O6-methylguanine DNA Research Articles

Survival in a consecutive series of 467 glioblastoma patients: Association with prognostic factors and treatment at recurrence at two independent institutions.

Therapy of recurrent glioblastoma (GBM) is challenging due to lack of standard treatment. We investigated physicians' treatment choice at recurrence and prognostic and predictive factors for survival in GBM patients from Norway's two largest regional hospitals. Clinicopathological data from n = 467 patients treated at Haukeland and Oslo university hospitals from January 2015 to December 2017 was collected. Data included tumour location, promoter methylation of O6 methylguanine-DNA methyltransferase (MGMT) and mutation of isocitrate dehydrogenase (IDH), patient age, sex, extent of resection at primary diagnosis and treatment at successive tumour recurrences. Cox-proportional hazards regression adjusting for multiple risk factors was used. Median overall survival (OS) was 12.1 months and 21.4% and 6.8% of patients were alive at 2 and 5 years, respectively. Median progression-free survival was 8.1 months. Treatment at recurrence varied but was not associated with difference in overall survival (OS) (p = 0.201). Age, MGMT hypermethylation, tumour location and extent of resection were independent prognostic factors. Patients who received 60 Gray radiotherapy with concomitant and adjuvant temozolomide at primary diagnosis had 16.1 months median OS and 9.3% were alive at 5 years. Patients eligible for gamma knife/stereotactic radiosurgery alone or combined with chemotherapy at first recurrence had superior survival compared to chemotherapy alone (p<0.001). At second recurrence, combination chemotherapy with or without bevacizumab were both superior to no treatment. Treatment at recurrence differed between the institutions but there was no difference in median OS, indicating that it is the disease biology that dictates patient outcome.

IM-2 Possibility of immunotherapy for the glioblastoma patients with O6-methyl-guanine DNA methyltransferase (MGMT) expression or promoter unmethylated

Background: MGMT is a DNA repair protein that removes the cytotoxic O6-methylguanine (O6MG) DNA lesions generated by TMZ; thereby, MGMT expression is mechanistically linked to TMZ resistance. However, thus far, there is no effective treatment for these patients with MGMT promoter unmethylated. Therefore, a new treatment for GBM patients with MGMT expression is urgently needed. To this end, we examined the tumor microenvironment in GBM with or without MGMT expression. Methods: Based on The Cancer Genome Atlas (TCGA) primary GBM cohort, the tumor-infiltrating lymphocytes (TILs) expression level was calculated using the CIBERSORTx algorithms and the single-sample Gene Set Enrichment Analysis (ssGSEA) method. Furthermore, the differential expression gene analysis was conducted and pathway analysis was performed using Ingenuity Pathway Analysis (IPA). The results were validated using the GBM cohort from the Chinese Glioma Genome Atlas (CGGA) database. In addition, TILs were isolated from 13 surgically removed primary GBM tumors in our institution. Their responses to autologous tumors were evaluated by IFNγ ELISA. Results: T cells CD8 score by CIBERSORTx was significantly higher in the MGMT-high tumor. Similarly, ssGSEA scores for activated CD8 T cell, Macrophage, activated B cell, and Type 1 T helper cell were significantly higher in the MGMT-high tumor. Conversely, T cells CD4 naive was significantly higher in the MGMT-low tumor. Consistently, tumor-reactive TILs were detected in the MGMT-high tumor. Pathway analysis showed that Rictor was highly enriched in the MGMT-high tumor and Rictor inhibited lymphocyte activation. Coclusion: In this study, we demonstrated that macrophage was highly activated in the MGMT-high tumors. Thus, CSF-1R inhibitor can be combined with immunotherapy in these MGMT-high tumors to enhance anti-tumor immune responses. In addition, TMZ + mTOR2 inhibitors + PD-1 inhibitors may be effective against the MGMT-L group.

Abstract 2596: Stage-specific inhibition of NNK-induced lung carcinogenesis by 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp)

