title: Digital PCR assessment of MGMT promoter methylation coupled with reduced protein expression optimises prediction of response to alkylating agents in metastatic colorectal cancer patients.
abstract: O(6)-methylguanine-DNA-methyltransferase (MGMT) is a repair protein, and its deficiency makes tumours more susceptible to the cytotoxic effect of alkylating agents. Five clinical trials with temozolomide or dacarbazine have been performed in metastatic colorectal cancer (mCRC) with selection based on methyl-specific PCR (MSP) testing with modest results. We hypothesised that mitigated results are consequences of unspecific patient selection and that alternative methodologies for MGMT testing such as immunohistochemistry (IHC) and digital polymerase chain reaction (PCR) could enhance patient enrolment.
  
  
  
  Formalin-fixed paraffin embedded archival tumour tissue samples from four phase II studies of temozolomide or dacarbazine in MGMT MSP-positive mCRCs were analysed by IHC for MGMT protein expression and by methyl-BEAMing (MB) for percentage of promoter methylation. Pooled data were then retrospectively analysed according to objective response rate, progression-free survival (PFS) and overall survival (OS).
  
  
  
  One hundred and five patients were included in the study. Twelve had achieved partial response (PR) (11.4%), 24 stable disease (SD; 22.9%) and 69 progressive disease (PD; 65.7%). Patients with PR/SD had lower IHC scores and higher MB levels than those with PD. MGMT expression by IHC was negatively and MB levels positively associated with PFS (p < 0.001 and 0.004, respectively), but not with OS. By combining both assays, IHC low/MB high patients displayed an 87% reduction in the hazard of progression (p < 0.001) and a 77% in the hazard for death (p = 0.001).
  
  
  
  In mCRC selected for MGMT deficiency by MSP, IHC and MB testing improve clinical outcome to alkylating agents. Their combination could enhance patient selection in this setting.
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sartore-Bianchi
authors.affiliation: Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Pietrantonio
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Amatu
authors.affiliation: Pathology and Laboratory Medicine Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Milione
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cassingena
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Ghezzi
authors.affiliation: Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Caporale
authors.affiliation: Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Berenato
authors.affiliation: Department of Oncology, University of Torino, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute-FPO, IRCCS, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Falcomatà
authors.affiliation: Pathology and Laboratory Medicine Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Pellegrinelli
authors.affiliation: Department of Oncology, University of Torino, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute-FPO, IRCCS, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Bardelli
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Nichelatti
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Tosi
authors.affiliation: Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: De Braud
authors.affiliation: Department of Oncology, University of Torino, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute-FPO, IRCCS, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Di Nicolantonio
authors.affiliation: Department of Oncology, University of Torino, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute-FPO, IRCCS, SP 142 Km 3.95, 10060 Candiolo, Torino, Italy. Electronic address: ludovic.barault@unito.it.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Barault
authors.affiliation: Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy; Università degli Studi di Milano, Milan, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Siena
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use*; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000556:secondary; D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000235:genetics; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D015999:Multivariate Analysis; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D012189:Retrospective Studies; D016019:Survival Analysis; D000077204:Temozolomide
chemical_list: D018906:Antineoplastic Agents, Alkylating; D003606:Dacarbazine; D015254:DNA Modification Methylases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide
keywords: Alkylating agent; DNA methylation; Digital PCR; Immunohistochemistry; MGMT; Metastatic colorectal cancer