Methylation-specific PCR method for MGMT coding gene silencing evaluation and its prognostic significance in alkylating antitumor treatment (original) (raw)
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The epigenotype of a tumor tissue containing methylated promoter of the MGMT encoding gene is considered presently as a good prognostic marker signifying an increase in the sensitivity to its treatment with alkylating agents such as temozolomide (TMZ). The principle of this biomarker application in pharmaco-epigenetics as well as its correct clinical significance is based on the correlation between the silencing methylation process in gene promoters and the DNA damaging activity of the classical alkylating drugs. Two methods that are currently used in genetic laboratories for methylation pattern based biomarkers: Methylation Sensible (MS)-Multiplex ligation-dependent probe amplification (MLPA) and Methylation Specific (MS)-PCR are described comparatively on two tumor types (glioma tumors and Ewing sarcoma-EWS) whose treatment with the alkylating drug TMZ requires an evaluation of its efficiency. The first method was chosen for its semiquantitative capacity as compared with the class...
The Journal of Molecular Diagnostics, 2008
Epigenetic silencing of the DNA repair protein O 6methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy , such as with temozolomide , in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test , suitable for high-throughput analysis of small amounts of formalin-fixed , paraffin-embedded tumor tissue. A direct , real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the -actin gene , provides a quantitative test result. We analyzed 134 clinical glioma samples , comparing the new test with the previously validated nested gel-based MSP assay , which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results , supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa ؍ 0.80; 95% confidence interval, 0.82؊0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gelbased MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy. (J Mol
BMC Cancer, 2010
Background: Epigenetic silencing of the MGMT gene by promoter methylation is associated with loss of MGMT expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference. Methods: An analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The SNURF promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization. Results: Concordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). MGMT promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with MGMT unmethylated and methylated, respectively. Considering MGMT methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring MGMT promoter unmethylated and other patients with any percentage of MGMT methylation (p = 0.003). This difference was retained using other cut off values for MGMT methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of MGMT methylated allele was used as cut-off.
Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2011
O 6 -Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, precarcinogenic and pre-toxic DNA damage O 6 -methylguanine. It also repairs larger adducts on the O 6 -position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O 6 -chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O 6 -alkylating drugs are still lacking.
BBA clinical, 2015
CpG methylation in the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors ha...
Clinical Cancer Research, 2004
Purpose: In the setting of a prospective clinical trial, we determined the predictive value of the methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter for outcome in glioblastoma patients treated with the alkylating agent temozolomide. Expression of this excision repair enzyme has been associated with resistance to alkylating chemotherapy. Experimental Design: The methylation status of MGMT in the tumor biopsies was evaluated in 38 patients undergoing resection for newly diagnosed glioblastoma and enrolled in a Phase II trial testing concomitant and adjuvant temozolomide and radiation. The epigenetic silencing of the MGMT gene was determined using methylation-specific PCR. Results: Inactivation of the MGMT gene by promoter methylation was associated with longer survival (P ؍ 0.0051; Log-rank test). At 18 months, survival was 62% (16 of 26) for patients testing positive for a methylated MGMT promoter but reached only 8% (1 of 12) in absence of methylation (P ؍ 0.002; Fisher's exact test). In the presence of other clinically relevant factors, methylation of the MGMT promoter remains the only significant predictor (P ؍ 0.017; Cox regression). Conclusions: This prospective clinical trial identifies MGMT-methylation status as an independent predictor for glioblastoma patients treated with a methylating agent. The association of the epigenetic inactivation of the DNA repair gene MGMT with better outcome in this homogenous cohort may have important implications for the design of future trials and supports efforts to deplete MGMT by O-6-benzylguanine, a noncytotoxic substrate of this enzyme.
Scientific reports, 2018
O-methylguanine-DNA methyltransferase (MGMT) promoter methylation and its subsequent loss of protein expression has been identified to have a variable impact on clinical outcome of glioma patients indicated for chemotherapy with alkylating agents (Temozolomide). This study investigated methylation status of MGMT gene along with in situ protein expression in malignant glioma patients of different histological types to evaluate the associated clinical outcome vis-a-vis use of alkylating drugs and radiotherapy. Sixty three cases of glioma were evaluated for MGMT promoter methylation by methylation-specific PCR (MS-PCR) and protein expression by immunostaining (IHC). Methylation status of MGMT and loss of protein expression showed a very high concordant association with better survival and progression free survival (PFS) (p < 0.0001). Multivariate Cox regression analysis showed both MGMT methylation and loss of protein as significant independent prognostic factors in glioma patients ...
The International Journal of Biological Markers, 2015
It is already well known that hypermethylation of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a predictive biomarker of response to temozolomide treatment and of favorable outcomes in terms of overall survival (OS) and progression-free survival (PFS) in glioblastoma (GBM) patients. Nevertheless, MGMT methylation status has not currently been introduced into routine clinical practice, as the choice of the ideal technique and tissue sample specimen is still controversial. The aim of this study was to compare 2 analytical methods, methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), and their use on 2 different tissue type samples, snap-frozen and formalin-fixed paraffin-embedded (FFPE), obtained from a single-center and uniformly treated cohort of 46 GBM patients. We obtained methylation data from all frozen tissues, while no results were obtained for 5 FFPE samples. The highest concordance for methylation was found on frozen tissues (8...
MGMT promoter methylation in plasma of glioma patients receiving temozolomide
Journal of Neuro-Oncology, 2014
Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays 2 a role in cellular response to alkylating agents. In the present study aimed to: i) evaluate the 3 concordance between MGMT promoter methylation status in tumor tissue and plasma; ii) 4 monitor MGMT promoter methylation status in plasma taken before and during 5 temozolomide treatment; iii) explore the value of MGMT promoter methylation status in 6 plasma as a prognostic/predictive biomarker in glioma patients. 7 We enrolled 58 patients with histologically confirmed glioma at different grades of 8 malignancy. All patients underwent surgical resection and temozolomide treatment. Paraffin-9 embedded tumor tissue was available for 48 patients. Blood samples were collected from all 10 patients before temozolomide treatment (baseline) and at each MRI examination for a 12-11 month period. MGMT promoter methylation status was assessed in both sample types by real 12 time PCR with a specific probe. 13 The frequency of MGMT promoter methylation was 60.4% in tumor tissue and 41.38% in 14 plasma. MGMT promoter methylation status was concordant in the two sample types 15 (Kappa=0.75, 95% confidence interval [CI] 0.57-0.93; p-value <0.001). Overall and 16 progression-free survival were longer in patients with methylated MGMT promoter. 17 Mortality was higher in patients with unmethylated MGMT promoter, whether in tumor 18 tissue (hazard ratio [HR]: 2.21; 95% CI 0.99-4.95) or plasma (HR: 2.19; 95% CI 1.02-4.68). 19 Progression-free survival was shorter in patients with unmethylated MGMT promoter, 20 whether in tissue (HR: 2.30; 95% CI 1.19-4.45) or plasma (HR: 1.77; 95% CI 0.95-3.30). 21 The cumulative incidence of unmethylated MGMT promoter in plasma at baseline was 58%, 22 and reached virtually 100% at 12 months. In conclusion MGMT promoter methylation status 23 in tumor tissue and plasma was highly concordant, and both were associated with longer treatment response. However we suggest caution in using plasma as a surrogate of tumor 1 tissue due to possible false-negative results.