Vascular Endothelial Growth Factor (VEGF) in Astrocytic Gliomas – A Prognostic Factor?

Survival in astrocytic gliomas is closely related to WHO tumor grade. Within one tumor grade, especially in grade II and III tumors, the clinical course is variable and can hardly be predicted by histological criteria. Neovascularization is a neuropathological hallmark in high grade gliomas and angiogenic factors may play an important role in malignant tumor progression. Therefore, 162 primary astrocytic gliomas (57 astrocytomas WHO grade II, 27 astrocytomas WHO grade III and 78 glioblastomas WHO grade IV) were investigated immunohistochemically for expression of vascular endothelial growth factor (VEGF), which is considered to represent the main angiogenic factor in astrocytic gliomas. Clinical data known to influence prognosis were documented. VEGF expression was found in 21 of 57 astrocytomas WHO grade II (36.8%), in 18 of 27 astrocytomas WHO grade III (66.7%) and in 50 of 78 glioblastomas (64.1%). A strong correlation between VEGF expression and survival was found within the whole study group, however, within one tumor grade no such correlation was obvious. In a multifactorial analysis VEGF expression was not found to be an independent prognostic factor in astrocytic gliomas.     

Nuclear karyopherin a2: a novel biomarker for infiltrative astrocytomas

The karyopherin (KPNA) protein family is involved in nucleocytoplasmic trafficking. Increased KPNA levels have been found to predict poor prognosis for a variety of solid tumors, including breast, ovarian, cervical, and prostate cancer, and melanoma. The purpose of this study was to evaluate karyopherin a2 as novel biomarker for astrocytic gliomas of WHO grades II-IV. We semiquantitatively measured nuclear expression of karyopherin a2 and the MIB1 labeling index, by immunohistochemical analysis, for 94 primary (23 astrocytomas WHO grade II, 24 astrocytomas WHO grade III, 47 glioblastomas) and 12 recurrent gliomas. In addition, IDH1 mutation status and Nijmegen breakage syndrome 1 protein expression were assessed, by immunohistochemical analysis, for all 71 malignant (WHO grade III and IV) and all 94 primary gliomas, respectively. Statistical analysis was performed by use of standard techniques. Karyopherin a2 expression correlated significantly with histological grade (p?5?years was seen only in those with KPNA2 expression levels ≤1?% (p?=?0.014). KPNA2 expression may have potential as a novel diagnostic and prognostic biomarker for astrocytic gliomas.     

Heme oxygenase-1 expression in human gliomas and its correlation with poor prognosis in patients with astrocytoma

In human glioma tumors, heme oxygenase-1 (HO-1) has been shown to be upregulated both when compared with normal brain tissues and also during oligodendroglioma progression. The cell types that express HO-1 have been shown to be mainly macrophages/microglia and T cells. However, many other reports also demonstrated that cell lines derived from glioma tumors and astrocytes express HO-1 after the occurrence of a wide variety of cell injuries and stressors. In addition, the significance of HO-1 upregulation in glioma had not, so far, been addressed. We therefore aimed at investigating the expression and significance of HO-1 in human glial tumors. For this purpose, we performed a wide screening of HO-1 expression in gliomas by using tissue microarrays containing astrocytomas, oligodendrogliomas, mixed tumors, and normal brain tissues. We subsequently correlated protein expression with patient clinicopathological data. We found differences in HO-1 positivity rates between non-malignant brain (22 %) and gliomas (54 %, p?=?0.01). HO-1 was expressed by tumor cells and showed cytoplasmic localization, although 19 % of tumor samples also depicted nuclear staining. Importantly, a significant decrease in the overall survival time of grade II and III astrocytoma patients with HO-1 expression was observed. This result was validated at the mRNA level in a cohort of 105 samples. However, no association of HO-1 nuclear localization with patient survival was detected. In vitro experiments aimed at investigating the role of HO-1 in glioma progression showed that HO-1 modulates glioma cell proliferation, but has no effects on cellular migration. In conclusion, our results corroborate the higher frequency of HO-1 protein expression in gliomas than in normal brain, demonstrate that HO-1 is expressed by glial malignant cells, and show an association of HO-1 expression with patients’ shorter survival time.     

