Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma

Vlassenbroeck, Ilse; Califice, Stéphane; Diserens, Annie‐Claire; Migliavacca, Eugenia; Straub, Josef; Stefano, Ivano Di; Moreau, Fabrice; Hamou, Marie‐France; Renard, Isabelle; Delorenzi, Mauro; Flamion, Bruno; Diguiseppi, James; Bierau, Katja; Hegi, Monika E.

doi:10.2353/jmoldx.2008.070169

Cited by 176 publications

(147 citation statements)

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“…Methylation seen in MSP but not in pyrosequencing may be because of the increased sensitivity of MSP detecting methylation in minor populations of tumour cells or false positives, whereas methylation obtained by pyrosequencing and not MSP may reflect the primer positions. More recently a quantitative real-time MSP assay, in which the copy number of methylated MGMT alleles is calculated, improved upon the gelbased MSP assay in terms of reproducibility and use with archival samples, but does not provide methylation data at individual CpG sites (Vlassenbroeck et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

“…The MSP assay is prone to lack of reliability attributed to poor quality archival tissue and small sample size and optimum results are usually obtained with cryopreserved tissues (Hegi et al, 2005;Yip et al, 2008). A number of improvements (Cankovic et al, 2007) and alternative methods including semi-quantative methods have been developed (Jeuken et al, 2007;Lorente et al, 2008;Vlassenbroeck et al, 2008). Pyrosequencing allows highly reproducible quantification of methylation at each CpG site within the chosen amplicon and has been found to be robust when applied to archival samples including glioblastomas (Mikeska et al, 2007), but use of pyrosequencing in analysis of extensive glioblastoma cohorts and comparison with outcome data has not been reported.…”

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Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

et al. 2009

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BACKGROUND: Epigenetic silencing of O 6 -methylguanine-DNA-methyltransferase (MGMT) by promoter methylation is associated with improved survival in glioblastomas treated with alkylating agents. In this study, we investigated MGMT promoter methylation in glioblastomas treated with temozolomide and radiotherapy in a single UK treatment centre. METHODS: Quantitative methylation data at individual CpG sites were obtained by pyrosequencing for 109 glioblastomas. RESULTS: Median overall survival (OS) was 12.4 months with 2-year survival of 17.9%. Pyrosequencing data were reproducible with archival samples yielding data for all glioblastomas. Variation in methylation patterns of discrete CpG sites and intratumoral methylation heterogeneity were observed. A total of 58 out of 109 glioblastomas showed average methylation 4non-neoplastic brain in at least one clinical sample; 86% had homogeneous methylation status in multiple samples. Methylation was an independent prognostic factor associated with prolonged progression-free survival (PFS) and OS. Cases with methylation more than 35% had the longest survival (median PFS 19.2; OS 26.2 months, 2-year survival of 59.7%). Significant differences in PFS were seen between those with intermediate or high methylation and unmethylated cases, whereas cases with low, intermediate or high methylation all showed significantly different OS. CONCLUSIONS: These data indicate that MGMT methylation is prognostically significant in glioblastomas given chemoradiotherapy in the routine clinic; furthermore, the extent of methylation may be used to provide additional prognostic stratification.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

et al. 2009

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“…This assay determines the number of copies of methylated MGMT, which is then normalized to the number of copies of the ACTB gene. 14 MGMT assessment at the protein level is usually done by immunohistochemistry (IHC), a low-cost method commonly used in diagnostic histopathology. Some studies of high-grade glioma patients treated with alkylating agents, including GBM patients, have reported significant association of MGMT expression by IHC with patient outcome.…”

Section: Introductionmentioning

confidence: 99%

Comparative assessment of 5 methods (methylation‐specific polymerase chain reaction, methylight, pyrosequencing, methylation‐sensitive high‐resolution melting, and immunohistochemistry) to analyze O6‐methylguanine‐DNA‐methyltranferase in a series of 100 glioblastoma patients

