Detailed Characteristics of Tonsillar Tumors with Extrachromosomal or Integrated Form of Human Papillomavirus.

Barbora Pokrývková,Martina Saláková,Ruth Tachezy,Vendula Novosadová,Jana Šmahelová,Zuzana Vojtěchová

doi:10.3390/v12010042

Barbora Pokrývková, Martina Saláková + Show 4 more

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https://doi.org/10.3390/v12010042

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Abstract

The human papillomavirus (HPV) integration, the critical step in viral carcinogenesis, most frequently occurs in the E2 gene, which results in its inactivation and in an increase of E6/E7 transcription. However, in a substantial number of tumors, the virus is present in an extrachromosomal form. For those tumors, the transformation mechanisms are not fully elucidated. Here we evaluated the possible mechanism of inactivating the E2 without interruption of the gene, methylation or mutation of the E2 binding sites (E2BSs) in HPV16-positive tonsillar tumors by next-generation and Sanger sequencing. Viral genome status was analyzed by the amplification of papillomavirus oncogene transcripts assay (APOT) and mRNA mapping, and expression of viral oncogenes was performed by qPCR. The methylation of E2BSs was significantly higher in tumors with an integrated, in comparison to extrachromosomal, form of the viral genome. No mutations were detected in the E2BSs. The viral oncogenes were equally expressed in samples with an integrated and extrachromosomal form of the virus. Only the nucleotide variants were identified in the E2 gene. No proposed mechanism of E2 inactivation was confirmed in tonsillar tumors with an extrachromosomal form of the HPV genome. The expression of E6/E7 genes seems to be sufficient to initiate and maintain the carcinogenic process

Highlights

Every year, more than 650,000 new cases of head and neck carcinomas (HNCs) are diagnosed worldwide [1]
The samples were grouped according to the presence and stable expression of complete E2 mRNA and viral–host fusion transcripts in two groups; group one represents samples with an expression of the E2 gene, the potential E6/E7 transcription repressor, as well as E4/E5 genes according to E2 breakpoint mapping and amplification of papillomavirus oncogene transcripts (APOT) assay, and samples with the extrachromosomal or mixed form of human papillomavirus (HPV), and group two with samples harboring an integrated form of HPV, which contained E6–E7-cell fusion transcripts only, and no E2 mRNA
Our results showed significantly higher methylation levels of the repressive E2BS2-4 in the samples with the integrated HPV genome where no E2 mRNA was detected, but the level of methylation of long control region (LCR) in our study did not affect the level of expression of viral E6/E7 mRNA

Summary

Introduction

More than 650,000 new cases of head and neck carcinomas (HNCs) are diagnosed worldwide [1]. Most HNCs are associated with smoking and alcohol consumption, but nearly 26% of HNCs are linked to infection by high-risk human papillomavirus (HR HPV) [2]. HPV-driven HNCs are mostly found in the oropharynx, with HR HPV prevalence varying from 7% to 88%, according to the sub-site of oropharyngeal cancer (OPC), the detection method and geographical area [3]. The incidence of OPC has been increasing markedly over the last decade [4,5], and it is widely accepted that. From the clinical point of view, the most important are the improved survival and response to treatment in HPV HNC patients [6].

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