Metabologenomic characterization uncovers a clinically aggressive IDH mutant glioma subtype.

Mutations in the pivotal metabolic isocitrate dehydrogenase (IDH) enzymes are recognized to drive the molecular footprint of diffuse gliomas, and patients with IDH mutant gliomas have overall favorable outcomes compared to patients with IDH wild-type tumors. However, survival still varies widely among patients with IDH mutated tumors. Here, we aimed to characterize molecular signatures that explain the range of IDH mutant gliomas. By integrating matched epigenome-wide methylome, transcriptome, and global metabolome data in 154 patients with gliomas, we identified a group of IDH mutant gliomas with globally altered metabolism that resembled IDH wild-type tumors. IDH-mutant gliomas with altered metabolism have significantly shorter overall survival from their IDH mutant counterparts that is not fully accounted for by recognized molecular prognostic markers of CDKN2A/B loss and glioma CpG Island Methylator Phenotype (GCIMP) status. IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles, providing a window to target novel dependencies in gliomas.

Integrated molecular analysis reveals hypermethylation and overexpression of HOX genes to be poor prognosticators in isocitrate dehydrogenase mutant glioma.

BACKGROUND: Diffuse gliomas represent over 80% of malignant brain tumors ranging from low-grade to aggressive high-grade lesions. Within isocitrate dehydrogenase (IDH)-mutant gliomas, there is a high variability in survival and a need to more accurately predict outcome. METHODS: To identify and characterize a predictive signature of outcome in gliomas, we utilized an integrative molecular analysis (using methylation, mRNA, copy number variation (CNV), and mutation data), analyzing a total of 729 IDH-mutant samples including a test set of 99 from University Health Network (UHN) and 2 validation cohorts including the German Cancer Research Center (DKFZ) and The Cancer Genome Atlas (TCGA). RESULTS: Cox regression analysis of methylation data from the UHN cohort identified CpG-based signatures that split the glioma cohort into 2 prognostic groups strongly predicting survival that were validated using 2 independent cohorts from TCGA and DKFZ (all P-values < .0001). The methylation signatures that predicted poor outcomes also exhibited high CNV instability and hypermethylation of HOX gene probes. Integrated multi-platform analyses using mRNA and methylation (iRM) showed that parallel HOX gene overexpression and simultaneous hypermethylation were significantly associated with increased mutational load, high aneuploidy, and worse survival (P-value < .0001). A 7-HOX gene signature was developed and validated using the most significantly associated HOX genes with patient outcome in both 1p/19q codeleted and non-codeleted IDHmut gliomas. CONCLUSIONS: HOX gene methylation and expression provide important prognostic information in IDH-mutant gliomas that are not captured by current molecular diagnostics. A 7-HOX gene signature of outcome shows significant survival differences in both 1p/19q codeleted and non-codeleted IDH-mutant gliomas.

Multiplatform molecular profiling uncovers two subgroups of malignant peripheral nerve sheath tumors with distinct therapeutic vulnerabilities.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive sarcoma, and a lethal neurofibromatosis type 1-related malignancy, with little progress made on treatment strategies. Here, we apply a multiplatform integrated molecular analysis on 108 tumors spanning the spectrum of peripheral nerve sheath tumors to identify candidate drivers of MPNST that can serve as therapeutic targets. Unsupervised analyses of methylome and transcriptome profiles identify two distinct subgroups of MPNSTs with unique targetable oncogenic programs. We establish two subgroups of MPNSTs: SHH pathway activation in MPNST-G1 and WNT/ß-catenin/CCND1 pathway activation in MPNST-G2. Single nuclei RNA sequencing characterizes the complex cellular architecture and demonstrate that malignant cells from MPNST-G1 and MPNST-G2 have neural crest-like and Schwann cell precursor-like cell characteristics, respectively. Further, in pre-clinical models of MPNST we confirm that inhibiting SHH pathway in MPNST-G1 prevent growth and malignant progression, providing the rational for investigating these treatments in clinical trials.

A primary DICER1-sarcoma with KRAS and TP53 mutations in a child with suspected ECCL.

