methylguanine-DNA Methyltransferase Status Research Articles

Preoperative chemoradiotherapy with capecitabine with or without temozolomide in patients with locally advanced rectal cancer: A prospective, randomized phase 2 study stratified by MGMT (O6-methylguanine DNA methyltransferase) status: KCSG-CO17-02.

3605 Background: We aimed to evaluate the clinical efficacy of adding temozolomide (TMZ) to preoperative capecitabine (CAP)-based chemoradiotherapy (CRT) in patients with locally advanced rectal cancer (LARC) and validate O6-methylguanine DNA methyltransferase (MGMT) methylation status as a predictive marker for TMZ combined regimens. Methods: : LARC patients with clinical stage II (cT3-4N0) or III (cTanyN+) disease were enrolled. They were stratified into MGMT unmethylated (uMGMT) and MGMT methylated (mMGMT) groups by methylation-specific PCR before randomization, and then were randomly assigned (1:1) to one of four treatment arms: uMGMT/CAP (arm A), uMGMT/TMZ+CAP (arm B), mMGMT/CAP (arm C), and mMGMT/TMZ+CAP (arm D). The primary endpoint was the pathologic complete response (pCR) rate. Results: Between November 2017 and July 2020, 64 patients were randomized. Slow accrual caused early study termination. After excluding 4 ineligible patients, 60 were included in the full analysis set. The pCR rate was 15.0% (9/60), 0%, 14.3%, 18.8%, and 26.7% for arms A, B, C, and D, respectively ( p= 0.0498 between arms A and D). The pCR rate was 9.7% in the CAP group (arms A+C), 20.7% in the TMZ+CAP group (arms B+D), 6.9% in the uMGMT group (arms A+B), and 22.6% in the mMGMT group (arms C+D). Grade 1–2 nausea or vomiting was significantly more frequent in the TMZ+CAP treatment groups (arms B+D) than in the CAP treatment groups (arms A+C, p< 0.001) with no difference in grade 3 adverse events (AEs). There were no grade 4 or 5 AEs. Conclusions: The addition of TMZ to CAP-based CRT tended to improve pCR rates, particularly in those with mMGMT LARC. MGMT status may warrant further investigation as a predictive biomarker for chemotherapeutic agents and radiotherapy. Clinical trial information: NCT03156036.

The Impact of Surgery in IDH 1 Wild Type Glioblastoma in Relation With the MGMT Deregulation.

Object: The treatment of choice in glioblastoma (GBM) is the maximal surgical extent of resection (EOR) followed by adjuvant chemo-radiotherapy. Furthermore, methylguanine-DNA methyltransferase (MGMT) promoter methylation is associated with prolonged overall survival (OS) and progression free survival (PFS). The objective of the present study is correlate the biomolecular aspects in relation with EOR.Materials and methods: We analyzed a series of 116 patients with IDH-1 wild type GBM and different EOR (Gross Total Resection—GTR-, Partial Resection—PR- and Biopsy), treated with adjuvant chemo-radiotherapy. The MGMT status was analyzed in terms of promoter methylation and protein expression.Results: When GTR was possible, OS and PFS were significantly better compared to the other two groups (p = 0.001 and p = 0.035, respectively). MGMT methylation was significantly associated with better OS in the biopsy group (p = 0.022) and better OS and PFS in PR (p = 0.02 and p = 0.012, respectively), but not in the GTR group (p = 0.252 for OS, p = 0.256 for PFS) nor the PFS in the biopsy group (p = 0.259). MGMT protein expression levels do not show any association with OS and PFS, regardless of the type of surgery.Conclusions: Our study confirms the positive association of a safe maximal EOR with better OS and PFS, and indicates a positive prognostic value of MGMT methylation status only in case of the presence of residual tumor tissue. MGMT protein expression seems not to play a clinical role in relation with the type of surgery.

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.

