methylguanine-DNA Methyltransferase Expression Research Articles

Impact of O6-methylguanine-DNA methyltransferase expression on the drug resistance of clear cell renal cell carcinoma.

The deoxyribonucleic acid-repair protein O(6)-methylguanine-deoxyribonucleic acid methyltransferase is a major determinant of resistance of cells to various alkylating drugs. Its expression profile is different in different cancer types. Here, we studied the expression and function of O(6)-methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma. The expression of O(6)-methylguanine-deoxyribonucleic acid methyltransferase was evaluated in clear cell renal cell carcinoma tissues and cell lines by quantitative real-time polymerase chain reaction and immunohistochemistry. The relationship between O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression and clinicopathological characteristics was analyzed. To further investigate the function of O(6)-methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma resistance to alkylating agents, siRNA targeting O(6)-methylguanine-deoxyribonucleic acid methyltransferase were used to silence the O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression. We found that O(6)-methylguanine-deoxyribonucleic acid methyltransferase is over-expressed in clear cell renal cell carcinoma tissues and cell lines. O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression is related with tumor progression in clear cell renal cell carcinoma patients. Up-regulation of O(6)-methylguanine-deoxyribonucleic acid methyltransferase plays a critical role in primary resistance to alkylating agents. The overexpression of O(6)-methylguanine-deoxyribonucleic acid methyltransferase contributes to resistance of clear cell renal cell carcinoma to standard chemotherapy. Our results have significance for understanding a new pathway of the development of drug resistance of clear cell renal cell carcinoma.

Alkylation and Carbamylation Effects of Lomustine and Its Major Metabolites and MGMT Expression in Canine Cells.

DNA Alkylation is thought to be the reason for the efficacy of lomustine while carbamylation has been implicated as the cause for the side effects seen with lomustine treatment such as hepatotoxicity. In the alkylation study we show that lomustine and its metabolites form similar levels of the DNA adducts N7 hydroxyethylguanine and O6 hydroxyethyldeoxyguanosine. In terms of carbamylation, lomustine showed greater extent of carbamylation in the canine hepatocytes and lymphoma cell lines. The DNA repair enzyme O6 methylguanine DNA methyltransferase (MGMT) causes resistance of tumor cells to bifunctional nitrosourea, like lomustine. There is no data available regarding MGMT expression/activity in canine cells or tissues. Our study shows that there is low MGMT activity in the canine lymphoid cell line 17–71 while the GL-1 cells did not show any detectable enzyme activity or mRNA expression. The MGMT enzyme activity measured in canine hepatocytes is about 250–350 fmol/mg protein as compared to about 90 fmol/mg protein in 17–71 cells. We also show that MGMT mRNA expression in 17–71 cells and canine hepatocytes positively correlates with its enzyme activity in these cells.

O(6) -methylguanine DNA methyltransferase repairs platinum-DNA adducts following cisplatin treatment and predicts prognoses of nasopharyngeal carcinoma.

Cisplatin (CDDP) is an important anti-cancer drug commonly used in various human cancers, including nasopharyngeal carcinoma (NPC). How to overcome the drug resistance of CDDP provides opportunities to improve clinical outcomes of NPC. O(6) -methylguanine-DNA methyltransferase (MGMT) has been well-characterized to be a therapeutic determinant of O(6) -alkylguanine alkylating drugs. However, the underlying mechanism and clinical relevance between MGMT and CDDP remain poorly defined in NPC. In this study, we showed that MGMT-proficient cells were highly resistant to the cytotoxic effects of CDDP as compared to MGMT-deficient cells. Further studies showed that the platinum level of DNA after CDDP exposure was significantly lower in MGMT-proficient cells than in MGMT-deficient cells. Host cell reactivation assay revealed that MGMT protected NPC cells from CDDP-induced DNA damage by enhancing DNA repair capacity. Importantly, we demonstrated for the first time that MGMT protein directly bound to CDDP-induced DNA damages. Subsequently, CDDP-bound MGMT protein became ubiquitinated and was degraded through ubiquitin-mediated proteasome system. We further analyzed the relationship between MGMT expression and clinical survivals in a cohort of 83 NPC patients. NPC patients who received CDDP-based concurrent chemoradiotherapy (CCRT), with high MGMT expression level, exhibited shorter progression-free survival (PFS; p = 0.022) and overall survival (OS; p = 0.015), than patients with low MGMT expression level. Furthermore, high MGMT expression level remained to be an independent prognostic factor for worse PFS (p = 0.01, hazard ratio 2.23) and OS (p = 0.018, hazard ratio 2.14). Our findings suggest that MGMT protein is important to determine the efficacy of CDDP in NPC.

