Next-Generation Sequencing analysis for detecting Human Papillomavirus in oral verrucous lesions


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Manar Samman1, Henry Wood1, Monica Pentenero3, Alec High2, Pamela Rabbitts1
1Leeds Institute of Cancer Studies and Pathology, Section of Pre-Cancer Genomics, University of Leeds, Leeds, UK, 2Department of Histopathology, St. James’s University Hospital and Leeds Dental Institute, Leeds, UK, 3Department of Oncology, Oral Medicine and Oral Oncology Unit, University of Torino, Turin, Italy

Background

Oral verrucous carcinoma (OVC) is a low-grade, slow-growing variant of squamous cell carcinoma. The aetiology of VC is unknown, and the suggested role of human papillomavirus (HPV) as a causative factor remains contentious. This dubiety can be attributed to varied detection procedures and difficulties in defining ‘gold-standard’ histological criteria for diagnosing verrucous lesions, including VC and verrucous hyperplasia (VH). Their rarity also makes them difficult to investigate, so most previous studies have been made on small numbers of cases. The aim of this study is to analyse oral verrucous lesions (including VC, and VH cases) for the presence of HPV subtype genomes.

Method

We used a recently developed and validated method, ‘next generation sequencing’ for the detection of HPV sequences identifying subtypes and computing viral loads from nanogram DNA quantities isolated from formalin-fixed, paraffin-embedded tissue. In this present study, we histologically identified a total of 61 oral verrucous cases: 48 OVCs, and 13 OVHs. DNA was extracted from all samples and sequenced at a coverage between 2.5% and 13%. Genomic copy number karyograms were produced and each sample was analysed for the presence of HPV subtypes and for all other sequenced human viral genomes.

Results

An HPV-16 sequence was detected in one OVH, and an HPV-2 sequence was detected in one OVC out of the 61 cases but at low viral loads of 2.24, and 0.33 viral genomes per cell respectively.

Conclusion

We confirm that NGS can be used as a precise method for detection of HPV subtypes and loads, and provide genomic copy number karyograms for FFPE verrucous samples in a single test. Our results indicate that there is no HPV involvement in oral verrucous lesions, nor are any other common human viruses implicated.