Low-grade Diffuse Astrocytomas Research Articles

The Definition of Primary and Secondary Glioblastoma

Glioblastoma is the most frequent and malignant brain tumor. The vast majority of glioblastomas (~90%) develop rapidly de novo in elderly patients, without clinical or histologic evidence of a less malignant precursor lesion (primary glioblastomas). Secondary glioblastomas progress from low-grade diffuse astrocytoma or anaplastic astrocytoma. They manifest in younger patients, have a lesser degree of necrosis, are preferentially located in the frontal lobe, and carry a significantly better prognosis. Histologically, primary and secondary glioblastomas are largely indistinguishable, but they differ in their genetic and epigenetic profiles. Decisive genetic signposts of secondary glioblastoma are IDH1 mutations, which are absent in primary glioblastomas and which are associated with a hypermethylation phenotype. IDH1 mutations are the earliest detectable genetic alteration in precursor low-grade diffuse astrocytomas and in oligodendrogliomas, indicating that these tumors are derived from neural precursor cells that differ from those of primary glioblastomas. In this review, we summarize epidemiologic, clinical, histopathologic, genetic, and expression features of primary and secondary glioblastomas and the biologic consequences of IDH1 mutations. We conclude that this genetic alteration is a definitive diagnostic molecular marker of secondary glioblastomas and more reliable and objective than clinical criteria. Despite a similar histologic appearance, primary and secondary glioblastomas are distinct tumor entities that originate from different precursor cells and may require different therapeutic approaches.

Gliomas and seizures

Glial neoplasms account for nearly 50% of all adult primary brain tumors. They originate from glial cells in the brain and/or spinal cord and include low-grade diffuse astrocytomas, anaplastic-astrocytomas, and glioblastomas. Of all brain tumors, glioblastoma multiforme (GBM) is the most aggressive and is characterized by rapid glial cell growth, resistance to radio- and chemo- therapies, and relentless infiltration and spreading throughout the central nervous system (CNS). In glioblastomas, primary tumor growth and CNS invasion are associated with the activation of complex structural molecular and metabolic changes within the tumor tissue, which profoundly affect the surrounding neuronal networks and may in part explain induction of epilepsy. In fact, epileptic seizures are very common among patients with glial tumors, reaching nearly 50% in glioblastoma patients and almost 90% in low-grade astrocytomas. The overall hypothesis presented here discusses the possibility that the aberrant tumor cell metabolism may act directly on neuronal network, and this leads to seizure susceptibility. Further invasion and growth of the malignant glial cells exacerbate this initial pathologic state which promotes recurrent seizures (epileptogenesis).

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.

Presentation patterns and outcome of gliomatosis cerebri

A total of 25 patients with gliomatosis cerebri (19 males and 6 females; median age 51 years, range 10-73 years) were diagnosed and treated at the Sheba Medical Center between 1995 and 2009. Of these, 3 patients were 10 years old at the time of diagnosis. Seizures were the initial clinical presentation in 19 patients, focal signs in 16 patients, headaches in 7 patients, cognitive disorder in 4 patients and rapidly progressive hemiparesis in 1 patient. Magnetic resonance imaging (MRI) was performed in the patients and demonstrated a diffuse infiltrative process with a hyperintensity signal on T2-weighted images and a minimal mass effect. Some level of enhancement on MRI was observed in 6 patients. The infiltrative process involved at least two lobes in each patient. Biopsy was performed for diagnosis in the majority of patients. In 1 patient with a markedly rapid deterioration, the diagnosis was established at autopsy. The pathology was compatible with gliomatosis with a diffuse infiltrative low-grade astrocytoma in 21 patients and anaplastic astrocytoma in 5 patients. The patients were treated with whole-brain radiation therapy and 7 patients were treated with combined whole-brain radiation therapy and chemotherapy. Treatment appeared to stabilize 6 patients or improve the clinical condition in 7 patients. Due to the small number of patients in the present study, however, further studies are required to determine the effect of treatment on the natural history of the disease.

Frequent epigenetic inactivation of the chaperone SGNE1/7B2 in human gliomas

In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.

