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Mapping of Human Papilloma Virus, p16, and Epstein-Barr Virusin Non-Malignant Tonsillar Disease
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.ORCID iD: 0000-0003-3522-1842
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology. Östersunds hospital, Sweden.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.ORCID iD: 0000-0001-6949-1213
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
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2019 (English)In: Laryngoscope Investigative Otolaryngology (LIO), E-ISSN 2378-8038, Vol. 4, no 3, p. 285-291Article in journal (Refereed) Published
Abstract [en]

Objectives: Due to their location in the entrance of the aero‐digestive tract, tonsils are steadily exposed to viruses. Human papilloma virus (HPV) and Epstein‐Barr virus (EBV) are two potentially oncogenic viruses that tonsils encounter. The incidence of HPV positive tonsillar cancer is on the rise and it is unknown when infection with HPV occurs.

Aim: To investigate if tonsils are infected with HPV and EBV, to study the co‐expression of HPV and its surrogate marker p16, and to evaluate the number of EBV positive cells in benign tonsillar disease.

Materials and Methods: Tonsils from 40 patients in a university hospital were removed due to hypertrophy, chronic or recurrent infection. These were analyzed for presence of HPV, its surrogate marker p16, and EBV. HPV was studied using PapilloCheck (a PCR method), while p16 was identified in epithelial and lymphoid tissue with immunohistochemistry and EBV using EBER‐ISH (Epstein‐Barr encoding region–in situ hybridization).

Results: HPV was not detected, and p16 was present at low numbers in all epithelial samples as well as in 92.5% of the lymphoid tonsillar samples. At least one EBER‐positive cell was seen in 65% of cases. Larger numbers of EBER‐expressing cells were only seen in two cases.

Conclusion: These findings demonstrate that EBV and HPV infect tonsils independently, but further studies are warranted to confirm their infectious relationship.

Level of Evidence: Cross‐sectional study

Place, publisher, year, edition, pages
Wiley-Blackwell, 2019. Vol. 4, no 3, p. 285-291
Keywords [en]
Human papillomavirus, Epstein-Barr virus, non-malignant tonsillar disease, EBER-ISH, PapilloCheck, immunohistochemistry
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
URN: urn:nbn:se:umu:diva-158485DOI: 10.1002/lio2.260ISI: 000471907200002PubMedID: 31236460OAI: oai:DiVA.org:umu-158485DiVA, id: diva2:1307659
Funder
Västerbotten County CouncilAvailable from: 2019-04-29 Created: 2019-04-29 Last updated: 2024-07-02Bibliographically approved
In thesis
1. Human papillomavirus in sinonasal inverted papilloma, recurrent respiratory papilloma and non-malignant tonsils
Open this publication in new window or tab >>Human papillomavirus in sinonasal inverted papilloma, recurrent respiratory papilloma and non-malignant tonsils
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Humant papillomvirus i respiratoriska papillom, inverterade näspapillom och vid godartad sjukdom i halsmandlarna
Abstract [en]

Background: Human papillomavirus (HPV) is known to cause recurrent respiratory papilloma (RRP) and certain types of oropharyngeal cancer. HPV has also been associated with sinonasal inverted papilloma (SIP). HPV transmission routes are under investigation and the conviction is that the infection occurs sexually at an adult stage, however, vertical transmission at birth with a dormant viral condition until disease eruption/co-activation has been stated as a possibility.

Purpose: The purpose of this work was to contribute to the understanding of HPV related chronic diseases in the airway. Specific aims were: 1. To increase understanding regarding changes in the immune system as well as of the glycosaminoglycan hyaluronan in patients with RRP. 2. To evaluate prevalence of HPV and its surrogate marker p16 in SIP as well as HPV, p16 and Epstein-Barr virus (EBV) in benign tonsillar disease. HPV and EBV in non-malignant tonsillar disease were studied due to the fact that incidence of HPV positive tonsillar cancer is increasing and the time of viral infection is unknown.

Methods: A phenotypic characterization of peripheral blood from 16 RRP patients and 12 age-matched controls, using immunoflow cytometry, and monoclonal antibodies against differentiation and activation markers, was performed. The cytokine mRNA profile of monocytes, T helper-, T cytotoxic-, and NK cells was assessed using RT-qPCR. 54 SIP samples were studied of which 53 were available for analyzation with PCR. Genotype screening for 18 high risk and six low risk HPV types was performed using the PapilloCheck® HPV-screening test (a PCR method). 54 samples were immunohistochemically (IHC) stained for p16. Biopsies from vocal folds (VFs) and false vocal folds (FVFs) were collected from 24 patients with RRP, 12 were randomly selected to histochemistry for Hyaluronan (HA) and IHC staining for CD44 in the epithelium, stroma and RRP lesions. The remaining 12 patients were analyzed for HA molecular mass distribution with a gas-phase electrophoretic molecular mobility analyzer (GEMMA). Eight VF samples and four FVF samples were successfully analyzed. Biopsies from 40 non-malignant tonsils were analyzed using Papillocheck® for HPV, IHC for p16 and EBER analysis for EBV.

