Low-grade Diffuse Astrocytomas Research Articles

Glioblastoma Multiforme Oncogenomics and Signaling Pathways

In the adult population, glioblastoma multiforme is one of the most common primary brain tumors encountered. Unfortunately, this highly malignant tumor represents over 50% of all types of primary central nervous system gliomas. The vast majority of GBMs develops quite rapidly without clinical, radiological, or morphologic evidence of a less malignant precursor lesion (primary or de novo GBMs), as compared to secondary GBMs that develop slowly by progression from diffuse low-grade astrocytomas. These GBM subtypes must be kept in mind because they may constitute distinct disease entities. Even though they look histologically quite similar, they likely involve different genetic alterations and signaling pathways. Decades of surgical therapy, radiotherapy, and chemotherapy have failed to drastically change survival. Clearly, we do not fully understand this tumor; however, the exciting genetic revolution in glioma research over the past decade is providing a promising outlook for exploring this tumor at the genetic level. Science has begun to elucidate the numerous genetic alterations and critical signaling pathways, and it has opened new exciting areas of research such as glioma stem cell biology and neoangiogenesis. This work has already begun to improve our understanding of GBM cell proliferation, migration, and invasion. Indeed, exciting novel targeted therapies are making their way to clinical trials based on this increased knowledge. This review provides the current understanding of GBM oncogenomics, signaling pathways, and glioma stem cell biology and discusses the potential new therapeutic targets on the horizon.

Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis.

BackgroundA verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations). In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells.MethodsRandom Amplified Polymorphic DNA (RAPD) analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA) and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM). The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s) scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation.ResultsThe overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods.ConclusionThis study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb.

Prognostic significance of loss of O6-methylguanine-DNA methyltransferase expression in supratentorial diffuse low-grade astrocytoma

BackgroundO6-Methylguanine-DNA methyltransferase is a DNA repair protein. Epigenetic silencing of MGMT function by its promoter hypermethylation is considered to contribute to carcinogenesis. If loss of function in MGMT is related to tumor progression, the immunohistochemical method may predict the malignant change of gliomas. MethodWe investigated the expression of MGMT by immunohistochemical method in 28 supratentorial hemispheric diffuse astrocytomas. The prognostic significance of MGMT expression, proliferation index (MIB-1), and various clinical factors was evaluated. ResultsThere were 19 MGMT-positive and 9 MGMT-negative astrocytomas. Their rates of malignant transformation at 5 years were 12.3% and 51.4%, respectively. The difference was significant in the univariate (P = .004) and multivariate analyses (P = .044). Age, sex, extent of surgery, MIB-1 value, and radiation therapy at initial treatment did not correlate with the malignant progression. The 10-year overall survival rates were 71.8% and 58.3% in the patients with MGMT-positive and MGMT-negative tumors, respectively, and were not significantly different between these 2 groups (P = .079). Two long-term survivors with MGMT-negative tumor responded well to nitrosourea-based chemotherapy and lived more than 8 years after malignant transformation. The patients' age (P = .0047) and the degree of surgical removal (P = .0082) affected the overall survival in the univariate analysis. In the multivariate analysis, none of these factors reached significance. ConclusionAlthough the status of MGMT did not affect the overall survival, immunohistochemical evaluation of MGMT expression may be a good marker for tumor progression.

Class III β-Tubulin and γ-Tubulin are Co-expressed and Form Complexes in Human Glioblastoma Cells

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.

Regression of Grade III Astrocytoma During the Treatment of CML with Imatinib Mesylate

Astrocytomas are central nervous system neoplasms, which are derived predominately from astrocytes. On the basis of the histopathologic characteristics astrocytomas are graded from I to IV. The cells that demonstrate the greatest degree of anaplasia are used to determine the histologic grade of the tumor. The mean age of survival are approximately 10 years from the time of diagnosis for pilocystic astrocytomas (World Health Organization grade I), more than 5 years for patients with low-grade diffuse astrocytomas (WHO grade II), 2 to 5 years for those with anaplastic astrocytomas (WHO grade III), and less than 1 year for patients with glioblastoma (WHO grade IV). The treatment is a combination of surgery, radiation, and chemotherapy depending of the grade of astrocytoma. We present a case of 31-year-old man with grade III astrocytoma with subsequent chronic myelogenous leukemia treated with imatinib mesylate as part of his chronic myelogenous leukemia treatment failing to show recurrence of the astrocytoma 10 years after standard treatment for astrocytoma.

