Unmethylated Tumors Research Articles

Glioblastoma gene expression subtypes and correlation with clinical, molecular and immunohistochemical characteristics in a homogenously treated cohort: GLIOCAT project.

2029 Background: Glioblastoma (GBM) gene expression subtypes have been described in last years, data in homogeneously treated patients is lacking. Methods: Clinical, molecular and immunohistochemistry (IHC) analysis from patients with newly diagnosed GBM homogeneously treated with standard radiochemotherapy were studied. Samples were classified based on the expression profiles into three different subtypes (classical, mesenchymal, proneural) using Support Vector Machine (SVM), the K-nearest neighbor (K-NN) and the single sample Gene Set Enrichment Analysis (ssGSEA) classification algorithms provided by GlioVis web application. Results: GLIOCAT Project recruited 432 patients from 6 catalan institutions, all of whom received standard first-line treatment (2004 -2015). Best paraffin tissue samples were selected for RNAseq and reliable data were obtained from 124. 82 cases (66%) were classified into the same subtype by all three classification algorithms. SVM and ssGEA algorithms obtain more similar results (87%). No differences in clinical variables were found between the 3 GBM subtypes. Proneural subtype was enriched with IDH1 mutated and G-CIMP positive tumors. Mesenchymal subtype (SVM) was enriched in unmethylated MGMT tumors (p = 0.008), and classical (SVM) in methylated MGMT tumors (p = 0.008). Long survivors ( > 30 months) were rarely classified as mesenchymal (0-7.5%) and were more frequently classified as Proneural (23.1-26.). Clinical (age, resection, KPS) and molecular ( IDH1, MGMT) known prognostic factors were confirmed in this serie. Overall, no differences in prognosis were observed between 3 subtypes, but a trend to worse survival in mesenchymal was observed in K-NN (9.6 vs 15 ). Mesenchymal subtype presented less expression of Olig2 (p < 0.001) and SOX2 (p = 0.003) by IHC, but more YLK-40 expression (p = 0.023, SVM). On the other hand, classical subtype expressed more Nestin (p = 0.004) compared to the other subtypes (K-NN). Conclusions: In our study we have not found correlation between glioblastoma expression subtype and outcome. This large serie provides reproducible data regarding clinical-molecular-immunohistochemistry features of glioblastoma genetic subtypes.

MGMT Protein Expression Adds Prognostic Value Beyond MGMT Promoter Methylation and Stratifies Survival Prognoses of Un-Methylated Glioblastoma Patients

In glioblastomas, one of the main mechanisms of resistance to treatment with radiation + temozolomide (Stupp protocol) is carried by the DNA-repair protein MGMT. Methylation of the MGMT promoter region is a key prognostic factor for glioblastoma, however, the current administered standard of care is the same for methylated and un-methylated tumors. Since MGMT protein expression is regulated by alternative post-transcriptional and post-translational mechanisms beyond promoter methylation, we hypothesized that MGMT protein expression assessed by immunohistochemistry could stratify the prognoses of patients with glioblastoma and add prognostic power to MGMT methylation, improving the decision-making process for glioblastoma treatment. We retrospectively analyzed an institutional cohort of 436 adult patients comparing MGMT immunohistochemistry, using a monoclonal antibody and ≥15% of positive nuclei as cut point for high MGMT expression, and MGMT promoter methylation, assessed by the platform EPIC, from FFPE glioblastomas. Log-rank test for the univariate analysis (UVA) and multivariable (MVA) Cox-regression models accounting for classical clinical factors (age, KPS, IDH1 status etc.), as well as MGMT expression and MGMT promoter methylation were employed to determine association with OS. Low MGMT expression according to the 15% cutoff was correlated with better OS and was a stronger predictor of survival than MGMT promoter methylation both in UVA [HR: 1.7(1.3-2.3 CI), p=0.00057 vs. HR: 1.6(1.2-2.3 CI), p=0.0028] and MVA [HR: 1.99(1.33-2.98 CI), p=0.001 vs. HR: 1.37(0.96-1.95 CI), p=0.081]. MGMT promoter methylation was strongly associated with low MGMT expression (p=0.005, Fisher’s exact test); nevertheless more than 70% of un-methylated tumors had low MGMT expression levels. In response to Stupp protocol, un-methylated tumors with low MGMT expression had significantly better OS than their counterparts with high MGMT expression (p=0.001), while rare methylated tumors with high MGMT expression had OS similar to the un-methylated tumors (p=0.270). MGMT promoter methylation significantly split OS only in the tumors with inherent low MGMT expression (p=0.02), but not when MGMT expression was ≥15% (p=0.270). Our data provides evidence that MGMT protein expression is an independent prognostic biomarker for newly diagnosed glioblastomas and the immunohistochemistry test may identify, upfront and at a lower cost, those patients who may not respond to the Stupp protocol. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and the Ohio State University CCC (all to AC).

Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival.

Between 2011 and 2016, O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation testing at University of California Los Angeles (UCLA) was performed through LabCorp, using a threshold of 2 to distinguish MGMT methylated from unmethylated tumors. In this study, we sought to determine whether the magnitude of the methylation score correlated with outcome. We identified 165 newly diagnosed glioblastoma (GBM) isocitrate dehydrogenase (IDH) wild-type and temozolomide-treated upfront patients at UCLA and Kaiser Permanente Los Angeles with LabCorp-derived quantitative MGMT scores obtained on pretreatment tissue samples. Using LabCorp's threshold, we found 102 unmethylated and 63 methylated patients. We then further substratified each group based on the magnitude of the score, and performed Kaplan-Meier and Cox regression analyses of overall survival (OS) and progression-free survival (PFS). We validated that the standard LabCorp threshold of 2 could separate our cohort by survival, showing longer OS and PFS for MGMT methylated patients vs unmethylated patients. Cox regression analysis confirmed that MGMT (<1) patients had worse outcome, with OS and PFS hazard ratios of 2.375 (P = .053) and 2.463 (P = .023), respectively, when compared to the MGMT (1-1.99) patients. Contrary to our expectation, when we substratified the ≥2 (methylated) group, we did not find a dose-dependent relationship between the magnitude of MGMT methylation and improved survival. The MGMT unmethylated group contains a partially methylated group (greater than 1) that shares survival benefits similar to the methylated group. However, we did not demonstrate an association of very high methylation scores with increased survival. These findings will require validation in additional independent clinical data sets.

MGMT pyrosequencing-based cut-off methylation level and clinical outcome in patients with glioblastoma multiforme.

MGMT promoter methylation has been associated with improved survival in glioblastoma multiforme treated with temozolomide. However, there is no consensus on specific cut-off levels of methylation. The aims of the study were to explore the prognostic impact of MGMT methylation status and to analyze the role of specific cut-off values. We analyzed 108 glioblastoma multiforme patients treated between 2008 and 2013 stratified according to three pyrosequencing-based quantitative methylation in: unmethylated (methylation <9%), intermediate (9-29%) and highly methylated (>29%). The three-class stratification has a prognostic impact (median progression-free survival: 7.97, 11.6 and 15months respectively; p=0.004; median OS: 13.2, 15.8 and 19.5months, respectively; p=0.0002), especially in patients exposed to temozolomide. Our study confirmed that the independent prognostic role of MGMT methylation status. An average level of methylation between all investigated CpGs of 9% may help discriminating between methylated and unmethylated tumors.

The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma

BackgroundThe use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity.MethodsTinostamustine has been tested in models of GBM by using 13 GBM cell lines and seven patient-derived GBM proliferating/stem cell lines in vitro. U87MG and U251MG (MGMT negative), as well as T98G (MGMT positive), were subcutaneously injected in nude mice, whereas luciferase positive U251MG cells and patient-derived GBM stem cell line (CSCs-5) were evaluated the orthotopic intra-brain in vivo experiments.ResultsWe demonstrated that tinostamustine possesses stronger antiproliferative and pro-apoptotic effects than those observed for vorinostat and bendamustine alone and similar to their combination and irrespective of MGMT expression. In addition, we observed a stronger radio-sensitization of single treatment and temozolomide used as control due to reduced expression and increased time of disappearance of γH2AX indicative of reduced signal and DNA repair. This was associated with higher caspase-3 activation and reduction of RT-mediated autophagy. In vivo, tinostamustine increased time-to-progression (TTP) and this was additive/synergistic to RT. Tinostamustine had significant therapeutic activity with suppression of tumor growth and prolongation of DFS (disease-free survival) and OS (overall survival) in orthotopic intra-brain models that was superior to bendamustine, RT and temozolomide and showing stronger radio sensitivity.ConclusionsOur data suggest that tinostamustine deserves further investigation in patients with glioblastoma.

