Madhuri Kambhampati

Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M—including H3.2K27M—mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.

Vamorolone, a dissociative steroidal compound, reduces pro-inflammatory cytokine expression in glioma cells and increases activity and survival in a murine model of cortical tumor

Corticosteroids, such as dexamethasone, are routinely used as palliative care in neuro-oncology for their anti-inflammatory benefits, however many patients experience dose limiting side effects caused by glucocorticoid response element (GRE)-mediated transcription. The purpose of this study was to use a murine model to investigate a new steroid alternative, vamorolone, which promises to reduce side effects through dissociating GRE-mediated transcription and NF-κB -mediated anti-inflammatory actions. To compare vamorolone to dexamethasone in reducing pro-inflammatory signals in vitro, murine glioma cells were treated with dexamethasone, vamorolone or vehicle control. Changes in mRNA expression were assessed using the nanostring inflammatory platform. Furthermore, drug efficacy, post-treatment behavioral activity and side effects were assessed by treating two cohorts of brain tumor bearing mice with dexamethasone, vamorolone, or vehicle control. Our investigation showed that treatment with vamorolone resulted in a reduction of pro-inflammatory signals in tumor cells in vitro similar to treatment with dexamethasone. Treatment with vamorolone resulted in a better safety profile in comparison to dexamethasone treatment. Vamorolone- treated mice showed similar or better activity and survival when compared to dexamethasone-treated mice. Our data indicate vamorolone is a potential steroid-sparing alternative for treating patients with brain tumors.

Histological and molecular analysis of a progressive diffuse intrinsic pontine glioma and synchronous metastatic lesions: a case report.

There is no curative treatment for patients with diffuse intrinsic pontine glioma (DIPG). However, with the recent availability of biopsy and autopsy tissue, new data regarding the biologic behavior of this tumor have emerged, allowing greater molecular characterization and leading to investigations which may result in improved therapeutic options. Treatment strategies must address both primary disease sites as well as any metastatic deposits, which may be variably sensitive to a particular approach. In this case report, we present a patient with DIPG treated with irradiation and serial investigational agents. The clinical, pathological and molecular phenotypes of both the progressive primary tumor as well as concomitant metastatic deposits obtained at autopsy are discussed. While some mRNA differences were demonstrated, all analyzed sites of disease shared similar mutational arrangements, suggesting that targeting the mutations of the primary tumor may be effective for all sites of disease.

Abstract 810: Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers

The recent surge in understanding genomic aberrations of some of the deadliest childhood brain cancers has highlighted the need for robust preclinical models. Such models will allow for robust drug screening and preclinical evaluation of efficacy, toxicity, and tumor penetrance in vivo. Given the rarity and importance of patient derived specimens, handling and processing methods are perhaps the most critical steps for successful establishment of viable and reproducible in vitro and in vivo models. Since specimen source varies (biopsy, autopsy, or cryo-preserved), processing methods should be refined to allow for optimal extraction of maximum numbers of viable cells from each specimen type. We have developed standardized procedures for handling and processing of tissue samples obtained from biopsy, autopsy, or cryo-preserved specimens as well as necropsy tissue obtained from existing xenograft models. Two processing methods for generating viable cell suspensions are described. The first method, which uses collagenase-DNAse mediated digestion of the tissue is efficient with bulky samples and can be used with tissues obtained at autopsy. The second method uses a commercially available enzymatic dissociation kit optimal for small volume samples such as biopsy, cryo-preserved and mouse necropsy specimens. We show that obtaining viable cell suspension from precious tumor tissue by these methods results in successful generation of pre-clinical in vitro and in vivo models of DIPG, pilocytic astrocytoma and medulloblastoma that represent the exact genetic makeup of the original patient tumor. We further demonstrate intracranial injections of these cells into P2 mice for generating orthotopic xenograft models of brainstem or cortical tumors. Our methods and results allow for rapid establishment of preclinical models using rare and valuable childhood brain tumor specimens. These pre-clinical models serve as valuable tools for understanding the molecular mechanisms of the disease, identifying targetable molecules, and screening of novel therapeutics. Citation Format: Sridevi Yadavilli, Madhuri Kambhampati, Jamila Gittens, Eshini Panditharatna, Mojca Stampar, Lindsay B. Kilburn, Suresh Magge, Roger J. Packer, Javad Nazarian. Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2017-810

