Localization of endostatin in rat and human gliomas

BACKGROUND: Endostatin is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. Its occurrence and localization has not yet been examined in human brain tumors. The authors report the production of a monoclonal antibody and detection of endostatin in rat and human gliomas by immunohistochemistry. METHODS: The authors analyzed localization and tissue distribution of endostatin in 41 paraffin embedded glioma samples (18 glioblastoma multiforme, 7 WHO Grade III astrocytomas, 13 fibrillary, and 3 protoplasmic WHO Grade II astrocytomas) of human origin and 21 rat C6 gliomas by immunohistochemistry. Double labeling experiments confirmed the origin of endostatin-labeled cells. RESULTS: Endostatin immunoreactivity was detected in tumor cells, endothelial cells, macrophages, and lymphocytes of both rat and human gliomas. The percentage of cells labeled with the endostatin antibody was significantly lower (P = 0.0126) in the tumor parenchyma of human glioblastomas than in WHO Grade II astrocytomas. CONCLUSIONS: Endostatin was present in various cell types in rat and human gliomas in vivo. Lower levels in glioblastomas than in WHO Grade II astrocytomas might have reflected the shift of a probable regulatory balance between promoters and inhibitors of angiogenesis towards facilitation of neovascularization.     

Significance of IDH mutations varies with tumor histology, grade, and genetics in Japanese glioma patients

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found frequently in malignant gliomas and are likely involved in early gliomagenesis. To understand the prevalence of these mutations and their relationship to other genetic alterations and impact on prognosis for Japanese glioma patients, we analyzed 250 glioma cases. Mutations of IDH1 and IDH2 were found in 73 (29%) and 2 (1%) cases, respectively. All detected mutations were heterozygous, and most mutations were an Arg132His (G395A) substitution. IDH mutations were frequent in oligodendroglial tumors (37/52, 71%) and diffuse astrocytomas (17/29, 59%), and were less frequent in anaplastic astrocytomas (8/29, 28%) and glioblastomas (13/125, 10%). The pilocytic astrocytomas and gangliogliomas did not have either mutation. Notably, 28 of 30 oligodendroglial tumors harboring the 1p/19q co-deletion also had an IDH mutation, and these alterations were significantly correlated (P < 0.001). The association between TP53 and IDH mutation was significant in diffuse astrocytomas (P = 0.0018). MGMT promoter methylation was significantly associated with IDH mutation in grade 2 (P < 0.001) and grade 3 (P = 0.02) gliomas. IDH mutation and 1p/19q co-deletion were independent favorable prognostic factors for patients with grade 3 gliomas. For patients with grade 3 gliomas and without 1p/19q co-deletion, IDH mutation was strongly associated with increased progression-free survival (P < 0.0001) and overall survival (P < 0.0001), but no such marked correlation was observed with grade 2 gliomas or glioblastomas. Therefore, IDH mutation would be most useful when assessing prognosis of patients with grade 3 glioma with intact 1p/19q; anaplastic astrocytomas account for most of these grade 3 gliomas.     

Expression and localization of scatter factor/hepatocyte growth factor in human astrocytomas

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic cytokine that has been implicated in glioma invasion and angiogenesis. The SF/HGF receptor, MET, has been found to be expressed in neoplastic astrocytes as well as in endothelial cells of the tumor vasculature. Both SF/HGF and MET expression have also been described to correlate with the malignancy grade of human gliomas. However, most glioblastoma cell lines lack SF/HGF expression, raising the question of the cellular origin of SF/HGF in vivo. Using in situ hybridization, we analyzed glioblastomas, anaplastic astrocytomas, diffuse astrocytomas, pilocytic astrocytomas, and normal brain for the expression of SF/HGF mRNA. We detected strong SF/HGF expression by the majority of the tumor cells and by vascular endothelial cells in all glioblastoma specimens analyzed. Combined use of in situ hybridization with fluorescence immunohistochemistry confirmed the astrocytic origin of the SF/HGF-expressiong cells. In contrast, CD68-immunoreactive microglia/macrophages, as well as vascular smooth muscle cells reactive to alpha-smooth muscle actin, lacked SF/HGF expression. In anaplastic, diffuse, and pilocytic astrocytomas, SF/HGF expression was confined to a subset of tumor cells, and signals were less intense than in glioblastomas. In addition, we detected SF/HGF mRNA in cortical neurons. SF/HGF expression was not up regulated around necroses or at tumor margins. MET immunoreactivity was observed in GFAP-expressing astrocytic tumor cells and endothelial cells as well as in a subset of microglia/macrophages. We conclude that in vivo, both autocrine and paracrine stimulation of tumor cells and endothelium through the SF/HGF-MET system are likely to contribute to tumor invasion and angiogenesis. Lack of SF/HGF expression by most cultured glioblastoma cells is not representative of the in vivo situation and most likely represents a culture artifact.     

