High MGMT Expression Research Articles

MGMT Promoter Methylation is a Poor Predictor of Gene Expression in Low Grade Gliomas

Radiation therapy with adjuvant temozolomide (TMZ) is the standard of care for glioblastoma multiforme (GBM). However, management of adult low grade gliomas (LGG) remains more complex. Expression of O6-methylyguanine-DNA-methyltransferase (MGMT) has been demonstrated to confer treatment resistance, while hypermethylation of the promoter is commonly used as a prognostic biomarker in GBM. In contrast, MGMT promoter methylation does not appear to improve progression-free survival in LGG. Here, we report an unexpected discordant relationship between MGMT promoter methylation and mRNA expression in LGG. This finding indicates that assays for MGMT promoter methylation may not properly identify treatment sensitive LGG. Grade II and III gliomas cases with clinical, methylation (450k infinium methylation beadchip) and gene expression (RNAseq) data were obtained from the TCGA-LGG dataset. Gene expression cutoffs were stratified by Youden index. The gplots R package was used to perform non-hierarchical cluster analysis using MGMT CpG sites for stratification. Differential methylation between samples was determined using t-tests with Bonferroni correction. In addition, differential methylation by two common MGMT promoter assays, DMR1 and MS-MLPA, was estimated using annotated CpG sites. Clinical variables were compared using t-tests for continuous variables and Fisher’s exact for categorical variables. Of the 516 TCGA-LGG cases eligible for analysis, 454 had complete clinical, methylation and sequencing data. Thirty-eight (8.4%) had high gene expression of MGMT. Non-hierarchical clustering analysis identified two distinct groups; 53% had hypermethylation of the MGMT promoter (Δ20% in methylation, q < 0.01), and by DMR1 (Δ13%, q = 0.02) and MS-MLPA sites (Δ31%, q < 0.01). There were no significant differences in average methylation across the entire MGMT gene (137 sites, Δ04%, q = 0.61) or the whole genome (38808 sites, Δ03%, q = 0.99). LGG cases with high MGMT expression and promoter methylation were four times more likely to have an IDH mutation (P < 0.01). Our analysis reveals an unexpected discordant relationship between MGMT promoter methylation and gene expression. Direct MGMT mRNA quantification may provide a better method for evaluating sensitivity to radiation and adjuvant TMZ in LGG.

DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma.

Wingless/β-catenin signaling as a modulator of chemoresistance in cancer

ABSTRACTO6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme involved in chemoresistance. We have shown that MGMT abundance is regulated by canonical Wingless (Wnt) signaling and that inhibition of Wnt signaling restores chemosensitivity in preclinical cancer models. These findings have direct therapeutic implications for the treatment of cancers with high MGMT expression.

Immunohistochemical analysis of O6-methylguanine-DNA methyltransferase (MGMT) protein expression as prognostic marker in glioblastoma patients treated with radiation therapy with concomitant and adjuvant Temozolomide

BackgroundO6-methylguanine-DNA methyltransferase (MGMT) protein expression using immunohistochemical analysis was proposed as a prognostic marker for patients with newly diagnosed glioblastoma (GBM) treated with radiation therapy with concurrent and adjuvant Temozolomide (TMZ). MethodsFrom April 2012 to October 2014, 73 patients with newly diagnosed GBM, MGMT protein expression were analyzed in formalin-fixed, paraffin-embedded tumor specimens. Patients received the radiation therapy plus concomitant and adjuvant TMZ chemotherapy. ResultsFor the whole cohort, the median overall survival (OS) was 15months, and the progression-free survival was 10months. Patients who had low MGMT protein expression (⩽15%) had a significantly improved OS and PFS compared with patients who had high MGMT expression (17.0months vs 14months; P value .006) and (15.0months vs 10months; P value .016) respectively. The age and extent of tumor resection were the strongest clinical predictors of outcome. In multivariate Cox models MGMT protein expression, extent of tumor resection and age were identified as independent prognostic factors. ConclusionsMGMT expression was identified as positive prognostic factor in patients with newly diagnosed glioblastoma who underwent surgical resection followed by adjuvant radiotherapy and concomitant oral TMZ chemotherapy (the Stupp protocol).

RTRB-01UTILIZING THE GENOMICALLY ADJUSTED DOSE (GAD) TO IDENTIFY PATIENTS FOR ADJUVANT RADIATION DOSE ESCALATION IN GLIOBLASTOMA

BACKROUND: We have previously validated a multi-gene model of tumor radiosensitivity (RSI) in glioblastoma (GBM). We propose a model to personalize radiotherapy dose utilizing the linear quadratic model. METHODS: As RSI models the surviving fraction at 2 Gy (SF2), we are able to derive a patient specific alpha. These terms were used in the linear quadratic model to calculate a genomically adjusted dose (GAD) to model radiation effect. Clinical and array-based gene expression (Affymetrix HT Human Genome U133 Array Plate Set) level 2 data were downloaded from The Cancer Genome Atlas (TCGA). Inclusion criteria were receipt of radiation and temozolomide, expression data to determine RSI and MGMT level, and dose and fractionation. RESULTS: Median follow-up in this cohort was 11.4 months (range: 1.5-91 months). In patients with low MGMT expression (dichotomized at the median), age (p = 0.03) and ECOG performance status (p < 0.001) were found to be predictors of overall survival (OS) while GAD (p = 0.98) was not. When restricting the analysis to patients with high MGMT expression (n = 98), GAD was found to be a predictor of OS on univariate (p = 0.039) and multivariate analyses (p = 0.01) when assessed by quartiles. Kaplan-Meier (KM) rates of OS at 12 and 24 months were 77.1% and 32.1% in quartile 4 (GAD ≥ 17.6) compared to 52.4% and 17.5% in quartile 1 (GAD <8.5). We then modeled GAD for an escalated dose to 70 Gy, 80 Gy, and 90 Gy, and found an additional 25, 29, and 38 patients, respectively, in the MGMT high cohort who could achieve GAD ≥ 17.6 (quartile 4) with dose escalation. CONCLUSIONS: Modeling dose escalation with GAD, we identified a select population of patients with high MGMT expression whom we hypothesize may benefit from genomically guided increased dose in the adjuvant setting. This population may represent a cohort for future clinical trial enrollment.

Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression.

BackgroundThe use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.MethodsFour nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.ResultsOur results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.ConclusionsThese results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.

The radiosensitivity index predicts for overall survival in glioblastoma.

We have previously developed a multigene expression model of tumor radiosensitivity (RSI) with clinical validation in multiple cohorts and disease sites. We hypothesized RSI would identify glioblastoma patients who would respond to radiation and predict treatment outcomes. Clinical and array based gene expression (Affymetrix HT Human Genome U133 Array Plate Set) level 2 data was downloaded from the cancer genome atlas (TCGA). A total of 270 patients were identified for the analysis: 214 who underwent radiotherapy and temozolomide and 56 who did not undergo radiotherapy. Median follow-up for the entire cohort was 9.1 months (range: 0.04-92.2 months). Patients who did not receive radiotherapy were more likely to be older (p < 0.001) and of poorer performance status (p < 0.001). On multivariate analysis, RSI is an independent predictor of OS (HR = 1.64, 95% CI 1.08-2.5; p = 0.02). Furthermore, on subset analysis, radiosensitive patients had significantly improved OS in the patients with high MGMT expression (unmethylated MGMT), 1 year OS 84.1% vs. 53.7% (p = 0.005). This observation held on MVA (HR = 1.94, 95% CI 1.19-3.31; p = 0.008), suggesting that RT has a larger therapeutic impact in these patients. In conclusion, RSI predicts for OS in glioblastoma. These data further confirm the value of RSI as a disease-site independent biomarker.

The expression profile of Dopamine D2 receptor, MGMT and VEGF in different histological subtypes of pituitary adenomas: a study of 197 cases and indications for the medical therapy.

BackgroundTo study the expression of D2R, MGMT and VEGF for clinical significance in pituitary adenomas, and to predict the potential curative medical therapy of dopamine agonists, temozolomide and bevacizumab on pituitary adenomas.MethodsImmunohistochemistry and western blot were performed to detect the expression of expression of D2R, MGMT and VEGF in pituitary adenoma tissue samples. The ratio of high expression of D2R, MGMT or VEGF in different subtypes of PA was compared by the use of chi-squared tests. The relationships between D2R, MGMT and VEGF expression were assessed by the Spearman rank correlation test. The association between their expression and clinical parameters was analyzed using a chi-squared test, or Fisher's exact probability test when appropriate.ResultsThe data showed that in 197 different histological subtypes of pituitary adenomas (PAs), 64.9% of them were D2R high expression, 86.3% were MGMT low expression and 58.9% were VEGF high expression. D2R high expression existed more frequently in PRL- and GH- secreting PAs. MGMT low expression existed in all PA subtypes. VEGF high expression existed more frequently in PRL, ACTH, FSH secreting and non-functioning PAs. The data of western blot also support the results. Spearman's rank correlation analysis showed that expression of MGMT was positively associated with D2R (r = 0.154, P = 0.031) and VEGF (r = 0.161, P = 0.024) in PAs, but no correlation was showed between D2R and VEGF expression (r = −0.025, P = 0.725 > 0.05). The association between their expression and clinical parameters was analyzed using a chi-squared test, or Fisher's exact probability test when appropriate, but the result showed no significant association.ConclusionsPRL-and GH-secreting PAs exist high expression of D2R, responding to dopamine agonists; Most PAs exist low expression of MGMT and high expression of VEGF, TMZ or bevacizumab treatment could be applied under the premise of indications.

