Supratentorial Pilocytic Astrocytomas, Astrocytomas, Anaplastic Astrocytomas and Glioblastomas are Characterized by a Differential Expression of S100 Proteins

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II-IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.     

WT1 expression distinguishes astrocytic tumor cells from normal and reactive astrocytes

Particularly in small brain biopsies, it might be difficult to distinguish reactive astrogliosis from low-grade or infiltration zones of high-grade astrocytomas. So far no immunohistochemical marker allows a reliable distinction. Recently, the over-expression of Wilms' tumor gene product WT1 was reported in astrocytic tumor cells. However, no sufficient data on WT1 expression in normal or reactive astrocytes are available. Therefore, we investigated WT1 expression in paraffin-embedded brain sections from 28 controls, 48 cases with astrogliosis of various etiology and 219 astrocytomas [World Health Organization (WHO) grades I-IV] by immunohistochemistry. In normal brains and in astrogliosis, expression of WT1 was restricted to endothelial cells. In astrocytomas, WT1-positive tumor cells were found in pilocytic astrocytomas (66.7% of cases), diffuse astrocytomas (52.7%) WHO grade II (52.7%), anaplastic astrocytomas (83.4%) and glioblastomas (98.1%). Overall, the majority of all astrocytic neoplasms (84.5%) expressed WT1. Establishing a cut-off value of 0% immunoreactive tumor cells served to recognize neoplastic astrocytes with 100% specificity and 68% sensitivity and was associated with positive and negative predictive values of 1 and 0.68, respectively. Therefore, WT1 expression in astrocytes indicates a neoplastic origin and might represent an important diagnostic tool to differentiate reactive from neoplastic astrocytes by immunohistochemistry.     

Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas

Frequent allelic losses on the long arm of chromosome 22 (22q) in gliomas indicate the presence of tumor suppressor gene (TSG) at this location. However, the target gene(s) residing in this chromosome are still unknown and their putative roles in the development of astrocytic tumors, especially in secondary glioblastoma, have not yet been defined. To compile a precise physical map for the region of common deletions in astrocytic tumors, we performed a high-density loss of heterozygosity (LOH) analysis using 31 polymorphic microsatellite markers spanning 22q in a series of grade II diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas, and secondary glioblastomas that had evolved from lower grade astrocytomas. LOH was found at one or more loci in 33% (12/36) of grade II diffuse astrocytomas, in 40% (4/10) of anaplastic astrocytomas, in 41% (26/64) of primary glioblastomas, and in 82% (23/28) of secondary glioblastomas. Characterization of the 22q deletions in primary glioblastomas identified two sites of minimally deleted regions at 22q12.3-13.2 and 22q13.31. Interestingly, 22 of 23 secondary glioblastomas affected shared a deletion in the same small (957 kb) region of 22q12.3, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. Investigation of the promoter methylation and expression of this gene indicated that frequent hypermethylation correlated with loss of TIMP-3 expression in secondary glioblastoma. This epigenetic change was significantly correlated to poor survival in eight patients with grade II diffuse astrocytoma. Our results suggest that a 957 kb locus, located at 22q12.3, may contain the putative TSG, TIMP-3, that appears to be relevant to progression to secondary glioblastoma and subsequently to the prognosis of grade II diffuse astrocytoma. In addition, the possibility of other putative TSGs on 22q12.3-13.2 and 22q13.31 that may also be involved in the development of primary glioblastomas cannot be ruled out.     

Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Real-time quantitative PCR analysis of regions involved in gene amplification reveals gene overdose in low-grade astrocytic gliomas.

We have studied gene amplification of genes located in 1q32 (GAC1, ELF3, MDM4, and ren1), 4q11 (PDGFR-alpha), and in 12q13-14 (MDM2 and CDK4) using quantitative real-time PCR in a group of 86 tumors consisting of 44 WHO grade IV glioblastomas (GBM) (34 primary and 10 secondary tumors), 21 WHO grade III anaplastic astrocytomas (AA), and 21 WHO grade II astrocytomas (AII). Gene amplification was present in 56 of the 86 samples (65%) in at least 1 gene in our series. GAC1 (51%) and MDM4 (27%) were the most frequently amplified genes within the 1q32 amplicon, and their higher amplification frequency was statistically significant (P<0.05, chi) in the low-grade astrocytomas. Concordant co-amplification was determined for ELF3 and ren1 or ren1 and MDM4 in the grade III-IV tumors. MDM2 amplification was significantly more frequent in primary GBM (16%) than was in secondary GBM (0%). The present study shows that gene amplification in the studied regions is already present in low-grade astrocytic tumors and that amplification of some genes may represent another molecular marker to differentiate primary from secondary GBM.     

