Galectin‐3: A useful biomarker for differential diagnosis of brain tumors

Galectin‐3 (gal‐3) is a 31 kDa β‐galactoside‐binding lectin that is immunohistochemically expressed in macrophages, lymphocytes, and endothelial cells, and also in some neoplastic cells. Gal‐3's expression in and significance to brain tumors has not been fully addressed. Here, we investigated its immunohistochemical expression in 409 cases of surgically resected primary brain tumors, including various glioneuronal tumors, pituitary adenomas, meningiomas and Schwannomas, among others. In normal brain tissues, gal‐3 was robustly expressed in normal astrocytes, endothelial cells and macrophages. It showed consistent and diffuse positivity in 100% of the pilocytic astrocytomas, pleomorphic xanthoastrocytomas (PXA), Schwannomas, meningiomas, capillary hemangioblastomas, as well as in ependymomas, but it was completely negative in the diffuse astrocytomas, anaplastic astrocytomas, both low‐ and high‐grades of the oligodendrogliomas, central neurocytomas, and medulloblastomas. Definitely positive but heterogeneous expression was found in various tumors including subependymal giant cell astrocytomas (SEGA), classic glioblastoma multiforme, anaplastic oligoastrocytomas, CNS primitive neuroectodermal tumors (CNS PNETs), and hemangiopericytomas. Eighty percent of small cell glioblstomas were completely negative, but 20% showed heterogeneous positivity for gal‐3. Focal positivity for gal‐3 was also found in dysembryoplastic neuroepithelial tumors (DNTs) and gangliogliomas, in which the positive cells were the astrocytic component. On the basis of our immunohistochemical data in conjunction with previous reports, we therefore conclude that gal‐3 is differentially expressed in various brain tumors, and thereby, is a helpful biomarker in making differential diagnoses, especially in cases where a morphological diagnosis is controversial.     

Immunohistochemical analysis of cyclin A in astrocytic tumours

Cyclins are important regulators of the cell cycle; there is increasing evidence that some cyclins are positively involved in carcinogenesis. Amplification and translocation of the cyclin genes and overexpression of their mRNAs and proteins have been observed in a variety of tumours. We studied cyclin A protein in astrocytic tumours by immunohistochemical analysis. Immunohistochemistry with microwave antigen retrieval was carried out on formalin fixed, paraffin embedded material from 15 glioblastomas (WHO grade IV), 10 anaplastic astrocytomas (WHO grade III), seven diffuse low grade astrocytomas (WHO grade II) and nine pilocytic astrocytomas (WHO grade I) using antibodies against cyclin A and a proliferation marker MIB1. Staining for these antibodies was seen mainly in the tumour cell nuclei; 66% of all cases showing staining for cyclin A and 95% of all cases staining for MIB1. Mean labelling indices (LI) for cyclin A were higher in glioblastoma (mean LI-6.7) and anaplastic astrocytoma (mean LI-5.9) than low grade diffuse astrocytoma (mean LI-1.7) and pilocytic astrocytoma (mean LI −0.12), although there was no clear cut off point between the various tumour types. A good correlation was seen between labelling indices of cyclin A and MIB1 (Pearson correlation coefficient r =0.59, P <0.0001). Cyclin A is variably expressed in astrocytic tumours, either reflecting increased tumour proliferation (cyclin A being an integral component of the cell cycle), an alteration of its gene, protein upregulation or regulation of apoptosis. The genetic basis of expression of cyclin A in astrocytic tumours remains to be determined.     

