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Human papillomavirus in sinonasal inverted papilloma, recurrent respiratory papilloma and non-malignant tonsils
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.ORCID iD: 0000-0003-3522-1842
2019 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Humant papillomvirus i respiratoriska papillom, inverterade näspapillom och vid godartad sjukdom i halsmandlarna (Swedish)
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 [en]
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: urn:nbn:se:umu:diva-158489ISBN: 978-91-7601-865-1 (print)OAI: oai:DiVA.org:umu-158489DiVA, id: diva2:1307744
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: 2019-05-08Bibliographically approved
List of papers
1. Lymphocyte profile and cytokine mRNA expression in peripheral blood mononuclear cells of patients with recurrent respiratory papillomatosis suggest dysregulated cytokine mRNA response and impaired cytotoxic capacity
Open this publication in new window or tab >>Lymphocyte profile and cytokine mRNA expression in peripheral blood mononuclear cells of patients with recurrent respiratory papillomatosis suggest dysregulated cytokine mRNA response and impaired cytotoxic capacity
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2017 (English)In: Immunity, Inflammation and Disease, E-ISSN 2050-4527, Vol. 5, no 4, p. 541-550Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: Recurrent respiratory papillomatosis (RRP) is a relatively rare, chronic disease caused by Human Papilloma Virus (HPV) 6 and 11, and characterized by wart-like lesions in the airway affecting voice and respiratory function. The majority of HPV infections are asymptomatic and resolve spontaneously, however, some individuals are afflicted with persistent HPV infections. Failure to eliminate HPV 6 and 11 due to a defect immune responsiveness to these specific genotypes is proposed to play a major role in the development of RRP.

METHODS: We performed a phenotypic characterization of peripheral blood mononuclear cells (PBMC) collected from 16 RRP patients and 12 age-matched healthy controls, using immunoflow cytometry, and monoclonal antibodies against differentiation and activation markers. The cytokine mRNA profile of monocytes, T helper-, T cytotoxic-, and NK cells was assessed using RT-qPCR cytokine analysis, differentiating between Th1-, Th2-, Th3/regulatory-, and inflammatory immune responses.

RESULTS: We found a dominance of cytotoxic T cells, activated NK cells, and high numbers of stressed MIC A/B expressing lymphocytes. There was an overall suppression of cytokine mRNA production and an aberrant cytokine mRNA profile in the activated NK cells.

CONCLUSION: These findings demonstrate an immune dysregulation with inverted CD4(+) /CD8(+) ratio and aberrant cytokine mRNA production in RRP patients, compared to healthy controls.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2017
Keywords
Human, T Cells, natural killer T cells, viral/retroviral
National Category
Immunology Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-142017 (URN)10.1002/iid3.188 (DOI)000424098900015 ()28805308 (PubMedID)
Available from: 2017-11-16 Created: 2017-11-16 Last updated: 2019-04-29Bibliographically approved
2. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma
Open this publication in new window or tab >>Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Objectives: Sinonasal inverted papilloma (SIP) is a relatively rare disease, and its etiology is not understood. It is characterized by locally aggressive growth and a strong tendency to recur despite its benign histology.

Aims: The aim of this study was to identify the presence of human papilloma virus (HPV) and its surrogate marker p16 in SIP tissue samples from a regional cohort.

Material and Methods: Subjects were identified from our regional center cohort of 88 SIP patients treated between 1984-2014. From these subjects, 54 were included in this study.  Of these, 53 biopsies were analyzed with PCR, and 54 samples were immunohistochemically stained for p16. DNA was extracted from histopathologically verified SIP.  Genotype screening for 13 high risk-, 5 oncogenic and 6 low risk HPV types was performed using the PapilloCheck® HPV-screening test.

Results: HPV analysis was successful for 38 of 53 samples. Of the 38 successfully analyzed samples, only 2 samples were positive for HPV 11.  Notably, p16 was present in the epithelia in all samples, and in the papilloma lesions in 37 samples.

Conclusion: Since only 2 out of 38 SIPs were positive for HPV (type 11), and at the same time p16 was positive in epithelia in all samples and in 37 of 38 papilloma lesions of the samples, it is concluded that p16 cannot be used as a surrogate marker for high-risk HPV-infection in SIP. We are currently planning a prospective, multicenter study in order to increase the study power and in order to be able to better evaluate the clinical implications of HPV-and p16 in SIP.

