Prognostic Significance of O6-Methylguanine-DNA Methyltransferase Determined by Promoter Hypermethylation and Immunohistochemical Expression in Anaplastic Gliomas

Abstract

Purpose: Anaplastic gliomas constitute a heterogeneous group of tumors with different therapeutic responses to adjuvant chemotherapy with alkylating agents. O 6 -Methylguanine-DNA methyltransferase (MGMT), a DNA repair protein, is one of the implicated factors in glioma chemoresistance.The prognostic value of MGMTremains controversial due in part to the fact that previous published studies included heterogeneous groups of patients with different tumor grades. The aim of this study was to evaluate the prognostic significance of MGMT in patients with anaplastic glioma. Experimental Design: Ninety-three patients with anaplastic glioma were analyzed for MGMT protein expression by immunohistochemistry. In addition, for those patients from whom a good yield of DNA was obtained (n = 40), MGMT promoter methylation profile was analyzed by methylation-specific PCR. MGMT prognostic significance was evaluated together with other well-known prognostic factors. Results: Fifty-one tumors (54.8%) showed nuclear staining of MGMT. There was a trend towards longer overall survival for those patients with negative MGMT immunostaining (hazard ratio,1.66; P = 0.066). In a secondary analysis including those patients who actually received chemotherapy (n = 72), the absence of MGMTexpression was independently associated with better survival (hazard ratio, 2.12; P = 0.027). MGMT promoter methylation was observed in 50% of the analyzed tumors. No statistical correlation between MGMT expression and MGMT promoter hypermethylation was observed. Conclusions: Unlike previous studies, we did not find a correlation between MGMT promoter methylation and survival. However, we observed a correlation between MGMT protein expression and survival in those patients who received chemotherapy thus suggesting that the absence of MGMTexpression is a positive predictive marker in patients with anaplastic glioma.Anaplastic gliomas (WHO grade 3) show a wide variability of clinical outcome. Despite optimal treatment, mainly consisting of gross total resection followed by radiotherapy and chemotherapy with alkylating agents (1), therapeutic response and survival times vary considerably. This fact suggests that a large number of factors, including patient, tumor, and treatment characteristics, may influence the outcome (2 -4).Alkylating agents cause cell death by forming cross-links between adjacent strands of DNA due to alkylation of the O 6 position of guanine. The cellular DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) inhibits the cross-linking of double-stranded DNA by removing alkylating lesions (5 -7). A direct relationship between MGMT activity and resistance to alkylating nitrosoureas and methylating agents (i.e., ionizing radiations) has been well documented in cell lines and xenografts derived from a variety of human tumors, including gliomas (8). Moreover, depletion of MGMT activity with the substrate analogue inhibitor O 6

Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas

“…On the other hand, heterogeneous MGMT expression within the tumors, high levels of MGMT protein in infiltrating lymphocytes, microglia and blood vessels, and the upregulation of the MGMT protein expression during the therapy (external beam radiation, chemotherapy, steroids) have been reported to limit the reproducibility of immunohistochemistry. 2,[10][11][12][13] In the current series, the MGMT protein expression profile was very similar in tumor tissue samples obtained from distant sites within the same tumor. This finding underscores the reproducibility of the applied method and indicates homogeneous protein expression throughout the viable areas of each tumor investigated.…”

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

“…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.…”

“…Wiewrodt et al (Wiewrodt et al, 2008) showed that patients expressing p30 fmol mg À1 MGMT protein in the pre-treatment tumour volume had a significantly better response to alkylating therapy than those with MGMT protein above this level. Others have shown that patients with low MGMT protein expression had significantly improved survival compared with those with high expression (Brell et al, 2005;Chinot et al, 2007;Nagane et al, 2007). Vlassenbroeck et al (Vlassenbroeck et al, 2008) used a real-time MSP assay to determine a clinically relevant cut-off for stratification of glioblastomas into two distinct populations, with prognostic significance in recurrent anaplastic astrocytoma but not glioblastomas treated with temozolomide (Sadones et al, 2009).…”

MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas

“…It has been reported that immunohistochemical detection or activity assays of the MGMT protein AGT can be used as tools to identify patients whose tumors are resistant to TMZ. 6,22 Immunohistochemistry has the advantage that it can be performed on archival, formalin-fixed, and paraffin-embedded tissue and may be helpful to sort out the contribution of 'contaminating' normal cells in the expression of MGMT. However, our results show that interpretation of the immunohistochemical staining results often remains difficult.…”