In this study, we have investigated the relation between MGMT promoter methylation status and protein expression and the association of these parameters with TMZ therapy as well as OS of glioma patients. To focus exclusively on the malignant cell compartment, we analyzed MGMT expression in primary tumor cells explanted from the surgical specimens instead of tissue extracts or tumor sections. Using this new approach, we prove that lack of MGMT protein expression is a strong predictive indicator for TMZ treatment response and the related survival benefit. Despite a significant correlation between the two MGMT parameters, protein expression was of superior predictive value in our patient cohort. This indicates that reliable MGMT protein detection methods have to be developed to identify GBM patients likely to benefit from TMZ therapy.
The optimal method for MGMT status determination and its predictive quality on TMZ response in GBM patients is a matter of debate.17,22,24–28
Since the promising results of the translational sub-study20
to the multicenter EORTC trial,1
detection of MGMT promoter methylation has been in the focus of interest. Methylation analysis can be performed using small amounts of tumor-derived DNA, and MGMT promoter methylation is a specific characteristic of tumor cells never observed in normal cell compartments.23
Thus, contaminations with nonmalignant cell-derived DNA should not lead to false-positive results. Based on these benefits, the use of promoter methylation as a parameter for patient stratification for TMZ therapy was recently suggested.28
Although the data by Hegi et al.20
are in accordance with some other studies,31
a lack of a significant association between MGMT promoter methylation and TMZ response and/or OS in primary or recurrent GBM was reported.17,32–34
In case of astrocytoma grade II, MGMT promoter methylation was even suggested as a negative prognostic marker for progression-free survival.35
Interestingly, the majority of studies using chloroethylating nitrosoureas demonstrated a significant association between MGMT promoter methylation and therapy response.15,36,37
Accordingly, MGMT promoter methylation was predictive for response to Carmustine (BCNU), but not to TMZ + cisplatin, in GBM patients.38
The reasons for the inconsistent results with regard to the predictive quality of MGMT promoter methylation might be based on the higher patient number analyzed by Hegi et al.20
However, the impressive correlation between MGMT protein detection by Western blot and patient survival in the TMZ-treated patient group in our study suggests that it is not the patient number but the parameter per se that determines the predictive value. Unfortunately, no MGMT protein detection method was used by Hegi et al.,20
making it impossible to establish a comparable analysis as done in this study. Additionally, it has to be mentioned that promoter methylation analysis by MSP, especially from paraffin-embedded tissues, is technically demanding. This is also obvious from the distinct center dependency of methylation detection in the translational sub-study leading to successful determination of this parameter in 36% of patients only.20
Technical problems with MSP can be ruled out in our study for two reasons: (i) for detection of MGMT promoter methylation, we used tumor cell culture–derived DNA, thus avoiding problems resulting from tissue fixation and paraffin embedding; and (ii) in a subgroup of tumors, MSP was done comparably from snap-frozen tissue and tumor cells, confirming in all cases the presence of methylated MGMT promoter sequences. The latter observation also excludes possible changes in the MGMT promoter methylation status during short-term in vitro cultivation of tumor cells.
In the present study, the two MGMT parameters showed strong correlation, confirming that promoter methylation is a major factor regulating protein expression. Nevertheless, as obvious from the scattergrams (Fig. C) and depicted by selected cases (Fig. ), there are several tumor cell samples not following this correlation. This indicates that in a subgroup of tumors with methylated MGMT promoter, the protein is still expressed and vice versa. Comparable observations have been reported consistently in repeated biopsies from inoperable GBM.26
High percentages of MGMT-negative tumor cells have also been detected by immunohistochemistry in tumors harboring only unmethylated DNA sequences.27,39
This might explain why the few studies addressing the correlation between MGMT promoter status and gene expression so far delivered contradictory results. Although some earlier reports found a good correlation,16,40,41
others described a rather loose association or no correlation at all.17,26,27,39,42
In agreement with our data, most of the studies suggested that these parameters cannot be used interchangeably for each other. Such observations might also explain why in the study of Hegi et al.20
TMZ treatment led to a borderline OS and significant progression-free survival benefit, even in the patient subgroup with unmethylated MGMT promoter.
The reasons for the discrepancy between promoter methylation and MGMT expression in a patient subgroup are unclear so far. Generally, epigenetic silencing via promoter methylation is an important but obviously not always decisive factor regulating MGMT expression in GBM cells. This assumption is supported by the data of Sasai et al.43
demonstrating activation of MGMT expression by demethylating agents at the mRNA but not protein level in transformed malignant astrocytes and GBM cells, suggesting posttranscriptional repression of MGMT expression. Additionally, a transcriptional activation of MGMT expression by wild-type p53 was reported in murine astrocytes and human gliomas also when harboring a methylated MGMT promoter.44,45
Accordingly, immunohistochemical staining of p53 and MGMT were mutually exclusive in astrocytoma sections.46
With regard to therapy response and OS, MGMT protein expression turned out to be of distinctly higher predictive power when compared with promoter methylation status in our patient samples. Accordingly, MGMT immunostaining, but not promoter methylation, was an independent prognostic factor with regard to OS of patients with anaplastic astrocytoma treated with TMZ.17
Predictive quality of MGMT protein expression with respect to TMZ response and/or disease progression was also reported in mixed gliomas,18
and recently in a small series of recurrent GBM by Western blot from tumor extracts.48
Accordingly, in a recent comparative analysis, MGMT immunostaining correlated with promoter methylation. However, only the protein levels correlated with MGMT activity in GBM tissues.27
Nevertheless, not all studies using MGMT immunostaining demonstrated correlations with TMZ response and/or patient survival.42
Additionally, several problems are associated with MGMT immunostaining, including tumor cell heterogeneity, infiltration of MGMT-positive inflammatory cells and microglia as well as induction of MGMT during therapy.28
All these factors restrain standardization and determination of cut-off levels.24,27,45
Accordingly, our attempts to analyze MGMT protein expression by immunohistochemistry in selected patient samples used in this study were inconclusive, and immunostaining neither correlated with MGMT promoter methylation nor MGMT expression detected by Western blot, even when using identical antibodies (data not shown). Nevertheless, our data based on tumor cell explants and Western blot analysis prove the high quality of MGMT protein expression as a predictive marker for TMZ response in GBM patients.
In summary, we used a completely new approach to analyze the impact of MGMT status on TMZ therapy response and survival of GBM patients. Determination of MGMT protein levels in GBM cell primo-cultures explanted from surgical specimens turned out to be a highly predictive factor for TMZ therapy-mediated survival benefit with superior information power as compared to the analysis of promoter methylation by MSP. Consequently, reliable and reproducible MGMT protein detection methods have to be developed and tested with regard to their predictive value for TMZ therapy response in prospective studies involving larger patient cohorts.