Abstract Chemopreventive efficacy of p-XSC is well documented but it failed due to systemic toxicity issues. This toxicity could partially be due to the release of poisonous hydrogen cyanide generated after p-XSC metabolizes to form active bis-selenol (p-XSeH). To address the said concern, we recently designed and developed p-XS-Asp, with the rationale that it would cleave in vivo to release the active p-XSeH and aspirin instead of undesired HCN, thus making the compound less toxic and possibly more potent than p-XSC. Indeed, we have shown previously that p-XS-Asp inhibits NNK-induced lung tumorigenesis in A/J mice more effectively than p-XSC in a complete A/J mouse model, and is better tolerable. The complete model, however, does not reveal at what stage of carcinogenesis does p-XS-Asp acts. In the current study, we evaluated the stage-specificity of chemopreventive efficacy of p-XS-Asp in NNK-induced lung carcinogenesis in A/J mice models. A/J mice were divided into 7 distinct groups (n=30 per group, half male, half female; except group 1 and 2 where n=10). The mice were fed AIN-93M diet (control) or p-XS-Asp diet until they reached termination end point of 26 weeks (adenomas) and 40 weeks (adenocarcinomas). Two weeks after the experiment started, all the groups, except for group 1 and 2, were given a single IP injection of 10 µmol (100 mg/kg) of NNK. Group 3 and 4 were on complete control AIN93M and p-XS-Asp diets, respectively. Group 5 (peri-initiation) was on p-XS-Asp for the first 3 weeks and then on the control diet till the end of the experiment. On the other hand, group 6 (post-initiation) was on control diet for 3 weeks and then was changed to the p-XS-Asp diet. In the progression group 7, mice were on control diet for 14 weeks and subsequently changed to p-XS-Asp diet. The Groups 4 and 5 mice showed a remarkable decrease in the tumor multiplicity (TM) and incidence (TI) as compared to the mice on control diet at both 26 and 40 week time-points. The TM in male mice for groups 1, 2, 3, 4, 5, 6 and 7 at 26 weeks was 0.6, 0, 5.7, 1, 1.54, 4.27, and 6.27, respectively, while at 40 weeks, it was 0.2, 0, 7.7, 1, 1.8, 4.1, and 4.9, respectively. A similar trend was also observed in female mice. These data clearly demonstrated that robust inhibition at peri-initiation stage is mainly responsible for the activity observed in the complete model. The inhibition of both O6-methylguanine and pyridoxobutyl mutagenic DNA adducts by p-XS-Asp further compliments that the compound acts at initial stages of carcinogenesis. Body weights comparison and the blood and tissue analyses showed no systemic toxicity for the p-XS-Asp fed groups. RNA-seq data of the tumor tissues showed numerous signaling pathways to be affected in p-XS-Asp treated mice. The exact mechanism of action is still under investigation. In summary, our results show that p-XS-Asp may be promising candidate for future clinical evaluation as a lung cancer preventive agent. Citation Format: Asif Raza, Amandeep Singh, Cesar Aliaga, Daniel Plano, Shantu Amin, Arun K. Sharma. Stage-specific inhibition of NNK-induced lung carcinogenesis by 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2596.

Predictors of Survival in Turkish Patients with Primary Glioblastoma.

An increasing number of biomarkers of primary glioblastoma (GBM) have recently been described. We aimed to investigate the biological and clinical factors that affect survival in Turkish patients with primary GBM. The clinical and demographic data of all patients with primary GBM diagnosed between 2007 and 2016 were evaluated. In all the patients? pathological specimens, O6 methylguanine-DNA methyltransferase (MGMT) methylation and isocitrate dehydrogenase (IDH) 1 mutation were detected retrospectively by immunohistochemistry. Kaplan-Meier survival analysis, log-rank test, and multivariate analyses of the Cox hazard proportional model for all the variables were performed using the SPSS statistical package. The treatment details and other patient-related factors were identified, and their correlations were analyzed. We enrolled 137 primary GBM patients to the study. Median progression free survival (PFS) was 8.57 months (95% CI:6.8-9.5) and median overall survival (OS) was 12 months (95% CI:10.8-13.3). IDH-1 mutations were detected in 21 primary GBMs (15.3%). PFS was 15.43 ± 1.95 months. Survival rates were higher, but no statistically significant difference (p=0.074). MGMT methylation was detected in 40 primary GBMs (29.2%). OS and PFS of MGMT (+) cases were higher than MGMT(-) cases (p=0.001; p=0.001 respectively). Ki67 (%) measurement (10%-90%) average is 32.64 ± 16.56. No statistically significant between higher and lower ki67 levels (p=0.510, p=0.505 respectively). KPS (%) more than 70 at the time of diagnosis statistically significant longer median OS and PFS (p=0.001). PFS and OS were higher in all treatment modalities. The most important factors that affected survival were performance score, MGMT methylation status, systemic oncologic therapy, and IDH mutation in the Turkish population with primary GBM. We demonstrated that MGMT methylation and higher KPS levels were associated with significiantly longer OS and PFS.

