Quality assessment of the MRI-radiomics studies for MGMT promoter methylation prediction in glioma: a systematic review and meta-analysis.

OBJECTIVES: To evaluate the methodological quality and diagnostic accuracy of MRI-based radiomic studies predicting O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in gliomas. METHODS: PubMed Medline, EMBASE, and Web of Science were searched to identify MRI-based radiomic studies on MGMT methylation in gliomas published until December 31, 2022. Three raters evaluated the study methodological quality with Radiomics Quality Score (RQS, 16 components) and Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis Or Diagnosis (TRIPOD, 22 items) scales. Risk of bias and applicability concerns were assessed with QUADAS-2 tool. A meta-analysis was performed to estimate the pooled area under the curve (AUC) and to assess inter-study heterogeneity. RESULTS: We included 26 studies, published from 2016. The median RQS total score was 8 out of 36 (22%, range 8-44%). Thirteen studies performed external validation. All studies reported AUC or accuracy, but only 4 (15%) performed calibration and decision curve analysis. No studies performed phantom analysis, cost-effectiveness analysis, and prospective validation. The overall TRIPOD adherence score was between 50% and 70% in 16 studies and below 50% in 10 studies. The pooled AUC was 0.78 (95% CI, 0.73-0.83, I2 = 94.1%) with a high inter-study heterogeneity. Studies with external validation and including only WHO-grade IV gliomas had significantly lower AUC values (0.65; 95% CI, 0.57-0.73, p < 0.01). CONCLUSIONS: Study RQS and adherence to TRIPOD guidelines was generally low. Radiomic prediction of MGMT methylation status showed great heterogeneity of results and lower performances in grade IV gliomas, which hinders its current implementation in clinical practice. CLINICAL RELEVANCE STATEMENT: MGMT promoter methylation status appears to be variably correlated with MRI radiomic features; radiomic models are not sufficiently robust to be integrated into clinical practice to accurately predict MGMT promoter methylation status in patients with glioma before surgery. KEY POINTS: • Adherence to the indications of TRIPOD guidelines was generally low, as was RQS total score. • MGMT promoter methylation status prediction with MRI radiomic features provided heterogeneous diagnostic accuracy results across studies. • Studies that included grade IV glioma only and performed external validation had significantly lower diagnostic accuracy than others.

[Analysis of the difference in MGMT promoter status in gliomas and its significance in prognosis assessment].

The data of 1 268 newly diagnosed gliomas from the Fourth Ward of Neurosurgery Department of Beijing Tiantan Hospital between April 2013 and March 2022 were retrospectively analyzed. Based on postoperative pathology, the gliomas were divided into groups: oligodendrogliomas (n=308), astrocytomas (n=337) and glioblastomas (n=623). According to the O6-methylguanine-DNA methyl transferase (MGMT) promoter status defined by the 12% of best cut-off value in previous research results, patients were divided into methylation group (n=763) and non-methylation group (n=505). Methylation level [M (Q1, Q3)] in patients with glioblastoma, astrocytoma and oligodendroglioma was 6% (2%, 24%), 17% (10%, 28%) and 29% (19%, 40%), respectively (P<0.001). Compared with non-methylation patients, the progression-free survival (PFS) and overall survival (OS) of glioblastomas patients with methylation of MGMT promoter demonstrated more favorable prognosis [M (Q1, Q3)]) of PFS: 14.0 (6.0, 36.0) months vs 8.0 (4.0, 15.0) months, P<0.001; M (Q1, Q3) of OS: 29.0 (17.0, 60.5) months vs 16.0 (11.0, 26.5) months, P<0.001]. In astrocytomas patients, the PFS was much longer for those with methylation [the median PFS of patients with methylation was not observed at the end of follow-up, but those without methylation showed a median PFS of 46.0 (29.0, 52.0) months] (P=0.001). However, no statistically significant difference was observed in OS [the median OS of patients with methylation was not observed at the end of follow-up, but those without methylation had a median OS of 62.0 (46.0, 98.0) months] (P=0.085). In oligodendrogliomas patients, no statistically significant differences of PFS and OS were observed between patients with methylation and those without methylation. MGMT promoter status was a related factor affecting PFS and OS in glioblastomas (PFS: HR=0.534,95%CI: 0.426-0.668, P<0.001; OS: HR=0.451, 95%CI: 0.353-0.576, P<0.001). Moreover, MGMT promoter status was also a related factor affecting PFS in astrocytomas (HR=0.462, 95%CI: 0.221-0.966, P=0.040), but not for OS (HR=0.664, 95%CI: 0.259-1.690, P=0.389). The methylation level of MGMT promoter differed substantially in different types of gliomas, and the status of MGMT promoter profoundly affected the prognosis of glioblastomas.