Pine (Pinus morrisonicola Hayata) needle extracts sensitize GBM8901 human glioblastoma cells to temozolomide by downregulating autophagy and O(6)-methylguanine-DNA methyltransferase expression.

Pine needle extracts of Pinus morrisonicola (Hayata) are commonly used as a functional health beverage. However, it remains unclear what the mechanism is underlying the antitumor activity of pine needle extract. The aims of present study were to investigate the anti-glioblastoma effects of pine needle extracts as well as its bioactive compounds. From three different solvent extracts of pine needles, the water extract displayed the strongest cytotoxicity effects on GBM8901 glioblastoma cells. The isolated compounds were identified as pinocembrin, chrysin, and tiliroside. Chrysin was the most active ingredient of pine needle extract for the induction of apoptosis and suppression of migration and invasion. It also markedly inhibited temozolomide (TMZ)-induced autophagy and O(6)-methylguanine-DNA methyltransferase (MGMT) expression. Because both autophagy and MGMT overexpression have been implicated to TMZ-induced drug resistance in glioblastoma, our results showed that pine needle extract and chrysin may serve as a potential anticancer agent against glioblastoma, especially with regard to sensitizing glioblastoma cells resistant to TMZ.

Progression of O⁶-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research.

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O(6)-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.

SDHB mutations are associated with response to temozolomide in patients with metastatic pheochromocytoma or paraganglioma.

Cyclophosphamide-dacarbazine-vincristine regimen is recommended for the treatment of malignant pheochromocytoma and paraganglioma (MPP); however, dacarbazine is the only recognized active drug in neuroendocrine tumours. We investigated the therapeutic benefit of temozolomide (TMZ), an oral alternative to dacarbazine, in patients with MPP. This is a retrospective study of consecutive patients with documented progressive MPP. We examined the correlation between Succinate dehydrogenase B (SDHB) mutation and O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation and MGMT expression in the French nation-wide independent cohort of 190 pheochromocytomas or paragangliomas (PP). Progression-free survival (PFS) according to RECIST 1.1 and PERCIST 1.0 criteria was the primary end point. Fifteen consecutive patients with MPP were enrolled; ten (67%) carried a mutation in SDHB. The mean dose intensity of TMZ was 172 mg/m(2) /d for 5 days every 28 days. Median PFS was 13.3 months after a median follow-up of 35 months. There were five partial responses (33%), seven stable (47%) and three progressive diseases (20%). Grade 3 toxicities were lymphopenia in two patients and hypertension in one. Partial responses were observed only in patients with mutation in SDHB. MGMT immunohistochemistry was negative in tumour samples from four patients who responded to treatment. SDHB germline mutation was associated with hypermethylation of the MGMT promoter and low expression of MGMT in 190 samples of the French nation-wide independent cohort. This study demonstrates that TMZ is an effective antitumour agent in patients with SDHB-related MPP. The silencing of MGMT expression as a consequence of MGMT promoter hypermethylation in SDHB-mutated tumours may explain this finding.

Immunohistochemical evaluation of O6‐methylguanine DNA methyltransferase (MGMT) expression in 117 cases of glioblastoma

