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Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma
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Original article
Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma A. Holm a,∗ , A. Allard b , I. Eriksson b , G. Laurell c , K. Nylander d , K. Olofsson a a
Department of Clinical Sciences, Division of Otorhinolaryngology, Umeå University, 90185 Umeå, Sweden Department of Clinical Microbiology, Division of Clinical Virology, Umeå University, 90185 Umeå, Sweden Department of Surgical Sciences, Division of Otorhinolaryngology, Uppsala University, 75236 Uppsala, Sweden d Department of Medical Bioscience, Division of Pathology, Umeå University, 90185 Umeå, Sweden b c
a r t i c l e
i n f o
Keywords: Human papilloma virus Inverted nasal papilloma PapilloCheck® Immunohistochemistry
a b s t r a c t 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. © 2019 Published by Elsevier Masson SAS.
1. Introduction Sinonasal inverted papilloma (SIP) is a relatively rare, benign neoplasm with an incidence of 1.5 per 100.000 inhabitants [1]. SIP is characterized by a locally aggressive growth and a profound tendency to recur, despite its benign histology [2]. SIP is associated synchronously or metachronously with invasive squamous cell carcinoma (SCC) [2]. SIP is one of three subtypes of Schneiderian papilloma, the other two are exophytic papilloma and oncocytic/columnar cell papilloma [3]. SIP commonly presents with nasal obstruction [4], epistaxis [5] and in some cases epiphora due to occlusion of the nasolacrimal duct [6]. The disease affects mainly men with an age at onset of 50-60 years [7]. The most important clinical
∗ Corresponding author. E-mail address: [email protected] (A. Holm).
characteristic is the unilaterality of inverted papilloma [8]. Diagnostically, SIP should be suspected when observing unilateral ‘nasal polyps’. SIP often presents in white, pink, or tan color and firm texture, in contrast to commonly translucent nasal polyps [8]. However, the macroscopic appearance of SIP lesions can be very heterogeneous, which highlights the importance of histopathology for differential diagnosis [9]. Treatment of choice for SIP is surgery; removing the lesion radically reduces the risk of recurrence [10]. For optimal access, radicality, and patient comfort, the endoscopic technique is preferred. The cause of SIP is poorly understood. Possible connections between SIP, occupational exposure to smoke [11] and chronic inflammation [12] have been studied. A report written by Dunn et al. [13] reported detection of EBV in 1 out of 25 SIP cases. Notably, the HPV prevalence in SIP has been reported to be as high as 37,8% [14]. HPV is strongly associated to SCCHN (squamous cell cancer of the head and neck) as well as oropharyngeal cancers, [15,16] but
https://doi.org/10.1016/j.anorl.2017.10.008 1879-7296/© 2019 Published by Elsevier Masson SAS.
Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008
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not to cancer of the mobile tongue [17]. The association between HPV, including the surrogate marker p16, and tumor development seems to be site-specific, and this has not been studied in SIP. If oncogenic HPV genotypes are strongly associated with p16 and SIP, this would affect both the clinical management of SIP patients in the operating theatre as well as the strength of the indication for preventive vaccination. The purpose of this study was to assess the prevalence and incidence of specific HPV genotypes and the HPV surrogate marker p16 in SIP samples in northern Sweden over time. We also aimed to assess HPV presence in SIP patients, SIP recurrence and development of malignancy, as well as p16 prevalence in regard to age. 2. Material and methods This retrospective cohort study included patients diagnosed with sinonasal inverted papilloma (SIP) between 1984-2014 at the University Hospital of Umeå. The study was approved by the Regional Ethical Review Board of Umeå University (Umeå, Sweden) and the Biobank North (County Council of Västerbotten, Sweden; approval nos. Dnr 2015-323-32 M (2012-379-31 M), 2015-10-19). Patient tissue samples were identified from 1984 to and including 2014. Biopsies from the pathology register at Clinical Pathology, University Hospital of Umeå, labeled with ICD-10 codes resembling inverted papilloma of the nose and sinus (80531/80531) and exophytic nasal papilloma/inverted urothelcellular papilloma (81210) were included. Exophytic nasal papilloma/inverted urothelcellular papilloma was included as a diagnosis since inverted sinonasal papillomas sometimes are labeled with this diagnosis. All biopsies were re-analyzed histopathologically to verify that no samples of exophytic nasal papilloma/inverted urothelcellular papilloma were included in this study. 