Following recent and validated cut-off values (12), a change in the category of MGMT promoter methylation status between primary and recurrent glioblastoma was noted in 10 (40 %)cases. Out of these, switching from higher to lower methylation levels was equally frequent than vice versa. These results partially agree with a recent meta-analysis (9) in which a change from methylated to unmethylated was found to be almost twice as frequent. It has to be taken into account that a high proportion of studies included in this meta-analysis use the MSP technique and dichotomize tumors into methylated or unmethylated without considering the intermediate methylation category. When analyzing the methylation level as a continuum, we observed increments or decrements nearly equally distributed in our cohort. However, the median value for the whole series dropped by approximately 6% between the first and second pathologic analysis.
Survival data of patients and proportion of tumors exhibiting higher degrees of methylation of MGMT promoter gene is higher in our series than in previous reports (12–14). This may reflect our highly selective indications for re-do procedures since we do not usually consider a reoperation for incomplete resections at first surgery or PFS intervals lower than 6–9 months. Thus, we might be excluding patients with more aggressive lesions (i.e., unmethylated). Survival benefit among patients whose tumors contain a methylated MGMT promoter is well established (2). Still, to our best knowledge, this is the first study that considers methylation status as a continuous variable and investigates its correlation with survival. Similar to previous reports (9, 15, 16), we observed that methylation was more linked to OS than PFS, although statistical significance was not achieved, possibly attributable to the small size of our cohort. The association between methylation status at recurrence and PPS was unexpected. Tumors whose methylation status was intermediate were more linked to post-progression survival than hypermethylated ones. This may be again influenced by other factors such as the limited size of our study group.
Methylation status results may be dependent on the laboratory test used for its determination. MSP has been to date, the most used technique to validate MGMT methylation status changes detection (15, 17–21) however, Park et al. (22) concluded that methylation changes might remain undetectable when analyzing tumors by MSP while other techniques, such as methylation-specific multiplex ligation probe amplification (MS-MLPA) can readily uncover the downward shift of methylation in recurrent tumors. In our study, pyrosequencing was used to detect these changes, but similar results to those previously published were obtained.
On the other hand, tumor samples for methylation analysis can provide different results if not previously selected. To avoid this kind of contamination in our study, areas with a higher amount of neoplastic cells were marked and manually dissected to exclude non-neoplastic elements as previously described (17). Intratumor heterogeneity concerning epigenetic silencing of the MGMT gene has been reported by some groups (17, 23) others have found that MGMT promoter methylation status is relatively homogenous within the tumor (18, 24) and therefore we have only examined one preselected area from each tumor. By contrast, MGMT protein expression evaluated by immunochemistry appears to have a decreasing gradient from the inner to the outer portion of the tumor (23). These data support that there is low concordance between MGMT promoter methylation and protein levels and that there may be other mechanisms involved in its expression apart from epigenetic silencing (25).
Another issue is the definition and validation of the technically and clinically relevant cut-off value for MGMT promoter methylation and the fact that most samples will be classified similarly by most tests, although different CpGs are interrogated (26).
Due to its mechanism of action, clonal selection by TMZ has been elucidated as a primary factor that could explain treatment resistance and methylation pattern reshaping within the clinical course. TMZ may deplete cell lines with low MGMT expression (i.e., hypermethylated promoter), inducing chemoresistance by selecting clones with low levels of methylation which are more resistant to alkylating drugs (16). Although exposed in a mathematical model (27), this feature remains controversial since changes from unmethylated to highly methylated status in paired samples after chemotherapy can be detected in several studies, including ours (15–17, 22, 28, 29). On the contrary, other authors found differences in the methylation pattern of recurrent glioblastoma depending on whether the patient was treated with RT followed by TMZ versus concurrent RT/TMZ (16, 20). Furthermore, it has been reported that switching from methylated to unmethylated status can happen without chemotherapy in lower-grade glioma cases (19). We consider our cohort to be very homogenous in terms of post-surgical treatment since all patients received simultaneous chemoradiotherapy, and 84% were treated with at least six cycles of adjuvant TMZ. These data suggest that epigenetic dynamics of MGMT promoter methylation status over the clinical course might be very complex and influenced by several processes that are still poorly understood. Additional mechanisms have been theorized in which chemotherapy with alkylating agents may promote de-differentiation from non-cancer stem cells to glioma stem cells, responsible for tumor growth and chemoresistance acquisition (30).
Several drawbacks need to be considered in the present study. Its small sample size limits the statistical power of our results. A future multicentre, more extensive study would be of interest, considering multi-area biopsies and other methylation measuring techniques that may disclose different conclusions regarding modifications in epigenetic changes.