methylguanine-DNA Methyltransferase Promoter Research Articles

MGMT promoter methylation and 1p/19q co-deletion of surgically resected pulmonary carcinoid and large-cell neuroendocrine carcinoma

BackgroundThe response to temozolomide (TMZ) treatment in small-cell lung cancer (SCLC) correlated with O(6)-methylguanine -DNA methyltransferase (MGMT) promoter methylation. 1p/19q co-deletion within oligodendroglioma is a responsive predictor for TMZ. Currently, the status of MGMT promoter methylation and 1p/19q co-deletion in pulmonary carcinoid (PC) and large-cell neuroendocrine carcinoma (LCNEC) is not reported.MethodsNine PC [two atypical carcinoids (AC), seven typical carcinoids (TC)] and six LCNEC patients were collected retrospectively. The pyrosequencing and fluorescence in situ hybridization were used to detect the MGMT promoter methylation and 1p/19q co-deletion in surgically resected specimens. Kaplan–Meier analysis was used to assess the rate of disease-free survival (DFS).ResultsMGMT promoter methylation was found in two (2/6, 15.3%) LCNEC patients but not in any PC patients. Three (3/6, 50%) 1p and two (2/6, 33.3%) 19q single deletions were found in LCNEC patients. One 1p single deletion was found in AC patients. One (1/7, 14.3%) 1p and two (2/7, 28.6%) 19q single deletions were found in TC patients. After a median follow-up of 38 months, three LCNEC patients developed distant metastasis and one patient died of LCNEC disease. The DFS of PC patients was much longer than LCNEC patients (χ2 = 7.565, P = 0.006).ConclusionsMGMT promoter methylation and 1p/19q co-deletion might not be the ideal biomarkers for TMZ treatment in TC/AC patients. Thus, the detection of MGMT promoter methylation and whether it can be used as a medication for TMZ in LCNEC patients necessitates investigation. Furthermore, 1p deletion could be a negative prognostic factor for LCNEC patients.

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.

Transcriptional control of O6 -methylguanine DNA methyltransferase expression and temozolomide resistance in glioblastoma.

O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma-initiating cells (GIC) with stem-like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real-time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF-κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S-to-M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF-κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF-κB by the small molecule inhibitor, BAY 11-7082, or siRNA-mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over-expression of MGMT which results from increased activity of the NF-κB pathway in this cell culture model of glioma stem-like cells. Read the Editorial Highlight for this article on page 688.

Treatment of glioblastoma in adults.

The diagnosis of a glioblastoma is mainly made on the basis of their microscopic appearance with the additional determination of epigenetic as well as mutational analyses as deemed appropriate and taken into account in different centers. How far the recent discovery of tumor networks will stimulate novel treatments is a subject of intensive research. A tissue diagnosis is the mainstay. Regardless of age, patients should undergo a maximal safe resection. Magnetic resonance imaging is the surrogate parameter of choice for follow up. Patients should receive chemoradiotherapy with temozolomide with the radiation schedule adapted to performance status, age and tumor location. The use of temozolomide may be reconsidered according to methylguanine DNA methyltransferase (MGMT) promoter methylation status; patients with an active promoter may be subjected to a trial or further molecular work-up in order to potentially replace temozolomide; patients with an inactive (hypermethylated) MGMT promoter may be counseled for the co-treatment with the methylating and alkylating compound lomustine in addition to temozolomide. Tumor-treating fields are an additive option independent of the MGMT status. Determination of recurrence is still challenging. Patients with clinical or radiographic confirmed progression should be counseled for a second surgical intervention, that is, to reach another macroscopic removal of the tumor bulk or to obtain tissue for an updated molecular analysis. Immune therapeutic approaches may be dependent on tumor types and molecular signatures. In newly diagnosed and recurrent glioblastoma, bevacizumab prolongs progression-free survival without affecting overall survival in an unselected population of glioblastoma patients. Whether or not selection can be made on the basis of molecular or imaging parameters remains to be determined. Some patients may benefit from a second radiotherapy. In our view, the near future will provide support for translating the amazing progress in understanding the molecular background of glioblastoma in to more complex, but promising therapy concepts

Repair gene O6 -methylguanine-DNA methyltransferase is controlled by SP1 and up-regulated by glucocorticoids, but not by temozolomide and radiation.

Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.

Genetics and Epigenetics of Glioblastoma: Therapeutic Challenges

Glioblastoma is a brain tumor that develops due to both genetic and epigenetic risk factors. Crosstalk between the genetic and the epigenetic offers new possibilities for therapy. Abnormal methylation of methylguanine-DNA methyltransferase (MGMT) promoter region and isocitrate dehydrogenase 1 (IDH1) mutations are prognostic and therapeutic response markers in glioblastoma. Mutations in genes such as epidermal growth factor receptor, TP53, and P16 have been reported in glioblastoma; therefore, they might associate with survival and worth to be used in estimating survival risks. MKI67 expression associates with posttreatment such as adjuvant radiotherapy results evaluation. On the other hand, monosomies, such as deletions of chromosome 10, especially q23 and q25–26, are good markers for estimating the progression and aggressiveness of glioblastoma. The profile of MGMT methylation is modified in glioblastoma and hence can be a good target for epigenetic drugs. Other useful strategies in the treatment of gliomas include several micro-RNAs (MiRs) which are alerted in glioblastoma and which affect the regulation of mRNAs are associated with gene expression profiles of the disease. Epigenetic drugs, such as azacitidine and decitabine, which belong to the DNA methyltransferases (DNMT) inhibitor 5-aza-2'deoxycytidine (5-aza-dC), can suppress DNMT1 and stimulate tumor suppressor genes expression. MGMT methylation status and IDH mutational status are two valuable prognosis and therapeutic response markers in glioblastoma. Regulation of glioblastoma through epigenetic drugs, such as not only inhibitors of EZH2, histone deacetylase, and DNMT, but also MiRs, are promising approaches in glioblastoma treatment. Improves in understanding cancer genetic and epigenetic disruptions is the key point in solving the puzzle of glioblastoma treatment.

Clinical and Prognostic Significance of O6-Methylguanine-DNA Methyltransferase Promoter Methylation in Patients with Melanoma: A Systematic Meta-Analysis.

Tumor suppressor gene O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been reported in melanoma. However, the clinical and prognostic significance of MGMT promoter methylation in patients with melanoma remained to be determined. A systematic search was performed to identify eligible papers published. The overall odds ratios (ORs) or hazard ratios and their 95% confidence intervals were calculated. Final 12 eligible publications involving Caucasian population were performed in this study, including 1,071 metastatic melanoma patients, 154 primary melanoma patients, and 211 normal controls. MGMT promoter methylation was significantly higher in primary or metastatic melanoma than in normal controls (p<0.05). No difference of MGMT promoter methylation was found in primary and metastatic melanoma (p=0.432). When metastatic melanoma was compared to normal controls, subgroup analysis showed the correlation between MGMT promoter methylation and different sample materials (tissue: OR=7.01, p<0.001 and blood: OR=12.04, p=0.005). MGMT promoter methylation was not associated with response to drug therapy and the prognosis in overall survival and progression-free survival for multivariate analysis. Our results show that MGMT promoter methylation may be correlated with the increased risk of primary or metastatic melanoma. Based on blood samples, MGMT promoter methylation may become a noninvasive biomarker for the detection of metastatic melanoma. Further additional clinical studies are necessary.

A 4-miRNAs signature predicts survival in glioblastoma multiforme patients.

Although O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation status is an important marker for glioblastoma multiforme (GBM), there is considerable variability in the clinical outcome of patients with similar methylation profles. We examined whether a MicroRNA (miRNA) signature can be identified for predicting clinical outcomes and helping in treatment decisions. The differentially expressed miRNAs were evaluated in 6 pairs of short- (⩽ 450 days) and long-term survivors (>450 days) by using microarray. Real time quantitative PCR (qRT-PCR) was applied to further verify screened miRNAs with a greater number of samples (n= 48). Meanwhile, functional interpretation of miRNA profile was carried out based on miRNA-target databases. In addition, MGMT promoter methylation status was tested by means of pyrosequencing (PSQ) testing. Six miRNAs were upregulated in the long-term survival group (fold change ⩾ 2.0, P< 0.05). The further verification by qRT-PCR indicated that the increase in let-7g-5p, miR-139-5p, miR-17-5p and miR-9-3p level in long-term survivors was statistically significant. Kaplan-Meier survival analysis showed that high expression of a prognostic 4-miRNA signature was significantly associated with good patient survival (p= 0.0012). The signature regulated signaling pathways including Calcium, MAPK, ErbB, mTOR and cell cycle involved in carcinogenesis from glial progenitor cell to primary GBM. The 4-miRNA signature was identified as an independent prognostic biomarker that identified patients who have a favorable outcome.

