Astrocytomas Of Different Grades Research Articles

Proteomic profiling distinguishes astrocytomas and identifies differential tumor markers

Methods to permit more precise delineation of astrocytomas of different grades may have therapeutic utility. The authors selectively microdissected pure populations of cells from normal brain and astrocytomas. They performed two-dimensional protein gel electrophoresis (2DGE) followed by protein sequencing. Differential expression was confirmed immunohistochemically. 2DGE identified proteomic patterns and proteins that differentiated normal brain from tumor and distinguished astrocytomas of increasing grade.

Immunocytochemical detection of 14-3-3 in primary nervous system tumors

14-3-3 proteins have attracted much recent interest in the etiopathogenesis of human cancers owing to their involvement in the prevention of apoptosis. However, the expression of 14-3-3 in primary nervous system tumors has not been previously characterized. In this paper, Immunohistochemistry using a specific anti-14-3-3 antibody was performed on formalin-fixed, paraffin embedded archival tissue from 124 primary human nervous system tumors and 10 normal brain tissues. In the normal control brains, 14-3-3 immunoreactivity was localized mainly in the neuronal somata and processes, and some glial cells showed only weak immunoreactivity. However, 14-3-3 immunoreactivity was seen in the majority of astrocytomas [grade I (9/11), II (16/21), III (13/17), IV (17/21)]. There was no difference between the positive expression rates of 14-3-3 in different grades of astrocytomas (P = 0.968). But the intensity and degree of 14-3-3 immunoreactivity in diffuse astrocytomas, anaplastic astrocytoma, and glioblastoma multiformes showed trends with tumor grade, with glioblastomas having the highest positivity (P = 0.048). The 14-3-3 immunoreactivity was also seen in the majority of other gliomas [oligodendroglioma (2/3), anaplastic oligodendroglioma (4/4), ependymoma (1/2), anaplastic ependymoma (2/2), choroid plexus papilloma (3/3), pineocytoma (2/2), medulloblastoma (5/8)]. All meningiomas [syncytical (3), fibrous/fibroblastic (4), angiomatous (4), transitional/mixed (3)] were intensely and diffusely positive. All schwannomas (4), neurofibromas (2), pituitary adenomas (6) and craniopharyngiomas(4) also showed intense positive staining. These results showed that 14-3-3 is expressed in the majority of the primary human nervous system tumors. The up-regulated expression of 14-3-3 may be a common mechanism for evading apoptosis in most primary human nervous system tumors, and targeting 14-3-3 may be a novel promising strategy for the treatment of these tumors, especially for malignant tumors.

Expression of Telomeric Repeat Binding Factor-1 in Astroglial Brain Tumors

In human somatic cells, telomeres shorten with successive cell divisions, resulting in progressive genomic instability, altered gene expression, and cell death. Recently, telomere-specific deoxyribonucleic acid-binding proteins, such as telomeric repeat binding factor-1 (TRF1), have been proposed as candidates for the role of molecules regulating telomerase activity, and they have been suggested to play key roles in the maintenance of telomere function. The present study was designed to assess TRF1 expression in human astroglial brain tumors and to speculate on the clinical implications of its expression. Twenty flash-frozen surgical specimens obtained from adult patients who underwent craniotomy for microsurgical tumor resection, histologically verified as World Health Organization Grade II to IV astrocytomas, were used. Expression of TRF1 in astrocytomas of different grades was studied by means of both immunohistochemical and Western blotting analysis. The correlation between the extent of TRF1 expression and histological grading, performance status, and length of survival of patients underwent statistical analyses. TRF1 was expressed in all tumor samples. The level of its expression was variable, decreasing from low-grade through high-grade astrocytomas (P = 0.0032). TRF1 expression correlated with the patient's length of survival (P < 0.001) and performance status (P < 0.001) and proved to be an independent indicator of length of survival. Our findings suggest that the loss of TRF1 expression capability, as a result of down-regulation of TRF1 expression in malignant gliomas cells, may play a role in the malignant progression of astroglial brain tumors.

Distinct methylation profiles of glioma subtypes.

