|Year : 2017 | Volume
| Issue : 2 | Page : 45-51
Prognostic effects of O6-Methylguanine DNA methyltransferase promoter hypermethylation in high-grade glioma patients with carmustine wafer implants
Mao-Yu Chen1, Ping-Ching Pai2, Shih Ming Jung3, Chi-Cheng Chuang1, Chen-Nen Chang1, Kuo-Chen Wei1
1 Department of Surgery, Division of Neurosurgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan
2 Department of Radiation Oncology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
3 Department of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
|Date of Submission||11-Jan-2016|
|Date of Decision||07-Mar-2016|
|Date of Acceptance||07-Jun-2016|
|Date of Web Publication||18-Apr-2017|
Department of Surgery, Division of Neurosurgery, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kwei-Shan, Taoyuan
Source of Support: None, Conflict of Interest: None
Background: Local chemotherapy with carmustine (BCNU) wafer implantation has survival benefits for malignant glioma patients. However, available data regarding its association with O6-methylguanine-DNA-methyltransferase (MGMT) are scant.
Purpose: To evaluate whether MGMT hypermethylation has prognostic effects in malignant glioma patients with interstitial BCNU wafer implants.
Methods: From September 2004 to August 2007, 32 patients with malignant gliomas underwent surgical resection plus interstitial BCNU wafer implantation at our hospital.
Results and Conclusion: BCNU wafer implantation was performed in 18 patients with newly diagnosed gliomas and in 14 with recurrent gliomas. All patients had a Karnofsky performance status of ≥70. The median age was 51 years. At a median follow-up of 31 months, the 1- and 2-year overall survival (OS) rate was 43% and 22%, respectively. OS rates did not significantly differ between the newly diagnosed and recurrent patients. Gross total tumor resection was achieved in 19 (59%) patients, and MGMT hypermethylation was noted in 13 (41%) tumor specimens. Multivariate analysis demonstrated that patients with MGMT hypermethylation in their tumors and gross total tumor removal have more favorable survival rates (P = 0.03).
Keywords: Carmustine wafer, glioblastoma multiforme, gross total resection, high-grade gliomas, O6-methylguanine-DNA-methyltransferase
|How to cite this article:|
Chen MY, Pai PC, Jung SM, Chuang CC, Chang CN, Wei KC. Prognostic effects of O6-Methylguanine DNA methyltransferase promoter hypermethylation in high-grade glioma patients with carmustine wafer implants. Formos J Surg 2017;50:45-51
|How to cite this URL:|
Chen MY, Pai PC, Jung SM, Chuang CC, Chang CN, Wei KC. Prognostic effects of O6-Methylguanine DNA methyltransferase promoter hypermethylation in high-grade glioma patients with carmustine wafer implants. Formos J Surg [serial online] 2017 [cited 2019 Jul 23];50:45-51. Available from: http://www.e-fjs.org/text.asp?2017/50/2/45/204657
| Introduction|| |
Malignant gliomas are the most common primary malignant brain tumors, with glioblastoma multiforme (GBM) being the most predominant. Although there have been several advances in the diagnostic techniques and treatment modalities for GBM, the management of these fatal neoplasms remains challenging, with the median survival of GBM patients being only 8–20 months , and most patients dying within 2 years of diagnosis as a result of the inability to achieve local tumor control.
The current standard treatment for malignant glioma comprises surgery and postoperative involved-field fractionated radiotherapy. Systemic chemotherapy regimens have moderate efficacy in primary and recurrent malignant gliomas., Carmustine (BCNU) has been frequently used because of its activity in malignant gliomas. The addition of systemic BCNU-based chemotherapy to radiotherapy slightly but significantly prolongs high-grade glioma patient survival. Temozolomide (TMZ), an oral alkylating agent, has antitumor activity as a monotherapy or in combination with BCNU in primary and recurrent malignant gliomas., Since high-grade gliomas have a high propensity for local failure within 2 cm of the initial tumor, local treatments such as interstitial brachytherapy , and stereotactic radiosurgery,, for local radiation dose escalation or controlled-release polymer for local BCNU delivery , have been proposed to improve local tumor control.
