Promising alternatives for assessing MGMT at the DNA level are real-time quantitative polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA)

Several independent clinical studies have shown a strong association between the methylation status of the 0-methylguanine-methyltransferase gene (MGMT) promoter and the response to temozolomide. MGMT, a DNA repair protein, is thought to protect tumor cells by removing methyl groups from the 0 position of guanine, thus acting as an antagonist against the DNAdamaging effects of alkylating agents. Methylation of MGMT promoter causes silencing of the MGMT gene, inhibiting MGMT protein expression. There is much interest in making MGMT assessment of tumor tissue specimens amenable for routine diagnostic evaluation of patients with glioblastoma. Previous studies have employed different methods and protocols for assessing MGMT. Most have used MGMT gene promoter methylation status, but others have analyzed MGMT expression at the protein or RNA levels. The optimal method for assessing MGMT should meet the standard requirements for any diagnostic test. It should be widely available, cost effective, and reproducible in the same laboratory and in different laboratories, and correlate consistently with patient outcomes. The processing of neurosurgical tumor samples begins with limitations. The standard formalin-fixing and paraffin-embedding techniques degrade the quality of DNA and RNA. Also, tissue fixation protocols are inconsistent. Different laboratories use different fixatives, and the duration of formalin preservation also varies. In addition, stereotactic biopsies may yield only small amounts of tumor tissue. Many studies have used methylation-specific polymerase chain reaction (MSP) to assess MGMT promoter methylation status. However, the high dependence of MSP on tissue quality limits the usefulness of this method for routine diagnosis. Results of repeat analysis from the same formalin-fixed and paraffin-embedded tumor tissue samples are often inconsistent. Reproducibility seems to be better with frozen tissue rather than formalin-fixed tissue. However, in the routine clinical setting, frozen tumor samples are usually not available in all cases. Promising alternatives for assessing MGMT at the DNA level are real-time quantitative polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA). Real-time quantitative PCR may be applied on formalin-fixed paraffin-embedded tissue; results are available within a few hours. This method provides quantitative results, in contrast to the semiquantitative results available with conventional MSP. One study involving a commercial company has shown results comparable to those obtained with MSP. This method has yet to be validated through interlaboratory comparisons in an academic setting and through prospective clinical trials. A disadvantage of real-time quantitative PCR is that the protocol, similar to conventional MSP, requires bisulfite modification, which is particularly problematic when using formalin-fixed material. Multiplex litigation-dependent probe amplification analysis also works with formalin-fixed paraffin-embedded tissue, but does not require bisulfite modification. It allows copy number detection, which might be beneficial in some cases. In one study, results were shown to agree highly with those from MSP testing. MLPA appears to be an interesting alternative, but still requires intraand interlaboratory comparison and validation in prospective clinical trials. Immunohistochemistry (IHC) is another possible alternative for MGMT testing. This diagnostic method is available in most laboratories and is usually technically reliable on formalin-fixed paraffin-embedded tissue. There are MGMT antibodies commercially available. The analytical and clinical performance of SUMMARIES