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Abstract: Objectives. : Although initiated by human papillomavirus (HPV), cervical carcinogenesis demands other cofactors to shape its natural course. Epigenetic effects such as DNA methylation, are considered to contribute to carcinogenesis process. Methods. : The methylation status of BLU and RASSF1A, as well as the HPV infection status, were assessed in a full spectrum of cervical neoplasia, including 45 low-grade squamous intraepithelial lesions (LSIL), 63 high-grade squamous intraepithelial lesions (HSIL), 107 squamous cell carcinomas (SCC), 23 adenocarcinomas (AC), and 44 normal control tissues. Results. : The BLU was methylated in 76.9% of SCC, 57.4% of HSIL, 20.0% of LSIL and 12.5% of normal tissues (P <0.001). The RASSF1A was methylated in 15% of SCC, 17.5% of HSIL, but not in LSIL or normal tissues (P <0.001). In AC, 43.5% of patients showed BLU methylation and 26.1% RASSF1A methylation, significantly higher than the corresponding control frequencies of 12.5% (P =0.005) and 0% (P =0.001), respectively. There was an insignificant trend toward loss of BLU methylation with advancing clinical stages of SCC (84.8%, 67.7%, and 63.6% in stages I, II, and III/IV, respectively; P =0.08). Patients with LSIL infected with high-risk HPV showed a higher rate of BLU methylation than those without HPV (38.8% vs 9.1%, respectively; P =0.057). The methylation of RASSF1A was inversely related to HPV infection in patients with HSIL/SCC (P =0.003). Conclusions. : These results suggest that the methylation of BLU and RASSF1A genes is associated with cervical carcinogenesis, which could be clinically important in the future molecular screening of cervical neoplasia. [Copyright &y& Elsevier]

Abstract: Objective.: Whereas human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis, host genetic variations may confer individual susceptibility. Resistance to apoptosis is a hallmark of cancer in which FAS/FAS ligand signaling plays an important role. The present study examines the hypothesis that genetic polymorphisms in FAS and FAS ligand genes, alone or in combination, are associated with cervical carcinogenesis. Methods.: The genotypes of FAS −670A/G, FAS −1377G/A, and FASL −844C/T were assessed in 143 patients with high-grade squamous intraepithelial lesions (HSIL), 175 patients with invasive squamous cell carcinomas (SCC), and in age-matched controls by real-time PCR with allele-specific TaqMan probes. The status of cervical high-risk HPV infection was determined and adjusted to test the independence of genotype in the risk assessment. Results.: The A-allele and AA-genotype frequencies of FASA −670G were significantly higher in HSIL/SCC than in controls (60% vs. 54%, P = 0.04, OR 1.26 [95% CI 1.01–1.57]; 38.0% vs. 28.6%, P = 0.02, OR 1.70 [95% CI 1.07–2.70]). No association between FAS −1377 or FASL −844 polymorphisms and HSIL/SCC could be identified. The FAS −1377A/−670A haplotype conferred a higher risk for HSIL/SCC (OR 3.05, 95% CI 1.28–7.30) than FAS −670A alone (OR 1.26, 95% CI 1.28–7.30). The interaction between FAS −670AA and FASL −844CC genotypes was associated with a risk of HSIL/SCC (OR 2.13, 95% CI 1.06–4.29) higher than that of the FAS −670AA genotype alone (OR 1.70, 95% CI 1.07–2.70). Conclusions.: The FAS −1377A/−670A haplotype in combination with FASL −844C is associated with cervical carcinogenesis. [Copyright &y& Elsevier]

Abstract: Objectives: Aberrant activation of the Wnt/β-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as Wnt antagonists and play important implications in carcinogenesis. Recently, we have shown that SFRP1 and SFRP2 are frequently downregulated through promoter hypermethylation. However, the function of SFRP1 and SFRP2 in cervical cancer remains unclear. Methods: To improve our understanding of the role of SFRP1 and SFRP2 in cervical cancer cells, we use overexpression or shRNA approach in cervical cancer cell lines. Results: Restoration of the expression of SFRP1 and SFRP2 attenuated Wnt signaling in CaSki cells, decreased abnormal accumulation of free β-catenin in the nucleus, and suppressed cancer cell growth. In addition, different statuses of β-catenin accumulation in the cytoplasm of CaSki or HeLa3rd cells were observed, suggesting that different Wnt pathways are executed. Furthermore, we demonstrated that SFRP1 and SFRP2 enhance the expression of the epithelial marker E-cadherin, through inhibition of the expression of SLUG, TWIST and SNAIL, three transcription factors involved in the epithelial mesenchymal transition (EMT) program. Finally, in a xenograft animal model, we showed that SFRP1 suppresses tumorigenicity of cancer cells in vivo. Conclusions: Taken together, these data strongly suggest that epigenetic silencing of SFRP genes leads to oncogenic activation of the Wnt pathway and contributes to cervical cancer progression through the EMT program. [Copyright &y& Elsevier]

