O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

Abstract

O(6)-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (chi(2) test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76-17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45-2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.

Fig. 1.

Detection of the MGMT status in glioblastoma-derived cell cultures. Representative examples are shown. (A) MGMT promoter methylation was detected by MSP. Amplification products with specific primers for methylated (m, 81 bp) and unmethylated (u, 93 bp) DNA sequences are shown, and scoring as methylated (m), unmethylated (u), and mixed (u/m) is indicated. Positive controls for methylated (pcm) and unmethylated (pcu) sequences were included. M, size marker. (B) MGMT protein expression was determined by Western blot (band at 25 kDa), and relative expression levels were obtained by the densitometric evaluation of immunoblots compared with the MGMT-overexpressing glioblastoma cell line GL80 included as positive control (pc) and set arbitrarily as 1. Probing with β-actin served as loading control. (C) Scatter gram analysis of MGMT protein expression in subgroups with methylated (m) and unmethylated (u) MGMT promoter.

Fig. 2.

Discrepancy between MGMT promoter methylation status and protein expression determined by methylation specific PCR (MSP) and Western blot (WB). Representative examples for promoter methylation and protein expression (BTL183, BTL35) as well as lack of promoter methylation and protein expression (BTL298, BTL365) are given. (A) Polyacrylamide gel showing amplification products of methylated (m, 81 bp) and unmethylated (u, 93 bp) DNA sequences. Positive controls for methylated (pcm) and unmethylated (pcu) sequences were included. (B) Western blot analysis for the corresponding samples. Data obtained by MSP and WB were expressed as relative (expression) levels as described in Patients and Methods.

Fig. 3.

MGMT promoter methylation and protein expression when compared with overall survival (OS) of patients. Results of Kaplan–Meier analyses of OS according to MGMT protein expression (A and B) or MGMT promoter methylation (C and D) of GBM patients treated (A and C) and untreated (B and D) with TMZ are shown.