ACTR-43. GENOMIC ANALYSIS OF RESPONDERS OF PHASE II TRIAL OF TEMOZOLOMIDE AND TRC-102 (BASE EXCISION REPAIR INHIBITOR) IN BEVACIZUMAB-NAÏVE GLIOBLASTOMA AT FIRST RECURRENCE

Abstract BACKGROUND Temozolomide forms O6-methylguanine (O6mG), 7-methylguanine (N7mG), and 3- methyladenine (N3mA) DNA adducts. The O6mG DNA adduct is repaired by MGMT. N7mG and N3mA DNA adducts are removed by the base excision repair (BER) pathway, initiated by N- methylpurine DNA glycosolase (MPG). TRC-102 is a BER inhibitor that binds to the apurinic site created through the action of MPG. METHODS A phase II study of adult glioblastoma in first recurrence was performed in the Adult Brain Tumor Consortium with temozolomide, 150 mg/ m2 and TRC-102, 150 mg (1–5/ 28 days). Primary objective included radiographic response rate. Secondary objectives included safety and PFS-6. Exploratory objectives included tumor expression of N-methylpurine DNA glycosylase (MPG). The study tested hypothesis that combination therapy will achieve 30% RR. To understand the context of vulnerability to TRC102 we performed RNA sequencing on treatment naïve tissue from 7 patients. RESULTS Nineteen patients were enrolled in first stage. Median age was 60 years (range: 48–76), 53% females, median KPS was 80 (range: 70–90). Median cycles of treatment was 2 (range: 1–12). No responses were observed. Median OS was 11.0 months (95% CI: 8–18 months), median PFS was 2.0 months (95% CI: 1.8–3.6 months). PFS-6 rate was 10.5 % (2/19). The combination was safe. MPG staining was negative in six, 1+ in five and 2+ in three patients. PFS of 11 + months in two patients (exceptional responders) was associated with MPG expression. Preliminary analysis on RNA sequencing revealed significant enrichment for DNA Damage Response pathways (MsigDB), chromosomal instability gene signature (CIN70 and CIN25), and proliferative gene signature (PCNA25) in these 2 patients. CONCLUSIONS TRC 102 with temozolomide has acceptable safety but did not meet the primary endpoint of response. Gene signature of MsigDB, CIN70, CIN25 and PCNA25 was seen in exceptional responders and biomarker driven study is planned.

Noninvasive O6 Methylguanine-DNA Methyltransferase Status Prediction in Glioblastoma Multiforme Cancer Using Magnetic Resonance Imaging Radiomics Features: Univariate and Multivariate Radiogenomics Analysis

This study aimed to predict methylation status of the O6 methylguanine-DNA methyltransferase (MGMT) gene promoter status by using magnetic resonance imaging radiomics features, as well as univariate and multivariate analysis. Eighty-two patients who had an MGMT methylation status were included in this study. Tumors were manually segmented in the 4 regions of magnetic resonance images, 1) whole tumor, 2) active/enhanced region, 3) necrotic regions, and 4) edema regions. About 7000 radiomics features were extracted for each patient. Feature selection and classifier were used to predict MGMT status through different machine learning algorithms. The area under the curve (AUC) of the receiver operating characteristic curve was used for model evaluations. Regarding univariate analysis, the Inverse Variance feature From Gray Level Co-occurrence Matrix in whole tumor segment with 4.5 mm Sigma of Laplacian of Gaussian filter with AUC of 0.71 (P value= 0.002) was found to be the best predictor. For multivariate analysis, the Decision Tree classifier with Select from Model feature selector and LOG (Laplacian of Gaussian) filter in edema region had the highest performance (AUC, 0.78), followed by Ada-Boost classifier with Select from Model feature selector and LOG filter in edema region (AUC, 0.74). This study showed that radiomics using machine learning algorithms is a feasible noninvasive approach to predict MGMT methylation status in patients with glioblastoma multiforme cancer.

Abstract B116: Nano-neo-mRNA vaccine: A novel platform technology for cancer-immunotherapy