Analysis of immunobiologic markers in primary and recurrent glioblastoma.

Glioblastoma (GBM) generates a varied immune response and understanding the immune microenvironment may lead to novel immunotherapy treatments modalities. The goal of this study was to evaluate the expression of immunologic markers of potential clinical significance in primary versus recurrent GBM and assess the relationship between these markers and molecular characteristics of GBM. Human GBM samples were evaluated and analyzed with immunohistochemistry for multiple immunobiologic markers (CD3, CD8, FoxP3, CD68, CD163, PD1, PDL1, CTLA4, CD70). Immunoreactivity was analyzed using Aperio software. Degree of strong positive immunoreactivity within the tumor was compared to patient and tumor characteristics including age, gender, MGMT promoter methylation status, and ATRX, p53, and IDH1 mutation status. Additionally, the TCGA database was used to perform similar analysis of these factors in GBM using RNA-seq by expectation-maximization. Using odds ratios, IDH1 mutated GBM had statistically significant decreased expression of CD163 and CD70 and a trend for decreased PD1, CTLA4, and Foxp3. ATRX-mutated GBMs exhibited statistically significant increased CD3 immunoreactivity, while those with p53 mutations were found to have significantly increased CTLA4 immunoreactivity. The odds of having strong CD8 and CD68 reactivity was significantly less in MGMT methylated tumors. No significant difference was identified in any immune marker between the primary and recurrent GBM, nor was a significant change in immunoreactivity identified among age intervals. TCGA analysis corroborated findings related to the differential immune profile of IDH1 mutant, p53 mutant, and MGMT unmethylated tumors. Immunobiologic markers have greater association with the molecular characteristics of the tumor than with primary/recurrent status or age.

Estrogen receptor-α promoter methylation is a biomarker for outcome prediction of cisplatin resistance in triple-negative breast cancer.

A number of previous studies have indicated the presence of a link between estrogen receptor-α (ERα) methylation and triple-negative breast cancer (TNBC). However, the association between ERα methylation and drug resistance during the treatment of TNBC remains unclear. Methylation-specific polymerase chain reaction was used to investigate the methylation of ERα in the genomic DNA of 35 patients with TNBC who were defined as cisplatin-based chemotherapy-resistant using chemosensitivity testing. Survival probabilities by covariates were assessed using Kaplan-Meier estimator survival analysis and Cox's proportional hazards models, adjusting for age, menopausal status, tumor size, lymph node metastasis and ERα promoter DNA methylation. Of the 35 patients with TNBC analyzed, 8 exhibited ERα promoter DNA methylation. Cisplatin resistance was confirmed to be overwhelmingly associated with ERα methylation by univariate and multivariate analysis. Even in a limited analysis in patients with ERα methylation, the results generated from methylated tumor tissue and unmethylated tumor tissue revealed that expression of breast cancer type 1/2 susceptibility proteins was increased in ERα-methylated breast tumor tissue compared with in unmethylated tissue. The ERα methylation group tended to have significantly shorter progression-free (P=0.010) and overall (P=0.023) survival times compared with those in the unmethylated group. Similarly, shorter progression-free (P=0.024) and overall (P=0.018) survival times were observed in the cisplatin-resistant group compared with the cisplatin-non-resistant group. ERα methylation predicts a poor clinical outcome for patients with TNBC. The results of the present study indicated that ERα methylation may be a candidate surrogate biomarker for outcome prediction and cisplatin resistance in TNBC. Further investigation is required to identify potential biomarkers in a larger cohort in a prospective study.

A 4-miRNA signature to predict survival in glioblastomas.

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06–23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93–33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45–0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.

Radiomics signature: A potential biomarker for the prediction of MGMT promoter methylation in glioblastoma.

