Glioblastoma (GB) is not only the most prevalent primary tumor of the brain, but also has an aggressive clinical course with poor prognosis [1, 2, 3], Standard management includes wide resection of the tumor and postoperative radiotherapy (PORT) with concomitant temozolomide (TMZ) followed by maintenance TMZ [4, 5, 6].
Although inclusion of TMZ in the treatment protocols was shown to improve survival in patients with GB, eventual progression occurs in approximately 70.0% of the subjects within 1 year [7]. Patients usually exhibit a variable response to TMZ [8], which induces apoptosis by damaging DNA through binding to alkyl groups responsible for DNA repair [9].
O6-Methyl guanine methyl transferaseis (MGMT), a repair protein for cellular DNA that quickly reverses alkyl-ation and methylation of guanine at the O6 position [9] , neutralizing the cytotoxic effects of alkylating agents [10]. O6-Methyl guanine methyl transferase is synthesized in all human tissues [11]. The majority of our current understanding of MGMT as a DNA repair protein is based on observations after exposure to alkylating agents [12]. The protective role of MGMT against cytotoxic effects of al-kylating agents has been shown in human cell line xenograft models and in MGMT transgenic mice [13,14]. The MGMT-mediated repair mechanism is unique and different from other DNA repair pathways in that it does not directly represent a component of the repair process and acts independently. It specifically separates the methyl group at the O6 position, thereby repairing the nucleotide without any breaks in DNA fiber congruous with its natural form. It is referred to as a self-destruction (suicide) enzyme [11], as the enzyme is irreversibly inactivated due to the transfer of the alkyl group to the cysteine residue in the active part of MGMT. Therefore, the maintenance of activity of the enzyme is dependent upon new protein synthesis. When the process is saturated, the excess O6-methyl guanine depletes MGMT [11]. Although the O6 position of guanine is not a general target for alkylating agents, the consequently occurring pro-mutagenic lesions represent important inducers of cytotoxicity and apoptosis. If left unrepaired, this modified guanine may be paired with thymine, which activates the mismatch repair (MMR) pathway, instead of pairing with cytosine. Mismatch repair discards O6-methyl guanine in the main strain, only focusing on the newly synthesized strain and repairing it. When the MMR pathway is attempting to repair this false pair, it enters into new cycles of synthesis and repair. The end result consists of double strain breaks in DNA, activating the apoptotic pathways and inducing cell death [14]. The increment in MGMT activity in tumor cells is related to the resistance to alkylating agents. Nevertheless, due to the promotor methylation in tumor cells, the epigenetic silence of MGMT results in low MGMT expression [15]. The MGMT is considered as a potential prognostic marker in patients with GB and it has also been shown to have a prognostic value in TMZ response [16]. In this study involving GB patients receiving adjuvant concomitant TMZ in addition to PORT and in patients receiving RT only, the effect of promotor methylation of MGMT on treatment response, its prognostic importance, as well as the effect of methylation of CpG1, CpG2, CpG3 and CpG4 were explored.
Median age was 53 years (18-73 years). DNA assessment of samples from patients yielded 52 patients (52.0%) with and 48 cases (48.0%) without promoter methylated MGMT (Table 1). Overall median, overall survival (OS) and progression free survival (PFS), were 10 and 6 months, respectively. Cox regression was used to evaluate the prognostic importance of MGMT-methylation, age, treatment and gender in terms of survival. Accordingly, MGMT-methylation emerged as a significant prognostic factor for OS and PFS [odds ratio (OR): 0.609,95% confidence interval (95% CI): 0.395-0.939,
Patient characteristics.
