Usefulness of MS-MLPA for detection of MGMT promoter methylation in the evaluation of pseudoprogression in glioblastoma patients

Neuro-Oncology, Feb 2011

Pseudoprogression is a major diagnostic dilemma in current treatment protocols for malignant gliomas that involve concurrent chemoradiotherapy. We hypothesized that methylation-specific multiplex ligation probe amplification (MS-MLPA), an assay that permits semiquantitative evaluation of promoter methylation, may be used to diagnose pseudoprogression based on the quantification of the methylation status of the O6-methylguanine DNA methyltransferase (MGMT) promoter. We examined the methylation ratio of the MGMT promoter with MS-MLPA in 48 samples from glioblastoma patients. The results were compared with those from methylation-specific polymerase chain reaction (MSP), and protein levels were confirmed by immunohistochemical staining. We then evaluated the correlation between those molecular signatures and clinical outcomes. With regard to radiological progression after chemoradiotherapy, the diagnostic accuracy of the MS-MLPA method was 80% (using a cut-off value of 0.2). These results are better than those obtained with MSP (diagnostic accuracy of 68%). Combining the MS-MLPA and MSP methods resulted in a diagnostic accuracy of 93% for the identification of pseudoprogression among patients to whom these results were coherent. These results demonstrate that MS-MLPA is a useful method to predict radiological progression vs pseudoprogression in glioblastoma patients and that the interpretation of these results in combination with MSP results will provide good practical guidelines for clinical decision making in glioblastoma treatment.

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Usefulness of MS-MLPA for detection of MGMT promoter methylation in the evaluation of pseudoprogression in glioblastoma patients

Advance Access publication November Usefulness of MS-MLPA for detection of MGMT promoter methylation in the evaluation of pseudoprogression in glioblastoma patients Chul-Kee Park 0 1 2 3 JinWook Kim 0 1 2 3 Su Youn Yim 0 1 2 3 Ah Reum Lee 0 1 2 3 Jung Ho Han 0 1 2 3 Chae-Yong Kim 0 1 2 3 Sung-Hye Park 0 1 2 3 Tae Min Kim 0 1 2 3 Se-Hoon Lee 0 1 2 3 Seung Hong Choi 0 1 2 3 Seung-Ki Kim 0 1 2 3 Dong Gyu Kim 0 1 2 3 Hee-Won Jung 0 1 2 3 0 Hospital, Seoul (S.H.C.); Division of Pediatric Neurosurgery, Seoul National University Children's Hospital 1 Pathology, Seoul National University Hospital , Seoul (S. -H.P.); Department of Internal Medicine , Seoul 2 Department of Neurosurgery, Clinical Research Institute, Seoul National University Hospital , Seoul (C.-K.P. 3 Seoul (S.-K.K.) , South Korea glioblastoma; MS-MLPA; MGMT; pseudoprogression - Pseudoprogression is a major diagnostic dilemma in current treatment protocols for malignant gliomas that involve concurrent chemoradiotherapy. We hypothesized that methylation-specific multiplex ligation probe amplification (MS-MLPA), an assay that permits semiquantitative evaluation of promoter methylation, may be used to diagnose pseudoprogression based on the quantification of the methylation status of the O6methylguanine DNA methyltransferase (MGMT) promoter. We examined the methylation ratio of the MGMT promoter with MS-MLPA in 48 samples from glioblastoma patients. The results were compared with those from methylation-specific polymerase chain reaction (MSP), and protein levels were confirmed by immunohistochemical staining. We then evaluated the correlation between those molecular signatures and clinical outcomes. With regard to radiological progression after chemoradiotherapy, the diagnostic accuracy of the MS-MLPA method was 80% (using a cut-off value of 0.2). These results are better than those obtained with MSP (diagnostic accuracy of 68%). Combining the MS-MLPA and MSP methods resulted in a diagnostic accuracy of 93% for the identification of pseudoprogression among patients to whom these results were coherent. These results demonstrate that MS-MLPA is a useful method to predict radiological progression vs pseudoprogression in glioblastoma patients and that the interpretation