2.1. Patients characteristics A total of 139 biopsies were identified as SIP. The 139 eligible samples belonged to 88 patients, 17 of which had more than one type of benign diagnosis (Fig. 1). In 2 cases, the first biopsy was too small, and the second registered biopsy was analyzed. Of the 88 biopsies, 19 were excluded due to insufficient material. Six biopsies were not included due to inaccessible material (second opinion referrals from another hospital), 6 due to old fragmented DNA samples, one due to the fact that the patient had been treated with radiotherapy in a different country. One patient was excluded due to analysis of wrong biopsy number (regarding analysis of HPV, p16), one due to none SIP histopathology and one because malignant conversion could be seen. This left a total of 54 biopsies stained with p16, and a total of 53 biopsies analyzed with PapilloCheck® (Fig. 1). Out of 54 analyzed patients, 16 were female (30%) and 38 were male (70%), and ages ranged from 18 to 88 with a mean of 59 years (infosup 1 et 2 supplementary Tables 1 and 2 Tables 1 and 2). The age of one patient was not registered. A minority of the patients (12/54) had more than 1 benign diagnosis registered. The diagnosis nasal inverted papilloma was found 37 times, whereas inverted papilloma of the sinus and inverted urothelcellular papilloma were used 24 and 6 times, respectively. For 19/54 (35%), there was more than 1 diagnosis registered with an interval of at least 100 days in between them, a cut- off time interval used to identify recurrence. 2.2. HPV detection and PCR Five 10 M sections/sample were cut from paraffin blocks. Thereafter the microtome and other accessories were cleaned with 70% alcohol and a fresh blade was installed before cutting each
block, in order to prevent cross contamination. After cutting, the sections were placed in 2.0 mL Eppendorf tubes and spinned briefly. DNA extraction was performed using the QIAamp® DNA FFPE Tissue Kit (QIAgen, Hilden, Germany). The process starts with deparaffinization with xylene and rehydration with 99.5% ethanol. After drying at 37 ◦ C in 10 minutes, tissue pellets were incubated with a solution of Proteinase K in ATL Buffer at 56 ◦ C overnight. On the second day, the samples were incubated at 90 ◦ C for 1 hour, after this a mixture of AL Buffer and 99.5% ethanol was added. After short centrifugation, the entire lysate was transferred to the QIAamp MinElute column, washed twice and centrifuged (13000 rpm, 3 min). DNA was eluted from the column by 100 l ATE buffer. Quantity of DNA was assessed spectrophotometrically and quality of DNA was measured based on its purity by spectrophotometric analysis of A260/280 ratio. In order to assess the degree of DNA fragmentation or integrity, amplifiability of housekeeping ß-globin genes of three different amplicon lengths (536 bp, 268 bp and 100 bp) were evaluated. For successful amplification and HPV typing a fragment size of about 350 bp was needed. Amplification of endogenous ß-globin DNA was performed by general PCR thermal cycling (M3000, Thermo Fisher Scientific, Waltham, USA), initiated with 3 min incubation at 95 ◦ C, followed by 40 cycles at 95 ◦ C for 30s, 72 ◦ C for 30s and 55 ◦ C for 1 min. The PapilloCheck® HPV-screening test system (Greiner BioOneGmbH, Frickenhausen, Germany) screens for 13 high risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), five possibly oncogenic HPV types (HPV 53, 66, 70, 73, and 82) and six HPV types that are considered being low risk types (HPV 6, 11, 40, 42, 43, and 44). The assay uses multiplex PCR with fluorescent primers to amplify a DNA fragment of about 350 nucleotides within the E1 open reading frame of the HPV genome. Amplification of an internal HPV template present in the PCR master-mix generates a signal on the PCR control spot on the chip. In addition, an internal PCR control which targets a region within the human ADAT-1 gene (adenosine deaminase tRNA specific 1) is used to assess cellularity. All samples were tested according to manufacturer’s instructions.
2.3. p16 immunohistochemistry and scoring system Detection of p16 was performed by staining in a Ventana staining machine (BenchMark ULTRA; Ventana Medical Systems, Tuscon, AZ, USA) following the supplier’s recommendations. Prior to staining, slides were pretreated in Tris-EDTA (10 mM Tris-HCl, 1 mM disodium EDTA; pH 8.0). After this, an antibody against p16 (monoclonal mouse anti-human; cat. no. sc-56330, Santa Cruz Biotechnology, Dallas/TX/USA) was used at a dilution of 1:200. The antibody was visualized using the Ultraview Universal DAB Detection Kit (Ventana Medical Systems) and staining was evaluated by using a light microscope (BX51; Olympus Corporation, Tokyo, Japan) and Spectrum online software (Spectrum Version 11.2.0.780 © Copyright 2006-2012 Aperio Technologies, Inc.). In order to score the percentage of cells expressing p16 and intensity of p16 staining, the quickscore system was used [18]. The proportion of cells expressing p16 was graded from 1 to 6 as follows: 1 = 0-4%, 2 = 5-19%, 3 = 20-39%, 4 = 40-59%, 5 = 60-79% or 6 = 80-100%. Staining intensity was evaluated with 4 grades: 0 = negative, 1 = weak, 2 = intermediate and 3 = strong. The product of the proportions’ score of cells expressing p16, multiplied by the intensity score equals a quickscore (QS) ranging from 0-18. Epithelium and inverted papilloma were scored separately. Two members of the research group scored the histopathological slides independently of one another. Thereafter scores were compared and consensus was reached.
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Fig. 1. Flowchart over patients biopsies analyzed with PapilloCheck® and p16 immunohistochemistry.