Noninvasive Assessment of O(6)-Methylguanine-DNA Methyltransferase Promoter Methylation Status in World Health Organization Grade II-IV Glioma Using Histogram Analysis of Inflow-Based Vascular-Space-Occupancy Combined with Structural Magnetic Resonance Imaging.

BACKGROUND: O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation is an important prognostic factor for gliomas and is associated with tumor angiogenesis. Arteriolar cerebral blood volume (CBVa) obtained from inflow-based vascular-space-occupancy (iVASO) magnetic resonance imaging (MRI) is assumed to be an indicator of tumor microvasculature. Its preoperative predictive ability for MGMT promoter methylation remains unclear. PURPOSE: To investigate the role of iVASO-CBVa histogram features in determining MGMT promoter methylation status of grade II-IV gliomas. STUDY TYPE: Retrospective SUBJECTS: Forty-six patients consisting of 20 MGMT methylated and 26 unmethylated gliomas. FIELD STRENGTH/SEQUENCE: 3.0 T magnetic resonance images containing iVASO MRI, T1 -weighted image (T1 WI), T2 -weighted image, T2 -weighted fluid attenuated inversion recovery image images, and enhanced T1 WI. ASSESSMENT: Sixteen structural imaging features were visually evaluated on structural MRI and 14 CBVa histogram features were extracted from iVASO-CBVa maps. STATISTICAL TESTS: Imaging features were screened and ranked using Fisher's exact test, Mann-Whitney U-test, and randomforest algorithm. Features with higher importance were selected to develop logistic regression models to determine MGMT methylation status. Receiver operating characteristics (ROC) curve with the area under the curve (AUC) and leave-one-out cross-validation (LOOCV) were used to assess effectiveness and stability. RESULTS: The top two CBVa histogram features were root mean squared (RMS) and variance. The top two structural imaging features were contrast-enhancing component of the tumor (CET) location and tumor location. Both the CBVa model of RMS and variance (ROC, AUC = 0.867; LOOCV, AUC = 0.819) and the model of structural features (ROC, AUC = 0.882; LOOCV, AUC = 0.802) accurately identified MGMT methylation. The fusion model of CBVa RMS and CET location improved diagnostic performance (ROC, AUC = 0.931; LOOCV, AUC =0.906). DATA CONCLUSION: iVASO-CBVa has potential in evaluating MGMT methylation status in grade II-IV gliomas. LEVEL OF EVIDENCE: 4 TECHNICAL EFFICACY: Stage 2.

Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma.

BACKGROUND: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values. RESULTS: We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.

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

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

Evaluating the impact of universal Lynch syndrome screening in a publicly funded healthcare system.

PURPOSE: Referrals for Lynch syndrome (LS) assessment have traditionally been based on personal and family medical history. The introduction of universal screening practices has allowed for referrals based on immunohistochemistry tests for mismatch repair (MMR) protein expression. This study aims to characterize the effect of universal screening in a publicly funded healthcare system with comparison to patients referred by traditional criteria, from January 2012 to March 2017. METHODS: Patient files from the time of initiation of universal screening from 2012 to 2017 were reviewed. Patients were sorted into two groups: (a) universally screened and (b) referred by traditional methods. Mutation detection rates, analysis of traditional testing criteria met, and cascade carrier testing were evaluated. RESULTS: The mutation detection rate of the universal screening group was higher than the traditionally referred group (45/228 (19.7%) vs 50/390 (12.5%), P = .05), though each were able to identify unique patients. An analysis of testing criteria met by each patient showed that half of referred patients from the universal screening group could not meet any traditional testing criteria. CONCLUSION: The implementation of universal screening in a publicly funded system will increase efficiency in detecting patients with LS. The resources available for genetic testing and counseling may be more limited in public systems, thus inclusion of secondary screening with BRAF and MLH1 promoter hypermethylation testing is key to further optimizing efficiency.

Identifying Disparities in Care in Treating Glioblastoma: A Retrospective Cohort Study of Patients Treated at a Safety-net Versus Private Hospital Setting.