Altered Cellular Distribution and Subcellular Sorting of γ-Tubulin in Diffuse Astrocytic Gliomas and Human Glioblastoma Cell Lines

Centrosome amplification is a pivotal mechanism underlying tumorigenesis but its role in gliomas is underinvestigated. The present study specifically examines the expression and distribution of the centrosome-associated cytoskeletal protein gamma-tubulin in 56 primary diffuse astrocytic gliomas (grades II-IV) and in 4 human glioblastoma cell lines (U87MG, U118MG, U138MG, and T98G). Monoclonal anti-peptide antibodies recognizing epitopes in C-terminal or N-terminal domains of the gamma-tubulin molecule were used in immunohistochemical, immunofluorescence, and immunoblotting studies. In tumors in adults (n = 46), varying degrees of localization were detected in all tumor grades, but immunoreactivity was significantly increased in high-grade anaplastic astrocytomas and glioblastomas multiforme as compared to low-grade diffuse astrocytomas (p = 0.0001). A similar trend was noted in diffuse gliomas in children but the sample of cases was too small as to be statistically meaningful. Two overlapping patterns of ectopic cellular localization were identified in both primary tumors and glioblastoma cell lines: A punctate pattern, in which gamma-tubulin was partially co-distributed with pericentrin in the pericentriolar region, and a diffuse pattern, independent of pericentrin staining, denoting a soluble pool of gamma-tubulin. Cellular gamma-tubulin was detected in both soluble and insoluble (nocodazole-resistant) fractions of glioblastoma cells. Divergent localizations of gamma-tubulin and pericentrin suggest a differential distribution of these 2 centrosome-associated proteins in glioblastoma cell lines. Our results indicate that overexpression and ectopic cellular distribution of gamma-tubulin in astrocytic gliomas may be significant in the context of centrosome protein amplification and may be linked to tumor progression and anaplastic potential.

Frequent HRK inactivation associated with low apoptotic index in secondary glioblastomas

To detect and identify the genetic alterations and methylation status of the HRK gene in human glioblastomas, we analyzed a cohort of astrocytic tumors for hypermethylation, loss of heterozygosity on 12q13.1, and gene expression. Our study examined a series of 36 diffuse low-grade astrocytomas, 32 anaplastic astrocytomas, 64 primary glioblastomas, and 28 secondary glioblastomas that had evolved from either 24 low-grade diffuse astrocytomas or 4 anaplastic astrocytomas. The region around the HRK transcription start site was methylated in 19% of diffuse astrocytomas, in 22% of anaplastic astrocytomas, in 27% of primary glioblastomas, and in 43% of secondary glioblastomas. HRK expression was significantly reduced in 61% of secondary glioblastomas as compared to other types of tumors, and aberrant methylation was closely associated with loss of expression. Reverse transcription-PCR analysis also demonstrated a clear agreement between reduced HRK protein levels and low or absent HRK transcripts. Lack of HRK immunoreactivity was significantly correlated with a low apoptotic index, whereas a strong association between methylation status and apoptosis was found only in secondary glioblastomas. Abnormal methylation of HRK was detected in astrocytic tumors concurrent with methylation of multiple genes, including p16(INK4a) and p14(ARF). Interestingly, these epigenetic changes in secondary glioblastoma were further associated with wild-type p53. Our findings suggest that HRK is inactivated mainly by aberrant DNA methylation in astrocytic tumors and that reduced HRK expression contributes to the loss of apoptotic control in high-grade tumors. Reduced expression of HRK may serve as one important molecular mechanism in progression to secondary glioblastoma.