The expression profile of Dopamine D2 receptor, MGMT and VEGF in different histological subtypes of pituitary adenomas: a study of 197 cases and indications for the medical therapy

To study the expression of D2R, MGMT and VEGF for clinical significance in pituitary adenomas, and to predict the potential curative medical therapy of dopamine agonists, temozolomide and bevacizumab on pituitary adenomas. Immunohistochemistry and western blot were performed to detect the expression of expression of D2R, MGMT and VEGF in pituitary adenoma tissue samples. The ratio of high expression of D2R, MGMT or VEGF in different subtypes of PA was compared by the use of chi-squared tests. The relationships between D2R, MGMT and VEGF expression were assessed by the Spearman rank correlation test. The association between their expression and clinical parameters was analyzed using a chi-squared test, or Fisher's exact probability test when appropriate. The data showed that in 197 different histological subtypes of pituitary adenomas (PAs), 64.9% of them were D2R high expression, 86.3% were MGMT low expression and 58.9% were VEGF high expression. D2R high expression existed more frequently in PRL- and GH- secreting PAs. MGMT low expression existed in all PA subtypes. VEGF high expression existed more frequently in PRL, ACTH, FSH secreting and non-functioning PAs. The data of western blot also support the results. Spearman's rank correlation analysis showed that expression of MGMT was positively associated with D2R (r = 0.154, P = 0.031) and VEGF (r = 0.161, P = 0.024) in PAs, but no correlation was showed between D2R and VEGF expression (r = −0.025, P = 0.725 > 0.05). The association between their expression and clinical parameters was analyzed using a chi-squared test, or Fisher's exact probability test when appropriate, but the result showed no significant association. PRL-and GH-secreting PAs exist high expression of D2R, responding to dopamine agonists; Most PAs exist low expression of MGMT and high expression of VEGF, TMZ or bevacizumab treatment could be applied under the premise of indications.

Optimizing Glioblastoma Temozolomide Chemotherapy Employing Lentiviral-based Anti-MGMT shRNA Technology

Despite treatments combining surgery, radiation-, and chemotherapy, patients affected by glioblastoma (GBM) have a limited prognosis. Addition of temozolomide (TMZ) to radiation therapy is the standard therapy in clinical application, but effectiveness of TMZ is limited by the tumor's overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). The goal of this study was to use the highly specific and efficient RNA interference (RNAi) pathway to modulate MGMT expression to increase TMZ efficiency in chemotherapy resistant GBM. Using lentiviral-based anti-MGMT small hairpin RNA (shRNA) technology we observed a specific inhibition of the MGMT expression in GBM cell lines as well as in subcutaneous tumors. Tumor growth inhibition was observed following TMZ treatment of xenografts with low MGMT expression in contrast to xenografts with high MGMT expression. Bioluminescence imaging (BLI) measurements indicated that luciferase and shRNA-expressing lentiviruses were able to efficiently transduce the GBM xenografts in vivo. Treatment combining injection of a lentivirus expressing an anti-MGMT shRNA and TMZ induced a reduction of the size of the tumors, in contrast with treatment combining the lentivirus expressing the control shRNA and TMZ. Our data suggest that anti-MGMT shRNA therapy could be used in combination with TMZ chemotherapy in order to improve the treatment of resistant GBM.

