Faculty Bibliography

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 BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.

Inactivation of NF2/Merlin causes the autosomal-dominant cancer predisposition syndrome familial neurofibromatosis type 2 (NF2) and contributes to the development of malignant pleural mesothelioma (MPM). To develop a targeted therapy for NF2-mutant tumors, we have exploited the recent realization that Merlin loss drives tumorigenesis by activating the E3 ubiquitin ligase CRL4^DCAF1, thereby inhibiting the Hippo pathway component Lats. Here, we show that MLN4924, a NEDD8-activating enzyme (NAE) inhibitor, suppresses CRL4^DCAF1 and attenuates activation of YAP in NF2-mutant tumor cells. In addition, MLN4924 sensitizes MPM to traditional chemotherapy, presumably as a result of collateral inhibition of cullin-RING ubiquitin ligases (CRL) involved in DNA repair. However, even in combination with chemotherapy, MLN4924 does not exhibit significant preclinical activity. Further analysis revealed that depletion of DCAF1 or treatment with MLN4924 does not affect mTOR hyperactivation in NF2-mutant tumor cells, suggesting that loss of Merlin activates mTOR independently of CRL4^DCAF1 Intriguingly, combining MLN4924 with the mTOR/PI3K inhibitor GDC-0980 suppresses the growth of NF2-mutant tumor cells in vitro as well as in mouse and patient-derived xenografts. These results provide preclinical rationale for the use of NAE inhibitors in combination with mTOR/PI3K inhibitors in NF2-mutant tumors. Mol Cancer Ther; 16(8); 1693-704. ©2017 AACR.

Optic gliomas are brain tumors characterized by slow growth, progressive loss of vision, and limited therapeutic options. Optic gliomas contain various amounts of myxoid matrix, which can represent most of the tumor mass. We sought to investigate biological function and protein structure of the myxoid matrix in optic gliomas to identify novel therapeutic targets. We reviewed histological features and clinical imaging properties, analyzed vasculature by immunohistochemistry and electron microscopy, and performed liquid chromatography-mass spectrometry on optic gliomas, which varied in the amount of myxoid matrix. We found that although subtypes of optic gliomas are indistinguishable on imaging, the microvascular network of pilomyxoid astrocytoma, a subtype of optic glioma with abundant myxoid matrix, is characterized by the presence of endothelium-free channels in the myxoid matrix. These tumors show normal perfusion by clinical imaging and lack histological evidence of hemorrhage organization or thrombosis. The myxoid matrix is composed predominantly of the proteoglycan versican and its linking protein, a vertebrate hyaluronan and proteoglycan link protein 1. We propose that pediatric optic gliomas can maintain blood supply without endothelial cells by using invertebrate-like channels, which we termed primitive myxoid vascularization. Enzymatic targeting of the proteoglycan versican/hyaluronan and proteoglycan link protein 1 rich myxoid matrix, which is in direct contact with circulating blood, can provide novel therapeutic avenues for optic gliomas of childhood.

Background: Bone sarcomas present a unique diagnostic challenge because of the considerable morphologic overlap between different entities. The choice of optimal treatment, however, is dependent upon accurate diagnosis. Genome-wide DNA methylation profiling has emerged as a new approach to aid in the diagnosis of brain tumors, with diagnostic accuracy exceeding standard histopathology. In this work we developed and validated a methylation based classifier to differentiate between osteosarcoma, Ewing's sarcoma, and synovial sarcoma. Methods: DNA methylation status of 482,421 CpG sites in 15 osteosarcoma, 10 Ewing's sarcoma, and 11 synovial sarcoma samples were measured using the Illumina HumanMethylation450 array. From this training set of 36 samples we developed a random forest classifier using the 400 most differentially methylated CpG sites (FDR q value < 0.001). This classifier was then validated on 10 synovial sarcoma samples from TCGA, 86 osteosarcoma samples from TARGET-OS, and 15 Ewing's sarcoma from a recently published series (Huertas-Martinez et al., Cancer Letters 2016). Results: Methylation profiling revealed three distinct molecular clusters, each enriched with a single sarcoma subtype. Within the validation cohorts, all samples from TCGA were correctly classified as synovial sarcoma (10/10, sensitivity and specificity 100%). All but one sample from TARGET-OS were classified as osteosarcoma (85/86, sensitivity 98%, specificity 100%) and all but one sample from the Ewing's sarcoma series was classified as Ewing's sarcoma (14/15, sensitivity 93%, specificity 100%). The single misclassified osteosarcoma sample was classified as Ewing's sarcoma, and was later determined to be a misdiagnosed Ewing's sarcoma based on RNA-Seq demonstrating high EWRS1 and ETV1 expression. An additional clinical sample that was misdiagnosed as a synovial sarcoma by initial histolopathology, was accurately recognized as osteosarcoma by the methylation classifier. Conclusions: Osteosarcoma, Ewing's sarcoma and synovial sarcoma have distinct epigenetic profiles. Our validated methylation-based classifier can be used to provide an accurate diagnosis when histological and standard techniques are inconclusive