A child had been followed since infancy by our multi-disciplinary neuro-oncology clinic with annual magnetic resonance imaging (MRI) under the presumed diagnosis of encephalocraniocutaneous lipomatosis (ECCL), with clinical features including nevus psiloliparus, scalp lipoma, nodular skin tag on and coloboma of the eyelid, cortical atrophy and meningeal angiomatosis. At the age of 4, she was found to have a large temporoparietal lesion causing elevated intracranial pressure requiring surgical resection. Histopathological exam of the tumor was suggestive of an intracranial sarcoma. Sequencing analysis of the tumor revealed mutations in DICER1, KRAS and TP53. Subsequent germline testing confirmed DICER1 syndrome and revealed an insignificant FGFR1 variant at a low frequency. Methylation profile of the tumor showed the tumor clustered most closely with sarcoma (rhabdomyosarcoma-like), confirming this tumor to be a primary DICER1-sarcoma. Compared to the previously reported cases, our unique case of primary DICER1-sarcoma also demonstrated neurofilament and chromogranin positivity, and genomic instability with loss of chromosome 4p, 4q, 8p, 11p, and 19p, as well as gains in chromosome 7p, 9p, 9q, 13q, and 15q on copy variant analysis. The detailed sequencing and methylation information discovered in this unique case of DICER1-sarcoma will hopefully help further our understanding of this rare and emerging entity.

Integrative analysis of non-small cell lung cancer patient-derived xenografts identifies distinct proteotypes associated with patient outcomes.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. Only a fraction of NSCLC harbor actionable driver mutations and there is an urgent need for patient-derived model systems that will enable the development of new targeted therapies. NSCLC and other cancers display profound proteome remodeling compared to normal tissue that is not predicted by DNA or RNA analyses. Here, we generate 137 NSCLC patient-derived xenografts (PDXs) that recapitulate the histology and molecular features of primary NSCLC. Proteome analysis of the PDX models reveals 3 adenocarcinoma and 2 squamous cell carcinoma proteotypes that are associated with different patient outcomes, protein-phosphotyrosine profiles, signatures of activated pathways and candidate targets, and in adenocarcinoma, stromal immune features. These findings portend proteome-based NSCLC classification and treatment and support the PDX resource as a viable model for the development of new targeted therapies.

Transcriptome and methylome analysis of CNS germ cell tumor finds its cell-of-origin in embryogenesis and reveals shared similarities with testicular counterparts.

BACKGROUND: CNS germ cell tumors (GCTs) predominantly develop in pediatric and young adult patients with variable responses to surgery, radiation, and chemotherapy. This study aimed to examine the complex and largely unknown pathogenesis of CNS GCTs. METHODS: We used a combined transcriptomic and methylomic approach in 84 cases and conducted an integrative analysis of the normal cells undergoing embryogenesis and testicular GCTs. RESULTS: Genome-wide transcriptome analysis in CNS GCTs indicated that germinoma had a transcriptomic profile representative of primitive cells during early embryogenesis with high meiosis/mitosis potentials, while nongerminomatous GCTs (NGGCTs) had differentiated phenotypes oriented toward tissue formation and organogenesis. Co-analysis with the transcriptome of human embryonic cells revealed that germinomas had expression profiles similar to those of primordial germ cells, while the expression profiles of NGGCTs were similar to those of embryonic stem cells. Some germinoma cases were characterized by extensive immune-cell infiltration and high expression of cancer-testis antigens. NGGCTs had significantly higher immune-cell infiltration, characterized by immune-suppression phenotype. CNS and testicular GCTs (TGCTs) had similar mutational profiles; TGCTs showed enhanced copy number alterations. Methylation analysis clustered germinoma/seminoma and nongerminoma/nonseminoma separately. Germinoma and seminoma were co-categorized based on the degree of the tumor microenvironment balance. CONCLUSIONS: These results suggested that the pathophysiology of GCTs was less dependent on their site of origin and more dependent on the state of differentiation as well as on the tumor microenvironment balance. This study revealed distinct biological properties of GCTs, which will hopefully lead to future treatment development.

Impact of the methylation classifier and ancillary methods on CNS tumor diagnostics.

BACKGROUND: Accurate CNS tumor diagnosis can be challenging, and methylation profiling can serve as an adjunct to classify diagnostically difficult cases. METHODS: An integrated diagnostic approach was employed for a consecutive series of 1258 surgical neuropathology samples obtained primarily in a consultation practice over 2-year period. DNA methylation profiling and classification using the DKFZ/Heidelberg CNS tumor classifier was performed, as well as unsupervised analyses of methylation data. Ancillary testing, where relevant, was performed. RESULTS: Among the received cases in consultation, a high-confidence methylation classifier score (>0.84) was reached in 66.4% of cases. The classifier impacted the diagnosis in 46.7% of these high-confidence classifier score cases, including a substantially new diagnosis in 26.9% cases. Among the 289 cases received with only a descriptive diagnosis, methylation was able to resolve approximately half (144, 49.8%) with high-confidence scores. Additional methods were able to resolve diagnostic uncertainty in 41.6% of the low-score cases. Tumor purity was significantly associated with classifier score (P = 1.15e-11). Deconvolution demonstrated that suspected glioblastomas (GBMs) matching as control/inflammatory brain tissue could be resolved into GBM methylation profiles, which provided a proof-of-concept approach to resolve tumor classification in the setting of low tumor purity. CONCLUSIONS: This work assesses the impact of a methylation classifier and additional methods in a consultative practice by defining the proportions with concordant vs change in diagnosis in a set of diagnostically challenging CNS tumors. We address approaches to low-confidence scores and confounding issues of low tumor purity.

