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Cervix uteri, Conization, Cervical intraepithelial neoplasia, Immunohistochemistry, What&,rsquo s Known The result of cervical conization could be negative due to the small size of the cervical lesion removed in the initial biopsy, spontaneous regression, the absence of transformation zone or denudation epithelium, and misdiagnosis of the initial cervical punch biopsy. There is a low diagnostic agreement on cervical punch biopsies among pathologists. What&,rsquo s New Conization biopsy can be associated with high inter-observer variability among the pathologists. Negative conization result following a positive punch biopsy could be due to misdiagnosis of the initial punch biopsy. The use of Ki-67 and p16 markers is recommended in punch biopsy to prevent unnecessary cone biopsy. IntroductionAs the fourth most prevalent type of cancer among women and the third deadly cancer worldwide among all other types of cancer, cervical cancer is a major global health concern, putting a huge financial burden on the affected patients. More than 85% of cervical cancer cases are reported in developing countries. In Iran, its prevalence has been on the rise during the last decades. 1, It is believed that early detection of cervical intraepithelial neoplasia (CIN) is critical in reducing the likelihood of developing cervical cancer. CIN is the precancerous epithelial transformation of invasive squamous cell carcinoma of the cervix. The role of human papillomavirus (HPV), a DNA virus, in causing cervical cancer and precancerous lesions is well recognized especially the high-risk types. The E6 and E7 viral oncoproteins can bind to host cell regulatory proteins and inactivate tumor suppressor P53 and Rb genes, respectively, leading to cell proliferation and increase of cell mutation. An immunohistochemical (IHC) study can reveal cellular dysregulation of antibodies such as Ki-67 and p16. 2,- 4, Cell proliferation in the G1-S phase is regulated by p16, which negatively impacts cell proliferation. There is a reciprocal relationship between p16 and pRb, which is a tumor suppressor protein. Inactivity of pRb results in the overexpression of p16 which, commonly presents in HPV infection. 3, Ki-67 is a nuclear protein expressed in the active phases of the cell cycle (G1, S, G2, and M phases), and its overexpression causes high cellular proliferation, commonly observed in HPV infection. 3, Therefore, examining the expression of biomarkers such as p16 and Ki-67 in pathological biopsies has been suggested as a method with high sensitivity to improve diagnostic accuracy. 4, Cervical conization is a widely used and efficient intervention in the diagnosis and control of precancerous cervical lesions. Its main advantages are low blood loss, shorter operating time, low cost, and high success rate. 5, The results of cervical conization could be negative despite the diagnosis of precancerous lesions on punch biopsy. The present study aimed to evaluate diagnostic accuracy and agreement between pathologists by adjunctive Ki-67 and p16 IHC staining. Materials and MethodsStudy DesignIn the current retrospective study, 118 pathology slides of formalin-fixed, paraffin-embedded specimens of cervical punch and cone biopsies were re-evaluated. The specimens were obtained from the pathology archives (dated 2007-2016) of Motahari Clinic and Shahid Faghihi Hospital, both affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. The protocol of the study was approved by the Ethics Committee of Shiraz University of Medical Sciences (number, IR.SUMS.MED.REC.1395.S181). All punch biopsies had been performed under colposcopic guidance, and all conization samples were taken via the loop electrosurgical excision procedure (LEEP) method. Out of the 118 specimens, 30 were excluded due to the missing data, unavailable slides, or tissue blocks. The remaining 88 punch and cone biopsy slides were evaluated. These slides were re-examined by two pathologists, unaware of the primary diagnosis, and the discrepancy between the initial diagnosis and re-evaluation was confirmed. Agreement between the initial diagnoses and re-evaluations as well as between the two pathologists was assessed with the kappa coefficient. The initial diagnosis with hematoxylin and eosin (H&,amp E) staining indicated 22 patients with negative cone biopsy results following a positive punch biopsy. Cone biopsies of the 22 specimens initially diagnosed as negative for dysplasia were completely sectioned, reviewed, and suspicious areas were stained with Ki-67 and p16. The absence of intraepithelial