Abstract Nanoparticle-based immunotherapeutic platforms are emerging as radically different, biocompatible alternatives for viral-vectors. Liposomal nanoplexes already showed potential for clinical translation of mutanome specific personalized cancer-vaccines in human. Here, we report a novel nanoparticle platform technology for neoantigen specific mRNA (neo-mRNA) vaccine delivery and demonstrated its potential for evoking T-cell mediated immune response against drug/radiation resistant glioma cells. Glioblastoma multiforme (GBM) is one of the most aggressive type primary brain tumors with high rate of recurrence and poor prognosis. Temozolomide (TMZ) chemotherapy in concurrence with radiation is less effective for patients with over-expression of DNA repair protein, O6-methyl guanine-DNA methyltransferase (MGMT) or epidermal growth factor receptor variant III (EGFRvIII) mutation. Here, we developed a unique pathogen mimicking nanoparticle platform that can effectively encapsulate and transfect dendritic cells with mRNA vaccine specific to MGMT or EGFRvIII. MGMT/EGFRvIII mRNA was synthesized by in vitro transcription (IVT) from respective cDNA plasmid and 100-150nm sized nanoplex was formed with mRNA at the core with &amp;gt;90% encapsulation efficiency. This Nano-neo-mRNA vaccineTM platform was successfully delivered to human monocyte derived DCs with a transfection efficiency of 35-50% compared to 3-5% in the case of naked mRNA. The transfected DCs showed significant phenotypic expression of MGMT-GFP with MHC I/II and CD80/86 co-activation compared to the naked mRNA or vehicle control. The efficiency of vaccinated DCs to prime cytotoxic T-cells was demonstrated in co-cultures where the nanoplex showed efficient activation of T-cell-mediated cytotoxicity on MGMT or EGFRvIII expressing, TMZ/radiation resistant glioblastoma cells . Biocompatibility of the nano-carrier was demonstrated in primary human cells and animal models. Thus, we present a new, biocompatible nanoparticle platform technology for IVT-mRNA based cancer-immunotherapy. Citation Format: Manzoor Koyakutty, Navyashree A. Ramesh, Ashwathy Nambiar, Najuma Nujum Ambili Anna, Manju C. Abraham, Minu Anoop, Anjana Ramkumar, Nair Shanti, Vijay Harish Harish, Anusha Ashokan. Nano-neo-mRNA vaccine: A novel platform technology for cancer-immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B116.

Abstract B082: PD-1 to CD8 ratio on tumor-infiltrating lymphocytes and peripheral blood mononuclear cells as a predictor for determining response of glioblastoma patients to radiation-induced ICD-based DC vaccine therapy

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive glioma within the central nervous system in adults. Radiation-induced ICD-based DC vaccine (RICD-DC vaccine) therapy plus conventional multi-modal regimen for GBM has been demonstrated with promising outcomes in clinical trials. However, some GBM patients received RICD-DC vaccine therapy did not increased survival rates than other GBM patients who only received conventional therapy. To investigate this issue, we conducted a retrospective study to analyze clinical and laboratory data to evaluate the factors which is critical for affecting the response rate of RICD-DC vaccine treatment. Patients with de novo GBM were enrolled (n=47) and divided into two subgroups: the first subgroup received post-surgical adjuvant immunotherapy with autologous RICD-DC vaccine (n=27) and the second received conventional treatment without immunotherapy (control, n=20). Quantitative immunohistochemistry for CD45, CD4, CD8, programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) was performed on patient tumor samples and peripheral blood mononuclear cells (PBMCs) at initial resection/biopsy before treatment. Pearson’s correlation, Cox proportional hazard model, and Kaplan-Meier analyses were performed to examine the correlations between these biomarkers expression and survival rates. In the RICD-DC vaccine group, patients with a lower PD-1+/CD8+ ratio (≤0.21) on tumor infiltrating lymphocytes (TILs) had longer overall survival (OS) (median 60.97 months, P &amp;lt; 0.001) than patients with a higher PD-1+/CD8+ ratio (median 20.07 months, , P &amp;lt; 0.001); 80% of the patients with a lower PD-1+/CD8+ ratio survived longer than 2 years whereas only 20% of patients with a higher PD-1+/CD8+ ratio. Similar results were observed in progression-free survival (PFS). Consistently, lymphocyte counts with lower PD-1+/CD8+ ratio (≤0.197) had much longer OS and PFS in patients’ PBMCs. In addition, there was a significant correlation of PD-1+/CD8+ ratio between TILs and PBMCs (Pearson’s correlation R2 = 0.6002, P &amp;lt; 0.001). By contrast, OS and PFS did not be impacted by CD4−, CD8−, but PD-1+, CD45+ TILs (P = 0.073 and P = 0.249, respectively). Moreover, the significant prognostic factors of OS and PFS also contained younger age (&amp;lt;57 years), gross total resection, and conventional treatment of concomitant chemoradiotherapy (CCRT) and PD-1+ lymphocyte counts in the RICD-DC vaccine group. In contrast to other factors, gender, counts of CD45+, CD4+ or CD8+ lymphocytes, tumoral PD-L1 expression, isocitrate dehydrogenase 1 mutation, and methylation status of O6 methylguanine-DNA methyltransferase promoter have no impact in both groups. Taken together, we found that TILs or PBMCs with lower PD-1+/CD8+ ratio in GBM patients with younger age, with gross tumor resection may be the desirable candidates who can be benefited from conventional multimodal regimen plus RICD-DC vaccine immunotherapy. Citation Format: Fang-Yu Lin, Chia-Ing Jan, Wan-Chen Tsai, Horng-Jyh Harn, Hsin-Man Lu, Ming-Chao Liu, Shao-Chih Chiu, Der-Yang Cho. PD-1 to CD8 ratio on tumor-infiltrating lymphocytes and peripheral blood mononuclear cells as a predictor for determining response of glioblastoma patients to radiation-induced ICD-based DC vaccine therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B082.