The Prognostic Value of the Pretreatment Neutrophil/Lymphocyte Ratio in Patients with Glioblastoma Multiforme Brain Tumors: A Retrospective Cohort Study of Patients Treated with Combined Modality Surgery, Radiation Therapy, and Temozolomide Chemotherapy

Background: Recent studies have shown that the peripheral blood pretreatment neutrophil/lymphocyte ratio (NLR) is a prognostic measure in various cancers. The few studies evaluating NLR in glioblastoma multiforme (GBM) patients yielded inconsistent results. Objectives: The primary objective of our study was to test the ability of pretreatment NLR to predict the overall survival (OS) and progression-free survival (PFS) of patients with GBM treated by combined modality therapy (surgery, radiation, and temozolomide chemotherapy). A secondary objective was to evaluate the toxicity of the combined modality protocol in a consecutive series of patients treated in our center, in the context of a real-world universal health-care setting. Methods: We analyzed 89 patients with GBM in a retrospective cohort analysis who were treated in Soroka University Medical Center’s Oncology Department between the years 2005–2016. We analyzed NLR as a dichotomous variable at 3 cut-off points, 2.5, 3 and 4, as a predictor of OS and PFS. Methylation status of the O⁶-methylguanine-DNA methyltransferase (MGMT) promoter was not determined. Results: No significant correlation was found between NLR and either OS or PFS. Factors that predicted a shorter OS were age and extent of surgery. Patients over 70 years of age had a statistically significant shorter OS, 12.5 months (95% CI: 10.4–14.5 months) versus 17.6 months (95% CI: 14.2–21.1 months) in those 70 years of age and younger (p = 0.004). The OS of patients undergoing partial resection (12.7 months 95% CI: 8.3–17.1 months) or biopsy only (9.3 months 95% CI: 7.8–24.6 months), was significantly shorter than that of patients undergoing total resection (18.9 months, 95% CI: 11.8–26.0 months; p = 0.035). There were no treatment-related deaths. The most common grade III–IV toxicities were thrombocytopenia, 12.4%, and fatigue, 13.5%. Conclusions: In our cohort of GBM patients treated with combined modality therapy, pretreatment NLR was not prognostic. Toxicity of treatment was acceptable. Investigation of the NLR with larger groups of patients selected by MGMT status is warranted.

Predictive MGMT status in a homogeneous cohort of IDH wildtype glioblastoma patients

Methylation of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter is predictive for treatment response in glioblastoma patients. However, precise predictive cutoff values to distinguish “MGMT methylated” from “MGMT unmethylated” patients remain highly debated in terms of pyrosequencing (PSQ) analysis. We retrospectively analyzed a clinically and molecularly very well-characterized cohort of 111 IDH wildtype glioblastoma patients, who underwent gross total tumor resection and received standard Stupp treatment. Detailed clinical parameters were obtained. Predictive cutoff values for MGMT promoter methylation were determined using ROC curve analysis and survival curve comparison using Log-rank (Mantel-Cox) test. MGMT status was analyzed using pyrosequencing (PSQ), semi-quantitative methylation specific PCR (sqMSP) and direct bisulfite sequencing (dBiSeq). Highly methylated (> 20%) MGMT correlated with significantly improved progression-free survival (PFS) and overall survival (OS) in our cohort. Median PFS was 7.2 months in the unmethylated group (UM, < 10% mean methylation), 10.4 months in the low methylated group (LM, 10-20% mean methylation) and 19.83 months in the highly methylated group (HM, > 20% mean methylation). Median OS was 13.4 months for UM, 17.9 months for LM and 29.93 months for HM. Within the LM group, correlation of PSQ and sqMSP or dBiSeq was only conclusive in 51.5% of our cases. ROC curve analysis revealed superior test precision for survival if additional sqMSP results were considered (AUC = 0.76) compared to PSQ (cutoff 10%) alone (AUC = 0.67). We therefore challenge the widely used, strict PSQ cutoff at 10% which might not fully reflect the clinical response to alkylating agents and suggest applying a second method for MGMT testing (e.g. MSP) to confirm PSQ results for patients with LM MGMT levels if therapeutically relevant.

