Survey of p 16 INK 4 a immunohistochemistry in diagnosis of dysplastic changes in cervix

© 2017 The Authors; Tabriz University of Medical Sciences This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Survey of p16INK4a immunohistochemistry in diagnosis of dysplastic changes in cervix

Cervical cancer ranks as the fourth most common malignancy of females worldwide, affecting 527,624 women every year and 265,672 annual deaths from the disease.Underdeveloped countries account for 84% of cervical cancer cases.Almost all cervical cancers are resulted from infection with human papillomavirus (HPV), which is the most common sexually transmitted infection (STD) all around the globe. 1 HPV is reported to be present in 99.7% of cervical cancers in the United States. 2 The incidence of cervical cancer can be reduced in two ways: preventing the pre-cancerous lesions in the first place, and detecting them before they turn into cancer. 1ervical intraepithelial neoplasia (CIN) is defined as intraepithelial squamous abnormalities which exhibit nuclear atypia in epithelial layers and have the potential of becoming invasive carcinoma if not diagnosed and removed well.CIN is subdivided into categories of high-and lowrisk of cancer, which is based on the association between CIN III and subsequent invasive carcinoma.However, in practice, a small proportion may become invasive carcinoma.The trend among pathologists is to classify CIN I, as a process either identical to or closely resembling low-grade, and CIN II and CIN III as lesions within the spectrum of high-grade CIN. 3 Cytological screening of cervical abnormalities has been shown to reduce mortality and morbidity of cervical cancer, yet cytology relies on some diagnostic parameters with variable sensitivity and specificity. 4,5In order to improve the detection of abnormalities on Pap smears and histopathology, additional biomarkers may need to be employed for better identifying the high-grade abnormalities.Detection of high-risk HPV DNA has also been reported to be highly sensitive, yet less specific than the cytological and histopathological examination. 1,5Women with abnormal screening test results for cervical cancer are commonly referred to colposcopy after some triage test. 6yclin dependent kinase (CDK) inhibitor 2A also known as p16 INK4a limits cell cycle through negative feedback control to check the cell proliferation by regulation of CDK4 and CDK6.Thus, in the cervical neoplasia and dysplasia, the overexpression of p16 INK4a happens. 7,8

