methylguanine-DNA Methyltransferase Research Articles

Clinical validation of a novel quantitative assay for the detection of MGMT methylation in glioblastoma patients

BackgroundThe promoter hypermethylation of the methylguanine-DNA methyltransferase gene is a frequently used biomarker in daily clinical practice as it is associated with a favorable prognosis in glioblastoma patients treated with temozolamide. Due to the absence of adequately standardized techniques, international harmonization of the MGMT methylation biomarker is still an unmet clinical need for the diagnosis and treatment of glioblastoma patients.ResultsIn this study we carried out a clinical validation of a quantitative assay for MGMT methylation detection by comparing a novel quantitative MSP using double-probe (dp_qMSP) with the conventional MSP in 100 FFPE glioblastoma samples. We performed both technologies and established the best cutoff for the identification of positive-methylated samples using the quantitative data obtained from dp_qMSP. Kaplan–Meier curves and ROC time dependent curves were employed for the comparison of both methodologies.ConclusionsWe obtained similar results using both assays in the same cohort of patients, in terms of progression free survival and overall survival according to Kaplan–Meier curves. In addition, the results of ROC(t) curves showed that dp_qMSP increases the area under curve time-dependent in comparison with MSP for predicting progression free survival and overall survival over time. We concluded that dp_qMSP is an alternative methodology compatible with the results obtained with the conventional MSP. Our assay will improve the therapeutic management of glioblastoma patients, being a more sensitive and competitive alternative methodology that ensures the standardization of the MGMT-biomarker making it reliable and suitable for clinical use.

Perfusion Parameter Obtained on 3-Tesla Magnetic Resonance Imaging and the Ki-67 Labeling Index Predict the Overall Survival of Glioblastoma

Pulsed arterial spin-labeling, diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS) are useful for predicting glioma survival. We performed a comparative review of multiple parameters obtained using these pulse sequences on 3-Tesla magnetic resonance imaging (MRI) including the molecular status and Ki-67 labeling index in newly diagnosed supratentorial glioblastomas. A total of 35 patients with glioblastomas underwent pulsed arterial spin-labeling, DTI, and MRS studies using 3-Tesla MRI preoperatively. The isocitrate dehydrogenase (IDH) mutation status, methylguanine-DNA methyltransferase methylation status, and Ki-67 labeling index were calculated from the tumor specimen. Cutoff values were identified by analyzing a receiver operating characteristic curve, and the multivariate survival statistical technique was performed to determine the significant and independent parameters for predicting overall survival. The multivariate Cox analysis showed that the maximum/mean relative cerebral blood flow (rCBF) ratio and the Ki-67 labeling index were significant and independent predictive parameters with a cutoff value of 1.589 for the maximum rCBF ratio, 1.286 for the mean rCBF ratio, and 19% for the Ki-67 labeling index and hazard ratios of 6.132 and 5.119, respectively. The Kaplan-Meier survival curves showed that patients with higher rCBF ratios and Ki-67 labeling indices had a shorter overall survival than others, with median overall survival durations of 479 (95% CI, 370-559) and 1243 (95% CI, 666-NA) days, respectively (P= 0.000167). Our findings indicate that the preoperative rCBF ratio and Ki-67 labeling index are useful parameters for predicting the overall survival of cerebral glioblastomas.

The Potential Use of Radiomics with Pre-Radiation Therapy MR Imaging in Predicting Risk of Pseudoprogression in Glioblastoma Patients.