Galectin-3: Cellular Distribution and Correlation with WHO-grade in Human Gliomas

In order to elucidate the reason for conflicting results that have been published previously on galectin-3 expression in human gliomas, we used single labeling and double labeling immunohistochemistry experiments to identify cellular origin and extent of galectin-3 positivity in 53 glioma-samples (16 glioblastomas, 21 anaplastic astrocytomas, 16 low-grade astrocytomas). Galectin-3 positivity was observed in neoplastic astrocytes, macrophages/microglial cells, endothelial cells and some B- and T-lymphocytes. The quantitative analysis showed that the percentage of galectin-3 positive cells was significantly higher in the tumor parenchyma of glioblastomas than in anaplastic (p = 0.0371) and low-grade astrocytomas (p = 0.0042). Single labeling with anti-CD68 antibodies revealed a significant correlation between CD68 and galectin-3 immunoreactivity (p = 0.0092). Endothelial cells were labeled in all low-grade and anaplastic astrocytomas, but only in 10/16 glioblastomas (p = 0.0003). This detailed analysis demonstrates that galectin-3 positivity in human gliomas is considerably influenced by tumor-infiltrating macrophages. The differential expression on endothelial cells raises the question if galectin-3 plays a role in tumor angiogenesis of human gliomas.     

KDR activation in astrocytic neoplasms.

BACKGROUND: The development of new capillary networks appears to be necessary for the growth of solid tumors. Tumor angiogenesis is believed to be mediated by soluble factors released from tumor cells that then act on endothelial cells in a paracrine manner. Vascular endothelial growth factor (VEGF) is a prime regulator of normal and tumor angiogenesis as well as vasculogenesis. VEGF is expressed in glioma cells and its receptors (Flt-1 and KDR) are expressed in the same gliomas. The two receptors are tyrosine kinases and have an extracellular domain containing seven immunoglobulin-like loops and a split tyrosine-kinase domain. KDR is a receptor for the various VEGF isoforms and for VEGF-C; Flt-1 is a receptor for the various isoforms. Studies suggest that the VEGF receptors are induced in endothelial cells during tumor angiogenesis. Stimulation of aortic endothelial cells results in receptor tyrosine phosphorylation (receptor activation). In this study the activation state of the KDR receptors was determined in low grade, anaplastic, and high grade gliomas. METHODS: A synthetic tyrosine phosphopeptide was used to raise an antibody that recognizes the phosphorylation state of tyrosine 1054/1059 in the KDR receptor. Western blot analysis was performed on 37 astrocytic neoplasms (7 low grade astrocytomas, 13 anaplastic astrocytomas, and 17 cases of glioblastoma multiforme). RESULTS: Immunoblotting with this antibody found that tyrosines 1054/1059 were phosphorylated constitutively within multiple fresh surgical specimens of glioblastomas (71%) and anaplastic gliomas (15%), but not in low grade gliomas. CONCLUSIONS: The findings of the current study strongly support the hypothesis that the onset of angiogenesis is an important event during the disease progression of gliomas.     

Biological role of thymidine phosphorylase in human astrocytic tumors

Thymidine phosphorylase (TP) has strong angiogenic activity and is overexpressed in a wide variety of malignant tumors. To elucidate the role of TP in human astrocytic tumors, we immunohistochemically investigated the expression of TP in 62 astrocytic tumors (12 astrocytomas, 12 anaplastic astrocytomas and 38 glioblastomas). Fifty-five astrocytic tumors (88.7%) were immunopositive for TP. The level of TP-expression was significantly correlated with the malignancy grade of astrocytic tumors; most of malignant gliomas highly expressed TP, while a small number of cells were positive in low grade astrocytomas (p < 0.001). Using double-immunostaining, we clarified that TP-expression was predominantly detectable in macrophages. There was no significant correlation between MIB-1 labeling index and TP-expression. However, TP-expression and the microvessel density were well correlated. These suggest that TP, mainly produced by the infiltrated macrophages, may play an important role in the progression of astrocytic tumors via neovascularization. Inhibitor of TP may represent a therapeutic modality for treating malignant gliomas.     

