Effect of Tumor Microenvironment and Angiogenesis on Clinical Outcomes of Primary Central Nervous System Lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare lymphoma, and the disease course is often aggressive with poor prognosis outcomes. PCNSL undergoes germinal center reactions and impairs B-cell maturation. However, angiogenesis is also involved in the tumorigenesis and progression of PCNSL. This study investigated the effects of the tumor microenvironment and angiogenesis-associated genomic alterations on the outcomes of PCNSL. The analysis also evaluated the influence of treatment modality and timing on PCNSL survival using partial least squares variance-based path modeling (PLS-PM). PLS-PM can be used to evaluate the complex relationship between prognostic variables and disease outcomes with a small sample of measurements and structural models. A total of 19 immunocompetent PCNSL samples were analyzed by exome sequencing. Our results suggest that the timing of radiotherapy and mutations of ROBO1 and KAT2B are potential indicators of PCNSL outcomes and may be affected by baseline characteristics such as age and sex. Our results also showed that patients with no mutations of ROBO1 and KAT2B, SubRT subgroup showed favorable survival outcomes compared with no SubRT subgroup in short-term follow-up. All SubRT patients have received high-dose methotrexate induction chemotherapy in the initial treatment. Therefore, initial induction chemotherapy combined with subsequent radiotherapy might improve survival outcomes in PCNSL patients who have no ROBO1 and KAT2B somatic mutations in short-term follow-up. The overall findings suggest that the tumor microenvironment and angiogenesis-associated genomic alterations and treatment modalities are potential indicators of overall survival and may be affected by the baseline characteristics of PCNSL patients.

Performance of diffusion and perfusion MRI in evaluating primary central nervous system lymphomas of different locations.

BACKGROUND: Diffusion and perfusion MRI can invasively define physical properties and angiogenic features of tumors, and guide the individual treatment. The purpose of this study was to investigate whether the diffusion and perfusion MRI parameters of primary central nervous system lymphomas (PCNSLs) are related to the tumor locations. METHODS: We retrospectively reviewed the diffusion, perfusion, and conventional MRI of 68 patients with PCNSLs at different locations (group 1: cortical gray matter, group 2: white matter, group 3: deep gray matter). Relative maximum cerebral blood volume (rCBVmax) from perfusion MRI, minimum apparent diffusion coefficients (ADCmin) from DWI of each group were calculated and compared by one-way ANOVA test. In addition, we compared the mean apparent diffusion coefficients (ADCmean) in three different regions of control group. RESULTS: The rCBVmax of PCNSLs yielded the lowest value in the white matter group, and the highest value in the cortical gray matter group (P < 0.001). However, the ADCmin of each subgroup was not statistically different. The ADCmean of each subgroup in control group was not statistically different. CONCLUSION: Our study confirms that rCBVmax of PCNSLs are related to the tumor location, and provide simple but effective information for guiding the clinical practice of PCNSLs.

Delayed Contrast Enhancement in Magnetic Resonance Imaging and Vascular Morphology of Primary Diffuse Large B-Cell Lymphoma (DLBCL) of the Central Nervous System (CNS): A Retrospective Study.

BACKGROUND This study aimed to compare the magnetic resonance imaging (MRI) findings of primary diffuse large B-cell lymphoma (DLBCL) of the central nervous system (CNS) with delayed contrast enhancement and histological microvessel density (MVD). T1-weighted and T2-weighted contrast-enhanced and non-enhanced brain imaging were used. CNS lymphoma tissue was evaluated using primary antibodies to endothelial cells and smooth muscle cells, and histochemical staining for reticulin fibers and basement membrane, which allowed quantification of the MVD. MATERIAL AND METHODS Twenty-one patients with histologically confirmed primary DLBCL of the CNS underwent pre-contrast-enhanced and postcontrast-enhanced MRI. Histology of the CNS lymphoma tissue included immunohistochemical staining with antibodies to CD34 for vascular endothelial cells and alpha smooth muscle actin (ASMA) for vascular smooth muscle cells, and histochemical staining included periodic acid-Schiff (PAS) and silver staining for reticulin fibers to evaluate microvessel density (MVD). RESULTS In primary DLBCL of the CNS, a positive correlation was found between the degree of necrosis and the size of the lymphoma (r=0.546, P=0.01). Delayed imaging enhancement was significantly correlated with the number of mature vessels, MVD, basement membrane, and reticulin fibers (r=0.593, 0.466, 0.446 and 0.497, respectively). Standardized ß regression coefficient analysis showed that the MVD, PAS-positive structures, the number of mature vessels, and reticulin fibers, were significantly associated with delayed enhancement on MRI (ß values, 0.425, 0.409, 0.295, and 0.188, respectively). CONCLUSIONS In primary DLBCL of the CNS, delayed imaging enhancement on MRI may be due to reduced neovascularization and vascular infiltration by lymphoma cells.