Prognostic relevance of programmed cell death ligand 1 expression in glioblastoma.

The aim of this study was to determine the clinicopathological significance of programmed cell death ligand 1 (PD-L1) expression in glioblastoma (GBM). In a retrospective cohort of 115 consecutive patients with GBM, PD-L1 expression was determined using immunohistochemistry (IHC). Membranous and fibrillary PD-L1 staining of any intensity in > 5% neoplastic cells and tumour infiltrating immune cells (TIIs) was considered positive staining. In addition, isocitrate dehydrogenase-1 (IDH-1) (R132H) expression and cluster of differentiation 3 (CD3)-positive T-cell infiltration were investigated using IHC. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation assay and fluorescence in situ hybridization (FISH) for the assessment of 1p/19q deletion were performed. Expression of PD-L1 in tumour cells and TIIs was found in 37 (32.2%) and 6 (5.2%) patients, respectively. Kaplan-Meier analysis indicated that PD-L1 expression in tumour cells was significantly associated with poor overall survival (OS) (P = 0.017), though multivariate Cox analysis did not confirm this association (hazard ratio 1.204; P = 0.615). PD-L1 expression in TIIs did not correlate with the patient prognosis (P = 0.545). In addition, MGMT methylation and IDH-1 (R132H) expression were associated with a better prognosis (P < 0.001 and P = 0.024, respectively). The expression of PD-L1 was associated with CD3-positive T-cell infiltration (P < 0.001), and IDH-1 wild type status (P = 0.008). A deeper insight into PD-L1 expression could help to ensure the success of future immunotherapy in GBM. Our study suggested that PD-L1 target therapy might be beneficial for PD-L1-expressing GBM patients with a poor prognosis.

Patients Affected by Unmethylated O(6)-Methylguanine-DNA Methyltransferase Glioblastoma Undergoing Radiochemotherapy May Benefit from Moderately Dose-Escalated Radiotherapy.

Purpose To compare the therapeutic results of two radiotherapy (RT) dose schedules in combined temozolomide- (TMZ-) RT treatment in newly diagnosed glioblastoma (GB), according to the O(6)-methylguanine-DNA methyltransferase (MGMT) methylation status. Material and Method Patients received either standard (60 Gy) or moderately escalated dose (70 Gy) radiotherapy (RT) with concomitant and adjuvant TMZ between June 2006 and October 2013. We retrospectively evaluated the therapeutic effectiveness of RT schedules in terms of Overall Survival (OS) and Progression-Disease Free Survival (PDFS) analyzing the MGMT methylation status. Results One hundred and seventeen patients were selected for the present analysis. Seventy-two out of the selected cases received the standard RT-TMZ course (SDRT-TMZ) whereas the remaining 45 underwent the escalated schedule (HDRT-TMZ). The analysis according to the MGMT promoter methylation status showed that, in unmethylated-MGMT GB patients, HDRT-TMZ and SDRT-TMZ groups had different median OS (p = 0,01) and PDFS (p = 0,007), that is, 8 months and 5 months for the SDRT-TMZ group and 14 months and 9 months for the HDRT-TMZ group, respectively. No difference in survival outcomes was found in methylated MGMT patients according to the two RT schedules (p = 0,12). Conclusions In our experience, unmethylated-MGMT GB patients benefited from a moderately escalated dose of RT plus TMZ.

MGMT expression predicts response to temozolomide in pancreatic neuroendocrine tumors.

