Loss of FUBP1 expression in gliomas predicts FUBP1 mutation and is associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity.

AIMS: The Far Upstream Element [FUSE] Binding Protein 1 (FUBP1) regulates target genes, such as the cell cycle regulators MYC and p21. FUBP1 is up-regulated in many tumours and acts as an oncoprotein by stimulating proliferation and inhibiting apoptosis. Recently, FUBP1 mutations were identified in approximately 15% of oligodendrogliomas. To date, all reported FUBP1 mutations have been predicted to inactivate FUBP1, which suggests that in contrast to most other tumours FUBP1 may act as a tumour suppressor in oligodendrogliomas. METHODS: As no data are currently available concerning FUBP1 protein levels in gliomas, we examined the FUBP1 expression profiles of human glial tumours by immunohistochemistry and immunofluorescence. We analysed FUBP1 expression related to morphological differentiation, IDH1 and FUBP1 mutation status, 1p/19q loss of heterozygosity (LOH) as well as proliferation rate. RESULTS: Our findings demonstrate that FUBP1 expression levels are increased in all glioma subtypes as compared with normal central nervous system (CNS) control tissue and are associated with increased proliferation. In contrast, FUBP1 immunonegativity predicted FUBP1 mutation with a sensitivity of 100% and a specificity of 90% in our cohort and was associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity (LOH). Using this approach, we detected a to-date undescribed FUBP1 mutation in an oligodendroglioma. CONCLUSION: In summary, our data indicate an association between of FUBP1 expression and proliferation in gliomas. Furthermore, our findings present FUBP1 immunohistochemical analysis as a helpful additional tool for neuropathological glioma diagnostics predicting FUBP1 mutation.

Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma.

BACKGROUND: microRNA-9 is a key regulator of neuronal development aberrantly expressed in brain malignancies, including medulloblastoma. The mechanisms by which microRNA-9 contributes to medulloblastoma pathogenesis remain unclear, and factors that regulate this process have not been delineated. METHODS: Expression and methylation status of microRNA-9 in medulloblastoma cell lines and primary samples were analysed. The association of microRNA-9 expression with medulloblastoma patients' clinical outcome was assessed, and the impact of microRNA-9 restoration was functionally validated in medulloblastoma cells. RESULTS: microRNA-9 expression is repressed in a large subset of MB samples compared with normal fetal cerebellum. Low microRNA-9 expression correlates significantly with the diagnosis of unfavourable histopathological variants and with poor clinical outcome. microRNA-9 silencing occurs via cancer-specific CpG island hypermethylation. HES1 was identified as a direct target of microRNA-9 in medulloblastoma, and restoration of microRNA-9 was shown to trigger cell cycle arrest, to inhibit clonal growth and to promote medulloblastoma cell differentiation. CONCLUSIONS: microRNA-9 is a methylation-silenced tumour suppressor that could be a potential candidate predictive marker for poor prognosis of medulloblastoma. Loss of microRNA-9 may confer a proliferative advantage to tumour cells, and it could possibly contribute to disease pathogenesis. Thus, re-expression of microRNA-9 may constitute a novel epigenetic regulation strategy against medulloblastoma.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

BCL-xL: time-dependent dissociation between modulation of apoptosis and invasiveness in human malignant glioma cells.

Conditionally BCL-xL-overexpressing LNT-229 Tet-On glioma cell clones were generated to investigate whether the 'antiapoptosis phenotype' and the 'motility phenotype' mediated by BCL-2 family proteins in glioma cells could be separated. BCL-xL induction led to an immediate and concentration-dependent protection of LNT-229 cells from apoptosis. BCL-xL induction for up to 3 days did not result in altered invasiveness. In contrast, long-term BCL-xL induction for 21 days resulted in increased transforming growth factor-beta2 expression, and in metalloproteinase-2 and -14 dependent, but integrin independent, increased invasiveness. Withdrawal of doxycycline (Dox) abolished the protection from apoptosis whereas the 'invasion phenotype' remained stable. Dox stimulation of BCL-xL-inducible LNT-229 cells conferred infiltrative growth to BCL-xL-positive glioma cells in vivo and reduced the survival of tumor-bearing mice. These data allow to dissect a direct antiapoptotic action of BCL-xL from an indirect effect, presumably mediated by altered gene expression, which modifies tumor cell invasiveness in vitro and in vivo.

