DNA methylation profile during multistage progression of pulmonary adenocarcinomas

Multiple genetic and epigenetic alterations are known to be involved in the carcinogenesis of peripheral pulmonary adenocarcinoma (ADC). However, epigenetic abnormalities have not been extensively investigated in the following multistage progression sequence: atypical adenomatous hyperplasia (AAH) to adenocarcinoma in situ (AIS), to invasive ADC. To determine the potential role of promoter methylation during ADC development of the lung, we examined methylation status in 20 normal, 20 AAH, 30 AIS, and 60 ADC lung tissues and compared methylation status among the lesions. The MethyLight assay was used to determine the methylation status of 18 CpG island loci, which were hypermethylated in ADC compared to noncancerous lung tissues. The mean number of methylated CpG island loci was significantly higher in ADC than in AAH and AIS, (p < 0.003 between ADC and AAH, p < 0.005 between ADC and AIS). Aberrant methylation of HOXA1, TMEFF2, and RARB was frequently observed in preinvasive lesions, including AAH and AIS. Furthermore, methylation of PENK, BCL2, RUNX3, DLEC1, MT1G, GRIN2B, CDH13, CCND2, and HOXA10 was significantly more frequent in invasive ADC than AAH or AIS. Our results indicate that epigenetic alterations are involved in the multistep progression of pulmonary ADC development, and aberrant CpG island methylation accumulates during multistep carcinogenesis. In addition, aberrant methylation of HOXA1, TMEFF2, and RARB occurred in preinvasive lesions, which indicates that epigenetic alterations of these genes are involved in the early stages of pulmonary ADC development. In contrast, hypermethylation of PENK, BCL2, RUNX3, DLEC1, MT1G, GRIN2B, CDH13, CCND2, and HOXA10 was more frequent in invasive ADC than in preinvasive lesions, which indicates that methylation of these genes occurs later during tumor invasion in the AAH–AIS–ADC sequence.