Anti-VEGF treatment improves neurological function in tumors of the nervous system.

Research of various diseases of the nervous system has shown that VEGF has direct neuroprotective effects in the central and peripheral nervous systems, and indirect effects on improving neuronal vessel perfusion which leads to nerve protection. In the tumors of the nervous system, VEGF plays a critical role in tumor angiogenesis and tumor progression. The effect of anti-VEGF treatment on nerve protection and function has been recently reported - by normalizing the tumor vasculature, anti-VEGF treatment is able to relieve nerve edema and deliver oxygen more efficiently into the nerve, thus reducing nerve damage and improving nerve function. This review aims to summarize the divergent roles of VEGF in diseases of the nervous system and the recent findings of anti-VEGF therapy in nerve damage/regeneration and function in tumors, specifically, in Neurofibromatosis type 2 associated schwannomas.

Activation of multiple angiogenic signaling pathways in hemangiopericytoma.

Hemangiopericytoma (HPC) is a highly vascularized mesenchymal tumor. Local recurrence and distant metastasis are common features of HPC. Considering the remarkable hyper-vasculature phenotype of HPC, we assumed that dysregulated angiogenic signaling pathways were involved in HPC. The key components of angiogenic signaling pathways including VEGF-VEGF-R2, EphrinB2-EphB4 and DLL4-Notch were examined by real-time RT-PCR, Western blotting and immunostaining in 17 surgical specimens of HPC patients and in 6 controls. A significant upregulation of VEGF and VEGF-R2 associated with elevated levels of p-Akt and proliferating cell nuclear antigen (PCNA) was detected in HPC. Moreover, a dramatic increase in the mRNA and protein expression of EphB4 and its downstream factor p-Erk1/2 was found in HPC. A massive activation of core-components of DLL4-Notch signaling was detected in HPC. Double-immunofluorescent staining confirmed the expression of these upregulated key factors in the endothelial cells of tumor vessels. The present study identified the activation of multiple and crucial angiogenic signaling pathways, which could function individually and/or synergistically to stimulate angiogenesis in HPC and eventually contribute to tumor growth and progression. Our findings emphasize the importance to target multiple angiogenic signaling pathways when an anti-angiogenic therapy is considered for this highly vascularized tumor.

Adaptation to antiangiogenic therapy in neurological tumors.

Because tumors require a vascular supply for their survival and growth, angiogenesis is considered an important therapeutic target in most human cancers including cancer of the central nervous system. Antiangiogenic therapy has focused on inhibitors of the vascular endothelial growth factor (VEGF) signaling pathway. VEGF pathway-targeted drugs have shown therapeutic efficacy in several CNS tumors and have been tried most frequently in glioblastoma. These therapies, however, have been less effective than anticipated as some patients do not respond to therapy and some receive only modest benefit. Underlying this suboptimal response are multiple mechanisms of drug resistance involving changes in both tumor cells and their microenvironment. In this review, we discuss the multiple proposed mechanisms by which neurological tumors evolve to become resistant to antiangiogenic therapies. A better understanding of these mechanisms, their context, and their interplay will likely facilitate improvements in pharmacological strategies for the targeted treatment of neurological tumors.

