BACKGROUND.: Despite many reports about the involvement of DNA methyltransferases (DNMTs) in human cancers, including nonsmall cell lung cancer (NSCLC), the clinicopathologic significance of DNMTs in primary NSCLC remains to be elucidated.METHODS.: The relation between the mRNA levels of DNMTs (1 and 3b) and the promoter methylation of the p16, RARβ2, H‐cadherin, GSTP1, RIZ, and FHIT genes and the clinicopathologic features in 102 fresh‐frozen tissues and paraffin blocks were retrospectively studied. The mRNA levels of the DNMTs were assessed via semiquantitative reverse‐transcription polymerase chain reaction (RT‐PCR), and the methylation status of the CpG islands were determined by methylation‐specific PCR.RESULTS.: The mRNA levels of DNMT1 and DNMT3b were elevated in 53% and 58% of 102 NSCLCs, respectively. Hypermethylation of p16, RARβ2, H‐cadherin, GSTP1, RIZ, and FHIT occurred in 37%, 38%, 34%, 18%, 9%, and 31% of patients, respectively. Univariate analysis showed that elevated DNMT mRNA levels were not significantly associated with the hypermethylation of 6 genes. However, the elevated mRNA levels of DNMT1 were determined to be significantly associated with the hypermethylation of the p16 promoter (odds ratio [OR] = 2.70, 95% confidence interval [95% CI], 1.02–7.15; P = .02), after controlling for age, gender, pack‐years smoked, histology, and pathologic stage. The hazard of failure in cases with elevated mRNA levels of DNMT1 was 3.51 (95% CI, 1.18–12.76; P = .02) times higher than that in those without. The elevated mRNA levels of DNMT3b were not ultimately associated with patient prognosis.CONCLUSIONS.: Elevated mRNA expression of DNMT1 may be an independent prognostic factor in NSCLC and CpG island hypermethylation in NSCLC may be maintained by a complex interaction of several factors rather than by a simple transcriptional up‐regulation of DNMT1. Cancer 2006. © 2006 American Cancer Society.

Fragile histidine triad ( FHIT ) gene plays an important role in the pathogenesis of lung cancer. However, the clinicopathological significance of CpG island hypermethylation of FHIT gene in non-small cell lung cancer (NSCLC) remains to be elucidated. We studied FHIT methylation in 254 NSCLCs in order to further understand the clinicopathological and prognostic significance of FHIT methylation in NSCLC. Methylation status of the FHIT gene was examined using Methylation-Specific PCR. All statistical analyses were two-sided, with a 5% type I error rate. Hypermethylation of the FHIT gene occurred more frequently in squamous cell carcinoma than adenocarcinoma. For 93 adenocarcinomas there was no statistically significant association between FHIT methylation and age, gender, smoking history, pathologic stage and p16 methylation. However, FHIT methylation in 125 squamous cell carcinomas was associated with exposure to tobacco smoke and p16 methylation, but not with age, gender and pathologic stage. Hypermethylation of FHIT in squamous cell carcinomas occurred more frequently in current smokers (45%) than in never-smokers (13%). FHIT methylation was significantly associated with p16 methylation in current- and ex-smokers ( P = 0.02 and P = 0.01, respectively) with squamous cell carcinoma and in patients with pathologic stage I squamous cell carcinoma ( P = 0.001). Patients with p16 methylation were 3.74 times [95% confidence interval (CI) = 1.62 − 7.95; P = 0.001] more likely to have FHIT methylation in squamous cell carcinoma. FHIT methylation in squamous cell carcinoma occurred at a 4.62 times (95% CI = 1.26 − 34.97; P = 0.02) higher prevalence in current smokers than in never-smokers. No prognostic effect of FHIT methylation was observed in stage I and stage II NSCLCs. In conclusion, hypermethylation of the FHIT gene did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinoma, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.

This study was aimed at investigating the functional significance of heparan sulfate (glucosamine) 3-O-sulfotransferase 2 (HS3ST2) hypermethylation in non-small cell lung cancer (NSCLC). HS3ST2 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting) and EpiTYPER^TM assays showed substantial hypermethylation of CpG island at the promoter region of HS3ST2 in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2′-deoxycytidine (5-Aza-dC). A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation in vitro. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, P = 0.009; Spearman’s rank correlation). HS3ST2 hypermethylation was found in 95 (32%) of 298 primary NSCLCs. Patients with HS3ST2 hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25–3.58, P = 0.005) compared to those without HS3ST2 hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation. The present study suggests that HS3ST2 hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrence-associated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif-1, sFRP1, integrin α4, CDH1, DAP kinase and RARβ2 genes in 251 ESCCs. The methylation status was determined by methylation-specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARβ2, 43% for CDH1, 21% for integrin α4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif-1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR = 5.26, 95% CI = 1.48–18.67; p = 0.01) and a poor recurrence-free survival after surgery (HR = 3.13, 95% CI = 1.21–8.09; p = 0.02). The hazard of failure after recurrence was about 13.17 (95% CI = 2.46–70.41; p = 0.003) times higher in patients with Wif-1 methylation than in those without. For stage II cancers, integrin α4 methylation was associated with an increased risk of recurrence (OR = 3.03, 95% CI = 1.09–8.37; p = 0.03) and a poor recurrence-free survival (HR = 2.12, 95% CI = 1.13–3.98; p = 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin α4 genes may be used as recurrence-associated prognostic indicators in stage I and stage II ESCCs, respectively.