Predictors of Response to Autologous Dendritic Cell Therapy in Glioblastoma Multiforme.

Glioblastoma (GBM) is the most common and lethal primary malignant glioma in adults. Dendritic cell (DC) vaccines have demonstrated promising results in GBM clinical trials. However, some patients do not respond well to DC therapy, with survival rates similar to those of conventional therapy. We retrospectively analyzed clinical and laboratory data to evaluate the factors affecting vaccine treatment. Forty-seven patients with de novo GBM were enrolled at China Medical University Hospital between 2005 and 2010 and divided into two subgroups. One subgroup of 27 patients received postsurgical adjuvant immunotherapy with autologous dendritic cell/tumor antigen vaccine (ADCTA) in conjunction with conventional treatment of concomitant chemoradiotherapy (CCRT) with temozolomide. The other 20 patients received only postsurgical conventional treatment without immunotherapy. Immunohistochemistry for CD45, CD4, CD8, programed death ligand 1 (PD-L1), and programed death 1 (PD-1) was performed on sections of surgical tumor specimens and peripheral blood mononuclear cells (PBMCs). Pearson's correlation, Cox proportional hazard model, and Kaplan-Meier analyses were performed to examine the correlations between the prognostic factors and survival rates. Younger age (<57 years), gross total resection, and CCRT and PD-1+ lymphocyte counts were significant prognostic factors of overall survival (OS) and progression-free survival (PFS) in the ADCTA group. Sex, CD45+ lymphocyte count, CD4+ or CD8+ lymphocyte count, tumor PD-L1 expression, isocitrate dehydrogenase 1 mutation, and O6 methylguanine-DNA methyltransferase promoter methylation status were not significant factors in both groups. In the ADCTA group, patients with tumor-infiltrating lymphocytes (TILs) with a lower PD-1+/CD8+ ratio (≤0.21) had longer OS and PFS (median OS 60.97 months, P < 0.001 and PFS 11.2 months, P < 0.008) compared to those with higher PD-1+/CD8+ ratio (>0.21) (median OS 20.07 months, P < 0.001 and PFS 4.43 months, P < 0.008). Similar results were observed in patients' PBMCs; lymphocyte counts with lower PD-1+/CD8+ ratio (≤0.197) had longer OS and PFS. There was a significant correlation of PD-1+/CD8+ ratio between TILs and PBMCs (Pearson's correlation R2 = 0.6002, P < 0.001). By contrast, CD4-, CD8-, but PD-1+, CD45+ tumor-infiltrating lymphocytes have no impact on OS and PFS (P = 0.073 and P = 0.249, respectively). For patients receiving DC vaccine adjuvant therapy, better outcomes are predicted in patients with younger age, with TILs or PBMCs with lower PD-1+/CD8+ ratio, with gross tumor resection, and receiving CCRT.

Effect of temozolomide on the viability of musculoskeletal sarcoma cells

Musculoskeletal sarcomas (MSS) are a heterogeneous group of malignancies with relatively high mortality rates. The prognosis for patients with MSS is poor, with few drugs inducing measurable activity. Alkylating agents, namely ifosfamide and dacarbazine, which act nonspecifically on proliferating cells, are the typical therapy prescribed for advanced MSS. A novel alkylating agent, temozolomide (TMZ), has several advantages over existing alkylating agents. TMZ induces the formation of O6-methylguanine in DNA, thereby inducing mismatches during DNA replication and the subsequent activation of apoptotic pathways. However, due to conflicting data in the literature, the mechanism of TMZ action has remained elusive. Therefore, the present study aimed to evaluate apoptosis in MSS cells treated with TMZ, and to evaluate the correlation between TMZ action and survival pathways, including the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen activated protein kinase (MAPK) pathways. Cell proliferation was evaluated by performing an XTT (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay. Apoptotic morphological changes, for example chromatin condensation, were evaluated by fluorescence confocal microscopy. The expression of the apoptosis-associated proteins caspase-3, poly adenosine diphosphate ribose polymerase (PARP), Akt and ERK1/2, was determined by western blotting. The results of the present study indicated that, in certain MSS cells, the IC50 value was lower than that in TMZ-sensitive U-87 MG cells. Furthermore, TMZ treatment was associated with apoptotic morphological changes and the expression levels of pro-apoptotic cleaved caspase-3 and PARP were also increased in TMZ-treated MSS cells. In addition, the results indicated that PI3K/Akt and ERK1/2 MAPK were constitutively phosphorylated in MSS cells, and phosphorylation of PI3K/Akt was suppressed in certain cells, and maintained in other cells, by TMZ. These observations emphasized the plasticity of MSS cells, and suggested that this plasticity may contribute to the variance in cell sensitivity to TMZ and TMZ-resistance in MSS.