Temozolomide (TEM) showed encouraging results in well-differentiated pancreatic neuroendocrine tumors (WDPNETs). Low O(6)-methylguanine-DNA methyltransferase (MGMT) expression and MGMT promoter methylation within tumors correlate with a better outcome under TEM-based chemotherapy in glioblastoma. We aimed to assess whether MGMT expression and MGMT promoter methylation could help predict the efficacy of TEM-based chemotherapy in patients with WDPNET. Consecutive patients with progressive WDPNET and/or liver involvement over 50% who received TEM between 2006 and 2012 were retrospectively studied. Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines. Nuclear expression of MGMT was assessed by immunochemistry (H-score, 0-300) and MGMT promoter methylation by pyrosequencing. Forty-three patients (21 men, 58years (27-84)) with grade 1 WDPNET (n=6) or 2 (n=36) were analyzed. Objective response, stable disease, and progression rates were seen in 17 patients (39.5%), 18 patients (41.9%), and 8 patients (18.6%), respectively. Low MGMT expression (≤50) was associated with radiological objective response (P=0.04) and better progression-free survival (PFS) (HR=0.35 (0.15-0.81), P=0.01). Disease control rate at 18months of treatment remained satisfying with an MGMT score up to 100 (74%) but dropped with a higher expression. High MGMT promoter methylation was associated with a low MGMT expression and longer PFS (HR=0.37 (0.29-1.08), P=0.05). Low MGMT score (≤50) appears to predict an objective tumor response, whereas an intermediate MGMT score (50-100) seems to be associated with prolonged stable disease.

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response

BackgroundThe DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value.ResultsWe analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan–Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP.ConclusionsCompared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0204-7) contains supplementary material, which is available to authorized users.

Glioblastoma in the Canton of Zurich, Switzerland revisited: 2005 to 2009.

A population-based analysis of patients with glioma diagnosed between 1980 and 1994 in the Canton of Zurich in Switzerland confirmed the overall poor prognosis of glioblastoma. To explore changes in outcome, registry data were reevaluated for patients diagnosed between 2005 and 2009. Patients with glioblastoma who were diagnosed between 2005 and 2009 were identified by the Zurich and Zug Cancer Registry. The prognostic significance of epidemiological and clinical data, isocitrate dehydrogenase 1 (IDH1)(R132H) mutation status, and O6 methylguanine DNA methyltransferase (MGMT) promoter methylation status was analyzed using the Kaplan-Meier method and the Cox proportional hazards model. A total of 264 patients with glioblastoma were identified, for an annual incidence of 3.9 compared with the previous incidence of 3.7. The mean age of the patients at the time of diagnosis was 59.5 years in the current cohort compared with 61.3 years previously. The overall survival (OS) rate was 46.4% at 1 year, 22.5% at 2 years, and 14.4% at 3 years in the current study compared with 17.7% at 1 year, 3.3% at 2 years, and 1.2% at 3 years as reported previously. The median OS for all patients with glioblastoma was 11.5 months compared with 4.9 months in the former patient population. The median OS was 1.9 months for best supportive care, 6.2 months for radiotherapy alone, 6.7 months for temozolomide alone, and 17.0 months for radiotherapy plus temozolomide. Multivariate analysis revealed age, Karnofsky performance score, extent of tumor resection, first-line treatment, year of diagnosis, and MGMT promoter methylation status were associated with survival in patients with IDH1(R132H) -nonmutant glioblastoma. The OS of patients newly diagnosed with glioblastoma in the Canton of Zurich in Switzerland markedly improved from 1980 through 1994 to 2005 through 2009. Cancer 2016;122:2206-15. © 2016 American Cancer Society.

Association between the methylation status of the MGMT promoter in bone marrow specimens and chemotherapy outcomes of patients with acute myeloid leukemia.

The O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a tumor suppressor gene that is associated with the risk of developing acute myeloid leukemia (AML). However, the association between the methylation status of the MGMT promoter and the chemotherapeutic outcomes of patients with AML remains unknown. In the present study, 30 bone marrow samples derived from patients with AML were collected prior and subsequent to chemotherapy. The methylation status of the MGMT promoter in the bone marrow specimens was determined by methylation-specific polymerase chain reaction. The results indicated that the methylation status of the MGMT promoter was influenced by different chemotherapeutic regimens. The MGMT methylation status of M4 patients (3 out of 6) were more chemosensitive, compared with that of patients with other AML subtypes (M1, 1 out of 3; M2, 0 out of 8; M3, 3 out of 7; M5, 0 out of 3; and M6, 1 out of 3). Age-based analysis revealed that the group aged ≤60 years (7 out of 24 patients) exhibited more methylation changes than patients aged >60 years (1 out of 6). Male patients (4 out of 13) were more susceptible to chemotherapy-induced methylation changes than female patients (4 out of 17). Thus, the methylation status of the MGMT promoter may serve as a potential biomarker to predict the therapeutic outcomes in male AML patients. However, further studies in larger sample sets are required to confirm the present findings.