Genetic alterations associated with the evolution and progression of astrocytic brain tumours

Diffusely infiltrating low-grade astrocytomas (WHO grade II) have an intrinsic tendency for progression to anaplastic astrocytoma (WHO grade III) and glioblastoma (WHO grade IV). This change is due to the sequential acquisition of genetic alterations, several of which have recently been identified. In low-grade astrocytomas, p53 mutations with or without loss of heterozygosity on chromosome 17p are the principal detectable change. Anaplastic astrocytomas contain p53 mutations at an overall incidence of 34% and, in addition, loss of heterozygosity on chromosome 19q and frequent homozygous deletion of the p16 tumor suppressor (MTS-1) gene. The most malignant astrocytic neoplasms, the glioblastoma, further shows loss of chromosome 10 and amplification of the epidermal growth factor receptor (EGF-R) gene at overall incidences of 66% and 34%, respectively. The type and distribution of p53 mutations in astrocytic brain tumours are not suggestive of specific environmental carcinogens operative in their aetiology. Analysis of 91 families with p53 germline mutations reported to date show that tumours of the nervous system account to 12% of all neoplasms. Of a total of 57 brain tumours reported, 30 were classified histologically and of these, 73% were of astrocytic origin. The observation that somatic p53 mutations in sporadic brain tumours are largely restricted to those of astrocytic origin and that astrocytomas also prevail among CNS neoplasms associated with p53 germline mutation strongly suggests, that p53 mutations are capable of initiating neoplastic transformation in astrocytes of the human nervous system.     

Detection of novel genomic aberrations in anaplastic astrocytomas by GTG-banding, SKY, locus-specific FISH, and high density SNP-array

Astrocytomas represent the largest and most common subgroup of brain tumors. Anaplastic astrocytoma (WHO grade III) may arise from low-grade diffuse astrocytoma (WHO grade II) or as primary tumors without any precursor lesion. Comprehensive analyses of anaplastic astrocytomas combining both cytogenetic and molecular cytogenetic techniques are rare. Therefore, we analyzed genomic alterations of five anaplastic astrocytomas using high-density single nucleotide polymorphism arrays combined with GTG-banding and FISH-techniques. By cytogenetics, we found 169 structural chromosomal aberrations most frequently involving chromosomes 1, 2, 3, 4, 10, and 12, including two not previously described alterations, a nonreciprocal translocation t(3;11)(p12;q13), and one interstitial chromosomal deletion del(2)(q21q31). Additionally, we detected previously not documented loss of heterozygosity (LOH) without copy number changes in 4/5 anaplastic astrocytomas on chromosome regions 5q11.2, 5q22.1, 6q21, 7q21.11, 7q31.33, 8q11.22, 14q21.1, 17q21.31, and 17q22, suggesting segmental uniparental disomy (UPD), applying high-density single nucleotide polymorphism arrays. UPDs are currently considered to play an important role in the initiation and progression of different malignancies. The significance of previously not described genetic alterations in anaplastic astrocytomas presented here needs to be confirmed in a larger series.     