Activation of STAT3, MAPK, and AKT in malignant astrocytic gliomas: correlation with EGFR status, tumor grade, and survival

Diffuse astrocytic gliomas are the most common human glial tumors with glioblastoma being the most malignant form. Epidermal growth factor receptor (EGFR) gene amplification is one of the most common genetic changes in glioblastoma and can lead to the activation of various downstream signaling molecules, including STAT3, MAPK, and AKT. In this study, we investigated the activation status of these 3 signaling molecules as well as wild-type (EGFRwt) and mutant (EGFRvIII) EGFR in 82 malignant astrocytic gliomas (55 glioblastomas and 27 anaplastic astrocytomas) using immunohistochemistry. The presence of EGFRwt, but not EGFRvIII, immunopositivity correlated significantly with prevalent EGFR gene amplification in glioblastomas. STAT3 and AKT activation correlated significantly with EGFR status, although the correlation for p-STAT3 was attributed exclusively to EGFRvIII. The distribution of these 3 activated molecules varied significantly with tumor grade; although activation of STAT3 was essentially identical between anaplastic astrocytomas and glioblastomas, an increase in the activation of MAPK and AKT appeared to correlate with the progression of anaplastic astrocytoma to glioblastoma. Finally, activated STAT3 and AKT were marginally predictive of improved and worse prognosis, respectively. Taken together, these findings begin to elucidate the interrelationship between these signaling pathways in astrocytic gliomas in vivo.     

J1‐31 protein expression in astrocytes and astrocytomas

J1‐31 is one of the astrocytic proteins, the expression of which has not been evaluated in astrocytomas. In the present study, we studied the expression of J1‐31 protein in astrocytes and astrocytomas in comparison with GFAP, p53 and Ki‐67. Materials consisted of formalin‐fixed paraffin‐embedded tissue specimens that included five cases of normal brain, 17 of gliosis, 15 of pilocytic astrocytoma (WHO grade I), 26 of low‐grade diffuse astrocytoma (WHO grade II), four of anaplastic astrocytoma (WHO grade III), and eight of glioblastoma (WHO grade IV). GFAP was highly expressed in all specimens examined. The anti‐J1‐31 antibody exhibited strong cytoplasmic staining of reactive gliosis in 17/17 (100%) cases with a higher intensity of staining than that observed in the adjacent normal astrocytes. The antibody showed reactivity with tumor cells in 12/15 (80%) cases of pilocytic astrocytoma, although intensity of staining was generally weaker and more focal than observed in reactive gliosis. J1‐31‐positive tumor cells were detected in only 9/26 (35%) cases of the low‐grade diffuse astrocytoma and none of the cases of anaplastic astrocytoma and glioblastoma. Increasing Ki‐67 indices paralleled advancing tumor grades. p53 protein was expressed more commonly in infiltrating astrocytomas compared to pilocytic astrocytoma. In conclusion, down‐regulation of J1‐31 expression correlates with advancing grade of astrocytomas. The result suggests this protein plays some role in astrocytes that is progressively lost in malignant progression. The anti‐J1‐31 antibody may help further our understanding of astrocytes in disease and may be useful as an aid in the pathologic diagnosis of astrocytic lesions.     

Elevated HLA-E levels in human glioblastomas but not in grade I to III astrocytomas correlate with infiltrating CD8+ cells

HLA-E is a ligand for the immune-inhibitory NKG2A receptor expressed on NK and T cells. To investigate HLA-E expression and immune cell infiltration in human astrocytic tumors in vivo, we analyzed normal CNS controls and astrocytomas of all WHO grades by immunohistochemistry. Both, CD8(+) immune cell infiltration and HLA-E expression were significantly higher in astrocytic tumors than in normal brain. Further, HLA-E expression levels and immune cell infiltration were significantly correlated in WHO grade IV glioblastomas. Thus, HLA-E overexpression in glioblastomas may be triggered by T and NK cell infiltration.     