Biodegradable polymer wafers are designed to release BCNU to the tumor resection cavity in a slow and sustained manner. After the wafers are placed on the surface of the surgical resection cavity, they can deliver high concentrations of BCNU, which can act directly on residual tumor cells and the glioma-infiltrated area of the normal brain, preventing systemic toxicity. Evidence from Phase III multicenter studies has suggested that the addition of BCNU wafers to radiotherapy significantly prolongs survival of patients with recurrent malignant gliomas or newly diagnosed GBM., By contrast, a randomized trial of GBM patients receiving systemic TMZ as a postoperative adjuvant treatment as well as a retrospective study on systemic BCNU promoters has reported that the methylation of the DNA repair gene; the methylation of the DNA repair gene by O 6-methylguanine-DNA-methyltransferase (MGMT) is associated with longer survival., However, few studies have reported regarding the association between the clinical effects and MGMTstatus in patients receiving local chemotherapy. We have been using BCNU wafers in patients with operable primary or recurrent high-grade gliomas for several years. Therefore, in this study, we evaluated whether MGMT promoter hypermethylation has prognostic effects in these patients.
| Methods|| |
Between September 2004 and August 2007, we enrolled 32 patients with newly diagnosed or recurrent malignant gliomas who were treated by one neurosurgeon using BCNU wafers as an adjuvant to surgery in our hospital. Eligible patients for this treatment were required to be aged over 18 years, have surgically resectable supratentorial high-grade glioma that did not cross the midline, have a Karnofsky performance status (KPS) of ≥70, and have adequate hematological, renal, and hepatic function. Informed consent was obtained before the procedure for local chemotherapy with BCNU wafers (Gliadel, 3.85% BCNU, Guilford Pharmaceuticals Inc., Baltimore, MA, USA).
After the histological analysis of intraoperative frozen sections supported the diagnosis of high-grade glioma, up to eight BCNU wafers were implanted in each resection cavity after maximal surgical tumor resection. The extent of tumor removal was recorded according to the surgical note and postoperative magnetic resonance imaging (MRI). Focal radiotherapy using a linear accelerator was delivered once daily at 1.8–2 Gy/fraction for 5 days/week, a total of 59.4–60 Gy for primary high-grade gliomas; furthermore, pre- and postoperative MR images were considered in the treatment fields. Concurrent TMZ was administrated orally at a dose of 75 mg/m 2/day, 7 days/week, during the entire radiation course for the primary gliomas. Adjuvant TMZ was administrated orally at a dose of 150–200 mg/m 2/day for 5 days of every 28-day cycle after radiotherapy, and up to 12 cycles were prescribed for primary and recurrent tumors.
The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the institute. Informed written consent was obtained from all patients prior to their enrollment in this study.
Laboratory procedures for methylation study
DNA extraction and purification from paraffin-embedded tissue blocks
Six 10 μm thick paraffin-embedded tissue sections were used, followed by deparaffinization with xylene and ethanol, treatment with lysis buffer (10 mM Tris-HCl [pH 8.3], 50 mM KC1, 2.5 mM MgCl2, and 0.45% Tween 20) and proteinase K (0.1 mg), incubation at 50°C, and finally centrifugation at 14,000 rpm to collect the DNA-containing supernatants; the lysate after purification was directly used for subsequent DNA methylation assays.
Methylation-specific polymerase chain reaction
Bisulfite modification of genomic DNA was performed using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA), according to manufacturer instructions: 2 μg of DNA was used in the bisulfite reaction; primers and hybridization probes, which bind specifically to bisulfite-converted sequences in the CpG islands in the MGMT promoter, were used. The MGMT forward primer was 5′-GCGTTTCGACGTTCGTAGGT-3′, the reverse primer was 5′-CACTCTTCCGAAAACGAAACG-3′, and the probe was FAM-5′-CGCAAACGATACGCACCGCGA-3′-TAMRA. Real-time polymerase chain reaction (PCR) was performed in a 20 μL reaction volume on 384-well plates on an ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Each PCR mixture contained 2 × TaqMan Universal PCR Master Mix, 250 nM probe, and 500 nM of each primer. Amplification and detection were performed using the following profile: One step at 50°C for 2 min, one step at 95°C for 10 min, 45 cycles at 95°C for 15 s, and 60°C for 1 min. β-actin (ACTB) was used as the internal reference in each amplification; in our assay, an amplification reaction was considered successful when the cycle threshold Ct for each bisulfite-converted sample was between 20 and 37 for ACTB. All samples were run in duplicate with standards, controls, and blanks on every plate, and samples with Ct value exceeding the assay range of sensitivity and reproducibility were repeated. Reactions using Sss I methylase-treated DNA were used to normalize differences in amplification efficiencies between MGMT and ACTB. The interpretation of methylated DNA was defined arbitrarily on the basis of whether the Ct value was <37 in MGMT or the percentage of methylated reference value was >4.