Glioblastoma (GBM) is the most common adult glioma. Differentiating post-treatment effects such as pseudoprogression from true progression is paramount for treatment. Radiomics has been shown to predict overall survival and MGMT (methylguanine-DNA methyltransferase) promoter status in those with GBM. A potential application of radiomics is predicting pseudoprogression on pre-radiotherapy (RT) scans for patients with GBM. A retrospective review was performed with radiomic data analyzed using pre-RT MRI scans. Pseudoprogression was defined as post-treatment findings on imaging that resolved with steroids or spontaneously on subsequent imaging. Of the 72 patients identified for the study, 35 were able to be assessed for pseudoprogression, and 8 (22.9%) had pseudoprogression. A total of 841 radiomic features were examined along with clinical features. Receiver operating characteristic (ROC) analyses were performed to determine the AUC (area under ROC curve) of models of clinical features, radiomic features, and combining clinical and radiomic features. Two radiomic features were identified to be the optimal model combination. The ROC analysis found that the predictive ability of this combination was higher than using clinical features alone (mean AUC: 0.82 vs. 0.62). Additionally, combining the radiomic features with clinical factors did not improve predictive ability. Our results indicate that radiomics is potentially capable of predicting future development of pseudoprogression in patients with GBM using pre-RT MRIs.

Automated machine learning to predict the co-occurrence of isocitrate dehydrogenase mutations and O6 -methylguanine-DNA methyltransferase promoter methylation in patients with gliomas.

Combining isocitrate dehydrogenase mutation (IDHmut) with O6 -methylguanine-DNA methyltransferase promoter methylation (MGMTmet) has been identified as a critical prognostic molecular marker for gliomas. The aim of this study was to determine the ability of glioma radiomics features from magnetic resonance imaging (MRI) to predict the co-occurrence of IDHmut and MGMTmet by applying the tree-based pipeline optimization tool (TPOT), an automated machine learning (autoML) approach. This was a retrospective study, in which 162 patients with gliomas were evaluated, including 58 patients with co-occurrence of IDHmut and MGMTmet and 104 patients with other status comprising: IDH wildtype and MGMT unmethylated (n=67), IDH wildtype and MGMTmet (n=36), and IDHmut and MGMT unmethylated (n=1). Three-dimensional (3D) T1-weighted images, gadolinium-enhanced 3D T1-weighted images (Gd-3DT1WI), T2-weighted images, and fluid-attenuated inversion recovery (FLAIR) images acquired at 3.0 T were used. Radiomics features were extracted from FLAIR and Gd-3DT1WI images. The TPOT was employed to generate the best machine learning pipeline, which contains both feature selector and classifier, based on input feature sets. A 4-fold cross-validation was used to evaluate the performance of automatically generated models. For each iteration, the training set included 121 subjects, while the test set included 41 subjects. Student's t-test or a chi-square test was applied on different clinical characteristics between two groups. Sensitivity, specificity, accuracy, kappa score, and AUC were used to evaluate the performance of TPOT-generated models. Finally, we compared the above metrics of TPOT-generated models to identify the best-performing model. Patients' ages and grades between two groups were significantly different (p=0.002 and p=0.000, respectively). The 4-fold cross-validation showed that gradient boosting classifier trained on shape and textual features from the Laplacian-of-Gaussian-filtered Gd-3DT1 achieved the best performance (average sensitivity=81.1%, average specificity=94%, average accuracy=89.4%, average kappa score=0.76, average AUC=0.951). Using autoML based on radiomics features from MRI, a high discriminatory accuracy was achieved for predicting co-occurrence of IDHmut and MGMTmet in gliomas. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 3.

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.

Disentangling the therapeutic tactics in GBM: From bench to bedside and beyond.

Glioblastoma multiforme (GBM) is one of the most common and malignant form of adult brain tumor with a high mortality rate and dismal prognosis. The present standard treatment comprising surgical resection followed by radiation and chemotherapy using temozolomide can broaden patient's survival to some extent. However, the advantages are not palliative due to the development of resistance to the drug and tumor recurrence following the multimodal treatment approaches due to both intra- and intertumoral heterogeneity of GBM. One of the major contributors to temozolomide resistance is O6 -methylguanine-DNA methyltransferase. Furthermore, deficiency of mismatch repair, base excision repair, and cytoprotective autophagy adds to temozolomide obstruction. Rising proof additionally showed that a small population of cells displaying certain stem cell markers, known as glioma stem cells, adds on to the resistance and tumor progression. Collectively, these findings necessitate the discovery of novel therapeutic avenues for treating glioblastoma. As of late, after understanding the pathophysiology and biology of GBM, some novel therapeutic discoveries, such as drug repurposing, targeted molecules, immunotherapies, antimitotic therapies, and microRNAs, have been developed as new potential treatments for glioblastoma. To help illustrate, "what are the mechanisms of resistance to temozolomide" and "what kind of alternative therapeutics can be suggested" with this fatal disease, a detailed history of these has been discussed in this review article, all with a hope to develop an effective treatment strategy for GBM.

