Analysis of the integration of human papillomaviruses in head and neck tumours in relation to patients' prognosis

Abstract

Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods-the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.Head and neck cancer (HNC) is the seventh most common cancer in men and the 13 th most common cancer in women worldwide and its incidence is on the rise in some anatomical sites 1 . HNC has at least two different etiologies. Most HNC cases are associated with smoking and alcohol consumption, which induce mutations in important pathways 2,3 . However, 26% of all HNC and up to 50% of oropharyngeal tumours are associated with the presence and expression of mucosal high-risk human papillomaviruses (Alfa HR-HPV; HR-HPVs) [4][5][6] . Mucosal HRHPVs also cause cervical cancer and other neoplasms of anogenital skin and mucosae. The most common mucosal HR-HPV is HPV16, which is found in almost 90% of HPV-positive HNC, other common types being HPV18, HPV33 and HPV52 6,7 . Patients with HPV-associated HNC have a better prognosis, overall survival, and response to treatment 4 . The mechanisms of Alfa HR-HPV-induced carcinogenesis, regardless of the anatomical site, rely on the functions of viral oncoproteins, E6 and E7, as they inactivate tumour

Integrated Analysis of Whole-Genome ChIP-Seq and RNA-Seq Data of Primary Head and Neck Tumor Samples Associates HPV Integration Sites with Open Chromatin Marks

“…The fact that the episomal and integrated HPV can co-exist in the same tissue or even the same cell (30, 32, 47, 48), and both can be transcribed, adds another twist to the complex picture of HPV infection in human cancers. Southern blots or FISH-based technologies followed by high-resolution microscopy might help to resolve this scientific dispute (32, 49), and should be evaluated in conjunction with H3K27ac binding sites in future studies.…”

“…Although the role of HPV-integration is controversial, recent studies have demonstrated that HPV+ HNSCC samples with episomal and integrated HPV have unique signatures of gene expression, and can be separated based on them (22, 30, 32, 47–49). Clustering of gene expression data described two HPV+ HNSCC subtypes: mesenchymal and classical (29).…”

Prevalence and Prognostic Significance of HPV in Laryngeal Squamous Cell Carcinoma in Northeast China

“…Neither the physical status of HPV DNA (integrated vs. episomal) nor the transcriptional activity of E6/E7 oncogenes was examined. Vojtechova et al reported that both integrated and episomal HPV was associated with a significantly better prognosis compared with HPV− cases of OPSCC [38]. HPV DNA alone is not sufficient to claim causality in LSCC.…”

Proportion of Transcriptionally Active Dna Virus Integrants: A Meta-Analysis

“…Transcription of the X gene was observed in two out of eight (25.00%) cell lines analyzed, but none expressed the S gene [46]. Nine out of nine (100%) HPV integrants were expressing E6 mRNAs but only seven (77.78%) were also transcribing for messenger RNAs bearing E7 [52]. Other studies reported integrants active only in certain tissues: in transgenic C57BL/6 mice, HBV particles or HBsAg were expressed in various amounts and in distinct sites in different subjects [53,54].…”

“…For instance, the amplification of papillomavirus oncogene transcripts (APOT) assay specifically amplifies viral messengers from transcriptionally active integrants but cannot detect the integrants directly [52], therefore silent integrants are missed. On the other end, detection of integrated papillomavirus sequences (DIPS) is used to characterize the sequence structure of the viral integrants, particularly at the virus-cell junctions, but it is not designed to query the transcriptional activity [75], thus it cannot assess the expression status of the proviruses.…”