P04.10 * PREDICTIVE VALUE OF MGMT PROMOTER METHYLATION QUANTITATIVE ASSESSMENT IN GLIOBLASTOMA, WHICH OPTIMAL CUT-POINT?

BACKGROUND: The methylation in the promoter region of the MGMT gene encoding the DNA repair protein O6-methyl guanine-DNA methyl transferase is a predictive marker for benefit from alkylating agent therapy and a prognostic factor in glioblastoma. Pyrosequencing-based assessement of MGMT promoter status is of particularly interest, but analytical methods and cut-points may varied from one center to another. We intend to establish a correlation between quantitative methylation of MGMT promoter values and overall survival (OS) or progression-free survival (PFS). METHODS: The clinical and biological data of 159 newly diagnosed glioblastoma recruited in Lille University Hospital between 2008 and 2011 were retrospectively collected. For the patients, the methylation status has been assessed by pyrosequencing on 5 CpG islands. Statistical analyses were performed using SAS software V9.3. RESULTS: Median age of the patients was 61 years. Sixty three patients were male. Of 159 patients, 156 underwent a surgery (40,4% of complete resection), 3 patients were diagnosed only by biopsies. 123 received concomitant radiotherapy and temozolomide and adjuvant temozolomide. The median PFS and the median OS were 9,9 and 15,9. The most significant cut-off methylation determined on 5 sites mean methylaion value was 12% for both predictive and pronostic evaluations. Site 1 methylation status was the best predictive value for both PFS and OS compared to the other CpG islands. On this site 1, a >3% methylation status was a significant predictive factor for reponse to standard treatment for PFS and a >25% site 1 methylation status was a strong pronostic factor for 2 years survival (OS) (47%). CONCLUSIONS: Even a low MGMT methylation (>3%) status on selected CpG site can already be a predictive factor for response to treatment in glioblastoma and allow us to reduce the cut-off values. Highest methylations status are correlated with the longest overall survival and therefore confirm the pronostic value of MGMT methylation. Establishing correlation between clinical data and biological assessment, such as pyrosequencing providing quantitative values for each CpG, would allow to lower and precise useful cut-points and to chose analysed site for a better predictive and pronostic significativity.

The medicinal chemistry of imidazotetrazine prodrugs.

Temozolomide (TMZ) is the standard first line treatment for malignant glioma, reaching “blockbuster” status in 2010, yet it remains the only drug in its class. The main constraints on the clinical effectiveness of TMZ therapy are its requirement for active DNA mismatch repair (MMR) proteins for activity, and inherent resistance through O6-methyl guanine-DNA methyl transferase (MGMT) activity. Moreover, acquired resistance, due to MMR mutation, results in aggressive TMZ-resistant tumour regrowth following good initial responses. Much of the attraction in TMZ as a drug lies in its PK/PD properties: it is acid stable and has 100% oral bioavailability; it also has excellent distribution properties, crosses the blood-brain barrier, and there is direct evidence of tumour localisation. This review seeks to unravel some of the mysteries of the imidazotetrazine class of compounds to which TMZ belongs. In addition to an overview of different synthetic strategies, we explore the somewhat unusual chemical reactivity of the imidazotetrazines, probing their mechanisms of reaction, examining which attributes are required for an active drug molecule and reviewing the use of this combined knowledge towards the development of new and improved anti-cancer agents.

Pathodiagnostic parameters and evaluation of O6– methyl guanine methyl transferase gene promoter methylation in meningiomas

Histopathological evaluation and grading of meningioma give important prognostic information. We evaluated retrospectively monotonous sheeting, necrosis, hypercellularity, nuclear pleomorphism, small cell changes, brain invasion, mitosis, mast cells, psammoma bodies, MIB-1 labeling index (MIB-1 LI) and histological grade of 230 primary meningioma tumors according to the latest World Health Organization (WHO) classification. To reveal any possible association between clinical features and promoter hypermethylation of O6–methylguanine-DNA methyltransferase (MGMT) as an important epigenetic modification in many human cancers, we also evaluated the methylation status of MGMT in meningiomas by a SYBR-green-based real-time PCR method. There was a female predominance (2.38 to 1) in the meningiomas. The mean age of the patients was 49.9±12.6years (range 16 to 78years). Transitional meningiomas were the most common subtype of the meningiomas (35.21%, n=81). Most of the meningiomas were located in the falx and parasagital area. There was a significant correlation between histopathological features of malignancy. These features were observed more frequently and with statistical relation to grade II rather than grade I. Mast cells, psammoma bodies and nuclear pleomorphism had poor associations (P>0.05). When we re-evaluated the tumor grading, 31 patients with grade I meningiomas were upgraded to grade II. None of the meningiomas tested by MSQP were methylated in MGMT promoter sequence. High MIB-1 LI could be indicative for higher grade of meningioma. Continuous revision of the classification system is needed to improve the accuracy of prognostic judgments in meningioma. The data confirm that there is no rationale to test meningiomas for MGMT methylation status.