Characteristics | All Patients | MGMT-Methylated | MGMT-Unmethylated | |||
---|---|---|---|---|---|---|
% | % | % | ||||
Gender: | ||||||
males | 65 | 65.0 | 36 | 69.2 | 29 | 60.4 |
females | 35 | 35.0 | 16 | 30.8 | 19 | 39.6 |
Age (years): | ||||||
<60 | 67 | 67.0 | 38 | 73.1 | 29 | 60.4 |
>60 | 33 | 33.0 | 14 | 26.9 | 19 | 39.6 |
Tumor location: | ||||||
frontal lobe | 17 | 17.0 | 11 | 21.2 | 6 | 12.5 |
parietal lobe | 25 | 25.0 | 13 | 25.0 | 12 | 25.0 |
temporal lobe | 17 | 17.0 | 8 | 15.4 | 9 | 18.8 |
occipital lobe | 3 | 3.0 | 1 | 1.9 | 2 | 4.2 |
basal ganglion | 2 | 2.0 | 1 | 1.9 | 1 | 2.1 |
brain stem | 2 | 2.0 | 1 | 1.9 | 1 | 2.1 |
cerebellum | 5 | 5.0 | 1 | 1.9 | 4 | 8.3 |
multi-lobar | 29 | 29.0 | 16 | 30.8 | 13 | 27.1 |
Number of tumors: | ||||||
single | 96 | 96.0 | 49 | 94.2 | 47 | 97.9 |
multiple | 4 | 4.0 | 3 | 5.8 | 1 | 2.1 |
Type of surgery: | ||||||
total resection | 79 | 79.0 | 41 | 78.8 | 38 | 79.2 |
subtotal resection | 17 | 17.0 | 7 | 13.5 | 10 | 20.8 |
biopsy | 4 | 4.0 | 4 | 7.7 | 0 | 00.0 |
Treatment: | ||||||
RT+TMZ | 55 | 55.0 | 23 | 44.2 | 32 | 66.7 |
RT only | 45 | 45.0 | 29 | 55.8 | 16 | 33.3 |
Recurrence location: | ||||||
in the area | 31 | 31.0 | 11 | 21.2 | 20 | 41.7 |
out of the area | 37 | 37.0 | 21 | 40.4 | 16 | 33.3 |
in+out of area | 14 | 14.0 | 9 | 17.3 | 5 | 10.4 |
Second series treatment | 21 | 21.0 | 15 | 28.8 | 6 | 12.5 |
2 | 2.0 | 1 | 1.9 | 1 | 2.1 | |
4 | 4.0 | 1 | 1.9 | 3 | 6.3 | |
73 | 73.0 | 24.0 | 67.3 | 38 | 79.2 |
MGMT: O6-methyl guanine-methyl transferase; RT: radiotherapy; TMZ: temozolomide.
Multivariate analysis according to risk factors for overall survival.
All Patients | MGMT-Methylated | MGMT-Unmethylated | |||||||
---|---|---|---|---|---|---|---|---|---|
Parameters | OR | 95% CI | OR | 95% CI | OR | 95% CI | |||
Age | 0.493 | 0.313-0.777 | 0,0002 | 0.524 | 0.267-1.031 | 0.05 | 0.485 | 0.256-0.920 | 0.02 |
Gender (males/females) | 0.955 | 0.618-1.476 | 0.8 | 1.066 | 0.564-2.015 | 0.8 | 0.999 | 0.541-1.844 | 0.9 |
TMZ treatment included | 0.546 | 0.353-0.845 | 0.006 | 0.293 | 0.146-0.587 | <0.0001 | 0.930 | 0.496-1.745 | 0.8 |
MGMT-methylation | 0.609 | 0.395-0.939 | 0.02 | - | - | - | - | - | - |
MGMT: O6-methyl guanine-methyl transferase; TMZ: temozolomide.
Multivariate analysis according to risk factors for progression-free survival.
All Patients | MGMT-Methylated | MGMT-Unmethylated | |||||||
---|---|---|---|---|---|---|---|---|---|
Parameters | OR | 95% CI | OR | 95% CI | OR | 95% CI | |||
Age | 0.809 | 0.521-1.254 | 0.3 | 0.872 | 0.451-1.687 | 0.6 | 0.952 | 0.516-1.756 | 0.8 |
Gender (males/females) | 1.033 | 0.665-1.605 | 0.8 | 1.3 | 0.658-2.568 | 0.4 | 1.04 | 0.564-1.920 | 0.8 |
TMZ treatment included | 0.515 | 0.332-0.798 | 0.03 | 0.285 | 0.142-0.575 | <0.0001 | 0.90 | 0.472-1.714 | 0.7 |
MGMT-methylation | 0.662 | 0.430-1.019 | 0.05 | - | - | - | - | - | - |
MGMT: O6-methyl guanine-methyl transferase; TMZ: temozolomide.