Gliomas are tumors of the central nervous system with a wide spectrum of different tumor types. They range from pilocytic astrocytoma, with a generally good prognosis, to the extremely aggressive malignant glioblastoma. In addition to these 2 types of contrasting neoplasms, several other subtypes can be distinguished, each characterized by specific phenotypic, as well as genotypic features. Recently, the epigenotype, as evident from differentially methylated DNA loci, has been proposed to be useful as a further criterion to distinguish between tumor types. In our study, we screened 139 tissue samples, including 33 pilocytic astrocytomas, 46 astrocytomas of different grades, 7 oligoastrocytomas, 10 oligodendrogliomas, 10 glioblastoma multiforme samples and 33 control tissues, for methylation at CpG islands of 15 different gene loci. We used the semiquantitative high throughput method MethyLight to analyze a gene panel comprising ARF, CDKN2B, RB1, APC, CDH1, ESR1, GSTP1, TGFBR2, THBS1, TIMP3, PTGS2, CTNNB1, CALCA, MYOD1 and HIC1. Seven of these loci showed tumor specific methylation changes. We found tissue as well as grade specific methylation profiles. Interestingly, pilocytic astrocytomas showed no evidence of CpG island hypermethylation, but were significantly hypomethylated, relative to control tissues, at MYOD1. Our results show that glioma subtypes have characteristic methylation profiles and, with the exception of pilocytic astrocytomas, show both locus specific hyper- as well as hypomethylation.

Comparative Genomic Hybridization Reveals Recurrent Enhancements on Chromosome 20 and in One Case Combined Amplification Sites on 15q24q26 and 20p11p12 in Glioblastomas

We examined homogenized tissue samples of biopsies from 19 astrocytomas of different grades for genetic imbalances using comparative genomic hybridization (CGH): three astrocytomas grade II, and 16 astrocytomas grade IV (glioblastoma multiforme), one of the glioblastomas representing the recurrence of a benign oligoastrocytoma. In two of three cases of astrocytoma grade II, a gain of chromosome 7 was found. The alterations in the glioblastomas were complex, and most frequently showed the characteristic gain of chromosome 7 and loss of chromosome 10. The single analyzed case of recurrence of an oligoastrocytoma was characterized by a unique CGH pattern. This tumor showed two distinct alterations: apart from an amplification on 15q24q26, we found a distinct amplification of a small region on 20p11.2p12, which has not been previously described in brain tumors. Partial or complete gains of chromosome 20 arose in six other tumors; we conclude that chromosome 20 in particular 20p11.2p12, may harbor relevant genes for glioma progression.

Quantitative analysis of NF1 and OMGP gene transcripts in sporadic gliomas, sporadic meningiomas and neurofibromatosis type 1-associated plexiform neurofibromas.

The close association of neurofibromatosis type 1 (NF1) with gliomas raises the question of whether the NF1 gene may be involved in the pathogenesis of sporadic astrocytic brain tumors. However, no frequent mutations within NF1 have been described in these tumors. Recent data on a limited series of gliomas indicate that NF1 expression may even be increased, thereby questioning the role of NF1 as a tumor suppressor in astrocytomas. In the present study, we examined the expression of NF1 in a series of 96 tumors including astrocytomas, meningiomas and plexiform neurofibromas. NF1 RNA transcription levels were compared to those of the reference genes B2M, ACTB and GAPD. The expression of OMGP, which is interposed in the NF1 gene, served as an additional control. NF1 expression did not significantly diverge among different malignancy stages of astrocytomas. As expected, the plexiform neurofibromas showed only very low NF1 expression. A striking finding was the highly variable expression of those genes selected to serve as references. While B2M and ACTB exhibited comparable levels of expression within different grades of astrocytomas and meningiomas, GAPD showed an inverse pattern in these tumors. In conclusion, NF1 expression is strongly reduced in NF1-associated plexiform neurofibromas but not in astrocytic tumors. The significant differences between B2M, ACTB and GAPD transcript levels brings into question the common practice of defining gene expression as a ratio between the transcripts of interest and those of these reference genes.

Protein and messenger RNA expression of connexin43 in astrocytomas: implications in brain tumor gene therapy.

