MGMT Pyrosequencing-Based Cut-Off Methylation Level and Clinical Outcome in Patients with Glioblastoma Multiforme

Gurrieri, Lorena; Carlo, Elisa De; Gerratana, Lorenzo; Maglio, Giovanna De; Macerelli, Marianna; Pisa, Federica Edith; Masiero, Elena; Aprile, Giuseppe; Follador, A.; Puglisi, Fabio; Fasola, Gianpiero; Rizzato, Simona; Pizzolitto, Stefano

doi:10.2217/fon-2017-0437

Cited by 31 publications

(32 citation statements)

References 38 publications

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“…Of note, qMSP is the only method with a comparatively high rate of invalid results (in our cohort: 4/76, 5.3%). Pyrosequencing protocols usually encompass several CpG sites within a differentially methylated region (in our case CpG sites 74‐80, in Felsberg et al 74‐78 22 ), and thus may produce more reliable and quantitative information on the MGMT promoter methylation status, but PSQ tests applied in different centers may interrogate different CpG sites and use distinct evaluation methods 22‐25 . Thus, the cutoff of a mean methylated allele frequency (here: 8%), may differ between different assays.…”

Section: Discussionmentioning

confidence: 99%

MGMT promoter methylation analysis for allocating combined CCNU/TMZ chemotherapy: Lessons learned from the CeTeG/NOA‐09 trial

Tzaridis

Schäfer

Weller

et al. 2020

Intl Journal of Cancer

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The CeTeG/NOA‐09 trial showed a survival benefit for combined CCNU/TMZ therapy in MGMT‐promoter‐methylated glioblastoma patients (quantitative methylation‐specific PCR [qMSP] ratio > 2). Here, we report on the prognostic value of the MGMT promoter methylation ratio determined by qMSP and evaluate the concordance of MGMT methylation results obtained by qMSP, pyrosequencing (PSQ) or DNA methylation arrays (MGMT‐STP27). A potential association of qMSP ratio with survival was analyzed in the CeTeG/NOA‐09 trial population (n = 129; log‐rank tests, Cox regression analyses). The concordance of MGMT methylation assays (qMSP, PSQ and MGMT‐STP27) was evaluated in 76 screened patients. Patients with tumors of qMSP ratio > 4 showed superior survival compared to those with ratios 2‐4 (P = .0251, log‐rank test). In multivariate analysis, the qMSP ratio was not prognostic across the study cohort (hazard ratio [HR] = 0.88; 95% CI: 0.72‐1.08). With different cutoffs for qMSP ratio (4, 9, 12 or 25), the CCNU/TMZ benefit tended to be larger in subgroups with lower ratios (eg, for cutoff 9: HR 0.32 for lower subgroup, 0.73 for higher subgroup). The concordance rates with qMSP were 94.4% (PSQ) and 90.2% (MGMT‐STP27). Discordant results were restricted to tumors with qMSP ratios ≤4 and PSQ mean methylation rate ≤25%. Despite a shorter survival in MGMT‐promoter‐methylated patients with lower methylation according to qMSP, these patients had a benefit from combined CCNU/TMZ therapy, which even tended to be stronger than in patients with higher methylation rates. With acceptable concordance rates, decisions on CCNU/TMZ therapy may also be based on PSQ or MGMT‐STP27.

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Section: Discussionmentioning

confidence: 99%

MGMT promoter methylation analysis for allocating combined CCNU/TMZ chemotherapy: Lessons learned from the CeTeG/NOA‐09 trial

Tzaridis

Schäfer

Weller

et al. 2020

Intl Journal of Cancer

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“…The literature surveys identified a lack of consistency in determining methylation cut-off values. A number of authors depend on three categories; unmethylated 0–9%, methylated 10–29% and highly methylated 30–100% ( 49 , 50 ), whereas others rely on two; unmethylated <15% and methylated >15% ( 62 – 64 ). In addition, several studies use ROC curve analysis to establish the optimal methylation threshold in discriminating diagnostic categories, cancer types, treatment decisions and outcomes, and patient survival ( 62 , 65 , 66 ).…”

Section: Discussionmentioning

confidence: 99%

“…The methylation index (MI) was calculated as the mean-percent-methylation across all gene CpG sites (three CpG sites of the HS3ST2 gene and six CpG sites of the KLF4 gene). The promoter methylation status was theoretically classified as unmethylated (0–9%), methylated (10–29%) and highly methylated (30–100%) ( 49 , 50 ). Multinomial logistic regression then modelled dependence of the diagnosis on age, BMI, menarche, parity, CpGs and smoking.…”

