The Expession Of P16, Ki-67 In Precancerous Lesion In Uterine Cervix And Their Practical Value

BackgroundCervical cancer is one of the leading causes of mortality in female malignant tumors. The International Agency for Research concluded that there is a strong association between Human papillomavirus (HPV) and cervical cancer development, and the risk of developing cancer depends on the HPV high risk type infection. However, not all women who have HPV infection develop cervical cancer, suggesting that HPV alone is not sufficient for cervical carcinogenesis. Researchers are currently focused on other important cell cycle factors such as loss of growth suppression, increased cell growth rates, and angiogenesis. Two of the cell cycle genes which may be involved include Ki-67 and P16.The early detection of precursors of invasive cervical cancerrep- resents an important advance in many developed countries to reduce significantly the morbidity and mortality of this kind of cancer. For this reason, it is basic to improve the histopathologic interpretation of cervical biopsies, which still shows considerable interobserver variations despite well-defined criteria. Ki-67 immunoquantitative features have been correlated with the grade of dysplasia in cervical epithelium, while P16 accumulation has been related to the presence of high grade intraepithelial lesions (HSIL) and detection of human papillomavirus (HPV). Being immunohist- ochemistry techniques widely available and feasible, the aim of this study was to assess the value of these markers in assisting CIN grading and HPV infection, which has been wellestablished as a necessary cause of cervical cancer.PATIENTS AND TISSUESA total of 60 formalin-fixed, paraffin-embedded samples, collect- ed from 60 women, were reviewed by two pathologists in order to obtain the consensus histologic diagnoses. Our series included 12 normal cases, 12 CIN I, 12 CIN II, 12 CIN III and 12 invasive cervical squam- ouscell carcinomas in our hospital from 10.2007 to 12.2008. The specimens had been collected from 60 women ranging in age from 24 to 58 years (mean 39 yrs). METHODSImmunohistochemical analysis was performed by ABC Immunoperoxidase staining, using mouse monoclonal antibodies Ki-67 and P16. Briefly, 4μm thick sections were treated with 0.1M citrate buffer, pH:6.0, for antigen retrieval, followed by endogenous peroxidase blocking, incubation with horse serum and incubation with unlabeled primary antibodies. Specifically bound antibodies are then visualized by incubation with a biotinylated secondary horse anti-mouse antibody (Vector Laboratories Inc., Burlingame, CA) (1:100 dilution) and a preformed avidin-biotin complex (Vectastain ABC PK4000 ST, Vector Laboratories) was applied at 1:100. Finally, diaminobenzidine (0.05 %) was used as the final chromogen and Harris- modified hematoxylin as counterstain. Positive controls that were cases known to have positive staining and negative controls in which the primary antibody was omitted were included in each experiment. Results were reviewed by two pathologists. Ki-67 was classified in four categories according to the extension of nuclear staining within the epithelial thickness. Based on previous reports, Ki-67 staining was defined as positive when a cluster of at least two strongly stained epithelial nuclei were present. Results were also reported in a semiquantitative fashion as when staining of cells was found in the upper 1/3 of the epithelium (3+), in the middle 1/3-2/3 (2+) or in the lower 1/3 (1+) (figure 1). Ki-67 0 category defined negative cases in which only staining of parabassal cells of the squamous epithelium was observed and served as internal control. Nuclear or nuclear and cytoplasmic diffuse strong staining was considered positive for P16, focal staining or absence of staining was considered negative. HPV detection and typing was performed using a commercial PCRbased assay (Clinical arrays HPV, Genomica).Statistical analysesFisher exact tests and exact confidence intervals for odd-ratios were used to assess statistical differences in P16 and Ki-67 detection amongst different grades of lesions, as well as HPV -DNA detection and the prevalence of high risk viruses. Predictive values less than 0.05 were considered statistically significant. All these statistical compu- tations were performed using the package. Kendall's Tauc was performed using Spss 11.5.RESULTSThe distribution of the expression of Ki-67 was the following: 3+category was observed in 55 cases which included 12 invasive cervi- cal squamous-cell carcinomas, 8 CIN III, 2 CIN II and 1 CIN I; 2+ category involved 14 cases corresponding to 3 CIN III, 8 CIN II, 2 CIN I and 1 normal case; 1+ was observed in 17 cases which were 2 CIN II, 298 CIN I, 5 HPV-suspicious and 1 normal case. Kendall's Tau-c showed a highly significant association (P<0.0001) between increasing grade of CIN and Ki-67 expression, two ordinal-level variables. Further Chi-squared analysis of the 2 by 2 contingency table proved Ki-67 especially useful in discriminating the presence of CIN (P=1.004e-09). Fifty out of 60 cases showed positive P16 staining, as defined above. In relation to the histologic diagnoses the positive results were found in 11 invasive cervical squamous-cell carcinomas, 8 CIN III, 6 CIN II, 1 CIN I. No P16 staining was observed in normal or HPV suspicious cases. A significant relation (P = 2.2 e-16) was found between P16 immunoexpression and the presence of HSIL lesions, with 75 % specificity, 85 % positive predictive value and 90 % negative pred- ictive value.DISCUSIONClassification of cervical dysplasia is based upon histologic features, with the primary goal of identifying and grading squamous epithelial lesions at increased risk for progression to invasive carcinoma. Criteria for the histologic diagnosis of these lesions are well established. The non-invasive lesions are microscopically evaluated taking into account the epithelial level involved and the nuclear cytologic abnormality in the cervical squamous epithelium. However, in the routinary cervical biopsies assessment may be especially difficult the distinction between lowgrade (CIN I) and benign no HPV related lesions. In addition may be also difficult in some cases of intermediate dysplastic pattern to decide their classif- ication as low or high grade lesions.As in previous studies, in our experience the pattern of Ki-67 staining in squamous intraepithelial lesions (SIL) differs signific- antly from that of normal cervical epithelium, in which positive cells are confined to the parabassal cell layer. In cervical SILs the presence of Ki-67 positive cells is increased from the parabassal areas into the intermediate and superficial layers conferring to this marker a potential value in the distinction between low-grade CIN and normal, metaplastic and reactive cervical epithelia. This observation was confirmed in the present study with the use of Kendall's Tau-c as statistical tool which reveal how samples with increasing grade of CIN had a tendency to contain Ki-67 positive cells in gradually more upper layers of their epithelium (P < 0.0001). It is well establi- shed that persistent infection with high-risk HPV types is associated with the subsequent development of high-grade dysplasia and invasive cervical carcinoma. Nevertheless, since many infections are transi- tory and a substantial proportion of low-grade dysplasias are also associated with infection by high-risk HPV types, another tool becomes necessary to help in the assessment of high grade lesions. Viral DNA integration characteristic of high-risk HPV types, and closely related to malignant transformation, results in increased expression of the E6 and E7 oncogenes. The E7 binds to and inactivates the host cell's retinoblastoma protein, which leads to an overproduction of P16. As confirmed in the present study, P16 diffuse strong staining is a useful predictor of the presence of high grade dysplasia and high-risk HPV infection. In fact it is remarkable that 40 out of the 60 cases showing P16 positivity were high grade lesions, and 51 also presented high-risk HPV infection.CONCLUSIONS The combined use of these two markers, Ki-67 and P16, provides a valuable tool to asses the grade of CIN, to predict the presence of high-risk HPV infection and to verify the diagnosis of equivocal cases.