Faculty Bibliography

AIMS/OBJECTIVE:Hippocampal findings are implicated in the pathogenesis of sudden unexplained death in childhood (SUDC), although some studies have identified similar findings in sudden explained death in childhood (SEDC) cases. We blindly reviewed hippocampal histology in SUDC and SEDC controls. METHODS:Hippocampal H&E slides (n=67; 36 SUDC, 31 controls) from clinical and forensic collaborators were evaluated by 9 blinded reviewers: 3 board-certified forensic pathologists, 3 neuropathologists, and 3 dual-certified neuropathologist/forensic pathologists. RESULTS:Among nine reviewers, about 50% of hippocampal sections were rated as abnormal (SUDC 52.5%, controls 53.0%), with no difference by cause of death (COD) (p=0.16) or febrile seizure history (p=0.90). There was little agreement among nine reviewers on whether a slide was within normal range (Fleiss' kappa=0.014, p=0.47). Within reviewer groups, there were no findings more frequent in SUDC compared to controls, with variability in pyramidal neuron and dentate gyrus findings. Across reviewer groups, there was concordance for bilamination and granule cell loss. Neither SUDC (51.2%) nor control (55.9%) slides were considered contributory to determining COD (p=0.41). CONCLUSIONS:The lack of an association of hippocampal findings in SUDC and controls, as well as inconsistency of observations by multiple blinded reviewers, indicates discrepancy with previous studies and an inability to reliably identify hippocampal malformation associated with sudden death (HMASD). These findings underscore a need for larger studies to standardize evaluation of hippocampal findings, identify the range of normal variation and, changes unrelated to SUDC or febrile seizures. Molecular studies may help identify novel immunohistological markers that inform on COD.

BACKGROUND: DNA methylation profiling has emerged as a useful tool for robust classification of rare CNS tumors with a broad morphological spectrum. Routine diagnostic molecular profiling performed in Heidelberg and at international collaborating centers revealed a small but recurring number of CNS tumors with fusions of the PATZ1 gene coupled to either MN1 or EWSR1, displaying a distinct genome-wide methylation profile; indicating that these tumors could form a seperate biological entity. MATERIAL AND METHODS: We obtained genome-wide DNA-methylation array profiling of 68 primary CNS tumors. RNA-sequencing was perfomed on (n=23/68, 34%) of the tumor samples, including (n=6) from fresh frozen tissue used for gene expression profiling. For n=3 cases, whole exome sequencing (WES) data was generated, and gene panel sequencing data was available for n=13 cases, We systematically reevaluated the histopahthological features of 14 tumors, while immunohistochemical (IHC) staining with Ki-67, GFAP, MAP2, NeuN, Olig-2, Synaptophysin, S-100 and Vimentin was performed for (n=12) tumors. We finally collected clinical data to preliminarily characterize this novel tumor entity.
RESULT(S): A selected analysis of the tumors in this novel cohort (n=68), compared with a reference cohort consisting of 15 other low-and high-grade glial and glioneuronal tumor classes, confirmed a clearly distinct grouping. No similarity was seen with the MN1:BEND2 and MN1:CXXC5-fused CNS-tumors. Analysis of Copy number profiles derived from the DNA-methylation data showed a mostly quite genome, with (n=64/65, 98%) of tumors showing copy number variations on Chromosome 22. RNA-sequencing detected PATZ1 fusions in all tumors sequenced (n=12; MN1:PATZ1, n=11; EWSR1:PATZ1). IGF2, PAX2 and GATA2, all genes involved in brain stem cell biology, were upregulated compared to a combined reference cohort of other glioma subtypes. DNA-sequencing showed no relevant alterations at the level of point mutations or small insertions/ deletions. The tumors in our cohort showed polyphenotypic histologies along the glial spectrum, with a subset of tumors being diagnosed as Gliobastoma, WHO Grade 4 and bi-and multiphasic differentaion patterns being evident. IHC performed on tissue available did not favor a particular lineage, with most tumors showing immunopositivity to GFAP. Reverse translation of the gene expression data showed a potential role for NG2 as immunostaining marker. The median age was 11.0 years (0-80), (MN1:PATZ1 manifested at a younger age (median = 4 years) vs EWSR1:PATZ1 (median = 14 years)). Median PFS was 12 months.
CONCLUSION(S): We describe here a novel, molecularly distinct CNS tumor class with strikingly variable histopathologic morphology. We postulate that the PATZ1 fusions are a key driver of tumor initiation. Preliminary indications suggest an intermediate prognosis