Aberrant localization of the neuronal class III beta-tubulin in astrocytomas

BACKGROUND: The class III beta-tubulin isotype (betaIII) is widely regarded as a neuronal marker in development and neoplasia. In previous work, we have shown that the expression of betaIII in neuronal/neuroblastic tumors is differentiation dependent. In contrast, the aberrant localization of this isotype in certain nonneuronal neoplasms, such as epithelial neuroendocrine lung tumors, is associated with anaplastic potential. OBJECTIVE: To test the generality of this observation, we investigated the immunoreactivity profile of betaIII in astrocytomas. DESIGN: Sixty archival, surgically excised astrocytomas (8 pilocytic astrocytomas, WHO grade 1; 18 diffuse fibrillary astrocytomas, WHO grade 2; 4 anaplastic astrocytomas, WHO grade 3; and 30 glioblastomas, WHO grade 4), were studied by immunohistochemistry using anti-betaIII monoclonal (TuJ1) and polyclonal antibodies. A monoclonal antibody to Ki-67 nuclear antigen (NC-MM1) was used as a marker for cell proliferation. Antibodies to glial fibrillary acidic protein (GFAP) and BM89 synaptic vesicle antigen/synaptophysin were used as glial and neuronal markers, respectively. RESULTS: The betaIII immunoreactivity was significantly greater in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas; median labeling index [MLI], 35%; interquartile range [IQR], 20%-47%) as compared with diffuse fibrillary astrocytomas (MLI, 4%; IQR, 0.2%-21%) (P <.0001) and was rarely detectable in pilocytic astrocytomas (MLI, 0%; IQR, 0%-0.5%) (P <.0001 vs high-grade astrocytomas; P <.01 vs diffuse fibrillary astrocytomas). A highly significant, grade-dependent relationship was observed between betaIII and Ki-67 labeling and malignancy, but this association was stronger for Ki-67 than for betaIII (betaIII, P <.006; Ki-67, P <.0001). There was co-localization of betaIII and GFAP in neoplastic astrocytes, but no BM89 synaptic vesicle antigen/synaptophysin staining was detected. CONCLUSIONS: In the context of astrocytic gliomas, betaIII immunoreactivity is associated with an ascending gradient of malignancy and thus may be a useful ancillary diagnostic marker. However, the significance of betaIII-positive phenotypes in diffuse fibrillary astrocytomas with respect to prognostic and predictive value requires further evaluation. Under certain neoplastic conditions, betaIII expression is not neuron specific, calling for a cautious interpretation of betaIII-positive phenotypes in brain tumors.     

Reverse transcriptase polymerase chain reaction as a reliable method to detect epidermal growth factor receptor exon 2-7 gene deletion in human glioblastomas

Epidermal growth factor receptor (EGFR) gene amplification has been reported to occur in diverse carcinoma types such as lung, ovarian, and breast carcinomas and in glioblastomas. A 801-bp in-frame deletion close to the aminoterminus of the receptor protein has been found to occur more or less frequently within at least three of these tumor entities. We studied EGFR gene alterations using the polymerase chain reaction and EGFR gene expression of 65 astrocytic tumors (51 glioblastomas World Health Organization [WHO] IV, five anaplastic astrocytomas WHO III, and nine astrocytomas WHO II). EGFR gene amplification, as determined by Southern blotting using a full-length cDNA probe, was observed in 22 of 51 glioblastomas (43%) but in none of the grade II astrocytomas. Two of five anaplastic astrocytomas at WHO III showed a considerable degree of EGFR amplification but, according to the neuroradiological data, these two tumors had to be considered as glioblastomas. The most frequently found genetic alteration was the 801-bp deletion near the receptor aminoterminus comprising a complete loss of exon 2 to exon 7 (del2-7). We showed that RT-PCR is superior to Southern blot analysis in detection of this type of deletion and can be assigned to 9 of 38 (24%) glioblastomas examined. Expression of a EGF receptor protein was enhanced in most of the tumors with gene amplification. However, 5 of 18 tumors that express a receptor protein in the absence of EGFR gene amplification also showed elevated levels of EGFR gene expression. In addition to the full-length receptor protein, a signal in the 140-kDa range was observed in 17 of 35 glioblastomas (49%). This fragment may correspond to the truncated de12-7 receptor protein or might be due to proteolysis of the full-length receptor protein.     

Expression of the epidermal growth factor receptor in astrocytic tumours is specifically associated with glioblastoma multiforme

Summary Epidermal growth factor and its receptor (EGFR) constitute an important and well-characterized mitogenic system in various ectodermal tissues including glial cells. Over-expression of the EGFR due to gene amplification has been reported in primary brain tumours of glial origin. Using a monoclonal antibody to the EGFR and immunohistochemical analysis, we examined the expression and distribution of EGFR in 103 astrocytic tumours. In addition, selected tumours were studied by Western blotting using a polyclonal antibody to EGFR and by Southern blot analysis. Glioblastomas (WHO grade IV) showed EGFR expression in 37% of cases, whereas pilocytic (WHO grade I), low-grade (WHO grade II) or anaplastic astrocytoma (WHO grade III) were invariably EGFR negative. Generally, there was a close correlation between the presence of EGFR gene amplification and over-expression of receptor protein. Different patterns of immunoreactive cells and significant intratumour heterogeneity of EGFR expression were observed in glioblastomas. The specific association of EGFR over-expression with glioblastoma may provide a useful diagnostic tool for distinguishing anaplastic astrocytoma (WHO grade III) and glioblastoma multiforme (WHO grade IV).     