CTNI-76. PHASE 2 CLINICAL TRIAL OF VAL-083 IN NEWLY DIAGNOSED MGMT-UNMETHYLATED GBM

Abstract Approximately 60% of glioblastoma multiforme (GBM) patients possess an unmethylated methylguanine DNA-methyltransferase (MGMT) promoter region, which confers a limited response to standard-of-care treatment with temozolomide (TMZ), resulting in shorter median survival when compared to patients with methylated MGMT promoter. VAL-083 is a novel bi-functional DNA targeting agent that induces inter-strand cross-links at N7-guanine, leading to DNA double-strand breaks and ultimately cell death. VAL-083 circumvents MGMT-mediated TMZ resistance in vitro and in vivo. A Phase 2 study has been initiated for VAL-083 in newly diagnosed MGMT unmethylated GBM. The study has 2 stages: Stage 1 is a dose-escalation safety and tolerability phase to confirm the phase 2 dose of VAL-083 when administered concurrently with radiation therapy (RT). Patients received VAL-083 at 20, 30, or 40 mg/m2/day x 3 days every 21 days along with standard radiation treatment (RT) (2 Gy/day, 5 days/week). The dose escalation stage is complete, and 30 mg/m2/day of VAL-083 in combination with RT was generally safe and well-tolerated. Stage 2 comprises an expansion phase to enroll up to 20 additional patients at the 30 mg/m2/day of VAL-083 in combination with RT. As of June 2, 2020, all patients have been enrolled, with a total of 29 patients in the study, and 25 patients receiving 30 mg/m2/day VAL-083. Of the 29 patients enrolled, 27 have completed their prospectively planned MRI scans and had their initial assessment for tumor response. Two additional patients died prior to their post-cycle 3 MRI. Consistent with our prior experience, myelosuppression was the most common adverse event. Three patients have experienced dose-limiting toxicities - one (1/3; 33%) at the 40 mg/m2/day and two (2/25; 8%) at the 30 mg/m2/day dose. Further safety and efficacy updates will be presented at the meeting. Clinicaltrials.gov identifier: NCT03050736.

NIMG-37. DELAYED PSEUDOPROGRESSION IN TWO PATIENTS UNDERGOING TTFIELDS TREATMENT FOR NEWLY DIAGNOSED GLIOBLASTOMA

Abstract PURPOSE Tumor treatment fields (TTFields) are approved by the FDA for newly diagnosed as well as recurrent glioblastoma (GBM). TTFields have been shown to extend survival by 4.9 months in newly diagnosed GBM, and a landmark overall survival rate of 13% at 5 years. However, the specific effects remain widely unknown, which has prevented widespread clinical use of this treatment. METHODS This case study examines two glioblastoma patients, IDH-1 wildtype, MGMT unmethylated, that received TTFields (Optune) in addition to maintenance temozolomide (TMZ) following radiation (RT). Both cases were followed using standard MR imaging. Second resections were performed due to radiographic progression of contrast enhancement. RESULTS Although imaging was concerning for tumor progression, pathology showed only treatment effect, ultimately leading to the diagnosis of pseudoprogression. Both patients fell outside the normal expected timeline for chemo-radiation induced pseudoprogression. Based on the pathology, both patients resumed treatment with TMZ/TTFields. One patient expired at 25 months and one is still alive. CONCLUSIONS Pathologic confirmation was essential for guiding further treatment and allowed patients to continue treatment that was effective despite contrary indications on imaging. These findings suggest that pathological confirmation should be strongly considered in patients receiving TMZ/TTFields who develop radiographic progression, potentially with a less invasive biopsy procedure. Future studies should look to characterize the underlying mechanism of interaction between TTFields/TMZ and quantify the prevalence, associated risk factors and whether there is a genotype more susceptible. Both patients reported here had O(6)-methylguanine-DNA methyltransferase (MGMT) unmethylated GBM, and while about 60% of glioblastomas are diagnosed likewise, it is possible that MGMT methylation status plays a role in TTFields response. Better characterization of this phenomenon will improve treatment guidance, potentially reducing unnecessary surgeries and the discontinuance of successful therapies.

