Careful Exclusion of Non-neoplastic Brain Components is Required for an Appropriate Evaluation of O6-methylguanine-DNA Methyltransferase Status in Glioma

Abstract

Evaluation of O6-methylguanine-DNA methyltransferase (MGMT) expression is important for antiglioma therapy as many clinical trials have demonstrated that promoter hypermethylation and low level expression of MGMT are associated with an enhanced response to alkylating agents. However, here we report that the current strategies used to evaluate MGMT status in gliomas are unreliable. We observed discordance in the MGMT expression status when immunohistochemical evaluation and polymerase chain reaction-based methylation assessments were used: 73% of gliomas with methylated MGMT promoter had substantial numbers of MGMT-immunopositive tumor cells. Furthermore, when MGMT expression was tested in tumor homogenates using reverse transcription-polymerase chain reaction, 43% of tumors were found positive, in comparison to only 24%, when histologic samples were assayed immunohistochemically. To explain these inconsistencies we undertook a detailed immunohistochemical evaluation of tumor samples and found that some gliomas demonstrated remarkably high expression of MGMT in the entire tumor whereas others contained only a small immunopositive area. Additionally, we found that gliomas contained various types of non-neoplastic cells expressing MGMT, including lymphocytes, vascular endothelial cells, and macrophages/microglias, which contribute to overall MGMT expression detected in tumor homogenates, and thus result in overestimation of tumor MGMT expression. Therefore, to correctly establish MGMT expression in the tumor, which could be informative of glioma sensitivity to alkylating agents, exclusion of non-neoplastic brain components from analysis is required.

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

“…As tumour samples were chosen to have 470% neoplastic cells, these data suggest variation in the numbers of methylated tumour cells in different regions of glioblastoma tissues. Similarly, glioblastomas show heterogeneous patterns of MGMT protein expression often with regions within the same tumour displaying widely different staining patterns (Juillerat-Jeanneret et al, 2008;Sasai et al, 2008). On the basis of the MSP assay and bisulphite sequencing some studies have claimed homogeneity in MGMT status within glioblastomas (Grasbon-Frodl et al, 2007), whereas others have reported a degree of intratumoral heterogeneity (Juillerat-Jeanneret et al, 2008;Parkinson et al, 2008).…”

“…Thus, the extent of methylation within the tumour impacts on associations with survival. This may be a reflection of the proportions of cells with silenced MGMT, but the relationship between methylation and expression of active enzyme has been questioned (Maxwell et al, 2006;Sasai et al, 2008). Alternatively, high numbers of methylated cells may be a marker of less aggressive biology, possibly in association with methylation of other gene promoters .…”

“…However, most studies report poor agreement between these methods (Brell et al, 2005;Maxwell et al, 2006;Preusser et al, 2008;Rodriguez et al, 2008;Sasai et al, 2008;Yachi et al, 2008) and most correlations with outcome have been obtained through investigation of promoter methylation Preusser et al, 2008). Unlike the MSP assay used in the majority of clinical studies, pyrosequencing allows highly reproducible quantitative evaluation of methylation at discrete CpG sites thereby providing more information on promoter methylation status and facilitating analysis of specific methylation patterns.…”

STAT3 Inhibition Overcomes Temozolomide Resistance in Glioblastoma by Downregulating MGMT Expression

“…Despite pseudosubstrates of MGMT such as O 6 -benzylguanine were expected to suppress resistance by depleting MGMT (8)(9)(10), clinical trials did not show significant restoration of temozolomide sensitivity in patients with temozolomide-resistant GBM (11). Therefore, other therapeutic agents which suppress MGMT expression and sensitize the efficacy of temozolomide are highly desired (12,13).…”

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma

“…Many glioblastomas contain a considerable proportion of non-neoplastic cells such as macrophages/microglia (tumor-associated macrophages) that can comprise over 70% of the tumor. [21][22][23][24][25] Interference of tumor-associated macrophages is illustrated by the improvement in interpretation of MGMT (O-6-methylguanine-DNA methyltransferase) status of glioblastoma using immunohistochemistry. 26 That tumor-associated macrophages obscure the telomere maintenance mechanism is suggested by molecular similarities between glioblastomas classified as having the non-defined telomere maintenance mechanism and those typed as alternative lengthening of telomeres or telomerase positive.…”