PCTAIRE3: a putative mediator of growth arrest and death induced by CTS-1, a dominant-positive p53-derived synthetic tumor suppressor, in human malignant glioma cells.

Chimeric tumor suppressor-1 (CTS-1) is based on the sequence of p53 and was designed as a therapeutic tool resisting various mechanisms of p53 inactivation. We previously reported that an adenovirus expressing CTS-1 (Ad-CTS-1) has superior cell death-inducing activity in glioma cells compared with wild-type p53. Here, we used cDNA microarrays to detect changes in gene expression preferentially induced by Ad-CTS-1. The putative serine threonine kinase, PCTAIRE3, and the quinone oxireductase, PIG3, were strongly induced by Ad-CTS-1 compared with wild-type p53. An adenoviral vector encoding PCTAIRE3 (Ad-PCTAIRE3) induced growth arrest and killed a minor proportion of the glioma cells. Ad-PIG3 alone affected neither growth nor viability. However, coinfection with Ad-PCTAIRE3 and Ad-PIG3 resulted in enhanced growth inhibition compared with Ad-PCTAIRE3 infection alone. Ad-CTS1, Ad-PCTAIRE3 or Ad-PIG3 induced the formation of free reactive oxygen species (ROS). However, the prevention of ROS formation induced by Ad-PCTAIRE3 and Ad-CTS-1 did not block growth arrest and cell death, suggesting that ROS formation is not essential for these effects. Altogether, these data identify PCTAIRE3 as one novel growth-inhibitory and death-inducing p53 response gene and suggest that changes in the expression of specific target genes contribute to the superior anti-glioma activity of CTS-1.

Lung fibre burden in lung cancer cases employed in the rock and slag wool industry.

OBJECTIVES: To evaluate the relationship between estimated exposure to man-made vitreous fibres (MMVF) and to asbestos fibres and their concentration in the lung tissue of lung cancer cases amongst MMVF production workers. METHODS: Retrospective retrieval of available lung tissue specimens was conducted following a case-control study that assessed estimated occupational exposures of MMVF workers. Fibre recovery and analysis by transmission electron microscopy (TEM) were conducted to determine fibre type, fibre dimension and numbers per gram of dry lung tissue. For cases with detailed exposure data, geometric mean (GM) concentrations were compared across the exposure categories, and regression models were used to investigate the relationship between the lung fibres and the variables of estimated exposure, with and without additional variables that may affect fibre retention. RESULTS: A total of 24 samples from 17 cases of lung cancer were available for analysis: MMVF were detected in all cases. Asbestos fibres were detected in 16. No difference or trend in GM MMVF concentration was observed across the estimated exposure categories. Odds ratio (OR) for MMVF g(-1) dry lung was 0.5 (95% confidence interval: 0.1-2.4) for the second, and 3.5 (0.6-18.9) for the third quartile of index of average exposure to MMVF in industry, compared with the first (lowest exposed) quartile (no cases in the highest quartile). CONCLUSIONS: No observable relationship existed between estimated exposure and directly-measured lung fibres among this sample of cases. Retrospective specimen collection, intra-individual variability in fibre concentration, effect of unknown factors on fibre retention and small sample size militated against this study providing evidence for or against a relationship between estimated exposure and lung fibre concentrations.

CP-31398, a novel p53-stabilizing agent, induces p53-dependent and p53-independent glioma cell death.