Increased β-Catenin/Tcf Signaling in Pilocytic Astrocytomas: A Comparative Study to Distinguish Pilocytic Astrocytomas from Low-Grade Diffuse Astrocytomas

Although pilocytic and diffuse grade II astrocytomas considered as low-grade tumors, the distinction between them is still a major clinical problem. Previously we reported the activation of Wnt/β-catenin/Tcf signaling pathway in diffuse astrocytomas, however its role in pilocytic astrocytomas is not well understood. In this study, we investigated the Wnt/β-catenin/Tcf pathway in pilocytic astrocytomas and compared with diffuse astrocytomas. We observed the differential expression of β-catenin, Tcf4, Lef1 and c-Myc in astrocytomas particularly higher levels were observed in pilocytic astrocytomas and GBM while very little expression was documented in grade II tumors. Further, immunohistochemical analysis revealed the strong positivity of β-catenin, Tcf4, Lef1 and c-Myc in pilocytic astrocytomas than that of grade II tumors and also exhibited the strong positivity in vascular endothelial cells of pilocytic astrocytomas and GBM. Hence, Wnt/β-catenin/Tcf signaling pathway is differentially expressed in astrocytomas, activation of this pathway might be helpful in separating pilocytic astrocytomas from low-grade diffuse astrocytomas.

Microtubule-Severing ATPase Spastin in Glioblastoma: Increased Expression in Human Glioblastoma Cell Lines and Inverse Roles in Cell Motility and Proliferation

We studied the expression and distribution of the microtubule-severing enzyme spastin in 3 human glioblastoma cell lines (U87MG, U138MG, and T98G) and in clinical tissue samples representative of all grades of diffuse astrocytic gliomas (n = 45). In adult human brains, spastin was distributed predominantly in neuronsand neuropil puncta and, to a lesser extent, in glia. Compared with normal mature brain tissues, spastin expression and cellular distribution were increased in neoplastic glial phenotypes, especiallyin glioblastoma (p < 0.05 vs low-grade diffuse astrocytomas). Overlapping punctate and diffuse patterns of localization wereidentified in tumor cells in tissues and in interphase and mitotic cells ofglioblastoma cell lines. There was enrichment of spastin in the leading edges of cells in T98G glioblastoma cell cultures and in neoplastic cell populations in tumor specimens. Real-time polymerase chain reaction and immunoblotting experiments revealed greater levels of spastin messenger RNA and protein expression in theglioblastoma cell lines versus normal human astrocytes. Functional experiments indicated that spastin depletion resulted in reduced cell motility and higher cell proliferation of T98G cells. Toour knowledge, this is the first report of spastin involvement incellmotility. Collectively, our results indicate that spastinexpression in glioblastomas might be linked to tumor cell motility, migration, and invasion.

Abstract 4797: A member of the SLC25 family is epigenetically inactivated in human gliomas and suppresses cell growth in vitro

Abstract In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG rich fragment upstream of the coding region of a member of the solute carrier family 25 (SLC25) that showed hypermethylation in gliomas compared to normal white matter controls. Pyrosequencing analysis was performed to confirm the methylation status of this gene in 76 gliomas and 7 glioma cell lines. Hypermethylation was detected in 14% of primary glioblastomas, 62% of secondary glioblastomas, 27% of anaplastic astrocytomas, 0.7% low-grade diffuse astrocytomas and in 71% of glioma cell lines. None of three investigated white matter samples showed hypermethylation of the investigated region. Hypermethylation of the SLC25 member was significantly more frequent in secondary glioblastomas compared to primary glioblastomas (P = 0.0066). Furthermore, the investigation of matched low grade and high grade glioma tissues revealed that this hypermethylation was associated with progression. Expression of the SLC25 member was investigated by real-time reverse transcription-PCR and found to be down regulated in primary and secondary glioblastomas as well as in anaplastic and low-grade astrocytomas when compared to white matter and total brain tissue. After treatment of glioma cell lines with 5-aza-2’-deoxycytidine, the transcription was restored. In functional investigations overexpression of exogenous SLC25 family member in glioblastoma cell lines lead to a significant reduction in focus formation. In summary, we describe for the first time the epigenetic silencing of a nuclear encoded member of the mitochondrial transporter family (SLC25) in high grade malignant human brain tumors and provide functional proof for its growth suppressive activity on glioma cells in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2011-4797