In glioblastoma (GBM), promoter methylation of the DNA repair gene O-methylguanine-DNA methyltransferase (MGMT) is associated with beneficial chemotherapy. To analyze radiomics features for utilizing the full potential of medical imaging as biomarkers of MGMT promoter methylation. Retrospective. In all, 98 GBM patients with known MGMT (48 methylated and 50 unmethylated tumors). 3.0T magnetic resonance (MR) images, containing T1 -weighted image (T1 WI), T2 -weighted image (T2 WI), and enhanced T1 WI. A region of interest (ROI) of the tumor was delineated. A total of 1665 radiomics features were extracted and quantized, and were reduced using least absolute shrinkage and selection operator (LASSO) regularization. After the support vector machine construction, accuracy, sensitivity, and specificity were computed for different sequences. An independent validation cohort containing 20 GBM patients was utilized to further evaluate the radiomics model performance. Radiomics features of T1 WI reached an accuracy of 67.54%. Enhanced T1 WI features reached an accuracy of 82.01%, while T2 WI reached an accuracy of 69.25%. The best classification system for predicting MGMT promoter methylation status originated from the combination of 36 T1 WI, T2 WI, and enhanced T1 WI images features, with an accuracy of 86.59%. Further validation on the independent cohort of 20 patients produced similar results, with an accuracy of 80%. Our results provide further evidence that radiomics MR features could predict MGMT methylation status in preoperative GBM. Multiple imaging modalities together can yield putative noninvasive biomarkers for the identification of MGMT. 4 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:1380-1387.

Molecular differences in IDH wildtype glioblastoma according to MGMT promoter methylation.

O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma. We investigated whether this marker furthermore defines a molecularly distinct tumor subtype with clinically different outcome. We analyzed copy number variation (CNV) and methylation profiles of 1095 primary and 92 progressive isocitrate dehydrogenase wildtype glioblastomas, including paired samples from 49 patients. DNA mutation data from 182 glioblastoma samples of The Cancer Genome Atlas (TCGA) and RNA expression from 107 TCGA and 55 Chinese Glioma Genome Atlas samples were analyzed. Among untreated glioblastomas, MGMT promoter methylated (mMGMT) and unmethylated (uMGMT) tumors did not show different CNV or specific gene mutations, but a higher mutation count in mMGMT tumors. We identified 3 methylation clusters. Cluster 1 showed the highest average methylation and was enriched for mMGMT tumors. Seventeen genes including gastrulation brain homeobox 2 (GBX2) were found to be hypermethylated and downregulated on the mRNA level in mMGMT tumors. In progressive glioblastomas, platelet derived growth factor receptor alpha (PDGFRA) and GLI2 amplifications were enriched in mMGMT tumors. Methylated MGMT tumors gain PDGFRA amplification of PDGFRA, whereas uMGMT tumors with amplified PDGFRA frequently lose this amplification upon progression. Glioblastoma patients surviving <6 months and with mMGMT harbored less frequent epidermal growth factor receptor (EGFR) amplifications, more frequent TP53 mutations, and a higher tumor necrosis factor-nuclear factor-kappaB (TNF-NFκB) pathway activation compared with patients surviving >12 months. MGMT promoter methylation status does not define a molecularly distinct glioblastoma subpopulation among untreated tumors. Progressive mMGMT glioblastomas and mMGMT tumors of patients with short survival tend to have more unfavorable molecular profiles.

Histopathological Insights on Imaging Results of Intraoperative Magnetic Resonance Imaging, 5-Aminolevulinic Acid, and Intraoperative Ultrasound in Glioblastoma Surgery

For appropriate use of available intraoperative imaging techniques in glioblastoma (GB) surgery, it is crucial to know the potential of the respective techniques in tumor detection. To assess histopathological basis of imaging results of intraoperative magnetic resonance imaging (iMRI), 5-aminolevulinic acid (5-ALA), and linear array intraoperative ultrasound (lioUS). We prospectively compared the imaging findings of iMRI, 5-ALA, and lioUS at 99 intraoperative biopsy sites in 33 GB patients during resection control. Histological classification of specimens, tumor load, presence of necrosis, presence of vascular malformations, and O6-methylguanin-DNA methyltransferase (MGMT) promoter state was correlated with imaging findings. Solid tumor was found in 57%, infiltration zone in 42%, and no tumor in 1% of specimens. However, imaging was negative in iMRI in 49%, using 5-ALA in 17%, and in lioUS in 21%. In positive imaging results, share of solid tumor was highest in 5-ALA (65%) followed by lioUS (60%) and lowest in iMRI (55%). In comparison to 5-ALA, iMRI had a high share of solid tumor in specimens when showing intermediate results. Sensitivity for invasive tumor was higher in 5-ALA (84%) and lioUS (80%) than in iMRI (50%). We found a significant correlation of 5-ALA with classification of specimen, presence of necrosis, and microproliferations. Methylated MGMT promoter correlated with positive findings in 5-ALA. lioUS and iMRI showed no correlations with histopathological findings. All of the assessed established imaging techniques detect infiltrating tumor only to a certain extent. Only 5-ALA showed a significant correlation with histopathological findings. Interestingly, tumor remnants in an MGMT-methylated tumor are more likely to be visible using 5-ALA as in unmethylated tumors.