DNA methylation profiling has emerged as a convincing tool to classify tumors of the central nervous system. However, for ependymomas of the lower spinal cord, neither histology nor DNA methylation can currently reliably predict progressionfree survival (PFS). To establish a clinically relevant classification, we analyzed 185 tumors classified as myxopapillary ependymoma (MPE) based on DNA methylation. Histology, global DNA methylation, copy number alterations, and global gene expression were correlated with clinical parameters and PFS. Patients with molecularly defined MPE ranged from 5-81 years of age, 58% being male. 55% of the tumors were initially diagnosed as myxopapillary ependymoma (WHO grade I), 34% as ependymoma (WHO grade II), 4% as anaplastic ependymoma (WHO grade III), and 7% with other diagnoses. 97% of the tumors with available material (n = 89) immunohistochemically expressed HOXB13, a feature that was not detected in tumors assigned to the methylation groups of spinal subependymomas (n = 4), ependymomas (n = 15), or NMYC-amplified ependymoma (n = 5). Based on DNA methylation, copy number alterations, and global gene expression, our series comprised two subgroups: MPE-A occurred in younger patients and were significantly enriched with tumors demonstrating papillary morphology and MGMT promoter methylation. These tumors occurred at lower parts of the spinal cord, could not be totally resected in many patients, and frequently relapsed. On the other hand, MPE-B included a significantly higher number of tumors with an initial diagnosis of ependymoma (WHO grade II) and tanycytic morphology. Patients within this subgroup had a significantly better outcome with a 5-year PFS of more than 90%

PURPOSE:), whereas no molecularly based stratification exists for the broad spectrum of patients with low- and intermediate-risk meningioma. METHODS:DNA methylation data and copy-number information were generated for 3,031 meningiomas (2,868 patients), and mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNVs), mutations, and WHO grading were analyzed. Prediction power for outcome was assessed in a retrospective cohort of 514 patients, validated on a retrospective cohort of 184, and on a prospective cohort of 287 multicenter cases. RESULTS:= .005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (hazard ratio 4.34 [2.48-7.57] and 3.34 [1.28-8.72] retrospective and prospective validation cohorts, respectively). CONCLUSION:Merging these layers of histologic and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision making for patients with meningioma on the basis of robust outcome prediction.

Glioblastoma (GBM) is the most common primary brain cancer in adults where tumor cell heterogeneity and sex differences influence clinical outcomes. Here, we functionally characterize three male and three female patient-derived GBM cell lines, identify protumorigenic BTICs, and create novel male and female preclinical models of GBM. Cell lines were evaluated on the following features: proliferation, stemness, migration, tumorigenesis, clinical characteristics, and sensitivity to radiation, TMZ, rhTNFSF10 (rhTRAIL), and rhBMP4 All cell lines were classified as GBM according to epigenetic subtyping, were heterogenous and functionally distinct from one another, and re-capitulated features of the original patient tumor. In establishing male and female preclinical models, it was found that two male-derived GBM cell lines (QNS108 and QNS120) and one female-derived GBM cell line (QNS315) grew at a faster rate in female mice brains. One male-derived GBM cell line (QNS108) decreased survival in female mice in comparison with male mice. However, no survival differences were observed for mice injected with a female-derived cell line (QNS315). In summary, a panel of six GBM patient-derived cell lines were functionally characterized, and it was shown that BTIC lines can be used to construct sex-specific models with differential phenotypes for additional studies.