BACKGROUND: Patients of lower socioeconomic status (SES) may experience barriers to their oncologic care, but current data conflict over whether SES affects the prognosis of patients with glioblastoma (GB). OBJECTIVE: We sought to determine whether SES disparities impaired delivery of neuro-oncologic care and affected the prognosis of GB patients. METHODS: The records of GB patients treated from 2010 to 2014 at a safety-net hospital (SNH) or private hospital (PH), both served by 1 academic medical institution, were retrospectively reviewed and compared. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. RESULTS: A total of 55 SNH and 39 PH GB patients were analyzed with median 11-month follow-up. SNH patients were predominantly Hispanic, low income, enrolled in Medicaid, were less likely to receive radiation (89% vs. 100%), took longer to start radiation (41 vs. 29 days), and were less likely to complete radiation treatment (80% vs. 95%). Concurrent and adjuvant temozolomide use were also lower (85% vs. 94% and 60% vs. 71%, respectively). OS and PFS were not significantly different (15 vs. 16 months and 8 vs. 11 months, respectively). On multivariate analysis, adjuvant chemotherapy and RT completion predicted for better OS, whereas hospital type, income, and insurance did not. CONCLUSION: Although GB patients at our SNH received less adjuvant treatment compared with PH, outcomes were similar. Access to multidisciplinary care staffed by academic physicians may play an important role in overcoming socioeconomic barriers to treatment availability and quality at SNHs.

A Four-gene Decision Tree Signature Classification of Triple-negative Breast Cancer: Implications for Targeted Therapeutics.

The molecular complexity of triple-negative breast cancers (TNBCs) provides a challenge for patient management. We set out to characterize this heterogeneous disease by combining transcriptomics and genomics data, with the aim of revealing convergent pathway dependencies with the potential for treatment intervention. A Bayesian algorithm was used to integrate molecular profiles in two TNBC cohorts, followed by validation using five independent cohorts (n = 1,168), including three clinical trials. A four-gene decision tree signature was identified, which robustly classified TNBCs into six subtypes. All four genes in the signature (EXO1, TP53BP2, FOXM1, and RSU1) are associated with either genomic instability, malignant growth, or treatment response. One of the six subtypes, MC6, encompassed the largest proportion of tumors (∼50%) in early diagnosed TNBCs. In TNBC patients with metastatic disease, the MC6 proportion was reduced to 25%, and was independently associated with a higher response rate to platinum-based chemotherapy. In TNBC cell line data, platinum sensitivity was recapitulated, and a sensitivity to the inhibition of the phosphatase PPM1D was revealed. Molecularly, MC6-TNBCs displayed high levels of telomeric allelic imbalances, enrichment of CD4+ and CD8+ immune signatures, and reduced expression of genes negatively regulating the MAPK signaling pathway. These observations suggest that our integrative classification approach may identify TNBC patients with discernible and theoretically pharmacologically tractable features that merit further studies in prospective trials.

MGMT Gene Promoter Methylation Status - Assessment of Two Pyrosequencing Kits and Three Methylation-specific PCR Methods for their Predictive Capacity in Glioblastomas.

BACKGROUND: Although methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter predicts response to temozolomide in patients with glioblastoma, no consensus exists as to which assay is best for its detection. MATERIALS AND METHODS: Methylation of MGMT promoter was examined by methylation-specific polymerase chain reaction (MSP), quantitative real-time MSP, methylation-sensitive high-resolution melting analysis, and two commercial pyrosequencing (PSQ) kits. Survival was compared among 48 patients with glioblastoma according to assay results. RESULTS: Only PSQ and MSP significantly separated patients who benefited from temozolomide, with PSQ being the superior method. For PSQ analysis, the cut-off value that best correlated with prognostic outcome was 7% methylation of MGMT. Median survival in patients with MGMT promoter methylation above this cut-off value was 7.8 months longer compared to those with less than 7% methylation. Two-year overall survival for the two groups was 42% and 7.4%, respectively. CONCLUSION: PSQ is the method of choice for MGMT promoter methylation analysis in routine clinical practice.

Assessment of DNA repair susceptibility genes identified by whole exome sequencing in head and neck cancer.

