Identification and validation of DNA methylation markers to predict lymph node metastasis of esophageal squamous cell carcinomas.
BACKGROUND
The presence of lymph node metastasis in esophageal squamous cell carcinoma (ESCC) patients is a critical factor for decision of treatment strategy. However, there have been no molecular markers to assess lymph node metastasis. In this study, we aimed to identify CpG islands (CGIs) whose DNA methylation statuses are associated with the presence of lymph node metastasis.


METHODS
A total of 96 ESCCs were divided into a screening set (n = 48) and a validation set (n = 48). Genome-wide methylation analysis was performed by methylated DNA immunoprecipitation-CGI microarray analysis. Methylation levels were analyzed by quantitative methylation-specific PCR (qMSP).


RESULTS
Genome-wide methylation analysis identified 25 CGIs differentially methylated between 8 ESCCs with lymph node metastasis and 4 without. In the screening set, 7 CGIs had significantly different methylation levels (P < 0.05) between the ESCCs with and without lymph node metastasis, and cut-off methylation levels for these CGIs were determined. The validation set was analyzed with the prefixed cut-offs, and methylation statuses of 2 CGIs in the vicinities of PAX6 and ENST00000363328 were validated to be associated with the presence of lymph node metastasis. Using these 2 markers, the presence was predicted with a sensitivity of 93% and specificity of 57%. In addition, the methylation statuses of the 2 CGIs were significantly associated with disease-free survival (P = 0.006).


CONCLUSIONS
Methylation statuses of these 2 CGIs were significantly associated with the presence of lymph node metastasis of ESCCs. These CGIs are promising markers to predict the presence of lymph node metastases.
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Ken
K
Gyobu
Satoshi
S
Yamashita
Yasunori
Y
Matsuda
Hiroyasu
H
Igaki
Tohru
T
Niwa
Daiji
D
Oka
Ryoji
R
Kushima
Harushi
H
Osugi
Shigeru
S
Lee
Shigefumi
S
Suehiro
Toshikazu
T
Ushijima
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology*; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000473:pathology*; D005260:Female; D006801:Humans; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D011379:Prognosis
D014408:Biomarkers, Tumor