squamous, glandular neoplasia, or invasive disease in the cone biopsy specimens was defined as negative. The patients&,rsquo age, the interval between punch and cone biopsy, the presence of transformation zone (TZ) in the cone biopsy samples, the method of conization, and denudation of squamous epithelium were recorded. Moreover, the corresponding punch biopsies were stained with Ki-67 and p16. The agreement between the two pathologists on the IHC study was assessed with the kappa coefficient.Immunohistochemical StainingIHC staining for Ki-67 and p16 antigens was performed on 5-&,micro m sections of formalin-fixed, paraffin-embedded blocks using the avidin-biotin-peroxidase complex method. Unstained tissue sections were coated on Poly-L-lysin slides (Samaatashkhis, Iran) for IHC staining, deparaffinized with xylene (Merck, Germany) for 30 minutes, and gradually rehydrated with ethanol (100%&,rarr 96%&,rarr 70%, every 20 seconds). They were placed in distilled water for two minutes, followed by a mixture of distilled water and H2O2, and washed in phosphate-buffered saline (PBS Samatashkhis, Iran), for five minutes. All slides were boiled in Tris buffer (PH=9.0 Samatashkhis, Iran) for 50 minutes, then cooled and put in PBS for five minutes. The slides were blocked with 10% goat serum and incubated for 20 minutes at room temperature, and then incubated in a humidified chamber for one hour. The following antibodies were used, Monoclonal rabbit anti-Ki-67 antigen, clone SP6 (Dako, code, N1633, USA ready to use), and mouse monoclonal anti p16INK4a (Biogenex, clone G175-405, USA diluted in PBS).The slides were washed in PBS for 20 minutes and incubated in a wet chamber. Then, one drop of HRP polymer was applied to the sections for 30 minutes at room temperature and washed in PBS buffer for 10 minutes. 3.3&,rsquo Diaminobenzidine (DAB) chromogen (Dako, code K3468, USA) was added, and then the slides were washed in PBS for five minutes. The slides were counterstained with hematoxylin (Dako, code, CS70030-2, USA ready-to-use), rinsed under running water for some minutes, dehydrated in graded ethanol solutions, cleared with xylene, and mounted.Immunohistochemical ScoringTo identify the precise location of the lesions, the IHC stained sections were examined alongside the H&,amp E stained slides. Ki-67 (MIB1) staining was classified as positive when a cluster of at least two strongly stained epithelial nuclei was present in the upper two-thirds of the epithelial thickness anywhere within the lesion. Parabasal cells staining was used as an internal positive control. The p16 was classified as positive when nuclear and continuous diffuse cytoplasmic staining of the cells appeared in the basal and parabasal cell layers of the squamous epithelium and reached an intermediate and superficial cell layer mostly recognized by diffuse staining pattern. It was considered negative when completely unstained or revealing focal or sporadic epithelial staining, especially not of the basal and parabasal cells. The distribution of immunoreactive cells was used as a basis to evaluate the scoring of IHC results. CIN III slides were employed as positive controls. To avoid subjective interpretation, it was decided that staining intensity not be graded. Statistical AnalysisData were analyzed using SPSS software (version 21.0). Quantitative and categorical variables were presented as mean&,plusmn SD and frequency (percentage), respectively. The inter-observer reliability was assessed using the kappa coefficient. P&,lt 0.05 were considered as statistically significant.ResultsThe mean age of the patients was 39&,plusmn 10.2 years (25-78 years). The mean interval between punch and cone biopsy was 8.5 weeks. The results of the initial diagnosis of cone and punch biopsies on H&,amp E slides are shown in table 1,. The overall agreement between the punch diagnoses by the original pathologists and those in the present study (the first and second pathologists) before IHC was 0.33 (SE, 0.051, P=0.001) and 0.43 (SE, 0.058, P=0.001), respectively. The kappa coefficient between punch biopsy diagnoses by the first and second pathologists before IHC staining was 0.73 (SE, 0.061, P=0.001).Primary punch diagnosisPrimary cone diagnosisTotal N (%)Negative N (%)CIN I N (%)CIN II N (%)CIN III N (%)Negative13 (72.2%)2 (11.1%)2 (11.1%)1 (5.6%)118 (100.0%)CIN I3 (27.3%)8 (72.7%) 0 (0.0%) 0 (0.0%)111 (100.0%)CIN II14 (58.4%)5 (20.8%)3 (12.5%)2 (8.3%)224 (100.0%)CIN II5 (14.2%)3 (8.6%)1 (2.9%)26 (74.3%)335 (100.0%)Total35 (39.8%)18 (20.4%)6 (6.8%)29 (33.0%)88 (100.0%)CIN, Cervical intraepithelial neoplasia |