Expression of mos in astrocytic tumors and its potential role in neoplastic progression

The c-mos gene and its protein product mos, components of the mitogen-activated protein kinase transduction pathway, are known to be involved in the control of meiosis and mitosis. Apart from a study on lung carcinomas, there is little information about its role in human neoplasia. The aim of this study was to investigate expression of mos in astrocytic tumors and to correlate it with accumulation of p53. We studied expression of mos in 62 cases of supratentorial astrocytic tumor. Intracytoplasmic immunostaining for mos was found in 28 (45%) cases: 3 of 20 (15%) grade 2 astrocytomas, 9 of 20 (45%) grade 3 anaplastic astrocytomas, and 16 of 22 (73%) glioblastomas. Immunopositivity for mos correlated significantly (P < 0.01) with tumor grade but not with p53 expression. In contrast to the findings in relation to lung tumors, immunopositivity for mos in astrocytic tumors did not predict recurrence-free or overall survival time. Cytoplasmic immunostaining was observed in scattered large cortical neurons adjacent to tumors, possibly due to stress-induced abortive entry into the cell cycle. The correlation of mos immunopositivity with tumor grade may reflect the expansion of more malignant mos-positive clones. This study provides evidence that mos may be involved in the neoplastic progression of a proportion of astrocytic tumors.     

TP53 gene mutations and 17p deletions in human astrocytomas.

Astrocytomas, including the most malignant form, glioblastoma multiforme, are the most frequent and deadly primary tumors of the human nervous system. Recent molecular genetic analyses of astrocytomas have demonstrated frequent chromosome 17 deletions involving the telomeric region of the short arm (17p12-pter). This region contains a candidate tumor suppressor gene, TP53, which has recently been implicated in the etiology of a broad array of human cancers. To study the possible role of TP53 in astrocytoma development, 24 randomly chosen human astrocytic tumors were examined for genomic TP53 sequence aberrations using primer-directed DNA amplification in conjunction with direct sequencing. Five of the 11 grade III astrocytomas (glioblastoma multiforme), but only one of seven grade II astrocytomas (anaplastic astrocytoma) and none of either the grade I astrocytomas or oligodendrogliomas demonstrated distinct point mutations involving the TP53 gene. These data suggest that TP53 mutations may play a role in astrocytoma development and are predominantly associated with higher grade tumors.     

High Frequency of TP53 Mutations in Juvenile Pilocytic Astrocytomas Indicates Role of TP53 in the Development of These Tumors

In adults, the TP53 tumor suppressor gene is frequently mutated in astrocytic brain tumors which is supposed to represent an early event in their development. In juvenile pilocytic and low-grade astrocytomas, however, TP53 mutations have until now been reported as rare, which has led to the suggestion that these tumors may follow a different molecular pathogenesis with an involvement of genes other than TP53. Our analysis of 20 pilocytic and two low-grade astrocytomas of childhood, based on a comprehensive denaturing gradient gel electrophoresis (DGGE) mutation detection assay of the entire coding region, including all splice site junctions of TP53, showed mutations considered as causative in 7 of the 20 (35%) pilocytic astrocytomas and in one of the two low-grade astrocytomas. Our finding is significantly different from the mutation frequency of 1.3% (2/155) previously reported for these tumor types. This may be attributed to the mutation detection system used, which also detects mutations occurring outside the evolutionary conserved region of TP53. Our results suggest that, contrary to the present notion, TP53 mutations may well play a role in the development of juvenile astrocytomas. Furthermore, no mutations were found in tumors of patients with progression of residual tumor after postoperative follow-up. This suggests that TP53 mutations may be associated with less aggressive forms of juvenile astrocytomas, analogous to the situation in adult astrocytomas.     

