title: Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia.
abstract: The cell cycle inhibitor, p16INK4a may be a useful surrogate biomarker of cervical intraepithelial neoplasia (CIN); however, there is currently no consensus of p16INK4a genetic alterations throughout the multiple step process of CIN. Our goal was to identify the methylation frequency of p16INK4a in each step of CIN that is associated with human papillomavirus (HPV) infection, using several different detection methods of p16INK4a methylation to correlate the data. The present study included a total of 43 patients, including 38 with CIN, and 5 normal patients. Three different methods were used to detect hypermethylation of CpG islands, methylation-specific PCR (MSP) amplification of different primer sets of M1, M2, and M3, pyrosequencing of each forward primer region, and immunohistochemistry of p16INK4a. Analysis of MSP showed that 20 of the 38 CIN patients (52.6%) revealed hypermethylation in at least 1 primer set of the p16INK4a promoter. A complete loss of p16INK4a protein expression was observed in 11 cases (28.9%). There was no observed association of methylation of the p16INK4a gene with either CIN grading (P=0.0698) or HPV status (P=0.2811): specifically 42.9% (3/7) was found in CIN 1, 57.1% (8/14) in CIN 2, and 52.9% (9/17) in CIN 3. In concordance with immunohistochemistry results, hypermethylation of the p16INK4a promoter was significantly correlated with a lack of p16 protein expression (P=0.0145). All positive peaks from pyrosequencing matched the MSP results, which ranged from 6.3% to 24.5%. In conclusion, p16INK4a gene silencing during CIN was not determined to be a particularly rare event; however, it does not correlate with either HPV status or CIN grading.
authors.affiliation: Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Suki
authors.initials: S
authors.lastname: Kang
authors.forename: Jungsik
authors.initials: J
authors.lastname: Kim
authors.forename: Hong Bae
authors.initials: HB
authors.lastname: Kim
authors.forename: Jung Won
authors.initials: JW
authors.lastname: Shim
authors.forename: Eunji
authors.initials: E
authors.lastname: Nam
authors.forename: Sung Hoon
authors.initials: SH
authors.lastname: Kim
authors.forename: Hee Jung
authors.initials: HJ
authors.lastname: Ahn
authors.forename: Yoon Pyo
authors.initials: YP
authors.lastname: Choi
authors.forename: Boxiao
authors.initials: B
authors.lastname: Ding
authors.forename: Kijun
authors.initials: K
authors.lastname: Song
authors.forename: Nam Hoon
authors.initials: NH
authors.lastname: Cho
mesh_terms: D001483:Base Sequence; D016022:Case-Control Studies; D018899:CpG Islands; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D020868:Gene Silencing; D019942:Genes, p16; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D027383:Papillomaviridae / Q000145:classification / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology / Q000821:virology
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins