Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

Annette M. Lim, Ida L.M. Candiloro, Nicholas Wong, Marnie Collins, Hongdo Do, Elena A. Takano, Christopher Angel, Richard J. Young, June Corry, David Wiesenfeld, Stephen Kleid, Elizabeth Sigston, Bernard Lyons, Danny Rischin, Benjamin Solomon, Alexander Dobrovic

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Article number22
Number of pages16
JournalClinical Epigenetics
Volume6
Issue number1
DOIs
Publication statusPublished - 1 Jan 2014
Externally publishedYes

Keywords

  • Head and neck cancer
  • Methylation
  • Quantitative
  • RUNX3
  • Tongue

Cite this

Lim, Annette M. ; Candiloro, Ida L.M. ; Wong, Nicholas ; Collins, Marnie ; Do, Hongdo ; Takano, Elena A. ; Angel, Christopher ; Young, Richard J. ; Corry, June ; Wiesenfeld, David ; Kleid, Stephen ; Sigston, Elizabeth ; Lyons, Bernard ; Rischin, Danny ; Solomon, Benjamin ; Dobrovic, Alexander. / Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome. In: Clinical Epigenetics. 2014 ; Vol. 6, No. 1.
@article{121620da58a04ebda3ba1be6255c5be0,
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.",
keywords = "Head and neck cancer, Methylation, Quantitative, RUNX3, Tongue",
author = "Lim, {Annette M.} and Candiloro, {Ida L.M.} and Nicholas Wong and Marnie Collins and Hongdo Do and Takano, {Elena A.} and Christopher Angel and Young, {Richard J.} and June Corry and David Wiesenfeld and Stephen Kleid and Elizabeth Sigston and Bernard Lyons and Danny Rischin and Benjamin Solomon and Alexander Dobrovic",
year = "2014",
month = "1",
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doi = "10.1186/1868-7083-6-22",
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Lim, AM, Candiloro, ILM, Wong, N, Collins, M, Do, H, Takano, EA, Angel, C, Young, RJ, Corry, J, Wiesenfeld, D, Kleid, S, Sigston, E, Lyons, B, Rischin, D, Solomon, B & Dobrovic, A 2014, 'Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome' Clinical Epigenetics, vol. 6, no. 1, 22. https://doi.org/10.1186/1868-7083-6-22

Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome. / Lim, Annette M.; Candiloro, Ida L.M.; Wong, Nicholas; Collins, Marnie; Do, Hongdo; Takano, Elena A.; Angel, Christopher; Young, Richard J.; Corry, June; Wiesenfeld, David; Kleid, Stephen; Sigston, Elizabeth; Lyons, Bernard; Rischin, Danny; Solomon, Benjamin; Dobrovic, Alexander.

In: Clinical Epigenetics, Vol. 6, No. 1, 22, 01.01.2014.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

AU - Lim, Annette M.

AU - Candiloro, Ida L.M.

AU - Wong, Nicholas

AU - Collins, Marnie

AU - Do, Hongdo

AU - Takano, Elena A.

AU - Angel, Christopher

AU - Young, Richard J.

AU - Corry, June

AU - Wiesenfeld, David

AU - Kleid, Stephen

AU - Sigston, Elizabeth

AU - Lyons, Bernard

AU - Rischin, Danny

AU - Solomon, Benjamin

AU - Dobrovic, Alexander

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

KW - Head and neck cancer

KW - Methylation

KW - Quantitative

KW - RUNX3

KW - Tongue

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U2 - 10.1186/1868-7083-6-22

DO - 10.1186/1868-7083-6-22

M3 - Article

VL - 6

JO - Clinical Epigenetics

JF - Clinical Epigenetics

SN - 1868-7075

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ER -