Iranian Society of Gynecology Oncology

Document Type : Original Research Article


1 Department of Obstetrics and Gynecology, School of Medicine, Solid Tumor Research Center, Research Institute on Cellular and Molecular Medicine, Shahid Motahari Hospital, Urmia University of Medical Sciences, Urmia, Iran

2 Department of Pathology, School of Medicine, Shahid Motahari Hospital, Urmia University of Medical Sciences, Urmia, Iran

3 Department of Community Medicine, School of Medicine, Reproductive Health Research Center, Urmia University of Medical Sciences, Urmia, Iran


Background and Objective: Cervical cancer is a common neoplasm in women, and the role of the HPV virus in the development of precancerous and cancerous cells has been established. There exist different strains of the HPV virus with varied functions. In the high-risk HPV strains, the p16 and ki67 proteins play a crucial role in regulating the cell cycle leading to cell proliferation and progression. P16 and ki67 proteins are positive in almost all lesions and indicate a high degree of malignancy. This study aims to investigate the predictive effect of p16 and ki67 on the progression of low-grade intraepithelial lesions to high-grade malignancy.
Methods: P16 and ki67 were measured on CIN1 lesions, and during the average two-year follow-up period, the outcome of positive cases was investigated. A total of 106 referred patients between the age of 15 to 75 years were examined from April 2015 to March 2019.
Results: Among the patients with progression of CIN1 to CIN2 and other severe lesions, p16 was positive in 14 cases (60.9%), and a significant difference between groups with positive and negative markers in the progression or regression of lesions was noticed. Ki67 frequently occurs in CIN2 and other severe lesions.
Conclusion: The use of p16 and ki67 as predictive markers is still under debate. In countries like the United States, these are not yet used separately for prediction but are being used in combination together. The authors of this study strongly recommend the conduction of further studies to assess the role of p16 in association with other markers and within a larger population so as to apply the functional role of p16 and ki67 in the clinical setting thereby effectively preventing it.


Main Subjects


Cervical cancer is a prevalent issue among adult women. Many industrialized countries have achieved significant success in reducing invasive cervical cancer over the past six decades, with an annual prevalence of 4 per 100000 to 14 per 100000 cases. In developed countries, approximately 15000 new cases of cervical cancer and 5000 yearly deaths occur (1). Cervical cancer, a neoplasm caused by the HPV virus, originates from premalignant lesions inside the epithelium and is referred to as intra-epithelial lesions of the squamous cell or cervical intraepithelial neoplasm (CIN 1-LSIL) (CIN 2-3 -HSIL) that are the precursors of cervical cancer (2).
Nearly 80% of low-grade SIL lesions (LSIL-CIN1) regress spontaneously within one to two years. HSIL-CIN2-3 management generally involves biopsy and removal of the lesion and the transformed zone to prevent the lesion from progressing. While LSIL and CIN1 management is supportive and necessitates follow-ups, 10% to 15% of LSIL / CIN1 lesions progress to HSIL, CIN2-3 (3, 4). Unfortunately, the available clinical, cytological, and histological meth-ods cannot determine the eventual transformation of the LSIL, CIN1 lesions to HSIL, CIN2-3. HPV has an etiological role in cervical cancer and is found in almost all premalignant and malignant lesions of cervical cancer (5).
Different types of HPV are broadly classified into two categories: high-risk and low-risk (6). High-risk HPV sequence accumulation in the cell genome plays an essential role in the progression of cervical neopl-asm lesions with E2 gene disruption (7). It results in excessive expression of E6 and E7 oncogenes, which impairs P53 and RB suppressor activity (8) and eventually destroys cellular control. High-risk HPV in CIN1 lesions is important and common, but only 10% of CIN1 lesions progress to CIN2-3. P16, a tumor suppressor protein, inhibits CPK activation and regulates cell cycle and proliferation by PRB phosph-orylation. Immunohistochemical expression of p16 may attenuate PRB activity in negative feedback.
The diffused and strong pattern of p16 expression is observed in CIN2-3 but cannot be seen in CIN1. P16 has varied expressions, and its overexpression leads to interference of viral oncoproteins in the cell cycle and ends up in cell cycle destruction. Tumor suppressor protein p16 demonstrates the substitute marker role in high-risk HPV lesions. P16 is valuable in the diagnostic process because it is distinguished nearly in all HSL and CIN2-3 lesions despite focal or negative staining in imitative reactions such as immature metaplasia or atrophy. So, P16 staining has a key role in the classification of progression of lesions to HSL, CIN2-3, or other severe lesions.
Studies carried out in recent decades concerning this issue suggest that LSIL, CIN1 lesions with high p16 expression progress to HSIL and CIN2-3; owing to the insufficiency of research articles pertaining to this issue, authentic validity, and value of p16 as a progressive marker in women remains unknown (9-11). On the other hand, ki67 usually is found in all active phases of the cell cycle but not in resting cells (12).
During HPV infection, the transition control between mitosis phase c1 and phase S1 is destroyed by the retinoblastoma protein; following this, retinoblastoma itself decomposes, resulting in excessive expression of HPV infection and, subsequently, over-expression of ki67 (13). Immunohistochemical analysis of ki67 in histological biopsy of CIN1-2 shows that it is an independent and strong predictor of CIN lesions (14).
Co-expression of p16 and ki67 in the cell occurs only under abnormal conditions (15). Since cervical cancer is one of the most important causes of death in women in developing countries, determining the progression of intraepithelial lesions in these patients is of primary concern. Therefore, the purpose of this study is to investigate the predictive effect of p16 and ki67 proteins in the progression of low-grade intraepithelial lesions of the cervix. The present study was conducted at Shahid Motahari Hospital, Urmia, Iran, from 2015 to 2019.