Apoptosis and proliferation: correlation with p53 in astrocytic tumours

Apoptosis and cell proliferation occur simultaneously in tumour tissue with tumour suppressor gene, p53 being one of the key players in the complex relationship between these two key phenomena. We, as well as several other groups, have earlier demonstrated the association of p53 immunopositivity with increased degree of cell proliferation in astrocytic tumours. Here we have studied the extent of apoptosis in 62 primary human astrocytic tumours [25 Diffuse Astrocytoma (DA), 9 Anaplastic Astrocytoma (AA) and 28 Glioblastoma multiforme (GBM)] in relation to tumour grade, proliferative status and p53 protein expression. Apoptosis was measured by the TUNEL assay while, cell proliferation (MIB-1 index) and p53 protein immunoreactivity were evaluated by immunohistochemical staining using MIB-1 and DO-1 monoclonal antibodies respectively. The apoptotic index (AI) was greater in GBM than in AA or DA, and more in tumours with p53 immunopositivity than in those without. The most striking observation was the strong correlation between Apoptotic index (AI) and proliferation index (PI) in p53 negative GBM (r=0.766, P < 0.005). However this was not observed in p53 +ve GBM or in low grade DA either p53 positive or negative. Taking p53 negativity in IHC as evidence of a functional gene/protein, this extends the link between proliferation and apoptosis, hitherto observed only in cultured cells with functional p53, to a subset of solid tumours.

PROGRESS IN CLINICAL NEUROSCIENCES: Advances in the Management of Low-Grade Gliomas

The management of low-grade gliomas represents one of the most challenging and controversial areas in neuro-oncology. Many aspects of the treatment of low-grade gliomas are debated, including the optimal timing of surgery and radiotherapy, the benefit of extensive surgery, and the impact of these variables on the natural history of these indolent and generally incurable tumours. The recently published results of several large multicentre trials addressing the timing and dose of radiotherapy have provided solid evidence for delayed and reduced dose irradiation. These studies have also confirmed prognostic variables that can be used to guide management of individual patients. Among these variables is the observation that tumours with oligodendroglial features have a better natural history and response profile. The recognition that as many as two thirds of low-grade gliomas have oligodendroglial features, advances in molecular diagnostics making accurate pathologic diagnosis of oligodendroglial tumours possible, and the established chemosensitivity of malignant oligodendrogliomas, have raised new issues surrounding the potential value of chemotherapy for low-grade gliomas. This review will be restricted to low-grade diffuse astrocytomas, oligodendrogliomas, and low-grade mixed oligoastrocytomas in adults, and provide evidence-based guidelines for the management of these tumours, including the emerging role of chemotherapy as initial treatment.

Cytologic features of reactive brain tissue in brain biopsies

Objective: Cytologic examination is a key element in the intraoperative assessment of central nervous system (CNS) mass lesions. However, differentiation of reactive, non-neoplastic tissue from low-grade neoplasms may be difficult. We reviewed cases on file to determine what cytologic features may be most helpful in distinguishing reactive tissue from cytologically similar appearing CNS tumors. Methods: Retrospective review of CNS biopsies on file between 1990 and 2002 with intraoperative cytologic evaluation and final diagnosis of reactive CNS tissue. H&E stained crush preparations were assessed on a scale of 0 (normal) to 3 in regards to tissue clumping, cellularity, presence of astrocytes with prominent cytoplasmic processes, granular eosinophilic background material, cellular heterogeneity, cytologic atypia, gemistocytes, Rosenthal fibers, foamy macrophages, lymphocytes, and neutrophils. The findings were compared with cytologic features found in representative cases of low-grade gliomas. Results: Materials were available for review in a variety of reactive biopsies ( n=33), low-grade diffuse astrocytomas ( n=11), pilocytic astrocytomas ( n=8), central neurocytomas ( n=3), and low-grade oligodendrogliomas ( n=3). Smears of reactive tissue showed a variety of features depending on the subtype of response present, including coagulative necrosis, subacute necrosis, radiation change, and nonspecific gliosis. The nonspecific gliosis most often showed low cellularity, evenly spread smears, retention of the eosinophilic background material, and variable accentuation of the astrocytic cytoplasmic processes. Oligodendrogliomas showed similar findings. Diffuse and pilocytic astrocytomas were usually distinguished from reactive tissue by the presence of coarse astrocytic cytoplasmic processes and loss of granular background material. However, some reactive biopsies showed similar features. Central neurocytomas were readily differentiated from other conditions due to its high cellularity, lack of cytoplasmic processes, and uniform cell population. Assessment of inflammatory cells, macrophages, cellular atypia, and Rosenthal fibers were not helpful in most cases. Conclusion: Cytologic features including the cellularity, resistance of the tissue to smearing, prominence of cytoplasmic processes, cellular heterogeneity, and retention of background granular eosinophilic material are helpful in most cases to differentiate reactive brain tissue from low-grade glial neoplasms. More cellular forms of gliosis may resemble diffuse or pilocytic astrocytomas and oligodendrogliomas.

