Prognosis of glioblastoma with faint MGMT methylation-specific PCR product.

Methylation-specific polymerase chain reaction (MSP) for the promoter methylation status of O(6)-methylguanine-DNA-methyltranferase (MGMT) gene theoretically provides a positive or negative result. However, the faint MSP product is difficult to interpret. The aim of this study was to evaluate the significance of faint MSP product in glioblastoma (GBM). Critical concentrations of methylated control DNA, i.e., 100, 1, 0.5 and 0 % were run parallel with 116 newly diagnosed GBMs in order to standardize the interpretation and to distinguish positive (+), equivocal (±), and negative (-; unmethylated) results. Cases with the faint MSP product and its intensity between those of 1 and 0.5 % DNA controls were considered equivocal (±). MGMT methylation quantifications were also determined by quantitative real-time MSP (qMSP) and pyrosequencing (PSQ), and protein expression was detected by immunohistochemistry. There were significant correlations between MSP and all the aforementioned studies. The concordance rates between the MSP+ and qMSP+ cases, as well as the MSP- and qMSP- cases were 100 %, and the MSP± cases comprised 76.5 % of qMSP+ cases and 23.5 % of qMSP- cases. PSQ study showed that heterogeneous methylation was more frequently encountered in the MSP± cases. Multivariate analyses disclosed that although the overall survival of the MSP± cases was indistinct from that of the MSP+ cases, its progression free survival was significantly worse and was indistinct from that of the MSP- cases. In conclusion, GBMs with faint MGMT MSP products should be distinguished from MSP+ cases as their behaviors were different.

MGMT promoter methylation in non-neoplastic brain.

O(6)-methylguanine-DNA-methyltransferase (MGMT) is mainly regulated by cytosine-guanine island promoter methylation that is believed to occur only in neoplastic tissue. The present study was undertaken to investigate whether methylation occurs also in non-neoplastic brains by collecting 45 non-neoplastic brains from autopsies and 56 lobectomy specimens from epileptic surgeries. The promoter methylation status of MGMT was studied by methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), while protein expression was studied by immunohistochemical stain (IHC). The methylation rates, as determined by MSP and PSQ, were 3.0 % (3/101) and 2.9 % (2/69), respectively. Of note, no case had positive result concomitantly from both MSP and PSQ (3 were MSP+/PSQ- and 2 were MSP-/PSQ+), and all the positive samples were further confirmed by cloning and Sanger sequencing. All the methylated cases, except for those having indeterminate IHC results from autopsy specimens, revealed no loss of MGMT protein expression and similar staining pattern to that of the unmethylated cases. In conclusion, the current study demonstrated that MGMT promoter methylation could occur in a low percentage of non-neoplastic brains but did not affect the status of protein expression, which could be regarded as a normal variation in non-neoplastic brains.

Molecular diagnostic algorithm for epidermal growth factor receptor mutation detection in Asian lung adenocarcinomas: comprehensive analyses of 445 Taiwanese patients with immunohistochemistry, PCR-direct sequencing and Scorpion/ARMS methods.

BACKGROUND AND OBJECTIVE: Therapeutic responses of lung adenocarcinoma patients to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) are closely associated with activating mutations within the EGFR tyrosine kinase domain. Screening activating EGFR mutations prior to selection for therapeutic strategy has been considered extremely valuable for clinical management of lung adenocarcinoma patients in Asian countries including Taiwan, where the EGFR mutation rate is higher than in the rest of the world. Currently there is no consensus on the method of choice to assess EGFR mutations in tumour tissue. METHODS: We enrolled 445 lung adenocarcinoma patients for analysis of tumour EGFR mutations using polymerase chain reaction (PCR)-direct sequencing, scorpion/amplified refractory mutation system (ARMS) technology and immunohistochemistry with mutation-specific antibodies. RESULTS: Two hundred forty-five patients (245/445; 55%) were found to harbour activating EGFR mutations using PCR-direct sequencing method, with a majority of patients (233/245; 95%) carrying exon 19 deletion or p.L858R point mutations. One hundred three of 200 patients were negative for EGFR mutations from PCR-direct sequencing were further analysed using Scorpion/ARMS technology. Up to 30% of the PCR-direct sequencing negative patients turned out to be positive in the Scorpion/ARMS EGFR mutation tests. For immunohistochemistry analysis of EGFR mutations, the p.E746_A750del specific antibody showed a sensitivity of 57% and a specificity of 100% for exon 19 deletions while the p.L858R point mutation specific antibody showed a sensitivity of 68% and a specificity of 95%. CONCLUSIONS: Based on this study, we proposed an algorithm for comprehensive and efficient testing of EGFR mutations on lung adenocarcinoma patients in Asia.

Exclusion of histiocytes/endothelial cells and using endothelial cells as internal reference are crucial for interpretation of MGMT immunohistochemistry in glioblastoma.

We evaluated the predictive value of O6-methylguanine-DNA methyltransferase (MGMT) protein expression and MGMT promoter methylation status in glioblastomas (GBM) treated with temozolomide (TMZ) in a Taiwan medical center. Protein expression by immunohistochemical analysis (IHC) and MGMT promoter methylation detected by methylation-specific polymerase chain reaction (MSP) were performed in a series of 107 newly diagnosed GBMs. We used endothelial cells as an internal reference for IHC staining because the staining intensities of the MGMT-expressing cells in different specimens varied considerably; a positive result was defined as the staining intensity of the majority of tumor cells similar to that of the adjacent endothelial cells. Immunostainings for microglial/endothelial markers were included as part of the MGMT IHC evaluation, and in cases that were difficult to interpret, double-labeling helped to clarify the nature of reactive cells. The MGMT protein expression was reversely associated with MGMT promoter methylation status in 83.7% of cases (MSP/IHC and MSP/IHC; Pearson r=-0.644, P<0.001). Twenty-two of 24 (91.7%) IHC tumors did not respond to TMZ treatment. Combining MSP and IHC results, all the 15 MSP/IHC GBMs were TMZ resistant. The MGMT status detected by either IHC or MSP was significantly correlated with the TMZ treatment response (both P<0.001) and survival of GBM patients (both P<0.05).