DNA methylation-based prognostic subtypes of chordoma tumors in tissue and plasma.

BACKGROUND: Chordomas are rare malignant bone cancers of the skull-base and spine. Patient survival is variable and not reliably predicted using clinical factors or molecular features. This study identifies prognostic epigenetic chordoma subtypes that are detected noninvasively using plasma methylomes. METHODS: Methylation profiles of 68 chordoma surgical samples were obtained between 1996 and 2018 across three international centers along with matched plasma methylomes where available. RESULTS: Consensus clustering identified two stable tissue clusters with a disease-specific survival difference that was independent of clinical factors in a multivariate Cox analysis (HR = 14.2, 95%CI: 2.1-94.8, P = 0.0063). Immune-related pathways with genes hypomethylated at promoters and increased immune cell abundance were observed in the poor-performing "Immune-infiltrated" subtype. Cell-to-cell interaction plus extracellular matrix pathway hypomethylation and higher tumor purity were observed in the better-performing "Cellular" subtype. The findings were validated in additional DNA methylation and RNA sequencing datasets as well as with immunohistochemical staining. Plasma methylomes distinguished chordomas from other clinical differential diagnoses by applying fifty chordoma-versus-other binomial generalized linear models in random 20% testing sets (mean AUROC = 0.84, 95%CI: 0.52-1.00). Tissue-based and plasma-based methylation signals were highly correlated in both prognostic clusters. Additionally, leave-one-out models accurately classified all tumors into their correct cluster based on plasma methylation data. CONCLUSIONS: Here, we show the first identification of prognostic epigenetic chordoma subtypes and first use of plasma methylome-based biomarkers to noninvasively diagnose and subtype chordomas. These results may transform patient management by allowing treatment aggressiveness to be balanced with patient risk according to prognosis.

Paediatric atypical choroid plexus papilloma: is adjuvant therapy necessary?

INTRODUCTION: Choroid Plexus Tumours (CPTs) account for 1-4% of all brain tumours in children. Atypical choroid plexus papillomas (aCPPs) are a subset of these tumours, defined over a decade ago, yet no consensus exists on the optimal approach to their management. METHODS: We conducted a retrospective analysis of all patients treated for CPTs at the Hospital for Sick Children between January 1, 2000, and December 31, 2018, and focused on patients with aCPP. Data extracted from the patient records for analysis included: demographic and clinical features, radiological imaging, surgical and adjuvant therapies, key pathological features, immunohistochemical staining for TP53 and tumour karyotype. Six of seven aCPP samples were profiled using Illumina HumanMethylationEPIC arrays and the top 10,000 most variably methylated probes were visualized using tSNE. Copy number inferencing was also performed. RESULTS: Twenty-nine patients were diagnosed with CPT, seven of whom had a diagnosis of aCPP as confirmed by histological review. Methylation profiling demonstrated that aCPPs clustered with both choroid plexus papillomas (CPPs) and choroid plexus carcinomas (CPCs). Complete resection of the tumour was pursued in all cases of aCPP and no patient received adjuvant therapy. All aCPP patients were alive at last follow up. CONCLUSIONS: This limited case series suggests that paediatric aCPP can be successfully managed with surgical resection alone, followed by a 'watch and wait' approach thus avoiding adjuvant therapies. A deeper understanding of the biology of aCPP is required to identify objective markers which can help provide robust risk stratification and inform treatment strategies.

A clinically applicable integrative molecular classification of meningiomas.