Section: Methodsmentioning

confidence: 99%

Methylation status of KLF4 and HS3ST2 genes as predictors of endometrial cancer and hyperplastic endometrial lesions

et al. 2018

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Endometrial carcinoma is one of the most common tumours in developed countries. In addition to the active role of genetic factors, epigenetic changes also have an important effect. The present study analysed the methylation status of kruppel like factor 4 (KLF4) and heparan sulfate-glucosamine 3-sulfotransferase 2 (HS3ST2) genes in three endometrial tissue types for carcinoma prediction. The sample comprised 91 women with histologically-confirmed endometrial carcinoma (64.16±9.64 years old), 36 women with hyperplasia (53.39±9.64 years old) and 45 with no signs or symptoms of malignancy (48.53±11.11 years old). The CpG dinucleotide methylation levels were examined by quantitative pyrosequencing, and the discrimination accuracy of the model was calculated using the Random Forest classification algorithm of the area under the ROC curve (AUC). The mean values of KLF4 and HS3ST2 methylation indices were 23.83±11.39 and 8.52±2.57 in the control samples; 30.40±8.52 and 33.76±20.66 in hyperplasia and 34.72±10.79 and 34.49±18.39 in the cancerous tissues. Multinomial logistic regression indicated that the HS3ST2 CpG1 methylation status is a predictor of hyperplasia (P<0.05) and that the KLF4 CpG2 dinucleotide can predict carcinoma formation (P<0.001). The AUC value of 0.95 indicates high discrimination accuracy of the CpG nucleotides methylation status model between the controls and the two other diagnoses. The results of the present study establish the likelihood that aberrations in KLF4 and HS3ST2 gene methylation levels are important in the development of endometrial hyperplasia and carcinoma, with hyperplasia an intermediate step between healthy and tumour tissues.

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“…Although the current TMZ based CRT has marginally improved survival in MGMT methylated (ie MGMT inactivated) GBM patients, its cytotoxicity is relatively nullified in unmethylated GBM patients [ 17 ]. Recent, recent analysis of quantitative methylation using pyrosequencing on 108 GBM patients revealed that degree of MGMT promoter methylation is directly associated with median progression free survival [ 18 ]. Currently, small molecule inhibitors targeting MGMT are being utilized prior to TMZ based therapy in many clinical trials [ 19 ].…”

Section: Natural Products and Gbmmentioning

confidence: 99%

Natural products: a hope for glioblastoma patients

et al. 2018

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Glioblastoma (GBM) is one of the most aggressive malignant tumors with an overall dismal survival averaging one year despite multimodality therapeutic interventions including surgery, radiotherapy and concomitant and adjuvant chemotherapy. Few drugs are FDA approved for GBM, and the addition of temozolomide (TMZ) to standard therapy increases the median survival by only 2.5 months. Targeted therapy appeared promising in in vitro monolayer cultures, but disappointed in preclinical and clinical trials, partly due to the poor penetration of drugs through the blood brain barrier (BBB). Cancer stem cells (CSCs) have intrinsic resistance to initial chemoradiation therapy (CRT) and acquire further resistance via deregulation of many signaling pathways. Due to the failure of classical chemotherapies and targeted drugs, research efforts focusing on the use of less toxic agents have increased. Interestingly, multiple natural compounds have shown antitumor and apoptotic effects in TMZ resistant and p53 mutant GBM cell lines and also displayed synergistic effects with TMZ. In this review, we have summarized the current literature on natural products or product analogs used to modulate the BBB permeability, induce cell death, eradicate CSCs and sensitize GBM to CRT.

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MGMT Pyrosequencing-Based Cut-Off Methylation Level and Clinical Outcome in Patients with Glioblastoma Multiforme

Abstract: 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.

Cited by 31 publications

References 38 publications

MGMT promoter methylation analysis for allocating combined CCNU/TMZ chemotherapy: Lessons learned from the CeTeG/NOA‐09 trial

MGMT promoter methylation analysis for allocating combined CCNU/TMZ chemotherapy: Lessons learned from the CeTeG/NOA‐09 trial

Methylation status of KLF4 and HS3ST2 genes as predictors of endometrial cancer and hyperplastic endometrial lesions

Natural products: a hope for glioblastoma patients

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