Temozolomide (TMZ) is an oral alkylating agent which is widely used in the treatment of glioblastoma (GBM) and is composed of astrocytic and/or oligodendroglial tumors, and the evaluation of O(6) -methylguanine DNA methyltransferase (MGMT) expression is important to predict the response to TMZ therapy. In this study, we conducted immunohistochemical analysis of 117 cases of Japanese GBM including 19 cases of GBM with oligodendroglioma component (GBMO), using a scoring system for quantitative evaluation of staining intensity and proportion of MGMT, and performed survival analysis of these patients. Immunohistochemically, 55 cases (47%) were positive for MGMT with various intensities and proportions (total score (TS) ≥ 2), while 62 cases (53%) were negative (TS = 0). The distribution of MGMT expression pattern was not affected by any clinicopathological parameters such as the histological subtype (GBM vs. GBMO), age and gender. The survival analysis of these patients revealed that the minimal expression of MGMT (TS ≥ 2) was a significant unfavorable prognostic factor (P < 0.001) as well as resectability (P = 0.004). Moreover, multivariate analysis showed that minimal MGMT expression in GBM was the most potent independent predictor for progression free survival (P < 0.001) and also overall patient survival (P < 0.001). This is the first report employing the scoring system for both staining intensity and proportion to evaluate immunohistochemical MGMT expression in GBM. In addition, our results emphases the clinicopathological values of the immunohistochemical approach for MGMT expression in glioma patients as a routine laboratory examination.

Role of MGMT as biomarker in colorectal cancer.

O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation plays an important role in colorectal carcinogenesis, occurring in about 30%-40% of metastatic colorectal cancer. Its prognostic role has not been defined yet, but loss of expression of MGMT, which is secondary to gene promoter methylation, results in an interesting high response to alkylating agents such as dacarbazine and temozolomide. In a phase 2 study on heavily pre-treated patients with MGMT methylated metastatic colorectal cancer, temozolomide achieved about 30% of disease control rate. Activating mutations of RAS or BRAF genes as well as mismatch repair deficiency may represent mechanisms of resistance to alkylating agents, but a dose-dense schedule of temozolomide may potentially restore sensitivity in RAS-mutant patients. Further development of temozolomide in MGMT methylated colorectal cancer includes investigation of synergic combinations with other agents such as fluoropyrimidines and research for additional biomarkers, in order to better define the role of temozolomide in the treatment of individual patients.

Immunohistochemical Assessment of O(6)-Methylguanine-DNA Methyltransferase (MGMT) and Its Relationship with p53 Expression in Endometrial Cancers.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein, the loss of MGMT expression was commonly known due to hypermethylation of CpG islands in its promoter region. Overexpression of p53 protein may be associated with downregulated MGMT expression in brain tumors. The aims of this study were to investigate the role of MGMT expression loss and its correlation with p53 overexpression in endometrial cancers. MGMT and p53 expression was examined in formalin-fixed, paraffin-embedded tissues from 36 endometrial cancer cases using immnunohistochemical staining. The loss of MGMT expression was detected in 11 (30.6%) out of the 36 endometrial cancers and p53 immunoreactivity was detected in 23 (63.9%) out of the 36 endometrial cancers. Ten (90.9%) of the 11 cases with negative MGMT immunoreactivity showed positive p53 expression, so the loss of MGMT expression was significantly associated with the p53 overexpression (P=0.03). These findings suggest that the loss of MGMT expression may be one of factors capable of p53 overexpression in endometrial cancer. Further studies are needed to define the relation between MGMT and p53 for examining the mechanisms of tissue-specific MGMT expression.

Gain of Function of Mutant TP53 in Glioblastoma: Prognosis and Response to Temozolomide

Our aim was to investigate the relationship between mutant p53 and the prognosis of malignant glioma treated with temozolomide, and the regulation of mutant TP53 induced drug resistance, by molecular experimentation and a clinical trial. Adult patients with newly surgical diagnosed glioblastoma were randomly assigned to receive either temozolomide or semustine after radiation treatment. The statuses of TP53 and expression of TP53 and O(6)-methylguanine DNA-methyltransferase (MGMT) were determined retrospectively in tumor tissue from enrolled patients. The primary end point was overall survival. Synthetic small interfering RNA was used to knock down mutant TP53 in T98G and U138 cells, which are human glioblastoma cells with a P53 mutation, by screening of exons 4-8. Viable cell survival was measured when these cells were exposed to temozolomide or semustine. Expression of MGMT at the messenger RNA level was also determined. The overall survival was 34.3 % at 2 years, 22.9 % at 3 years, 11.4 % at 4 years, and 8.6 % at 5 years with temozolomide, versus 18.2, 12.1, 3.0, and 0 %, respectively, with semustine. TP53 mutation and expression of mutant TP53 and MGMT showed significant inverse correlations with overall survival. Knockdown of mutant TP53 led to a fivefold increase in chemosensitivity to temozolomide but not semustine. Mutant TP53 knockdown induced down-regulation of MGMT expression. Mutant TP53 is strongly associated with a poor prognosis for overall survival in patients with glioblastoma. Also, TP53 mutation may decrease the chemosensitivity of glioblastoma to temozolomide by increasing MGMT expression.

Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O 6 -methylguanine DNA methyltransferase promoter methylation via c-Myc signaling

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3β inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3β inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor, AR-A014418 or GSK3β-specific small interfering RNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-PCR. c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.

Global and MGMT promoter hypomethylation independently associated with genomic instability of lymphocytes in subjects exposed to high-dose polycyclic aromatic hydrocarbon

Global hypomethylation, gene-specific methylation, and genome instability are common events in tumorigenesis. To date, few studies have examined the aberrant DNA methylation patterns in coke oven workers, who are highly at risk of lung cancer by occupational exposure to polycyclic aromatic hydrocarbons (PAHs). We recruited 82 PAH-exposed workers and 62 unexposed controls, assessed exposure levels by urinary 1-hydroxypyrene, and measured genetic damages by comet assay, bleomycin sensitivity, and micronucleus assay. The PAHs in coke oven emissions (COE) were estimated based on toxic equivalency factors. We used bisulfite-PCR pyrosequencing to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and O(6)-methylguanine-DNA methyltransferase (MGMT). Further, the methylation alteration was also investigated in COE-treated human bronchial epithelial (16HBE) cells. We found there are higher levels of PAHs in COE. Among PAH-exposed workers, LINE-1 and MGMT methylation levels (with CpG site specificity) were significantly lowered. LINE-1, MGMT, and its hot CpG site-specific methylation were negatively correlated with urinary 1-hydroxypyrene levels (r = -0.329, p < 0.001; r = -0.164, p = 0.049 and r = -0.176, p = 0.034, respectively). In addition, LINE-1 methylation was inversely associated with comet tail moment and micronucleus frequency, and a significant increase of micronucleus in low MGMT methylation group. In vitro study revealed that treatment of COE in 16HBE cells resulted in higher production of BPDE-DNA adducts, LINE-1 hypomethylation, hypomethylation, and suppression of MGMT expression. These findings suggest hypomethylation of LINE-1 and MGMT promoter could be used as markers for PAHs exposure and merit further investigation.

Management and survival rates in patients with glioma in China (2004–2010): a retrospective study from a single-institution

To analyze the clinical characteristics and prognostic factors in patients with glioma in an academic institute in China. From October 2004 to August 2010, total 1,285 patients were diagnosed as glioma at the Glioma Center of Beijing Tiantan Hospital. Clinical and molecular pathology features and survival rates were analyzed. The median overall survival (OS) times were 78.1, 37.6 and 14.4 months for low-grade glioma (WHO grade II), anaplastic glioma (WHO grade III) and glioblastoma (WHO grade IV), respectively. In patients with low-grade glioma, age, preoperative Karnofsky performance scale (KPS), pathological type, radiotherapy, O(6)-methylguanine-DNA methyltransferase (MGMT) expression and Ki-67 expression, were significantly associated with OS in multivariate analyses; and preoperative KPS and radiotherapy were significantly associated with progression-free survival (PFS). For anaplastic gliomas, age, preoperative KPS, pathological type, extent of resection, radiotherapy, p53 expression and phosphatase and tensin homolog (PTEN) expression were associated with OS. For glioblastomas, age, preoperative KPS, pathology type, extent of resection, radiotherapy and chemotherapy were associated with OS; and age, gender, preoperative KPS, extent of resection, radiotherapy and chemotherapy were associated with PFS. This is the largest survey for glioma management in China to date. We found significant differences in age, presenting symptoms and the expression of p53, MGMT, PTEN, and Ki-67 among patients with different types of glioma. Age, preoperative KPS, tumor grades, radiotherapy, chemotherapy and Ki-67 expression were significantly associated with clinical prognosis.

Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents

The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Mutant TP53 enhances the resistance of glioblastoma cells to temozolomide by up-regulating O6-methylguanine DNA-methyltransferase

The "gain of function" of mutant TP53 is an important determinant in human tumor development and progression. This study aimed to investigate the possible mechanism of mutant TP53 inducing temozolomide resistance in glioblastoma cells. Three established human glioma cell lines, T98G, U87, and U138, were chemoresistant cells. The mRNA of cells was sequenced to confirm the status of TP53. Synthetic small interfering RNA (siRNA) was used to knock down TP53 in cells. TP53 mRNA was detected "silenced" by reverse transcriptase-polymerase chain reaction (RT-PCR) in five consecutive days. Viable cell survival was measured when these cells were exposed to temozolomide or semustine in step-up concentrations. The expression of O(6)-methylguanine DNA-methyltransferase (MGMT) at mRNA level was also determined. T98G, U87, and U138 cells were resistant to temozolomide. T98G and U138 cells expressed mutant-type TP53 with positive MGMT, while U87 cell expressed wild-type TP53 with negative MGMT. TP53-siRNA knocked down TP53 effectively (P = 0.021) in five consecutive days. Knockdown of mutant TP53 in T98G and U138 cells led to a fivefold increase in chemosensitivity to temozolomide, but not semustine. Knockdown of wild TP53 in U87 cell did not affect the chemoresistance. In addition, mutant TP53 knockdown induced a dramatic decrease of MGMT expression (P = 0.0000034). TP53 mutation decreases the chemosensitivity of malignant gliomas to temozolomide. This "gain of function" in drug resistance may be obtained by increasing MGMT expression.

A genome-wide association analysis of temozolomide response using lymphoblastoid cell lines shows a clinically relevant association with MGMT.

Recently, lymphoblastoid cell lines (LCLs) have emerged as an innovative model system for mapping gene variants that predict the dose response to chemotherapy drugs. In the current study, this strategy was expanded to the in-vitro genome-wide association approach, using 516 LCLs derived from a White cohort to assess the cytotoxic response to temozolomide. Genome-wide association analysis using ∼2.1 million quality-controlled single-nucleotide polymorphisms (SNPs) identified a statistically significant association (P<10(-8)) with SNPs in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene. We also show that the primary SNP in this region is significantly associated with the differential gene expression of MGMT (P<10(-26)) in LCLs and differential methylation in glioblastoma samples from The Cancer Genome Atlas. The previously documented clinical and functional relationships between MGMT and temozolomide response highlight the potential of well-powered genome-wide association studies of the LCL model system to identify meaningful genetic associations.

Methylguanine DNA methyl transferase (MGMT) expression to predict response to temozolomide (TMZ) in patients with digestive neuroendocrine tumors (NETs).

4133 Background: TMZ is an orally given drug used in patients (pts) with NETs, with encouraging results in pancreatic NETs in a recent study (Strosberg et al, 2011). Higher treatment efficacy seems to be correlated to MGMT tumor deficiency (Kulke et al, 2010). Aim-To assess MGMT expression in digestive NETs and to correlate with the efficacy of TMZ-based therapies. Methods: All pts with well differentiated, progressive, non resectable NETs treated with TMZ-based chemotherapy were included. Treatment efficacy was defined according to RECIST. Pts with progression or only stable disease were considered as “non-responders”. Nuclear expression of MGMT was assessed by immunohistochemistry on primary tumors or metastases and graded according the product of the intensity of staining (0 to 3) and the rate of positive cells (%), leading to a score comprised between 0 and 300. A score ≥ 80 was defined as “high” staining. Results: 22 pts (age 59 years (36-81)) with pancreatic (14 pts), small bowel (5 pts) or other (3 pts) NETs, grade 1 (5 pts) or 2 (17 pts) (WHO 2010 classification) were included. They received TMZ alone (19 pts) or combined with capecitabine (3 pts) as first line (3 pts) or 2+ line (19 pts).After a median of 6 cycles (3-16), objective response, stable disease and progression rates were seen in 32 % (7 pts, all with pancreatic NETs), 41% (9 pts, 5 pancreatic) and 27% (6 pts, 4 small bowel), respectively. Median (range) MGMT score was 10 (0-300). A “High” MGMT score was seen in 36% of pts (small bowel 4, pancreas 3 pts) ; it was correlated with primary tumor location (more frequent in small bowel NETs, p=0.02) and predictive of the absence of response (p=0.02). A “Low “MGMT score tended to be associated with objective response (p=0.06) whereas none of the pts with “high” score had tumor response. Response rate in pts with “low” MGMT score was 50%. Conclusions: MGMT deficiency is more frequent in pancreatic than small bowel NETs. Patients with pancreatic NET and low MGMT score are good candidates for TMZ, whereas those with high score should be treated with other drug in first intention. In patients with small bowel NET with most often high MGMT score, tumor stabilization using TMZ seems to be rare.

