Immunohistochemistry (IHC) for p16ink4a expression

Five of the eight HPV73-positive tumors (62.5%) had formalin-fixed paraffin-embedded cervical tissue available for IHC assay. All tissue sections were hematoxylin and eosin-stained and reviewed by an expert pathologist (FCBM). Three µm sections of each paraffin-embedded biopsy were stained for the qualitative detection of p16^ink4a protein with CINtec p16 Histology (Ventana Medical System, Oro Valley, AZ, USA) according to standard protocols and were visualized using 3.3’-diaminobenzidine (DAB) (DAKO, Glostrup, Denmark) with an exposure time of 3 minutes. Positive controls included five HPV16-positive CxCa specimens that were matched by FIGO staging to the HPV73-positive CxCa cases to compare the expression of the p16^ink4a marker.