CP-31398 is a prototype small molecule that stabilizes the active conformation of p53 and promotes p53 activity in cancer cell lines with mutant or wild-type p53. Here, we report that CP-31398 induces p53 reporter gene activity and p21 expression in all of 11 glioma cell lines harboring wild-type or mutant p53, but not in p53-null LN-308 cells. Upon prolonged exposure to CP-31398, all glioma cell lines undergo caspase-independent and bcl-x(L)-insensitive cell death with EC(50) concentrations of 10-36 microM. By comparing p53 wild-type U87MG and p53-null LN-308 cells expressing the temperature-sensitive p53(V135A) mutant, we delineate two pathways of CP-31398-induced cell death: an early, p53-dependent pathway that requires (new p53) protein synthesis and a late, p53-independent pathway characterized by aurintricarboxylic acid -sensitive calcium release and epiphenomenal free radical formation. Post-transcriptional repression of p53 synthesis by an intracellularly transcribed short interfering RNA confirmed the presence of these two pathways of cell death. These observations point out some of the liabilities of CP-31398 as a prototype p53-based therapeutic and define a rationale for further refinement of small molecules that specifically target the p53 pathway, but lack the p53-independent effects.

Genetic evidence of the neoplastic nature of gemistocytes in astrocytomas.

Gemistocytic astrocytoma is characterized by a predominance of large astrocytes with plump processes and massive accumulation of glial fibrillary acidic protein (gemistocytes). This histological variant of low-grade diffuse astrocytoma (WHO grade II) is prone to more rapid progression to anaplastic astrocytoma and glioblastoma than the ordinary fibrillary astrocytoma. The biological basis of this unfavorable prognosis is unclear, since gemistocytes themselves have low proliferative activity, even if present in anaplastic astrocytomas or glioblastomas. This has raised the question of whether gemistocytes are neoplastic cells or dysplastic reactive astrocytes. In this study, gemistocytes and non-gemistocytic neoplastic cells were separated by laser-assisted microdissection from six gemistocytic astrocytomas carrying TP53 mutations. In all cases, identical TP53 mutations were identified in both cell types, indicating that gemistocytes are indeed neoplastic cells. Their lack of proliferative activity may indicate terminal differentiation.

Promoter methylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G:C --> A:T mutations of the TP53 tumor suppressor gene.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes promutagenic alkyl groups from the O(6) position of guanine in DNA. Repair of O(6)-alkylguanine adducts by tumour cells has been implicated in drug resistance since it reduces the cytotoxicity of alkylating chemotherapeutic agents. We assessed promoter methylation of the MGMT gene in astrocytic brain tumours by methylation-specific PCR. MGMT promoter methylation was detected in 26 of 54 (48%) low-grade diffuse astrocytomas (WHO grade II) and in 12 of 16 (75%) of secondary glioblastomas (WHO grade IV) that had progressed from low-grade astrocytomas. The frequency of MGMT methylation was significantly lower in primary (de novo) glioblastomas (13 of 36, 36%, P = 0.0155). The majority of low-grade astrocytomas with MGMT methylation (24/26, 92%) contained a TP53 mutation, whereas only 11 out of 28 (39%) cases without MGMT methylation carried a TP53 mutation (P < 0.0001). Furthermore, G:C --> A:T transition mutations at CpG sites were significantly more frequent in low-grade astrocytomas with MGMT methylation (15/26, 58%) than in those without (3/28, 11%, P = 0.0004). These results suggest that loss of MGMT expression as a result of promoter methylation, which frequently occurs at an early stage in the pathway leading to secondary glioblastomas, appears to be associated with increased frequency of TP53 mutations, in particular G:C --> A:T transitions.

Germline SDHD mutation in paraganglioma of the spinal cord.

Hereditary paraganglioma of the head and neck is associated with germline mutations in the SDHD gene, which encodes a mitochondrial respiratory chain protein. Paragangliomas of the central nervous system are very rare, occur almost exclusively in the cauda equina of the spinal cord and are considered non-familial. In the present study, we screened 22 apparently sporadic paragangliomas of the cauda equina for SDHD mutations. One spinal paraganglioma and similar cerebellar tumours that developed 22 years later in the same patient contained a missense mutation at codon 12 (GGT-->AGT, Gly-->Ser) and a silent mutation at codon 68 (AGC-->AGT, Ser-->Ser). There was no family history of paragangliomas but DNA from white blood cells of this patient showed the same sequence alterations, indicating the presence of a germline mutation. All other cases of spinal paraganglioma had the wild-type SDHD sequence, except one case with a silent mutation at codon 68 (AGC-->AGT, Ser-->Ser). This is the first observation indicating that inherited SDHD mutations may occasionally cause the development of paragangliomas in the central nervous system.