5-day dosing schedule of temozolomide in relapsed sensitive or refractory small cell lung cancer (SCLC) and methyl-guanine-DNA methyltransferase (MGMT) analysis in a phase II trial.

7052 Background: MGMT promoter methylation and loss of MGMT activity are associated with improved sensitivity to alkylating agents. We recently reported that the oral alkylating agent temozolomide is active in 2nd- and 3rd-line treatment of relapsed SCLC at 75mg/m2/day for 21 out of 28 days (Pietanza et al, Clin Can Res 2012). Here we evaluate the 5-day dosing schedule of temozolomide in a second cohort of patients and analyze MGMT activity in both cohorts of patients on the same study. Methods: Patients with disease progression after 1 or 2 prior chemotherapy regimens received temozolomide at 200mg/m2/day for 5 out of 28 days (n=25). Those with sensitive (S-SCLC, n=16) and refractory (R-SCLC, n=9) disease were enrolled to assess safety. Available tumor tissue from patients treated according to either schedule was assessed for MGMT promoter methylation status by PCR and MGMT expression by immunohistochemistry (IHC). Results: An overall response rate of 12% was noted (3 partial responses: 1 S-SCLC, 2 R-SCLC). 4 patients had stable disease for at least 3 cycles. Median progression-free survival and overall survival for all patients were 1.3 months and 7.9 months, respectively. Grade ≥3 thrombocytopenia and neutropenia was observed in 20% with a shorter mean duration of myelosuppression compared to the 21-day schedule. Results from MGMT evaluation of tumor samples from both dosing schedules were combined. Promoter methylation of MGMT was not significantly associated with response (p=0.23). However, patients that lacked MGMT expression by IHC had a higher response rate compared to those with MGMT-positive tumors (43% vs. 13%, p = 0.052; see table). Conclusions: The 5-day dosing schedule of temozolomide is both active and safe in patients with relapsed SCLC. A strong trend toward increased response was demonstrated in patients with MGMT-negative tumors compared to patients with MGMT-positive tumors by IHC. A trial combining temozolomide with the PARP inhibitor, ABT888, is planned. [Table: see text]

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.

Pituitary carcinomas and aggressive pituitary tumours: merits and pitfalls of temozolomide treatment

Pituitary carcinomas are rare, accounting for about 0.2% of all pituitary tumours. They represent a challenge to clinical practice in both diagnosis and treatment. They may present initially as typical pituitary adenomas, with a delayed appearance of aggressive signs, or as aggressive tumours from the outset. Predicting the pituitary tumour behaviour remains difficult: increased mitotic, Ki-67 and P53 indices might be associated with tumour aggression. The treatment of pituitary carcinomas and aggressive pituitary tumours includes surgery, adjuvant medical treatment, external beam radiotherapy and chemotherapy. Until recently, the treatment of pituitary carcinomas was mainly palliative and did not seem to increase overall survival. Recent case reports have detailed the successful use of temozolomide, an orally administered alkylating agent used to treat malignant gliomas, in the management of pituitary carcinomas and aggressive pituitary tumours. The outcome of treatment might depend on the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that potentially interferes with drug efficacy. This review describes the clinical presentation and response to temozolomide in 44 patients with pituitary carcinomas or aggressive pituitary tumours reported in the literature. The results suggest that temozolomide should be considered a drug of major importance in the therapeutic algorithm of aggressive pituitary tumours and pituitary carcinomas. Because of the inconsistency of published data, MGMT expression should probably not be taken as a reason to deny these patients the potential benefit of temozolomide treatment, taking into account the paucity of other available treatments.