Meningiomas are the most common primary intracranial tumour in adults1. Patients with symptoms are generally treated with surgery as there are no effective medical therapies. The World Health Organization histopathological grade of the tumour and the extent of resection at surgery (Simpson grade) are associated with the recurrence of disease; however, they do not accurately reflect the clinical behaviour of all meningiomas2. Molecular classifications of meningioma that reliably reflect tumour behaviour and inform on therapies are required. Here we introduce four consensus molecular groups of meningioma by combining DNA somatic copy-number aberrations, DNA somatic point mutations, DNA methylation and messenger RNA abundance in a unified analysis. These molecular groups more accurately predicted clinical outcomes compared with existing classification schemes. Each molecular group showed distinctive and prototypical biology (immunogenic, benign NF2 wild-type, hypermetabolic and proliferative) that informed therapeutic options. Proteogenomic characterization reinforced the robustness of the newly defined molecular groups and uncovered highly abundant and group-specific protein targets that we validated using immunohistochemistry. Single-cell RNA sequencing revealed inter-individual variations in meningioma as well as variations in intrinsic expression programs in neoplastic cells that mirrored the biology of the molecular groups identified.

TNFα secreted by glioma associated macrophages promotes endothelial activation and resistance against anti-angiogenic therapy.

One of the most prominent features of glioblastoma (GBM) is hyper-vascularization. Bone marrow-derived macrophages are actively recruited to the tumor and referred to as glioma-associated macrophages (GAMs) which are thought to provide a critical role in tumor neo-vascularization. However, the mechanisms by which GAMs regulate endothelial cells (ECs) in the process of tumor vascularization and response to anti-angiogenic therapy (AATx) is not well-understood. Here we show that GBM cells secrete IL-8 and CCL2 which stimulate GAMs to produce TNFα. Subsequently, TNFα induces a distinct gene expression signature of activated ECs including VCAM-1, ICAM-1, CXCL5, and CXCL10. Inhibition of TNFα blocks GAM-induced EC activation both in vitro and in vivo and improve survival in mouse glioma models. Importantly we show that high TNFα expression predicts worse response to Bevacizumab in GBM patients. We further demonstrated in mouse model that treatment with B20.4.1.1, the mouse analog of Bevacizumab, increased macrophage recruitment to the tumor area and correlated with upregulated TNFα expression in GAMs and increased EC activation, which may be responsible for the failure of AATx in GBMs. These results suggest TNFα is a novel therapeutic that may reverse resistance to AATx. Future clinical studies should be aimed at inhibiting TNFα as a concurrent therapy in GBMs.

Epigenomic, genomic, and transcriptomic landscape of schwannomatosis.

Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome.

Transcription factor networks of oligodendrogliomas treated with adjuvant radiotherapy or observation inform prognosis.

BACKGROUND: Recent international sequencing efforts have allowed for the molecular taxonomy of lower-grade gliomas (LGG). We sought to analyze The Cancer Genome Atlas (TCGA, 2015) gene expression datasets on molecularly defined oligodendrogliomas (IDH-mutated and 1p/19q-codeleted) patients treated with adjuvant radiation or those observed to discover prognostic markers and pathways. METHODS: mRNA expression and clinical information of patients with oligodendroglioma were taken from the TCGA "Brain Lower Grade Glioma" provisional dataset. Transcription factor network reconstruction and analysis were performed using the R packages "RTN" and "RTNsurvival." Elastic net regularization and survival modeling were performed using the "biospear," "plsRCox," "survival" packages. RESULTS: From our cohort of 137 patients, 65 received adjuvant radiation and 72 were observed. In the cohort that received adjuvant radiotherapy, a transcription factor activity signature, that correlated with hypoxia, was associated with shorter disease-free survival (DFS) (median = 45 months vs 108 months, P < .001). This increased risk was not seen in patients who were observed (P = .2). Within the observation cohort, a transcription factor activity signature was generated that was associated with poor DFS (median = 72 months. vs 143 months., P < .01). CONCLUSIONS: We identified a transcription factor activity signature associated with poor prognosis in patients with molecular oligodendroglioma treated with adjuvant radiotherapy. These patients would be potential candidates for treatment intensification. A second signature was generated for patients who were more likely to progress on observation. This potentially identifies a cohort who would benefit from upfront adjuvant radiotherapy.

Programmed death ligand-1 (PD-L1) expression in meningioma; prognostic significance and its association with hypoxia and NFKB2 expression.

Management of clinically aggressive meningiomas is a considerable challenge. PD-L1 induced immune suppression has increasingly gained attention in clinical management of cancer; however, to date, the clinical significance and regulatory mechanisms of PD-L1 in meningioma is not yet fully characterized. We sought to characterize PD-L1 expression in meningioma and elucidate its regulatory mechanisms. Immunohistochemical staining of PD-L1 expression in meningiomas showed 43% positivity in both tumor and immune cells and we observed intra and inter tumoral heterogeneity. Univariate and multivariate analyses confirmed that PD-L1 protein expression is an independent prognostic marker for worse recurrence free survival in meningioma. Furthermore, our transcriptomic analysis revealed a strong association between PD-L1 expression and that of NFKB2 and carbonic anhydrase 9 (CA9). We also demonstrated that both of these markers, when co-expressed with PD-L1, predict tumor progression. Our studies on several meningioma cell lines cultured in hypoxic conditions validated the association of CA9 and PD-L1 expression. Here we show the clinical significance of PD-L1 in meningioma as a marker that can predict tumor recurrence. We also show an association PD-L1 expression with NFKB2 expression and its induction under hypoxic conditions. These findings may open new avenues of molecular investigation in pathogenesis of meningioma.