Reduced expression of the Aalpha subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the Aalpha and Abeta subunit genes.

Protein phosphatase 2A (PP2A) consists of 3 subunits: the catalytic subunit, C, and the regulatory subunits, A and B. The A and C subunits both exist as 2 isoforms (alpha and beta) and the B subunit as multiple forms subdivided into 3 families, B, B' and B". It has been reported that the genes encoding the Aalpha and Abeta subunits are mutated in various human cancers, suggesting that they may function as tumor suppressors. We investigated whether Aalpha and Abeta mutations occur in human gliomas. Using single strand conformational polymorphism analysis and DNA sequencing, 58 brain tumors were investigated, including 23 glioblastomas, 19 oligodendrogliomas and 16 anaplastic oligodendrogliomas. Only silent mutations were detected in the Aalpha gene and no mutations in the Abeta gene. However, in 43% of the tumors, the level of Aalpha was reduced at least 10-fold. By comparison, the levels of the Balpha and Calpha subunits were mostly normal. Our data indicate that these tumors contain very low levels of core and holoenzyme and high amounts of unregulated catalytic C subunit.

PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes. By screening a human GBM cDNA library with autologous patient serum (SEREX-approach), we isolated a gene termed PHF3 (PHD finger protein 3). The gene product of PHF3 is immunogenic in GBM as tested in an allogenic patient serum screening demonstrating antibodies in 24 of 39 (61.53%) sera, whereas none of the 14 healthy persons had antibodies against PHF3. While previous SEREX studies revealed allogenic antibody responses up to 40%, our results for PHF3 represent the highest reported rate for a specific antibody response. We show that GBM patients with an antibody response against PHF3 show significant better survival than patients without PHF3-specific antibodies. Because the amino acid sequence of PHF3 contains a PHD finger (also termed LAP motif), a TFIIS homology, a proline rich region and nuclear localization signals, it supposedly functions as a transcription factor. A polyclonal antibody generated against PHF3 shows nuclear expression in most investigated formalin-fixed, paraffin embedded tissues. In GBM, PHF3 expression is concentrated in cells surrounding necroses.

Mutation analysis of hBUB1, hBUBR1 and hBUB3 genes in glioblastomas.

Glioblastomas, the most malignant human brain tumors, are characterized by marked aneuploidy, suggesting chromosomal instability which may be caused by a defective mitotic spindle checkpoint. We screened 22 glioblastomas for mutations in the mitotic spindle check-point genes hBUB1, hBUBR1 and hBUB3. DNA sequencing revealed a silent mutation at codon 144 of hBUB1 (CAG-->CAA, Gln-->Gln) in one glioblastoma, a silent mutation at codon 952 of hBUBR1 (GAC-->GAT, Asp-->Asp) in another glioblastoma, and a silent mutation at codon 388 of the hBUBR1 gene (GCG-->GCA, Ala-->Ala) in 8 glioblastomas. We also observed a known polymorphism at hBUBR1 codon 349 (CAA/CGA, Gln/Arg), with an allelic frequency of 0.75 for Gln and 0.25 for Arg, which is similar to that among healthy Caucasian individuals (0.73 vs 0.27). The coding sequence of the hBUB3 gene did not contain any mutation, but in 4 glioblastomas (18%), a C-->T point mutation was detected at position -6 (6 nucleotides upstream of the ATG initiator codon). Analysis of blood DNA of these patients showed identical sequence alterations, indicating that this is a polymorphism. Again, the frequency in glioblastomas was similar to that in healthy Caucasians (15%). We further screened hBUB1 in 18 cases of giant cell glioblastoma, a variant characterized by a predominance of bizarre, multinucleated giant cells. There were no changes, except for a silent mutation at codon 144 in two cases. These results suggest that mutations in these mitotic spindle checkpoint genes do not play a significant role in the causation of chromosomal instability in glioblastomas.

Promoter hypermethylation and homozygous deletion of the p14ARF and p16INK4a genes in oligodendrogliomas.

