Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression

Abstract

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Methylation status of HPV16 E2‐binding sites classifies subtypes of HPV‐associated oropharyngeal cancers

“…The integration status of the virus was analyzed by means of amplification of papillomavirus oncogene transcripts (APOT) and detection of integrated papillomavirus sequences by PCR (DIPS-PCR) as published previously 18 from fresh-frozen tumor biopsies. For both assays, bands typical for episomal and integrated genomes were cut out, purified using the Qiagen Gel Extraction Kit (Qiagen), and sequenced (GATC Biotech AG, Konstanz, Germany).…”

“…15 However, several findings have strongly suggested that as well as integration-mediated E2 gene disruption, other mechanisms may contribute to the inhibition of E2 functions. First, it was reported that in approximately 50% of HPV16-associated cervical cancers 16,17 and OPSCC 18,19 only episomal HPV genomes and no integration-mediated disruption of the E2 gene was observed. Second, a substantial percentage of cell lines and tumors with integrated HPV genomes carry additional viral genomes with intact E2 gene sequences, either as episomes or integrated head-to-tail concatemers.…”

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

“…On the other hand, transcription from individual integrants (type I) might be assumed to be restricted by the functional integrity of the viral sequence, although host's promoters might compensate for the loss of the viral ones. There was no direct data in the present work that could corroborate such assumption: the scientific publications recording clinical samples with high viral copy number only reported the VL data for the overall group rather than for each sample, therefore it was not possible to assess whether there was a relation between viral copy numbers and transcriptional status [79][80][81]. Furthermore, it has been suggested that type II pro-viruses might undergo epigenetic silencing [19]; this process can overcome the physical status of the integrants and disrupt an association between VL and transcriptional activity.…”

Genotipificación, niveles de expresión y estado físico del virus del papiloma humano en pacientes colombianos con cáncer de células escamosas en la cavidad oral

“…Inicialmente, se amplificó la región L1 del HPV mediante una reacción en cadena de la polimerasa (PCR) múltiple que contenía nueve cebadores sentido y tres antisentido 'biotinilados', y para evaluar la calidad del ADN, se incluyó un par de cebadores para amplificar el gen de ß-globina; en este caso, el cebador antisentido también estaba 'biotinilado'. Posteriormente, se hizo la hibridación de los productos de PCR desnaturalizados para su conversión en microesferas que detectan 24 tipos virales: seis de bajo riego (6,11,42,43, 44 y 70), 15 de alto riesgo (16,18,31,33,35,39,45, 51, 52, 56, 58, 59, 68, 73 y 82) y tres posiblemente de alto riesgo (26, 53 y 66). Para evidenciar la hibridación se añadieron el conjugado y el sustrato y, finalmente, las lecturas se hicieron en el equipo Luminex 100 ® bajo las condiciones sugeridas por el fabricante.…”

“…En Colombia solo se ha publicado el estudio sobre cáncer de cabeza y cuello asociado a HPV llevado a cabo por Quintero, et al, en el cual se registró una prevalencia de HPV de 18,9 % (13). Este virus presenta más de 100 tipos virales y, aproximadamente, 40 genotipos se han asociado hasta la fecha con las infecciones anales y genitales; estos tipos de HPV se dividen, a su vez, en tres grupos según su asociación epidemiológica con el cáncer: tipos de alto riesgo: 16,18,31,33,35,39,45, 51, 52, 56, 58, 59, 68, 73, y 82; posiblemente de alto riesgo: 26, 53 y 66, y de bajo riesgo: 6, 11, 40, 42, 43, 44 y 70 (14-16). El genotipo de HPV encontrado con mayor frecuencia en el cáncer de células escamosas en la cavidad oral en todo el mundo, es el HPV-16 (12,17,18).…”