Identification of novel MET fusion transcript amplification in glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary CNS malignant tumor with a 15.2% 2 year survival rate for tumors diagnosed between 2008 and 2012. Amplification of MET proto-oncogene has been identified in 4% of glioblastomas, leading to high expression and ligand independent activation in some cases. Our goal was to develop MET-amplified GBM models to study signaling and investigate response to MET inhibitors as a therapy for GBMs. We identified 2 GBM patients (HF3035 and HF3077) with MET gene amplifications after low-pass whole genome DNA sequencing of 13 cases. Fluorescent in situ hybridization (FISH) analyses confirmed heterogeneous MET amplification in HF3035 and HF3077 tumors, in 63.5% and 83.0% of nuclei, respectively. In vitro neurosphere cultures derived from these tumors showed drastic depletion of MET amplicons, to 15.5% MET in HF3035 (P7) and 1.5% in HF3077 (P11). FISH in the metaphase neurosphere spreads showed that MET amplification was extrachromosomal. Interestingly, MET-amplified neurospheres were strongly selected for after intracranial (IC) implant in immunocompromised mice. HF3035 and HF3077 PDX presented MET amplified in high frequency: 79.5-86.5% for HF3035 and 47-65% for HF3077. Met expression levels by RNAseq were congruent with the oscillating gene amplification pattern. In depth RNA sequencing analysis using PRADA has revealed genomic rearrangements involving MET, yielding three novel MET-CAPZA2 fusion transcripts. For both cell lines exon 1 of CAPZA2 was fused to exon 2 of MET, resulting in full length MET coding region, with altered 5\u27 cis-regulatory sequences. For HF3035 samples, we observed an additional in frame fusion of exon 1 of CAPZA2 to exon 6 of MET, resulting in a truncated MET transcript with 13 codons from CAPZA2. Co-expression of the wild-type and fusion MET transcripts in the tumors and PDXs were validated using PCR. MET and p-MET levels were high thoughout the parental and PDX tumors. Capmatinib, which is a selective c-MET inhibitor was administered to the PDXs orally 5days/week. The treatment was effective in improving survival of HF3077 IC PDXs (p=0.028) and decreasing subcutaneous tumor size to 30% of the controls after 2 week treatment (t-test, p=0.017). However, treatment of HF3035 IC PDXs did not significantly improve survival (p=0.313). Kaplan-Meier survival curves were compared by log-rank (Mantel-Cox) test, sig. set at p\u3c0.05. MET and p-MET detection by IHC of control and capmatinib treated xenografts show complete inhibition of p-MET, but did not affect MET overexpression in HF3035 PDX. Our results show that highly amplified regions are susceptible to genomic arrangements and the formation of fusion genes. Under investigation, is the basis for the strong selection for MET expressing cells in vivo and potential novel roles for MET in tumor progression

Optimization of Glioblastoma Mouse Orthotopic Xenograft Models for Translational Research

Glioblastoma is an aggressive primary brain tumor predominantly localized to the cerebral cortex. We developed a panel of patient-derived mouse orthotopic xenografts (PDOX) for preclinical drug studies by implanting cancer stem cells (CSC) cultured from fresh surgical specimens intracranially into 8-wk-old female athymic nude mice. Here we optimize the glioblastoma PDOX model by assessing the effect of implantation location on tumor growth, survival, and histologic characteristics. To trace the distribution of intracranial injections, toluidine blue dye was injected at 4 locations with defined mediolateral, anterioposterior, and dorsoventral coordinates within the cerebral cortex. Glioblastoma CSC from 4 patients and a glioblastoma nonstem-cell line were then implanted by using the same coordinates for evaluation of tumor location, growth rate, and morphologic and histologic features. Dye injections into one of the defined locations resulted in dye dissemination throughout the ventricles, whereas tumor cell implantation at the same location resulted in a much higher percentage of small multifocal ventricular tumors than did the other 3 locations tested. Ventricular tumors were associated with a lower tumor growth rate, as measured by in vivo bioluminescence imaging, and decreased survival in 4 of 5 cell lines. In addition, tissue oxygenation, vasculature, and the expression of astrocytic markers were altered in ventricular tumors compared with nonventricular tumors. Based on this information, we identified an optimal implantation location that avoided the ventricles and favored cortical tumor growth. To assess the effects of stress from oral drug administration, mice that underwent daily gavage were compared with stress-positive and -negative control groups. Oral gavage procedures did not significantly affect the survival of the implanted mice or physiologic measurements of stress. Our findings document the importance of optimization of the implantation site for preclinical mouse models of glioblastoma

