Defining the frequency of human papillomavirus and polyomavirus infection in urothelial bladder tumours

Matthew A Llewellyn,Nicholas D James,Ben Abbotts,Richard T Bryan,Sally Roberts,K K Cheng,Joanna L Parish,Maurice P Zeegers,Naheema S Gordon,Douglas G Ward,Andrew Macdonald

doi:10.1038/s41598-018-29438-y

Abstract

Given the contradictory nature of the literature regarding the role of human papillomaviruses and polyomaviruses in the pathogenesis of urothelial bladder cancer (UBC), we sought to investigate the frequency of their involvement in a large cohort of primary UBCs. DNA was extracted from 689 fresh-frozen UBC tissues and screened for the presence of high-risk human papillomavirus (HPV) types 16 and 18 and BKV/JCV genomic DNA by qPCR. In positive cases, viral identity was confirmed by Sanger sequencing and viral gene expression was analysed by RT-PCR or immunohistochemistry. All 689 UBCs were negative for HPV18. One UBC from a female patient with areas of squamous differentiation was positive for HPV16. The qPCR data indicated variable levels of polyomavirus in 49 UBCs. In the UBCs with low Cts we were able to confirm that 23 were BKV and 6 were JCV by Sanger sequencing. Polyomavirus large T antigen expression was low but detectable in 70% of the sequencing-confirmed polyomavirus positive samples. Thus, in United Kingdom patients, the presence of HPV DNA sequences is extremely rare in UBC (<1% of cases). Polyomavirus DNA (predominantly BKV) is more common in UBC, but still only detectable in 7% of cases and in many of these cases at low copy number. We have performed the largest virus screening to date in UBC, finding that HPV16, HPV18 and HPyV are unlikely to be common causative agents in UBC.

Highlights

Bladder cancer is estimated to be the 9th most common cancer worldwide[1]
Using the combined assay for HPV16/HPV18 sequences to analyse the DNA extracted from fresh frozen tissue specimens, we obtained a positive result for human papillomavirus (HPV) (Ct = 24) in 1 out of the 689 urothelial bladder cancer (UBC) tested (0.1%) (Table 1) whereas the internal control (GAPDH) was positive in all cases
RT-PCR was used to determine if the HPV16 oncogenes E6 and E7 were expressed in the HPV16 positive UBC: both E6 and E7 mRNAs were detectable (Fig. 1)

Summary

Introduction

Bladder cancer is estimated to be the 9th most common cancer worldwide[1]. Over 90% of bladder tumours are classed as urothelial bladder carcinomas (UBCs), with 75–80% presenting at non-muscle-invasive (NMIBC) stages and the remainder presenting at muscle-invasive (MIBC) and metastatic stages[2]. Risk factors which increase the chance of developing bladder cancer include (but are not limited to) age, being male, smoking, occupational exposure to aromatic amines, polycyclic hydrocarbons or heavy metals and ionising radiation[5]. Chronic inflammation such as that caused by the parasite Schistosoma haematobium is a risk factor usually resulting in squamous cell rather than urothelial carcinoma[5]. Viral transcripts were detected in the BKV positive tumour and one of the HPV16 positive tumours In these 2 cases, BKV and HPV16 sequences were integrated into GRB14 and BCL2L1, indicating that viruses might play a role in a small percentage of UBCs. We sought to clarify the role of HPVs and HPyVs in the United Kingdom UBC population.

Methods

Results

Conclusion