylation array or DNA panel data. The use of both methods is recommended for the most sensitive detection of this diagnostically and therapeutically important marker.

Genetic and epigenetic characterization of posterior pituitary tumors.

Pituicytoma (PITUI), granular cell tumor (GCT), and spindle cell oncocytoma (SCO) are rare tumors of the posterior pituitary. Histologically, they may be challenging to distinguish and have been proposed to represent a histological spectrum of a single entity. We performed targeted next-generation sequencing, DNA methylation profiling, and copy number analysis on 47 tumors (14 PITUI; 12 GCT; 21 SCO) to investigate molecular features and explore possibilities of clinically meaningful tumor subclassification. We detected two main epigenomic subgroups by unsupervised clustering of DNA methylation data, though the overall methylation differences were subtle. The largest group (n = 23) contained most PITUIs and a subset of SCOs and was enriched for pathogenic mutations within genes in the MAPK/PI3K pathways (12/17 [71%] of sequenced tumors: FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1)) and two with accompanying TERT promoter mutation. The second group (n = 16) contained most GCTs and a subset of SCOs, all of which mostly lacked identifiable genetic drivers. Outcome analysis demonstrated that the presence of chromosomal imbalances was significantly associated with reduced progression-free survival especially within the combined PITUI and SCO group (p = 0.031). In summary, we observed only subtle DNA methylation differences between posterior pituitary tumors, indicating that these tumors may be best classified as subtypes of a single entity. Nevertheless, our data indicate differences in mutation patterns and clinical outcome. For a clinically meaningful subclassification, we propose a combined histo-molecular approach into three subtypes: one subtype is defined by granular cell histology, scarcity of identifiable oncogenic mutations, and favorable outcome. The other two subtypes have either SCO or PITUI histology but are segregated by chromosomal copy number profile into a favorable group (no copy number changes) and a less favorable group (copy number imbalances present). Both of the latter groups have recurrent MAPK/PI3K genetic alterations that represent potential therapeutic targets.

Molecular profiling of pediatric meningiomas shows tumor characteristics distinct from adult meningiomas.

In contrast to adults, meningiomas are uncommon tumors in childhood and adolescence. Whether adult and pediatric meningiomas differ on a molecular level is unclear. Here we report detailed genomic analyses of 37 pediatric meningiomas by sequencing and DNA methylation profiling. Histologically, the series was dominated by meningioma subtypes with aggressive behavior, with 70% of patients suffering from WHO grade II or III meningiomas. The most frequent cytogenetic aberrations were loss of chromosomes 22 (23/37 [62%]), 1 (9/37 [24%]), 18 (7/37 [19%]), and 14 (5/37 [14%]). Tumors with NF2 alterations exhibited overall increased chromosomal instability. Unsupervised clustering of DNA methylation profiles revealed separation into three groups: designated group 1 composed of clear cell and papillary meningiomas, whereas group 2A comprised predominantly atypical meningiomas and group 2B enriched for rare high-grade subtypes (rhabdoid, chordoid). Meningiomas from NF2 patients clustered exclusively within groups 1 and 2A. When compared with a dataset of 105 adult meningiomas, the pediatric meningiomas largely grouped separately. Targeted panel DNA sequencing of 34 tumors revealed frequent NF2 alterations, while other typical alterations found in adult non-NF2 tumors were absent. These data demonstrate that pediatric meningiomas are characterized by molecular features distinct from adult tumors.

Clear cell meningiomas are defined by a highly distinct DNA methylation profile and mutations in SMARCE1.

Clear cell meningioma represents an uncommon variant of meningioma that typically affects children and young adults. Although an enrichment of loss-of-function mutations in the SMARCE1 gene has been reported for this subtype, comprehensive molecular investigations are lacking. Here we describe a molecularly distinct subset of tumors (n = 31), initially identified through genome-wide DNA methylation screening among a cohort of 3093 meningiomas, of which most were diagnosed histologically as clear cell meningioma. This cohort was further supplemented by an additional 11 histologically diagnosed clear cell meningiomas for analysis (n = 42). Targeted DNA sequencing revealed SMARCE1 mutations in 33/34 analyzed samples, accompanied by a nuclear loss of expression determined via immunohistochemistry and a decreased SMARCE1 transcript expression in the tumor cells. Analysis of time to progression or recurrence of patients within the clear cell meningioma group (n = 14) in comparison to those with meningioma WHO grade 2 (n = 220) revealed a similar outcome and support the assignment of WHO grade 2 to these tumors. Our findings indicate the existence of a highly distinct epigenetic signature of clear cell meningiomas, separate from all other variants of meningiomas, with recurrent mutations in the SMARCE1 gene. This suggests that these tumors may arise from a different precursor cell population than the broad spectrum of the other meningioma subtypes.

