title: Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome.
abstract: BACKGROUND
  DNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC).
  
  
  RESULTS
  The methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing. In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated. Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76).
  
  
  CONCLUSIONS
  We demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.
authors: Department of Medical Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; The University of Melbourne, Parkville, Victoria 3010 Australia ; Research Division, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Annette M
authors: AM
authors: Lim
authors: Research Division, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3010 Australia.
authors: Ida Lm
authors: IL
authors: Candiloro
authors: The University of Melbourne, Parkville, Victoria 3010 Australia ; Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Health, 145-163 Studley Road, Heidelberg, Victoria 3084 Australia.
authors: Nicholas
authors: N
authors: Wong
authors: Department of Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Marnie
authors: M
authors: Collins
authors: Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Health, 145-163 Studley Road, Heidelberg, Victoria 3084 Australia ; Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; School of Cancer Medicine, La Trobe University, Bundoora, Victoria 3084 Australia.
authors: Hongdo
authors: H
authors: Do
authors: Research Division, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Elena A
authors: EA
authors: Takano
authors: Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Christopher
authors: C
authors: Angel
authors: Research Division, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Richard J
authors: RJ
authors: Young
authors: The University of Melbourne, Parkville, Victoria 3010 Australia ; Department of Radiation Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: June
authors: J
authors: Corry
authors: Department of Surgery, Royal Melbourne Hospital, 300 Grattan St, Parkville, Victoria 3050 Australia.
authors: David
authors: D
authors: Wiesenfeld
authors: Department of Surgical Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Stephen
authors: S
authors: Kleid
authors: Department of Surgery, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168 Australia.
authors: Elizabeth
authors: E
authors: Sigston
authors: Department of Surgery, St Vincent's Hospital, PO Box 2900, Fitzroy, Victoria 3065 Australia.
authors: Bernard
authors: B
authors: Lyons
authors: Department of Medical Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; The University of Melbourne, Parkville, Victoria 3010 Australia.
authors: Danny
authors: D
authors: Rischin
authors: Department of Medical Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; The University of Melbourne, Parkville, Victoria 3010 Australia ; Research Division, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia.
authors: Benjamin
authors: B
authors: Solomon
authors: Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Health, 145-163 Studley Road, Heidelberg, Victoria 3084 Australia ; Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A'Beckett Street, Melbourne, Victoria 8006 Australia ; Department of Pathology, The University of Melbourne, Parkville, Victoria 3010 Australia ; School of Cancer Medicine, La Trobe University, Bundoora, Victoria 3084 Australia.
authors: Alexander
authors: A
authors: Dobrovic
keywords: Head and neck cancer; Methylation; Quantitative; RUNX3; Tongue