Epidermal growth factor receptor expression in oligodendroglial tumors.

A series of 13 oligodendrogliomas (WHO grade II) and 20 anaplastic oligodendrogliomas (WHO grade III) was studied for gene amplification and expression of the epidermal growth factor receptor gene (EGFR). EGFR gene amplification was found in only one case of anaplastic oligodendroglioma, which additionally showed a deletion/rearrangement at the 5' end of the gene. Northern blot analysis, however, revealed increases of EGFR mRNA expression relative to non-neoplastic control brain in 6 of 13 oligodendrogliomas and 10 of 18 anaplastic oligodendrogliomas. All cases with increased mRNA expression showed strong immunoreactivity for EGFR protein. Our findings thus indicate that increased expression of EGFR mRNA and protein is common in low-grade and high-grade oligodendroglial tumors and in the vast majority of cases is not caused by gene amplification.     

Molecular Genetic Aspects of Oligodendrogliomas Including Analysis by Comparative Genomic Hybridization

Oligodendroglial neoplasms are a subgroup of gliomas with distinctive morphological characteristics. In the present study we have evaluated a series of these tumors to define their molecular profiles and to determine whether there is a relationship between molecular genetic parameters and histological pattern in this tumor type. Loss of heterozygosity (LOH) for 1p and 19q was seen in 17/23 (74%) well-differentiated oligodendrogliomas, in 18/23 (83%) anaplastic oligodendrogliomas, and in 3/8 (38%) oligoastrocytomas grades II and III. LOH for 17p and/or mutations of the TP53 gene occurred in 14 of these 55 tumors. Only one of the 14 cases with 17p LOH/TP53 gene mutation also had LOH for 1p and 19q, and significant astrocytic elements were seen histologically in the majority of these 14 tumors. LOH for 9p and/or deletion of the CDKN2A gene occurred in 15 of these 55 tumors, and 11 of these cases were among the 24 (42%) anaplastic oligodendrogliomas. Comparative genomic hybridization (CGH) identified the majority of cases with 1p and 19q loss and, in addition, showed frequent loss of chromosomes 4, 14, 15, and 18. These findings demonstrate that oligodendroglial neoplasms usually have loss of 1p and 19q whereas astrocytomas of the progressive type frequently contain mutations of the TP53 gene, and that 9p loss and CDKN2A deletions are associated with progression from well-differentiated to anaplastic oligodendrogliomas.     

IDH mutations in older patients with diffuse astrocytic gliomas

In 2016, the World Health Organization recommended that isocitrate dehydrogenase (IDH) mutation status be included in the classification of diffuse astrocytic gliomas. IDH mutations are part of the current definition of oligodendrogliomas and are predictive of a better outcome in diffuse astrocytic gliomas. A few studies, examining the role of routine IDH testing in older patients, came to differing conclusions and made differing recommendations regarding a routine IDH testing algorithm with respect to patient age. The purpose of this study was to examine IDH mutations in a series of diffuse astrocytic gliomas [N = 381; 53 diffuse astrocytomas (WHO grade II), 66 anaplastic astrocytomas (WHO grade III) and 262 glioblastomas (WHO grade IV)], paying particular attention to age of patient and any relationship between age and IDH status. IDH status was evaluated by immunohistochemistry with IDH-1 (R132H) antibody and if negative staining was noted, followup polymerase chain reaction (PCR) testing assessing for IDH-1 and IDH-2 mutations was performed. Overall, IDH mutations were discovered in 50.1% of grade II tumors, 54.4% of grade III tumors and 15.1% of grade IV tumors. Of tumors studied, 224 tumors (58.8%) arose in patients 55 years or older. Higher rates of IDH mutations were observed in the patient group less than 55 years of age versus those 55 years or older. By PCR testing in patients 55 years or older, non IDH-1 (R132H) mutations were noted in 0/4 grade II tumors, 3/11 grade III tumors and 26/37 grade IV tumors. The results of this study suggest that although IDH mutations in diffuse astrocytic gliomas are more frequently encountered in patients less than 55 years of age, a significant subset of older patients have mutations that would not be discovered on routine immunohistochemistry and therefore, followup PCR testing is recommended for all patients whose tumors are negative by immunohistochemistry.