We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.Gliomas are the most common group of primary brain tumors consisting of clinically and biologically distinct subtypes. The three main subgroups of glioma are astrocytic, oligodendroglial and mixed gliomas, which are further malignancy graded into up to four grades according to the WHO classification. 1 Glioblastomas (WHO grade IV) are the most malignant and common astrocytic brain tumor in adults, the overall median survival being only just over 14 months despite modern combined therapies. 2 Most glioblastomas are primary glioblastoma (pGB), while secondary glioblastoma (sGB) generally develops by progression from astrocytomas WHO grade II or III. Patients with astrocytic or oligodendroglial tumors WHO grade II and III survive longer, however they always recur and are ultimately fatal.Among the many genetic and epigenetic abnormalities commonly found in adult gliomas, three have been reported to predict response to treatment and/or prognosis: IDH1/ IDH2 mutations, concurrent deletion of the entire 1p and 19q chromosomal arms (total 1p/19q loss) and MGMT methylation. 3,4 With the exception of pGB, mutations of either IDH1 or IDH2 occur in more than 50% of adult astrocytic, oligodendroglial and oligoastrocytic tumors. [5][6][7][8] It has been shown that the presence of IDH1/2 mutations is associated with longer patient survival. 5-7,9,10 IDH1/2 mutations have been proposed as the earliest genetic changes in a ...
Copy number alterations are frequently found in colorectal cancer (CRC), and recurrent gains or losses are likely to correspond to regions harbouring genes that promote or impede carcinogenesis respectively. Gain of chromosome 13q is common in CRC but, because the region of gain is frequently large, identification of the driver gene(s) has hitherto proved difficult. We used array comparative genomic hybridization to analyse 124 primary CRCs, demonstrating that 13q34 is a region of gain in 35% of CRCs, with focal gains in 4% and amplification in a further 1.6% of cases. To reduce the number of potential driver genes to consider, it was necessary to refine the boundaries of the narrowest copy number changes seen in this series and hence define the minimal copy region (MCR). This was performed using molecular copy-number counting, identifying IRS2 as the only complete gene, and therefore the likely driver oncogene, within the refined MCR. Analysis of available colorectal neoplasia data sets confirmed IRS2 gene gain as a common event. Furthermore, IRS2 protein and mRNA expression in colorectal neoplasia was assessed and was positively correlated with progression from normal through adenoma to carcinoma. In functional in vitro experiments, we demonstrate that deregulated expression of IRS2 activates the oncogenic PI3 kinase pathway and increases cell adhesion, both characteristics of invasive CRC cells. Together, these data identify IRS2 as a likely driver oncogene in the prevalent 13q34 region of gain/amplification and suggest that IRS2 over-expression may provide an additional mechanism of PI3 kinase pathway activation in CRC.
Diffuse astrocytomas (WHO grade II) typically present as slow-growing tumours showing significant cellular differentiation, but possessing a tendency towards malignant progression. They account for ~10% of all astrocytic tumours, with a peak incidence between 30 and 40 years of age. Median survival is reported as around 6-8 years. Mutations of TP53 and IDH1 have been described as genetic hallmarks, while copy number alterations are also relatively common. However, there is some evidence to suggest that these characteristics may vary with age. Here, we present an integrated clinicopathologic, genomic and transcriptomic analysis suggesting that paediatric and adult tumours are associated with distinct genetic signatures. For example, no childhood tumour showed mutation of IDH1/2 or TP53, virtually no copy number changes were seen, and MGMT methylation was absent. In contrast, adult tumours showed IDH1/2 mutation in 94% and TP53 mutation in 69% of cases, with multiple copy number alterations per case and hypermethylation of MGMT in the majority of tumours. These differences were associated with a worse prognosis in the adult patients. The expression array data also revealed a significant difference in the expression of a number of genes putatively involved in neural stem cell maintenance and CNS development, including DLL3, HES5, BMP2, TIMP1 and BAMBI. Genes involved in DNA replication and the cell cycle were also enriched in the adult tumours, suggesting that their more aggressive behaviour may be due to derivation from a more rapidly dividing, less differentiated cell type.
Background: Astrocytic, oligodendroglial and oligoastrocytic tumors are major subtypes of human gliomas, which are the most common malignant primary brain tumor. IDH1/IDH2 mutations are found in more than 50% of WHO grade II and III tumors in each of these three subtypes and is considered to be an early change. Presence of MGMT methylation has been shown to predict a good response to temozolomide and improved survival. Various protocols for MGMT methylation testing are utilized in the clinic, however there is a lack of consensus on how MGMT CpG island (CGI) should be analysed. The aim of this study was to 1) define the optimal target region for MGMT methylation-testing by bisulfite modification and pyrosequencing 2) profile methylation at each CpG in MGMT CGI in a large cohort of gliomas and 3) correlate MGMT methylation with other genetic abnormalities including IDH1/IDH2 mutations. Method: The methylation status of each CpG in the MGMT CGI was determined and compared with MGMT mRNA expression in 22 glioblastoma xenografts, 13 glioma cell lines and 6 normal brain tissues. A luciferase assay was used to investigate selected CpGs for their role in transcription. An optimized pyrosequencing assay was then used to investigate 406 astrocytic and oligodendroglial tumors of all major types. A normal mixture model was applied to the data points and the optimal cut-off value to judge MGMT methylation in clinical samples was determined. Results: We identified a 120 bp region containing 16 CpG sites to be most critical in transcriptional control. We determined an optimal cut-off of value of 22% to judge MGMT methylation in clinical samples. Using this criterion, methylation in this region was found in 58% of 406 gliomas. Pilocytic astrocytomas had a low incidence of MGMT methylation (6.8%). All the other tumor types had methylation incidence of 50-87%. When compared with the pattern of genetic alterations, we found that all tumors with IDH1/IDH2 mutations had MGMT methylation. In astrocytoma grade II and all oligodendrogliomas, MGMT methylation and IDH mutations showed 100% concordance. Conversely, the majority of glioblastomas with MGMT methylation did not have IDH1/IDH2 mutations, suggesting MGMT methylation can occur independently of IDH mutation in glioblastoma. Conclusion: We propose a robust pyrosequencing assay to accurately assess MGMT methylation: 16 CpGs sites using a cut-off value of 22%. We showed that all IDH mutations were associated with MGMT methylation, while some tumors with MGMT methylation did not harbor IDH mutations. We therefore propose that MGMT methylation may be the earliest change in the development of astrocytomas and oligodendrogliomas, preceding IDH1/IDH2 mutations. This research highlights the importance of integrating epigenetic and genetic alteration data to study the pathogenesis of a disease. 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 2794. doi:10.1158/1538-7445.AM2011-2794
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