Increased endothelial expression of transglutaminase in glioblastomas

D. A. Hilton, S. Love and R. Barber (1997) Neuropathology and Applied Neurobiology , 23, 507–511
Increased endothelial expression of transglutaminase in glioblastomas
Transglutaminases are a family of calcium-dependent enzymes that catalyse the formation of covalent cross-links between proteins. They have several diverse functions and are thought to be involved in cell differentiation, apoptosis and blood coagulation. We have investigated the expression of tissue transglutaminase in five fibrillary astrocytomas, five anaplastic astrocytomas and seven glioblastomas by immunohistochemistry. Strongly labelled tumour cells were seen in most of the fibrillary and anaplastic astrocytomas and all of the glioblastomas. Labelling was particularly prominent in the pseudopalisading tumour cells that surrounded foci of necrosis and apoptosis in glioblastomas. Most of the immunostained cells did not themselves show morphological features of apoptosis. In addition, apoptotic cells were demonstrated using in situ end-labelling and by in situ hybridization with digoxigenin-labelled poly(A) oligonucleotide probes. Apoptotic cells demonstrated by both of these methods were most numerous in anaplastic astrocytomas and glioblastomas. However, their distribution did not correlate with that of the tumour cells showing transglutaminase labelling. Strong transglutaminase labelling was also observed in the endothelial cells of vessels showing microvascular proliferation in all of the glioblastomas studied. In contrast, endothelial transglutaminase labelling was weak or absent in lower grade astrocytic tumours. Enhanced expression of transglutaminase by endothelial cells in glioblastomas may contribute to the high prevalence of vascular thrombosis and necrosis in these tumours.     

Podoplanin expression in primary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma

Podoplanin, a mucin-like transmembrane sialoglycoprotein, promotes platelet aggregation and may be involved in cancer cell migration, invasion, metastasis, and malignant progression. Podoplanin/aggrus is highly expressed in testicular seminoma, suggesting that it may be a sensitive marker for testicular seminomas. Here we investigated the expression of podoplanin in central nervous system (CNS) germ cell tumors (GCTs) by immunohistochemical staining of tumor samples from 62 patients. In 40 of 41 (98%) germinomas (including germinomatous components in mixed GCTs), podoplanin was diffusely expressed on the surface of germinoma cells; lymphocytes, interstitial cells, and syncytiotrophoblastic giant cells were negative for podoplanin. Except for immature teratomas (12/17; 71%), podoplanin expression was absent in non-germinomatous GCTs, including seven teratomas, seven embryonal carcinomas, seven yolk sac tumors, and seven choriocarcinomas. In immature teratomas, focal podoplanin staining was observed in fewer than 10% of immature squamous and columnar epithelial cells. Thus, podoplanin expression may be a sensitive immunohistochemical marker for germinoma in CNS GCTs. As such, it may be useful for diagnosis, for monitoring the efficacy of treatment, and as a potential target for antibody-based therapy.Kazuhiko Mishima and Yukinari Kato contributed equally to this work.     

Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age: implications for classification of gliomas

WHO grading of human brain tumors extends beyond a strictly histological grading system by providing a basis predictive for the clinical behavior of the respective neoplasm. For example, patients with glioblastoma WHO grade IV usually show a less favorable clinical course and receive more aggressive first-line treatment than patients with anaplastic astrocytoma WHO grade III. Here we provide evidence that the IDH1 status is more prognostic for overall survival than standard histological criteria that differentiate high-grade astrocytomas. We sequenced the isocitrate dehydrogenase 1 gene (IDH1) at codon 132 in 382 patients with anaplastic astrocytoma and glioblastoma from the NOA-04 trial and from a prospective translational cohort study of the German Glioma Network. Patients with anaplastic astrocytomas carried IDH1 mutations in 60%, and patients with glioblastomas in 7.2%. IDH1 was the most prominent single prognostic factor (RR 2.7; 95% CI 1.6-4.5) followed by age, diagnosis and MGMT. The sequence from more favorable to poorer outcome was (1) anaplastic astrocytoma with IDH1 mutation, (2) glioblastoma with IDH1 mutation, (3) anaplastic astrocytoma without IDH1 mutation and (4) glioblastoma without IDH1 mutation (p < 0.0001). In this combined set of anaplastic astrocytomas and glioblastomas both, IDH1 mutation and IDH1 expression status were of greater prognostic relevance than histological diagnosis according to the current WHO classification system. Our data indicate that much of the prognostic significance of patient age is due to the predominant occurrence of IDH1 mutations in younger patients. Immunohistochemistry using a mutation-specific antibody recognizing the R132H mutation yielded similar results. We propose to complement the current WHO classification and grading of high-grade astrocytic gliomas by the IDH1 mutation status and to use this combined histological and molecular classification in future clinical trials.     

