Reid C. Thompson

Proteomic-Based Prognosis of Brain Tumor Patients Using Direct-Tissue Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Clinical diagnosis and treatment decisions for a subset of primary human brain tumors, gliomas, are based almost exclusively on tissue histology. Approaches for glioma diagnosis can be highly subjective due to the heterogeneity and infiltrative nature of these tumors and depend on the skill of the neuropathologist. There is therefore a critical need to develop more precise, non-subjective, and systematic methods to classify human gliomas. To this end, mass spectrometric analysis has been applied to these tumors to determine glioma-specific protein patterns. Protein profiles have been obtained from human gliomas of various grades through direct analysis of tissue samples using matrix-assisted laser desorption ionization mass spectrometry (MS). Statistical algorithms applied to the MS profiles from tissue sections identified protein patterns that correlated with tumor histology and patient survival. Using a data set of 108 glioma patients, two patient populations, a short-term and a long-term survival group, were identified based on the tissue protein profiles. In addition, a subset of 57 patients diagnosed with high-grade, grade IV, malignant gliomas were analyzed and a novel classification scheme that segregated short-term and long-term survival patients based on the proteomic profiles was developed. The protein patterns described served as an independent indicator of patient survival. These results show that this new molecular approach to monitoring gliomas can provide clinically relevant information on tumor malignancy and is suitable for high-throughput clinical screening.

Inhibition of BET Bromodomain Targets Genetically Diverse Glioblastoma

Purpose: Glioblastoma is refractory to conventional therapies. The bromodomain and extraterminal domain (BET) proteins are epigenetic readers that selectively bind to acetylated lysine residues on histone tails. These proteins recently emerged as important therapeutic targets in NUT midline carcinoma and several types of hematopoietic cancers. In this study, the therapeutic potential of a novel BET bromodomain inhibitor, JQ1, was assessed in a panel of genetically heterogeneous glioblastoma samples. Experimental Design: The antineoplastic effects of JQ1 were shown using ex vivo cultures derived from primary glioblastoma xenograft lines and surgical specimens of different genetic background. The in vivo efficacy was assessed in orthotopic glioblastoma tumors. Results: We showed that JQ1 induced marked G1 cell-cycle arrest and apoptosis, which was phenocopied by knockdown of individual BET family members. JQ1 treatment resulted in significant changes in expression of genes that play important roles in glioblastoma such as c-Myc, p21CIP1/WAF1, hTERT, Bcl-2, and Bcl-xL. Unlike the observations in some hematopoietic cancer cell lines, exogenous c-Myc did not significantly protect glioblastoma cells against JQ1. In contrast, ectopically expressed Bcl-xL partially rescued cells from JQ1-induced apoptosis, and knockdown of p21CIP1/WAF1 attenuated JQ1-induced cell-cycle arrest. Cells genetically engineered for Akt hyperactivation or p53/Rb inactivation did not compromise JQ1 efficacy, suggesting that these frequently mutated signaling pathways may not confer resistance to JQ1. Furthermore, JQ1 significantly repressed growth of orthotopic glioblastoma tumors. Conclusion: Our results suggest potentially broad therapeutic use of BET bromodomain inhibitors for treating genetically diverse glioblastoma tumors. Clin Cancer Res; 19(7); 1748–59. ©2013 AACR.

Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells.

The hedgehog (Hh) signaling pathway regulates progenitor cells during embryogenesis and tumorigenesis in multiple organ systems. We have investigated the activity of this pathway in adult gliomas, and demonstrate that the Hh pathway is operational and activated within grade II and III gliomas, but not grade IV de novo glioblastoma multiforme. Furthermore, our studies reveal that pathway activity and responsiveness is confined to progenitor cells within these tumors. Additionally, we demonstrate that Hh signaling in glioma progenitor cells is ligand-dependent and provide evidence documenting the in vivo source of Sonic hedgehog protein. These findings suggest a regulatory role for the Hh pathway in progenitor cells within grade II and III gliomas, and the potential clinical utility of monitoring and targeting this pathway in these primary brain tumors.

Direct and combined revascularization in pediatric moyamoya disease.

OBJECTIVE Surgical revascularization of moyamoya disease can improve neurological outcomes, compared with the natural history of the disease or the results of medical treatment. Controversy exists regarding whether direct or indirect revascularization yields better outcomes. This study involves a single-center experience with direct anastomosis and is the first North American series using direct revascularization for pediatric patients with moyamoya disease. METHODS Twelve patients (age range, 5-17 yr; mean age, 10.2 yr) underwent direct revascularization of 21 hemispheres. Two patients had experienced failure of previous indirect revascularization procedures, with continued clinical deterioration. Superficial temporal artery-middle cerebral artery anastomosis was performed in 19 hemispheres (with concurrent encephaloduroarteriosynangiosis in 6). Middle meningeal artery-middle cerebral artery anastomosis and omental transposition were each performed in one hemisphere. Follow-up periods ranged from 12 to 65 months (mean, 35 mo), and monitoring included neurological examinations, angiography, magnetic resonance imaging, and cerebral blood flow studies. RESULTS The neurological conditions of all patients were stable or improved after surgery. None of the patients developed new strokes, and no new ischemic lesions were seen in magnetic resonance imaging scans. All grafts evaluated by follow-up angiography were patent. Postoperative cerebral blood flow studies showed significantly improved blood flow (54.4 versus 42.5 ml/100 g/min; P = 0.017, n = 4) and hemodynamic reserve (70.3 versus 43.9 ml/100 g/min; P = 0.009, n = 4), compared with preoperative studies. CONCLUSION Surgical revascularization by direct anastomosis in pediatric patients is technically feasible, is well tolerated, and can improve the progressive natural history, the angiographic appearance, and the cerebral blood flow abnormalities associated with the disease. Direct revascularization has the advantage of providing immediate and high-flow revascularization and is particularly useful for patients who have experienced failure of previous indirect revascularization procedures.