Perivascular microenvironment in primary central nervous system lymphomas: the role of chemokines and the endothelin B receptor.

Chemokines are peptides that function as chemoattractant cytokines in cell activation, differentiation and trafficking. Endothelin B receptor (ETBR) is a receptor for endothelin, which is known to function as a vasoconstrictor. In the present study, to clarify the immune escape mechanism of primary central nervous system lymphomas (PCNSLs), the expression of ETBR and of subsets of chemokines (CXCL12, 13) in 24 PCNSLs was investigated. CXCL12 was expressed by lymphoma cells in different resident brain cell populations in 22/24 cases. CXCL13 expression was identified in tumor cells in 19/24 cases, but was only expressed by tumor cells and by proliferating vascular endothelial cells. In addition, tumor-infiltrated lymphocytes (TILs) accumulated in areas with expression of chemokines, particularly of CXCL13. ETBR expression was detected in 12/24 cases. Positive ETBR cases were associated with a paucity of TILs, particularly of cytotoxic T cells, whereas negative ETBR cases were associated with an abundance of TILs. The combined data indicate that CXCL12 and CXCL13 up-regulation may be differently linked to the development of PCNSLs and to the accumulation of TILs. In addition, ETBR expression by lymphoma and endothelial cells may mediate trafficking of TILs, which may explain the immune escape processes of PCNSLs.

Vascular normalization in cerebral angiogenesis: friend or foe?

Current antiangiogenic therapies have led to the observation that such agents can lead to improved tumor vessel structure and function termed "vascular normalization" which reduces tumor burden. However, vessel normalization is a transient process, and patients often develop resistance/poor response to anti-vascular strategies that remains an important clinical challenge. Therefore, increasing effort has been made to better understand the cellular and molecular mechanisms of vascular normalization and its contribution to immunomodulation. Herein, we summarize the recent effort to better understand the cellular and molecular mechanisms of vascular normalization with a focus on preclinical genetic models. These studies remain important directions for a mechanistic understanding of the complexities of the maintenance of BBB integrity and the impact of its breakdown on tumor dissemination and pharmaco-distribution of therapeutics.

Cerebral cavernous malformation proteins at a glance.

Loss-of-function mutations in genes encoding KRIT1 (also known as CCM1), CCM2 (also known as OSM and malcavernin) or PDCD10 (also known as CCM3) cause cerebral cavernous malformations (CCMs). These abnormalities are characterized by dilated leaky blood vessels, especially in the neurovasculature, that result in increased risk of stroke, focal neurological defects and seizures. The three CCM proteins can exist in a trimeric complex, and each of these essential multi-domain adaptor proteins also interacts with a range of signaling, cytoskeletal and adaptor proteins, presumably accounting for their roles in a range of basic cellular processes including cell adhesion, migration, polarity and apoptosis. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of current models of CCM protein function focusing on how known protein-protein interactions might contribute to cellular phenotypes and highlighting gaps in our current understanding.

Update on anti-angiogenic treatment for malignant gliomas.

Malignant gliomas are the most common primary brain tumor found in adults. Unfortunately, the prognosis for these type of tumors remains dismal despite aggressive treatment with surgical resection, radiation and chemotherapy. Therefore, therapeutics aimed at disrupting the angiogenesis of these tumors is being utilized in to improve survival outcomes and quality of life. This paper reviews the history of antiangiogenic agents in malignant gliomas, discusses the FDA approval of bevacizumab as monotherapy in recurrent glioblastoma and the subsequent controversy, and analyzes the most recent newly diagnosed trials of RTOG 0825 and AVAglio. Additionally, the results of the latest trials with antiangiogenic agents and possible biomarkers are reviewed. Multiple questions remain regarding the potential benefit of antiangiogenic treatments in patients with glioblastoma. Future clinical trials should be designed to learn more about these drugs, to optimize their future use.

Tenascin-C is expressed by human glioma in vivo and shows a strong association with tumor blood vessels.