Apoptosis in cerebral astrocytic tumours and its relationship to expression of the bcl-2 and p53 proteins

Apoptosis is an important determinant of tumour growth which can be regulated by the bcl-2 and p53 genes. This study examines the relationship between apoptosis, growth fraction (Ki-67 immunolabelling index), and accumulation of the bcl-2 and p53 proteins in a spectrum of cerebral astrocytic tumours (n=81). including fibrillary astrocytomas (n=16), anaplastic astrocytomas (n=19), and glioblastomas (n=46). Median apoptosis indices (AIs) increased across this spectrum of tumours, and a significant (P<0.0001) correlation was demonstrated between A1 and Ki-67 labelling index (LI). Immunolabelling with the bcl-2 antibody was found in 44% of fibrillary astrocytomas, 42% of anaplastic astrocytomas, and 28% of glioblastomas. It was also found in the vascular endothelial proliferation typically seen in glioblastomas, and in the giant, multinucleated cells of some glioblastomas. No clear relationship between AI and bcl- 2 accumulation was evident. Immunolabelling with the p53 antibody was found in 56% of fibrillary astrocytomas, 79% of anaplastic astrocytomas, and 50% of glioblastomas. No clear relationship between AI and patterns of p53 immunolabelling was evident. Equal proportions of p53-positive tumours were bcl- 2 positive and bcl- 2 negative, but a small proportion of p53-negative tumours was bcl- 2 positive. The correlation between A1 and Ki-67 LI is in line with findings in other malignant tumours. We suggest that the regulation of apoptosis in astrocytic tumours is too complex for a clear association between A1 and bcl- 2 and p53 protein expression to be demonstrated.     

Expression and activity of l-isoaspartyl methyltransferase decrease in stage progression of human astrocytic tumors

Protein l-isoaspartyl methyltransferase (PIMT) functions as a repair enzyme that acts upon damaged proteins bearing abnormal aspartyl residues. We previously reported that PIMT expression and activity are reduced by half in human epileptic hippocampus. Here we investigated PIMT regulation in astrocytic tumors, which are the most common human brain tumors. PIMT expression and enzyme activity were significantly decreased in all grades of human astrocytic tumors. More precisely, PIMT levels were significantly lower by 76% in pilocytic astrocytomas (grade I), 46% in astrocytomas (grade II), 69% in anaplastic astrocytomas (grade III), and a marked 80% in glioblastomas (grade IV) as compared to normal brains. RT-PCR analysis showed that levels of type I PIMT mRNA were up-regulated while those of type II PIMT mRNA were down-regulated in glioblastomas. Furthermore, the reduced PIMT levels correlated closely with a decrease in the number of neuron cells in astrocytic tumors as assessed by measuring the neuron-specific enolase level. Many proteins with abnormal aspartyl residues accumulated in brain tumors and some were specific to individual grades of astrocytic tumors. Similar results were obtained, either by measuring the reduction in PIMT activity and expression or by measuring the formation of abnormal proteins, in an orthotopic rat brain tumor model implanted with invasive CNS-1 glioma cells. The novelty of these findings was to provide the first evidence for a marked reduction of PIMT expression and activity during stage progression of astrocytic tumors in humans.     