MGMT expression and pituitary tumours: relationship to tumour biology

Over the past half decade, temozolomide, an oral akylating chemotherapeutic agent, has been shown to have significant activity in the management of aggressive pituitary tumours. The expression of 06-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is an important predictor of response to therapy. Low MGMT expression has been reported with a higher frequency amongst more aggressive pituitary tumours, suggesting MGMT may play a role in pituitary tumour progression. In this study, we performed a microarray analysis to determine whether there was a distinct gene expression profile between tumours with low MGMT and high MGMT expression. Overall, 1,403 differentially expressed genes were identified with raw p values less than 0.05. Gene set enrichment analysis (GSEA) revealed significant differences in the gene expression profile between high and low MGMT expressing pituitary tumours. High MGMT expressing pituitary tumours were found to have upregulation of components of the FGFR family and downstream signaling cascades such as PI3K/Akt and MAPK pathways. Activation of genes involved in the DNA damage response and DNA repair pathways, as well as genes involved in transcription, were identified in pituitary tumours with low MGMT expression. These results form the basis of our proposed model to describe the role of MGMT in pituitary tumorigenesis.

Effect of VAL-083, a novel N7 alkylating agent, on growth of temolozomide-resistant primary adult glioblastoma multiforme (GBM) cells providing a new potential treatment option for GBM.

e13123 Background: Glioblastoma (GBM) remains one of the most difficult tumors to treat first because many new agents fail to cross the blood brain barrier (BBB), and second due to intrinsic drug resistance. Temozolomide (TMZ) is a front-line therapy for the treatment of GBM; however, it is often ineffective due to drug inactivation by O6-methylguanine-DNA methyltransferase (MGMT). Cancer stem cells (CSC) are a subpopulation of the tumor that resist therapy and give rise to relapse. Here we describe VAL-083, a novel alkylating agent that creates N7 methylation on DNA, and readily crosses the BBB. VAL-083 is currently undergoing human clinical trials in the USA in refractory GBM patients. We previously described how VAL-083 can overcome resistance associated with MGMT in cell lines, and targets brain tumor CSCs. This demonstrates that VAL-083 has the potential to surpass the standard-of-care. Methods: To provide further preclinical support for VAL-083 we examined the effect of VAL-083 on primary adult GBM cells in culture, isolated fresh following surgical resection, in cases where clinical activity of TMZ was known to be limited and high expression of MGMT had been observed. Results: VAL-083 (5uM) inhibited cell growth in these primary adult GBMs that did not show significant sensitivity to TMZ in vitro. VAL-083 also inhibited the growth of CSCs by 100% in neurosphere growth assays. Conclusions: In summary, VAL-083 demonstrates in vitro efficacy against primary adult GBM cells where TMZ has limited activity thereby further supporting the potential of VAL-083 to surpas the standard-of-care.

MGMT CpG island is invariably methylated in adult astrocytic and oligodendroglial tumors with IDH1 or IDH2 mutations

We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.

Pituitary Carcinoma

Pituitary tumors are amongst the most common intracranial neoplasms and are generally benign. However, some pituitary tumors exhibit clinically aggressive behavior that is characterized by tumor recurrence and continued progression despite repeated treatments with conventional surgical, radiation and medical therapies. More recently, temozolomide, a second generation oral alkylating agent, has shown therapeutic promise for aggressive pituitary adenomas and carcinomas with favorable clinical and radiographic responses. Temozolomide causes DNA damage by methylation of the O6 position of guanine, which results in potent cytotoxic DNA adducts and consequently, tumor cell apoptosis. The degree of MGMT expression appears to be inversely related to therapeutic responsiveness to temozolomide with a significant number of temozolomide-sensitive pituitary tumors exhibiting low MGMT expression. The presence of high MGMT expression appears to mitigate the effectiveness of temozolomide and this has been used as a marker in several studies to predict the efficacy of temozolomide. Recent evidence also suggests that mutations in mismatch repair proteins such as MSH6 could render pituitary tumors resistant to temozolomide. In this article, the authors review the development of temozolomide, its biochemistry and interaction with O6-methylguanine-DNA methyltransferase (MGMT), its role in adjuvant treatment of aggressive pituitary neoplasms, and future works that could influence the efficacy of temozolomide therapy.