The extracellular matrix (ECM) protein tenascin-C (TN-C) is upregulated within glioma tissues and cultured glioma cell lines. TN-C possesses a multi-modular structure and a variety of functional properties have been reported for its domains. We describe five novel monoclonal antibodies identifying different domains of TN-C. The epitopes for these antibodies were investigated by using recombinantly expressed fibronectin type III domains of TN-C. The biological effects of TN-C fragments on glioma cell proliferation and adhesion were analyzed. The expression pattern of TN-C in human glioma tissue sections and in glioma cell lines was studied with the novel library of monoclonal antibodies. The immunocytochemical analyses of the established human glioma cell lines U-251-MG, U-373-MG and U-87-MG revealed distinct staining patterns for each antibody. Robust expression of TN-C was found within the tumor mass of surgery specimens from glioblastoma. In many cases, the expression of this ECM molecule was clearly associated with blood vessels, particularly with microvessels. Three of the new antibodies highlighted individual TN-C-expressing single cells in glioma tissues. The effect of TN-C domains on glioma cells was examined by a BrdU-proliferation assay and an adhesion assay. Short fragments of constitutively expressed TN-C-domains did not exert significant effects on the proliferation of glioma cells, whereas the intact molecule increased cell division rates. In contrast, the long fragment TNfnALL containing all of the FNIII domains of TN-C decreased proliferation. Additionally, we found strong differences between the adhesion-influencing properties of the recombinant fragments on glioma cells.

Distinct functions for Rap1 signaling in vascular morphogenesis and dysfunction.

Rap1 signaling is important for both major processes of vessel formation: vasculogenesis, or de novo vessel formation, and angiogenesis, sprouting of new vessels from pre-existing ones. We provide an overview of genetic studies in mice and zebrafish and discuss some of the proposed underlying mechanisms derived from cellular models, with particular emphasis on Rap1's role in angiogenesis, maintenance of endothelial barrier and connection with cerebral cavernous malformation (CCM), a neurological deficit that leads to seizures and lethal stroke. Lastly, we provide a brief summary of studies in cardiac and smooth muscle cells, where the Epac-Rap1 signaling axis is emerging as an important regulator of contractility.

High-resolution steady-state cerebral blood volume maps in patients with central nervous system neoplasms using ferumoxytol, a superparamagnetic iron oxide nanoparticle.

Cerebral blood volume (CBV) measurement complements conventional magnetic resonance imaging (MRI) to indicate pathologies in the central nervous system (CNS). Dynamic susceptibility contrast (DSC) perfusion imaging is limited by low resolution and distortion. Steady-state (SS) imaging may provide higher resolution CBV maps but was not previously possible in patients. We tested the feasibility of clinical SS-CBV measurement using ferumoxytol, a nanoparticle blood pool contrast agent. SS-CBV measurement was analyzed at various ferumoxytol doses and compared with DSC-CBV using gadoteridol. Ninety nine two-day MRI studies were acquired in 65 patients with CNS pathologies. The SS-CBV maps showed improved contrast to noise ratios, decreased motion artifacts at increasing ferumoxytol doses. Relative CBV (rCBV) values obtained in the thalamus and tumor regions indicated good consistency between the DSC and SS techniques when the higher dose (510 mg) ferumoxytol was used. The SS-CBV maps are feasible using ferumoxytol in a clinical dose of 510 mg, providing higher resolution images with comparable rCBV values to the DSC technique. Physiologic imaging using nanoparticles will be beneficial in visualizing CNS pathologies with high vascularity that may or may not correspond with blood-brain barrier abnormalities.

Endothelial Wnt/ß-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression.

Endothelial Wnt/ß-catenin signaling is necessary for angiogenesis of the central nervous system and blood-brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/ß-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active ß-catenin specifically in the endothelium. Enforced endothelial ß-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/ß-catenin-Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that ß-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/ß-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.

Morphometric study of microvessels in primary CNS tumors and its correlation with tumor types and grade.