Clinical impact of combined epigenetic and molecular analysis of pediatric low-grade gliomas.

BACKGROUND: Both genetic and methylation analysis have been shown to provide insight into the diagnosis and prognosis of many brain tumors. However, the implication of methylation profiling and its interaction with genetic alterations in pediatric low-grade gliomas (PLGGs) are unclear. METHODS: We performed a comprehensive analysis of PLGG with long-term clinical follow-up. In total 152 PLGGs were analyzed from a range of pathological subtypes, including 40 gangliogliomas. Complete molecular analysis was compared with genome-wide methylation data and outcome in all patients. For further analysis of specific PLGG groups, including BRAF p.V600E mutant gliomas, we compiled an additional cohort of clinically and genetically defined tumors from 3 large centers. RESULTS: Unsupervised hierarchical clustering revealed 5 novel subgroups of PLGG. These were dominated by nonneoplastic factors such as tumor location and lymphocytic infiltration. Midline PLGG clustered together while deep hemispheric lesions differed from lesions in the periphery. Mutations were distributed throughout these location-driven clusters of PLGG. A novel methylation cluster suggesting high lymphocyte infiltration was confirmed pathologically and exhibited worse progression-free survival compared with PLGG harboring similar molecular alterations (P = 0.008; multivariate analysis: P = 0.035). Although the current methylation classifier revealed low confidence in 44% of cases and failed to add information in most PLGG, it was helpful in reclassifying rare cases. The addition of histopathological and molecular information to specific methylation subgroups such as pleomorphic xanthoastrocytoma-like tumors could stratify these tumors into low and high risk (P = 0.0014). CONCLUSION: The PLGG methylome is affected by multiple nonneoplastic factors. Combined molecular and pathological analysis is key to provide additional information when methylation classification is used for PLGG in the clinical setting.

Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt.

Glioblastoma multiforme (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds Frizzled class receptor 4 (FZD4) to activate canonical Wnt signaling. Although Norrin, encoded by NDP, has a well-described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-suppressive and -promoting effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor-suppressive effect of Norrin suggested by the tumor outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor-suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch-mediated function of Norrin in regulating cancer stem cell biology. This study identifies an unanticipated role of Norrin in human brain cancer progression. In addition, we provide preclinical evidence suggesting Norrin and canonical Wnt signaling as potential therapeutic targets for GBM subtype-restricted cancer stem cells.

c-Src Phosphorylates and Inhibits the Function of the CIC Tumor Suppressor Protein.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes in response to receptor tyrosine kinase (RTK)/Ras/ERK signaling. Following activation of RTK, ERK enters the nucleus and serine-phosphorylates CIC, releasing it from its targets to permit gene expression. We recently showed that ERK triggers ubiquitin-mediated degradation of CIC in glioblastoma (GBM). In this study, we examined whether another important downstream effector of RTK/EGFR, the non-RTK c-Src, affects CIC repressor function in GBM. We found that c-Src binds and tyrosine-phosphorylates CIC on residue 1455 to promote nuclear export of CIC. On the other hand, CIC-mutant allele (CIC-Y1455F), that escapes c-Src-mediated tyrosine phosphorylation, remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic transcription factors ETV1 and ETV5. Furthermore, we show that the orally available Src family kinase inhibitor, dasatinib, which prevents EGF-mediated tyrosine phosphorylation of CIC and attenuates elevated ETV1 and ETV5 levels, reduces viability of GBM cells and glioma stem cells (GSC), but not of their control cells with undetectable c-Src activity. In fact, GBM cells and GSC expressing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity to dasatinib, further endorsing the effect of dasatinib on Src-mediated tyrosine phosphorylation of CIC. These findings elucidate important mechanisms of CIC regulation and provide the rationale to target c-Src alongside ERK pathway inhibitors as a way to fully restore CIC tumor suppressor function in neoplasms such as GBM. IMPLICATIONS: c-Src tyrosine-phosphorylates CIC exports to cytoplasm and inactivates its repressor function in GBM.