Expression of Cathepsin B and microvascular density increases with higher grade of astrocytomas

Samples were taken from supratentorial gliomas border and normal brain autopsy which were divided into four groups, these including eight cases normal brain tissues, 30 cases of astrocytomas, 25 cases of anaplastic astrocytomas and 22 cases of glioblastomas. Cathepin B (CB) expression and microvessel density (MVD) were determined with immunohistochemical studies. Staining results of CB was scored according to the percentage of positive cells, graded as negative (–), weak (+), moderate (++), and strong (+ + +). MVD was analyzed by Weidners revised technique. CB positive staining was negative in eight cases of normal brain tissue. Only 9 out of 30 cases of astrocytomas showed a low percentage of positive cells that were stained in a light, diffuse cytoplasmic pattern (score +). Twenty-two out of 35 cases of anaplastic astrocytomas showed positive light, granular staining pattern, it including five samples (score +), and 17 samples (score + +). In contrast, all 22 cases of glioblastomas were stained all, and it was present in a course, granular staining pattern with an intensity of score (+ +) of two sample, and score (+ + +) of 20 samples. Positive staining tumor cells were found in extracellular matrix (ECM), basement membrane (BM), and the endothelial cells of blood vessels were also positive stained. Along with elevating glioma grade, CB expression and MVD value were both increased. Therefore, it showed MVD value was positive correlated with expression of CB. It highly suggested that CB and angiogenesis plays an important role in glioma progression.     

Upregulation of vasohibin-1 expression with angiogenesis and poor prognosis of hepatocellular carcinoma after curative surgery

Vasohibin-1 has recently been found and is known as an endogenous angiogenesis inhibitor, but the role of vasohibin-1 in hepatocellular carcinoma (HCC) is unknown. This study investigated the expression pattern of vasohibin-1, its correlation with clinicopathological features, and its potential role in tumor angiogenesis and prognosis of HCC. Expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and intratumoral microvessel density (MVD, labeled by CD34) were assessed by immunohistochemistry in 117 HCC specimens and adjacent nontumor liver tissues (ANLT). Correlation between vasohibin-1 and VEGF-A, MVD, and clinicopathological features was then investigated. Prognostic value of these factors was determined using Kaplan-Meier analysis and a Cox proportional hazards regression model. Cytoplasm high expression of vasohibin-1 was detected in 38.5% (45/117) of the HCC tissues, which was significantly higher than that in 16.2% (19/117) of ANLT (P?相似文献   

VEGF-A和VEGF-C在乳腺癌组织中的表达及其意义

背景与目的:VEGF家族都与血管生成相关,血管内皮生长因子-A(vascularendothelialgrowthfactorA,VEGF-A)和血管内皮生长因子-C(vascularendothelialgrowthfactorC,VEGF-C)与肿瘤的生长和转移关系密切。本研究探讨乳腺癌组织中VEGF-A、VEGF-C的表达与癌细胞增殖、微血管密度(microvesseldensity,MVD)和淋巴结转移的关系。方法:采用免疫组织化学方法观察98例乳腺癌组织中VEGF-A、VEGF-C、增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)、CD34的表达情况。结果:98例乳腺癌组织中,VEGF-A阳性率为85.7%(84/98),VEGF-C阳性率90.8%(89/98),两者在淋巴结转移组表达均高于未转移组,差异具有显著性(P<0.05)。PCNA的表达随着VEGF-A、VEGF-C表达强度增强,肿瘤细胞增殖活性也随之增强(r=0.432,P=0.000;r=0.294,P=0.001)。淋巴结转移组MVD值(64.26±26.40)明显高于未转移组(50.29±29.35)(P<0.05),且随着VEGF-A表达增强,MVD也随之增高(r=0.327,P<0.001),VEGF-C表达与MVD无相关性(r=0.123,P>0.05)。结论:VEGF-A主要介导了血管生成、细胞增殖和转移;VEGF-C促进乳腺癌细胞增殖,与血管密度无关,与淋巴结转移密切相关。     

Combined VEGF-A and VEGFR-2 concentrations in plasma: diagnostic and prognostic implications in patients with advanced NSCLC

Introduction

The vascular endothelial growth factor (VEGF) family of ligands and receptors (VEGFR) play an important role in tumor angiogenesis. Increased expression of angiogenic factors in tumors or in blood is associated with poor prognosis. The aim of this study was to investigate the role of VEGF-A and soluble VEGFR-2 (sVEGFR-2) as biomarkers in advanced non-small-cell lung cancer (NSCLC).

