Faculty Bibliography

The cerebellum is a prominent part of the vertebrate hindbrain that is critically involved in the regulation of important body functions such as movement coordination, maintenance of balance and posture, and motor control. Here, we describe a cerebellar window that provides access to the mouse cerebellum for intravital imaging, thereby allowing for a detailed characterization of the dynamic processes in this region of the brain. First, the skull overlying the cerebellum is removed, and then the window is applied to the region of interest. Windows may be exchanged depending on the desired imaging modality. This technique has a variety of applications. In the setting of medulloblastoma, spontaneous or orthotopically implanted lesions can be imaged, and tumor morphology and size can be monitored using ultrasonography. Multiphoton laser-scanning microscopy (MPLSM) or optical-frequency-domain imaging (OFDI) can be applied for in vivo visualization and analysis of cellular and vascular structures in a variety of disease states, including malignancies and ataxia telangiectasia. This protocol describes a novel and rapid method for cerebellar window construction that can be set up in under an hour.

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

Background: Osteosarcoma is one of the most common bone malignancies in the pediatric population, although it affects a wide age range. While pathognomonic genomic alterations have been identified in other pediatric bone and soft tissue tumors such as Ewing sarcoma and synovial sarcoma, no such alterations are seen in osteosarcoma. Epigenetic modifications such as global or specific changes in DNA methylation are gaining recognition as a primary mechanism of oncogenesis in pediatric and adult cancers. Identifying unique epigenetic modifications in tumors lacking known fusions could contribute to both diagnosis and selection of potential therapeutic targets. Methods: Using the Illumina Infinium Human Methylation450 BeadChip Array (450K array) platform, we performed genome-wide DNA methylation analysis on 15 osteosarcomas with tissue meeting criteria for methylation analysis, including formalin-fixed paraffin-embedded, frozen, and fresh tissue obtained from NYU and Memorial Sloan Kettering Cancer Center (mean age = 26 years; range 6-80 years). Comparison was made to 10 Ewing sarcomas and 11 synovial sarcomas in the same pilot cohort. Diagnosis was based on histologic criteria and, where available, absence of a known non-osteosarcoma genomic fusion. Unsupervised hierarchical clustering analysis was performed to classify tumor type and to assess for differentially methylated target regions. Results: Osteosarcomas formed a unique subtype on unsupervised hierarchical clustering analysis of DNA methylation. Of the 15 tumors profiled, molecular testing confirming the absence of a known fusion was previously done on 5, and fusion status did not impact clustering. Pathway analysis through MSig

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.

PURPOSE: Lower grade gliomas (WHO grade II/III) have been classified into clinically-relevant molecular subtypes based on IDH and 1p/19q mutation status. The purpose was to investigate whether T2/FLAIR MRI features could distinguish between lower grade glioma molecular subtypes

Experimental Design: MRI scans from the TCGA/TCIA lower grade glioma database (n=125) were evaluated by 2 independent neuroradiologists to assess: 1) presence/absence of homogenous signal on T2WI; 2) presence/absence of "T2-FLAIR mismatch" sign; 3) sharp or indistinct lesion margins; 4) presence/absence of peritumoral edema. Metrics with moderate-substantial agreement underwent consensus review, and were correlated with glioma molecular subtypes. Somatic mutation, DNA copy number, DNA methylation, gene expression, and protein array data from the TCGA lower grade glioma database were analyzed for molecular-radiographic associations. A separate institutional cohort (n=82) was analyzed to validate the T2-FLAIR mismatch sign.

Results: Among TCGA/TCIA cases, inter-reader agreement was calculated for lesion homogeneity (k=0.234 [0.111-0.358]), T2-FLAIR mismatch sign (k=0.728 [0.538-0.918]), lesion margins (k=0.292 [0.135-0.449]), and peritumoral edema (k=0.173 [0.096-0.250]). All 15 cases that were positive for the T2-FLAIR mismatch sign were IDH-mutant, 1p/19q-non-codeleted tumors (p<0.0001; PPV=100%, NPV=54%). Analysis of the validation cohort demonstrated substantial inter-reader agreement for the T2-FLAIR mismatch sign (k=0.747 [0.536 - 0.958]); all 10 cases positive for the T2-FLAIR mismatch sign were IDH-mutant, 1p/19q non-codeleted tumors (p<0.00001; PPV=100%, NPV=76%).

Conclusion: Among lower grade gliomas, T2-FLAIR mismatch sign represents a highly specific imaging biomarker for the IDH-mutant, 1p/19q-non-codeleted molecular subtype.