Heterogeneous extrachromosomal amplification of mutant PDGFRA is associated with an aggressive phenotype in glioblastoma

Background & Objective: Receptor tyrosine kinase (RTK) signaling is altered in over 80% of glioblastoma (GBM) cases, frequently through gene amplification. About 14% of GBMs carry amplification in the gene coding for platelet-derived growth factor receptor A (PDGFRA), according to TCGA. PDGFRA plays a key role in brain development and is associated with GBM proneural (PN) subtype. Despite the frequency of oncogenic RTK signaling in GBMs, RTK inhibitors have not yet achieved sufficient efficacy in clinical trials to earn FDA approval. Imatinib, a multi-kinase inhibitor that targets PDGFRA, has not shown efficacy in earlier clinical trials for GBM, in which amplification status was not an inclusion criteria. It has been reported that treatment of GBMs carrying extrachromosomal (ecDNA) amplification with EGFR inhibitors leads to a decrease in ecDNA copy number as a mechanism of resistance. Here, our objective was to assess the heterogeneity of ecDNA PDGFRA amplification in a newly diagnosed PN GBM tumor (HF3253), and to evaluate whether similar modulation of ecDNA copy number could be attained by Imatinib treatment of patient-derived xenografts (PDX). Experimental Approaches & Results: PDGFRA amplification was detected by low pass whole genome sequencing and confirmed to be extrachromosomal by fluorescent in situ hybridization (FISH) in metaphase spreads using PDGFRA and centromere 4 labeled DNA probes. The amplified PDGFRA also harbored a novel missense mutation corresponding to the extracellular domain. PDGFRA FISH signals/number ranged from 3 to 100 in the HF3253 samples, with high signal (\u3e 20) evident in 67%, 39%, and 43% and low amplification signal (6 \u3c x \u3c 9) evident in only 0%, 6%, and 19% of nuclei from biopsy, neurosphere, and xenograft respectively. HF3253 neurospheres were orthotopically implanted into the brains of immunocompromised nude mice. Imatinib mesylate was administered by oral gavage at a 75 mg/kg/day dosage in 5-day cycles with 2-day drug holiday intervals, starting 2 weeks post implant. Control mice received vehicle gavage under the same schedule. Mice were monitored daily and sacrificed on the basis of weight loss or neurological symptoms. HF3253 PDX treated with Imatinib mesylate did not have a significant survival advantage (median survival: 47.2 days) relative to control mice (median survival: 45.8 days; log rank test p value = 0.7825). The untreated and treated PDXs exhibited high levels of phospho-PDGFRA by IHC. Two independent in vitro dose response assays showed no difference in the IC50 for Imatinib between PDGFRA positive and negative GBM neurospheres: (9.447 μ M, 8.384 μ M) and (8.972 μ M, 6.896 μ M). Conclusions: We have developed a patient derived model of glioblastoma that retains ecDNA PDGFRA amplification and high levels of expression and RTK activation. Although PDGFRA is a driver of malignancy, our results show that better inhibitors are needed

Magnetization transfer and T2-weighted MRI studies are useful for visualizing phenotypic presentations of orthotopic, patientderived xenograft mouse models of glioblastoma