Prognostic value of test(s) for O6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide

Background Glioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine-DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide. Objectives To determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who are treated with temozolomide. Search methods We searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies, we additionally searched NHS Economic Evaluation Database (EED) up to December 2014. Selection criteria Eligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiotherapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies and cost analysis. Data collection and analysis Two review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios (RHR), using an imputed value of the correlation between results based on the same individuals. Main results We included 32 independent cohorts involving 3474 people that compared two or more methods. We found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds (RHR 1.31, 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR 1.36, 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR 1.14, 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarised 190 articles describing a single method for measuring MGMT promoter methylation status. Authors' conclusions PSQ and MSP appear more prognostic for overall survival than IHC. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.

Prognostic Value of O-(2-[18F]Fluoroethyl)-L-Tyrosine PET/CT in Newly Diagnosed WHO 2016 Grade II and III Glioma.

The use of [18F]fluoroethyl)-L-tyrosine ([18F]FET) positron emission tomography/computed tomography (PET/CT) has proven valuable in brain tumor management. This study aimed to investigate the prognostic value of radiotracer uptake in newly diagnosed grade II or III gliomas according to the current 2016 World Health Organization (WHO) classification. A total of 35 treatment-naive patients (mean age, 48 ± 17years) with histologically proven WHO grade II or III gliomas as defined by the current 2016 WHO classification were included. Static PET/CT imaging was performed 20min after intravenous [18F]FET injection. Images were assessed visually and semi-quantitatively using regions of interest for both tumor (SUVmax, SUVmean) and background (BKGmean) to calculate tumor-to-background (TBR) ratios. The association among histological results, molecular markers (including isocitrate dehydrogenase enzyme and methylguanine-DNA methyltransferase status), clinical features (age), and PET findings was tested and compared with outcome (progression-free [PFS] and overall survival [OS]). Fourteen patients presented with grade II (diffuse astrocytoma n = 10, oligodendroglioma n = 4) and 21 patients with grade III glioma (anaplastic astrocytoma n = 15, anaplastic oligodendroglioma n = 6). Twenty-seven out of the 35 patients were PET-positive (grade II n = 8/14, grade III n = 19/21), with grade III tumors exhibiting significantly higher amino acid uptake (TBRmean and TBRmax; p = 0.03 and p = 0.02, respectively). PET-negative lesions demonstrated significantly prolonged PFS (p = 0.003) as compared to PET-positive gliomas. PET-positive disease had a complementary value in prognostication in addition to patient age, glioma grade, and molecular markers. Amino acid uptake as assessed by [18F]FET-PET/CT imaging is useful as non-invasive read-out for tumor biology and prognosis in newly diagnosed, treatment-naive gliomas according to the 2016 WHO classification.

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

Multi-institutional external validation of a novel glioblastoma prognostic nomogram incorporating MGMT methylation

A recent nomogram for glioblastoma (GBM) was designed to incorporate methylguanine-DNA methyltransferase (MGMT) methylation status in trial patients receiving temozolomide. Since clinical trial patients are strictly selected, compared to the general population, we performed a multi-institutional, external, independent assessment of the nomogram. Consecutive adult patients with supratentorial GBM diagnosed between June 2007 and December 2014 who initiated TMZ-based concurrent chemoradiotherapy (CRT) and were not enrolled on RTOG 0525 or 0825 were eligible. We collected age, gender, MGMT status, performance status, resection extent, race, and tumor site and Cox regression analysis of overall survival (OS) was conducted with the 1-year nomogram-predicted survival (NPS). The predictive accuracy was quantified by the concordance index (c-index) as well as by separating patients into quintile-groups of the population distribution of NPS and comparing mean NPS and observed OS. Of 514 patients with GBM, 309 had all nomogram factors. Median OS was 18.7months. NPS and observed OS demonstrated a c-index of 0.695. On univariate analysis, the NPS and all included factors except gender were significant. On multivariable analysis (MVA) the only significant factor for worse survival was lower NPS. When separated into quintile-groups of NPS, the observed survival was slightly better than the predicted survival for all but the worst prognostic group. Our multi-institutional cohort provides independent external validation of a novel GBM nomogram incorporating MGMT methylation status. No individual factor included in the nomogram retained significance on MVA after adjusting for NPS.