Sensitivity Analysis of the MGMT-STP27 Model and Impact of Genetic and Epigenetic Context to Predict the MGMT Methylation Status in Gliomas and Other Tumors

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Our model MGMT-STP27 allows prediction of the methylation status of the MGMT promoter using data from the Illumina's Human Methylation BeadChips (HM-27K and HM-450K) that is publically available for many cancer data sets. Here, we investigate the impact of the context of genetic and epigenetic alterations and tumor type on the classification and report on technical aspects, such as robustness of cutoff definition and preprocessing of the data. The association between gene copy number variation, predicted MGMT methylation, and MGMT expression revealed agene dosage effect on MGMT expression in lower grade glioma (World Health Organization gradeII/III) that in contrast to glioblastoma usually carry two copies of chromosome 10 on which MGMT resides (10q26.3). This implies some MGMT expression, potentially conferring residual repair function blunting the therapeutic effect of alkylating agents. A sensitivity analyses corroborated the performance of the original cutoff for various optimization criteria and for most data preprocessing methods. Finally, we propose an R package mgmtstp27 that allows prediction of the methylation status of the MGMT promoter and calculation of appropriate confidence and/or prediction intervals. Overall, MGMT-STP27 is a robust model for MGMT classification that is independent of tumor type and is adapted for single sample prediction.

The progress in molecular biomarkers of gliomas

Malignant glioma, a common form of central nervous system tumor, has a poor prognosis. The overall survival of these patients is as low as 12–14 months only. In general, the progress in personal precision medication has been gradually directed toward the molecular profiling of the tumors. Malignant glioma is one such tumor in which treatment response relies largely on its molecular characteristics, thus making the understanding of these markers essential to deliver the best treatment possible. Representative molecular markers (isocitrate dehydrogenase 1 mutation, 1p19q codeletion, epidermal growth factor receptor variant III amplification, human telomerase reverse transcriptase promoter mutations, alpha thalassemia/mental retardation syndrome X-linked mutation, and O[6]-methylguanine DNA methyltransferase promoter methylation) are described/discussed in this article. Furthermore, the research prospects of cell-free DNA, regarded as a new developing trend of molecular markers, are discussed. There is an immense hope in these promising molecular markers which are expected to improve the overall survival and quality of life of malignant glioma patients.

The tumoral A genotype of the MGMT rs34180180 single-nucleotide polymorphism in aggressive gliomas is associated with shorter patients' survival.

Malignant gliomas are the most common primary brain tumors. Grade III and IV gliomas harboring wild-type IDH1/2 are the most aggressive. In addition to surgery and radiotherapy, concomitant and adjuvant chemotherapy with temozolomide (TMZ) significantly improves overall survival (OS). The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter is predictive of TMZ response and a prognostic marker of cancer outcome. However, the promoter regions the methylation of which correlates best with survival in aggressive glioma and whether the promoter methylation status predictive value could be refined or improved by other MGMT-associated molecular markers are not precisely known. In a cohort of 87 malignant gliomas treated with radiotherapy and TMZ-based chemotherapy, we retrospectively determined the MGMT promoter methylation status, genotyped single nucleotide polymorphisms (SNPs) in the promoter region and quantified MGMT mRNA expression level. Each of these variables was correlated with each other and with the patients' OS. We found that methylation of the CpG sites within MGMT exon 1 best correlated with OS and MGMT expression levels, and confirmed MGMT methylation as a stronger independent prognostic factor compared to MGMT transcription levels. Our main finding is that the presence of only the A allele at the rs34180180 SNP in the tumor was significantly associated with shorter OS, independently of the MGMT methylation status. In conclusion, in the clinic, rs34180180 SNP genotyping could improve the prognostic value of the MGMT promoter methylation assay in patients with aggressive glioma treated with TMZ.