Effects of treatment on long-term survivors with malignant astrocytomas

We reviewed the records of 160 consecutive patients with glioblastoma and anaplastic astrocytoma to evaluate the long-term consequences of radiation therapy and chemotherapy. We defined long-term survivors as those patients with glioblastoma or anaplastic astrocytoma who lived at least 100% longer than median survival of historical controls, for example, 2 years for patients with glioblastoma and 4 years for patients with anaplastic astrocytoma. There were 9 (5.6%) long-term survivors. Three (30%) became demented and died without evidence of tumor recurrence. One, after survival of 10 years, died of tumor recurrence. Of the remaining survivors, 2 (22%) have significantly impaired short-term memory function and other neurological deficits such as gait apraxia. Three (30%) can function independently. It is likely but cannot be proved that it is radiotherapy and not chemotherapy that is the causal factor of this dismal therapeutic outcome. Our study suggests restraint in the use of radiotherapy for patients with brain tumors that have more favorable prognoses than glioblastomas and anaplastic astrocytomas, such as low-grade astrocytomas and oligodendrogliomas.     

The glucose transporter and blood-brain barrier of human brain tumors

The glucose transporter of the human brain has been localized to endothelial cells expressing the blood-brain barrier, but little is known regarding its mechanism of induction or whether its expression is exclusively linked with restricted vascular permeability. We investigated glucose transporter expression by vessels in human astrocytic tumors and pulmonary metastases to the brain using immunohistochemical techniques. Vessels in 9 of 10 low-grade astrocytomas and 8 of 10 anaplastic astrocytomas were positive for glucose transporter. Glioblastoma vessels were transporter-positive in only 2 of 10 specimens. Vessels in all three metastatic tumors were negative for the glucose transporter. The decrease in transporter expression observed in higher-grade tumors occurred independently of increases in vascular permeability. In low-grade astrocytomas and glioblastomas transporter expression and contrast enhancement were inversely related, but vessels in 6 of 9 anaplastic astrocytomas were transporter-positive despite contrast enhancement. These findings suggest that separate mechanisms induce the glucose transporter and the permeability restrictions of the human blood-brain barrier. They also have potential implications for the therapy and prognosis of astroglial neoplasms.     

APO2L/TRAIL expression in human brain tumors

APO2 ligand (APO2L)/TRAIL is a novel member of the tumor necrosis factor cytokine family and a potent inducer of apoptosis in tumor cell lines. We recently reported that APO2L is consistently expressed in low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, and cell lines derived thereof, and that malignant glioma cell lines are susceptible to APO2L-induced apoptosis. In this study, we investigated whether APO2L is expressed in medulloblastoma or neuroblastoma cell lines and whether these cells are sensitive to APO2L-induced apoptosis. Immunoblot analyses revealed full-length APO2L protein expression in one (DAOY) of three medulloblastoma cell lines but not in two neuroblastoma cell lines (SKN-BE and SKN-LE). Viability assay performed after exposure to soluble APO2L for 16 h showed that DAOY medulloblastoma cells were the most sensitive and that apoptosis induced by APO2L was greatly enhanced when protein synthesis was inhibited by cycloheximide. Neuroblastoma cell lines were almost completely resistant to APO2L-induced apoptosis. We also carried out APO2L immunohistochemistry in a total of 115 tumors of the nervous system with different histogenesis and biological behavior. In all 9 pilocytic astrocytomas, the areas of dense fibrillary network showed diffuse and strong APO2L expression. In oligodendrogliomas, APO2L expression was observed in areas with a significant admixture of astrocytic cells, but was absent in neoplastic oligodendrocytes. In 13 of 14 ependymomas, APO2L was expressed in perivascular pseudorosettes. In all 12 medulloblastomas, strong APO2L expression was observed in intra-tumoral-reactive astrocytes, but neoplastic cells did not show APO2L immunoreactivity. Thus, the pattern of APO2L expression was largely similar to that of glial fibrillary acidic protein (GFAP), except for choroid plexus tumors and 3 of 8 anaplastic meningiomas, in which APO2L was focally expressed without concomitant GFAP expression. APO2L expression was absent in meningiomas, neurocytomas, and schwannomas. Thus, there is considerable heterogeneity of APO2L expression and susceptibility to APO2L-induced apoptosis among human brain tumors. Received: 18 August 1999 / Revised, accepted: 29 September 1999     