The Effects of O6-methyl Guanine DNA-methyl Transferase Promotor Methylation and CpG1, CpG2, CpG3 and CpG4 Methylation on Treatment Response and their Prognostic Significance in Patients with Glioblastoma.

This retrospective study examined the prognostic significance and treatment effect of promoter methylation of O6- methyl guanine methyl transferase (MGMT) and meth-ylation of CpG 1, CpG2, CpG3 and CpG4 in glioblastoma (GB) patients received postoperative radiotherapy (PORT), with or without adjuvant temozolomide (TMZ). One hundred patients with GB who received PORT with concomitant TMZ plus adjuvant TMZ or PORT alone, were included. The MGMT promoter methylation of CpG1, CpG2, CpG3 and CpG4 islands were examined. Overall, MGMT-methylation emerged as a significant prognostic factor for better overall survival (OS) and progression-free survival (PFS) [odds ratio (OR): 0.609, 95% confidence interval (95% CI): 0.395-0.939, p = 0.02; OR: 0.662,95% CI: 0.430-1019, p = 0.5, respectively]. The methylation of each CpG1, CpG2, CpG3 and CpG4 islands was found to have no significant effects on OS and the methylation of each CpGl, CpG2 and CpG4 islands had no significant effect on PFS (p <0.05 for all). On the other hand, the methylation of CpG3 had a positive prognostic effect on PFS (OR: 2.1, 95% CI: 0.99-4.67, p = 0.04). In the group that only received radiotherapy (RT), CpG1 and CpC3 methylations were found to have a positive prognostic significance in terms of PFS (OR: 266, 95% CI: 1.05-6.75, p -0.03 for CpG1; OR: 2.4, 95% CI: 1.01-5.92, p = 0.04 for CpG3). The MGMT promoter methylation represents an important biomarker for predicting response to therapy. Individual islands, particularly CpG3, deserves further investigation as a prognostic marker. Further studies need to be done with larger sample sizes to clarify the results.

Preconditioning with INC280 and LDK378 drugs sensitizes MGMT-unmethylated glioblastoma to temozolomide: Pre-clinical assessment

Temozolomide (TMZ) therapy is the standard of care for patients with glioblastoma (GBM). Clinical studies have shown that elevated levels of DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) or deficiency/defect of DNA mismatch repair (MMR) genes is associated with TMZ resistance in some, but not all, GBM tumors. Another reason for GBM treatment failure is signal redundancy due to coactivation of several functionally linked receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK) and c-Met (hepatocyte growth factor receptor). As such, these tyrosine kinases serve as potential targets for GBM therapy. Thus, we tested two novel drugs: INC280 (Capmatinib: a highly selective c-Met receptor tyrosine kinase-RTK inhibitor) and LDK378 (Ceritinib: a highly selective anaplastic lymphoma kinase-ALK inhibitor), aiming to overcome TMZ resistance in MGMT-unmethylated GBM cells in in vitro cell culture models. Treatments were examined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, caspase-3 assay and western blot analysis. Results obtained from our experiments demonstrated that preconditioning with INC280 and LDK378 drugs exhibit increased MMR protein expression, specifically MMR protein MLH1 (MutL Homolog 1) and MSH6 (MutS Homolog 6) and sensitized TMZ in MGMT-unmethylated GBM cells via suppression of ALK and c-Met expression. INC280 and LDK378 plus TMZ also induced apoptosis by modulating downstream signaling of PI3K/AKT/STAT3. Taken together, this data indicates that co-inhibition of ALK and c-MET can enhance growth inhibitory effects in MGMT-unmethylated cells and enhance TMZ sensitivity in-vitro, suggesting c-Met inhibitors combined with ALK-targeting provide a therapeutic benefit in MGMT-unmethylated GBM patients.

TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT.

BackgroundOur previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.MethodsThe distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.ResultsOverexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.ConclusionsOur results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.

Adaptive Radiotherapy for Glioblastoma Multiforme - The Impact on Disease Outcome.

To study the changes of glioblastoma multiforme during chemoradiotherapy (CRT) and to evaluate the impact of changes on dosimetry and clinical outcomes. Forty-three patients underwent volumetric imaging-based replanning. Prognostic factors and gross tumor volume changes in relation to overall survival and the effect of adaptive replanning were statistically analyzed. Patients with total tumor removal, with shorter time to CRT (<27 days), with methylated O-6 methylguanine DNA methyltransferase and good performance status (>60%) had better survival. Tumor shrinkage in 24 patients resulted in improved survival compared to 19 in whom tumor was unchanged or progressed (25.3 vs. 11.1 months, p=0.04). Adapted planning target volume allowed a reduction in irradiated volume, while increasing survival (12.06 vs. 28.98 months, p=0.026). Tumor response during CRT has significant impact on the outcome. Adaptation of the planning target volume to the tumor changes proved to be beneficial and warrants further investigation.

MGMT-inhibitor in combination with TGF-βRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells.

Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-βRI inhibitor) seeking to overcome GB treatment resistance. Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-β-dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells.

A Ki-67 Index to Predict Treatment Response to the Capecitabine/Temozolomide Regimen in Neuroendocrine Neoplasms: A Retrospective Multicenter Study

Objective: The efficacy of the capecitabine/temozolomide (CAPTEM) regimen has been demonstrated in metastatic neuroendocrine neoplasms (NENs), but because of varying response rates among the patients, biomarkers to predict its response are greatly needed. Here, we investigated the clinical utility of a Ki-67 index to predict the CAPTEM regimen objective responses and select patients who could benefit from this regimen. Methods: Metastatic NENs patients treated with the CAPTEM regimen from 4 high-volume medical centers were selected and grouped in a training and validation cohort. The classification and regression tree (CART) was generated to identify the optimal threshold of Ki-67 for stratifying the patients into different Ki-67 range groups based on their response to the CAPTEM regimen. Results and Conclusions: The overall response rate (ORR) and disease control rate of the entire cohort (N = 151) were 26.5 and 76.2%, respectively, with a median progression-free survival (PFS) of 12.0 months. CART analysis showed that patients in the Ki-67 range group 10–40% demonstrated a significantly higher ORR than those in Ki-67 >40 and <10% groups (p < 0.001 in the training cohort and p = 0.036 in the validation cohort). Response to the CAPTEM regimen was not influenced by the expression of O⁶-methylguanine-DNA methyltransferase or primary tumor location. Multivariate analysis identified the Ki-67 index as the only independent prognostic factor for overall survival (p = 0.031) and PFS (p = 0.006). The proposed Ki-67 index was externally validated and could be used to clinically identify suitable metastatic NENs patients who could achieve an optimal cytoreduction using the CAPTEM regimen.

Pulsed and Discontinuous Electromagnetic Field Exposure Decreases Temozolomide Resistance in Glioblastoma by Modulating the Expression of O6-Methylguanine-DNA Methyltransferase, Cyclin-D1, and p53.

Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O6-methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.

Stereotactic reirradiation with temozolomide in patients with recurrent aggressive pituitary tumors and pituitary carcinomas.

To evaluate the efficacy of a second course of fractionated stereotactic radiotherapy (re-SRT) and temozolomide (TMZ) as salvage treatment option in patients with aggressive pituitary tumors (APTs) and pituitary carcinomas (PCs). Twenty-one patients with recurrent or progressive APTs (n = 17) and PCs (n = 4) who received combined TMZ and re-SRT, 36Gy/18fractions or 37.5Gy/15fractions, were retrospectively evaluated. TMZ was given at a dose of 75mg/m2 given concurrently to re-SRT, and then 150-200mg/m2/day for 5days every 4weeks or 50mg/m2 daily for 12months. Local control (LC) and overall survival (OS) were calculated from the time of re-SRT by Kaplan-Meier method. With a median follow-up of 27months (range 12-58months), 2-year and 4-year LC rates were 73% and 65%, respectively; 2-year and 4-year survival rates were 82% and 66%, respectively. A complete response was achieved in 2 and partial response in 11 patients. Six patients recurred with a median time to progression of 14months. O(6)-Methylguanine-DNA methyltransferase (MGMT) status and tumor volume emerged as prognostic factors. Grade 3 radiation-related toxicities occurred in 3 (14%) patients. Grade 2 or 3 hematologic toxicities during chemotherapy occurred in 8 (38%) patients. Re-SRT and TMZ is a safe treatment offering high LC in patients with progressive APTs and PCs. The potential advantages of combined chemoradiation as up-front or salvage treatment need to be explored in prospective trials.

Curcumin Administered as Micellar Solution Suppresses Intestinal Inflammation and Colorectal Carcinogenesis

Colorectal cancer (CRC) is one of the most common cancers and preventive strategies based on natural compounds are highly desirable. Curcumin, the principal bioactive compound in Curcuma longa, was described to have multiple beneficial health effects. A drawback, however, is the low bioavailability due to its insolubility in water. Here, we studied whether nanoscaled micellar curcumin with improved bioavailability administered in drinking water reduces inflammation and CRC formation in a mouse model. C57BL6 wild-type (WT) mice and a strain defective in the DNA repair enzyme O6 -methylguanine-DNA methyltransferase (MGMT) were used, in which tumors were induced by azoxymethane (AOM) followed by dextran sodium sulfate (DSS). Inflammation and tumor formation were determined by mini-colonoscopy. Micellar curcumin (mCur) administered in drinking water significantly reduced AOM/DSS-induced colorectal inflammation in both WT and MGMT-deficient mice as compared to animals receiving drinking water with micelles not containing curcumin. In line with this, the tumor yield and tumor score were significantly lower in mCur-treated mice compared to the control group. No adverse effects were observed in animals receiving mCur daily for at least three months. Overall, our data show that chronic oral administered micellar curcumin is well tolerated and reduces chemical-induced gut inflammation and CRC formation in mice. Impact: The study shows that micellar curcumin with high bioavailability chronically administered at low and physiologically relevant concentration suppresses inflammation and carcinogenesis in a mouse colorectal tumor model.

Direct DNA Methylation Profiling with an Electric Biosensor.

DNA methylation is one of the principal epigenetic mechanisms that control gene expression in humans, and its profiling provides critical information about health and disease. Current profiling methods require chemical modification of bases followed by sequencing, which is expensive and time-consuming. Here, we report a direct and rapid determination of DNA methylation using an electric biosensor. The device consists of a DNA-tweezer probe integrated on a graphene field-effect transistor for label-free, highly sensitive, and specific methylation profiling. The device performance was evaluated with a target DNA that harbors a sequence of the methylguanine-DNA methyltransferase, a promoter of glioblastoma multiforme, a lethal brain tumor. The results show that we successfully profiled the methylated and nonmethylated forms at picomolar concentrations. Further, fluorescence kinetics and molecular dynamics simulations revealed that the position of the methylation site(s), their proximity, and accessibility to the toe-hold region of the tweezer probe are the primary determinants of the device performance.