The expression of connexin43, the primary gap-junction constituent of glial cells, was evaluated at the messenger RNA and protein levels in different grades of astrocytoma to investigate the relevance of gap junctions in herpes simplex virus-thymidine kinase (HSV-tk)-mediated gene therapy of brain tumors. Transduction of the retroviral-mediated HSV-tk gene into tumor cells with subsequent administration of ganciclovir has recently been used as an experimental therapeutic strategy for treatment of brain tumors. One aspect of this approach is the bystander effect, which augments the efficacy of this therapeutic approach. Glioblastoma cells with minimum levels of connexin43 protein were transfected with a connexin43 complementary DNA. These cells manifested a marked increase in the in vitro bystander effect, supporting the contention that the in vitro bystander effect is a consequence of metabolic cooperation between cells mediated by gap junctions. To assess relative levels of gap-junction protein expression in the relevant tumor type, we examined primary astrocytomas, primary astrocytoma cell cultures, and glioblastoma cell lines. Although most astrocytoma tumor samples expressed connexin43, they differed in the level of expression, with the greatest variation exhibited in high-grade astrocytomas. Primary glioblastoma cell cultures and established glioblastoma cell lines also displayed some variability in connexin43 levels. In aggregate, our results anticipate that glioblastomas will have a varied bystander effect during HSV-tk gene therapy depending on the level of connexin43 expression.

PROGNOSTIC IMPLICATION OF ESTRAMUSTINE-BINDING PROTEIN IN ASTROCYTOMA

Estramustine-binding protein (EMBP) is a glycoprotein shown to be expressed in the cytoplasm of astrocytoma cells. Originally, it was found in rat prostatic tissue and described as a secretory protein. Its expression has been demonstrated to positively correlate with the degree of neoplastic transformation in astrocytoma tissue. EMBP has been proposed to be responsible for a specific and high affinity binding of estramustine in astrocytoma tumor tissue. In this study the expression of EMBP was evaluated by immunohistochemistry and radioimmunoassay in a series of astrocytoma of different grades. Staining intensity and the number of stained cells increased with the degree of malignancy. The levels of EMBP were 1.3-6.2 ng/g tumor tissue with higher levels in tumors of grade III to IV compared to grade I and II. It was found that a high expression of EMBP always implied a short survival of the patients. On the other hand, a low expression of EMBP did not always assure a favorable prognosis. It is proposed that EMBP might have a value to predict survival in patients with astrocytoma, especially if estramustine is to be included in the treatment schedule. However, further extended studies are needed before final conclusions can be made.

Choline‐Containing Compounds in Human Astrocytomas Studied by 1H NMR Spectroscopy In Vivo and In Vitro

We have studied 14 patients with different grades of astrocytomas using 1H NMR spectroscopy in vivo. Typically, astrocytomas exhibited a low N-acetylaspartate peak, a prominent signal from choline group-containing compounds, and lactate in the 1H NMR spectra in vivo. The uncorrected choline/creatine + phosphocreatine peak area ratios were higher in tumors than in normal brain tissue. Absolute concentration of choline-containing compounds (1.74 +/- 0.09 mmol/L) in the normal brain tissue was not different in any grade of astrocytoma, but total creatine concentration in healthy brain (7.49 +/- 0.30 mmol/L) was higher than that in grade IV astrocytomas (4.84 +/- 0.89 mmol/L). Relaxation constants of choline-containing compounds did not differ in tumors from those determined in normal brain. Perchloric acid extracts of biopsy samples from 35 astrocytomas and 13 samples of normal temporal white matter were analyzed with 1H NMR. Total concentration of choline-containing compounds did not differ between controls and any grade of astrocytoma when the quantification was done in vitro. It is interesting that phosphorylcholine concentration was about twofold greater in grade IV astrocytomas than in controls or other grades of astrocytomas. We conclude that high phosphorylcholine in grade IV astrocytomas may be an indicator of degree of malignancy. The proportional changes within the group of choline-containing compounds observed in vitro were not reflected in the NMR properties of choline signal in vivo.

