Faculty Bibliography

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 to 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 non-neoplastic factors such as tumor location and lymphocytic infiltration. Midline PLGG clustered 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 to 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 (PXA)-like tumors could stratify these tumors into low and high risk (p = 0.0014). CONCLUSION/CONCLUSIONS:The PLGG methylome is affected by multiple non-neoplastic factors. Combined molecular and pathological analysis is key to provide additional information when methylation classification is used for PLGG in the clinical setting.

Myxopapillary ependymoma (MPE) is a relatively common neoplasm arising primarily in the filum terminale/lumbosacral region of the spinal cord. It is designated as a grade I tumor in the most recent WHO Classification of Tumours of the CNS, although aggressive clinical behavior can be observed, especially in cases arising in an extradural location. Anaplastic transformation in MPE is exceedingly rare with <20 examples reported in the English literature, and consensus on diagnostic features and definitive grading remain to be determined. Here, we present 2 cases of recurrent MPE with anaplastic features, both of which had histology consistent with conventional MPE as well as areas with significant atypia, frequent mitotic figures, elevated Ki-67 proliferation indices (>10%-50%), necrosis, and focal vascular proliferation. Targeted next-generation sequencing panels revealed no definitive pathogenic mutations or fusion proteins in either case. Copy number profiling, methylation profiling, and t-Distributed Stochastic Neighbor Embedding were performed to investigate the molecular characteristics of these tumors. To the best of our knowledge, these are the first reported cases of MPE with anaplastic features with methylation profiling data. In addition, we review the literature and discuss common histologic and molecular findings associated with anaplastic features in MPE.

We report a case of a slow-growing, diffuse, infiltrating glioma in the right brainstem of an 9 year-old boy. The tumor was negative by immunohistochemical staining for histone H3 K27M, BRAF V600E, and IDH1 R132H mutations. Fluorescence in situ hybridization did not reveal a BRAF duplication. Genomic profiling of the tumor, by DNA methylation array and cancer whole exome and transcriptome sequencing, was performed. This analysis showed copy number alterations, including gains of several chromosomes. In addition, a novel fusion involving the first 17 exons of FGFR2 fused to exon 2 of VPS35 was identified. This novel fusion is predicted to result in activation of FGFR signaling, and is potentially targetable using FGFR inhibitors. This tumor expands the spectrum of pediatric diffuse gliomas.

Programmed cell death protein-1 (PD-1) checkpoint immunotherapy efficacy remains unpredictable in glioblastoma (GBM) patients due to the genetic heterogeneity and immunosuppressive tumor microenvironments. Here, we report a microfluidics-based, patient-specific 'GBM-on-a-Chip' microphysiological system to dissect the heterogeneity of immunosuppressive tumor microenvironments and optimize anti-PD-1 immunotherapy for different GBM subtypes. Our clinical and experimental analyses demonstrated that molecularly distinct GBM subtypes have distinct epigenetic and immune signatures that may lead to different immunosuppressive mechanisms. The real-time analysis in GBM-on-a-Chip showed that mesenchymal GBM niche attracted low number of allogeneic CD154+CD8+ T-cells but abundant CD163+ tumor-associated macrophages (TAMs), and expressed elevated PD-1/PD-L1 immune checkpoints and TGF-β1, IL-10, and CSF-1 cytokines compared to proneural GBM. To enhance PD-1 inhibitor nivolumab efficacy, we co-administered a CSF-1R inhibitor BLZ945 to ablate CD163+ M2-TAMs and strengthened CD154+CD8+ T-cell functionality and GBM apoptosis on-chip. Our ex vivo patient-specific GBM-on-a-Chip provides an avenue for a personalized screening of immunotherapies for GBM patients.

Early detection and serial therapeutic monitoring for pediatric brain tumors are essential for diagnosis and therapeutic intervention. Currently, neuropathological diagnosis relies on biopsy of tumor tissue and surgical intervention. There is a great clinical need for less invasive methods to molecularly characterize the tumor and allow for more reliable monitoring of patients during treatment and to identify patients that might potentially benefit from targeted therapies, particularly in the setting where diagnostic tissue cannot be safely obtained. In this literature review, we highlight recent studies that describe the use of circulating tumor DNA, circulating tumor cells, circulating RNA and microRNA, and extracellular vesicles as strategies to develop liquid biopsies in pediatric central nervous system tumors. Liquid biomarkers have been demonstrated using plasma, urine, and cerebrospinal fluid. The use of liquid biopsies to help guide diagnosis, determine treatment response, and analyze mechanisms of treatment resistance is foreseeable in the future. Continued efforts to improve signal detection and standardize liquid biopsy procedures are needed for clinical application.

