Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

Background O6-Methyl-Guanine-Methyl-Transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. Patients and methods We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (Methyl-BEAMing). Results were compared to two established techniques, Methylation specific PCR (MSP) and Bs-pyrosequencing. Results Thresholds for MGMT methylated status for each technique were established in a training-set of 98 glioblastoma patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 glioblastoma patients treated with temozolomide in which Methyl-BEAMing displayed a better specificity than the other techniques. Cut-off values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays Methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS)(p<0.001). Ability of Methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and pre-selected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by Methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (p=0.008). Conclusions Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin fixed paraffin embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.