Of the 52 patients (52.0%) with MGMT-methylation, 23 (44.2%) received RT+TMZ, and 29 (55.8%) received RT alone (Table 1). Median age of the patients was 51 years (19-69 years). In patients with MGMT promotor methylation, median OS and PFS were 12 and 6 months, respectively. Overall and progression-free survival among patients with methylation who received RT plus TMZ or RT only were compared using Kaplan-Meier curves. The mean OS for the group receiving RT+TMZ was 27 months (95% CI: 16.91-37.08)
Based on Cox regression model, addition of TMZ to RT was found to have prognostic significance in terms of OS and PFS in patients with MGMT-methylation (OR: 0.293, 95% CI: 0.146-0.587,
Of the 48 cases (48%) without MGMT-methylation; 32 (66.7%) received RT+TMZ and 16 (33.3%) received RT alone (Table 1). Median age of the cases was 56 (19-73 years). Median OS and PFS were 10 and 6 months, respectively. The OS and PFS were determined using Kaplan-Meier estimates in the patients receiving either RT+TMZ or RT alone who had no MGMT-methylation. Those receiving RT+TMZ or RT alone, had a median OS of 10 and 7 months, respectively (
A comparison of methylated vs. unmethylated MGMT patients receiving RT+TMZ in terms of OS and PFS showed a significant difference for the combination of methylated MGMT and RT+TMZ (
The effect of the methylation/unmethylation of CpG1, CpG2, CpG3 and CpG4 islands on OS and PFS were assessed by Kaplan-Meier analysis (log rank) in the RT+ TMZ group. Methylation/unmethylation of CpGl, CpG2, or CpG3 islands did not have a significant effect on OS (
Similarly, the effect of methylation status (methylated/unmethylated) of CpG1, CpG2, CpG3 and CpG4 islands on OS and PFS was evaluated by Kaplan-Meier analysis (log rank) in groups receiving RT alone. The effect of methylation of CpG1, CpG2, and CpG3 islands on PFS was significant (
In the evaluation of the MGMT methylated group with 52 cases, within group adjustments were made and the effect of methylation/unmethylation status of CpG1, CpG2, CpG3 and CpG4 islands on OS and PFS were estimated by Kaplan-Meier analysis (log rank). Only methylation in CpG3 had a significant effect (
Prognostic factors were assessed using the Cox regression test. Among patients receiving RT+CT (chemotherapy), the methylation of CpG1, CpG2, CpG3 and CpG4 islands did not have a significant impact on OS and PFS (
Multivariate analysis according to CpG island methylation status for overall survival.
Methylated MGMT Patients Receiving RT+TMZ | Methylated MGMT Patients Receiving RT | Methylated MGMT Patients | All Patients | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
OR | 95% CI | OR | 95% CI | OR | 95% CI | OR | 95% CI | |||||
CpG1 | 0.243 | 0.044-1.332 | 0.07 | 2.077 | 0.867-4972 | 0.09 | 1.552 | 0.701-3.437 | 0.2 | 1.552 | 0.701-3.437 | 0.2 |
CpG2 | 0.243 | 0.044-1.332 | 0.07 | 1.490 | 0.598-3.713 | 0.3 | 1.121 | 0.498-2.523 | 0.7 | 1.121 | 0.498-2.523 | 0.71 |
CpG3 | 0.533 | 0.115-2.477 | 0.4 | 1.818 | 0.788-4.193 | 0.1 | 1.479 | 0.692-3.158 | 0.3 | 1.479 | 0.692-3.158 | 0.3 |
CpG4 | - | - | - | 0.325 | 0.041-2.603 | 0.3 | 0.325 | 0.041-2.603 | 0.2 | 0.325 | 0.041-2.603 | 0.2 |
MGMT: O6-methyl guanine-methyl transferase; RT: radiotherapy; TMZ: temozolomide.