Aggressive pituitary tumours: the role of temozolomide and the assessment of MGMT status

Aggressive pituitary tumours are associated with substantial morbidity and mortality. Treatment options are often limited, and chemotherapy has been reserved as salvage therapy although historically results have often been disappointing. However, temozolomide, an oral alkylating agent, has recently demonstrated significant activity against these tumours. A DNA repair protein, 06-methylguanine-DNA methyltransferase (MGMT) has been suggested as a biomarker to predict response to temozolomide in pituitary tumours. This paper will review the current literature on temozolomide and pituitary tumours and discuss the recent controversy surrounding the value of determining the MGMT status in this tumour group. A PubMed search was performed to retrieve articles, using the terms 'pituitary tumour' and 'temozolomide'. Overall, 24/40 (60%) of the published cases demonstrated a response to temozolomide therapy. The highest response rates were seen amongst prolactinomas (73%) and ACTH-secreting tumours (60%), whilst nonfunctioning pituitary tumours exhibit lower response rates (40%). Responsivity is typically evident in the first 3 months of therapy and may be dramatic and sustained. Low MGMT expression, as determined by immunohistochemistry, is associated with a high response rate (76%), whilst high MGMT expression has not been associated with responses. MGMT promoter methylation does not correlate with temozolomide response. Temozolomide is the first chemotherapeutic agent to show substantial response rates in aggressive pituitary tumours. MGMT immunohistochemistry, but not MGMT methylation analysis, shows promise as a predictive tool. Prospective clinical trials are now necessary to more accurately determine the efficacy of this agent in this patient group.

Expression of MGMT and its clinopathological significance in thyroid carcinoma.

To study the expression of O(6)-methylguanine-DNA methyltransferase (MGMT) and its clinicopathological significance in thyroid cancer. Immunohistochemistry was used to determine the expression of MGMT in 61 thyroid cancer tissues, 21 thyroid adenomas, 15 Hashimoto's thyroiditis, 8 nodular goiter, and 12 peri-tumor tissues. There was statistic difference in the expression of MGMT between the normal tissues and thyroid cancers (P<0.05). Expression of MGMT increased from the normal tissue (16.67%, 10/12), nodular goiter (25.00%, 2/8), Hashimoto's thyroiditis (60.00%, 9/15), and thyroid adenoma (52.38%, 11/21)to thyroid cancer (60.66%, 38/61). Expression of MGMT in papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) had significant difference (P<0.05), and the expression level of MGMT decreased with the malignancy of thyroid cancer, such as in PTC (72.22%, 26/36), and FTC (50.00%, 8/16). There was no statistic difference in MGMT expression in sex, age, and nationality (P>0.05). High expression of MGMT might be related to the malignancy of thyroid cancer, which may be one of the diagnosis indexes for thyroid cancer. It will be a common clinical index in diagnosing thyroid cancer since there is no difference in MGMT expression among sexes, ages, and nationalities.

Can high-dose fotemustine reverse MGMT resistance in glioblastoma multiforme?

Glioblastoma multiforme (GBM), the highest grade malignant glioma, is associated with a grim prognosis-median overall survival is in the range 12-15months, despite optimum treatment. Surgery to the maximum possible extent, external beam radiotherapy, and systemic temozolomide chemotherapy are current standard treatments for newly diagnosed GBM, with intracerebral delivery of carmustine wafers (Gliadel). Unfortunately, the effectiveness of chemotherapy can be hampered by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT), which confers resistance both to temozolomide and nitrosoureas, for example fotemustine and carmustine. MGMT activity can be measured by PCR and immunohistochemistry, with the former being the current validated technique. High-dosechemotherapycan deplete MGMT levels in GBM cells and has proved feasible in various trials on temozolomide, in both newly diagnosed and recurrent GBM. We here report the unique case of a GBM patient, with high MGMT expression by immunohistochemistry, who underwent an experimental, high-dose fotemustine schedule after surgery and radiotherapy. Although treatment caused two episodes of grade 3-4 thrombocytopenia, a complete response and survival of more than three years were achieved, with a 30% increase in dose intensity compared with the standard fotemustine schedule.

Abstract 4354: Celecoxib reduces MGMT expression and potentiates the effect of temozolomide in childhood medulloblastoma

Abstract Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects DNA from the biological effects of alkylating agents. MGMT has been implicated in resistance to alkylating drugs such as temozolomide used in the clinic against brain tumors. Temozolomide has been shown to be ineffective in tumors expressing MGMT and in medulloblastoma high expression of MGMT is correlated with poor prognosis. Therefore, the demand to find agents that deplete MGMT is warranted. Methods: medulloblastoma cell cytotoxicity of chemotherapeutic drugs in combination with Celecoxib was examined in MGMT positive cell lines using fluorometric microculture cytotoxicity assay. In vivo, nude NMRI mice carrying MGMT positive D283 MED xenografts were treated with celecoxib in combination with temozolomide. For mechanistic studies, western blot and quantitative PCR were used. Results: the clinically available COX-2 inhibitor Celecoxib induced a synergistic or an additive cytotoxic effect in combination with Temozolomide, Cyclophosphamide, Doxorubicin, Irinotecan, Vincristine, Rapamycin or CCI-779 in vitro. In vivo, Celecoxib synergistically potentiates the effect of Temozolomide through downregulation of MGMT expression. Conclusions: this study shows that Celecoxib reduces the DNA repair capacity of MGMT in medulloblastoma and increases the susceptibility to temozolomide. These findings support the use of pharmacological MGMT depletion as a rational approach for intensification of temozolomide therapy in the treatment of medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4354.