Biological Basis for Threshold Responses to Methylating Agents.

The cellular outcomes of chemical exposure are as much about the cellular response to the chemical as it is an effect of the chemical. We are growing in our understanding of the genotoxic interaction between chemistry and biology. For example, recent data has revealed the biological basis for mutation induction curves for a methylating chemical, which has been shown to be dependent on the repair capacity of the cells. However, this is just one end point in the toxicity pathway from chemical exposure to cell death. Much remains to be known in order for us to predict how cells will respond to a certain dose. Methylating agents, a subset of alkylating agents, are of particular interest, because of the variety of adverse genetic end points that can result, not only at increasing doses, but also over time. For instance, methylating agents are mutagenic, their potency, for this end point, is determined by the cellular repair capacity of an enzyme called methylguanine DNA-methyltransferase (MGMT) and its ability to repair the induceed methyl adducts. However, methyl adducts can become clastogenic. Erroneous biological processing will convert mutagenic adducts to clastogenic events in the form of double strand breaks (DSBs). How the cell responds to DSBs is via a cascade of protein kinases, which is called the DNA damage response (DDR), which will determine if the damage is repaired effectively, via homologous recombination, or with errors, via nonhomologous end joining, or whether the cell dies via apoptosis or enters senescence. The fate of cells may be determined by the extent of damage and the resulting strength of DDR signaling. Therefore, thresholds of damage may exist that determine cell fate. Such thresholds would be dependent on each of the repair and response mechanisms that these methyl adducts stimulate. The molecular mechanism of how methyl adducts kill cells is still to be fully resolved. If we are able to quantify each of these thresholds of damage for a given cell, then we can ascertain, of the many adducts that are induced, what proportion of them are mutagenic, what proportion are clastogenic, and how many of these clastogenic events are toxic. This review examines the possibility of dose and damage thresholds for methylating agents, from the perspective of the underlying evolutionary mechanisms that may be accountable.

Association between glioblastoma cell-derived vessels and poor prognosis of the patients.

BackgroundVessels with different microcirculation patterns are required for glioblastoma (GBM) growth. However, details of the microcirculation patterns in GBM remain unclear. Here, we examined the microcirculation patterns of GBM and analyzed their roles in patient prognosis together with two well‐known GMB prognosis factors (O6‐methylguanine DNA methyltransferase [MGMT] promoter methylation status and isocitrate dehydrogenase [IDH] mutations).MethodsEighty GBM clinical specimens were collected from patients diagnosed between January 2000 and December 2012. The microcirculation patterns, including endothelium‐dependent vessels (EDVs), extracellular matrix‐dependent vessels (ECMDVs), GBM cell‐derived vessels (GDVs), and mosaic vessels (MVs), were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) staining in both GBM clinical specimens and xenograft tissues. Vascular density assessments and three‐dimensional reconstruction were performed. MGMT promoter methylation status was determined by methylation‐specific PCR, and IDH1/2 mutations were detected by Sanger sequencing. The relationship between the microcirculation patterns and patient prognosis was analyzed by Kaplan‐Meier method.ResultsAll 4 microcirculation patterns were observed in both GBM clinical specimens and xenograft tissues. EDVs were detected in all tissue samples, while the other three patterns were observed in a small number of tissue samples (ECMDVs in 27.5%, GDVs in 43.8%, and MVs in 52.5% tissue samples). GDV‐positive patients had a median survival of 9.56 months versus 13.60 months for GDV‐negative patients (P = 0.015). In MGMT promoter‐methylated cohort, GDV‐positive patients had a median survival of 6.76 months versus 14.23 months for GDV‐negative patients (P = 0.022).ConclusionGDVs might be a negative predictor for the survival of GBM patients, even in those with MGMT promoter methylation.