Fluorescent In Situ Hybridization on Isolated Tumor Cell Nuclei: A Sensitive Method for 1p and 19q Deletion Analysis in Paraffin-Embedded Oligodendroglial Tumor Specimens

In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP—Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P = .001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.

Loss of heterozygosity of a locus in the chromosomal region 17p13.3 is associated with increased cell proliferation in astrocytic tumors

We had previously reported that loss of heterozygosity (LOH) of the D17S379 locus on 17p13.3 was significantly more frequent in high-grade gliomas (anaplastic astrocytoma, AA; glioblastoma multiforme, GBM) than in those of a low-grade diffuse astrocytoma (DA); however, this was independent of alterations at the TP53 locus, We also showed that LOH of D17S379 was associated with positive staining for p53 protein on immunohistochemistry, but LOH of the TP53 gene had no such association. In this work we show that cell proliferation as determined by MIB-1 labeling index (LI) was significantly higher in tumors with LOH of D17S379 than those with no LOH (NLOH). In accord with our previous results, p53 protein immunopositivity was also associated with increased MIB-1 LI; however, we observed no such association of LI with TP53 LOH. The results further confirm that alteration of one or more putative tumor suppressor loci at 17p13.3 is associated with increased proliferation in astrocytic tumors.

Promoter hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase is an independent predictor of shortened progression free survival in patients with low-grade diffuse astrocytomas.

The O6-methylguanine-DNA methyltransferase (MGMT) plays a major role in repairing DNA damage from alkylating agents. In several human neoplasms including low-grade diffuse astrocytomas, promoter hypermethylation of MGMT has been shown to correlate with an increased frequency of p53 mutation. In the present study, we analyzed MGMT promoter methylation by the methylation-specific PCR in 49 newly diagnosed WHO grade II astrocytomas and evaluated its clinical usefulness. MGMT promoter methylation was found in 21 (43%) of the 49 tumors. A tight correlation existed between MGMT methylation and p53 protein accumulation (P=0.0424). The presence of MGMT methylation was significantly associated with a shorter progression free survival (PFS) on both univariate analysis (P=0.0014) and multivariate analysis (P=0.0081). It was a more powerful determinant of the PFS than age, sex, performance status, proliferative activity, or p53 expression, and was independent of the extent of surgery. In terms of the overall survival, MGMT methylation demonstrated a prognostic utility in the univariate analysis but not in the multivariate analysis. The present findings indicate that aberrant methylation of the MGMT promoter independently augurs for an unfavorable clinical course in patients with low-grade diffuse astrocytomas. Since the presence of MGMT methylation is expected to predict an increased sensitivity to alkylating chemotherapeutic agents, earlier chemotherapy could serve to improve an unfavorable natural history in tumors with MGMT methylation.

KLF6, a putative tumor suppressor gene, is mutated in astrocytic gliomas.

Gliomas are the most common tumors of the central nervous system and have a grave prognosis. Deletion of chromosome 10p15 is one of the most common chromosomal alterations in gliomas. Recently, a candidate tumor suppressor gene, KLF6, which is mapped to chromosome 10p, was found to be frequently mutated in prostate cancer. KLF6 is a zinc finger transcription factor and transactivates p21/WAF1/CIP expression. To elucidate the role of genetic alterations of KLF6 in gliomas, we analyzed the 4 exons of the gene by direct DNA sequencing in 155 gliomas. Of these, mutations of KLF6 were found in 9 of 76 (11.8%) glioblastomas multiforme, 2 of 28 (7.1%) anaplastic astrocytomas, 2 of 36 (5.5%) low-grade diffuse astrocytomas and in none of the 15 oligodendrogliomas. All 13 mutations were located in the transactivation domain and most of them affected either serine residues or codons next to serine residues. Of the 13 cases with KLF6 mutation, loss of heterozygosity (LOH) at the KLF6 locus was inferred from the LOH displayed by the flanking microsatellite markers in 11 cases. We conclude that mutations of the KLF6 gene play a role in the pathogenesis of astrocytic gliomas.

Treatment of low-grade diffuse astrocytomas by surgery and human fibroblast interferon without radiation therapy.

Low-grade diffuse astrocytomas are slowly growing tumors with a relatively long overall survival. However, a substantial proportion undergoes dedifferentiation to a more malignant phenotype. Considerable controversy exists as to the best therapeutic management for patients with such tumors. Over the past decade, we have applied human fibroblast interferon (HFIF) therapy without radiation therapy to low-grade astrocytomas. We investigated 28 patients with WHO grade II astrocytomas of the cerebral hemispheres treated by surgery plus HFIF therapy. The overall response rate to the HFIF therapy was 36%. All side-effects of HFIF were transient, tolerable and manageable. The 5-year progression free survival and overall survival probabilities were 65% and 96%, respectively. Although our data from small cohort of patients may have modest value, our results suggest that HFIF may be useful in treating low-grade diffuse astrocytomas.

Effect of surgery on tumor progression and malignant degeneration in hemispheric diffuse low-grade astrocytomas

The aim of this study is to determine the impact of surgery on tumor progression and malignant degeneration in hemispheric diffuse astrocytoma WHO grade II. Twenty-eight patients who were operated or underwent stereotactic biopsy for hemispheric diffuse astrocytoma WHO grade II at Marmara University between January 1987 and January 1996, were prospectively reviewed for the presence of recurrence and histopathological dedifferentiation at their fourth years after the initial treatment. Twenty-two patients underwent surgical resection. Of this group, 7 patients had a total, 11 had a subtotal and 4 patients had a partial resection. Six patients underwent stereotactic biopsy. All patients, except for the ones in whom a radiological total surgical removal could be achieved, received postoperative radiotherapy. In the total surgical-removal group only one patient had recurrence, while no upgrade was noted. All of the patients in the partial resection and stereotactic biopsy groups recurred at a higher grade. Our results indicate that both tumor progression and histopathological dedifferentiation were less commonly seen when a total or subtotal resection could be achieved. So, surgery, as radical as possible, should be the choice of treatment in low-grade hemispheric astrocytomas.

Methylation of the p73 gene in gliomas

The p73 gene encodes a protein structurally and functionally homologous to TP53, and maps to chromosomal band 1p36.33, where loss of heterozygosity has been observed in up to 90% of oligodendrogliomas and in 10-25% of diffuse astrocytomas. We assessed the methylation status of the CpG islands in the promoter region of the p73 gene by methylation-specific PCR in 117 glioma biopsies. Methylation was detected in 5 out of 28 (18%) glioblastomas and in 4/26 (15%) anaplastic oligodendrogliomas (WHO grade III) but not in grade II oligodendrogliomas, low-grade diffuse astrocytomas (grade II), and anaplastic astrocytomas (grade III). To assess whether p73 methylation leads to loss of expression, we carried out reverse transcription-PCR and methylation-specific PCR in 10 glioblastoma cell lines. Two lines (U87MG and T98G) showed p73 methylation. U87MG had no unmethylated p73 (complete methylation), and showed loss of expression. T98G had methylated and unmethylated p73 (partial methylation), and retained p73 expression. A third cell line (LN-308) showed loss of p73 expression without p73 methylation. These results suggest that complete p73 methylation is associated with loss of expression, but that additional mechanisms may cause loss of p73 expression. Analysis of a polymorphic site in exon 2 further showed that p73 was mono-allelically expressed in 6 out of 7 primary gliomas with heterozygous GC/TA polymorphism.

Evaluation of molecular markers in low-grade diffuse astrocytomas: loss of p16 and retinoblastoma protein expression is associated with short survival.