of these results in combination with MSP results will provide good practical guidelines for clinical decision making in glioblastoma treatment. Cchemoradiotherapy for malignant gliomas have urrent treatment protocols involving concurrent led to novel insights and demonstrated an increased incidence of pseudoprogression or radiation necrosis.1,2 The differentiation of these treatment side effects from the true tumor recurrence that frequently occurs during the management period is a major diagnostic dilemma, especially because they have a similar appearance on standard gadolinium-based MRI. Inaccurate diagnosis of pseudoprogression in the early period of chemoradiation may lead to an unwanted discontinuation of effective treatment. Several reports have demonstrated that some clinical, radiological, and biological indicators can be helpful in predicting pseudoprogression.1,3 – 5 However, no single modality can reliably diagnose pseudoprogression. One reason for the lack of definite diagnostic criteria for pseudoprogression is that variables associated with pseudoprogression are difficult to quantify. The prognostic value of O6-methylguanine DNA methyltransferase (MGMT) promoter methylation in glioblastoma patients treated with concurrent radiotherapy and temozolomide followed by adjuvant temozolomide is well established, and its association with an incidence of pseudoprogression has also been documented.3 However, methylation-specific polymerase chain reaction (MSP), a standard method used to test the methylation status of the MGMT promoter, does not provide quantitative results. This results in some inaccuracy in determining the prognosis, including the occurrence of pseudoprogression. Methylation-specific multiplex ligation probe amplification (MS-MLPA) is a semiquantitative method that has been recently introduced for testing the methylation status of multiple genes simultaneously and also proved as a reliable method for the determination of MGMT promoter methylation in glioma patients.6,7 In the present study, we used MS-MLPA to evaluate the MGMT promoter methylation status in tumor tissues. We compared these results with those obtained from MSP and immunohistochemical (IHC) staining to determine the efficacy of MS-MLPA in predicting treatment outcomes in glioblastoma patients, with particular attention to the development of pseudoprogression. Materials and Methods Study Population and Preparation of Tissue Samples Clinical data and tissue samples were obtained from 48 newly diagnosed supratentorial glioblastoma patients following surgical treatment and the standard regimen of concurrent radiotherapy and temozolomide, followed by adjuvant temozolomide.8 Thirty-eight patients were treated at Seoul National University Hospital, and 10 were treated at Seoul National University Bundang Hospital. All patients were adults with a mean age of 53.4 years (range, 28 – 74); 30 were males, and 18 were females. Based on pre- and postsurgical T1-enhanced MRI, more than 95% of the tumor was surgically removed in 18 patients (38%), and less than 95% of the tumor was removed in 18 patients (38%). A small group of 12 patients (24%) had undergone biopsy only. Every patient maintained good performance status after surgery (Eastern Cooperative Oncology Group performance status grade ≤2). Follow-up brain MRIs were taken 4 weeks after the completion of concurrent chemoradiotherapy and subsequently for every 3 months. The early-response evaluation was carried out with postchemoradiotherapy MRIs according to Macdonald’s criteria.9 The adjuvant temozolomide was administered to all patients as planned regardless of the outcome of the early-response evaluation, excluding 3 patients who demonstrated radiological progression after chemoradiotherapy and were unable to continue further chemotherapy due to clinical deterioration. Pseudoprogression was diagnosed if radiological disease progression was observed after chemoradiotherapy, and the disease was stable or had improved after 3 months without switching to a regimen other than the planned adjuvant temozolomide. If the lesions that worsened after