In glioblastoma methylation of pMGMT is predictive of
In glioblastoma, methylation of pMGMT is predictive of the efficacy of temozolomide with an increased survival , , , . As in glioblastoma , , , , pMGMT methylation assessed by pyrosequencing (with immunochemistry, IHC) is the most effective and reproducible technique to predict the response to ALKY in NETs . pMGMT is methylated in 25%–50% of NETs in general, but it SB 204070 mg should be noted that a variation exists depending on the site of primitive cancer. Current data estimate a methylation of pMGMT in about 50% of pancreatic NETs and 0%–15% of lung and gastro-intestinal NETs , , . This variation could also contribute to the different chemosensitivities of these tumors. To the best of our knowledge no prospective study investigating MGMT status in NET has been published; currently, two studies are registered in ClinicalTrials.gov that evaluates MGMT as a secondary endpoint (NCT02698410 and NCT01824875). The latter was recently presented by Kunz at the ASCO 2018 annual meeting, but results regarding MGMT are currently pending .
Furthermore, although ALKY are commonly recommended upfront in NET , , , oxaliplatin (Ox), either with 5-fluorouracile , , , , , ,  or with gemcitabine , , has shown interesting activity with response rates ranging from 17% to 30%. In a retrospective study, we demonstrated that GEMOX is effective in NET and that its activity is similar to ALKY, but irrespective of the MGMT status . Prospective studies are needed, but the data suggests that ALKY should be offered first to patients with pMGMT methylation while Ox-based chemotherapy should be offered first to patients with unmethylated pMGMT tumors.
In this context, the purpose of this prospective study is to evaluate the contribution of the pMGMT methylation, in predicting the objective response (OR) in patients treated with ALKY and to evaluate a treatment with ALKY versus Ox in patients with a NET.
Study design The MGMT-NET study is a national, prospective, open-label, randomized, controlled and multicenter trial assessing the value of using the MGMT status in clinical practice to predict OR of patients suffering from NET. Patients included will first undergo analysis to determine the MGMT status before randomization. Patients with unmethylated pMGMT NETs will be randomly assigned (1:1) to either the ALKY-based chemotherapy arm or to the Ox-based chemotherapy arm. Patients with methylated pMGMT NETs will be randomly assigned (2:1) to either the ALKY-based chemotherapy arm or to the Ox-based chemotherapy arm (Fig. 1). As current data suggests that patients with methylated pMGMT respond better to ALKY ,  we considered that a 1:1 randomization would not have been ethical for such patients. In addition, an Ox arm was included to facilitate recruitment by offering another option of treatment to patients. However, we considered that a phase III study designed to evaluate the best chemotherapy (Ox vs. ALKY) according to MGMT status in each arm would have been impossible to complete given the high number of patients needed and the rarity of the disease. Methylation tests and IHC will be centralized and conducted on the most recent available histological material. pMGMT methylation status will be evaluated using two techniques: methylation-specific polymerase chain reaction (MS-PCR) and pyrosequencing, which will provide, respectively, qualitative and quantitative information. The techniques used are derived from the procedures employed in our institution for gliomas. All examined samples must contain more than 80% tumor cells. DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue is performed after deparaffinization using a purification kit (Promega, Madison, WI, USA, ref AS1450). Genomic DNA is modified by bisulfite conversion (EZ DNA Methylation Gold Kit, Zymo, Irvine, CA, US). For MS-PCR, we use the primers described by Dong et al. . For pyrosequencing analysis, an 8% cutoff is used . pMGMT promoter is therefore scored “methylated” if more than 8% methylated alleles are detected compared to unmethylated alleles. The results are presented as methylated, not methylated, or not interpretable. The MGMT protein expression will be evaluated by an immunohistochemical technique for which an automated immunostaining system will be used (Ventana Benchmark, Tucson, AZ, US). MGMT expression will be assessed on a whole slide using a score based on nuclear staining intensity (0–3) multiplied by the proportion of stained cells (0–100%). The score ranges from 0 to 300 and tumors are considered MGMT negative (loss of expression) if the score is ≤50 . pMGMT methylation test and IHC will be carried out in parallel, but only the result of the methylation test will be considered for randomization unless the result is not interpretable; in which case the IHC result will be used, considering that a loss of MGMT expression in IHC corresponds to a methylated pMGMT. If the pMGMT methylation test and the IHC result are both not interpretable, the patients will be randomized (ratio 1:1 between the two arms), but these patients will not be included in the analysis of the main outcome. Furthermore, a blood sample will be collected specifically for circulating free DNA (cfDNA) analyses.