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.

MGMT in colorectal cancer: a promising component of personalized treatment.

The identification of new, effective drugs is a pressing need in colorectal cancer (CRC) rescue therapy. Data examining O (6)-methylguanine-DNA-methyl transferase (MGMT) and its predictive role in temozolomide (TMZ) treatment in CRC are scarce. In this study, the effect of MGMT status on the cytotoxic sensitivity caused by TMZ was analyzed using cytology proliferation assays in colon cancer cell lines. MGMT protein expression was assessed with immunohistochemistry in 385 patients. Concordance between primary and metastatic sites and the role of MGMT status on survival were statistically analyzed. TMZ sensitivity was significantly affected by the level of MGMT protein expression. Of 385 cases, 13 (3.4%) demonstrated loss of MGMT expression. However, low MGMT expression levels were significantly more common in signet ring cell carcinomas (p = 0.011). In 111 of 385 cases, the overall concordance of MGMT status between primary tumor and metastatic sites was 66.67% (κ = 0.271, p < 0.001). The median progression-free survival was significantly different between groups with low or high MGMT expression for the irinotecan-based regimen (p = 0.025), but MGMT protein expression was not observed to be a prognostic factor. In conclusion, MGMT was an important in vitro predictor of TMZ activity in CRC. The rate of MGMT protein loss was low in metastatic CRC patients from China, and MGMT might be more commonly lost in signet ring cell carcinoma. The MGMT status at primary and metastatic sites was consistent, but the power of concordance was poor. Further study into these topics is warranted.

Gemcitabine and oxaliplatin or alkylating agents for neuroendocrine tumors: Comparison of efficacy and search for predictive factors guiding treatment choice.

The alkylating agents (ALKYs) streptozotocin, dacarbazine, and temozolomide currently are the main drugs used in systemic chemotherapy for neuroendocrine tumors (NETs). The promising activity shown by gemcitabine and oxaliplatin (GEMOX) in previous studies prompted this study 1) to confirm the use of GEMOX in a larger population of NET patients, 2) to compare its efficacy with that of ALKYs, and 3) to explore whether the O(6) -methylguanine-DNA methyltransferase (MGMT) status could help in selecting the chemotherapy regimen. One hundred four patients with metastatic NETs (37 pancreatic NETs, 33 gastrointestinal NETs, 23 bronchial NETs, and 11 NETs of other/unknown origin) were treated with GEMOX between 2004 and 2014. Among these patients, 63 also received ALKYs. MGMT promoter gene methylation was assessed via pyrosequencing in 42 patients. Patients received a median of 6 courses of GEMOX. Twenty-four (23%) had an objective response (OR). The median progression-free survival (PFS) and overall survival were 7.8 and 31.6 months, respectively. In the 63 patients treated with both ALKYs and GEMOX, the ORs (22% and 22%) and the PFSs (7.5 and 7.3 months) were similar. The response was concordant in 53% of the patients. Promoter gene methylation of MGMT was associated with better outcomes with ALKYs (P = .03 for OR and P = .04 for PFS) but not GEMOX. GEMOX is effective against NETs; its activity is comparable to that of ALKYs, and it is not influenced by the MGMT status. Our data suggest that GEMOX might be preferred for patients with unmethylated MGMT tumors. Cancer 2015;121:3435-43. © 2015 American Cancer Society.

Elderly patients with glioblastoma multiforme treated with concurrent temozolomide and standard- versus abbreviated-course radiotherapy.