2.4. Analysis and statistics Recurrence was defined as two or more samples registered with an interval of 100 days or more between them. This interval was chosen to verify that the material actually resembled recurrence and not merely an initial diagnostic sample and material from following larger surgery. We did not have ethical permission to acquire more precise information regarding recurrence in patients’ medical records. In order to determine the oncogenic potential of SIP we analyzed a concomitant presence of malignancy in the sinonasal samples. Specifically, we searched for ‘squamous cell cancer of the nose’, ‘squamous cell cancer of the sinus’, ‘suspicious of squamous cell cancer of the nose’, ‘suspicious of squamous cell cancer of the sinus’, ‘recurrence of squamous cell cancer of the nose’ and ‘recurrence of squamous cell cancer of the sinus’ in our register of pathological diagnoses. Descriptive statistics are presented. A Pearson’s correlation analysis is performed and correlation coefficient presented for age
in regard to histopathological findings. A Poisson regression was used to describe illness incidences during the inclusion period, and across the regional population.
3. Results 3.1. HPV and sinonasal inverted papilloma/inverted urothelcellular papilloma The PapilloCheck® analysis for the qualitative type-specific identification of 24 HPV genotypes on SIP samples was performed successfully in all but one of the biopsies. Of 53 patients, 36 (68%) were negative for HPV and two were positive for HPV 11 (4%). Of the remaining, 15 (28%) biopsy analyses failed due to fragmented DNA (info suppl 2: Supplementary Table 2). The reason for this was old sample age. Thus, 2 out of 38 (5%) were successfully analyzed samples and HPV positive (info suppl 1: Supplementary Table 1).
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Table 1 p16 score in papilloma parts of the specimen for all 54 patients.
Table 4 p16 scores in epithelial parts of specimen with successful HPV analysis.
p16 in sinonasal inverted papilloma (SIP) # of patients p16 Intensity
Percent
0 2 4 6 8 10 12 15 18 Total
3,7 3,7 13,0 31,5 29,6 9,3 3,7 1,9 3,7 100,0
HPV
2 2 7 17 16 5 2 1 2 54
p16 epithelium
1 2 4 6 8 9 10 12 15 18
Total Table 2 p16 score in the epithelium of all 54 patients. p16 in the epithelium p16 Intensity
# of patients
Percent
1 2 4 6 8 9 10 12 15 18 Total
2 1 9 27 7 1 2 2 2 1 54
3,7 1,9 16,7 50,0 13,0 1,9 3,7 3,7 3,7 1,9 100,0
p16 SIP
0 2 4 6 8 10 12 15 18
Total
Total
Negative
HPV 11
1 1 5 11 11 4 1 1 1 36
0 1 1 0 0 0 0 0 0 2
HPV 11
1 0 7 19 4 1 1 1 2 0 36
0 0 1 0 1 0 0 0 0 0 2
1 0 8 19 5 1 1 1 2 0 38
and p16 in papilloma and epithelium were 0.13 and − 0.03, respectively. For patients with successful HPV analysis in which age was registered (n = 38), the Pearson r for age and p16 in papilloma and epithelium was 0.19 and − 0.08, respectively (Info suppl. 3: figures supplémentaires 1-4). 3.5. Recurrence There were 19 of 54 patients which had 2 or more further diagnoses registered with at least 100 days in between. One patient with HPV 11 had 1 diagnosis registered, the other had 2 diagnoses registered within 73 days. 3.6. Development of SCC
Table 3 p16 scores in papilloma parts of specimen with successful HPV analysis. HPV
Total
Negative
1 2 6 11 11 4 1 1 1 38
3.2. p16 expression Expression of p16 was scored in all 54 samples. This showed p16 expression in 52 papilloma lesions of the specimen, whereas the epithelium showed p16 in all 54 biopsies. The mean score in the SIP parts was 7.17 and 6.65 in the surrounding epithelium (Tables 1 and 2). 3.3. p16 expression and HPV When separately analyzing p16 expression in the 38 samples with successful HPV analysis, p16 was present in the epithelia in all samples and in the papilloma lesions in 37 samples (Tables 3 and 4). Mean score for p16 in papilloma lesions for HPV negative and HPV positive specimens was 7.25 and 3.00, respectively. Mean value for p16 in SIP epithelium for HPV negative and HPV positive specimens were 6.56 and 6.00, respectively.