Abstract 4887: Comprehensive molecular analysis of pediatric thalamic tumors

Childhood thalamic tumors are relatively rare cancers, accounting for 5% of all pediatric brain tumors and categorized as midline gliomas such as diffuse intrinsic pontine gliomas (DIPGs). We and others have shown that mutations in genes encoding for histone 3.3 (H3F3A), histone 3.2 (HIST2H3C) and histone 3.1 (HIST1H3B) along with their obligate partner mutations are the major driver mutations in DIPGs. Where recent studies have identified major histone partner mutations associated with DIPGs, more research is required to provide a clear landscape of genomic aberrations associated with thalamic tumors. We hypothesize that comprehensive whole genome sequence, methylation and proteome analysis of a large cohort of thalamic tumors will map differentially regulated pathways and identify potential novel driver and obligate partner mutations associated with thalamic gliomas. We have established a cohort (CNHS and CBTTC) of 128 thalamic specimens, including 56 pediatric and adolescent primary thalamic tumors with median age at diagnosis of 5.6 years (range 0-20 years); 40 normal controls with matched age and gender; and 32 midline tumors with potential thalamic involvement with median age at diagnosis of 7.6 years (range 0-19 years). Our cohort of primary thalamic tumors contained 31 (55.3%) and 19 (33.9%) tumors reviewed as high and low grade gliomas, respectively. From our midline tumors with potential thalamic involvement, 22 (68.7%) were classified as primary DIPG and 10 (31.2%) were other midline gliomas. Where available, MRI reviews and histopathological analysis were performed. Preliminary results showed that 7 of the extended tumors presented hypercellularity and positive histone 3 K27M staining, confirming that these tumors in fact extended to the thalamus as MRI showed. Additionally, Whole Exome Sequencing (WES) from one DIPG sample extending to the thalamus showed the same mutations found on the primary pons tumor: H3.1 K27M; ACVR1 G328V; PIK3CA H1047R; MAX R51Q and PTEN A126S. The remaining primary and extended thalamic tumors will be analyzed by WES to understand the molecular changes associated with this disease. In addition to our results, we analyzed the genomic landscape from pediatric thalamic tumors previously published (188), showing H3.3/H3.1 K27M (51%); BRAF (10.6%) and TP53 (8%) as the most frequent mutations among thalamic high and low grade astrocytomas. Further studies will allow us to compare comprehensive molecular analysis of thalamic tumors (primary and metastatic) and non-thalamic midline tumors (specimen and data already in hand) and identify similarities and differences in genomic, epigenomic and proteomic expression pattern which may guide a better characterization of thalamic tumor as a separate entity. Citation Format: Heloisa H. Moser, Susanne Yoon, Madhuri Kambhampati, Sridevi Yadavilli, Angela J. Waanders, Adam Resnick, Roger J. Packer, Javad Nazarian. Comprehensive molecular analysis of pediatric thalamic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4887. doi:10.1158/1538-7445.AM2017-4887

Abstract 5004: NG2 upregulation and its defective asymmetric distribution in pediatric brainstem glioma and diffuse intrinsic pontine glioma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Pediatric brainstem glioma (BSG) is one of the most difficult cancers to treat accounting for 10-20% of all pediatric central nervous system (CNS) tumors. BSGs may occur throughout the brainstem and are categorized into two main groups: diffuse intrinsic pontine gliomas (DIPGs) and focal brainstem gliomas. DIPGs represent about 80% of BSG and have a peak onset of six to nine years of age. DIPGs invade throughout the pons and may spread to other portions of brainstem. To better understand the pathophysiology of the disease, a genetically engineered (PDGFα-expressing) BSG mouse and a xenograft model have been established. Results: We recently reported proteome profiling of DIPG CSF and formalin fixed specimens. To expand our molecular studies of the disease, we generated complete protein profiles of specimens obtained from BSG mouse tumor and aged-matched healthy controls. Neuroglia 2 (NG2), also known as chondroitine sulfate proteoglycan 4 (CSPG4) was selected for further analysis based on its exclusive expression in BSG specimens. Although NG2 has been previously implicated in adult gliomas, its role in pediatric gliomas has not been investigated and currently there are no publications on the role of NG2 in DIPGs. Recently, NG2 expression has been shown to play a significant role in neoplastic transformation of glioma precursor cells. Gliomas are thought to originate from cells including astrocytes, stem cells, and glioma progenitor cells. Glial progenitor cells are specifically important since several groups have reported detection of oligodendrocyte markers including NG2, PDGFRα, and Olig-2 in gliomas. Our preliminary data shows high expression of NG2 in murine model of brainstem glioma as well as 80% of pediatric DIPG specimens tested. We show that shRNA-mediated knockdown of NG2 reduces cellular migration in vitro. NG2 expression is defective (symmetric) in dividing cells in vitro and in vivo. The defective NG2 expression is consistent with a recent observation in adult high grade gliomas. Injection of NG2 expressing neurospheres (NS) into brainstems of 2 day old mice (P2) results in highly aggressive brainstem tumors resulting in death within 3-7 weeks post-injection. Therefore the NS injected mouse model of brainstem glioma provides a solid model for testing therapeutics and evaluating interventions. Furthermore, we show selective delivery of liposomal nanoparticles to brainstem of our robust BSG mouse model. We also show that nanoparticle-mediated delivery of doxorubicin will induce apoptosis in tumor and not the adjacent normal region. Conclusion: We introduce a robust murine model of brainstem glioma that is developed using NG2 expressing cells. High expression of NG2 in a subset of DIPGs and its defective expression may provide novel approaches for treating DIPGs and BSGs. Citation Format: Sridevi Yadavilli, Madhuri Kambhampati, Oren J. Becher, Tobey MacDonald, Ravi Bellamkonda, Roger J. Paker, Javad Nazarian. NG2 upregulation and its defective asymmetric distribution in pediatric brainstem glioma and diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2013-5004

Abstract C30: Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma

Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p 2 or 2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p 2 or 2). Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy. Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.