title: Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.
abstract: BACKGROUND
  O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide.
  
  
  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.
authors.affiliation: Experimental Clinical Molecular Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin).
authors.forename: L
authors.initials: L
authors.lastname: Barault
authors.affiliation: Niguarda Cancer Center, Ospedale Niguarda Ca' Granda, Milan, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Amatu
authors.affiliation: Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: F E
authors.initials: FE
authors.lastname: Bleeker
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Moutinho
authors.affiliation: Experimental Clinical Molecular Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin).
authors.forename: C
authors.initials: C
authors.lastname: Falcomatà
authors.affiliation: Department of Medical Sciences, University of Turin, Città Della Salute e Della Scienza Hospital, Turin.
authors.forename: V
authors.initials: V
authors.lastname: Fiano
authors.affiliation: Niguarda Cancer Center, Ospedale Niguarda Ca' Granda, Milan, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Cassingena
authors.affiliation: Experimental Clinical Molecular Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin); Department of Oncology, University of Torino, Candiolo (Turin); FIRC Institute of Molecular Oncology (IFOM), Milan.
authors.forename: G
authors.initials: G
authors.lastname: Siravegna
authors.affiliation: Departments of Pathology and Laboratory Medicine.
authors.forename: M
authors.initials: M
authors.lastname: Milione
authors.affiliation: Department of Medical Sciences, University of Turin, Città Della Salute e Della Scienza Hospital, Turin.
authors.forename: P
authors.initials: P
authors.lastname: Cassoni
authors.affiliation: Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan.
authors.forename: F
authors.initials: F
authors.lastname: De Braud
authors.affiliation: Department of Neuro-Oncology, University of Turin and Città Della Salute e Della Scienza Hospital, Turin, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Rudà
authors.affiliation: Department of Neuro-Oncology, University of Turin and Città Della Salute e Della Scienza Hospital, Turin, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Soffietti
authors.affiliation: Experimental Clinical Molecular Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin).
authors.forename: T
authors.initials: T
authors.lastname: Venesio
authors.affiliation: Experimental Clinical Molecular Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin); Department of Oncology, University of Torino, Candiolo (Turin).
authors.forename: A
authors.initials: A
authors.lastname: Bardelli
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam; Department of Pathology, Radboud University Medical Center, Nijmegen.
authors.forename: P
authors.initials: P
authors.lastname: Wesseling
authors.affiliation: Department Neurosurgery, Neurosurgical Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: de Witt Hamer
authors.affiliation: Departments of Pathology and Laboratory Medicine.
authors.forename: F
authors.initials: F
authors.lastname: Pietrantonio
authors.affiliation: Niguarda Cancer Center, Ospedale Niguarda Ca' Granda, Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Siena
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain; Department of Physiological Sciences II, School of Medicine, University of Barcelona, Catalonia; Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Esteller
authors.affiliation: Niguarda Cancer Center, Ospedale Niguarda Ca' Granda, Milan, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Sartore-Bianchi
authors.affiliation: Experimental Clinical Molecular Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin); Department of Oncology, University of Torino, Candiolo (Turin). Electronic address: federica.dinicolantonio@unito.it.
authors.forename: F
authors.initials: F
authors.lastname: Di Nicolantonio
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use*; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000401:mortality; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000401:mortality; D004247:DNA / Q000097:blood / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000627:therapeutic use; D018572:Disease-Free Survival; D005909:Glioblastoma / Q000188:drug therapy* / Q000401:mortality; D006801:Humans; D016133:Polymerase Chain Reaction; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D000077204:Temozolomide; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D004247:DNA; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; D000077204:Temozolomide
keywords: DNA methylation; MGMT; alkylating agent; cell free circulating DNA; digital PCR; metastatic colorectal cancer