One patient diagnosed with inverted papilloma of the nose and sinus also had an initial sinonasal SCC diagnosis, a second nasal SCC diagnosis in this patient was negative for HPV. One patient with inverted urothelcelluclar papilloma was initially diagnosed with ‘suspicious of nasal SCC’, which was later confirmed. There was an insufficient amount of benign material for analysis, and the malignant biopsy was not further studied. One patient diagnosed with inverted urothelcellular cancer of the nose was initially registered as nasal SCC. The first registered specimen, was categorized as both benign and malignant, and was positive for HPV 18. One HPV negative patient with inverted papilloma of the nose and sinus and inverted urothelcellular papilloma was diagnosed with ‘suspicious of nasal SCC’ but no confirming malignant transformation was registered. 3.7. Development of SIP incidence We had no information regarding patients’ residence apart from the fact that 6 patients’ biopsies were sent from the county of Norrbotten to Umeå for second opinion. Nevertheless, we wanted to study the incidence rates over time. We chose to assume that patients during this time period were referred to Umeå from the northern region (counties of Västerbotten, Västernorrland, Norrbotten, Jämtland), and that the per capita rate in each county is the same. Poisson regression was performed with time as the covariate and using the relative population size of Västerbotten as an offset. Here, the incidence rate was estimated to increase with 7.6% per year (approximate 95% Confidence Interval: 4.7%-10.6%). 4. Discussion
3.4. p16 and age Age was registered in 53 of 54 patients. In patients tested for HPV in which age was registered (n = 53), the Pearson r for age
The purpose of this study was to assess the prevalence of specific HPV genotypes and the HPV surrogate marker p16 in SIP samples in northern Sweden over time. We also wanted to know if there
Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008
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is a connection between HPV in SIP patients and the tendency for development of malignancy and recurrence. We analyzed 53 biopsies with PCR, and 54 biopsies with immunohistochemistry that stained for p16. 38 PCR analyses succeeded whereas all 54 samples could be tested for p16. Out of 38 PCR samples 2 were positive for HPV 11. In these 38 samples p16 in epithelia was present in all cases and p16 in papilloma in 37 cases. Our results indicate that there is no correlation between p16 and prevalence of HPV in SIP. These findings support the idea that the surrogate marker p16 used for detection of HPV is organ specific [16,17] and that p16 cannot be used in the detection of HPV in SIP. Out of 3 specimens diagnosed with both inverted papilloma and nasal SCC, the first sample was negative for HPV, the second was positive for HPV 18 and the third was not analyzed. During our study period (1984-2014) SIPs incidence rate increased by 7.6%. It has to be noted that research on the causative agents of SIP so far have not been conclusive, though many different potential risk factors (e.g. EBV, chronic inflammation, exposure to occupational smoke) are suspected to play a role. In our SIP study, only 2 out of 38 samples (5%) were HPV positive. These findings diverge from those presented by Syrjanen et al. which observed a prevalence of HPV in SIP of 37.8% [14]. The reason for this disparity might be due to sample sizes, disease classification, or detection methods. Sample sizes of patients with SIP in Syrjanen’s meta-analysis ranged from case reports [19] to larger studies with 101 [20] cases and detection rates varied from 0%-100% [14]. In Syrjanen’s metaanalysis, different types of detection methods were used (PCR, in situ hybridization, southern blot hybridization and dot blot hybridization) which is, along with varying geographic locations, a plausible reason for the large variation in HPV detection outcome. Considering the clinical applicability of mapping HPV status in oropharyngeal SCC with PapilloCheck® , that provides a sensitivity of 89% and 92.5% for CIN2+ (cervical intraepithelial neoplasia) and CIN3+ respectively [21], along with a high level of reliability for detection of 6 low- and 18 high-risk HPV types, we were encouraged to proceed with this analysis. Although this study indicates that SIPs generally express low numbers of HPV, p16 was expressed throughout the epithelia in all samples and in the papilloma parts in all but two samples. p16 is often used as a surrogate marker for high risk HPV, as it is overexpressed by a negative feedback loop when HPV E7 binds to pRb (retinoblastoma protein). However, increased levels of p16 could also originate from p16 pathway changes that are independent of HPV [22]. LaPak et al. discussed infection among other causes as source of p16 elevation [22]. In contrast to previous studies revealing that p16 could be used as a marker for senescence [23], we were unable to correlate p16 expression to age. Lin et al. [24] found that p16 expression usually is preserved in SIP, whereas p16 expression is more likely to be lost when the papilloma lesions undergo malignant change. A ‘hit and run’ hypothesis has previously been postulated. This theory implies that HPV may induce SIP formation, however, the non- keratinizing SIP may not be capable of harbouring an HPV infection [25]. The correlation between the HPV surrogate marker p16 and HPV is clearly site specific in the head and neck region, and in this sense SIP mucosa resembles SCC mucosa in the mobile tongue [17]. Although SIP is a histologically benign disease, it exhibits features similar to mobile tongue cancer, such as properties of local invasion, regional relapse rate, HPV negativity and lack of correlation between p16 and HPV.
