Genome–Wide Hypomethylation and Specific Tumor-Related Gene Hypermethylation are Associated with Esophageal Squamous Cell Carcinoma Outcome Sojun Hoshimoto,

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Genome–Wide Hypomethylation and Specific Tumor-Related Gene Hypermethylation are Associated with Esophageal Squamous Cell Carcinoma Outcome Sojun Hoshimoto, MD, PhD, Hiroya Takeuchi, MD, PhD, Shigeshi Ono, MD, PhD, Myung Shin Sim, DrPH, Jamie L. Huynh, Sharon K. Huang, PhD, Diego M. Marzese, PhD, Yuko Kitagawa, MD, PhD, Dave S.B. Hoon, MSc, PhD Journal of Thoracic Oncology Volume 10, Issue 3, Pages 509-517 (March 2015) DOI: 10.1097/JTO.0000000000000441 Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Comparison of LINE-1 U index between: A, Normal epithelium versus ESCC. B, Normal epithelium versus primary ESCC versus lymph node metastases. C, Normal epithelium versus T1 tumors versus T2/3/4 tumors. p values were obtained by Wilcoxon test. D, ROC curve for the discrimination between primary ESCC and adjacent noncancerous epithelium. AUC value reflecting group discrimination was indicated. ESCC, esophageal squamous cell carcinoma; LINE-I, long interspersed nucleotide element-1; ROC, receiver operating characteristics; AUC, area under the curve. Journal of Thoracic Oncology 2015 10, 509-517DOI: (10.1097/JTO.0000000000000441) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Genome-wide DNA methylation analysis of ESCC and normal esophagus tissues identifies differential methylation of 1631 CpG sites surrounding 160 TRGs. A, Volcano plot depicting CpG sites showing significant (orange line; FDR-p = 0.05) hypomethylation (green dots) and hypermethylation (red dots) in ESCC specimens. B, The terrain map analysis using Euclidean metric distance revealed a complete separation between normal tissues and ESCC. C, Unsupervised hierarchical cluster analysis. The methylation status in ESCC (black bar) and normal esophagus specimens (grey bar) were identified in two independent clusters (bootstrap = 100). ESCC, esophageal squamous cell carcinoma; TRG, tumor-related gene; FDR, false discovery rate. Journal of Thoracic Oncology 2015 10, 509-517DOI: (10.1097/JTO.0000000000000441) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Comparison of LINE-1 U index between RARB hypermethylated group versus RARB unmethylated group. p values were obtained by Wilcoxon test. LINE-I, long interspersed nucleotide element-1. Journal of Thoracic Oncology 2015 10, 509-517DOI: (10.1097/JTO.0000000000000441) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Kaplan–Meier curves of ESCC patients for OS. Patients are divided into subgroups according to: A, RASSF1 promoter methylation status. B, APC promoter methylation status. C, The number of methylated TRGs (greater than two versus less than or equal to two). ESCC, esophageal squamous cell carcinoma; OS, overall survival; TRG, tumor-related gene. Journal of Thoracic Oncology 2015 10, 509-517DOI: (10.1097/JTO.0000000000000441) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions