定量方法对于评估DNA甲基化及其对与患者预后相关性的影响至关重要。
Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome.
作者信息
Lim Annette M, Candiloro Ida Lm, 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
机构信息
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.
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.
出版信息
Clin Epigenetics. 2014 Dec 9;6(1):22. doi: 10.1186/1868-7083-6-22. eCollection 2014.
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.
背景
DNA高甲基化在头颈部鳞状细胞癌（HNSCC）中是一种常见事件和预后标志物。甲基化通常采用非定量方法进行评估，如甲基化特异性PCR（MSP）。我们采用定量方法对先前报道的口腔舌鳞状细胞癌（OTSCC）中的高甲基化基因进行了研究。
结果
通过甲基化敏感高分辨率熔解（MS-HRM）、实时甲基化特异性PCR后的敏感熔解分析（SMART-MSP）和亚硫酸氢盐焦磷酸测序这三种定量分析方法中的一种或多种，评估了115例OTSCC样本中12个基因的甲基化状态。与许多文献不同，MS-HRM未检测到或仅偶尔检测到DAPK1、RASSF1A、MGMT、MLH1、APC、CDH1、CDH13、BRCA1、ERCC1和ATM基因座特异性甲基化。最常发生甲基化的基因座是RUNX3（18/108甲基化）和ABO（22/107甲基化）。对癌症基因组图谱（TCGA）HNSCC队列的分析证实了所研究基因座显著甲基化的频率。MS-HRM检测到的RUNX3（18/108）和ABO（22/107）的异质性甲基化与较差的生存率显著相关（P = 0.01和P = 0.03）。然而，在使用焦磷酸测序对甲基化水平进行定量后，只有4个肿瘤的RUNX3甲基化水平显著（>15%），这与较差的患者预后相关（P <0.001），而ABO高甲基化的预后意义消失。RUNX3甲基化对TCGA队列无预后价值（P = 0.76）。
结论
我们证明了甲基化水平定量及其对相关性分析影响的迫切需求。在OTSCC中，我们几乎没有发现许多报道中常见的显著或频繁高甲基化的证据。由于使用了非定量方法，先前的报道可能高估了显著甲基化事件的频率。
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