Glioblastoma multiforme (GBM) is an aggressive neoplasm, with controversy regarding treatment in elderly patients. To review outcomes of elderly patients aged ≥ 65 with newly diagnosed GBM treated with concurrent temozolomide and either standard-course radiotherapy (SRT) or abbreviated-course radiotherapy (ART). Retrospective review from 2003 to 2012. Survival, comparing treatment regimens. One hundred patients received SRT (median dose = 60 Gy), and 29 received ART (median dose = 35 Gy). O6- methylguanine-DNA methyltransferase (MGMT) status was available for 26 SRT and 13 ART recipients. Median age was 70 years. Median follow-up was 11 months. At analysis, 3 patients were alive. Multivariate analysis of the entire cohort found SRT (hazard ratio [HR] = 0.421, p = 0.0001), Karnofsky Performance Score of 70 or higher (HR = 1.894, p = 0.0031), and more extensive surgery (HR = 0.466, p = 0.0023) were associated with longer survival time, but age was not. Median time to death with SRT was 13 months versus 5.4 months with ART, but the latter had worse prognostic factors, including lower Karnofsky Performance Scores, fewer gross total resections, and higher recursive partitioning analysis class. Recipients of SRT with methylated MGMT promoter had a trend toward longer survival compared with unmethylated MGMT (p = 0.06), but ART recipients had shorter survival with MGMT methylation (p = 0.02). Elderly patients with multiple poor prognostic factors given ART had short survival times. Relative to other variables, MGMT status may not predict outcome for these patients.

O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents.

Background:O6-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins.Methods:A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status.Results:MGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O6-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, P<0.001 and 105 vs 34 months, P=0.006) or MGMT promoter methylation assessed by pyrosequencing (respectively, 26.4 vs 10.8 months, P=0.002 and 77 vs 43 months, P=0.026).Conclusions:Our results suggest that MGMT status is associated with response to alkylant-based chemotherapy in NETs.

Somatic mutations in glioblastoma are associated with methylguanine-DNA methyltransferase methylation.

The high level of methylguanine-DNA methyltransferase (MGMT) in glioblastoma is responsible for resistance to alkylating agents, such as temozolomide (TMZ). In glioblastomas with a methylated MGMT promoter, MGMT deficiency is presumed, resulting in an enhanced effect of TMZ. The aim of the present study was to investigate whether genomic alterations work synergistically with MGMT methylation status and contribute to the response to treatment and overall prognosis in glioblastoma. The current study included a cohort of 35 glioblastoma patients, with MGMT promoter methylation present in 48% of tumors. MGMT methylation was associated with significantly longer median survival (29.0 months) compared with patients without MGMT methylated tumors (12.0 months), as well as longer median time to progression following TMZ treatment (13.2 months, compared with 5.6 months for patients with an unmethylated MGMT status). In addition, somatic variants in hot spot exonic regions of 50 key cancer genes were examined in these glioblastomas. Non-synonymous mutations in methylated MGMT glioblastomas were four times higher compared with unmethylated MGMT glioblastomas. Furthermore, significantly increased frequencies of mutations in the TP53, CDKN2A, PTEN and PIK3CA genes were detected in MGMT methylated glioblastomas. The relative significance of these mutations, and their contribution to TMZ sensitivity, adjunct to MGMT methylation, require further investigation in a larger cohort.

Long-term therapy with temozolomide is a feasible option for newly diagnosed glioblastoma: a single-institution experience with as many as 101 temozolomide cycles.