Quillien

Lavenu

Karayan‐Tapon

et al. 2012

Cancer

187

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BACKGROUND: There is a strong need to determine the best technique for O 6 -methylguanine-DNA-methyltranferase (MGMT) analysis, because MGMT status is currently used in clinical trials and occasionally in routine clinical practice for glioblastoma patients. METHODS:The authors compared analytical performances and predictive values of 5 techniques in a series of 100 glioblastoma patients who received standard of care treatment (Stupp protocol). RESULTS: MGMT promoter was considered methylated in 33%, 33%, 42%, and 60% of patients by methylation-sensitive high-resolution melting, MethyLight, pyrosequencing (with an optimal risk cutoff at 8% for the average percentage of the 5 CpGs tested), and methylation-specific polymerase chain reaction (MS-PCR), respectively. Fifty-nine percent of the samples had <23% (the optimal risk cutoff) of MGMT-positive tumor cells. The best predictive values for overall survival (OS), after adjustment for age and performance status, were obtained by pyrosequencing (hazard ratio [HR], 0.32; P < .0001), MS-PCR (HR, 0.37; P < .0001), and immunohistochemistry (HR, 0.43; P ¼ .0005) as compared with methylation-sensitive high-resolution melting (HR, 0.52 P ¼ .02) and MethyLight (HR, 0.6; P ¼ .05). For progression-free survival (PFS), the best predictive values were obtained with pyrosequencing (HR, 0.35; P < .0001), methylation-sensitive high-resolution melting (HR, 0.46; P ¼ .002), and MS-PCR (HR, 0.49; P ¼ .002). Combining pyrosequencing and immunohistochemistry slightly improved predictive power for OS, but not for PFS. Poor reproducibility and interobserver variability were, however, observed for immunohistochemistry. CONCLUSIONS: Good prediction of survival in addition to high reproducibility and sensitivity made pyrosequencing the best among the 5 techniques tested in this study. Cancer 2012;118:4201-11.

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“…The small number of patients in each cancer cohort does not permit efficacy conclusions; the overall low RR does not permit conclusions to be drawn about influence of MMR status. The type of assay used for promoter methylation status may underestimate the prevalence of methylation detected by the serum assay (33). The source of serum DNA methylation in the absence of tumor methylation, which is an important aspect to be considered in the development of serum-based assays, was not explored.…”

Section: Discussionmentioning

confidence: 99%

“…One central laboratory conducted all MGMT analyses (OncoMethylome Sciences Testing Laboratory) in all screened patients using a direct, real-time methylationspecific PCR assay (MSP) to determine methylation status of the MGMT gene promoter (33). This assay detects CpG island methylation in the same MGMT promoter region as Molecular Cancer Therapeutics 810 the nested, gel-based assay used in the phase III glioblastoma trial (7).…”

Section: Dna Extraction and Methylation-specific Pcr Assaymentioning

confidence: 99%

A Phase II Study of Temozolomide in Patients with Advanced Aerodigestive Tract and Colorectal Cancers and Methylation of theO6-Methylguanine-DNA Methyltransferase Promoter

Hochhauser

Glynne‐Jones

Potter

et al. 2013

Molecular Cancer Therapeutics

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Responses of patients with gliomas to temozolomide are determined by O 6 -methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) pathways. This phase II study (NCT00423150) investigated whether MGMT promoter methylation predicts response in patients with advanced aerodigestive tract and colorectal cancers (CRC). Tumor and serum samples were screened for MGMT promoter methylation. In methylation-positive patients, 150 mg/m 2 temozolomide was administered daily on a seven-day-on, sevenday-off schedule for each 28-day cycle. The primary efficacy endpoint was response rate (RR). MMR status was determined by a microsatellite instability assay. Among 740 patients screened, 86 were positive for MGMT promoter methylation and enrolled. Nineteen percent of the screened population (137/740) had confirmed tissue and/or serum MGMT promoter methylation, including 25% (57 of 229) for CRC, 36% (55 of 154) for esophageal cancer, 11% (12 of 113) for head and neck cancer, and 5% (13 of 242) for non-small cell lung carcinoma. Among patients with valid methylation results in both tissue and serum samples, concordance was 81% (339 of 419). The majority of enrolled patients (69 of 86; 80%) had microsatellite stable cancer. Overall RR was 6% (5 of 86 partial responses); all responders had microsatellite stable cancer. Temozolomide resulted in low RRs in patients enriched for MGMT methylation. MGMT methylation status varied considerably in the patient population. Although serum methylation assay is an option for promoter methylation detection, tissue assay remains the standard for methylation detection. The low RR of this cohort of patients indicates that MGMT methylation as a biomarker is not applicable to heterogeneous tumor types, and tumor-specific factors may override validated biomarkers. Mol Cancer Ther; 12(5); 809-18. Ó2013 AACR.

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Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma

Cited by 176 publications

References 21 publications

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

Comparative assessment of 5 methods (methylation‐specific polymerase chain reaction, methylight, pyrosequencing, methylation‐sensitive high‐resolution melting, and immunohistochemistry) to analyze O6‐methylguanine‐DNA‐methyltranferase in a series of 100 glioblastoma patients

A Phase II Study of Temozolomide in Patients with Advanced Aerodigestive Tract and Colorectal Cancers and Methylation of theO6-Methylguanine-DNA Methyltransferase Promoter

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