The INK4a/ARF locus on chromosome 9p21 encodes two gene products that are involved in cell cycle regulation through inhibition of CDK4-mediated RB phosphorylation (p16INK4a) and binding to MDM2 leading to p53 stabilization (p14ARF). The locus is deleted in up to 25% of oligodendrogliomas and 50% of anaplastic oligodendrogliomas, but little is known on the frequency of gene silencing by DNA methylation. We assessed promoter hypermethylation of p14ARF and p16INK4a using methylation-specific PCR, and homozygous deletion of the p14ARF and p16INK4a genes by differential PCR in 29 oligodendrogliomas (WHO grade II) and 20 anaplastic oligodendrogliomas (WHO grade III). Promoter hypermethylation of the p14ARF gene was detected in 6/29 (21%) oligodendrogliomas and 3/20 (15%) anaplastic oligodendrogliomas. None of the oligodendrogliomas and only 1 out of 20 anaplastic oligodendrogliomas showed hypermethylation of p16INK4a. Homozygous deletion was not detected in any of the WHO grade II oligodendrogliomas but was present in 5/20 (25%) anaplastic oligodendrogliomas and always affected both genes. In one tumor containing distinct areas with and without anaplasia, p14ARF hypermethylation was detected in the WHO grade II area, while homozygous co-deletion of p14ARF and p16INK4a was found in the region with anaplastic features (grade III). These data suggest that aberrant p14ARF expression due to hypermethylation is the earliest INK4a/ARF change in the evolution of oligodendrogliomas, while the presence of p14ARF and p16INK4a deletions indicates progression to anaplastic oligodendroglioma.

Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis.

Decoy receptor 3 (DcR3) is a newly identified soluble protein that binds to CD95 ligand (CD95L) and inhibits its proapoptotic activity. Here we report that DcR3 is expressed by the majority of long-term and ex vivo malignant glioma cell lines as well as in human glioblastoma in vivo. Expression of DcR3 correlates with the grade of malignancy: 15 of 18 (83%) glioblastomas (WHO grade IV) but none of 11 diffuse astrocytomas (WHO grade II) exhibited DcR3 immunoreactivity. We also demonstrate that human malignant glioma cells engineered to release high amounts of DcR3 into the cell culture supernatant are protected from CD95L-induced apoptotic cell death. In contrast, DcR3 does not confer protection from the death ligand Apo2 ligand (TRAIL). Importantly, ectopic expression of DcR3 resulted in substantial differences in immune cell infiltration in the 9L rat gliosarcoma model. Thus, the infiltration of CD4+ and CD8+ T cells as well as microglia/macrophages into glioma was substantially decreased in DcR3-producing tumors compared with control tumors. Chemotaxis assays revealed that DcR3 counteracts the chemotactic activity of CD95L against microglial cells in vitro. These findings suggest that DcR3 may be involved in the progression and immune evasion of malignant gliomas.

p14ARF deletion and methylation in genetic pathways to glioblastomas.

The CDKN2A locus on chromosome 9p21 contains the p14ARF and p16INK4a genes, and is frequently deleted in human neoplasms, including brain tumors. In this study, we screened 34 primary (de novo) glioblastomas and 16 secondary glioblastomas that had progressed from low-grade diffuse astrocytomas for alterations of the p14ARF and p16INK4a genes, including homozygous deletion by differential PCR, promoter hypermethylation by methylation-specific PCR, and protein expression by immunohistochemistry. A total of 29 glioblastomas (58%) had a p14ARF homozygous deletion or methylation, and 17 (34%) showed p16INK4a homozygous deletion or methylation. Thirteen glioblastomas showed both p14ARF and p16INK4a homozygous deletion, while nine showed only a p14ARF deletion. Immunohistochemistry revealed loss of p14ARF expression in the majority of glioblastomas (38/50, 76%), and this correlated with the gene status, i.e. homozygous deletion or promoter hypermethylation. There was no significant difference in the overall frequency of p14ARF and p16INK4a alterations between primary and secondary glioblastomas. The analysis of multiple biopsies from the same patients revealed hypermethylation of p14ARF (5/15 cases) and p16INK4a (1/15 cases) already at the stage of low-grade diffuse astrocytoma but consistent absence of homozygous deletions. These results suggest that aberrant p14ARF expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary glioblastomas, and that p14ARF promoter methylation is an early event in subset of astrocytomas that undergo malignant progression to secondary glioblastoma.