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5. Conclusion In summary, the study outcome suggests an incidence increase with 7.6% per year. To assess the etiology of sinonasal inverted papilloma (SIP), we studied the presence of human papilloma virus (HPV) and its surrogate marker p16 in 54 retrospectively-identified SIP patients using PCR and immunohistochemistry. Out of 38 successfully analyzed samples, only 2 samples were positive for HPV 11 whereas p16 was expressed in 37 papilloma parts of the samples and in all epithelial parts of the samples. We conclude that p16 cannot be used as a surrogate marker for high-risk HPV-infection in SIP. Our research group is currently planning more rigorous prospective, multicenter studies in order to further map the observed HPV-negative and p16 positive findings in SIP. We are especially interested in studying p16 levels in healthy sinonasal mucosa. Disclosure of interest The authors declare that they have no competing interest. Acknowledgements This investigation was supported by grants from Lion’s Cancer Research Foundation (KO), Umeå University and Västerbotten County Council (KO), A Allard, I Eriksson, A Holm and K Nylander performed the experiments, K Olofsson, A Holm, G Laurell, K Nylander designed the study; A Holm, A Allard, K Olofsson, K Nylander and G Laurell wrote the paper. Michael Haney assisted with text revision. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.anorl.2017.10.008. References [1] Outzen KE, et al. Inverted papilloma: incidence and late results of surgical treatment. Rhinology 1996;34(2):114–8. [2] Mirza S, et al. Sinonasal inverted papillomas: recurrence, and synchronous and metachronous malignancy. J Laryngol Otol 2007;121(9):857–64. [3] Shanmugaratnam K, Sobin LH. The World Health Organization histological classification of tumours of the upper respiratory tract and ear. A commentary on the second edition. Cancer 1993;71(8):2689–97. [4] van Samkar A, Georgalas C. Long-term quality of life after endoscopic removal of sinonasal inverted papillomas: a 6-year cohort analysis in a tertiary academic hospital. Eur Arch Otorhinolaryngol 2016;273(6):1433–7. [5] Vrabec DP. The inverted Schneiderian papilloma: a clinical and pathological study. Laryngoscope 1975;85(1):186–220. [6] Bewes T, et al. Incidence of neoplasia in patients with unilateral epiphora. J Laryngol Otol 2015;129:S53–7. [7] Bhandary S, et al. Sinonasal inverted papilloma in eastern part of Nepal. Kathmandu Univ Med J (KUMJ) 2006;4(4):431–5. [8] Barnes L. Schneiderian papillomas and nonsalivary glandular neoplasms of the head and neck. Mod Pathol 2002;15(3):279–97. [9] Tritt S, McMains KC, Kountakis SE. Unilateral nasal polyposis: clinical presentation and pathology. Am J Otolaryngol 2008;29(4):230–2. [10] Lombardi D, et al. Limitations and complications of endoscopic surgery for treatment for sinonasal inverted papilloma: a reassessment after 212 cases. Head Neck 2011;33(8):1154–61. [11] Deitmer T, Wiener C. Is there an occupational etiology of inverted papilloma of the nose and sinuses? Acta Otolaryngol 1996;116(5):762–5. [12] Papon JF, et al. Matrix metalloproteinase-2 and -9 expression in sinonasal inverted papilloma. Rhinology 2006;44(3):211–5. [13] Dunn ST, et al. Survey of sinonasal inverted papillomata for Epstein-Barr virus. Head Neck 1997;19(2):98–106. [14] Syrjanen K, Syrjanen S. Detection of human papillomavirus in sinonasal papillomas: systematic review and meta-analysis. Laryngoscope 2013;123(1):181–92. [15] Marur S, et al. HPV-associated head and neck cancer: a virus-related cancer epidemic. Lancet Oncol 2010;11(8):781–9. [16] Loizou C, et al. Incidence of tonsillar cancer in northern Sweden: Impact of human papilloma virus. Oncol Lett 2015;10(6):3565–72.
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[17] Sgaramella N, et al. Expression of p16 in squamous cell carcinoma of the mobile tongue is independent of HPV infection despite presence of the HPV-receptor syndecan-1. Br J Cancer 2015;113(2):321–6. [18] Detre S, Saclani Jotti G, Dowsett M. A “quickscore” method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol 1995;48(9):876–8. [19] Luceno V. Naso-sinusal inverse papilloma: a case report. Rev Laryngol Otol Rhinol (Bord) 1999;120(2):103–9. [20] Sun P, et al. [Relationship between nasal inverted papilloma and human papillomavirus subtypes]. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2010;45(4):310–3. [21] Crosbie EJ, et al. The PapilloCheck Assay for Detection of High-Grade Cervical Intraepithelial Neoplasia. J Clin Microbiol 2015;53(11):3553–9.
[22] LaPak KM, Burd CE. The molecular balancing act of p16(INK4a) in cancer and aging. Mol Cancer Res 2014;12(2):167–83. [23] Pustavoitau A, et al. Role of senescence marker p16 INK4a measured in peripheral blood T-lymphocytes in predicting length of hospital stay after coronary artery bypass surgery in older adults. Exp Gerontol 2016;74:29–36. [24] Lin GC, et al. Epidermal growth factor receptor, p16, cyclin D1, and p53 staining patterns for inverted papilloma. Int Forum Allergy Rhinol 2013;3(11):885–9. [25] Lawson W, Schlecht NF, Brandwein-Gensler M. The role of the human papillomavirus in the pathogenesis of Schneiderian inverted papillomas: an analytic overview of the evidence. Head Neck Pathol 2008;2(2):49–59.
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Original article
Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma A. Holm a,∗ , A. Allard b , I. Eriksson b , G. Laurell c , K. Nylander d , K. Olofsson a a
Department of Clinical Sciences, Division of Otorhinolaryngology, Umeå University, 90185 Umeå, Sweden Department of Clinical Microbiology, Division of Clinical Virology, Umeå University, 90185 Umeå, Sweden Department of Surgical Sciences, Division of Otorhinolaryngology, Uppsala University, 75236 Uppsala, Sweden d Department of Medical Bioscience, Division of Pathology, Umeå University, 90185 Umeå, Sweden b c
a r t i c l e
i n f o
Keywords: Human papilloma virus Inverted nasal papilloma PapilloCheck® Immunohistochemistry
a b s t r a c t 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. © 2019 Published by Elsevier Masson SAS.