O 6-methylguanine-DNA-methyltransferase immunohistochemistry
Paraffinized tumor sections were immunostained using a commercially available anti-MGMT antibody, MT23.2 (Zymed Laboratories, Carlsbad, CA, USA). The sections were deparaffinized with xylene for 20 min and rehydrated in decreasing concentrations of ethanol. The primary antibodies were incubated for 30 min at room temperature with the anti-MGMT antibody (1:100 dilution). Antibody binding was observed using a Zymed polymer detection system (Zymed Laboratories), according to manufacturer protocol. Immunoreactivity was visualized with 3,3′-diaminobenzidine as the chromogen. All sections were counterstained with hematoxylin. One neuropathologist, blinded to clinical data and MGMT promoter methylation status, evaluated anti-MGMT immunohistochemistry in the tissue sections and scored MGMT expression as low and high expression (<10% and >10% MGMT immunoreactive tumor cells, respectively).
Patient follow-up and statistical analysis
Patients underwent physical and neurological examination as well as tumor bed imaging every 3 months for 2 years and every 4–6 months thereafter. Once the evidence of clinical deterioration was identified, brain MRI or computed tomography (if MRI was medically contraindicated or not feasible) was performed. The primary endpoint was overall survival (OS). OS was measured from the date of BCNU wafer implantation to the date of death or final follow-up visit; patients who were alive at the final follow-up visit were censored then.
For statistical analysis, Student's t-test and Chi-squared test were used to compare differences in continuous and categorical data, respectively. Kaplan–Meier production limit estimation method was used to obtain the survival rates of different prognostic factors. A log-rank test was used to detect the differences among them. A Cox's proportional hazards model was used for multivariate analysis. P < 0.05 was considered statistically significant.
| Results|| |
The median patient age was 51 years (range, 24–84 years). Sixteen patients (50%) were men. Eighteen patients (56%) had newly diagnosed malignant gliomas, of whom 15 had GBM, 2 had anaplastic oligoastrocytomas, and 1 had anaplastic oligodendroglioma; the remaining 14 patients had recurrent high-grade tumors (13 had GBM and 1 had anaplastic oligoastrocytoma1). Of the recurrent patients, the median time to BCNU wafer treatment was 16 months from the original surgery (range, 2–94 months). The median follow-up time for surviving patients was 31 months (range, 16–48 months).
Of the patients with newly diagnosed malignant gliomas, initial management comprised gross total tumor removal and subtotal tumor resection in 11 and 7 patients, respectively. Local radiotherapy was administered to all patients; the median dose was 60 Gy (range, 55.8–61.2 Gy). Four patients received concomitant chemoradiotherapy and eight received adjuvant TMZ for 2–12 cycles. Of the recurrent patients, eight and six patients underwent gross total tumor removal and subtotal tumor resection, respectively. Systemic TMZ was used as a salvage treatment in 13 patients; only one patient could not be administered TMZ because of mortality soon after surgery [Table 1].
Two of the 32 patients died within 1 month of BCNU implantation – one because of bilateral pneumonia and the other because of sudden onset of cardiac arrest 4 days after implantation; both patients were included in our survival analysis. Twenty-five patients (78%) died, whereas seven remained alive at the time of the analysis. The median OS time for all patients was 9.8 months, with 1- and 2-year OS rates of 44% and 23%, respectively [Figure 1]. The OS did not differ significantly between BCNU wafer-implanted patients undergoing treatment for primary and recurrent tumors (median OS time: 9.8 vs. 9 months; 1-year OS rate: 39% vs. 43%; all P = 0.24).