Cerebellar astrocytomas: a 24-year experience

INTRODUCTION: Cerebellar astrocytomas are the most benign tumors of the CNS. Seventy to eighty percent are found in children. METHODS AND RESULTS: We report on 38 children under 18 who had cerebellar astrocytoma in the posterior fossa and were treated by a multidisciplinary team in our Neurosurgical Department from January 1974 to December 1997. We included all patients in whom the histopathological diagnosis was astrocytoma, regardless of malignancy. The diagnostic methods used were pneumoventriculography, cranial X-rays, CT scan, and MRI. All patients were treated surgically. Neither radiotherapy nor chemotherapy was indicated in patients with pilocytic or fibrillary astrocytomas. A greater prevalence was observed in female (25/38; 66%) than in male (13/38; 34%) patients. Histopathological results revealed 27 (71%) pilocytic astrocytomas, 8 (21%) diffuse fibrillary astrocytomas, 1 (2%) anaplastic astrocytoma and 2 (6%) glioblastomas. These tumors were more frequently located in the right cerebellar hemisphere; increased intracranial pressure syndrome was the most frequent form of clinical presentation. Total tumor resection was obtained in 29 (83%) cases and subtotal resection in 9 (17%). In 6 (16%) cases, ventriculoperitoneal shunts were placed to control persistent hydrocephalus after tumor excision. CONCLUSION: The most frequent complication was increased ataxia. The mortality rate was 8.5%.     

Immunohistochemical study of human brain tumors with vimentin and astroprotein (GFAP)

Distributions of two different subclasses of intermediate filaments, vimentin and glial filaments, were studied immunohistochemically in human brain tumors using specific antiserum to each protein subunit, vimentin and astroprotein (GFAP), Surgical specimens (5 meningiomas, 4 ependymomas, 5 benign astrocytomas, 5 anaplastic astrocytomas and 7 glioblastomas) were fixed in 95% ethanol or ethanol-acidic acid (95:5) and embedded in paraffin Avidin biotin peroxidase-complex (ABC) method (Vectastain) was carried out on 6 microns-thick paraffin sections. All meningioma cells were negative for astroprotein (GFAP) and positive for vimentin. Ependymoma cells showed various patterns of immunoreaction for astroprotein (GFAP) but were invariably positive for vimentin. In benign astrocytomas, many cells (or cell body and processes) were positive for astroprotein (GFAP). Immunoreaction for vimentin was, however, less frequent and intense. In anaplastic astrocytomas, population of astroprotein (GFAP)-positive cells decreased and vimentin-positive cells increased. Astroprotein (GFAP)-positive cells were further decreased in glioblastomas and the population of vimentin-positive cells varied among tissues. The present study suggests that the anaplastic change of astrocytoma cells were associated with decreased expression of glial filaments and increased expression of vimentin filaments. It was also suggests that the expression of both intermediate filaments may be suppressed in highly-malignant glial tumor cells.     

Amplification of the platelet-derived growth factor receptor-A (PDGFRA) gene occurs in oligodendrogliomas with grade IV anaplastic features

Activation of the platelet-derived growth factor (PDGF) signaling system has been implicated in the development and malignant progression of diffuse gliomas. Overexpression of PDGF system components, particularly the alpha subtype receptor (PDGFRA), is common in glial tumors, and PDGFRA gene amplification has been reported in glioblastomas. In order to address the incidence of PDGFRA gene amplification in a broad set of diffuse gliomas, we used Southern and fluorescence in situ hybridization (FISH) analyses to examine 167 astrocytic gliomas (20 grade III and 147 grade IV), 41 anaplastic oligodendrogliomas, and 29 anaplastic oligoastrocytomas. PDGFRA gene amplification was identified in 4 anaplastic oligodendrogliomas and in a single case of anaplastic oligoastrocytoma, but in none of the malignant astrocytomas. Each of the 5 tumors with PDGFRA amplification displayed features generally associated with grade IV malignancy in astrocytic tumors. Consequently, our data indicate that this gene alteration is restricted to tumors having oligodendroglial differentiation and highly anaplastic features.