1. Introduction Sinonasal inverted papilloma (SIP) is a relatively rare, benign neoplasm with an incidence of 1.5 per 100.000 inhabitants [1]. SIP is characterized by a locally aggressive growth and a profound tendency to recur, despite its benign histology [2]. SIP is associated synchronously or metachronously with invasive squamous cell carcinoma (SCC) [2]. SIP is one of three subtypes of Schneiderian papilloma, the other two are exophytic papilloma and oncocytic/columnar cell papilloma [3]. SIP commonly presents with nasal obstruction [4], epistaxis [5] and in some cases epiphora due to occlusion of the nasolacrimal duct [6]. The disease affects mainly men with an age at onset of 50-60 years [7]. The most important clinical
∗ Corresponding author. E-mail address: [email protected] (A. Holm).
characteristic is the unilaterality of inverted papilloma [8]. Diagnostically, SIP should be suspected when observing unilateral ‘nasal polyps’. SIP often presents in white, pink, or tan color and firm texture, in contrast to commonly translucent nasal polyps [8]. However, the macroscopic appearance of SIP lesions can be very heterogeneous, which highlights the importance of histopathology for differential diagnosis [9]. Treatment of choice for SIP is surgery; removing the lesion radically reduces the risk of recurrence [10]. For optimal access, radicality, and patient comfort, the endoscopic technique is preferred. The cause of SIP is poorly understood. Possible connections between SIP, occupational exposure to smoke [11] and chronic inflammation [12] have been studied. A report written by Dunn et al. [13] reported detection of EBV in 1 out of 25 SIP cases. Notably, the HPV prevalence in SIP has been reported to be as high as 37,8% [14]. HPV is strongly associated to SCCHN (squamous cell cancer of the head and neck) as well as oropharyngeal cancers, [15,16] but
https://doi.org/10.1016/j.anorl.2017.10.008 1879-7296/© 2019 Published by Elsevier Masson SAS.
Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008
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not to cancer of the mobile tongue [17]. The association between HPV, including the surrogate marker p16, and tumor development seems to be site-specific, and this has not been studied in SIP. If oncogenic HPV genotypes are strongly associated with p16 and SIP, this would affect both the clinical management of SIP patients in the operating theatre as well as the strength of the indication for preventive vaccination. The purpose of this study was to assess the prevalence and incidence of specific HPV genotypes and the HPV surrogate marker p16 in SIP samples in northern Sweden over time. We also aimed to assess HPV presence in SIP patients, SIP recurrence and development of malignancy, as well as p16 prevalence in regard to age. 2. Material and methods This retrospective cohort study included patients diagnosed with sinonasal inverted papilloma (SIP) between 1984-2014 at the University Hospital of Umeå. The study was approved by the Regional Ethical Review Board of Umeå University (Umeå, Sweden) and the Biobank North (County Council of Västerbotten, Sweden; approval nos. Dnr 2015-323-32 M (2012-379-31 M), 2015-10-19). Patient tissue samples were identified from 1984 to and including 2014. Biopsies from the pathology register at Clinical Pathology, University Hospital of Umeå, labeled with ICD-10 codes resembling inverted papilloma of the nose and sinus (80531/80531) and exophytic nasal papilloma/inverted urothelcellular papilloma (81210) were included. Exophytic nasal papilloma/inverted urothelcellular papilloma was included as a diagnosis since inverted sinonasal papillomas sometimes are labeled with this diagnosis. All biopsies were re-analyzed histopathologically to verify that no samples of exophytic nasal papilloma/inverted urothelcellular papilloma were included in this study. 2.1. Patients characteristics A total of 139 biopsies were identified as SIP. The 139 eligible samples belonged to 88 patients, 17 of which had more than one type of benign diagnosis (Fig. 1). In 2 cases, the first biopsy was too small, and the second registered biopsy was analyzed. Of the 88 biopsies, 19 were excluded due to insufficient material. Six biopsies were not included due to inaccessible material (second opinion referrals from another hospital), 6 due to old fragmented DNA samples, one due to the fact that the patient had been treated with radiotherapy in a different country. One patient was excluded due to analysis of wrong biopsy number (regarding analysis of HPV, p16), one due to none SIP histopathology and one because malignant conversion could be seen. This left a total of 54 biopsies stained with p16, and a total of 53 biopsies analyzed with PapilloCheck® (Fig. 1). Out of 54 analyzed patients, 16 were female (30%) and 38 were male (70%), and ages ranged from 18 to 88 with a mean of 59 years (infosup 1 et 2 supplementary Tables 1 and 2 Tables 1 and 2). The age of one patient was not registered. A minority of the patients (12/54) had more than 1 benign diagnosis registered. The diagnosis nasal inverted papilloma was found 37 times, whereas inverted papilloma of the sinus and inverted urothelcellular papilloma were used 24 and 6 times, respectively. For 19/54 (35%), there was more than 1 diagnosis registered with an interval of at least 100 days in between them, a cut- off time interval used to identify recurrence. 2.2. HPV detection and PCR Five 10 M sections/sample were cut from paraffin blocks. Thereafter the microtome and other accessories were cleaned with 70% alcohol and a fresh blade was installed before cutting each