Glioblastoma care in the elderly

Glioblastoma is common among elderly patients, a group in which comorbidities and a poor prognosis raise important considerations when designing neuro-oncologic care. Although the standard of care for nonelderly patients with glioblastoma includes maximal safe surgical resection followed by radiotherapy with concurrent and adjuvant temozolomide, the safety and efficacy of these modalities in elderly patients are less certain given the population's underrepresentation in many clinical trials. The authors reviewed the clinical trial literature for reports on the treatment of elderly patients with glioblastoma to provide evidence-based guidance for practitioners. In elderly patients with glioblastoma, there is a survival advantage for those who undergo maximal safe resection, which likely includes an incremental benefit with increasing completeness of resection. Radiotherapy extends survival in selected patients, and hypofractionation appears to be more tolerable than standard fractionation. In addition, temozolomide chemotherapy is safe and extends the survival of patients with tumors that harbor O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation. The combination of standard radiation with concurrent and adjuvant temozolomide has not been studied in this population. Although many questions remain unanswered regarding the treatment of glioblastoma in elderly patients, the available evidence provides a framework on which providers may base individual treatment decisions. The importance of tumor biomarkers is increasingly apparent in elderly patients, for whom the therapeutic efficacy of any treatment must be weighed against its potential toxicity. MGMT promoter methylation status has specifically demonstrated utility in predicting the efficacy of temozolomide and should be considered in treatment decisions when possible. Cancer 2016;122:189-197. © 2015 American Cancer Society.

METB-07CLASSIFICATION OF HIGH GRADE GLIOMA USING MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY IMAGING (MALDI MSI): INTERIM RESULTS OF THE GLIOMIC STUDY

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) allows to determine spatial localization of molecules along tissue sections. The aim of the Gliomic study is to classify high-grade gliomas (HGG) by MALDI-MSI according to their prognosis. Until now, samples from 13 patients have been analyzed after tumor resection. According to the OMS 2007 classification, 3 tumors were grade 3 (2 oligoastrocytoma and 1 astrocytoma) and 10 were grade 4. An isocitrate dehydrogenase 1 (IDH1) mutation was found in 31% of the cases, a O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in 20% (median value: 5%, range 2-93%). Frozen tissue sections of 12 µm thickness were realized and matrix was then deposited homogeneously with a manual sprayer to cover the entire surface. Tissues were prepared for analysis of low-weighted molecules, particularly lipids. Tumor-specific mass spectra were acquired with 70 µm spatial resolution. After molecular image acquisition, matrix was removed, tissues were stained and annotated by a pathologist. The imaging datasets were spatially segmented by clustering all spectra by their molecular profiles similarity. Individual tissue segmentations were compared with histological annotations and revealed new regions of interest. A low concordance between the histological annotations and the MS imaging data was found for tumor regions. Global segmentations were generated using all the tissues and revealed 3 molecularly different groups. Known low infiltrated regions and necrotic regions were still grouped together respectively after this global segmentation. Pre-necrotic regions were divided between necrotic and tumor regions. Moreover, we highlighted sub-regions within the tumor area which were not histologically annotated. This analysis provided new insights into HGG tissue organization. The data obtained from MALDI MSI analysis may provide specific information and allow a more accurate diagnosis. A total of 21 patients have been enrolled and updated results will be presented taking account histological and molecular biology data.

MGMT testing allows for personalised therapy in the temozolomide era.

Adjuvant temozolomide (TMZ)-based chemoradiation is the standard of care for most glioblastoma patients (GBMs); however, a large proportion of these patients do not respond to TMZ. Silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter is thought to induce chemosensitivity, and testing for methylation may allow for patient stratification; however, this has yet to become routine clinical practice despite an abundance of literature on the subject. The databases PubMed, Embase, The Cochrane Library, Science Direct and Medline were searched for relevant articles published between 1999 and 2015. Articles utilising MGMT testing in glioblastomas, and treatment of glioblastomas with temozolomide were assessed. Immunohistochemistry, methylation-specific PCR (MSP), reverse transcriptase PCR, pyrosequencing and bisulphite sequencing were the main testing methods identified. Nested-MSP techniques produced poor correlation with survival, whilst bisulphite sequencing showed no evident benefit over MSP. Testing is limited by sample quality and contamination; however, efforts are made to minimise this. Strong evidence for MGMT-based personalised therapy was presented in the elderly but remains controversial in the entire GBM population. MGMT testing presents many obstacles yet to be overcome, and these warrant attention prior to the routine implementation of MGMT testing to aid decision making in GBMs. However, there is evidence to support its use, particularly in the elderly.