A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacological inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggests that PHGDH inhibitors may be useful in the treatment of brain metastasis.

The dynamics of viral load (VL) of the 2019 novel coronavirus (severe acute respiratory syndrome coronavirus 2) and its association with different clinical parameters remain poorly characterized in the US patient population. Herein, we investigate associations between VL and parameters, such as severity of symptoms, disposition (admission versus direct discharge), length of hospitalization, admission to the intensive care unit, length of need for oxygen support, and overall survival in a cohort of 205 patients from a tertiary care center in New York City. VL was determined using quantitative PCR and log10 transformed for normalization. Univariate and multivariate regression models were used to test these associations. We found that diagnostic viral load is significantly lower in hospitalized patients than in patients not hospitalized (log10 VL = 3.3 versus 4.0; P = 0.018) after adjusting for age, sex, race, body mass index, and comorbidities. Higher VL was associated with shorter duration of the symptoms in all patients and hospitalized patients only and shorter hospital stay (coefficient = -2.02, -2.61, and -2.18; P < 0.001, P = 0.002, and P = 0.013, respectively). No significant association was noted between VL, admission to intensive care unit, length of oxygen support, and overall survival. Our findings suggest a higher shedding risk in less symptomatic patients, an important consideration for containment strategies in severe acute respiratory syndrome coronavirus 2. Furthermore, we identify a novel association between viral load and history of cancer. Larger studies are warranted to validate our findings.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 236 SARS-CoV2 sequences from cases in the New York City metropolitan area during the initial stages of the 2020 COVID-19 outbreak. The majority of cases throughout the region had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that the majority were most related to cases from Europe. Our data are consistent with numerous seed transmissions from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of real-time genomic surveillance in addition to traditional epidemiological indicators.

The tumor microenvironment (TME) plays critical roles in tumor growth and progression, however key regulators of gene expression in the TME of cutaneous malignant peripheral nerve sheath tumor (C-MPNST) and spindle cell melanoma (SCM) have not been well elucidated. Herein, we investigate the epigenetic regulation of promoters and gene bodies and their effect on the TME composition of C-MPNSTs and SCMs. A cohort of 30 patients was analyzed using differential gene expression (DGE) and gene set enrichment analysis (GSEA) using the Nanostring platform. Methylation analysis was carried out utilizing an Infinium Methylation EPIC array targeting 866,562 methylation site (CpG) islands. DGE revealed overexpression of genes related to mast cells in the TME of SCMs, and a predominance of exhausted CD8⁺ T cells and macrophages in the TME of C-MPNSTs. Interestingly, we further observed promoter hypermethylation in key overexpressed genes and corresponding gene body hypomethylation. Analysis using ENCODE ChIP-sequencing data identified CTCF as the common transcription factor at the site of the hypomethylated probe. These findings support that the TME composition of C-MPNSTs and SCMs is at least partially independent on promoter methylation status, suggesting a possible relationship between gene body enhancers and expression of key TME genes in both entities.

Pleomorphic xanthoastrocytoma (PXA) is a rare type of brain tumor that affects children and young adults. Molecular prognostic markers of PXAs remain poorly established. Similar to gangliogliomas, PXAs show prominent immune cell infiltrate, but its composition also remains unknown. In this study, we correlated DNA methylation and BRAF status with clinical outcome and explored the tumor microenvironment. We performed DNA methylation in 21 tumor samples from 18 subjects with a histological diagnosis of PXA. MethylCIBERSORT was used to deconvolute the PXA microenvironment by analyzing the associated immune cell-types. Median age at diagnosis was 16 years (range 7-32). At median follow-up of 30 months, 3-year and 5-year overall survival was 73% and 71%, respectively. Overall survival ranged from 1 to 139 months. Eleven out of 18 subjects (61%) showed disease progression. Progression-free survival ranged from 1 to 89 months. Trisomy 7 and CDKN2A/B (p16) homozygous deletion did not show any association with overall survival (p = 0.67 and p = 0.74, respectively). Decreased overall survival was observed for subjects with tumors lacking the BRAF V600E mutation (p = 0.02). PXAs had significantly increased CD8 T-cell epigenetic signatures compared with previously profiled gangliogliomas (p = 0.0019). The characterization of immune cell-types in PXAs may have implications for future development of immunotherapy.