Assessment of gene promoter hypermethylation for detection of cervical neoplasia

Abstract

Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot-study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 patients diagnosed with cervical cancer (20 squamous cell carcinomas and 10 adenocarcinomas) and 19 women with histologically normal cervices. The scraped cells were used for determination of promoter hypermethylation by QMSP for 12 genes and for morphological assessment. Overall, CALCA, DAPK, ESR1, TIMP3, APC and RAR-b 2 promoters were significantly more often hypermethylated in cancers than in controls, while adenocarcinomas were more often hypermethylated above the highest control ratio for APC, TIMP3 and RASSF1A promoters. Combining 4 genes (CALCA, DAPK, ESR1 and APC) yielded a sensitivity of 89% (with all adenocarcinomas identified), equal to cytomorphology (89%) and high-risk human papilloma virus (Hr-HPV; 90%). The 4-gene QMSP proved theoretically superior to cytomorphology as well as Hr-HPV in specificity (100% vs. 83 and 68%, respectively), because cytology identified 3 controls as moderate or severe dyskaryosis and 6 controls were positive for Hr-HPV. In conclusions, QMSP of 4 gene promoters combined appears to have comparable sensitivity and potentially better specificity in comparison to ''classic'' cytomorphological assessment and Hr-HPV detection. QMSP holds promise as a new diagnostic tool for both squamous cell carcinoma and adenocarcinoma of the cervix. ' 2006 Wiley-Liss, Inc. Key words: methylation; cervical cancer; QMSP; DAPK; ESR1Cervical cancer is an important cause of death in women worldwide. 1 Cervical carcinogenesis is highly associated with (high-risk) human papilloma virus (HPV) infections. 2 Cytomorphological examination of cervical smears is a widely applied, though not ideal screening method for cervical cancer and its precursors, since the Pap smear has false negative rates of 2-40%, due to a combination of sampling error, processing artifacts and the nature of subjective interpretation. 1,3,4 False-negative cytology can also be found in about 50% of cases when previous negative smears are reviewed from the small proportion of screened women who develop invasive cancer. 4 Moreover, as many as 20% of all Pap smears are interpreted as atypical squamous cells of undetermined significance (ASCUS) or borderline dyskaryotic, leading to increased surveillance and more invasive tests in many of these patients. 3,5 The incidence of squamous cell carcinoma of the cervix has decreased since introduction of nation wide screening programs, compared to a relative increased incidence of adenocarcinoma of the cervix. 6 The efficacy of ...

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

“…The detection of distinct methylation profiles between SCCs and AdCAs is in concordance with literature data, showing higher frequencies of DAPK1 promoter methylation in SCCs compared with AdCAs (Dong et al, 2001;Narayan et al, 2003;Yang et al, 2004;Kang et al, 2006) and increased rates of APC, RASSF1A and TIMP3 promoter methylation in AdCAs compared with SCCs (Dong et al, 2001;Narayan et al, 2003;Jeong et al, 2006;Kang et al, 2006;Wisman et al, 2006).…”

“…In several of these studies, methylation of up to 16 gene promoters has been tested on cervical cancer biopsies, showing that up to 93% of them were positive for at least one of the methylation markers tested (Dong et al, 2001;Virmani et al, 2001;Narayan et al, 2003;Gustafson et al, 2004;Widschwendter et al, 2004;Feng et al, 2005;Zambrano et al, 2005;Wisman et al, 2006). However, for most of the genes studied, it is still unclear to what extent and at what stage promoter methylation reflects a functionally important step in the transformation process.…”

Correlation of promoter hypermethylation in hTERT, DAPK and MGMT genes with cervical oncogenesis progression

“…In the present study, we investigated the methylation profiles in the promoter region of hTERT, MGMT and DAPK genes in normal, premalignant and cervical cancer specimens, as the existing data on the methylation status of these genes is limited and results are conflicting (22,(32)(33)(34)(35)(36). hTERT gene plays a critical role in cell division and serves as a mitotic clock for cell proliferation (17).…”

Host-cell DNA methylation patterns during high-risk HPV-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer

“…Quality control was routinely performed using a methylation-independent assay for the housekeeping gene ACTB [13]. …”