|Figure 1: Kaplan–Meier estimates of progression-free survival and overall survival of all 32 patients with high-grade gliomas|
Click here to view
MGMT promoter hypermethylation was noted in 13 (41%) tumor specimens; 39% and 43% hypermethylation was noted in primary and recurrent tumors, respectively. MGMT immunoreactivity was low in 12 (38%) tumor specimens, and low MGMTexpression of 28% and 50% was noted in primary and recurrent tumors, respectively. The association between MGMT promoter methylation status and MGMT expression was highly correlated (P = 0.001): 9 of 13 hypermethylated MGMT samples had low MGMT immunoreactivity and 16 of 19 unmethylated MGMT samples had high MGMT immunoreactivity. Both MGMT promoter methylation status and MGMT expression did not significantly affect the survival of the entire cohort (P = 0.15 and P = 0.96, respectively). Nevertheless, MGMT hypermethylation demonstrated a significant survival benefit in patients with primary high-grade gliomas [P = 0.03; [Figure 2]a and [Figure 2]b: The 1-year OS rate was 71% in patients with MGMT methylation, but only 18% in patients with unmethylated MGMT. The effect of MGMT hypermethylation on the survival of patients with recurrent tumors was nonsignificant (P = 0.48).
|Figure 2: Kaplan–Meier estimates of overall survival, stratified by O6-methylguanine-DNA-methyltransferase promoter methylation status in the entire cohort (a) and only in those with primary tumors (b)|
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Gross total tumor removal was performed in 19 of 32 patients, but a nonsignificant survival difference was noted in the entire cohort, regardless of whether the tumor was primary or recurrent (median survival time, 9.8 months vs. 9.0 months, respectively, P = 0.44). However, considering both the surgical extent and MGMT promoter methylation status, of the patients with totally resected tumors, MGMT hypermethylation patients had a more favorable OS than those with unmethylated MGMT did (P = 0.03); however, of the patients with subtotally resected tumors, MGMT methylation status did not have a prognostic effect on OS (P = 0.81). Unmethylated MGMT or subtotally resected tumors led to poor OS [P = 0.05; [Figure 3]. We compared patients aged <50 and ≥50 years, including male and female patients as well as patients with KPS of >90 and ≤90 but observed no significant difference in their OS (P = 0.16, P = 0.61, and P = 0.21, respectively). Multivariate analysis using a Cox regression model including the aforementioned OS-affecting factors revealed the following: gross total tumor removal and MGMT methylation versus subtotal tumor resection or unmethylated MGMT (P = 0.03), age (P = 0.06), performance status (P = 0.45), recurrence (P = 0.96), and sex (P = 0.11).
|Figure 3: Kaplan–Meier estimates of overall survival, stratified by gross total tumor removal versus subtotal resection and hypermethylated O6-methylguanine-DNA-methyltransferase (M) versus unmethylated O6-methylguanine-DNA-methyltransferase (U)|
Click here to view
| Discussion|| |
The treatment outcomes of patients with malignant gliomas remain poor although significant efforts have been made with regard to the applicable therapeutic approaches. Studies have shown that adjuvant radiation therapy for malignant gliomas results in increased tumor control, thus prolonging patient survival; however, the median survival rate of GBM patients is approximately 12 months, with nearly all patients dying because of local disease progression. Nitrosoureas, particularly BCNU and TMZ, are the agents most frequently used in systemic chemotherapy. Before the introduction of TMZ, nitrosourea-based adjuvant systemic chemotherapy could moderately improve survival. In a meta-analysis conducted by the Glioma Meta-analysis Trialist Group, which collected 12 trials' information from 3004 patients with high-grade gliomas and explored the effects of systemic chemotherapy, the 2-year survival rate for GBM patients was increased from 9% to 13%. Furthermore, in the EORTC and NCICs randomized Phase III GBM trial, Stupp et al. compared the effects of concurrent administration of TMZ (as an adjuvant) with radiotherapy on the survival of GBM patients with those of radiotherapy alone; the 2-year survival rate was 10% with radiotherapy alone and significantly increased to 26% in patients undergoing radiotherapy plus TMZ. In addition to systemic chemotherapy, local chemotherapy has survival benefits in high-grade glioma patients. The efficacy of BCNU polymers in patients with recurrent high-grade gliomas was demonstrated in a placebo-controlled trial. This study showed that median survival time was significantly prolonged from 23 weeks in the placebo wafer group to 31 weeks in the BCNU wafer group. Subsequently, similar survival benefits were observed in patients with newly diagnosed high-grade gliomas: one Phase III trial enrolling 240 newly diagnosed malignant glioma patients demonstrated a 27% reduction in the mortality risk in the BCNU wafer group compared with the placebo wafer group; the survival rates of the BCNU wafer and placebo groups were 16% and 8%, respectively, and at 2 years, the median survival time was 60 and 50 weeks, respectively. In the current study, all patients received BCNU wafers, and the 2-year OS rates were 23% and 7% in the primary and recurrent high-grade glioma patients, respectively; this result is comparable with those of previous randomized studies.