Modification of the association between recreational physical activity and survival after breast cancer by promoter methylation in breast cancer-related genes

BackgroundMechanisms underlying the inverse association between physical activity and survival after breast cancer are unresolved, but DNA methylation may play a role. We hypothesized that promoter methylation of breast cancer-related genes, as well as global methylation, may modify the association between prediagnostic recreational physical activity (RPA) and breast cancer mortality.MethodsUsing a population-based sample of 1254 women diagnosed with first primary breast cancer, we examined modification of the RPA-mortality association by gene-specific promoter methylation and global methylation. Average lifetime RPA was assessed from menarche to diagnosis through structured in-home interviews. Promoter methylation of 13 breast cancer-related genes was evaluated in archived tumor by methylation-specific polymerase chain reaction and MethyLight assay. Global methylation in white blood cell DNA was determined at long interspersed nucleotide element 1 and by the luminometric methylation assay. After approximately 15 years of follow-up, 486 patients had died, and 186 of the deaths were breast cancer-related. We used Cox proportional hazards regression to estimate HRs and 95% CIs as well as likelihood ratio tests to assess multiplicative interactions.ResultsAll-cause mortality was lower only among physically active women with methylated promoter of APC (HR 0.60, 95% CI 0.40–0.80), CCND2 (HR 0.56, 95% CI 0.32–0.99), HIN (HR 0.55, 95% CI 0.38–0.80), and TWIST1 (HR 0.28, 95% CI 0.14–0.56) in tumors, but not among those with unmethylated tumors (significant interaction p < 0.05). We found no interaction between RPA and global methylation.ConclusionsThe improved survival after breast cancer that is associated with RPA may be more pronounced in women with promoter tumor methylation in biologically plausible genes.

The Response to Chemo Radiation Therapy in Unresectable Glioblastoma Multiforme Patients in Relation to MGMT Promoter Methylation Status: A Study from a Single Saudi Center

Background: The optimal therapy for Glioblastoma Multiforme (GBM) constitutes of maximal safe resection followed by adjunctive concurrent radiation and chemotherapy with Temozolomide (TMZ). However, in certain cases safe maximal resection is not practically amenable. The methylation of the MGMT promoter is being used as a prognostic and predictive factor for the GBM response to TMZ therapy. In the present study, we aimed to measure the outcome of GBM Saudi patients who underwent tumor biopsy followed by radiation therapy with or without chemotherapy based on the MGMT promoter methylation status. Methods: The methylation-specific PCR assay was used to study the methylation status of the MGMT promoter in 77 formalin-fixed paraffin-embedded tissues of high grade glioblastoma. Results: MGMT promoter methylation was detected in 48 samples (62%). Interestingly, significant difference (p=0.0073) was found in overall survival between patients treated with surgery, radiotherapy and temozolomide and those treated with surgery and radiotherapy only, irrespective of MGMT promoter status. The survival rate for patients with MGMT methylated tumor was significantly different favoring the plus temozolomide group (p=0.0107). However, for the patients with MGMT unmethylated tumor, there was no significant difference (p=0.1453) between the two groups. Using the Cox proportional-hazards model, the methylation status of the MGMT promoter emerged as a significant (p=0.0234) clinically relevant predictor of benefit from temozolomide. Conclusion: Here, we have demonstrated that MGMT methylation has significantly improved the survival rate in Saudi patients with unresectable GBM who received concomitant radiation therapy and TMZ. However, MGMT methylation status did not impact the response to radiation therapy.