Introduction: Patient-derived xenograft (PDX) models for glioblastoma (GBM) from resected tumor tissues replicate several features of the original tumor. They are considered to be representative models to study tumor progression, and to test responses to putative therapies. Longitudinal noninvasive imaging can be useful in such investigations. To that end, we employed magnetic resonance imaging (MRI) to visualize and measure tumor burden in four different PDX models of GBM. Experimental procedures: Four orthotopic mouse PDX models, HF2587, HF2927, HF3077 and HF3253, developed from neurosphere cultures of four different human glioblastoma samples were used in the study. The neurosphere cells were implanted into the right striatum in immunocompromised nude mice (n=5-8 per model) and allowed to grow for 2-8 weeks, depending on their known growth rates from previous studies. They were imaged in a Varian 7T MRI system with the following weightings: T2 , T1 , magnetization transfer (MT), and contrast enhanced MRI (CE-MRI) with Magnevist as the contrast agent (CA). Following imaging, all the mice were sacrificed and their brains processed for hematoxylin and eosin (H&E) histology and human major histocompatibility complex (MHC) immunohistochemistry. Results: Tumor masses were visible as hyperintense regions on MT and T2-weighted images. The extent of such masses matched the H&E and MHC staining patterns. Ventricle enlargements seen on MRI in several mice were also confirmed by histology. Necrotic cores, when present, were observed on both imaging and on histopathology with good spatial correlations. Surprisingly, post-contrast T1 imaging did not enhance in the tumor mass or peritumorally, except in one mouse in which some intratumoral enhancement was observed. In all other instances from the four PDX models tested, enhancement was observed only when the tumor tissue or parts of it were contiguous with pial or dural vasculature. Conclusions: At 7 Tesla, MT-MRI and T2-weighted imaging, rather than CE-MRI, appear to be of better utility in visualizing these PDX models of GBM if MRI is chosen as the imaging modality. Since the parent tumors imaged at lower field strengths showed contrast enhancement, absence of a similar feature in these models needs additional studies to understand their vascular characteristics. Such properties may include low level of vascularization and/or relatively less leaky tumor vasculature. Another possible reason may be that the models tested represent the invasive features of GBM better than the vascular features, e.g. peritumoral ring enhancement, of larger clinical tumors with increased exposure to hypoxia

Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation

The high-mobility group–box transcription factor sex-determining region Y–box 2 (Sox2) is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM), Sox2 is a marker of cancer stemlike cells (CSCs) in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications

The impact of initial tumor microenvironment on imaging phenotype.

Models of human cancer, to be useful, must replicate human disease with high fidelity. Our focus in this study is rat xenograft brain tumors as a model of human embedded cerebral tumors. A distinguishing signature of such tumors in humans, that of contrast-enhancement on imaging, is often not present when the human cells grow in rodents, despite the xenografts having nearly identical DNA signatures to the original tumor specimen. Although contrast enhancement was uniformly evident in all the human tumors from which the xenografts\u27 cells were derived, we show that long-term contrast enhancement in the model tumors may be determined conditionally by the tumor microenvironment at the time of cell implantation. We demonstrate this phenomenon in one of two patient-derived orthotopic xenograft (PDOX) models using cancer stem-like cell (CSC)-enriched neurospheres from human tumor resection specimens, transplanted to groups of immune-compromised rats in the presence or absence of a collagen/fibrin scaffolding matrix, Matrigel. The rats were imaged by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and their brains were examined by histopathology. Targeted proteomics of the PDOX tumor specimens grown from CSC implanted with and without Matrigel showed that while the levels of the majority of proteins and post-translational modifications were comparable between contrast-enhancing and non-enhancing tumors, phosphorylation of Fox038 showed a differential expression. The results suggest key proteins determine contrast enhancement and suggest a path toward the development of better animal models of human glioma. Future work is needed to elucidate fully the molecular determinants of contrast-enhancement

Detection of diagnostic and prognostic methylation-based signatures in liquid biopsy specimens from patients with meningiomas.

Recurrence of meningiomas is unpredictable by current invasive methods based on surgically removed specimens. Identification of patients likely to recur using noninvasive approaches could inform treatment strategy, whether intervention or monitoring. In this study, we analyze the DNA methylation levels in blood (serum and plasma) and tissue samples from 155 meningioma patients, compared to other central nervous system tumor and non-tumor entities. We discover DNA methylation markers unique to meningiomas and use artificial intelligence to create accurate and universal models for identifying and predicting meningioma recurrence, using either blood or tissue samples. Here we show that liquid biopsy is a potential noninvasive and reliable tool for diagnosing and predicting outcomes in meningioma patients. This approach can improve personalized management strategies for these patients

Detection of diagnostic and prognostic methylation-based signatures in liquid biopsy specimens from patients with meningiomas

Abstract Recurrence of meningiomas is unpredictable by current invasive methods based on surgically removed specimens. Identification of patients likely to recur using noninvasive approaches could inform treatment strategy, whether intervention or monitoring. In this study, we analyze the DNA methylation levels in blood (serum and plasma) and tissue samples from 155 meningioma patients, compared to other central nervous system tumor and non-tumor entities. We discover DNA methylation markers unique to meningiomas and use artificial intelligence to create accurate and universal models for identifying and predicting meningioma recurrence, using either blood or tissue samples. Here we show that liquid biopsy is a potential noninvasive and reliable tool for diagnosing and predicting outcomes in meningioma patients. This approach can improve personalized management strategies for these patients