Genotyping of high-risk human papillomaviruses in p16/Ki-67-positive urothelial carcinoma cells: even a worm will turn

Objective Co‐expression of p16INK4a protein and Ki‐67 (p16/Ki‐67) is noted in almost all high‐grade urothelial lesions. However, the aetiological role or, conversely, the absence of causative effect of high‐risk human papillomaviruses (hr‐HPVs) has not been documented. The purpose of this study is to evaluate HPV DNA in p16/Ki‐67‐positive, high‐grade urothelial tumour cells. Methods Fifty‐seven urine samples collected from 50 patients, including 55 histologically proven high‐grade proliferations and two cases with clinical evidence of malignancy, were analysed for p16/Ki‐67. Immunolabelling was performed in destained Papanicolaou‐stained slides after ThinPrep® processing. HPV genotyping was performed by polymerase chain reaction (PCR) using a DNA microarray for 35 HPV types. Confirmation of the presence (or absence) of HPV in tissue samples was verified using a reasoned approach combining PCR and in situ hybridization (ISH) for hr‐HPVs. Results Co‐expression of p16/Ki‐67 was noted in 43 of 57 (75.4%) cases. In these, hr‐HPVs 16, 31 and 70, and low risk HPV 84, were detected in the urine in four patients (8%). Upregulation of p16INK4a protein was confirmed on bladder biopsy or transurethral resection specimens, but PCR and ISH for hr‐HPVs were both negative on the tissue sections. Conclusion Our results show a low prevalence of HPV infection in the urinary tract of patients with p16/Ki‐67‐positive urothelial malignancy. The study confirms that the deregulated cell cycle, as demonstrated by p16/Ki‐67 dual labelling, is independent of the oncogenic action of hr‐HPVs in high‐grade urothelial proliferations.