In high-grade glioma patients, the association of the extent of tumor removal with patient survival has been suggested; in one series comprising 416 consecutive GBM patients who underwent tumor resection between 1993 and 1999, resection of 98% or more of the T1-weighted MRI-enhanced tumor volume was associated with survival advantage: the median survival time was 13 months compared with the 8.8 months for patients with <98% resected tumor volume. Another single institution series that included 1215 primary and recurrent malignant brain astrocytoma patients who underwent surgery from 1996 to 2006 reported improved survival with increasing extent of tumor resection; GBM patients with gross total resection had higher median survival times (13 and 11 months for primary and recurrent GBM, respectively) than those with near-total resection (11 and 9 months for primary and recurrent GBM, respectively) or subtotal resection (8 and 5 months for primary and recurrent GBM, respectively). Our patients with gross total tumor resection exhibited a nonsignificant increase in survival compared with patients with subtotal tumor resection (9.8 months vs. 9 months); this may be attributable to the small sample size and the smearing effect by patients with near-total tumor resection.
Recently, the correlation of the methylation status of MGMT promoter with the outcome of malignant gliomas has been increasingly investigated. MGMT is a DNA repair enzyme that can reverse O 6-methylation caused by alkylating agents, such as nitrosoureas and TMZ, and then mitigate cytotoxicity to the tumor. MGMT inactivation, assessed using MGMT promoter methylation, or low MGMT expression measured through immunohistochemistry, has been shown to be related to favorable outcome in patients with high-grade gliomas.,,, In a randomized trial, Hegi et al. reported that 44.7% of their tumor samples contained hypermethylated MGMT promoter and noted a significant difference in OS favoring patients with MGMT hypermethylation; the median OS time of patients with hypermethylation was 18.2 months compared with 12.2 months of those without hypermethylation. A similar correlation was observed in primary high-grade glioma patients treated by systemic BCNU, in which 40% of the samples demonstrated MGMT hypermethylation, and the median time to disease progression was 21 months for methylated gliomas and 8 months for unmethylated gliomas. In addition to the survival benefit of MGMT promoter hypermethylation in patients receiving systemic chemotherapy, Smith et al. reported a similar benefit in 27 primary GBM patients who underwent gross total tumor resection and Gliadel wafer implantation, followed by 12-Gy gamma knife radiosurgery and 60-Gy fractionated radiotherapy as the initial treatment; the median survival time for all patients was 50 weeks and the 2-year survival rate was 22%, and the survival of patients with and without MGMT promoter hypermethylation was 103 and 45 weeks, respectively. Similarly, in our patients who had primary high-grade gliomas and were treated using similar modalities (except for radiosurgery boost), the median OS time was 9.8 months and the 2-year OS rate was 23%. Patients with MGMT promoter hypermethylation had a 1-year OS rate of 71%, which is a highly favorable OS rate compared with 18% for those with unmethylated MGMT.
The limitations of this study are as follows: in Taiwan, TMZ was not covered as a first-line chemotherapeutic agent in general health insurance until 2008; thus, the treatment protocol considerably differed among enrolled patients. In addition, the patient number was insufficient for the analysis of independent variables to achieve statistical significance and power.
| Conclusion|| |
In this small series of patients with primary or recurrent high-grade gliomas and favorable prognosis, OS remained poor. In addition, intraoperative BCNU wafer implantation aided the treatment of only the patients that had high-grade gliomas with totally resected tumors and MGMT promoter hypermethylation. The additional benefits of adjuvant systemic chemotherapy including nitrosoureas (e.g., TMZ) in patients with tumors containing MGMT promoter hypermethylation but with gross residual tumor warrant further study.
Financial support and sponsorship
This study was sponsored and funded by National Health Research Institutes, Taiwan (Contract Grant No.: NHRI-ex95-9507NI).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]