A Phase 2 Trial of Neoadjuvant Temozolomide Followed by Hypofractionated Accelerated Radiation Therapy With Concurrent and Adjuvant Temozolomide for Patients With Glioblastoma

We performed a phase 2 trial of neoadjuvant temozolomide (TMZ), followed by hypofractionated accelerated radiation therapy (HART) with concurrent TMZ, and adjuvant TMZ in patients with newly diagnosed glioblastoma to determine whether neoadjuvant TMZ would safely improve outcomes in this group of patients prior to subsequent cytotoxic therapy. Adult patients with newly diagnosed glioblastoma and a Karnofsky Performance Status >60 were eligible. Neoadjuvant TMZ administration started 2 to 3weeks from surgery at a daily dose of 75mg/m2 for 2weeks prior to delivery of HART (60Gy in 20 daily fractions) with concurrent and adjuvant TMZ. The primary endpoints were feasibility and toxicity. The secondary endpoints included overall survival (OS) and progression-free survival. Fifty patients were accrued. The median follow-up period was 44.0months for patients at risk and 22.3months for all 50 patients. Except for 1 patient in whom infection developed and another patient with progression during HART, all patients completed protocol therapy as planned. The median OS and progression-free survival were 22.3months (95% confidence interval, 14.6-42.7months) and 13.7months (95% confidence interval, 8.0-33.3months), respectively. The 4-year OS rates were 30.4% for the entire cohort and 53.3% and 14.0% for patients with methylated (n=21) and unmethylated (n=27) MGMT gene promoter tumors, respectively. One patient had grade 5 pancytopenia during HART, and another patient had transient grade 4 hepatotoxicity. A second surgical procedure was performed in 13 patients: 2 had intracranial infection, 3 had recurrences, 4 had recurrences and radiation-induced damage, and 4 had only radiation-induced damage. This novel approach of neoadjuvant TMZ is associated with an encouraging favorable long-term survival with acceptable toxicity. A future comparative trial of the efficacy of this regimen is warranted.

Promoter CpG island methylation in ion transport mechanisms and associated dietary intakes jointly influence the risk of clear-cell renal cell cancer

Sodium intake, but not potassium or fluid intake, has been associated with higher renal cell cancer (RCC) risk. However, risk factors may differ by molecular subtypes of the tumour. In renal physiology, electrolyte and water homeostasis is facilitated by ion transport mechanisms (ITM). Aberrant regulation of ITM genes, for example by promoter CpG island methylation, may modify associations between sodium, potassium and fluid intake and RCC risk. We identified ARHGDIG , ATP1A1 , SCNN1B and SLC8A3 as ITM genes exhibiting RCC-specific promoter methylation and down-regulation. Methylation-specific polymerase chain reaction (PCR) was used to analyse promoter CpG island methylation in tumour DNA of 453 RCC cases from the Netherlands Cohort Study ( n = 120852) after 20.3 years of follow-up. Diet was measured at baseline using food-frequency questionnaires. Cox regression analyses were restricted to clear-cell (cc)RCC ( n = 306) and stratified by tumours with no, low (1 gene) and high (≥ 2 genes) methylation. Sodium intake (high vs low) increased ccRCC risk particularly in tumours with a high methylation index: hazard ratio (HR) [95% confidence interval (CI)]: 2.04 (1.16-3.58), whereas heterogeneity across the methylation index was not significant ( P -heterogeneity = 0.26). Potassium intake was differentially associated with ccRCC risk ( P -heterogeneity = 0.008); the risk for high (vs low) potassium intake was low for unmethylated tumours [HR (95% CI): 0.60 (0.36-1.01)], but high for tumours with a high methylation index [HR (95% CI): 1.60 (0.96-2.65)]. Risks similarly differed for fluid intake, though not significantly ( P -heterogeneity = 0.54). Our findings suggest for the first time that dietary intakes are differentially associated with ccRCC risk according to molecular subtypes defined by ITM gene-specific promoter methylation.

Frontal glioblastoma multiforme may be biologically distinct from non-frontal and multilobar tumors

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a grim prognosis. Lobar GBM, notably those localized to the frontal lobe, are generally more amenable to complete surgical resection, and may carry a better prognosis. The biology of differently localized GBM has been reported scarcely in terms of prognostic markers, including isocitrate dehydrogenase 1 (IDH1) mutation and O(6)-methylguanine-methyltransferase (MGMT) methylation. To our knowledge, there has been no evaluation in the literature of different proliferation indexes in different GBM locations in the brain. We performed a retrospective evaluation of our prospectively collected database to assess the rate of IDH1 positivity, MGMT methylation and Ki67 index for GBM located in the frontal lobes alone, lobar GBM in other supra-tentorial lobes and multilobar GBM. IDH1 mutated tumors were localized in the frontal lobes in 50.0%, whereas only 20.3% of IDH1 wild-type tumors were localized in the frontal lobe (p=0.006); MGMT methylated tumors were localized in the frontal lobe in 32.0% of the cases. Only 13.75% of the MGMT unmethylated tumors were localized to the frontal lobe (p=0.005); Tumors with higher Ki67 proliferation index were more likely to be localized in the frontal lobe (40.6% vs. 19.5%, p=0.019). This is the largest cohort of GBM assessed for these purposes in the literature. Frontal lobe GBMs may be intrinsically biologically distinct from GBM in other lobes and from multilobar tumors.