block, in order to prevent cross contamination. After cutting, the sections were placed in 2.0 mL Eppendorf tubes and spinned briefly. DNA extraction was performed using the QIAamp® DNA FFPE Tissue Kit (QIAgen, Hilden, Germany). The process starts with deparaffinization with xylene and rehydration with 99.5% ethanol. After drying at 37 ◦ C in 10 minutes, tissue pellets were incubated with a solution of Proteinase K in ATL Buffer at 56 ◦ C overnight. On the second day, the samples were incubated at 90 ◦ C for 1 hour, after this a mixture of AL Buffer and 99.5% ethanol was added. After short centrifugation, the entire lysate was transferred to the QIAamp MinElute column, washed twice and centrifuged (13000 rpm, 3 min). DNA was eluted from the column by 100 l ATE buffer. Quantity of DNA was assessed spectrophotometrically and quality of DNA was measured based on its purity by spectrophotometric analysis of A260/280 ratio. In order to assess the degree of DNA fragmentation or integrity, amplifiability of housekeeping ß-globin genes of three different amplicon lengths (536 bp, 268 bp and 100 bp) were evaluated. For successful amplification and HPV typing a fragment size of about 350 bp was needed. Amplification of endogenous ß-globin DNA was performed by general PCR thermal cycling (M3000, Thermo Fisher Scientific, Waltham, USA), initiated with 3 min incubation at 95 ◦ C, followed by 40 cycles at 95 ◦ C for 30s, 72 ◦ C for 30s and 55 ◦ C for 1 min. The PapilloCheck® HPV-screening test system (Greiner BioOneGmbH, Frickenhausen, Germany) screens for 13 high risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), five possibly oncogenic HPV types (HPV 53, 66, 70, 73, and 82) and six HPV types that are considered being low risk types (HPV 6, 11, 40, 42, 43, and 44). The assay uses multiplex PCR with fluorescent primers to amplify a DNA fragment of about 350 nucleotides within the E1 open reading frame of the HPV genome. Amplification of an internal HPV template present in the PCR master-mix generates a signal on the PCR control spot on the chip. In addition, an internal PCR control which targets a region within the human ADAT-1 gene (adenosine deaminase tRNA specific 1) is used to assess cellularity. All samples were tested according to manufacturer’s instructions.
2.3. p16 immunohistochemistry and scoring system Detection of p16 was performed by staining in a Ventana staining machine (BenchMark ULTRA; Ventana Medical Systems, Tuscon, AZ, USA) following the supplier’s recommendations. Prior to staining, slides were pretreated in Tris-EDTA (10 mM Tris-HCl, 1 mM disodium EDTA; pH 8.0). After this, an antibody against p16 (monoclonal mouse anti-human; cat. no. sc-56330, Santa Cruz Biotechnology, Dallas/TX/USA) was used at a dilution of 1:200. The antibody was visualized using the Ultraview Universal DAB Detection Kit (Ventana Medical Systems) and staining was evaluated by using a light microscope (BX51; Olympus Corporation, Tokyo, Japan) and Spectrum online software (Spectrum Version 11.2.0.780 © Copyright 2006-2012 Aperio Technologies, Inc.). In order to score the percentage of cells expressing p16 and intensity of p16 staining, the quickscore system was used [18]. The proportion of cells expressing p16 was graded from 1 to 6 as follows: 1 = 0-4%, 2 = 5-19%, 3 = 20-39%, 4 = 40-59%, 5 = 60-79% or 6 = 80-100%. Staining intensity was evaluated with 4 grades: 0 = negative, 1 = weak, 2 = intermediate and 3 = strong. The product of the proportions’ score of cells expressing p16, multiplied by the intensity score equals a quickscore (QS) ranging from 0-18. Epithelium and inverted papilloma were scored separately. Two members of the research group scored the histopathological slides independently of one another. Thereafter scores were compared and consensus was reached.
Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008
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Fig. 1. Flowchart over patients biopsies analyzed with PapilloCheck® and p16 immunohistochemistry.
2.4. Analysis and statistics Recurrence was defined as two or more samples registered with an interval of 100 days or more between them. This interval was chosen to verify that the material actually resembled recurrence and not merely an initial diagnostic sample and material from following larger surgery. We did not have ethical permission to acquire more precise information regarding recurrence in patients’ medical records. In order to determine the oncogenic potential of SIP we analyzed a concomitant presence of malignancy in the sinonasal samples. Specifically, we searched for ‘squamous cell cancer of the nose’, ‘squamous cell cancer of the sinus’, ‘suspicious of squamous cell cancer of the nose’, ‘suspicious of squamous cell cancer of the sinus’, ‘recurrence of squamous cell cancer of the nose’ and ‘recurrence of squamous cell cancer of the sinus’ in our register of pathological diagnoses. Descriptive statistics are presented. A Pearson’s correlation analysis is performed and correlation coefficient presented for age
in regard to histopathological findings. A Poisson regression was used to describe illness incidences during the inclusion period, and across the regional population.