Phase II trial of temozolomide (TMZ) plus irinotecan (CPT-11) in adults with newly diagnosed glioblastoma multiforme before radiotherapy

This phase II trial evaluated efficacy and safety of temozolomide (TMZ) in combination with irinotecan (CPT-11) before radiotherapy in patients with newly diagnosed glioblastoma multiforme (GBM). Prior to radiotherapy, patients were treated with a maximum of three 6-week cycles of TMZ and CPT-11. Patients received TMZ at a dose of 200 mg/m(2)/day on days 1-5 and CPT-11 on days 1, 8, 22, and 29, with a dose adjustment for enzyme-inducing antiepileptic drug use. The primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS), overall survival (OS), safety, and tumor O(6)-methylguanine-DNA methyltransferase (MGMT) expression. Of the 42 patients treated, 8 (19%) patients achieved a partial response. Median PFS and median OS were 3.1 and 13.8 months, respectively. Grade 3 or 4 AEs were documented in 36% of patients, most of which were hematologic (29%). Twenty-four percent of patients had grade 3 or 4 non-hematologic AEs, with gastrointestinal AEs being the most common (12%) Two patients died, one of intracranial hemorrhage and one of treatment-related renal failure. Low MGMT expression, compared with high MGMT expression, showed no significant difference in ORR (25 vs. 8%), median PFS (14 vs. 5 months) or OS (21 vs. 15 months). Although TMZ plus CPT-11 is at least comparable in efficacy to TMZ alone, this combination appears more toxic and poorly tolerated. The lack of correlation of activity with MGMT expression is intriguing, but needs further evaluation in subsequent trials.

IFN-β sensitizes neuroblastoma to the antitumor activity of temozolomide by modulating O6-methylguanine DNA methyltransferase expression

Although temozolomide has shown clinical activity against neuroblastoma, this activity is likely limited by the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT). We hypothesized that IFN-beta could sensitize neuroblastoma cells to the cytotoxic effects of temozolomide through its ability to down-regulate MGMT expression. In vitro proliferation of three neuroblastoma cell lines treated with IFN-beta and temozolomide alone or in combination was examined. Antitumor activity was assessed in both localized and disseminated neuroblastoma xenografts using single-agent and combination therapy, with continuous delivery of IFN-beta being established by a liver-targeted adeno-associated virus-mediated approach. Two neuroblastoma cell lines (NB-1691 and SK-N-AS) were found to have high baseline levels of MGMT expression, whereas a third cell line (CHLA-255) had low levels. Temozolomide had little effect on in vitro proliferation of the neuroblastoma cell lines with high MGMT expression, but pretreatment with IFN-beta significantly decreased MGMT expression and cell counts (NB-1691: 36 +/- 3% of control, P = 0.0008; SK-N-AS: 54 +/- 7% control, P = 0.003). In vivo, NB-1691 tumors in CB17-SCID mice treated with the combination of IFN-beta and temozolomide had lower MGMT expression and a significantly reduced tumor burden, both localized [percent initial tumor volume: 2,516 +/- 680% (control) versus 1,272 +/- 330% (temozolomide), P = 0.01; 1,348 +/- 220%, P = 0.03 (IFN-beta); 352 +/- 110%, P = 0.0001 (combo)] and disseminated [bioluminescent signal: control (1.32e10 +/- 6.5e9) versus IFN-beta (2.78e8 +/- 3.09e8), P = 0.025, versus temozolomide (2.06e9 +/- 1.55e9), P = 0.1, versus combination (2.13e7 +/- 7.67e6), P = 0.009]. IFN-beta appears to sensitize neuroblastoma cells to the cytotoxic effects of temozolomide through attenuation of MGMT expression. Thus, IFN-beta and temozolomide may be a useful combination for treating children with this difficult disease.