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

Recurrent fusions in PLAGL1 define a distinct subset of pediatric-type supratentorial neuroepithelial tumors.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Subgroup and subtype-specific outcomes in adult medulloblastoma.

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed.

PATZ1 fusions define a novel molecularly distinct neuroepithelial tumor entity with a broad histological spectrum.

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens.

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy-number profile with inactivation of TP53, PTEN, and RB1.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

Mosaic trisomy of chromosome 1q in human brain tissue associates with unilateral polymicrogyria, very early-onset focal epilepsy, and severe developmental delay.

Polymicrogyria (PMG) is a developmental cortical malformation characterized by an excess of small and frustrane gyration and abnormal cortical lamination. PMG frequently associates with seizures. The molecular pathomechanisms underlying PMG development are not yet understood. About 40 genes have been associated with PMG, and small copy number variations have also been described in selected patients. We recently provided evidence that epilepsy-associated structural brain lesions can be classified based on genomic DNA methylation patterns. Here, we analyzed 26 PMG patients employing array-based DNA methylation profiling on formalin-fixed paraffin-embedded material. A series of 62 well-characterized non-PMG cortical malformations (focal cortical dysplasia type 2a/b and hemimegalencephaly), temporal lobe epilepsy, and non-epilepsy autopsy controls was used as reference cohort. Unsupervised dimensionality reduction and hierarchical cluster analysis of DNA methylation profiles showed that PMG formed a distinct DNA methylation class. Copy number profiling from DNA methylation data identified a uniform duplication spanning the entire long arm of chromosome 1 in 7 out of 26 PMG patients, which was verified by additional fluorescence in situ hybridization analysis. In respective cases, about 50% of nuclei in the center of the PMG lesion were 1q triploid. No chromosomal imbalance was seen in adjacent, architecturally normal-appearing tissue indicating mosaicism. Clinically, PMG 1q patients presented with a unilateral frontal or hemispheric PMG without hemimegalencephaly, a severe form of intractable epilepsy with seizure onset in the first months of life, and severe developmental delay. Our results show that PMG can be classified among other structural brain lesions according to their DNA methylation profile. One subset of PMG with distinct clinical features exhibits a duplication of chromosomal arm 1q.

Infratentorial IDH-mutant astrocytoma is a distinct subtype.

Diffuse IDH-mutant astrocytic tumors are rarely diagnosed in the cerebellum or brainstem. In this multi-institutional study, we characterized a series of primary infratentorial IDH-mutant astrocytic tumors with respect to clinical and molecular parameters. We report that about 80% of IDH mutations in these tumors are of non-IDH1-R132H variants which are rare in supratentorial astrocytomas. Most frequently, IDH1-R132C/G and IDH2-R172S/G mutations were present. Moreover, the frequencies of ATRX-loss and MGMT promoter methylation, which are typically associated with IDH mutations in supratentorial astrocytic tumors, were significantly lower in the infratentorial compartment. Gene panel sequencing revealed two samples with IDH1-R132C/H3F3A-K27M co-mutations. Genome-wide DNA methylation as well as chromosomal copy number profiling provided further evidence for a molecular distinctiveness of infratentorial IDH-mutant astrocytomas. Clinical outcome of patients with infratentorial IDH-mutant astrocytomas is significantly better than that of patients with diffuse midline gliomas, H3K27M-mutant (p < 0.005) and significantly worse than that of patients with supratentorial IDH-mutant astrocytomas (p = 0.028). The presented data highlight the very existence and distinctiveness of infratentorial IDH-mutant astrocytomas that have important implications for diagnostics and prognostication. They imply that molecular testing is critical for detection of these tumors, since many of these tumors cannot be identified by immunohistochemistry applied for the mutated IDH1-R132H protein or loss of ATRX.