Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions

Abstract

The p16 protein (p16) is a cyclin-dependent kinase (CDK) inhibitor that decelerates the cell cycle by inactivating the CDKs that phosphorylate retinoblastoma (Rb) protein. Recent biological studies have revealed that p16 expression is markedly influenced by the status of Rb expression, and p16 overexpression has been demonstrated in cervical cancers because of functional inactivation of Rb by human papillomavirus (HPV) E7 protein. To clarify the relationship between p16 overexpression and HPV infection in cervical carcinogenesis, immunohistochemical analysis of p16 and detection of HPV by in situ hybridization and polymerase chain reaction were performed on 139 formalin-fixed and paraffin-embedded samples of cervical and genital condylomatous and neoplastic lesions. Marked overexpression of p16 protein, ie, diffuse and strong immunostaining, was observed in all cervical cancers and preneoplastic lesions with infection by high- and intermediate-risk HPVs, ie, subtypes 16, 18, 31, 33, 52, and 58. Condylomata acuminata and low-grade squamous intraepithelial lesions with infection by low-risk HPV such as HPV-6/11 showed focal and weak immunohistochemical staining for p16. Our results clearly showed that the mode of p16 expression in lesions with high- and intermediate-risk HPVs differed from its expression in lesions with low-risk HPVs and thus might be attributable to differences in functional inactivation of Rb protein by different HPVs.

Figure 1.

Immunohistochemical staining using anti-p16 antibody JC8 in cell lines. A and B: Diffuse and strong staining was observed in SiHa (A) and LAN1 cells (B) with intact p16 expression. C: LAN2 cells with inactivated p16 gene by hypermethylation showed no positive staining. D: NB69 cells that had lacked p16 expression by Western and Northern blotting showed almost no expression, but a few positive cells were observed.

Figure 2.

ISH for HPV (A, C, and E) and immunohistostaining for p16 (B, D, and F). A and B, C and D, and E and F are in the same areas of each lesion. A and B: HPV-16-positive cervical cancer case. A: ISH pattern showing the dot-type signals. B: Clear and distinct positive staining for p16 was seen throughout the nuclei and cytoplasm of the tumor cells. No positive staining was seen in the stroma. C and D: HPV-16-positive HSIL case. C: ISH pattern showing a mixture of dotted and diffuse signals. D: Intense and diffuse p16 reactivity in both nuclei and cytoplasm was confined to the dysplastic lesion. E and F: HPV-6-positive condyloma acuminatum case. E: ISH pattern showing a mixture of many diffuse signals and a few dots. F: Positive stained tumor cells were heterogeneously scattered among the many negative cells within the lesion.

Figure 3.

HSIL case double positive for HPV-16 and -6 in different areas within the same section. A: ISH for HPV-16. B: ISH for HPV-6. C and D: Immunostaining for p16 in the same lesion. Diffuse and intense staining corresponding to the HPV-16-positive lesion was seen (left side of the lesion), whereas the HPV-6-positive lesion was negative for p16 expression (right side; C). D: Higher magnification of the border between the positive and negative staining.