Factors predicting progression of low-grade diffusely infiltrating astrocytoma

Low-grade diffuse astrocytoma (DA) is considered benign tumor (World Health Organization [WHO] grade II), but it has an inherent tendency for malignant progression, which is quite variable. To identify malignant progression in an individual case of DA, we studied the clinico-radiological and immunohistochemical factors and correlated with progression of DA at a dedicated tertiary level neurosciences centre NIMHANS, Bangalore, India. Consecutive adult patients who had undergone tumor decompression for lobar supratentorial DA at our institute from 1994 to 1998 were retrospectively selected and followed up for clinico-radiological progression. The clinico-radiological and histomorphological features were studied. With the use of immunohistochemistry, proliferation index [MIB-1 labeling index (LI)], p53 protein expression, microvessel density (MVD) count [assessed using anti-CD34 antibody] were analyzed and correlated with progression-free survival (PFS) Results: There were 13 patients. Mean age was 34 years. The most common presenting symptom was seizures. The median follow-up was 54 months. There were four recurrences, with median interval of 75 months. Eight patients received radiotherapy. Younger patients (<40 years), seizure as the presenting symptom and postoperative radiotherapy were associated with longer PFS, while gemistocytic morphology (>20% gemistocytic cells), MVD value >20 correlated with shorter PFS, albeit statistically insignificant. MIB1 LI did not correlate with recurrence pattern. Moreover, p53 LI > 10% correlated with early progression (P = 0.04). Our study highlights some of the clinical, histological and immunohistochemical parameters that predict progression on DA. Validation on a larger sample may be useful to plan appropriate treatment in an individual case.

Practical value of MIB-1 index in predicting behavior of astrocytomas

The MIB-1 labeling index (LI) has proved to be useful in assigning grading and prognosis to astrocytomas. The purpose of our study was to analyze the utility of MIB-1 LI in differentiating astrocytomas of varying grades and the possible relationships of MIB-1 LI with clinical parameters like age and sex. We also wanted to study the prognostic role of MIB-1 index in predicting behavior of astrocytomas. Our study included 145 patients with astrocytic tumors of varying grades. Immunolabeling for all patients was done using MIB-1 antibody. Survival data could be obtained for 64 patients. A Mann-Whitney U test was used to test the difference in MIB-1 LI between different histological grades. The univariate analysis was done by the Kaplan-Meier method, and the multivariate analysis for survival was performed using the Cox proportional hazard model. Significant differences were noted in mean MIB-1 LI of high-grade and low-grade diffuse astrocytomas. MIB-1 LI did not vary significantly with age and sex. Univariate analysis showed favorable prognostic factors for low histopathological grade, young patient age and low MIB-1 LI; however, multivariate analysis showed that only histopathological grade had independent prognostic significance. Our study proves that MIB-1 LI is not dependent on factors like age and sex and is solely dependent on histological grade. Though the average level of MIB-1 LI varies considerably in the different grades of astrocytomas, considerable overlap can be observed between them. MIB-1 LI is a very useful adjunct to the histopathological diagnosis and can be of great help in situations where the clinical and radiological findings do not correlate with histological diagnosis.

MAPK pathway activation through BRAF gene fusion in pilocytic astrocytomas; a novel oncogenic fusion gene with diagnostic, prognostic, and therapeutic potential

Recently, a new mechanism for activation of B-RAF was identified resulting from a tandem duplication, generating a fusion protein with constitutive BRAF activity and thereby activating the MAPK pathway. Different fusion variants involving BRAF and KIAA1549 were demonstrated, present in 80% of pilocytic astrocytomas in children. As the KIAA1549-BRAF fusion gene is detected at a much lower frequency in diffuse low-grade astrocytomas and survival was much longer than expected in the patients with a 'non-pilocytic' astrocytoma carrying the fusion gene, identification of this fusion gene can be of diagnostic and prognostic value. In the near future, interference with the (fusion gene causing) activation of the MAPK signalling cascade may open new therapeutic avenues for children with pilocytic astrocytomas, as a first line of defence against tumour growth or in situations where the tumour has become refractory to other therapeutic modalities.

HHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33

The completion of the Human Genome Project resulted in discovery of many unknown novel genes. This feat paved the way for the future development of novel therapeutics for the treatment of human disease based on novel biological functions and pathways. Towards this aim, we undertook a bioinformatics analysis of in-house microarray data derived from purified hematopoietic stem cell populations. This effort led to the discovery of HSS1 (Hematopoietic Signal peptide-containing Secreted 1) and its splice variant HSM1 (Hematopoietic Signal peptide-containing Membrane domain-containing 1). HSS1 gene is evolutionarily conserved across species, phyla and even kingdoms, including mammals, invertebrates and plants. Structural analysis showed no homology between HSS1 and known proteins or known protein domains, indicating that it was a truly novel protein. Interestingly, the human HSS1 (hHSS1) gene is located at chromosome 19q13.33, a genomic region implicated in various cancers, including malignant glioma. Stable expression of hHSS1 in glioma-derived A172 and U87 cell lines greatly reduced their proliferation rates compared to mock-transfected cells. hHSS1 expression significantly affected the malignant phenotype of U87 cells both in vitro and in vivo. Further, preliminary immunohistochemical analysis revealed an increase in hHSS1/HSM1 immunoreactivity in two out of four high-grade astrocytomas (glioblastoma multiforme, WHO IV) as compared to low expression in all four low-grade diffuse astrocytomas (WHO grade II). High-expression of hHSS1 in high-grade gliomas was further supported by microarray data, which indicated that mesenchymal subclass gliomas exclusively up-regulated hHSS1. Our data reveal that HSS1 is a truly novel protein defining a new class of secreted factors, and that it may have an important role in cancer, particularly glioma.

MGMT promoter hypermethylation is a frequent, early, and consistent event in astrocytoma progression, and not correlated with TP53 mutation

Hypermethylation of the MGMT gene promoter and mutation of the TP53 tumor-suppressor gene are frequently present in diffuse astrocytomas. However, there is only anecdotal information about MGMT methylation status and TP53 mutations during progression of low-grade diffuse astrocytoma (AII) to anaplastic astrocytoma (AIII) and secondary glioblastoma (sGB). In this study biopsy specimens from 51 patients with astrocytic tumors with radiologically proved progression from low to high-grade malignancy were investigated for the presence and consistency of MGMT promoter hypermethylation and TP53 mutations. For 27 patients biopsy samples both of primary tumors and their recurrences were available. For the other 24 patients histology of either the low-grade lesion or the high-grade recurrence was available. It was found that MGMT promoter hypermethylation and TP53 mutations are both frequent and early events in the progression of astrocytomas and that their status is consistent over time. No correlation was found between MGMT methylation status and the presence of TP53 mutations. In addition, no correlation was found between MGMT promoter hypermethylation and the type of TP53 mutations. These results argue against the putative TP53 G:C>A:T transition mutations suggested to occur preferentially in MGMT hypermethylated tumors.

Expression of PROX1 Is a Common Feature of High-Grade Malignant Astrocytic Gliomas

PROX1 is a prospero-related transcription factor that plays a critical role in the development of various organs including the mammalian lymphatic and central nervous systems; it controls cell proliferation and differentiation through different transcription pathwaysand has both oncogenic and tumor-suppressive functions. We investigated PROX1 expression patterns in 56 human astrocytic gliomas of different grades using immunohistochemistry. An average of 79% of cells in World Health Organization Grade IV (glioblastoma, n = 15) and 57% of cells in World Health Organization Grade III (anaplastic astrocytoma, n = 13) were strongly PROX1 positive; low-grade diffuse astrocytomas (Grade II, n = 13) had 21% of cells that were strongly positive; Grade I tumors (n = 15) had 1.5%; and non-neoplastic brain tissue (n = 15) had 3.7% of cells that were PROX1 positive. Double immunolabeling showed that PROX1+ cells in glioblastomas frequently coexpressed early neuronal proteins MAP2 and betaIII-tubulin but not the mature neuronal marker protein NeuN. Analyses of coexpression with proliferation markers suggest that PROX1+ cells have a marginally lower rate of proliferation than other tumor cells but are still mitotically active. We conclude that PROX1 may constitute a useful tool for the diagnosis and grading ofastrocytic gliomas and for distinguishing Grade III and Grade IV tumors from Grade I and Grade II tumors.

Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes

It has been suggested that intrinsic brain tumours originate from a neural stem/progenitor cell population in the subventricular zone of the post-natal brain. However, the influence of the initial genetic mutation on the phenotype as well as the contribution of mature astrocytes to the formation of brain tumours is still not understood. We deleted Rb/p53, Rb/p53/PTEN or PTEN/p53 in adult subventricular stem cells; in ectopically neurografted stem cells; in mature parenchymal astrocytes and in transplanted astrocytes. We found that only stem cells, but not astrocytes, gave rise to brain tumours, independent of their location. This suggests a cell autonomous mechanism that enables stem cells to generate brain tumours, whereas mature astrocytes do not form brain tumours in adults. Recombination of PTEN/p53 gave rise to gliomas whereas deletion of Rb/p53 or Rb/p53/PTEN generated primitive neuroectodermal tumours (PNET), indicating an important role of an initial Rb loss in driving the PNET phenotype. Our study underlines an important role of stem cells and the relevance of initial genetic mutations in the pathogenesis and phenotype of brain tumours.

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.

Chronic epilepsy associated with temporal tumors: long-term surgical outcome

The authors undertook a study to review the clinical features and outcome in patients who underwent surgery for intractable chronic epilepsy caused by temporal lobe tumors. The Rush Surgical Epilepsy Database was queried to identify patients with chronic intractable epilepsy who underwent resection of temporal lobe tumors between 1981 and 2005 at Rush University Medical Center. Medical records were reviewed for age of the patient at seizure onset, delay to referral for surgery, seizure frequency and characteristics, preoperative MR imaging results, extent of resection, pathological diagnosis, complications, duration of follow-up period, and seizure improvement. Thirty-eight patients were identified, all with low-grade tumors. Gangliogliomas were the most common (36.8%), followed in descending order by dysembryoplastic neuroepithelial tumors (26.3%) and low-grade diffuse astrocytoma (10.5%). The mean duration between seizure onset and surgery was 15.4 years. Complex partial seizures were the most common presenting symptom. Detailed operative data were available for 28 patients; of these, 89.3% underwent complete resection of the amygdala, and 82.1% underwent partial or complete resection of hippocampus, in addition to lesionectomy. The mean follow-up duration was 7.7 years (range 1.0-23.1 years), with 78.9% of patients having seizure status that improved to Engel Class I, 15.8% to Engel Class II, and 5.3% to Engel Class III. Permanent complications were noted in 2.6% of patients. The authors' examination of the long-term follow-up data in patients with temporal lobe tumors causing chronic intractable epilepsy demonstrated excellent results in seizure improvement after surgery.

IDH1 Mutations Are Early Events in the Development of Astrocytomas and Oligodendrogliomas

IDH1 encodes isocitrate dehydrogenase 1, which participates in the citric acid cycle and was recently reported to be mutated in 12% of glioblastomas. We assessed IDH1 mutations in 321 gliomas of various histological types and biological behaviors. A total of 130 IDH1 mutations was detected, and all were located at amino acid residue 132. Of these, 91% were G-->A mutations (Arg-->His). IDH1 mutations were frequent in low-grade diffuse astrocytomas (88%) and in secondary glioblastomas that developed through progression from low-grade diffuse or anaplastic astrocytoma (82%). Similarly, high frequencies of IDH1 mutations were found in oligodendrogliomas (79%) and oligoastrocytomas (94%). Analyses of multiple biopsies from the same patient (51 cases) showed that there were no cases in which an IDH1 mutation occurred after the acquisition of either a TP53 mutation or loss of 1p/19q, suggesting that IDH1 mutations are very early events in gliomagenesis and may affect a common glial precursor cell population. IDH1 mutations were co-present with TP53 mutations in 63% of low-grade diffuse astrocytomas and with loss of heterozygosity 1p/19q in 64% of oligodendrogliomas; they were rare in pilocytic astrocytomas (10%) and primary glioblastomas (5%) and absent in ependymomas. The frequent presence of IDH1 mutations in secondary glioblastomas and their near-complete absence in primary glioblastomas reinforce the concept that despite their histological similarities, these subtypes are genetically and clinically distinct entities.

P53-mediated inhibition of angiogenesis in diffuse low-grade astrocytomas

The p53 tumour suppressor protein has long been recognized as the central factor protecting humans from cancer. In this study we evaluated the associations of p53 status and vessel density (angiogenesis) in a set of diffuse low-grade astrocytomas. Immunohistochemistry was performed on 23 diffuse low-grade astrocytomas for CD31 and p53. Mutations in the TP53 gene were identified by PCR amplification of genomic DNA extracted from the indicated tumour tissues. Microvessel counts were done by computer analyses. Intratumoural or peritumoural microvascular hot spots were assessed and analysed from an image taken with a 200× fold magnification. Statistical analysis was performed with Pearson correlation coefficient and Student's t-test. We found that 9/23 (39%) of the astrocytomas stained positive for p53 in the immunohistochemistry. We identified TP53 mutations in 11/23 (47%) of the astrocytomas. No association between micro vessel density (MVD) and p53 immunohistochemical status was found. However, the MVD was significantly increased in p53 mutated low-grade astrocytomas. Furthermore, the absolute vessel number was significantly higher in p53 mutated than in p53 wild-type low-grade astrocytomas. To analyse the molecular background for that epiphenomenon LN229 glioma cells which harbour a TP53 mutation were transfected with a plasmid encoding p53 wild-type and an angiogenesis protein array was performed. We detected a significant increase for thrombospondin-1, coagulation factor III and serpin E1 and a significant decrease of MMP-9 in wild-type p53 transfected LN229 cells. The higher microvessel density and the increased absolute vessel number in p53 mutated tumours supports the importance of p53 for tumour angiogenesis in diffuse low-grade astrocytomas. Our results support the hypothesis that p53 regulates angiogenesis in low-grade astrocytomas.

Frequent loss of heterozygosity and altered expression of the candidate tumor suppressor gene 'FAT' in human astrocytic tumors

BackgroundWe had earlier used the comparison of RAPD (Random Amplification of Polymorphic DNA) DNA fingerprinting profiles of tumor and corresponding normal DNA to identify genetic alterations in primary human glial tumors. This has the advantage that DNA fingerprinting identifies the genetic alterations in a manner not biased for locus.MethodsIn this study we used RAPD-PCR to identify novel genomic alterations in the astrocytic tumors of WHO grade II (Low Grade Diffuse Astrocytoma) and WHO Grade IV (Glioblastoma Multiforme). Loss of heterozygosity (LOH) of the altered region was studied by microsatellite and Single Nucleotide Polymorphism (SNP) markers. Expression study of the gene identified at the altered locus was done by semi-quantitative reverse-transcriptase-PCR (RT-PCR).ResultsBands consistently altered in the RAPD profile of tumor DNA in a significant proportion of tumors were identified. One such 500 bp band, that was absent in the RAPD profile of 33% (4/12) of the grade II astrocytic tumors, was selected for further study. Its sequence corresponded with a region of FAT, a putative tumor suppressor gene initially identified in Drosophila. Fifty percent of a set of 40 tumors, both grade II and IV, were shown to have Loss of Heterozygosity (LOH) at this locus by microsatellite (intragenic) and by SNP markers. Semi-quantitative RT-PCR showed low FAT mRNA levels in a major subset of tumors.ConclusionThese results point to a role of the FAT in astrocytic tumorigenesis and demonstrate the use of RAPD analysis in identifying specific alterations in astrocytic tumors.