The objective of this study was to report the authors' experience with the long-term administration of temozolomide (TMZ; > 6 cycles, up to 101) in patients with newly diagnosed glioblastoma and to analyze its feasibility and safety as well as its impact on survival. The authors also compared data obtained from the group of patients undergoing long-term TMZ treatment with data from patients treated with a standard TMZ protocol. A retrospective analysis was conducted of 37 patients who underwent operations for glioblastoma between 2004 and 2012. Volumetric analysis of postoperative Gd-enhanced MR images, obtained within 48 hours, confirmed tumor gross-total resection (GTR) in all but 2 patients. All patients received the first cycle of TMZ at a dosage of 150 mg/m(2) starting on the second or third postsurgical day. Afterward, patients received concomitant radiochemotherapy according to the Stupp protocol. With regard to adjuvant TMZ therapy, the 19 patients in Group A, aged 30-72 years (mean 56.1 years), received 150 mg/m(2) for 5 days every 28 days for more than 6 cycles (range 7-101 cycles). The 18 patients in Group B, aged 46-82 years (mean 64.8 years), received the same dose, but for no more than 6 cycles. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was analyzed for both groups and correlated with overall survival (OS) and progression-free survival (PFS). The impact of age, sex, Karnofsky Performance Scale score, and Ki 67 staining were also considered. All patients but 1 in Group A survived at least 18 months (range 18-101 months), and patients in Group B survived no more than 17 months (range 2-17 months). The long-term survivors (Group A), defined as patients who survived at least 12 months after diagnosis, were 51.3% of the total (19/37). Kaplan-Meier curve analysis showed that patients treated with more than 6 TMZ cycles had OS and PFS that was significantly longer than patients receiving standard treatment (median OS 28 months vs 8 months, respectively; p = 0.0001; median PFS 20 months vs 4 months, respectively; p = 0.0002). By univariate and multivariate Cox proportional hazard regression analysis, MGMT methylation status and number of TMZ cycles appeared to be survival prognostic factors in patients with glioblastoma. After controlling for MGMT status, highly significant differences related to OS and PFS between patients with standard and long-term TMZ treatment were still detected. Furthermore, in Group A and B, the statistical correlation of MGMT status to the number of TMZ cycles showed a significant difference only in Group A patients, suggesting that MGMT promoter methylation was predictive of response for long-term TMZ treatment. Prolonged therapy did not confer hematological toxicity or opportunistic infections in either patient group. This study describes the longest experience so far reported with TMZ in patients with newly diagnosed glioblastomas, with as many as 101 cycles, who were treated using GTR. Statistically significant data confirm that median survival correlates with MGMT promoter methylation status as well as with the number of TMZ cycles administered. Long-term TMZ therapy appears feasible and safe.

HOXA10 is associated with temozolomide resistance through regulation of the homologous recombinant DNA repair pathway in glioblastoma cell lines.

Temozolomide resistance is associated with multiple DNA repair pathways. We investigated homeobox (HOX) genes for their role in temozolomide resistance, focusing on the homologous recombination (HR) pathway, and we tested their therapeutic implications in conjunction with O(6)-methylguanine DNA methyltransferase (MGMT) status. Two glioblastoma cell lines with different MGMT statuses were used to test the augmented anticancer effect of temozolomide with HOXA10 inhibition. In vitro experiments, including gene expression studies with RNA interference, were performed to verify the related pathway dynamics. HOXA10 inhibition reinforced temozolomide sensitivity independent of MGMT status and was related to the impaired double-strand DNA breakage repair process resulting from the downregulation of Rad51 paralogs. Early growth response 1 (EGR1) and phosphatase and tensin homolog (PTEN) were the regulatory mediators between HOXA10 and the HR pathway. Moreover, HOXA10 inhibition selectively affected the nuclear function of PTEN without interfering with its cytoplasmic function of suppressing the phosphoinositide 3-kinase/Akt pathway. The mechanism of HR pathway regulation by HOXA10 harbors another target mechanism for overcoming temozolomide resistance in glioblastoma patients.

MGMT testing--the challenges for biomarker-based glioma treatment.

