Methylation Markers for CCNA1 and C13ORF18 Are Strongly Associated with High-Grade Cervical Intraepithelial Neoplasia and Cervical Cancer in Cervical Scrapings

Abstract

Purpose: Recently, we reported 13 possible cervical cancer-specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance. Experimental Design and Results: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high. Conclusion: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3000-7)

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

“…Other unidentified underlying abnormalities may have resulted in increased DNA methylation levels. Considering a potential effect of age, i.e., 55 year for this control, DNA methylation levels of the markers tested were not or minimally affected by age in our previous studies using large cohorts [8–11,42,43]. Therefore, solely age is very unlikely to explain the cancer-like methylation-high pattern in this hrHPV-positive control.…”

CCNA1 Promoter Methylation: a Potential Marker for Grading Papanicolaou Smear Cervical Squamous Intraepithelial Lesions

“…Here, we propose to add CCNA1 methylation as a useful marker, especially in the event of HSIL+ detection. Moreover, there is a report in earlier published study with a rather similar strategy in European people (Yang et al, 2009). This will bring us to explore more details in Asian population.…”

C13orf18 and C1orf166 (MULAN) DNA Genes Methylation are Not Associated with Cervical Cancer and Precancerous Lesions of Human Papillomavirus Genotypes in Iranian Women

“…® , Germany), according to manufacturer's protocol. HeLa S3 (cervical cancer cell line) and health women pooled plasma are used amplifications were performed in a 20µl reaction volume containing 12.5µl 2X Premix Ex Taq (Probe qPCR) (TaKaRa, Japan), 10 pmol/µl each primers and probes (C13orf18: FP; 5'-TTTTTAGGGAAGTAAAGCGTCG-3', RP; 5'-ACGTAATACTA AACCCGAACGC-3' and TaqMan Probe; Yakima Yellow 5'-AGATGGAAG AAATT TTGGAGAT GC GCGTT-3' BHQ1) (C1orf166: FP; 5'-TGGCGAGTTAGTTATGGTT AGT G C-3', RV; 5'-TCTCCCCAA CCGAATATCGA-3' and TaqMan Probe; FAM 5'-TTATGGAGCGGTGG GTGGTCGTTTAG-3' BHQ1) (TIB MOLBIOL ® , Germany) (Yang et al, 2009;Eijsink et al, 2011;Eijsink et al, 2012), and 5-6µl DNA extracted. These reaction following conditions: 95ºC; 90s for initial enzyme activation, for 50 cycles 95ºC; 5s,…”

“…HPVs 16(HR), 18(HR), 31(HR), 6(LR), 11(LR), 33(HR) are the most common HPV genotypes in Iranian women with and without cervical dysplasia (WHO 2010;Laudadido 2013;Shayanfar et al, 2013). It is proposed that individual genetic and epigenetic characteristics accompanied with HPV infection in cervical cancer have important role as main risk factors in cancer progression (Szalmás and Kónya., 2009;Wentzensen et al, 2009;Mohiuddin et al, 2013;Wang ., 2014 (Yang et al, 2009;Eijsink et al, 2011;Eijsink et al, 2012;Limpaiboon., 2012;Al-Shabanah et al, 2014;Ma et al, 2014). Hence, we assessed the DNA methylation status of C13orf18 and C1orf166 genes as novel potential candidate diagnostic biomarkers in Iranian female population suffering cervical malignancies.…”