Cyclooxygenase-2 in oligodendroglial neoplasms

BACKGROUND: Although increased expression of cyclooxygenase-2 (COX-2) has been described in association with a variety of neoplasms, including tumors of astrocytic derivation, limited data are available on COX-2 expression in oligodendrogliomas. METHODS: The current study retrospectively reviewed 53 oligodendrogliomas and 7 oligodendroglioma-predominant oligoastrocytomas (mixed gliomas) for COX-2 expression and MIB-1 proliferative index (by immunohistochemistry) and for chromosome 1p status (by fluorescence in situ hybridization). RESULTS: Patients included 35 males and 25 females, with a mean age of 41 years (range, 12-73 years) at the time of surgery. Forty-four tumor specimens were classified as World Health Organization (WHO) Grade II neoplasms and 16 as WHO Grade III tumors. MIB-1 labeling indices (marker of cell proliferation) ranged from 0 to 22.3 (mean 4.5). Twenty-eight tumor specimens demonstrated allelic loss on chromosome 1p. Positive staining was observed in 17 tumor specimens with COX-2 antibody. COX-2-positive tumor specimens were also evaluated with CD68 (macrophage/microglial cell marker) by coimmunolabeling to confirm that the observed COX-2 immunostaining was not due to immunoreactive macrophages or microglial cells. COX-2 expression, lack of allelic loss at chromosome 1p, and high proliferation indices were associated with decreased survival (P = 0.002, P = 0.009, and P = 0.015, respectively). No correlation with outcome was found with patient gender, age at diagnosis, or histologic grade. CONCLUSIONS: Chromosome 1p, COX-2 immunoreactivity, and MIB-1 labeling indices correlated with outcome and were associated with decreased survival. There was not a one-to-one correspondence between COX-2 immunoreactivity and lack of allelic loss at chromosome 1p. Tumors with expression of COX-2 by immunohistochemistry may, in theory, benefit from treatment with therapeutic agents that inhibit COX-2.     

Clinicopathological evaluation of 78 astrocytomas in Taiwan with emphasis on a simple grading system

Summary The grading systems of astrocytic tumors have long been the subject of controversy. A simple, objective and effective grading method is urgently needed for the evaluation of prognosis and the planning of treatment. This study investigated the relationship of clinical prognostic factors to the grading method of Daumas-Duport. This method determines the grade of tumor based on the presence or absence of four morphological criteria: nuclear atypia, mitosis, endothelial proliferation, and necrosis. A total of 143 astrocytic tumors were reviewed and screened, of which 65 ordinary and 13 pilocytic astrocytomas were selected for grading and comparison. Among ordinary astrocytomas, the grading method distinguished 9.2% grade 1, 26.2% grade 2, 36.9% grade 3, and 27.6% grade 4. At least 2-year follow-up was available on all surviving patients. Median survival was 57, 32, 12.5, and 8 months in grades 1, 2, 3, and 4 tumors, respectively. By a multiple regression model and analysis of variance, grade is significantly associated with survival (total regression coefficient r = 0.711). Age lost its significance on survival after multiple regression analysis. Sex, location, and surgical procedure were all unrelated to survival after regression. The age distribution and survival of patients with pilocytic astrocytomas revealed that this is a distinct disease entity and should not be admixed with ordinary astrocytomas in a grading scheme.This paper is part of his master thesis.     

Gene expression profiles of human glioblastomas are associated with both tumor cytogenetics and histopathology

Despite the increasing knowledge about the genetic alterations and molecular pathways involved in gliomas, few studies have investigated the association between the gene expression profiles (GEP) and both cytogenetics and histopathology of gliomas. Here, we analyzed the GEP (U133Plus2.0 chip) of 40 gliomas (35 astrocytic tumors, 3 oligodendrogliomas, and 2 mixed tumors) and their association with tumor cytogenetics and histopathology. Unsupervised and supervised analyses showed significantly different GEP in low- vs high-grade gliomas, the most discriminating genes including genes involved in the regulation of cell proliferation, apoptosis, DNA repair, and signal transduction. In turn, among glioblastoma multiforme (GBM), 3 subgroups of tumors were identified according to their GEP, which were closely associated with the cytogenetic profile of their ancestral tumor cell clones: (i) EGFR amplification, (ii) isolated trisomy 7, and (iii) more complex karyotypes. In summary, our results show a clear association between the GEP of gliomas and tumor histopathology; additionally, among grade IV astrocytoma, GEP are significantly associated with the cytogenetic profile of the ancestral tumor cell clone. Further studies in larger series of patients are necessary to confirm our observations.     

