In advanced myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML), aberrant methylation targets genes involved in apoptosis, cell cycle, DNA repair or adherence, or silencing tumor suppressor genes. 1–3 The correlation between DNA hypermethylation and disease progression provides a rationale for the therapeutic use of Dnmt inhibitors. Azacitidine induces a hematological response in 40–50% of high-risk (HR)-MDS patients, delays the disease progression to sAML and significantly prolongs overall survival. 4 FAS/TNFRSF6/APO1 that belongs to a set of genes involved in the control of apoptosis is hypermethylated in tumors. 5–7 We have previously shown that Fas is overexpressed in low/int-1-risk (LR)-MDS. 8 Conversely, decreased expression of pro-apoptotic Fas, or Fas-associated via death domain (FADD) or increased anti-apoptotic Bcl2 protein in int-2 or HR-MDS could participate in resistance to apoptosis and contribute to disease progression. Consecutive patients (n¼ 169) with a WHO diagnosis of MDS (n¼ 136), including 33 refractory anemia (RA)/refractory cytopenia (RC)/MDS-unclassified (MDS-U), 9 5q-, 17 RC with multilineage dysplasia (RCMD), 30 RA with ring sideroblasts (RARS)/RCMD with ring sideroblasts (RCMD-RS), 32 RA with excess of blasts (RAEB)-1, 15 RAEB-2, or 33 sAML and 25 age-matched controls with normal bone marrow (BM) were recorded between 2005 and 2011 for flow cytometry analysis of Fas expression on BM CD45lo/CD34þ cell population. The median ratio of fluorescence intensity (RFI) was significantly increased in LR-MDS (n¼ 104) or HR-MDS (n¼ 32) compared with a control group, and lower in sAML compared with LR-MDS or HR-MDS (Figure 1a). Sequential analysis of Fas expression in 26 MDS patients whose disease had progressed identified a significant decrease of the median Fas RFI from 2.1 (interquartile range (IQR) 25–75%: 1.8–2.8) at diagnosis to 1.2 (IQR 25–75%: 1.0–1.8) at later time points (Student t-test; P¼ 0.003), whereas the median percentage of blasts concomitantly increased from 5 (4–9) to 18%(12–24; Po0. 0001; Supplementary Table S1). The threshold of Fas protein expression positivity predictive of MDS, deduced from a receiver operating characteristics analysis of Fas expression, was an RFI of 1.8. We then investigated whether the FAS gene expression was epigenetically regulated along the MDS disease progression to AML. Variations of the methylation level at FAS promoter have already been described in one CpG island located over the transcription start site (þ 1) and in a 50-regulatory region located upstream the CpG island in the NIH3T3 cell line transformed by KRAS or in primary tumor cells. 5–7, 9 Thus, we extensively studied DNA methylation of the promoter region spanning between À 899 and þ 231, which contains 35 CpG dinucleotides, of which 26 belong to a CpG island. The FAS promoter was amplified from bisulfite-treated genomic DNA extracted from CD34þ cells in 18 LR-MDS, 8 HR-MDS, 11 sAML and 8 controls (Supplementary Table S2 for primers). The PCR products were cloned in bacteria, and at least eight clones per case were sequenced. Global level of methylation expressed as the percentage of methylated CpG at each dinucleotide was significantly higher in sAML compared with LR-MDS (P ¼ 0.01), HR-MDS (P ¼ 0.008), and LR-or HR-MDS (P ¼ 0.004). The most heavily and differentially methylated CpGs 1–8 were located in the 50-region upstream of the CpG island (Figure 1b). The Fas RFI was inversely correlated with the percentage of methylated CpG1-8 (Figure 1c). These results indicate that the FAS …