chemoradiotherapy showed continuous progression in follow-up images, then they were considered as true progression. Overall survival was measured from the time of surgery to death or date of last follow-up. We used a group of 24 snap-frozen tumor tissues and another group of 24 paraffin-embedded tissues for MS-MLPA and MSP. The clinical characteristics were not significantly different between these 2 groups (Table 1). Tumor tissues obtained during surgery and then snap-frozen in liquid nitrogen were stored at 2808C. DNA was isolated from tissue samples using the DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. CpGenome Universal Methylated DNA and Unmethylated DNA (Chemicon, Millipore) were included as controls in each set of MS-MLPA and MSP experiments. IHC staining was performed on 38 paraffin-embedded tissues. Informed consent was obtained from patients prior to resection in accordance with the local Institutional Review Board guidelines. Methylation-Specific Multiplex Ligation Probe Amplification The methylation status of the MGMT promoter was tested using the MS-MLPA probe mix prepared by MRC-Holland (Salsa MS-MLPA Kit ME011 MMR), which includes 3 probes specific for the MGMT promoter region containing an HhaI recognition site. The procedure was performed according to the manufacturer’s protocol. HhaI (R6441; Promega), a methylation-sensitive restriction enzyme that cuts unmethylated GCGC sites was applied to each set of samples. The resultant PCR Samples P-value Fresh frozen tissue (n 5 24) Paraffin-embedded tissue (n 5 24) ECOG, Eastern Cooperative Oncology Group. fragments were separated by capillary gel electrophoresis (ABI Prism 7000/7700, Applied Biosystems). The methylation status was quantified using GeneMarker software (version 1.5, Soft Genetics). To compensate for the differences in the efficiency of the PCR for the individual samples, the peak value of each probe was normalized by dividing it by the peak of the control probes. To evaluate the methylation status, the methylation ratio (MR) was calculated by the average of dividing each normalized peak value of the digested sample by that of the corresponding undigested sample. This value corresponds to the percentage of methylated sequences. Methylation-Specific Polymerase Chain Reaction The methylation status of the MGMT promoter was also analyzed by MSP, and the results were compared with those of MS-MLPA. Prepared DNA was modified by sodium bisulfite treatment using an EZ DNA Methylation-Gold Kit (Catalog No. D5005, Zymo Research). The primer sequences for the MGMT were as follows: methylated forward: 5′-TTT CGA CGT TCG TAG GTT TTC GC-3′, methylated reverse: 5′-GCA CTC TTC CGA AAA CGA AAC G-3′, unmethylated forward: 5′-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3′, unmethylated reverse: 5′-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3′. The annealing temperature was 648C. The obtained PCR products were electrophoresed in 2% agarose gels and visualized under ultraviolet illumination after staining with ethidium bromide. For the evaluation of the assay results, the products from the controls were examined first. The MGMT gene promoter fragments in the controls were observed at 80 and 92 bp for the methylated DNA – methylated primer and unmethylated DNA – unmethylated primer combinations, respectively. The methylated DNA – unmethylated primer and unmethylated DNA – methylated primer controls were not expected to show any bands. If the control results were acceptable, patient samples were evaluated for the presence of amplification with the methylated and unmethylated primers. The results were interpreted as positive if the MGMT gene promoter methylation was detected as a fragment of 80 bp observed in the gel, and they were negative if MGMT gene promoter methylation was not detected with the methylated primers. IHC Staining Tissue expression of MGMT protein was confirmed by IHC staining using conventional methods according to the manufacturer’s protocols. The used antibody was MGMT (MS-470-P, 1:100, Neomarkers). IHC staining results were semiquantitatively graded as ,5% (negative), 5% – 10% (1+), or .10% (2+), based on the percentage of tumor cells showing immunoreactivity.10 Grades greater than 1+ were considered positive for the corresponding markers. Statistical Analysis The independent t-test, x-square test, and Mann – Whitney test were used for parametric comparisons between the methods. Survival curves were estimated according to the MGMT methylation status by the Kaplan – Meier technique and compared using the log-rank test for MSP and MS-MLPA. Statistical significance was accepted at probability values of less than 0.05. These statistical analyses were performed with the aid of Statistical Package for the Social Sciences software (version 12.0, SPSS). Correlation of MS-MLPA Results with Those Obtained by MSP in the Evaluation of the MGMT Promoter Methylation Status For the 48-patient samples analyzed by MSP, the MGMT promoter was unmethylated in 34 (70.8%) and methylated in 14 (29.2%) samples. There was a significant difference (P ¼ .001) in the distribution of MRs (mean + SD: 0.20 + 0.18 vs 0.55 + 0.30) detected by MS-MLPA between the unmethylated and methylated samples that were classified by the MSP results (Fig. 1). This correlation between MS-MLPA and MSP was significant in both the frozen sample group (mean + SD: 0.12 + 0.12 vs 0.39 + 0.34; P ¼ .028) and the paraffin sample group (mean + SD: 0.29 + 0.19 vs 0.73 + 0.14; P ¼ .000). At the time of analysis, a total of 22 patients had died from their disease: the remaining 26 patients were alive, with a mean follow-up period of 16 months. The MGMT promoter methylation status measured by MSP showed significant impact on overall survival (Fig. 2A). However, stratification of the patients into multilevel prognostic groups by quantification of the MGMT promoter methylation status determined using MS-MLPA failed to show a significant difference in overall survival among groups, due largely to insufficient sample numbers to test multilevel category (Fig. 2B). Usefulness of MS-MLPA in the Early-Response Evaluation At the early-response MRIs taken 4 weeks after radiotherapy, complete or partial response was observed in 12 patients (25%), stable disease in 11 patients (23%), and progression in 25 patients (52%). Among those 25 patients with radiological progression, 14 (29% of total) were proved to exhibit true progressions, whereas 11 (23% of total) were better classified as pseudoprogressions. From the results of MSP evaluation of the MGMT promoter methylation status, we observed that the methylated group had a more favorable response rate (Table 2). In the case of radiological progression, the MSP-determined MGMT promoter methylation status was unable to predict whether a patient was experiencing pseudoprogression or true progression (P ¼ .070). The sensitivity, specificity, and accuracy of MSP in diagnosing pseudoprogression in cases of radiological progression were calculated as 80%, 65%, and 68%, respectively. Using quantifiable results, such as the MR calculated from MS-MLPA, more sophisticated analyses of the MGMT promoter methylation status could be applied to the evaluation of early response. Figure 3 shows the MRs for all cases in order of intensity. Only 2 cases were true progressions at the time of early-response evaluation if the MR was greater than 0.2. Using this cut-off value, we were able to very accurately predict the early-response rate and better diagnose pseudoprogression (Table 3). In the case of radiological progression with an MR .0.2, the MS-MLPA-determined MGMT promoter methylation status showed a significant power to predict whether a patient was experiencing pseudoprogression or true progression (P ¼ .003). The sensitivity, specificity, and accuracy of MS-MLPA were 80%, 67%, and 80%, respectively. The diagnosis of pseudoprogression using MS-MLPA was consistent between frozen and paraffin-embedded samples (Table 4). Among the 25 patients with radiological progression after chemoradiotherapy, the MS-MLPA and MSP MGMT promoter methylation status Methylated (n 5 14) Unmethylated (n 5 34) Radiological response Radiologically controlled Response Stable disease Radiologically progressed Pseudoprogression True progression 5 (35%) 4 (29%) 7 (21%) 7 (21%) P-value .070 results were in agreement for 14 patients (MR . 0.2 from MS-MLPA and methylated from MSP or MR ≤ 0.2 and unmethylated). The MS-MLPA and MSP results were not in agreement for the other 11 patients (MR . 0.2 from MS-MLPA and unmethylated from MSP or MR ≤ 0.2 and methylated). For patients for the results of the MGMT promoter MS-MLPA and MSP were coherent, the diagnostic accuracy for pseudoprogression increased to 93% (P ¼ .003). If the results of the MGMT promoter MS-MLPA and MSP were incoherent, the diagnostic accuracy for pseudoprogression by MS-MLPA was 64% and that of MSP was 36%, but this difference was not statistically significant (P ¼ .782). Correlation with IHC Staining IHC staining for MGMT was performed on 38 available paraffin-embedded tissue samples. Table 5 shows the results of IHC staining and their correlation with MSP and MS-MLPA. There was no significant correlation between MSP results and MGMT IHC staining, with a correlation rate of 39% (P ¼ .897). With an MR cut-off value of 0.2, the correlation between MS-MLPA results and MGMT expression determined by IHC staining (66%) was insignificant (P ¼ .088). Discussion Extensive studies on MGMT over the past decade have led to an accumulation of evidence suggesting that it could serve as a biomarker for glioblastoma prognosis and treatment. MGMT has recently been shown to be able to stratify or even select glioblastoma patients for clinical trials.11 In general, MSP has been accepted as the most reliable and readily available clinical method for the analysis of the MGMT promoter methylation status in glioblastoma patients. However, the Radiologically controlled Response Stable disease Radiologically progressed Pseudoprogression True progression Radiological response MR > 0.2 (n 5 23) 5 (21%) 8 (35%) MR ≤ 0.2 (n 5 25) 7 (28%) 3 (12%) MGMT promoter methylation status MR > 0.2 MR ≤ 0.2 Table 3. Distribution of the early responses of patients according to the MGMT promoter methylation status determined by MS-MLPA Radiological response MGMT promoter methylation status Frozen sample group (n ¼ 15) Pseudoprogression True progression Paraffin sample group (n ¼ 10) Pseudoprogression True progression 3 0 Table 5. Correlation of MGMT IHC staining and the MGMT promoter methylation status analyzed by MSP and MS-MLPA, respectively IHC staining Correlation P-value Expression Methylated Unmethylated MR ≤0.2 prognostic value of MSP at the time of diagnosis is still not sufficient to provide a solid background for clinical decision making due to nonnegligible false negatives or false positives in clinical outcome prediction. Among the many possible factors that contribute to this inaccuracy, the nonquantitative data resulting from MSP may play an important role. Previous studies have shown that the ambiguous results of MGMT MSP are due to the different degrees of methylation.6 We chose MS-MLPA, a recently introduced method for the detection of the methylation status, to improve on the shortcomings of MSP and to obtain more precise information about the MGMT promoter status, with the aim of achieving a better clinical application. This method can detect aberrant methylation at specific CpG sites based on digestion with a methylationsensitive restriction enzyme and yields semiquantitative results. The semiquantitative nature of MS-MLPA has been demonstrated by titration experiments that show a gradual increase in signal that correlates with the degree of sample methylation. These studies concluded that the calculated MR reflects the degree of methylation in the sample.6 The results of this study (Fig. 1) and other studies have shown that MS-MLPA data are concordant with those obtained from MSP for the specific CpG sites.6,12 Moreover, we were able to show the possibility of subdividing the prognostic groups according to the degree of MGMT promoter methylation determined by MS-MLPA, although differences in survival probability according to promoter methylation failed to reach statistical significance due to insufficient sample numbers (Fig. 2). Other advantages of MS-MLPA include the following: (1) it can bypass the bisulfite conversion reaction, the most troublesome step of most assays for the methylation status, (2) different CpG dinucleotides can be analyzed simultaneously, (3) multiple genomic DNA analyses can be performed during a single session within a day, (4) copy number analysis is possible by comparison with control DNA, and (5) only a small volume of DNA (20 ng) is required.6,7,12,13 This study demonstrates the utility of the semiquantitative results obtained with MS-MLPA in clinical decision making for early-response evaluation. This is an important step forward in meeting the diagnostic challenge posed by pseudoprogression in glioblastoma patients. Brandes et al.3 reported the incidence of pseudoprogression as 31% of 103 patients treated with combination chemoradiotherapy, and 64% of 50 patients demonstrated radiological progression. They also demonstrated the value of the MGMT promoter methylation status analyzed by MSP in the prediction of pseudoprogression. Among patients with radiological progression, pseudoprogression/true progression was confirmed in 66%/11% of methylated cases and 34%/89% of unmethylated cases, respectively.3 The diagnostic value of MGMT MSP for pseudoprogression agrees with the value of MS-MLPA in the present study (Table 2). Although the specificity of the test remains a problem in the prediction of pseudoprogression, we further improved the accuracy using MRs calculated from MS-MLPA (Table 3). In spite of various efforts to distinguish treatment effects from tumor growth, no noninvasive tests have been developed with proven efficacy in this context of pseudoprogression.14 Moreover, the diagnostic value of MS-MLPA is not influenced by the sample type (Table 4). Therefore, the MGMT promoter MS-MLPA can provide crucial information for clinical decision making on early-response evaluation. Nonetheless, the cut-off values for methylation (0.2) should be confirmed by interinstitute validation. Additionally, the combination of MS-MLPA and MSP can further improve the diagnostic accuracy of pseudoprogression up to 93% for some patients, an accuracy rate that can clearly help to make clinical decisions. There have been conflicting reports on the prognostic significance of MGMT immunohistochemistry for high-grade gliomas.10,15 – 17 Preusser et al.18 analyzed the samples from the European Organization for Research and Treatment of Cancer and the National Cancer Institute of Canada (EORTC/NCIC) trial 26981/22981 and concluded that IHC assessment of MGMT expression is not recommended due to the lack of a significant association of MGMT expression with patient outcomes. As in the present study, many previous studies have reported a poor correlation between MGMT IHC analysis and MGMT promoter MSP.6,10,19 – 22 Possible explanations for the disagreement between immunohistochemically assessed MGMT expression and the MGMT promoter methylation status include the following: (1) MGMT protein expression in nonneoplastic cells, (2) monoallelic methylation of the MGMT promoter, (3) negatively stained neoplastic cells that can be triggered to produce MGMT on exposure to genotoxic drug, (4) upregulation of MGMT protein by steroids and radiation, and (5) unknown control mechanisms underlying MGMT protein expression other than promoter silencing.18,20 – 22 However, there was a better correlation between the MGMT promoter methylation status analyzed by MS-MLPA and MGMT expression assessed by immunohistochemistry (Table 5), although these results are statistically insignificant and should be confirmed in a further study, because contradictory data have also been reported.6 We believe that the semiquantitative nature of the MS-MLPA results can be important in resolving the dilemma between degrees of MGMT promoter methylation and MGMT protein expression. In summary, MS-MLPA is a useful method that can simultaneously produce semiquantitative results on the methylation status of MGMT gene promoters. This method has a great predictive value in glioblastoma management outcomes, including pseudoprogression. Moreover, the combination of MS-MLPA and MSP provided a strong basis for clinical decision making regarding pseudoprogression. Further validation of MS-MLPA for MGMT will provide good practical guidelines for clinical decision making in glioblastoma treatment. Conflict of interest statement. None declared. Funding This study was partially supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (Study No. A090098 to C.