Head and neck cancer (HNC), the sixth most common cancer globally, stands second in India. In Northeast (NE) India, it is the sixth most common cause of death in males and seventh in females. Prolonged tobacco and alcohol consumption constitute the major etiological factors for HNC development, which induce DNA damage. Therefore, DNA repair pathway is a crucial system in maintaining genomic integrity and preventing carcinogenesis. The present work was aimed to predict the consequence of significant germline variants of the DNA repair genes in disease predisposition. Whole exome sequencing was performed in Ion Proton™ platform on 15 case-control samples from the HNC-prevalent states of Manipur, Mizoram, and Nagaland. Variant annotation was done in Ion Reporter™ as well as wANNOVAR. Subsequent statistical and bioinformatics analysis identified significant exonic and intronic variants associated with HNC. Amongst our observed variants, 78.6% occurred in ExAC, 94% reported in dbSNP and 5.8% & 9.3% variants were present in ClinVar and HGMD, respectively. The total variants were dispersed among 199 genes with DSBR and FA pathway being the most mutated pathways. The allelic association test suggested that the intronic variants in HLTF and RAD52 gene significantly associated (P < 0.05) with the risk (OR > 5), while intronic variants in PARP4, RECQL5, EXO1 and PER1 genes and exonic variant in TDP2 gene showed protection (OR < 1) for HNC. MDR analysis proposed the exonic variants in MSH6, BRCA2, PALB2 and TP53 genes and intronic variant in RECQL5 genetic region working together during certain phase of DNA repair mechanism for HNC causation. In addition, other intronic and 3'UTR variations caused modifications in the transcription factor binding sites and miRNA target sites associated with HNC. Large-scale validation in NE Indian population, in-depth structure prediction and subsequent simulation of our recognized polymorphisms is necessary to identify true causal variants related to HNC.

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization.

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5-methylcytosines (5-mC) are used for the capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The method is applied in the 45-min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step.

Genetic assessment and folate receptor autoantibodies in infantile-onset cerebral folate deficiency (CFD) syndrome.

INTRODUCTION: Cerebral folate deficiency (CFD) syndromes are defined as neuro-psychiatric conditions with low CSF folate and attributed to different causes such as autoantibodies against the folate receptor-alpha (FR) protein that can block folate transport across the choroid plexus, FOLR1 gene mutations or mitochondrial disorders. High-dose folinic acid treatment restores many neurologic deficits. STUDY AIMS AND METHODS: Among 36 patients from 33 families the infantile-onset CFD syndrome was diagnosed based on typical clinical features and low CSF folate. All parents were healthy. Three families had 2 affected siblings, while parents from 4 families were first cousins. We analysed serum FR autoantibodies and the FOLR1 and FOLR2 genes. Among three consanguineous families homozygosity mapping attempted to identify a monogenetic cause. Whole exome sequencing (WES) was performed in the fourth consanguineous family, where two siblings also suffered from polyneuropathy as an atypical finding. RESULTS: Boys (72%) outnumbered girls (28%). Most patients (89%) had serum FR autoantibodies fluctuating over 5-6 weeks. Two children had a genetic FOLR1 variant without pathological significance. Homozygosity mapping failed to detect a single autosomal recessive gene. WES revealed an autosomal recessive polynucleotide kinase 3´phosphatase (PNKP) gene abnormality in the siblings with polyneuropathy. DISCUSSION: Infantile-onset CFD was characterized by serum FR autoantibodies as its predominant pathology whereas pathogenic FOLR1 gene mutations were absent. Homozygosity mapping excluded autosomal recessive inheritance of any single responsible gene. WES in one consanguineous family identified a PNKP gene abnormality that explained the polyneuropathy and also its contribution to the infantile CFD syndrome because the PNKP gene plays a dual role in both neurodevelopment and immune-regulatory function. Further research for candidate genes predisposing to FRα-autoimmunity is suggested to include X-chromosomal and non-coding DNA regions.

Comparative assessment of three methods to analyze MGMT methylation status in a series of 350 gliomas and gangliogliomas.