Multivariate analysis according to CpG island methylation status for progression-free survival.
Methylated MGMT Patients Receiving RT+TMZ | Methylated MGMT Patients Receiving RT | Methylated MGMT Patients | All Patients | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
OR | 95% CI | OR | 95% CI | OR | 95% CI | OR | 95% CI | |||||
CpG1 | 0.482 | 0.103-2.255 | 0.3 | 2.666 | 1.052-6.757 | 0.03 | 1.977 | 0.866-4.513 | 0.1 | 1.977 | 0.866-4.513 | 0.1 |
CpG2 | 0.482 | 0.103-2.255 | 0.3 | 2.838 | 0.958-8.404 | 0.05 | 1.931 | 0.779-4.791 | 0.1 | 1.931 | 0.779-4.791 | 0.1 |
CpG3 | 1.365 | 0.301-6.198 | 0.6 | 2.453 | 1.015-5.926 | 0.04 | 2.156 | 0.994-4.678 | 0.04 | 2.156 | 0.994-4.678 | 0.04 |
CpG4 | - | - | - | - | - | - | 1.099 | 0.147-8.212 | 0.9 | 1.099 | 0.147-8.212 | 0.9 |
MGMT: O6-methyl guanine-methyl transferase; RT: radiotherapy; TMZ: temozolomide.
Among the overall group of 100 patients, the methylation of CpG1, CpG2, CpG3 and CpG4 was found to have no significant effects on OS, and the methylation of CpG1, CpG2 and CpG4 islands had no significant effect on PFS. The methylation of CpG3, on the other hand, had a positive prognostic effect on PFS (p = 0.04) (Tables 4 and 5).
In the current study, OS and PFS for patients with methylation receiving RT+TMZ or RT alone was 27 and 9 months (OS) and 13 and 6 months (PFS), respectively, with a significant difference. On the other hand, no such differences could be detected for patients without methylation receiving RT+TMZ or RT alone as the corresponding figures were 10 and 7 months (OS) and 6 and 6 months (PFS), respectively.
Hegi
In the study by Dunn
In the current study involving 100 patients, MGMT-methylation and inclusion of TMZ in the treatment protocol had a prognostic significance for both OS and PFS, while age (<60 years) had prognostic importance only for OS, and gender had no impact on prognosis. Among subjects with MGMT-methylation, addition of TMZ in the treatment was an independent prognostic factor for improved OS and PFS, and age (<60 years) had a positive prognostic effect only for OS; gender had no prognostic significance. In cases without MGMT-methylation, only age (<60 years) was a good independent prognostic factor for OS, whereas gender and inclusion of TMZ in the treatment protocol were not significant prognostic factors.
In a study by Binabaj
Previously, Karayan-Tapon
In this study, among patients treated with RT+TMZ, methylation status of CpG1, CpG2, CpG3 and CpG4 islands had no effect on OS or PFS as examined by Kaplan-Meier analysis, while in the RT only group, methylation of CpG1, CpG2, CpG3 and CpG4 islands had significant effects on PFS. In the methylated group including 52 cases, methylation of the CpG3 island had a significant effect on PFS. With respect to prognostic factors, methylation of CpG1 and CpG3 islands had a positive prognostic effect on PFS. In the overall group of 100 patients, methylation of CpG3 island again was a good prognostic factor for PFS.
In the abovementioned study by Karayan-Tapon
In conclusion, MGMT promotor methylation represents an important biomarker influencing the response to RT and concomitant and adjuvant TMZ treatment as well as RT alone in GB patients. Currently, RT and concomitant TMZ followed by adjuvant TMZ, represents the standard approach until further treatments are defined. In patients without methylation, studies examining the molecular pathways to explore potential means for reduced resistance are warranted. Again, we believe further prospective studies with larger sample size are needed to improve clinical outcomes and to determine alternative therapeutic regimens and their clinical response rates.