All the samples were cervical (punch) biopsy or endocervical curettage specimens. The patients aged from 20 to 80 years (mean 39.8 years). Initial diagnosis comprised 10 negative dysplasia (NEG), 21 ISM with or without reactive atypia, and 46 CIN, (18 CIN1, 11 CIN2, and 17 CIN3). All H&E stained sections were first reviewed by 2 independent pathologists blinded to the initial diagnosis. The consensus diagnosis was a gold Inhibitors,research,lifescience,medical standard, and defined as diagnostic agreement between the pathologists concerned. For patients with diagnostic disagreement, 12 of 77 cases, a third review was obtained from a gynecopathologist. All the specimens were immunostained for Ki-67,

p16 and CK17 antigens. Immunohistochemical Inhibitors,research,lifescience,medical (IHC) Staining IHC staining for Ki-67, p16 and CK-17 antigens was performed on 5 µm sections obtained from formalin-fixed, paraffin embedded blocks, using avidin-biotin peroxidase complex method. The primary antibodies were monoclonal mouse anti Ki-67

antigen, clone MIB-1 (Dako, code: N1633, Denmark; diluted 1:2); mouse monoclonal anti p16INK4a, (Santa cruz, (JC8) SC-56330, USA; diluted 1:50) and monoclonal mouse anti-CK17, clone E3 (Dako, code: M7046, Denmark; diluted 1:30). Secondary antibodies included goat anti-mouse and anti-rabbit immunoglobulines (Dako, code: K4061, Denmark; Ready to use) and DAB (3,3’ Diaminobenzidine; Inhibitors,research,lifescience,medical chromogen Inhibitors,research,lifescience,medical (Dako). Immunohistochemical Scoring The sections stained by IHC were examined alongside H&E stained specimens, to identify the precise Protein Tyrosine Kinase inhibitor locations of the lesions. Ki-67 (MIB-1) staining was interpreted positive when a cluster of at least 2 strongly stained epithelial nuclei were present in the upper two thirds of the epithelial thickness anywhere within the lesion. Presence of the para-basal cells staining was used as an internal positive control.4,7 The Inhibitors,research,lifescience,medical p16 was considered positive when it showed nuclear, as well as continuous diffuse cytoplasmic staining of the cells in the basal

and para-basal cell layers of the squamous epithelium, variably reaching intermediate and superficial cell layer characterized by diffuse staining pattern. p16 was considered negative when it was completely Calpain unstained, or showing focal or sporadic epithelial staining, particularly not of the basal and para-basal cells (focal staining pattern). Scoring of IHC results was evaluated on the basis of distribution of immunoreactive cells. However, staining intensity was not graded to avoid subjective interpretation.1 CIN3 specimens were used as positive controls. CK17 staining was considered positive, when cytoplasmic staining involved all squmous cell layers. Focal staining or completely unstained cell layers was considered as negative.13 Statistical Analysis Histologic diagnoses were categorized as (NEG), CIN1 (LG-SIL), CIN2 & CIN3 (HG-SIL), and ISM.