Expression of O6 Methyl Guanine Methyl Transferase (Mgmt) in Oral Squamous Cell Carcinoma Patients in Alexandria, Egypt (Smokers and Non-Smokers)

Objectives: O6-methylguanine-DNA methyltransferase (MGMT) removes mutagenic, carcinogenic, and cytotoxic adducts from O6-methylguanine in DNA through a direct reversal mechanism. Decreased expression of MGMT has been reported in a variety of human malignant tumors. The purpose of this study was to clarify the correlation of MGMT expression levels in oral squamous cell carcinoma (OSCC) and cigarette smoking. Study Design: MGMT protein expression in 22 cases of oral squamous cell carcinoma by immunohistochemistry was investigated. Correlation with detailed tobacco history was then tested by statistical analysis. Results: All the non smoker OSCC cases showed positive immunoexpression. However 3 cases of the smoker patient revealed a weak positive immunostaining with only sporadic cells at the periphery. Conclusion: The results suggest that the absence of MGMT expression is related strongly to tobacco smoking and, thus, might be a significant event in oral carcinogenesis.

Stabilization of MAPO1 by specific binding with folliculin and AMP-activated protein kinase in O6-methylguanine-induced apoptosis

When DNA is damaged by alkylating agents, apoptosis is induced to exclude cells carrying DNA lesions in order to prevent mutations and cancer. MAPO1, identified as a component involved in the induction of apoptosis, interacts with AMP-activated protein kinase (AMPK) and folliculin (FLCN). We herein report that MAPO1 is stabilized during the course of apoptosis, triggered by alkylation-induced O6-methylguanine in DNA. An immunoblotting analysis revealed that the amount of MAPO1 increased gradually after treatment with N-methyl-N-nitrosourea (MNU), although the level of mRNA for MAPO1 was unchanged. When cells were exposed to a proteasome inhibitor, MG132, the MAPO1 level significantly increased. On the other hand, application of a protein synthesis inhibitor, cycloheximide, caused a decrease in the MAPO1 content, implying that proteasome-mediated degradation is involved. In FLCN-knockdown cells, the MAPO1 level decreased, and no increases occurred even after MNU treatment. In contrast, stabilization of MAPO1 occurred in AMPKα-knockdown cells even without MNU treatment. While MAPO1 retains its ability to stably bind to FLCN, it dissociates gradually from AMPK after exposure to MNU. It seems that the proapoptotic function of MAPO1 may be regulated by AMPK and FLCN through stabilization of MAPO1 itself.

O6-Methylguanine DNA lesions induce an intra-S-phase arrest from which cells exit into apoptosis governed by early and late multi-pathway signaling network activation

The O(6)-methylguanine (O(6)MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O(6)MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O(6)MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O(6)MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O(6)MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.

Abstract 5465: Role of CYP2A13 in NNK bioactivation and lung tumorigenesis: In vivo studies using a CYP2A13-humanized mouse model