-K.P.), the Seoul National University Hospital Research Fund (Study No. 04-2009-0310 to C.-K.P.), the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (Study No. 2008-0061821 to S.-K.K.), and the Seoul National University Bundang Hospital Research Fund (Study No. 06-2008-050 to C.-Y.K.). 7. Nygren AO , Ameziane N , Duarte HM , et al. Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences . Nucleic Acids Res . 2005 ; 33 ( 14 ): e128 . 8. Stupp R , Mason WP , van den Bent MJ , et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma . N Engl J Med . 2005 ; 352 ( 10 ): 987 - 996 . 9. Macdonald DR , Cascino TL , Schold SC , Jr, Cairncross JG . Response criteria for phase II studies of supratentorial malignant glioma . J Clin Oncol . 1990 ; 8 ( 7 ): 1277 - 1280 . 10. Brell M , Tortosa A , Verger E , et al. Prognostic significance of O6-methylguanine-DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas . Clin Cancer Res . 2005 ; 11 ( 14 ): 5167 - 5174 . 11. Weller M , Stupp R , Reifenberger G , et al. MGMT promoter methylation in malignant gliomas: ready for personalized medicine ? Nat Rev Neurol . 2010 ; 6 ( 1 ): 39 - 51 . 12. Scott RH , Douglas J , Baskcomb L , et al. Methylation-specific MLPA (MS-MLPA) robustly detects and distinguishes 11p15 abnormalities associated with overgrowth and growth retardation . J Med Genet . 2008 ; 45 : 106 - 113 . 13. Jeuken J , Cornelissen S , Boots-Sprenger S , Gijsen S , Wesseling P. Multiplex ligation-dependent probe amplification: a diagnostic tool for simultaneous identification of different genetic markers in glial tumors . J Mol Diagn . 2006 ; 8 ( 4 ): 433 - 443 . 14. Clarke JL , Chang S. Pseudoprogression and pseudoresponse: challenges in brain tumor imaging . Curr Neurol Neurosci Rep . 2009 ; 9 ( 3 ): 241 - 246 . 15. Cahill DP , Levine KK , Betensky RA , et al. Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment . Clin Cancer Res . 2007 ; 13 ( 7 ): 2038 - 2045 . 16. Chinot OL , Barrie M , Fuentes S , et al. Correlation between O6-methylguanine-DNA methyltransferase and survival in inoperable newly diagnosed glioblastoma patients treated with neoadjuvant temozolomide . J Clin Oncol . 2007 ; 25 ( 12 ): 1470 - 1475 . 17. Nakasu S , Fukami T , Baba K , Matsuda M. Immunohistochemical study for O6-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas . J Neuro-Oncol . 2004 ; 70 ( 3 ): 333 - 340 . 18. Preusser M , Charles Janzer R , Felsberg J , et al. Anti-O6-methylguanine-methyltransferase (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as clinical biomarker . Brain Pathol . 2008 ; 18 ( 4 ): 520 - 532 . 19. Lee SM , Reid H , Elder RH , Thatcher N , Margison GP . Inter- and intracellular heterogeneity of O6-alkylguanine-DNA alkyltransferase expression in human brain tumors: possible significance in nitrosourea therapy . Carcinogenesis . 1996 ; 17 ( 4 ): 637 - 641 . 20. Maxwell JA , Johnson SP , Quinn JA , et al. Quantitative analysis of O6-alkylguanine-DNA alkyltransferase in malignant glioma . Mol Cancer Ther . 2006 ; 5 ( 10 ): 2531 - 2539 . 21. Grasbon-Frodl EM , Kreth FW , Ruiter M , et al. Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas . Int J Cancer . 2007 ; 121 ( 11 ): 2458 - 2464 . 22. Yachi K , Watanabe T , Ohta T , et al. Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas . Int J Oncol . 2008 ; 33 ( 3 ): 469 - 475 .


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Chul-Kee Park, JinWook Kim, Su Youn Yim, Ah Reum Lee, Jung Ho Han, Chae-Yong Kim, Sung-Hye Park, Tae Min Kim, Se-Hoon Lee, Seung Hong Choi, Seung-Ki Kim, Dong Gyu Kim, Hee-Won Jung. Usefulness of MS-MLPA for detection of MGMT promoter methylation in the evaluation of pseudoprogression in glioblastoma patients, Neuro-Oncology, 2011, 195-202, DOI: 10.1093/neuonc/noq162