Methods

We studied 432 patients with advanced NSCLC (stages IIIB-IV) treated with cisplatin and docetaxel and 89 healthy age-matched controls. Blood samples were collected before chemotherapy, and VEGF-A and sVEGFR-2 levels were determined by ELISA.

Results

VEGF-A and sVEGFR-2 levels were higher in NSCLC patients than in the controls, but VEGF-A behaves as a better diagnostic biomarker. There were no significant associations between VEGF-A and sVEGFR-2 concentrations and clinical characteristics, such as ECOG-PS, gender, stage, histology, metastases, and treatment response. A patient subgroup characterized by a combination of high VEGF-A and low sVEGFR-2 levels exhibited the worst patient prognoses in terms of TTP and OS.

Conclusions

VEGF-A and sVEGFR-2 levels were significantly higher in patients than in the controls. A combination of VEGF-A and sVEGFR-2 can be used as an independent prognostic biomarker in advanced NSCLC.     

Histological classification of human gliomas: state of art and controversies

The histological classification of human gliomas remains in 2005 a challenge. The aim is to define the histological type of glioma (astrocytic, oligodendrocytic or mixed) and the grade in order to classify the patients and give them an accurate treatment. Although the standard remains the WHO classification, this classification suffered from lack of reproducibility among pathologists. In particular this classification does not take into account the intrinsic morphological heterogeneity of infiltrative gliomas and does not discriminate the tumour cells from the residual brain parenchyma. According to the WHO classification, infiltrative gliomas encompass astrocytic gliomas (diffuse astrocytomas grade II, anaplastic astrocytomas grade III and glioblastomas grade IV), oligodendroglial tumours (oligodendrogliomas grade II, anaplastic oligodendrogliomas grade III) and mixed gliomas (oligoastrocytomas grade II and anaplastic oligoastrocytomas grade III). Circumscribed gliomas mainly corresponds to pilocytic astrocytomas (grade I). In contrast, the Sainte Anne classification takes into account the macroscopic informations provided by imaging techniques and the tumour growth patterns. Three distinct tumour growth patterns may be seen in gliomas, type I: tumor tissue only, type II: tumour tissue and isolated tumor cells permeating the brain parenchyma (ITC) and type III: ITCs only and no tumor tissue. According to the Sainte Anne classification, gliomas are divided into astrocytic gliomas (pilocytic astrocytomas, structure type I, glioblastomas structure type II) and oligodendrogliomas and mixed oligoastrocytomas (grade A: lack of contrast enhancement and lack of endothelial hyperplasia, structure type III; and grade B: contrast enhancement or endothelial hyperplasia, structure type II and III). In the future the glioma classification has to be unique and should take into account clinical data, neuroradiological and histological features and results of molecular biology.     

S100A13, a new marker of angiogenesis in human astrocytic gliomas

S100 proteins are Ca²⁺-binding polypeptides involved in the tumourigenesis of several human neoplasms. S100A13 is a key regulator of the stress-dependent release of FGF1, the prototype of the FGF protein family involved in angiogenesis. Indeed, S100A13 is a copper binding protein able to enhance the export of FGF1 in response to stress in vitro and to induce the formation of a multiprotein aggregate responsible for FGF1 release. We investigated the expression of S100A13 in human astrocytic gliomas in␣relation to tumour grading and vascularization. A series of 26 astrocytic gliomas was studied to evaluate microvessel density and to assess FGF1, S100A13 and VEGF-A expression. FGF1 was equally expressed in the vast majority of tumours, whereas S100A13 and VEGF-A were significantly up-regulated in high-grade vascularized gliomas. Moreover, both S100A13 and VEGF-A expression significantly correlated with microvessel density and tumour grading. These data suggest that the up-regulation of S100A13 and VEGF-A expression correlates with the activation of angiogenesis in high-grade human astrocytic gliomas.     