Keywords
human papillomavirus, inverted nasal papilloma, PapilloCheck, immunohistochemistry
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:umu:diva-158487 (URN)
Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-04-29
3. Hyaluronan in vocal folds and false vocal folds in patients with recurrent respiratory papillomatosis
Open this publication in new window or tab >>Hyaluronan in vocal folds and false vocal folds in patients with recurrent respiratory papillomatosis
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2018 (English)In: Acta Oto-Laryngologica, ISSN 0001-6489, E-ISSN 1651-2251, Vol. 138, no 11, p. 1020-1027Article in journal (Refereed) Published
Abstract [en]

Background: Hyaluronan (HA) is a glycosaminoglycan with viscoelastic properties necessary for vocal fold (VF) vibration and voice production. Changes in HAs molecular mass, possibly related to human papilloma virus, could affect formation/persistence of recurrent respiratory papillomatosis (RRP).

Aims/Objective: Describing mass and localization of HA and localization of HA receptor CD44 in VF and false vocal folds (FVF) in RRP.

Materials and Methods: Biopsies from VF and FVF from 24 RRP patients. Twelve were studied with histo-/immunohistochemistry for HA and CD44 in epithelium, stroma and RRP lesions. Twelve samples were analyzed for HA molecular mass distribution with gas-phase-electrophoretic-molecular-mobility-analyzer (GEMMA).

Results: Three of 23 stains (VF and FVF combined) showed faint HA staining in the epithelium; there was more extensive staining in the stroma. CD44 was present throughout all areas in FVF and VF, it did not concur with HA. GEMMA analysis revealed very high mass HA (vHMHA) with more varying amounts in VF.

Conclusions/Significance: HA was mainly distributed in the stroma. CD44 not binding to HA might explain the non-inflammatory response described in RRP. Possibly crosslinked vHMHA was seen in VF and FVF, with more variable amounts in VF samples. Counteracting HA crosslinking could become a treatment option in RRP.

Abstract [zh]

背景:透明质酸(HA)是一种糖胺聚糖, 具有声带(VF)振动和发声所必需的粘弹性。HA 分子量的变化可能与人乳头瘤病毒相关, 还可能影响复发性呼吸道乳头状瘤病(RRP)的形成或持续。

目的:描述HA的量和定位, HA受体CD44在VF中的定位和假性声带(FVF)在RRP中的定位。

材料和方法:24名RRP患者的VF和FVF的活组织检查。用组织/免疫组织化学方法研究12个样品的上皮、基质和RRP病变中的HA和CD44。用气-相-电泳 - 分子-迁移率-分析仪(GEMMA)分析另12个样品的HA分子量分布。

结果:23个染色中的3个(VF和FVF组合)在上皮细胞中显示出微弱的HA染色;基质中有更强的染色。 CD44存在于FVF和VF的所有区域, 它与HA不同时存在。 GEMMA分析显示非常高量的HA(vHMHA), 它在VF中的量多变。

结论/意义:HA主要分布在基质中。 CD44不与HA接合可能解释所描述的RRP中的非炎症反应。在VF和FVF中观察到可能交合的vHMHA, 而在VF样品中具有更多的变量。抗HA交合可能成为RRP的治疗选择。

Place, publisher, year, edition, pages
Taylor & Francis, 2018
Keywords
Vocal folds, hyaluronan, CD44, human papilloma virus, recurrent respiratory papillomatosis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156749 (URN)10.1080/00016489.2018.1500712 (DOI)000459000600012 ()30776265 (PubMedID)2-s2.0-85061778147 (Scopus ID)
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2019-04-29Bibliographically approved
4. Mapping of Human Papilloma Virus, p16, and Epstein-Barr Virusin Non-Malignant Tonsillar Disease
Open this publication in new window or tab >>Mapping of Human Papilloma Virus, p16, and Epstein-Barr Virusin Non-Malignant Tonsillar Disease
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2019 (English)In: Laryngoscope Investigative Otolaryngology, E-ISSN 2378-8038, Vol. 4, no 3, p. 285-291Article in journal (Refereed) Epub ahead of print
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
Keywords
Human papillomavirus, Epstein-Barr virus, non-malignant tonsillar disease, EBER-ISH, PapilloCheck, immunohistochemistry
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:umu:diva-158485 (URN)10.1002/lio2.260 (DOI)000471907200002 ()
Funder
Västerbotten County Council
Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-07-11Bibliographically approved

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