Diffuse astrocytomas have a median survival of 6-8 years. However, in a minority of cases that are histologically low grade, progression is rapid, leading to death within 2 years. Loss of p16, retinoblastoma protein, and deleted-in-colon-carcinoma protein expression, and monosomy of chromosome 10 have been shown to occur in malignant astrocytic tumors. We have investigated the prognostic value of expression of these markers, using techniques applicable in many histopathology laboratories, in diffuse astrocytomas that are histologically low grade. Paraffin sections from 71 diffuse, supratentorial, low-grade astrocytomas, from patients with at least 8-year survival data, were immunostained with antibodies to p16, deleted-in-colon-carcinoma protein, p53, Ki67, and retinoblastoma protein. In situ hybridization with a digoxigenin-labeled probe to chromosome 10 was used to assess chromosomal loss. In most cases there was immunostaining of virtually all tumor cell nuclei with antibodies to p16 and retinoblastoma protein. Three of the 68 tumors in which assessment of p16 was possible included discrete foci with lack of detectable immunoreactivity in tumor cells. The three patients concerned had a significantly shortened median survival (1.1 years vs 4.4 years in those without loss of p16; p <0.01). In six of the 61 cases where assessment of retinoblastoma protein was possible, <70% of tumor cell nuclei showed immunoreactivity. These six patients had a shorter survival (4.0 years) than had the remaining patients (5.4 years), although this difference was not statistically significant. The tumor from one of these patients included areas where only 36% of tumor cells showed retinoblastoma protein immunoreactivity, and this patient survived only 1.5 years. Tumors showing loss of both p16 and retinoblastoma were not seen. p53 and deleted-in-colon-carcinoma protein expression was highly variable and did not correlate with survival. Tumors with monosomy for chromosome 10 were not identified. Both polyploidy and the Ki67 labeling index were significantly associated with the p53 labeling index but not with survival. Focal loss of p16 or retinoblastoma protein is demonstrable in approximately 5% and 10% of diffuse low-grade diffuse astrocytomas, respectively. Tumors with focal loss of immunoreactivity for these proteins are associated with shorter survival than those without, suggesting that immunohistochemistry for p16 and retinoblastoma protein may be a useful adjunct to other methods for assessing the prognosis of astrocytomas.

Microtubule-associated protein 2 (MAP-2) is expressed in low and high grade diffuse astrocytomas

The expression of neuronal antigens in diffuse astrocytomas has not been thoroughly evaluated. We have investigated the expression of microtubule associated protein 2 (MAP-2), synaptophysin and non-phosphorylated epitopes of neurofilament protein (NFP) by immunohistochemistry in 15 low grade diffuse astrocytomas and 15 glioblastomas. MAP-2 was strongly expressed in 97% of cases, using an antibody to both low and high molecular weight isoforms. An antibody specific to high molecular weight isoforms of MAP-2 (hmw-MAP-2) revealed weaker, focal staining in 60% of cases with greater expression in the glioblastomas (P=0.027). NFP was expressed in 50% of cases, but was generally weak and focal. There was little evidence of synaptophysin expression. We conclude that MAP-2 expression in astrocytomas is due predominantly to low molecular weight isoforms, which may be expressed in astrocytes as well as neurons. Focal expression of hmw-MAP-2 and NFP, however, suggest that neuronal antigens may be expressed, particularly in high grade astrocytomas. Immunopositivity for these antigens should not preclude the diagnosis of diffuse astrocytoma.

Ki-67 immunoreactivity, basic fibroblastic growth factor (bFGF) expression, and microvessel density as supplementary prognostic tools in low-grade astrocytomas. An immunohistochemical study with special reference to the reliability of different Ki-67 antibodies.

Low-grade diffuse astrocytomas are generally slow-growing tumors; however, they may progress to anaplastic astrocytomas or glioblastomas. As existing grading systems fail to distinguish these tumors, ongoing research strives to identify new prognostic markers. In this study, 22 adult patients with supratentorial diffuse astrocytomas, WHO grade II, were investigated to clarify whether proliferative activity, assessed by different Ki67 antibodies (MIB-1, NC-MM1, NC-Ki67p, rahKi67), expression of basic fibroblast growth factor (bFGF) or microvessel density have prognostic power. The Ki67 antibodies revealed low proliferation indices (PI); however, there was a wide spread of values, ranging from 0.1% to about 10%. In general, a positive correlation between the different Ki67 PIs was found. In 12 of 22 cases, bFGF immunoreactivity was recorded. Microvessel density was generally low. MIB-1 PI was the only prognostic factor of statistical significance. Ki67 PI, obtained by using the monoclonal antibody MIB-1, can thus serve as a prognostic factor capable of identifying subsets of low-grade diffuse astrocytomas with a potentially more aggressive clinical behavior.