Survivin expression and its relation with proliferation, apoptosis, and angiogenesis in brain gliomas

BACKGROUND: An unbalance of cell proliferation and cell apoptosis is an important mechanism in carcinogenesis, and angiogenesis also plays a crucial role in tumorigenesis. Recently, survivin has been identified as an important member of the inhibitor of apoptosis protein (IAP) family. Although it has been shown that survivin is highly expressed in gliomas, and is associated with tumorigenesis, progression, and poor prognosis of gliomas, as yet the relation of survivin expression with proliferation, apoptosis, and angiogenesis of gliomas it is still unclear. METHODS: Eighty-three cases of brain glioma were chosen and protein expressions of survivin and proliferating cell nuclear antigen (PCNA) in glioma cells and Factor VIII-related antigen (FVIII-RAg) in vascular endothelial cells were investigated by immunohistochemistry. Apoptotic cells of brain glioma were screened by TdT-mediated dUTP nick end-labeling (TUNEL), and survivin immunoreactivity score (IRS), proliferative index (PI), apoptotic index (AI), overall daily growth (ODG), and microvessel density (MVD) in brain gliomas were measured. RESULTS: The survivin IRS, PI, AI, ODG, and MVD of brain gliomas were 3.75 +/- 3.89, 28.39 +/- 19.49%, 1.00 +/- 0.80%, 12.19 +/- 10.21%, and 62.75 +/- 31.50, respectively, and all of them increased markedly with an increase in the pathologic grade of brain gliomas (P < 0.001 for all). PI, ODG, and MVD in the survivin-positive group were significantly higher than those in the survivin-negative group (P < 0.001 for all). PI, ODG, and MVD were positively correlated with survivin IRS (P < 0.001 for all). Although there was no significant difference between AI in the survivin-positive group or in the survivin-negative group (P = 0.108), AI was inversely correlated with survivin IRS (P = 0.005). CONCLUSIONS: Survivin is overexpressed in brain gliomas, which may play an important role in malignant proliferation, antiapoptosis, and angiogenesis of brain gliomas.     

Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas.

Macrophages are key participants in angiogenesis. In this study on human brain tumors, we first investigated whether macrophage infiltration is associated with angiogenesis and malignant histological appearance. Immunostaining of macrophages and small vessels in resected glioma specimens indicated that numbers of infiltrating macrophages and small vessel density were higher in glioblastomas than in astrocytomas or anaplastic astrocytomas. Macrophage infiltration was closely correlated with vascular density in human gliomas. Heme oxygenase-1 (HO-1), which is the rate-limiting enzyme in heme catabolism, was also associated with activated macrophages. Expression of mRNA encoding HO-1 was correlated with macrophage infiltration and vascular density in human glioma samples. Infiltrating macrophages were positively stained with anti-HO-1 antibody by immunohistochemical analysis, and in situ hybridization for HO-1 indicated that HO-1 was expressed in infiltrating macrophages in gliomas. HO-1 gene may be a useful marker for macrophage infiltration as well as neovascularization in human gliomas.     

Determination of microvessel density by quantitative real-time PCR in esophageal cancer: correlation with histologic methods, angiogenic growth factor expression, and lymph node metastasis.

PURPOSE: Angiogenesis and lymphangiogenesis are important steps in tumor growth and dissemination and are of prognostic importance in solid tumors. The determination of microvessel density (MVD) by immunohistology is subject to considerable variability between different laboratories and observers. We compared MVD determination by immunohistology and quantitative real-time PCR and correlated the results with clinical variables. EXPERIMENTAL DESIGN: The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2, and lymphatic endothelial markers VEGFR-3, Prox, and LYVE was assessed by quantitative PCR (qPCR) in primary surgical samples. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was quantified by PCR and correlated with MVD and clinical variables. RESULTS: The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2 correlated with each other in 54 samples of primary esophageal cancer (P < 0.0001 for all comparisons). MVD determined immunohistologically by CD31 staining in a subgroup of 35 patients correlated significantly with the qPCR method. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was significantly associated with MVD (P < 0.0001 for all comparisons). Analysis of the expression of lymphendothelial markers VEGFR-3, Prox, and LYVE revealed concordant results, indicating that quantification of lymphendothelial cells is possible by qPCR. The presence of lymph node metastasis on surgical specimens was significantly correlated with MVD (P < 0.003), VEGFR-2 (P < 0.048), and VEGF-C (P < 0.042) expression. CONCLUSIONS: These results indicate that quantification of MVD by qPCR in surgical samples of esophageal carcinoma yields similar results with immunohistology. Interestingly, the extent of angiogenesis and lymphangiogenesis was not related in individual tumor samples. Lymph node metastases could be predicted by MVD and VEGF-C expression.