INTRODUCTION: Alterations of microvasculature are integral to CNS neoplasia, and a diagnostic feature of high-grade gliomas. The objectives of this study were two fold: First, to correlate morphometrically measured microvessel density (MVD), microvessel caliber (VC), and percentage of total microvessel area (%TVA) with WHO histologic grade in various types of primary CNS tumors. Second, to evaluate if such a correlation could be further refined by using mathematical derivatives of measured parameters namely coefficient of variation of VC (COofVC), microvessel cross-sectional area (VCSA), and percentage of total VCSA (%TVCSA). MATERIALS AND METHODS: Various microvessel parameters were assessed in a variety of 30 primary CNS tumors as consecutively encountered in routine surgical pathology practice including gliomas, meningiomas and others by image morphometry using CD34-immunostained sections. We introduced a novel method of effectively determining VC. Results were correlated with tumor type and grade. Appropriate statistical analysis was performed. RESULTS: Microvessel characteristics, especially VC (p<0.0022), VCSA (p<0.0164), CVofVC (p<0.0001), %TVCSA (p<0.0002) and %TVA (p<0.0003) of tumors were significantly greater than normal tissue. MVD increased in all tumors, excepting meningiomas, and was significantly higher in gliomas (p<0.0062). MVD showed negative correlation with VC (r=-0.808) and VCSA (r=-0.848) in the normal brain but was less significant in tumors. Unlike tumors, caliber distribution of microvessels in normal brain was noted to follow a Gaussian pattern. Histological grades of tumors showed positive correlation with MVD (r=0.547), VC (r=0.606), CVofVC (r=0.623), VCSA (r=0.485), %TVCSA (r=0.783) and %TVA (r=0.603). Calculated scores, estimated from multiple regressions of vessel parameters, correlated well with histological grade, with S2 (calculated using all measured as well as mathematically derived microvessel parameters) being better than S1 (calculated using measured parameters: MVD and VC). CONCLUSION: Tumor grades positively correlated with all microvessel parameters, with %TVCSA displaying the best. The correlation of %TVA with tumor grade was weaker than %TVCSA mainly due to the impact of MVD. These findings emphasize the value of VC as effectively measured using our novel method and best illustrated by its derivative %TVCSA (an indicator of blood flow), in addition to the well-recognized value of MVD in tumor prognostication. Multiple regressions of microvessel parameters provided the best correlation with grade. Morphometric analysis of microvessels in CNS tumor facilitates a better understanding of the tumor grade, tumor progression and overall prognosis.

Retrovirus-transformed erythroleukemia cells induce central nervous system failure in a new syngeneic mouse model of meningeal leukemia.

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.

Aquaporin-4 expression in primary human central nervous system lymphomas correlates with tumour cell proliferation and phenotypic heterogeneity of the vessel wall.

No literature data are available concerning the expression of aquaporin-4 in primary central nervous system lymphomas and the relationship between aquaporin-4 expression and the morphological characteristics of blood vessels. Here, we have investigated this relationship in 24 human diffuse large B-cell primary central nervous system lymphomas by means of immunocytochemistry and confocal laser microscopy. Results have shown that: (i) a high aquaporin-4 expression correlated with a high Ki-67 index and aquaporin-4 marked tumour and endothelial cells in cytoplasm and plasma membranes, while aquaporin-4 expression was low in tumour areas with a low Ki-67 index where few tumour cells were positive to aquaporin-4, and endothelial cells showed aquaporin-4 expression on their abluminal side. (ii) Different type of cells participated in vessels formation: CD20(+) tumour cells and factor VIII(+) endothelial cells; aquaporin-4(+) tumour cells and CD31(+) endothelial cells; CD20(+) and aquaporin-4(+) tumour cells; glial fibrillary acidid protein(+) endothelial cells surrounded by glial fibrillary acidic protein(+) tumour cells. Overall, these data suggest the importance of aquaporin-4 in primary central nervous system lymphomas due to its involvement in cerebral oedema formation and resolution and tumour cell migratory activity, and have documented that tumour microvasculature in lymphomas is extremely heterogeneous, confirming the importance of neoangiogenesis in the pathogenesis of lymphomas.