Expression of neu/c-erbB-2 in human brain tumors

The neu/c-erbB-2 oncogene encodes a 185 kd transmembrane protein (p185). Here we have used the monoclonal antibody (mAb) 3B5 to determine the expression of p185 in a series of fixed biopsy specimens of 180 human brain tumors, including the most frequent entities and, in addition, 18 recurrent gliomas with malignant progression. In summary, 3B5 immunoreaction was most prominent in astrocytomas of different grades of malignancies and in meningiomas. In World Health Organization (WHO) grade II astrocytomas mab 3B5-immunoreaction was related to the cytomorphological phenotype. Fibrillary astrocytomas showed no or only a weak immunoreaction (four of five, 80%) in contrast with protoplasmic or gemistocytic astrocytomas, where a strong reaction was observed in most cases (six of nine, 66.6%, and four of five, 80%, respectively). In WHO grade II to WHO grade IV astrocytomas a trend towards higher scores with increasing grade was found. In a limited number of cases (18 gliomas and two meningiomas) of the tumor series tested other mAbs against neu/c-erbB-2 epitopes, especially the mabs 9G6 and CB11, gave qualitatively comparable results. In WHO grade I pilocytic astrocytomas a wide range of 3B5 immunoreactivity has been observed. The results of in situ hybridization using a 32P-labeled neu/erbB-2 RNA probe performed on four WHO grade I and II astrocytomas, seven WHO grade IV glioblastomas, one WHO grade II oligoastrocytoma, one WHO grade III anaplastic astrocytoma, and three WHO grade I meningiomas were consistent with these immunomorphological data, and Northern blot analysis also indicated an overexpression of neu/c-erbB-2 mRNA in gliomas of different grades of malignancy and in meningiomas. These elevated neu-erbB-2 transcript levels occurred in the absence of gene amplification. In a second series of recurrent gliomas with malignant progression (n = 18) the higher 3B5-immunoreaction scores were apparent in the more malignant recurrent gliomas. In this series the overexpression of neu/c-erbB-2 parallels glioma progression. In our cases it was not, however, correlated with the postoperative relapse-free interval or with the overall length of survival.

Morphological appearance, growth kinetics and glial fibrillary acidic protein (GFAP) expression in primary in vitro explant culture of astrocytic neoplasms.

Astrocytomas of different grades of malignancy were cultured as primary explant and their sequential growth pattern, glial fibrillary acidic protein (GFAP) expression and labelling index (LI) using bromodeoxyuridine (BrdU) were assessed and correlated with the grade of malignancy of the original tumor tissue. Low-grade astrocytomas showed patterns of growth that diverged from anaplastic astrocytomas and glioblastoma multiforme. The GFAP expression decreased with increasing time in culture in all astrocytomas irrespective of the grading. Maximum GFAP was, however, expressed in the morphologically well-differentiated stellate cells. Contrary to expectations, lower BrdU LI was observed in glioblastoma multiforme in comparison to low-grade astrocytomas, which suggests some unidentified mechanism of differentiation in vitro for astrocytomas of higher grade of malignancy. Hence, in contrast to reported literature on the prognostic value of studies on primary cultures, the present study cautions the extrapolation of the in vitro findings for astrocytomas.

Expression of ETS proto-oncogenes in astrocytes in human cortex

In order to investigate a possible function of ETS proto-oncogenes in human brain, we incubated a polyclonal antibody raised against the viral region of E26 homologous to ETS1 and ETS2 with human brain frontal cortex sections. Our results show that this antibody decorates astrocytes but not neurons. By using astrocytomas of different grades as a source of astrocytes, we demonstrate the presence of ETS1 and ETS2 messenger RNAs and proteins. This leads to the idea that ETS genes are expressed in cells with dividing potentialities in human cortex and that they could provide a new marker for glial cells. Recently, a microduplication on chromosome 21 including ETS2 locus was described in karyotypically ‘normal’ Down's syndrome and suspected in Alzheimer's disease; when testing Alzheimer's disease-affected brain cortex sections, no obvious difference was observed with the technique used.

Local cerebral blood flow and partition coefficients measured in cerebral astrocytomas of different grades of malignancy

Local cerebral blood flow and local partition coefficients were measured in patients with different grades of malignant cerebral astrocytomas (n=5) who inhaled 35% stable xenon during computed tomography scanning. Results were compared with those in age-matched normal subjects (n = 5). Mean values for local cerebral blood flow in the gray matter in patients with astrocytomas were decreased throughout the tumor mass and surrounding brain that was apparently free of tumor. Patients with highly malignant glioblastoma multiforme (astrocytoma grade IV; n=2) showed more variable values for local cerebral blood flow and local partition coefficients compared to those with astrocytomas of lower grades (grades I-II; n = 3). Local partition coefficients in gray matter invaded by grade IV astrocytoma were significantly higher than those in gray matter invaded by grade I-III astrocytomas. Local cerebral blood flow and local partition coefficients in the brain tissue surrounding grade IV astrocytomas were reduced to a greater extent than those in more benign tumors.