Many patients with malignant gliomas do not respond to alkylating agent chemotherapy. Alkylator resistance of glioma cells is mainly mediated by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Epigenetic silencing of the MGMT gene by promoter methylation in glioma cells compromises this DNA repair mechanism and increases chemosensitivity. MGMT promoter methylation is, therefore, a strong prognostic biomarker in paediatric and adult patients with glioblastoma treated with temozolomide. Notably, elderly patients (>65-70 years) with glioblastoma whose tumours lack MGMT promoter methylation derive minimal benefit from such chemotherapy. Thus, MGMT promoter methylation status has become a frequently requested laboratory test in neuro-oncology. This Review presents current data on the prognostic and predictive relevance of MGMT testing, discusses clinical trials that have used MGMT status to select participants, evaluates known issues concerning the molecular testing procedure, and addresses the necessity for molecular-context-dependent interpretation of MGMT test results. Whether MGMT promoter methylation testing should be offered to all individuals with glioblastoma, or only to elderly patients and those in clinical trials, is also discussed. Justifications for withholding alkylating agent chemotherapy in patients with MGMT-unmethylated glioblastomas outside clinical trials, and the potential role for MGMT testing in other gliomas, are also discussed.

Outcome-based determination of optimal pyrosequencing assay for MGMT methylation detection in glioblastoma patients.

The methylation of O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a key biological marker in clinical neuro-oncology. Nevertheless, there is no consensus concerning the best technique for its assessment. In a recent study comparing five methods to analyze MGMT status, we found that the best prediction of survival was obtained with a pyrosequencing (PSQ) test assessing methylation of 5 CpGs (CpGs 74–78). In the present study we extended our PSQ analysis to 16 CpGs (CpGs 74–89) identified as critical for transcriptional control of the gene. The predictive value of the methylation levels at each CpG, as well as the mean methylation levels of selected sets of consecutive CpGs was tested in a cohort of 89 de novo glioblastoma patients who had received standard of care treatment (Stupp protocol). Using an optimal risk cut-off, each CpG or combination of CpGs, was associated with overall survival (OS) and progression free survival. The best predictive models for OS after stratification on performance score and age were obtained with CpG 89, CpG 84 and mean methylation of CpG 84–88 (Hazard ratio (HR), 0.31; p < 0.0001). The improvement compared to the predictive value of the test analyzing average methylation of CpG 74–78 (HR, 0.32; p < 0.0001) was however marginal. We recommend to test CpGs 74–78 when analyzing MGMT methylation status by PSQ because a commercial kit that has successfully been used in several studies is available, allowing reproducible and comparable results from one laboratory to another.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-013-1332-y) contains supplementary material, which is available to authorized users.

Prognostic or predictive value of MGMT promoter methylation in gliomas depends on IDH1 mutation

To explore whether the isocitrate dehydrogenase 1 (IDH1) or 1p/19q status determines the prognostic vs predictive role of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in the Neuro-Oncology Working Group of the German Cancer Society (NOA)-04 trial anaplastic glioma biomarker cohort. Patients (n = 183) of the NOA-04 trial with known MGMT and IDH1 status were analyzed for interdependency of the prognostic vs predictive role of MGMT promoter methylation from IDH1 or 1p/19q status and treatment, using progression-free survival (PFS) as an endpoint. An independent validation cohort of the German Glioma Network (n = 75) and the NOA-08 trial (n = 34) served as a confirmation cohort. In tumors with IDH1 mutation, MGMT promoter methylation was associated with prolonged PFS with chemotherapy ± radiotherapy (RT) or RT-only groups, and is thus prognostic. In tumors without IDH1 mutation, MGMT promoter methylation was associated with increased PFS in patients treated with chemotherapy, but not in those who received RT alone as the first-line treatment, and is thus chemotherapy-predictive. In contrast, 1p/19q codeletions showed no such association with the prognostic vs predictive value of MGMT. MGMT promoter methylation is a predictive biomarker for benefit from alkylating agent chemotherapy in patients with IDH1-wild-type, but not IDH1-mutant, malignant gliomas of World Health Organization grades III/IV. Combined IDH1/MGMT assessment may help to individualize clinical decision-making in neuro-oncology.