Abstract The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung procarcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis depends on target tissue bioactivation by pulmonary P450 enzymes (Weng et al., 2007, Cancer Res 67:7825-7832). However, the specific P450 enzyme(s) responsible for NNK bioactivation in the lung have not been identified. The aim of the present study was to test the hypotheses that 1) mouse CYP2A5 plays an essential role in NNK bioactivation and tumorigenesis in mouse lung, and 2) transgenic expression of human CYP2A13, known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, can increase the rates of NNK bioactivation and incidence of tumorigenesis in the mouse lung. Kinetic parameters of microsomal NNK bioactivation in vitro, tissue levels of O6-methylguanine (O6-mG) DNA adducts formed in vivo, and incidence of NNK-induced lung tumors were determined for wild-type, Cyp2a5-null, and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. The results indicated that, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the Km value for the formation of 4-oxo-4-(3-pyridyl)-butanal (OPB), which represents the reactive intermediate that can lead to the formation of O6-mG DNA adducts; but, the gain of CYP2A13 led to recovery of the activity in the lung, but not in the liver. The levels of O6-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of the wild-type mice and CYP2A13-humanized mice, than in the Cyp2a5-null mice. Furthermore, lung tumor bioassays, performed for the three mouse strains on A/J background, and using NNK at various doses (30 -200 mg/kg; single i.p. dose), revealed that lung tumor multiplicity determined at 4 months post-treatment was significantly greater in wild-type and CYP2A13-humanized mice than in Cyp2a5-null mice, for all NNK doses tested. The difference in lung tumor multiplicity between CYP2A13-humanized mice and Cyp2a5-null mice was greater at low (30 mg/kg) NNK dose (∼70%) than at high (200 mg/kg) NNK dose (∼30%). In control experiments, rates of systemic clearance of NNK and its major circulating metabolite, NNAL, were confirmed to be not different among the three mouse strains. These results indicated that CYP2A13 is a low-Km enzyme in catalyzing NNK bioactivation in vivo, and they provide strong support for the idea that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans. Moreover, the CYP2A13-humanized mouse model will be valuable for testing new, CYP2A13-targeted chemopreventive agents for lung carcinogenesis. (Supported in part by NIH grant CA092596) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5465. doi:1538-7445.AM2012-5465

Western Pacific ALS-PDC: a prototypical neurodegenerative disorder linked to DNA damage and aberrant proteogenesis?

Western Pacific ALS-PDC: a prototypical neurodegenerative disorder linked to DNA damage and aberrant proteogenesis?

Development and Validation of a New, Sensitive Immunochemical Assay for O6-Methylguanine in DNA and Its Application in a Population Study

Investigations of the presence of the precarcinogenic DNA adduct O⁶-methylguanine (O6-meG) in humans and its association with exposure or cancer risk have been hindered by the absence of analytic methods of adequate sensitivity and throughput. We report the development, validation, and application of an ELISA-type assay for O6-meG appropriate for large-scale population studies. In the new analytic method, restriction enzymes are used to digest DNA to fragments of size expected to contain no more than one O6-meG residue. Anti-adduct antisera are used to transfer O6-meG-containing fragments to a solid surface, where they are detected using anti-ssDNA antisera, the high ratio of normal nucleotides to adducts providing a strong signal enhancement. An assay with a limit of detection of 1.5 adducts/10⁹ nucleotides using 10 μg of DNA, a dynamic range of approximately two orders of magnitude and satisfactory precision and accuracy characteristics was established and validated. Analysis of samples from 120 subjects from the Rhea mother-child cohort in Crete led to the detection of O6-meG in 70% of maternal and 50% of cord blood buffy coat samples at mean levels of 0.65 and 0.38 adducts/10⁸ nucleotides, respectively. The frequent observation of O6-meG in human DNA is compatible with dietary compounds (e.g. N-nitroso compounds or their precursors), or endogenous processes being responsible for the formation of this adduct. The new assay opens the way for large-scale population studies of O6-meG as a biomarker of exposure or risk. The approach used in this assay can, in principle, be extended to any DNA adduct for which suitable antisera are available.

O6-methylguanine-DNA methyltransferase gene coding region polymorphisms and oral cancer risk

O (6) -methylguanine-DNA methyltransferase (MGMT) is a specific DNA direct reversal repair protein which ameliorates mutagenic, carcinogenic and cytotoxic adducts from O(6) -methylguanine in DNA. The aim of this study was to investigate the potential association between six polymorphisms in the coding region of the MGMT gene and oral cancer risk in Taiwanese population. In this hospital-based case-control study, the MGMT genotypes were determined by using DNA sequencing in 176 patients with oral squamous cell carcinoma (OSCC), 77 patients with oral premalignant lesions and 110 normal individuals. Although L53L and L84F polymorphisms were in complete linkage disequilibrium, no statistically significant association was found between the MGMT genotypes and haplotypes and oral cancer risk. We could also not detect any variations in the codon 65, 143, 160 and 178 within the coding region of the MGMT gene in both patients and controls. After stratification of OSCC patients by their mean age (54 years old), however, a higher overall survival rate with CT genotype than CC genotypes of the L53L polymorphisms was found in older patients (P=0.031). A borderline significance (P=0.074) between these genotypes and recurrence-free survivals has also been noted. The same results were also observed in L84F polymorphism. The present study did not find statistically significant association between six common MGMT polymorphisms and oral cancer risk, however, both MGMT L53L and L84F polymorphisms in old patients with CT genotype have higher overall survival rates than patients with CC genotype. Because of limited power of the present study, further study may be warranted in ethnically different populations to explore outcomes of these polymorphisms in the oral cancer risk.