Abstract: Objective.: This study was to correlate high-risk human papillomavirus (HR-HPV) viral load to p16INK4A (p16) expression in atypical glandular cell (AGC)-categorized Pap smears with follow-up biopsies for elucidating their relationships. Methods.: We enrolled 36 AGC-categorized Pap smears with subsequent follow-up biopsies. HR-HPV viral load was determined by Hybrid Capture II assay in each AGC-diagnosed Pap smear. Both smears and biopsies were immunostained with a primary anti-p16 antibody, clone E6H4. Correlations between HR-HPV viral load in each AGC-diagnosed Pap smear and p16 expression of smears with follow-up biopsies were performed. Results.: Comparative analysis of two tests disclosed both consistencies and discrepancies. There were significant differences (P = 0.02) between negative or weak p16 expression of Pap smears with the presence of reactive lesion or LSILs/CIN1s in follow-up biopsies and negative HR-HPV viral load. However, no significant difference (P = 0.317) was found between p16 expression of Pap smears with the presence of HSIL/CIN2, 3 and AIS or adenocarcinoma in follow-up biopsies and high HR-HPV viral load. In addition, there were significant differences (P = 0.012) in specificity, but no significant differences were found in sensitivity (P = 0.604), positive and negative predictive value (P = 0.066 and 0.264) between p16 immunoexpression and HR-HPV viral load. Conclusions.: Pathogenic activity of HR-HPV was indicated by p16 expression on smears and tissue sections, which appears to be a better strategy than HR-HPV viral load test for the detection of clinically insignificant lesions from AGC-categorized Pap smears. [Copyright &y& Elsevier]

Objectives: Oncogenic activation of the Wnt/beta-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and play an important role in carcinogenesis. Frequent promoter hypermethylation of SFRPs has been identified in human cancers; however, the precise role of SFRPs in cervical cancer is not clear., Methods: The methylation status of SFRPs gene family was analyzed in two cervical cancer cell lines and a full spectrum of cervical neoplasia, including 45 low-grade squamous intraepithelial lesions (LSIL), 49 high-grade squamous intraepithelial lesions (HSIL), 109 squamous cell carcinomas (SCC), and 45 normal controls., Results: The SFRP1 promoter was hypermethylated in 33.9% of SCC, 8.2% of HSIL, 2.2% of LSIL, but not in normal tissues. The SFRP2 promoter was hypermethylated in 80.7% of SCC, 16.3% of HSIL, 15.6% LSIL and 4.4% normal tissues. The SFRP4 promoter was hypermethylated in 67.9% of SCC, 36.7% of HSIL, 4.4% of LSIL, but not in normal tissues. The SFRP5 promoter was hypermethylated in 10.1% of SCC, 4.1% of HSIL, 13.3% of LSIL and 4.4% normal tissues. The frequency of SFRP1, SFRP2 and SFRP4 promoter methylation in tumors was significantly higher than in normal, LSIL, and HSIL samples (P<0.0001). SFRP5 methylation was significantly different in patients with or without lymph-node metastases (0% vs 15.2%, respectively, P<0.05)., Conclusions: Our data suggest that promoter hypermethylation of SFRP1, SFRP2 and SFRP4 is associated with cervical carcinogenesis, which could be used for molecular screening of cervical neoplasias in future.

Objective: This study was to correlate high-risk human papillomavirus (HR-HPV) viral load to p16(INK4A) (p16) expression in atypical glandular cell (AGC)-categorized Pap smears with follow-up biopsies for elucidating their relationships., Methods: We enrolled 36 AGC-categorized Pap smears with subsequent follow-up biopsies. HR-HPV viral load was determined by Hybrid Capture II assay in each AGC-diagnosed Pap smear. Both smears and biopsies were immunostained with a primary anti-p16 antibody, clone E6H4. Correlations between HR-HPV viral load in each AGC-diagnosed Pap smear and p16 expression of smears with follow-up biopsies were performed., Results: Comparative analysis of two tests disclosed both consistencies and discrepancies. There were significant differences (P=0.02) between negative or weak p16 expression of Pap smears with the presence of reactive lesion or LSILs/CIN1s in follow-up biopsies and negative HR-HPV viral load. However, no significant difference (P=0.317) was found between p16 expression of Pap smears with the presence of HSIL/CIN2, 3 and AIS or adenocarcinoma in follow-up biopsies and high HR-HPV viral load. In addition, there were significant differences (P=0.012) in specificity, but no significant differences were found in sensitivity (P=0.604), positive and negative predictive value (P=0.066 and 0.264) between p16 immunoexpression and HR-HPV viral load., Conclusions: Pathogenic activity of HR-HPV was indicated by p16 expression on smears and tissue sections, which appears to be a better strategy than HR-HPV viral load test for the detection of clinically insignificant lesions from AGC-categorized Pap smears.