Histone deacetylase 3 expression correlates with vasculogenic mimicry through the phosphoinositide3‐kinase / ERK–MMP–laminin5γ2 signaling pathway

Vasculogenic mimicry (VM) refers to the process by which highly aggressive tumor cells mimic endothelial cells to form vessel‐like structures that aid in supplying enough nutrients to rapidly growing tumors. Histone deacetylases (HDACs) regulate the expression and activity of numerous molecules involved in cancer initiation and progression. Notably, HDAC3 is overexpressed in the majority of carcinomas. However, thus far, no data are available to support the role of HDAC3 in VM. In this study, we subjected glioma specimens to immunohistochemical and histochemical double‐staining methods and found that VM and HDAC3 expression were related to the pathological grade of gliomas. The presence of VM correlated with HDAC3 expression in glioma tissues. The formation of tubular structures, as determined by the tube formation assay to evaluate VM, was impaired in U87MG cells when transfected by siRNA or treated with an HDAC3 inhibitor. Importantly, the expression of VM‐related molecules such as MMP‐2/14 and laminin5γ2 was also affected when HDAC3 expression was altered. Furthermore, U87MG cells were treated with a phosphoinositide 3‐kinase (PI3K) inhibitor or/and ERK inhibitor and found that the PI3K and ERK signaling pathways play key roles in VM; whereas, in VM, the two signaling pathways did not act upstream or downstream from each other. Taken together, our findings showed that HDAC3 contributed to VM in gliomas, possibly through the PI3K/ERK–MMPs–laminin5γ2 signaling pathway, which could potentially be a novel therapeutic target for gliomas.     

Proliferation and Apoptosis in Long-term Surviving Low Grade Gliomas in Relation to Radiotherapy

Identification of patients with a low grade glioma with a long-term recurrence-free survival is of clinical value as radiotherapy can be postponed until recurrence. The recurring glioma may increase in malignancy compared to the original tumor, which is possibly related to radiotherapy. We studied proliferation by counting mitotic figures and by MIB-1 labeling, apoptosis by TUNEL and expression of proteins related to cell cycle regulation by immunohistochemical analysis of p53, p21, bcl-2 and bax expression in 48 low grade gliomas. Astrocytomas (A, n = 14) and oligodendrogliomas (O, n = 4) with a recurrence-free survival of more than 9 years after surgery had a significantly lower p53 index compared to A (n = 18) and O (n = 12) with a histopathologically documented recurrence. Additionally, the recurrence-free A had a higher p21 index. No significant differences were observed in MIB-LI, TUNEL-LI, bcl-2 and bax expression. Initially low grade gliomas and their corresponding recurrences were compared (n = 30). In the gliomas without radiotherapy (n = 15), no differences in mitotic rate, TUNEL-LI, p53, p21, bcl-2 and bax expression were found between primary tumors and their recurrences. Only MIB-LI was higher in the recurrent tumors. In the gliomas with radiotherapy (n = 15) no differences were detected in these parameters between the original tumor and the recurrent tumor except for a higher number of mitoses in the recurrent tumors. We conclude that low grade gliomas with a long-term recurrence-free survival were characterized by a low p53 protein expression and, in the case of A, a higher p21 index. We found no evidence that radiotherapy is involved in changes of proliferation, apoptosis or expression of proteins related to cell cycle regulation in recurring gliomas.     

Tumor suppressor gene alterations in malignant gliomas: histopathological associations and prognostic evaluation.

We have examined a series of 135 gliomas for alterations of the p53, CDKN2A (p16) and PTEN tumor suppressor genes (TSGs) in order to evaluate the incidence of their inactivation as a function of tumor malignancy and cellular differentiation, and to examine potential associations with patient outcome. The composition of this series, classified using WHO criteria, is as follows: 27 grade 2 tumors (11 astrocytomas, 12 oligoastrocytomas, 4 oligodendrogliomas), 42 grade 3 tumors (22 astrocytomas, 16 oligoastrocytomas, 4 oligodendrogliomas), and 66 grade 4 tumors (63 astrocytomas and 3 oligoastrocytomas). Similar frequencies of p53 mutation were observed among grade 2 (37.0%), and grade 3 tumors (38.1%), as well as between astrocytomas and mixed tumors. CDKN2A and PTEN mutations were clearly associated with increasing tumor malignancy (occurring in 0% of grade 2 tumors, 14.3% and 4.8% respectively of grade 3 tumors, and 27.3% and 30.3% respectively of grade 4 tumors) and were observed at substantially higher rates among astrocytomas. For the tumor suppressor genes examined, there was no relationship between the occurrence of any two TSG inactivation events. With regard to outcome, the p53 genetic status showed no significant relationship with patient survival. The CDKN2 and PTEN alterations were negative prognostic indicators of survival when evaluated in all 135 gliomas, but failed to predict outcome when evaluated in either of the high grade (3 or 4) tumor groups.     

Anomalous p27kip1 expression in a subset of malignant gliomas

p27^Kip1 (p27) expression was immunohistochemically investigated in 28 astrocytic tumors, and compared with the cell proliferation index (MIB-1 staining index). Normal rat brains and surgical specimens from human nonneoplastic brain lesions were used as controls. In the rat brains, the astrocytes were exclusively p27-positive. The reactive astrocytes in various disease processes sometimes lacked p27 expression. The distribution of p27-positive cells was uniform in low-grade astrocytomas and heterogeneous in high-grade tumors. Double staining of p27 and MIB-1 showed a reciprocal pattern in most cases. The frequency of p27 expression was inversely correlated with MIB-1 staining index and tumor grade. However, several malignant gliomas showed high p27 expression in spite of high MIB-1 staining indices. In such cases, MIB-1-positive cells were occasionally p27-positive. In this paper we discuss the etiology of the anomalous p27 expression in a subset of malignant gliomas.     

Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer: specific role of class I histone deacetylases in vitro and in vivo.

PURPOSE: Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of tumors of the large intestine. However, despite the importance of these molecules, comparably little is known on expression patterns and functions of specific HDAC isoforms in colorectal cancer. EXPERIMENTAL DESIGN: We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model. RESULTS: We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA-based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did. CONCLUSIONS: The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.     

PAI-1 and EGFR expression in adult glioma tumors: toward a molecular prognostic classification

PURPOSE: Molecular classification of gliomas is a major challenge in the effort to improve therapeutic decisions. The plasminogen activator system, including plasminogen activator inhibitor type 1 (PAI-1), plays a key role in tumor invasion and neoangiogenesis. Epidermal growth factor receptor (EGFR) is involved in the control of proliferation. The contribution of PAI-1 and EGFR to the survival of gliomas was retrospectively investigated. METHODS AND MATERIALS: Fifty-nine adult gliomas treated by neurosurgery and conventional irradiation were analyzed, including 9 low-grade (2) and 50 high-grade (3-4) tumors (WHO classification). PAI-1 was measured on cytosols and EGFR on solubilized membranes using ELISA methods. RESULTS: High PAI-1 levels were strongly associated with high histologic grade (p < 0.001) and histologic necrosis (p < 0.001). PAI-1 also correlated positively with patient age (p = 0.05) and negatively with Karnofsky index (p = 0.01). By univariate analysis of the high-grade population, higher PAI-1 (p < 0.0001) and EGFR values (p = 0.02) were associated with shorter overall survival. Only PAI-1 was an independent factor in multivariate analysis. Grade 3 tumors with low PAI-1 (100% 3-year overall survival rate) presented the same clinical outcome as the low-grade tumors. CONCLUSIONS: In this prognostic study, PAI-1 and EGFR expression revealed similarities and differences between high-grade gliomas that were not apparent by traditional clinical criteria. These data strongly support that biologic factors should be included in glioma classification and the design of clinical trials to treat more homogeneous populations.