In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 70, No. 8_Supplement ( 2010-04-15), p. 4905-4905
Abstract:
Hypermethylation of the DNA repair gene 06-Methylguanosinemethyltransferase (06-MGMT) is an important prognostic and predictive marker for the management of glioblastoma patients. However, assessment of O6-MGMT gene methylation is not yet standardized and a comprehensive comparison of different methods described in the literature is missing. Therefore, the methylation status of the MGMT gene was analyzed in a large series of glioblastoma specimens, WHO grade IV (n = 92) using nested conventional methylation-specific PCR (MSP), real-time PCR using either SybrGreen or a sequence specific probe for detection and pyrosequencing. All specimens were routinely processed formalin-fixed paraffin-embedded tumor biopsies. For a subset of samples (n = 30) three different protocols for the treatment of genomic DNA with bisulfite were compared. As a control, 20 brain biopsies from 10 patients without glioblastoma were analyzed. MGMT mRNA level was measured using quantitative real-time RT-PCR in n = 39 samples and the mRNA expression levels were correlated to the methylation status of these samples. The three different bisulfite protocols did not show statistically significant differences concerning quality and yield of recovered DNA. A region in the MGMT promoter in which many published primers bind, displayed constitutively elevated methylation levels also in healthy brain tissue samples, thereby leading to false-positivity under many circumstances. Nested MSP and pyrosequencing correlate fairly well in scoring samples as “methylated” versus “unmethylated”, but a considerable number of samples scored “methylated” by nested MSP (n = 7) displayed only background methylation when analyzed by pyrosequencing. Only methylation levels measured by pyrosequencing correlated with mRNA expression levels (r2 = 0.32, p = 0.0002). Also, threshold definition was much more straightforward and easier to standardize using pyrosequencing. Real-time PCR based methods did not correlate very well with nested PCR or pyrosequencing. In conclusion, pyrosequencing is the most reliable methodology for methylation analysis of O6-MGMT gene methylation analysis in routinely processed glioblastoma specimens. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4905.
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM10-4905
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2010
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
