O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

Background: We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. 12 Hide Figures Abstract Introduction Methods Results Discussion Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings: Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance: MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined

Iodine-125 brachytherapy for brain tumours - a review

Iodine-125 brachytherapy has been applied to brain tumours since 1979. Even though the physical and biological characteristics make these implants particularly attractive for minimal invasive treatment, the place for stereotactic brachytherapy is still poorly defined

Very‐low‐carbohydrate diet enhances human T‐cell immunity through immunometabolic reprogramming

Abstract Very‐low‐carbohydrate diet triggers the endogenous production of ketone bodies as alternative energy substrates. There are as yet unproven assumptions that ketone bodies positively affect human immunity. We have investigated this topic in an in vitro model using primary human T cells and in an immuno‐nutritional intervention study enrolling healthy volunteers. We show that ketone bodies profoundly impact human T‐cell responses. CD4+, CD8+, and regulatory T‐cell capacity were markedly enhanced, and T memory cell formation was augmented. RNAseq and functional metabolic analyses revealed a fundamental immunometabolic reprogramming in response to ketones favoring mitochondrial oxidative metabolism. This confers superior respiratory reserve, cellular energy supply, and reactive oxygen species signaling. Our data suggest a very‐low‐carbohydrate diet as a clinical tool to improve human T‐cell immunity. Rethinking the value of nutrition and dietary interventions in modern medicine is required

Study population.

<p>Abbreviations: KPS, Karnofsky performance score; PE, stereotactic biopsy; OP, open tumor resection.</p><p>*after 3 months.</p

MGMT mRNA expression and MGMT promoter methylation status in malignant glioma.

<p>Horizontal bars indicate medians. The dotted line indicates the cut-off value distinguishing low from high MGMT expression values. <b>A:</b> Expression of MGMT determined with quantitative real-time PCR in non-cancerous brain tissue specimen (1, N = 10), in malignant glioma (2, N = 63), and in the glioma group stratified by <i>MGMT</i> promoter methylation status (methylated, 3, N = 32, and unmethylated, 4, N = 31). cDNA was synthesized from amplified RNA purified from tumor tissue obtained by stereotactic biopsy or open surgery and relative expression of MGMT with respect to expression of the reference genes <i>SDHA</i> and <i>TBP</i> was determined using real-time PCR. <b>B:</b> Validation set obtained from the TCGA database. All data were derived from array analyses and expression levels of methylated (3, N = 105) and unmethylated (4, N = 104) GBM tissue samples are shown.</p

Kaplan-Meier estimates of 63 patients with malignant glioma.

<p>Tumor tissue obtained either by stereotactic biopsy or by open surgery. <b>A:</b> Progression free survival and overall survival of the whole study population, <b>B:</b> Overall survival stratified by the <i>MGMT</i> promoter methylation status.</p

Expression of DNMTs in non-cancerous brain tissue specimen and in malignant glioma.

<p>cDNA was synthesized from amplified RNA purified from tumor tissue obtained by stereotactic biopsy or open surgery and expression of DNMTs was determined using real-time PCR. <b>A:</b> Expression pattern of DNMTs in normal brain tissue (1, N = 10) and in high grade glioma (2, N = 63). DNMT mRNA expression is calculated relative to the reference genes <i>SDHA</i> and <i>TBP</i> (*, p>0.01). <b>B:</b> Expression of DNMTs in non-cancerous brain tissue specimen (1, N = 10), in high grade glioma (2, N = 63), and in the glioma group stratified by <i>MGMT</i> promoter methylation status (methylated, 3, N = 32; unmethylated, 4, N = 31). All data were normalized to the reference genes SDHA and TBP, and the fold change of every DNMT relative to the median expression in the normal brain samples (arbitrarily set to 1) was calculated. Horizontal bars indicate medians.</p

Favorable prognostic factors (uni- and multivariate models).

<p>Favorable prognostic factors (uni- and multivariate models).</p