Results: We found a dominance of cytotoxic T cells, activated NK cells, and high numbers of stressed MIC A/B (MHC class I chain-related molecule A/B) expressing lymphocytes. The HPV analysis was successful for 38 SIP samples and two (5%) were positive for HPV 11. Notably, p16 was present in the epithelia of all samples and in the papilloma portions in 37 of 38 samples. We found extensive HA staining in the stroma of both VFs and FVFs. CD44 was expressed throughout the epithelium, stroma, and RRP lesions in both FVFs and VFs, it did however, not concur with the expression of HA. Very high mass HA was found in both VFs and FVFs, though more variation regarding amounts of HA was seen in the VFs compared to FVFs. No HPV was found in non-malignant tonsils, the p16 levels were low and the counted EBER positive cells showed great variation in numbers.

Conclusions: Our findings demonstrate an immune dysregulation with inverted CD4+/CD8+ ratio and aberrant cytokine mRNA production in RRP patients, compared to healthy controls. We concluded that p16 cannot be used as a surrogate marker for high-risk HPV-infection in SIP and that HPV incidence was low (5%). CD44 does not seem to bind to HA, which might explain the noninflammatory response previously described in RRP. Very high mass HA possibly crosslinked was seen in both VFs and FVFs. A possibility to counteract inflammatory crosslinking of HA may be found for medical treatment options in RRP.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2019. p. 56
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1955
Keywords
Human papillomavirus, recurrent respiratory papillomatosis, sinonasal inverted papilloma, non-malignant tonsillar disease, Epstein-Barr virus, immune system, p16, Hyaluronan
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:umu:diva-158489 (URN)978-91-7601-865-1 (ISBN)
Public defence
2019-05-24, Sal D, Unod T9, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Note

Titel enligt titelblad: Human papillomavirus in recurrent respiratory papilloma, sinonasal inverted papilloma, and non-malignant tonsils

Available from: 2019-05-03 Created: 2019-04-29 Last updated: 2024-07-02Bibliographically approved
2. Mapping viruses in non-malignant tonsils, nasal polyps, sinonasal inverted papilloma and laryngeal cancer
Open this publication in new window or tab >>Mapping viruses in non-malignant tonsils, nasal polyps, sinonasal inverted papilloma and laryngeal cancer
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Kartläggning av virus i godartade halsmandlar, näspolyper och inverterat papillom i näsa-bihålor samt cancer i struphuvudet
Abstract [en]

Background: The upper respiratory tract is exposed to viruses, which can lead to infection and cancer development. We chose to study common and/or chronic diseases along with common and cancer related viruses in the upper airway. High-risk human papillomavirus (HPV) causes cancer in tonsils and base of tongue, and Epstein-Barr virus (EBV) in the nasopharynx. p16 is used as a site-specific tumor marker for HPV. Human cytomegalovirus (HCMV) and human adenovirus (HAdV) are proposed to be oncomodulatory. It is unclear what significance these viruses have in benign tonsillar disease, chronic rhinosinusitis with nasal polyps (CRSwNP), sinonasal inverted papillomas (SIP) and laryngeal squamous cell carcinoma (LSCC). If virus is identified, it could make possible the use of current vaccines in prevention and treatment, as well as protection of healthcare providers.

Material and Methods: We analyzed 40 benign tonsils, 45 paired nasal polyp and healthy nasal mucosa samples, 53 SIP and 78 LSCC samples. We used PCR/microarrays (PapilloCheck®) for HPV detection and genotyping, immunohistochemistry (IHC) for p16 expression and real-time PCR for EBV, HCMV and HAdV detection. Additionally, Epstein-Barr encoding region (EBER) in situ hybridization (ISH) was used for EBV localization and count.

Results: HPV and p16 were not co-expressed, and p16 levels were low in benign tonsils, nasal polyps, and paired controls. Also, 9% of LSCC samples were high-risk HPV 16 positive and over-expressed p16.

EBV-positive cells were detected in 65% of the tonsils, nasal polyps (36%) versus controls (12%), 30% of SIP cases and 33% of LSCC samples.

Conclusions: EBV is commonly identified in benign tonsils, nasal polyps, SIP and LSCC, when using sensitive and robust detection methods. At the same time, viral infection with HPV, HCMV or HAdV appears to be uncommon in these conditions. p16 does not emerge as a reliable marker for HPV infection in LSCC.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2022. p. 72
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2188
Keywords
Human papilloma virus, Epstein-Barr virus, Human cytomegalovirus, Human adenovirus, p16 tumor suppressor protein, Non-malignant tonsillar disease, Chronic rhino sinusitis with nasal polyps, sinonasal inverted papilloma, laryngeal cancer, EBER-ISH
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:umu:diva-200180 (URN)978-91-7855-841-4 (ISBN)978-91-7855-842-1 (ISBN)
Public defence
2022-11-25, Hörsalen Snäckan, Hus 16, Östersunds Sjukhus, Östersunds sjukhus, Kyrkgatan 16, Östersund, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2022-11-04 Created: 2022-10-12 Last updated: 2022-10-13Bibliographically approved

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Holm, AnnaSchindele, AlexandraAllard, AnnikaEriksson, IreneNylander, KarinOlofsson, Katarina

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