Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading ...necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding (t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5 years. The most frequent localization was the posterior fossa (74%). Deletions of
CDKN2A/B
(66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting
NF1
, followed by
BRAF
and
FGFR1
) and mutations of
ATRX
or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were
IDH1/2
wildtype. The
MGMT
promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than
IDH
wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of
CDKN2A/B
and
ATRX
, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.
Epithelioid glioblastoma (eGBM) is a newly defined and rare GBM variant in the current WHO 2016 classification. BRAF V600E mutation is overrepresented in these tumors and there is known some ...morphological overlap with anaplastic epithelioid PXA (ePXA). In order to further elucidate this diagnostic category, we molecularly characterized 64 pediatric and adult examples initially diagnosed as “eGBM.” Tumors were analyzed using array based methylation and direct sequencing of the BRAF and TERT genes. Our results demonstrated considerable molecular and clinical heterogeneity among eGBM cohort. Methylation patterns, copy number alterations, and mutational analysis data, in combination with clinical findings disclosed three different, well established tumor subtypes: (i) PXA‐like tumors with favorable prognosis, predominantly in children and young adults (38), (ii) IDHwt GBM‐like tumors with poor prognosis, mainly occurring in older adults, albeit with more frequent BRAF mutations (17), and (iii) RTK1 pediatric GBM‐like neoplasms of intermediate prognosis in children and young adults, associated with chromothripsis and frequent PDGFRA amplifications (9). We conclude that the histopathologically defined eGBM do not represent a single diagnostic entity, but rather at least three molecularly and biologically distinct categories. Therefore, additional molecular testing through genome‐wide molecular profiling is recommended to further stratify these rare cases.
The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, ...microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.
The “isomorphic subtype of diffuse astrocytoma” was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain ...infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric
MYB/MYBL1
-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of
MYBL1
or
MYB
(13/25, 52%). Gene fusions of
MYBL1
or
MYB
with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective
MYB
-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated
MYBL1
(54%) or
MYB
(23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of
MYB/MYBL1
-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent
MYBL1
and
MYB
alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric
MYB/MYBL1
-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.
Abstract
Background
Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor ...receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern.
Methods
Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas.
Results
EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations.
Conclusions
Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.
Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal ...chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.
The large diversity of central nervous system (CNS) tumor types in children and adolescents results in disparate patient outcomes and renders accurate diagnosis challenging. In this study, we ...prospectively integrated DNA methylation profiling and targeted gene panel sequencing with blinded neuropathological reference diagnostics for a population-based cohort of more than 1,200 newly diagnosed pediatric patients with CNS tumors, to assess their utility in routine neuropathology. We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%). Discrepant results by neuropathological WHO-based and DNA methylation-based classification (30%) were enriched in histological high-grade gliomas, implicating relevance for current clinical patient management in 5% of all patients. Follow-up (median 2.5 years) suggests improved survival for patients with histological high-grade gliomas displaying lower-grade molecular profiles. These results provide preliminary evidence of the utility of integrating multi-omics in neuropathology for pediatric neuro-oncology.
Risk prediction for meningioma tumors was until recently almost exclusively based on morphological features of the tumor. To improve risk prediction, multiple models have been established that ...incorporate morphological and molecular features for an integrated risk prediction score. One such model is the integrated molecular‐morphologic meningioma integrated score (IntS), which allocates points to the histological grade, epigenetic methylation family and specific copy‐number variations. After publication of the IntS, questions arose in the neuropathological community about the practical and clinical implementation of the IntS, specifically regarding the calling of CNVs, the applicability of the newly available version (v12.5) of the brain tumor classifier and the need for incorporation of TERT‐promoter and CDKN2A/B status analysis in the IntS calculation. To investigate and validate these questions additional analyses of the discovery (n = 514), retrospective validation (n = 184) and prospective validation (n = 287) cohorts used for IntS discovery and validation were performed. Our findings suggest that any loss over 5% of the chromosomal arm suffices for the calling of a CNV, that input from the v12.5 classifier is as good or better than the dedicated meningioma classifier (v2.4) and that there is most likely no need for additional testing for TERT‐promoter mutations and/or homozygous losses of CDKN2A/B when defining the IntS for an individual patient. The findings from this study help facilitate the clinical implementation of IntS‐based risk prediction for meningioma patients.
In this manuscript, questions regarding the clinical application of the integrated molecular‐morphologic meningioma integrated score (IntS) are investigated and addressed. For example, here we investigate the influence of using results from the v12.5 methylation brain tumor classifier versus the dedicated v2.4 meningioma classifier on the predictive accuracy of the IntS.
Desmoplastic/nodular medulloblastomas (DNMB) and medulloblastomas with extensive nodularity (MBEN) were outlined in the current WHO classification of tumors of the nervous system as two distinct ...histological MB variants. However, they are often considered as cognate SHH MB entities, and it is a reason why some clinical MB trials do not separate the patients with DNMB or MBEN histology. In the current study, we performed an integrated DNA/RNA-based molecular analysis of 83 DNMB and 36 MBEN to assess the etiopathogenetic relationship between these SHH MB variants. Methylation profiling revealed "infant" and "children" SHH MB clusters but neither DNMB nor MBEN composed separate epigenetic cohorts, and their profiles were intermixed within the "infant" cluster. In contrast, RNA-based transcriptional profiling disclosed that expression signatures of all MBEN were clustered separately from most of DNMB and a set of differentially expressed genes was identified. MBEN transcriptomes were enriched with genes associated with synaptic transmission, neuronal differentiation and metabolism, whereas DNMB profiling signatures included sets of genes involved in phototransduction and NOTCH signaling pathways. Thus, DNMB and MBEN are distinct tumor entities within the SHH MB family whose biology is determined by different transcriptional programs. Therefore, we recommend a transcriptome analysis as an optimal molecular tool to discriminate between DNMB and MBEN, which may be of benefit for patients' risk stratification in clinical trials. Molecular events identified in DNMB by RNA sequencing could be considered in the future as potent molecular targets for novel therapeutic interventions in treatment-resistant cases.
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