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DNA methylation contributes to tumor formation, development and metastasis. Epigenetic dysregulation of stem cells is thought to predispose to malignant development. The clinical significance of DNA methylation in ovarian tumor-initiating cells (OTICs) remains unexplored. We analyzed the methylomic profiles of OTICs (CP70sps) and their derived progeny using a human methylation array. qRT-PCR, quantitative methylation-specific PCR (qMSP) and pyrosequencing were used to verify gene expression and DNA methylation in cancer cell lines. The methylation status of genes was validated quantitatively in cancer tissues and correlated with clinicopathological factors.ATG4AandHIST1H2BNwere hypomethylated in OTICs. Methylation analysis ofATG4AandHIST1H2BNby qMSP in 168 tissue samples from patients with ovarian cancer showed thatHIST1H2BNmethylation was a significant and independent predictor of progression-free survival (PFS) and overall survival (OS). Multivariate Cox regression analysis showed that patients with a low level ofHIST1H2BNmethylation had poor PFS (hazard ratio (HR), 4.5; 95% confidence interval (CI), 1.4-14.8) and OS (HR, 4.3; 95% CI, 1.3-14.0). Hypomethylation of bothATG4AandHIST1H2BNpredicted a poor PFS (HR, 1.8; 95% CI, 1.0-3.6; median, 21 months) and OS (HR, 1.7; 95% CI, 1.0-3.0; median, 40 months). In an independent cohort of ovarian tumors, hypomethylation predicted early disease recurrence (HR, 1.7; 95% CI, 1.1-2.5) and death (HR, 1.4; 95% CI, 1.0-1.9). The demonstration that expression ofATG4Ain cells increased their stem properties provided an indication of its biological function. Hypomethylation ofATG4AandHIST1H2BNin OTICs predicts a poor prognosis for ovarian cancer patients.