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

Ann Oncol. 2015 Sep;26(9):1994-1999. doi: 10.1093/annonc/mdv272. Epub 2015 Jun 25.


Background: O(6)-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 with 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 (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff 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 preselected 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.

Keywords: DNA methylation; MGMT; alkylating agent; cell free circulating DNA; digital PCR; metastatic colorectal cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Alkylating / therapeutic use*
  • Brain Neoplasms / drug therapy*
  • Brain Neoplasms / mortality
  • Colorectal Neoplasms / drug therapy*
  • Colorectal Neoplasms / mortality
  • DNA / blood
  • DNA / metabolism
  • DNA Methylation / genetics*
  • DNA Modification Methylases / genetics
  • DNA Modification Methylases / metabolism*
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism*
  • Dacarbazine / analogs & derivatives
  • Dacarbazine / therapeutic use
  • Disease-Free Survival
  • Glioblastoma / drug therapy*
  • Glioblastoma / mortality
  • Humans
  • Polymerase Chain Reaction
  • Prognosis
  • Promoter Regions, Genetic / genetics
  • Temozolomide
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*


  • Antineoplastic Agents, Alkylating
  • Tumor Suppressor Proteins
  • Dacarbazine
  • DNA
  • DNA Modification Methylases
  • MGMT protein, human
  • DNA Repair Enzymes
  • Temozolomide