Materials and Methods

This descriptive cross-sectional study consisted of outpatients in the age bracket of 15 to 70 years, who were examined and biopsied for CIN1, and were referred to the gynecology oncology clinic of Shahid Motahari hospital from April 2015 to March 2018. A selection of 115 patients with CIN1 diagnosis was done, and their cervical biopsy samples were reviewed by a common pathologist. Out of these 115 patients, nine were excluded, among which six cases had distorted slides upon review, two due to hysterectomy with CIN1 pathology, one because of history of breast cancer, the other because of a history of the disease and the last due to pregnancy.
A total of 106 patients with a definitive diagnosis of CIN1 were selected, and p16 and ki67 dual-staining was done (DBS-USA). Cytological slides were examined by a common pathologist and reported according to the BETHESDA system. The results of the HPV high-risk test were confirmed from the patients' files, and the patients subsequently underwent histological examination as well as P16 and ki67 immunohistochemistry tests under the following steps: four-micron thickness tissue incisions were made and inserted into 1/50 diluted histogrip solution for one minute, kept at 60 to 80 C for one hour, and then paraffin-disinfected with xylol.
Then the alcohol dehydration was done from one to seventy percent. The ANTIGEN RETRIEVAL stage was performed by EDTA solution. The slides were placed in the EDTA buffer, heated to 90 C for two minutes, and finally rinsed with TBS (Tris Buffered Saline). In the chromogen complex stage, the antibody-antigen adherence and staining were performed. Diagnosis of CIN1 was made by staining with H&E. The identification criteria are based on the Female Reproductive System Tumor Pathology and Genetic classification criteria (WHO 2014). For each slide, a control slide with squamous cell cancer (SCC) detection and a negative control slide (normal cervical epithelial cells) were kept. According to the LAST project:

  1. Positive finding: It is defined as nuclear or cytoplasmic staining, and in the case of positive p16, basal and parabasal cells are stained either with or without upper cell layers.
  2. Negative finding: It is defined as resistance to staining or trace staining with a focal dye in a few cells.

In addition to the basal layer, the degree of positivity is divided into three groups: one to ten percent (+), ten to thirty percent (++), and more than thirty percent (+++).
Follow-up was done at an interval of at least 6 months and a maximum of 4 years after diagnosis. Patients underwent colposcopy, cervical biopsy, and hrHPV testing at each follow-up, with all slides being examined by the previous pathologist. Progression of lesions was defined as a histologic diagnosis of CIN2 or more severe lesions. Regression was defined as negative hrHPV testing and grade less than that of CIN1, and persistent was determined as CIN1.

Ethical Issues

The present research strictly observed the principles of the Helsinki Declaration of 1975, as revised in 1983. The study was approved by the Forensic Medicine Ethics Committee, Bushehr (Ir.umsu.rec.1398.313), and is extracted from Samira Jahangards' residency research thesis (#9648).

Statistical Analysis

After data collection, patients were divided into two groups based on the positive and negative p16/ki67 and age, following which comparisons were drawn in each group concerning their frequency of outcomes. Descr-iptive statistical methods, including frequency-perce-ntage and average +/- standard deviation, have been used for statistical analysis. Chi-Square test (and if necessary, Fisher's exact test) was employed to compare frequency. SPSS 20 (SPSS Inc., Chicago, Ill., USA) was also brought to use, and a statistical significance was observed when P-value<0.05.


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