Aspects of intracranial and spinal tumors in patients with Down syndrome and report of a rapidly progressing Grade 2 astrocytoma.

BACKGROUND: Brain tumors in patients with Down syndrome (DS) rarely are reported, and their behavior is not well known. METHODS: The authors report on a male patient age 19 years who had DS with diffuse astrocytoma (World Health Organization Grade 2) that recurred twice despite treatment, leading to a glioblastoma and, finally, to death in just over 2 years. The literature on brain tumors in patients with DS is reviewed. RESULTS: Although brain neoplasms were suspected to be in excess in patients with DS, the authors found only 36 patients with brain neoplasms and 2 spinal tumors. An unusual distribution of histologic tumor types, with an over-representation of germ cell and mesenchymal tumors and a lack of embryonal tumors, was observed, in agreement with what is known currently about the tumor profile of patients with DS. CONCLUSIONS: Cerebral tumors in patients with DS have a specific distribution and may behave differently compared with the general population. These features may be related to the gene dosage effect of oncogenes, antioncogenes, and genes involved in cerebral development due to the supernumerary chromosome 21.

Second primary glioblastoma.

Although characterized by a highly variable phenotype and multiple genetic alterations, glioblastomas are considered monoclonal in origin. We here report on a 64-yr-old patient who developed a second glioblastoma in the left frontal lobe 10 yr after surgical resection of a glioblastoma of right frontal lobe. The first tumor contained 2 p53 mutations, in codon 213 (CGA-->TGA, Arg-->stop) and codon 306 (CGA-->TGA, Arg-->stop), further, 1 missense PTEN mutation (codon 257, TTC-->TTA, Phe-->Leu) and a silent PTEN mutation (codon 154, TTC-->TTT, Phe-->Phe). The second glioblastoma also contained multiple, but different mutations: p53 mutations in codons 158 (CGC-->CAC, Arg-->His) and 273 (CGT-->TGT, Arg-->Cys), and a PTEN mutation in codon 233 (CGA-->TGA, Arg-->Stop). Both neoplasms had a homozygous p16 deletion. The discordant pattern of mutations indicates that the second glioblastoma was not a recurrence but an independent second glioblastoma. The presence in these neoplasms of multiple mutations in tumor suppressor genes suggests the involvement of a novel disease mechanism but there was no indication of a DNA mismatch repair deficiency or of an inherited tumor syndrome.

Promoter hypermethylation of the RB1 gene in glioblastomas.

Loss of expression of the retinoblastoma gene (RB1) has been shown to occur in up to 25% of glioblastomas (WHO Grade IV). To elucidate the underlying mechanism, we assessed RB1 promoter hypermethylation using methylation-specific polymerase chain reaction and RB1 expression by immunohistochemistry in 35 primary (de novo) glioblastomas and in 21 secondary glioblastomas that had progressed from low-grade diffuse astrocytoma (WHO Grade II) or anaplastic astrocytoma (WHO Grade III). Promoter hypermethylation was significantly more frequent in secondary (9 of 21, 43%) than in primary glioblastomas (5 of 35, 14%; p = 0.0258). There was a clear correlation between loss of RB1 expression and promoter hypermethylation. In the majority of glioblastomas with loss of RB1 expression, there was promoter hypermethylation (11 of 13, 85%), whereas 93% of tumors with RB1 expression had a normal RB1 gene status (p < 0.0001). In three glioblastomas, areas with and without RB1 expression were microdissected; promoter hypermethylation was detected only in areas lacking RB1 expression. In patients with multiple biopsies, methylation of the RB1 promoter was not detectable in the less malignant precursor lesions, ie, low-grade diffuse and anaplastic astrocytoma. These results indicate that promoter hypermethylation is a late event during astrocytoma progression and is the major mechanism underlying loss of RB1 expression in glioblastomas.