Progressing Bevacizumab-Induced Diffusion Restriction Is Associated with Coagulative Necrosis Surrounded by Viable Tumor and Decreased Overall Survival in Patients with Recurrent Glioblastoma.

Patients with recurrent glioblastoma often exhibit regions of diffusion restriction following the initiation of bevacizumab therapy. Studies suggest that these regions represent either diffusion-restricted necrosis or hypercellular tumor. This study explored postmortem brain specimens and a population analysis of overall survival to determine the identity and implications of such lesions. Postmortem examinations were performed on 6 patients with recurrent glioblastoma on bevacizumab with progressively growing regions of diffusion restriction. ADC values were extracted from regions of both hypercellular tumor and necrosis. A receiver operating characteristic analysis was performed to define optimal ADC thresholds for differentiating tissue types. A retrospective population study was also performed comparing the overall survival of 64 patients with recurrent glioblastoma treated with bevacizumab. Patients were separated into 3 groups: no diffusion restriction, diffusion restriction that appeared and progressed within 5 months of bevacizumab initiation, and delayed or stable diffusion restriction. An additional analysis was performed assessing tumor O6-methylguanine-DNA-methyltransferase methylation. The optimal ADC threshold for differentiation of hypercellularity and necrosis was 0.736 × 10-3mm2/s. Progressively expanding diffusion restriction was pathologically confirmed to be coagulative necrosis surrounded by viable tumor. Progressive lesions were associated with the worst overall survival, while stable lesions showed the greatest overall survival (P < .05). Of the 40% of patients with O6-methylguanine-DNA-methyltransferase methylated tumors, none developed diffusion-restricted lesions. Progressive diffusion-restricted lesions were pathologically confirmed to be coagulative necrosis surrounded by viable tumor and associated with decreased overall survival. Stable lesions were, however, associated with increased overall survival. All lesions were associated with O6-methylguanine-DNA-methyltransferase unmethylated tumors.

Association of treatment for late-onset tMDS with glioblastoma recurrence in a patient displaying long-term disease response and survival.

e13506Background: Gene therapy with autologous, mutant methylguanine methyltransferase (MGMT) modified blood cells prevents myelosuppression from O6-benzylguanine and temozolomide (O6BG/TMZ) chemot...

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.

Which elderly newly diagnosed glioblastoma patients can benefit from radiotherapy and temozolomide? A PERNO prospective study.

The role of temozolomide concurrent with and adjuvant to radiotherapy (RT/TMZ) in elderly patients with glioblastoma (GBM) remains unclear. We evaluated the outcome of patients >70years in the context of the Project of Emilia-Romagna Region in Neuro-Oncology (PERNO), the first Italian prospective observational population-based study in neuro-oncology. For this analysis the criteria for selecting patients enrolled in the PERNO study were: age >70years; PS 0-3; histologically confirmed GBM; postoperative radiotherapy (RT) after surgery with or without concomitant temozolomide (TMZ) or postsurgical TMZ alone. Between January 2009 and December 2010, 76 GBM elderly patients were identified in the prospective PERNO study. Twenty-three patients did not receive any treatment after surgery, and 53 patients received postsurgical treatments (25 patients received RT alone and 28 patients RT/TMZ). Median survival was 11.1months (95% CI 8.8-13.5), adding temozolomide concomitant and adjuvant to radiotherapy it was 11.6months (95% CI 8.6-14.6), and 9.3months (95% CI 8.1-10.6) in patients treated with RT alone (P=0.164). However, patients with MGMT methylated treated with RT/TMZ obtained a better survival (17.2months, 95% CI 11.5-22.9) (P=0.042). No difference in terms of survival were observed if patients with MGMT unmethylated tumor received RT alone, or RT/TMZ or, in MGMT methylated tumor, if patients received radiotherapy alone. In elderly patients RT/TMZ represent a widely used approach but it is effective with methylated MGMT tumors only.