BACKGROUND: Epigenetic silencing of O 6 -methylguanine-DNA-methyltransferase (MGMT) by promoter methylation is associated with improved survival in glioblastomas treated with alkylating agents. In this study, we investigated MGMT promoter methylation in glioblastomas treated with temozolomide and radiotherapy in a single UK treatment centre. METHODS: Quantitative methylation data at individual CpG sites were obtained by pyrosequencing for 109 glioblastomas. RESULTS: Median overall survival (OS) was 12.4 months with 2-year survival of 17.9%. Pyrosequencing data were reproducible with archival samples yielding data for all glioblastomas. Variation in methylation patterns of discrete CpG sites and intratumoral methylation heterogeneity were observed. A total of 58 out of 109 glioblastomas showed average methylation 4non-neoplastic brain in at least one clinical sample; 86% had homogeneous methylation status in multiple samples. Methylation was an independent prognostic factor associated with prolonged progression-free survival (PFS) and OS. Cases with methylation more than 35% had the longest survival (median PFS 19.2; OS 26.2 months, 2-year survival of 59.7%). Significant differences in PFS were seen between those with intermediate or high methylation and unmethylated cases, whereas cases with low, intermediate or high methylation all showed significantly different OS. CONCLUSIONS: These data indicate that MGMT methylation is prognostically significant in glioblastomas given chemoradiotherapy in the routine clinic; furthermore, the extent of methylation may be used to provide additional prognostic stratification.

Angiogenesis and extracellular matrix degradation are key events in tumour progression, and factors regulating stromal -epithelial interactions and matrix composition are potential targets for the development of novel anti-invasive/antiangiogenic therapies. Here, we examine the expression of ADAMTS-8, a secreted protease with antiangiogenic properties, in brain tissues. Using quantitative RTpolymerase chain reaction (PCR), high, equivalent expression of ADAMTS-8 was found in normal whole brain, cerebral cortex, frontal lobe, cerebellum and meninges. ADAMTS-8 expression in 34 brain tumours (including 22 high-grade gliomas) and four glioma cell lines indicated at least two-fold reduction in mRNA compared to normal whole brain in all neoplastic tissues, and no detectable expression in 14 out of 34 (41%) tumours or four out of four (100%) cell lines. In contrast, differential expression of TSP1 and VEGF was seen in nine out of 15 (60%) and seven out of 13 (54%) tumours, with no relationship in the expression of these genes. Immunohistochemistry and Western analysis indicated downregulation of ADAMTS-8 protein in 477% tumours. Methylationspecific PCR analysis of ADAMTS-8 indicated promoter hypermethylation in one out of 24 brain tumours (a metastasis) and three out of four glioma cell lines suggesting an alternative mechanism of downregulation. These data suggest a role for ADAMTS-8 in brain tumorigenesis, warranting further investigation into its role in regulation of tumour angiogenesis and local invasion.

The antiangiogenic factor METH-2 (ADAMTS-8) was identified in a previous dual-channel cDNA microarray analysis to be at least two-fold under-represented in 85% (28 out of 33) of primary non-small-cell lung carcinomas (NSCLCs). This observation has been validated in an independent series of NSCLCs and adjacent normal tissues by comparative multiplex RT-PCR, and METH-2 mRNA expression was dramatically reduced in all 23 tumour samples analysed. Immunohistochemical analysis of the same sample set demonstrated that METH-2 was strongly expressed in 14 out of 19 normal epithelial sites examined but only one out of 20 NSCLCs. DNA methylation analysis of the proximal promoter region of this gene revealed abnormal hypermethylation in 67% of the adenocarcinomas and 50% of squamous cell carcinomas, indicating that epigenetic mechanisms are involved in silencing this gene in NSCLC. No homozygous deletions of METH-2 were found in lung cancer cell lines. Allelic imbalance in METH-2 was assessed by an intronic single nucleotide polymorphism (SNP) assay and observed in 44% of informative primary samples. In conclusion, the downregulation of METH-2 expression in primary NSCLC, often associated with promoter hypermethylation, is a frequent event, which may be related to the development of the disease.