MGMT promoter methylation is considered as a prognostic and predictive biomarker indicating response to chemotherapy and radiotherapy in glioblastoma. A number of different methods and platforms including pyrosequencing (PSQ), quantitative methylation-specific PCR (qMSP) and immunohistochemistry (IHC), methylation-sensitive high resolution melting (MS-HRM) and NGS (Next Generation Sequencing) have been used to detect MGMT promoter methylation in gliomas. However, controversy remains about the most appropriate method to use for analyzing MGMT status. The MGMT promoter methylation status of a total of 350 gliomas and gangliogliomas was examined using PSQ, qMSP and IHC in parallel. Using PSQ as a recommended standard method, the sensitivity, specificity, positive/negative predictive value and correlation with the other assays were calculated. Among 350 glioma and ganglioglioma cases, the MGMT promoter tested positive for methylation in 53.1%, 55.4%, and 70.3% of the cases by PSQ, qMSP and IHC, respectively. The sensitivity and specificity of qMSP were 97.8% and 92.7%, respectively. Twelve cases that tested positive for methylation using qMSP were negative according to PSQ, and four cases that were negative according to qMSP tested positive according to PSQ. The concordance rate between PSQ and qMSP was 90.8%. The sensitivity and specificity of IHC for the detection of MGMT at the protein level were 84.4% and 45.7%, respectively. The concordance rate between PSQ and IHC was 30.8%. This study demonstrated that qMSP is an effective and rapid detection method for routine use in pathology laboratories for the identification of MGMT promoter methylation. A combination of IHC and qMSP assays can provide high sensitivity and specificity for the prediction of MGMT status. A few cases that tested negative with PSQ did harbor MGMT promoter methylation, as confirmed by qMSP and sequencing, and this subgroup of patients may benefit from temozolomide.

Review of the current medical literature and assessment of current utilization patterns regarding mismatch repair protein immunohistochemistry in cutaneous Muir-Torre syndrome-associated neoplasms.

Muir-Torre syndrome is a clinical variant of Lynch syndrome defined by the synchronous or metachronous occurrence of at least one sebaceous neoplasm and at least one Lynch syndrome-related internal cancer. Although screening guidelines for patients with colorectal carcinomas have been established, screening guidelines for cutaneous Muir-Torre associated neoplasms are not currently available. As such, we reviewed the current evidence for the use of MLH1, MSH2, MSH6 and PMS2 immunohistochemistry when cutaneous Muir-Torre associated neoplasms are encountered. We identified weak to moderate support overall for the global use of these assays, with some evidence suggesting a tailored approach using clinical parameters as an adjunct. We also assessed the current utilization patterns of attendees of the American Society of Dermatopathology Annual Meeting (Chicago, 2016). We found that 91% of respondents utilize mismatch repair immunohistochemistry, with the majority utilizing these tests only when requested by the submitting clinician.

Assessment of molecular markers demonstrates concordance between samples acquired via stereotactic biopsy and open craniotomy in both anaplastic astrocytomas and glioblastomas.

The classification, treatment and prognosis of high-grade gliomas has been shown to correlate with the expression of molecular markers (e.g. MGMT promotor methylation and IDH1 mutations). Acquisition of tumor samples may be obtained via stereotactic biopsy or open craniotomy. Between the years 2009 and 2013, 22 patients initially diagnosed with HGGs via stereotactic biopsy, that ultimately underwent open craniotomy for resection of their tumor were prospectively included in an institutional glioma database. MGMT promotor analysis was performed using methylation-specific (MS)-PCR and IDH1R132H mutation analysis was performed using immunohistochemistry. Three patients (13.7%) exhibited IDH1R132H mutations in samples obtained via stereotactic biopsy. Tissue derived from stereotaxic biopsy was demonstrated to have MGMT promotor methylation in ten patients (45.5%), while a non-methylated MGMT promotor was demonstrated in ten patients (45.5%); inconclusive results were obtained for the remaining two patients (9%) within our cohort. The initial histologic grading, IDH1R132H mutation and MGMT promotor methylation results were confirmed using samples obtained during open craniotomy in all but one patient; here inconclusive MGMT promotor analysis was obtained in contrast to that which was obtained via stereotactic biopsy. Tumor samples acquired via stereotactic biopsy provide accurate information with regard to clinically relevant molecular markers that have been shown to impact patient care decisions. The profile of markers analyzed in our cohort was nearly concordant between those samples obtained via stereotactic biopsy or open craniotomy thereby suggesting that clinical decisions may be based on the molecular profile of the tumor samples obtained via stereotactic biopsy.

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

BACKGROUND: The DNA repair protein O(6)-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. RESULTS: We 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. CONCLUSIONS: Compared 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.

Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

Evolution of cancer risk assessment and counseling related to psychological, financial and legal implications.

Cancer risk assessment, genetic counseling and genetic testing have experienced advances and changes over the past two decades due to improved technology, legal movements to protect those at an increased risk for cancer due to genetics, as well as advances in detection, prevention and treatment. This brief article will provide a summary of these advances over three eras of cancer genetics: pre-discovery of the more common high impact genes, namely BRCA1/BRCA2 and the mismatch repair genes associated with Lynch syndrome; the time during which the genes were being discovered; and current day.