HN 08-02Integration of human papillomavirus type 11 into FGFR3 gene and long non-protein coding RNA LINC00486 in a patient with sinonasal carcinoma

22. HPV and oropharynx / Head and neck cancer
L. Hošnjak 1, B.J. Kocjan 1, T.M. Zorec 1, N. Gale 2, N. Zidar 2, P. Strojan 3, M. Poljak 1.
1Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia (Slovenia), 2Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia (Slovenia), 3Department of Radiation Oncology, Institute of Oncology, Ljubljana, Slovenia (Slovenia)

Background / Objectives

Low-risk human papillomavirus types 6 and 11 (HPV6 and HPV11), etiological agents of genital warts and laryngeal papillomas, are rarely associated with human cancers. However, HPV6 and HPV11 may incidentally integrate into the human genome and cause malignant transformation by insertional inactivation of particular human oncogenes (1) or intergenic regulatory regions (2). The objective of the present study was to investigate further the molecular mechanism(s) of low-risk HPV-mediated carcinogenesis by studying a case of HPV11-positive sinonasal carcinoma.


Methods

Archival tissue specimen of the sinonasal carcinoma, HPV11-positive with INNO-LiPA HPV Genotyping Extra test (Innogenetics), was tested for the presence of HPV6/11 DNA, HPV6/11 E6/E7 mRNA and high-risk HPV E6/E7 mRNA by in-situ hybridization (ISH), using INFORM HPV II Family 6 Probe (B) (Roche Diagnostics), Probe-HPV11 and Probe-HPV HR7 (Advanced Cell Diagnostics), respectively. Immunohistochemical stainings for p16INK4A, p53 and pRB proteins were performed using CINtec p16 Histology (Ventana Medical Systems), Monoclonal Mouse Anti-Human p53 Protein Clone DO-7 (Dako) and Rb Antibody (Santa Cruz Biotechnology), respectively. For next generation sequencing (NGS), 2 μg of extracted DNA were used to generate TruSeq Nano paired-end libraries and sequenced using Illumina NextSeq instrument.


Results

Using NGS complete HPV11 genome sequence (7,933-bp), showing 99.7% nucleotide similarity to the HPV11 reference sequence, was obtained from the sinonasal carcinoma. None of the identified nucleotide/amino acid changes in the LCR genomic region and E7, E1, E2 and E5a proteins clustered within their functional domains. HPV6/11 DNA ISH showed nuclei harboring a single punctate signal, indicating HPV integration. HPV6/11 E6/E7 mRNA ISH revealed active expression of viral oncogenes, high-risk HPV E6/E7 mRNA ISH was negative, and p16INK4A, p53 and pRB were highly expressed. NGS data analysis revealed that HPV11 was integrated in the chromosome 2 (2p22.3), within the Long Intergenic Non-protein Coding RNA 486 (LINC00486) region. Additionally, short parts of the HPV11 LCR were integrated in the chromosome 4 (4p16.3), within or in the close proximity of the FGFR3 gene.


Conclusion

HPV11 integration upstream of and within the FGFR3 gene and additionally in the LINC00486 region, and overexpression of p53 and pRB which are normally downregulated in high-risk HPV-induced malignant tumors, were detected in the sinonasal carcinoma. Alternations in expression and structural changes of the FGFR3 protein and long non-coding RNA complexes have been previously associated with some types of human cancers, thus our case warrants further research.


References

(1) Huebbers CU, Preuss SF, Kolligs J, Vent J, Stenner M, Wieland U, Silling S, Drebber U, Speel EJ, Klussmann JP. Integration of HPV6 and downregulation of AKR1C3 expression mark malignant transformation in a patient with juvenile-onset laryngeal papillomatosis. PLoS One 2013;8(2):e57207.

(2) Scheel A, Lin GC, McHugh JB, Komarck CM, Walline HM, Prince ME, Zacharek MA, Carey TE. Human papillomavirus infection and biomarkers in sinonasal inverted papillomas: clinical significance and molecular mechanisms. Int Forum Allergy Rhinol 2015;5(8):701-7.