Introduction

Annually, a total of 330,000 new cervical intraepithelial neoplasia (CIN) cases is reported in the European Union, with relatively half of them diagnosed as CIN I (1). A usually long pre-invasive process capable of developing into invasive cervical carcinoma over time; CIN is microscopically considered as a series of events which progress from cellular atypia to various grades of dysplasia (2).

In accordance with new WHO classification, CIN is categorized into Low-Grade Squamous Intraepithelial Lesion (LSIL) and High-Grade Squamous Intraepithelial Lesion (HSIL) terminology (2). However, based on the degree of dysplasia, previous classifications grouped it into CIN I, CIN II, and CIN III (3). As CIN lesions were monitored and treated in different ways, precise histological grading of CIN lesions assumed of great significance with regard to clinical management of patients. For example, due to its regression in about 80% of cases, CIN I was usually regarded as benign and no therapy was indicated (4). Nevertheless, CIN II and CIN III were considered as precursors to invasive carcinomas and therapy (conization or other less invasive procedures) was indicated since 0.2% to 4.0% of cases with CIN II and CIN III can progress to cervical carcinoma within 12 months (4). It is worth noting that there were no specific clinical symptoms to demonstrate the presence of CIN (5).

In fact, diagnosis of these precursor forms can result in inter-observer variability in comparison with its reactive mimics, such as atypical squamous metaplasia (ASM), immature squamous metaplasia (ISM), reactive/reparative atypia (RA), atrophy, reserve cell/basal cell hyperplasia, etc. Overall, this highlights the need for specific biomarkers to contribute to objective CIN grading and differentiation of true high-grade cervix dysplasia from its mimics (6).

Human papillomavirus (HPV) infection of any type was shown to be related to a 498-fold increase in the risk of cervical cancer development. In comparison to females infected with HPV-16, patients infected with HPV-18 had a higher risk of cervical cancer. The high-risk viruses have a crucial role in the carcinogenic process through production of two oncoproteins encoded by the viral E6 and E7 genes. Directly involved in inactivation of p53 and pRb, respectively, these oncoproteins promote progressive cell cycle and DNA synthesis by blocking apoptosis, thus contributing to viral replication (7).

A tumor suppressor protein, P16 is biologically tasked with regulating cell cycle progression at the G1/S boundary (8), thus demonstration the value of P16 would be as a diagnostic marker for cervical dysplasia and cervical carcinoma (9).

Cytokeratin-17 (CK17), which is an efficient marker for detection of cervical stem cells, is expressed in reserve cells and immature metaplastic cells. However, its expression is not reported in cervical glandular epithelial cells, squamous cells in the portio, or mature squamous metaplastic cells (10).

The areas where P63, a member of the P53 gene family, is expressed include the basal and parabasal cells of mature cervical, vaginal and vulvar squamous epithelium, as well as cervical reserve cells at the transformation zone, immature metaplastic cells, and atrophic cervical squamous epithelium. Instead of Ki67, the present study considers P63 as CIN and squamous metaplasia, e.g., basal cell hyperplasia are mostly the result of basal cell, reserve cell, and stem cell hyperplasia (6). 

In order to evaluate the status of HPV infection in cervix, the present study analyzed the most common histological alterations occurring in the cervix, such as squamous metaplasia, CIN 1, CIN 2, CIN 3, as well as expression of the  biomarkers of P16, CK17, and P63.

Patients

Based on the pathological reports available in the laboratory of Afzalipour Hospital and Besat Clinic, 50 patients with ASM and 50 patients with CIN were enrolled in the study. The study protocol was approved by the Ethics Committee of Kerman University of Medical Sciences, Iran.

Immunohistochemical (IHC) Staining

The tissue samples were histopathologically examined by two experts. Formalin-fixed paraffin-embedded tissue was cut in sections with 4μm thickness, which were then placed on slides, dehydrated by alcohol washes, and cleared using a detergent like xylene before being imaged under a microscope. Sections were incubated in 5.0% H₂O₂ to quench endogenous peroxidase activity. Furthermore, antigens were also retrieved by digesting the tissue sections with a proteolytic enzyme like trypsin. To decrease the degree of background staining in IHC, the samples were incubated with a buffer, blocking the non-specific sites to which the primary or secondary antibodies may otherwise bind such as normal serum. The slides were then incubated 30 minutes at room temperature with primary antibodies against the antigens including, P16 (rabbit monoclonal), P63, and CK17. In order to detect a biotinylated secondary antibody, the study adopted streptavidin for biotin. To visualize bonding of antibodies, 3, 3μ- diaminobenzidine tetrahydrochloride (DAB) was applied as chromogen for 5 minutes, and the sections were counterstained in hematoxylin for 2 minutes.

Evaluation of P16, CK17 and P63 Expression

The immunoreactivity of P16 and P63 was shown to be positive since more than 50% and 10% of the tumor cell nuclei indicated a strong intensity, respectively. The P16 was considered as positive when it showed nuclear and continuous cells diffuse cytoplasmic staining in the basal and para-basal squamous epithelium cell layers, which variably reached intermediate and superficial cell layer characteristic of a diffuse staining pattern. Additionally, P16 was shown to be negative when it was completely unstained or demonstrated a focal or sporadic epithelial staining, not characterized by basal and para-basal cells (focal staining pattern). CK17 staining indicated to be positive when cytoplasmic staining involved all squamous cell layers. Furthermore, focal staining or completely unstained cell layers were considered as negative.

Statistical Analysis

The data were analyzed by IBM SPSS Statistics for Windows, version 20 (IBM Corp., Armonk, N.Y., USA) using the statistical methods such as Chi square test and screening test. The statistical significance was considered to be 0.05.