Hematoxylin and eosin staining (H&E):
The paraffin-embedded tissues were subjected to H&E in order to confirm the histopathological findings.Tissue sections were prepared with 3 µm thickness using microtome device and the staining procedure was performed as described before. 9 IHC: IHC for p16 INK4a was performed on all of the samples (100 samples).The procedure of IHC has performed according to the manuals of antibody detection systems.Tissue sections with 4 µm thickness were prepared on the positive charged adhesive slides (Leica Biosystems, USA) and incubated overnight at 37 °C for the tissues to be adhered on the slide.The tissue sections underwent deparaffinization with xylene and hydration with ethanol 100%, 96% and 7%.The antigen retrieval process was done using antigen retrieval solution citrate buffer (AR Citra Plus, pH:6.2,BioGenex ® , USA) in 95 °C for 25 minute, cooled in room temperature (RT) and endogenous peroxidase was blocked using 30% H2O2 for 10 min.Anti-p16 INK4a (BioGenex ® , USA) was added to the slides as the first antibody and incubated for 20 min at RT, washed twice in IHC wash buffer (Trisbuffered saline Tween-20) each 7 min and once in phosphate buffered saline (PBS) for 5 min.For visualization, the slides were incubated with anti-mouse/rabbit EnVision ® Duo FLEX double stain system, horseradish peroxidase (HRP)/3,3'diaminobenzidine (DAB) (Dako, Denmark) for 30 min.The washing procedure was repeated again as the previous washing step.DAB was diluted (50 µl in 1000 µl) and added to the slides and incubated for 7 min for enzymatic reaction to develop.The slides were washed in distilled water, dehydrated and mounted.IHC staining for p16 INK4a was interpreted as positive when a strong and diffuse nuclear and/or cytoplasmic staining in cells was observable with the cytomorphologic features of CIN (Figure 1).Among the studied women, 13 out of 30 (43.0%) low-grade and 35 of 39 (89.7%) high-grade CIN lesions were positive for p16 INK4A .Considering the high-grade CIN lesions, 12 out of 16 (75.0%)and 23 out of 23 (100) CIN II and CIN III were positive for p16 INK4A , respectively (Table 1).None of the normal samples were positive for p16 INK4A .The difference between the expression of p16 INK4A among different grades of CIN lesions were statistically significant (P = 0.001) (Table 2).
Sensitivity of p16INK4A IHC for detection of CIN I, CIN II, CIN III was calculated as 63%, 80% and 100%, respectively.The overall sensitivity of the biomarker for detection of CIN lesions was 76.6% and the specificity was 100% for all CIN grades (Table 2).The sensitivity and specificity of p16INK4A for distinguishing the high-grade lesions from low-grade ones was 94.64% and 69.76%, respectively (Table 1).
Using one-sample Kolmogorov-Smirnov test showed that the data for the women's age was not normally distributed (P = 0.037).Thus the non-parametric Mann-Whitney test was used for estimating the P-value for mean ages among positive and negative P16 INK4a IHC.The results showed a significant difference (P = 0.003) which is available in table 3.  Cervical cancer is the third most common cancer among women and the second most frequent cause of mortality from malignancies of genital organs in women, which can be prevented by diagnosis of preneoplastic changes in cervix. 10The present study aimed to evaluate the diagnostic value of p16 INK4a biomarker in different grades of CIN using IHC method.Results of the preset study showed that 13 out of 30 (43.0%) low-grade and 35 out of 39 (89.7%) high-grade CIN lesions were positive for p16 INK4A .Sensitivity of p16 INK4A for detection of CIN I, CIN II, CIN III was calculated as 63%, 80% and 100%, respectively.The overall sensitivity of the biomarker for detection of CIN lesions was 76.6% and the specificity was 100% for all CIN grades.The sensitivity and specificity of p16 INK4A for distinguishing the high-grade lesions from low-grade ones was 94.64% and 69.76%, respectively (Table 1).
We found that p16 INK4a IHC is a great tool for detection of high-grade CIN lesions (CIN III), yet it is not absolutely sensitive for diagnosis of low-grade ones (CIN I, CIN II).Moreover, the specificity of p16 INK4A IHC was 100%, which means there were no false positives in all grades of CIN lesions.The difference between p16 INK4A positivity among different CIN lesions was statistically significant, which shows that the detection of the biomarker is far different among different lesions (CIN I < CIN II < CIN II).p16 INK4a IHC can be recommended for the diagnosis of high-grade lesions; however, it is not recommended to be the only diagnostic method for low-grade CIN lesions.Yet, it can be used as a helpful diagnostic assistant beside histopathological findings.
There are several studies that evaluated p16 INK4a as a diagnostic marker for cervical abnormalities and some are mentioned below.Tabrizi et al. 5 11 Similar to the findings of the present study, all of the normal myometrium were negative for p16 INK4a .According to their report, 4 out of 62 (6.5%) leiomyoma and 12 out of 12 (100%) leiomyosarcoma (LMS) samples were positive for p16 INK4a .They concluded that p16 INK4a is a good biomarker for differentiation of normal from tumoral tissue.In another study, Kava et al. evaluated p16 INK4a IHC for detection of cervical intraepithelial neoplasm.Based on their report, p16 INK4a was positive in 8 out of 15 (53.3%) samples of cervical intraepithelial neoplasms. 12n 2017, da Costa et al. 13 discussed p16 INK4a , Ki-67 and cytokeratin 7 (CK7) as potential markers for low-grade CIN progression.The highest frequency of positivity of each marker was associated with progression to high-grade squamous intraepithelial lesion (HSIL).The percentage of positivity of each marker was reported higher in the low-grade squamous intraepithelial lesion (LSIL) group who showed progression to HSIL (p16 INK4a 45.4%, Ki-67 54.5%, and CK7 63.7%), in comparison with women who kept the primary LSIL diagnosis (32%, 40%, and 56%, respectively) and those in whom the lesion regressed (15.8%, 42.1%, and 42.1%, respectively).They concluded that p16 INK4a , CK7, and Ki-67 may be useful biomarkers to identify LSIL lesions that require special attention. 1316 INK4a is reported as a potential diagnostic complementary for prediction of high-grade cervical lesions in cytology (liquid-based) with HPV testing and histopathological correlation.Wong et al. 14 reported that, 36 out of 57 (63.2%) liquid-based cervical cytology samples showed immunoreactivity for p16 INK4a and 43 out of 57 (75.4%) were infected by high-risk HPV.For prediction of CIN grade II and higher, p16 INK4a showed a sensitivity and specificity of 93.5% and 60.0%, respectively.They concluded that p16 INK4a is useful for prediction of severity of cytological abnormalities.However, they mentioned that p16 INK4a is more specific, yet less sensitive than high-risk HPV in detecting high-grade cervical lesions.Furthermore, combination of tests, high-risk HPV and p16 INK4a , failed to show significant improvement in diagnostic values, such as sensitivity, specificity and predictive value. 14In the present study, the sensitivity and specificity of p16 INK4a IHC for detection of CIN II was 80% and 100%, respectively, and for CIN III, both were 100%, which was higher that the report form Wong et al. 14 The p16 INK4A biomarker is a suitable diagnostic tool for high-grade CIN lesions, yet for low-grade ones it has lower sensitivity.p16 INK4A can be a helpful tool beside histopathology for diagnosis of CIN lesions.

Figure 1 .
Figure 1.Positive immunohistochemistry (IHC) staining of P16 INK4a basal and parabasal cells in cervix epithelium with cervical intraepithelial neoplasia (CIN) II lesion (100X magnification) The authors would like to acknowledge the Vice Chancellor of Research and Technology, Tabriz University of Medical Sciences.Heidar Ali Esmaieli designed the study, analyzed the data and prepared the manuscript.Siamak Berenjian carried out the laboratory work and cooperated in manuscript preparation.We have received financial support from Deputy of Research and Technology of Tabriz University of Medical Sciences.Authors have no conflict of interest.This study was approved by the Medical Ethics Committee of Tabriz University of Medical Sciences.

Table 1 .
p16 INK4a immunohistochemistry (IHC) among low-grade and high-grade CIN lesions and its sensitivity and specificity to distinguish the high-grade lesions from low-grade ones

Table 3 .
Estimated P-value for mean ages among positive and negative p16 INK4a immunohistochemistry (IHC)