Cytogenetic Prognostication Within Medulloblastoma Subgroups

PURPOSE Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. PATIENTS AND METHODS Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. RESULTS Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. CONCLUSION Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

CXCR4 mediates the proliferation of glioblastoma progenitor cells

Increasing evidence points to a fundamental role for cancer stem cells (CSC) in the initiation and propagation of many tumors. As such, in the context of glioblastoma multiforme (GBM), the development of treatment strategies specifically targeted towards CSC-like populations may hold significant therapeutic promise. To this end, we now report that the cell surface chemokine receptor, CXCR4, a known mediator of cancer cell proliferation and invasion, is overexpressed in primary glioblastoma progenitor cells versus corresponding differentiated tumor cells. Furthermore, administration of CXCL12, the only known ligand for CXCR4, stimulates a specific and significant proliferative response in progenitors but not differentiated tumor cells. Taken together, these results implicate an important role for the CXCR4 signaling mechanism in glioma CSC biology and point to the therapeutic potential of targeting this pathway in patients with GBM.

An atlas-based method to compensate for brain shift: preliminary results.

Compensating for intraoperative brain shift using computational models has shown promising results. Since computational time is an important factor during neurosurgery, a priori knowledge of the possible sources of deformation can increase the accuracy of model-updated image-guided systems. In this paper, a strategy to compensate for distributed loading conditions in the brain such as brain sag, volume changes due to drug reactions, and brain swelling due to edema is presented. An atlas of model deformations based on these complex loading conditions is computed preoperatively and used with a constrained linear inverse model to predict the intraoperative distributed brain shift. This relatively simple inverse finite-element approach is investigated within the context of a series of phantom experiments, two in vivo cases, and a simulation study. Preliminary results indicate that the approach recaptured on average 93% of surface shift for the simulation, phantom, and in vivo experiments. With respect to subsurface shift, comparisons were only made with simulation and phantom experiments and demonstrated an ability to recapture 85% of the shift. This translates to a remaining surface and subsurface shift error of 0.7+/-0.3 mm, and 1.0+/-0.4 mm, respectively, for deformations on the order of 1cm.

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Endothelial cell expression of intercellular adhesion molecule 1 in experimental posthemorrhagic vasospasm

OBJECTIVE The exposure of large intracranial arteries to blood after an aneurysmal subarachnoid hemorrhage leads to a cascade of morphological and physiological changes in the vessels, a condition generally described as vasospasm. This response to the periadventitial deposition of blood is mediated in part by the endothelial layer of the vessel. This study was undertaken to examine the role of endothelial cell expression of intercellular adhesion molecule 1 (ICAM-1) in the initiation and regulation of this response. METHODS The femoral artery model of vasospasm was used in rats (65 animals, 130 arteries). In each rat, one artery was exposed to blood and the contralateral vessel was exposed to saline, so that each animal served as its own control. Animals were perfused and killed at sequential time points, from 1 hour to 20 days after blood exposure. The vessels were examined immunohistochemically and histologically for the presence of ICAM-1 and morphological features of vasospasm, respectively. RESULTS Endothelial cell ICAM-1 immunoreactivity was extensively increased in only the blood-exposed vessels, beginning 3 hours after clot placement and persisting for 24 hours. ICAM-1 immunoreactivity returned to baseline by 48 hours after blood exposure. The influx of inflammatory cells correlated directly with the time and location of increased ICAM-1 expression. Peak arterial remodeling was observed on the blood-exposed side 8 to 12 days after clot placement, as quantified by measurements of increased wall thickness, decreased lumen size, and increased collagen content. CONCLUSION Endothelial cell ICAM-1 expression seems to be an early and specific signal used by a vessel in response to the deposition of blood periadventitially. This molecule may be a marker for vessels likely to undergo subsequent morphological remodeling and vasospasm.

Liquid-crystal tunable filter spectral imaging for brain tumor demarcation

Past studies have demonstrated that combined fluorescence and diffuse reflectance spectroscopy can successfully discriminate between normal, tumor core, and tumor margin tissues in the brain. To achieve efficient, real-time surgical resection guidance with optical biopsy, probe-based spectroscopy must be extended to spectral imaging to spatially demarcate the tumor margins. We describe the design and characterization of a combined fluorescence and diffuse reflectance imaging system that uses liquid-crystal tunable filter technology. Experiments were conducted to quantitatively determine the linearity, field of view, spatial and spectral resolution, and wavelength sensitivity of the imaging system. Spectral images were acquired from tissue phantoms, mouse brain in vitro, and human cortex in vivo for functional testing of the system. The spectral imaging system produces measured intensities that are linear with sample emission intensity and integration time and possesses a 1 in. (2.54 cm) field of view for a 7 in. (18 cm) object distance. The spectral resolution is linear with wavelength, and the spatial resolution is pixel-limited. The sensitivity spectra for the imaging system provide a guide for the distribution of total image integration time between wavelengths. Functional tests in vitro demonstrate the capability to spectrally discriminate between brain tissues based on exogenous fluorescence contrast or endogenous tissue composition. In vivo imaging captures adequate fluorescence and diffuse reflectance intensities within a clinically viable 2 min imaging time frame and demonstrates the importance of hemostasis to acquired signal strengths and imaging speed.