PURPOSE/OBJECTIVE(S): Despite maximal surgical resection, radiotherapy, chemotherapy and re-treatment at re-occurrence, median overall survival time of glioblastoma (WHO grade IV, IDH wild-type) is estimated to be ~16 months. In glioma, DNA methylation states are the most predictive marker of overall survival and response to therapy. Our understanding of how epigenetic states, such as DNA methylation, are "mis-repaired" after DNA damage repair is scant, hampering our ability to understand how treatment associated DNA methylation alterations may drive tumor resistance and growth. MATERIALS/METHODS: Three different patient derived glioma stem cell (GSC) lines, in duplicates, were treated with 20 Gy in 10 fractions and allowed to recover prior to DNA methylation analysis with 850K methylation arrays. To analyze the methylation array data, we used RnBeads (version 2.4.0) and R (version 3.6.1) packages. We further focused our analysis to various genomic regions, including CpG islands, promoters, gene bodies and CTCF motifs to understand how methylation alterations may differ between these and other genomic contexts.
RESULT(S): We found differential methylation (pre-treatment vs. radiation treatment) changes among the genomic regions examined. Interestingly, we found differential methylation changes at CTCF motifs, which play important DNA-methylation dependent roles in gene expression and chromatin architecture regulation. Hierarchical clustering, PCA and MDS analysis amongst CpG islands, promoters, gene bodies and CTCF domains did not reveal strong inter-sample differences that segregated the samples on the basis of treatment status, suggesting radiation associated methylation alterations are context dependent.
CONCLUSION(S): Radiation treatment is associated with wide-spread alterations of DNA methylation states in this patient derived glioblastoma model. Such alterations may drive gene expression changes, or genomic architecture alterations, that lead to treatment resistance in the recurrent setting. AUTHOR DISCLOSURE: A.S. Modrek: None. R. Ezhilarasan: None. M. Snuderl: None. E.P. Sulman: None.
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PURPOSE/OBJECTIVE(S): Olfactory neuroblastoma (ONB) and sinonasal undifferentiated carcinoma (SNUC) are rare sinonasal cancers with distinct clinical courses. Methylomic differences between these entities have not been previously explored. The aims of this study were to compare genome-wide methylomes of ONB and SNUC, and to explore candidate tissue of origin of SNUC using a comparative methylomics approach. MATERIALS/METHODS: We included a total of 87 sinonasal malignancy samples. Genome-wide methylome data were obtained with an SNP-based human microarray for 71 ONB-like samples (44 "definitive" ONB samples, 27 ONB-resembling samples) and 10 IDH2-mutatnt SNUC samples. RNA sequencing data for a further 3 ONB and 3 SNUC samples were included. Methylomes were compared between ONB and SNUC samples and to publicly available methylation data for 195 tissue samples representing an array of tumors and normal tissues using R. Strict quality control was performed.
RESULT(S): A total of 41 ONB and 9 SNUC samples passed QC and were included in the final analysis. A total of 89,016 differentially methylated probes had adjusted P-value < 0.01, the majority (90.4%) of which were hypermethylated in SNUC and located predominantly in regulatory regions, likely reflecting IDH2 mutation. The remaining 9.6% were hypomethylated in SNUC samples and were commonly located in intergenic regions. Top differentially methylated regions were GATA3, IGF2, LSP1, NKX6-2 and UNCX. NKX6-2 and UNCX were not expressed in SNUC RNAseq data. Gene set enrichment analysis (GSEA) of differentially hypermethylated CpG-island associated probes with at least 2.5-fold-decrease in SNUC RNA-seq data showed enrichment of EZH2 targets, H3K27 bound genes consistent with previous GSEA in IDH2-mutat sarcomas, in addition to WNT pathway, olfactory transduction, lipid & carbohydrate metabolism and genes pertaining to the nervous system (P < 0.0001). On Pearson's correlation-based analysis, ONB clustered with neuronal samples while SNUC clustered with medulloblastoma (MB) and malignant peripheral nerve sheath tumor (MPNST). On separate correlation, SNUC clustered with T-cell lymphoma, MPNST and MB. On examination of the top 300 differentially methylated probes, SNUC again clustered with MPNST and MB and with a neural crest cluster. On deconvolution analysis, both ONB and SNUC samples showed a predominant T-cell presence, which was significantly higher in SNUC (P < 0.001).
CONCLUSION(S): ONB and SNUC possess distinct methylomes. PRC2 complex dysregulation may represent a key driver of a genome-wide repressive phenotype in IDH2-mutant SNUC samples leading to a dedifferentiated phenotype and may be a potential avenue for targeted therapies in the future. The presence of T-cell infiltration in both tumors represents a potential avenue for exploration of therapeutic checkpoint inhibition. Genome-wide methylomics suggests SNUC may be derived from neuronal tissue and we are currently involved in further transomic approaches to independently validate this hypothesis.
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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.

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.

Emerging evidence from clinical and preclinical studies suggests that the imipridone ONC201 is well tolerated and may have some clinical impact in discrete diffuse intrinsic pontine glioma patients (DIPG). A primary goal of our work is to determine if DIPG are uniquely sensitive to ONC201 and if so, whether ONC201 itself can be used as a tool to illuminate novel vulnerabilities in DIPG. To accomplish this, we are utilizing a combination of patient-derived cell lines as well as mouse xenografts that dovetail with a variety of molecular, epigenetic and metabolomic tools. A central finding from our work is that ONC201 primarily activates the mitochondrial protease, ClpP in DIPG patient-derived cell lines, an effect consistent with recently described ONC201 mechanism of action in other tumors. We further demonstrate that activation of ClpP by ONC201 leads to a host of downstream effects in DIPG model systems including distinctive effects on the metabolome leading to direct alterations in the unique epigenetic signature of DIPG. By directly manipulating these metabolic and epigenetic factors we provide prospective mechanistic insight into how ONC201 as well as ClpP activity impacts DIPG growth and tumorigenicity. These preclinical research findings shed light on potential therapeutic vulnerabilities in DIPG as well as ways that these strategies may be combined to enhance their potential