3. Results 3.1. HPV and sinonasal inverted papilloma/inverted urothelcellular papilloma The PapilloCheck® analysis for the qualitative type-specific identification of 24 HPV genotypes on SIP samples was performed successfully in all but one of the biopsies. Of 53 patients, 36 (68%) were negative for HPV and two were positive for HPV 11 (4%). Of the remaining, 15 (28%) biopsy analyses failed due to fragmented DNA (info suppl 2: Supplementary Table 2). The reason for this was old sample age. Thus, 2 out of 38 (5%) were successfully analyzed samples and HPV positive (info suppl 1: Supplementary Table 1).
Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008
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Table 1 p16 score in papilloma parts of the specimen for all 54 patients.
Table 4 p16 scores in epithelial parts of specimen with successful HPV analysis.
p16 in sinonasal inverted papilloma (SIP) # of patients p16 Intensity
Percent
0 2 4 6 8 10 12 15 18 Total
3,7 3,7 13,0 31,5 29,6 9,3 3,7 1,9 3,7 100,0
HPV
2 2 7 17 16 5 2 1 2 54
p16 epithelium
1 2 4 6 8 9 10 12 15 18
Total Table 2 p16 score in the epithelium of all 54 patients. p16 in the epithelium p16 Intensity
# of patients
Percent
1 2 4 6 8 9 10 12 15 18 Total
2 1 9 27 7 1 2 2 2 1 54
3,7 1,9 16,7 50,0 13,0 1,9 3,7 3,7 3,7 1,9 100,0
p16 SIP
0 2 4 6 8 10 12 15 18
Total
Total
Negative
HPV 11
1 1 5 11 11 4 1 1 1 36
0 1 1 0 0 0 0 0 0 2
HPV 11
1 0 7 19 4 1 1 1 2 0 36
0 0 1 0 1 0 0 0 0 0 2
1 0 8 19 5 1 1 1 2 0 38
and p16 in papilloma and epithelium were 0.13 and − 0.03, respectively. For patients with successful HPV analysis in which age was registered (n = 38), the Pearson r for age and p16 in papilloma and epithelium was 0.19 and − 0.08, respectively (Info suppl. 3: figures supplémentaires 1-4). 3.5. Recurrence There were 19 of 54 patients which had 2 or more further diagnoses registered with at least 100 days in between. One patient with HPV 11 had 1 diagnosis registered, the other had 2 diagnoses registered within 73 days. 3.6. Development of SCC
Table 3 p16 scores in papilloma parts of specimen with successful HPV analysis. HPV
Total
Negative
1 2 6 11 11 4 1 1 1 38
3.2. p16 expression Expression of p16 was scored in all 54 samples. This showed p16 expression in 52 papilloma lesions of the specimen, whereas the epithelium showed p16 in all 54 biopsies. The mean score in the SIP parts was 7.17 and 6.65 in the surrounding epithelium (Tables 1 and 2). 3.3. p16 expression and HPV When separately analyzing p16 expression in the 38 samples with successful HPV analysis, p16 was present in the epithelia in all samples and in the papilloma lesions in 37 samples (Tables 3 and 4). Mean score for p16 in papilloma lesions for HPV negative and HPV positive specimens was 7.25 and 3.00, respectively. Mean value for p16 in SIP epithelium for HPV negative and HPV positive specimens were 6.56 and 6.00, respectively.
One patient diagnosed with inverted papilloma of the nose and sinus also had an initial sinonasal SCC diagnosis, a second nasal SCC diagnosis in this patient was negative for HPV. One patient with inverted urothelcelluclar papilloma was initially diagnosed with ‘suspicious of nasal SCC’, which was later confirmed. There was an insufficient amount of benign material for analysis, and the malignant biopsy was not further studied. One patient diagnosed with inverted urothelcellular cancer of the nose was initially registered as nasal SCC. The first registered specimen, was categorized as both benign and malignant, and was positive for HPV 18. One HPV negative patient with inverted papilloma of the nose and sinus and inverted urothelcellular papilloma was diagnosed with ‘suspicious of nasal SCC’ but no confirming malignant transformation was registered. 3.7. Development of SIP incidence We had no information regarding patients’ residence apart from the fact that 6 patients’ biopsies were sent from the county of Norrbotten to Umeå for second opinion. Nevertheless, we wanted to study the incidence rates over time. We chose to assume that patients during this time period were referred to Umeå from the northern region (counties of Västerbotten, Västernorrland, Norrbotten, Jämtland), and that the per capita rate in each county is the same. Poisson regression was performed with time as the covariate and using the relative population size of Västerbotten as an offset. Here, the incidence rate was estimated to increase with 7.6% per year (approximate 95% Confidence Interval: 4.7%-10.6%). 4. Discussion
3.4. p16 and age Age was registered in 53 of 54 patients. In patients tested for HPV in which age was registered (n = 53), the Pearson r for age
The purpose of this study was to assess the prevalence of specific HPV genotypes and the HPV surrogate marker p16 in SIP samples in northern Sweden over time. We also wanted to know if there
Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008
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is a connection between HPV in SIP patients and the tendency for development of malignancy and recurrence. We analyzed 53 biopsies with PCR, and 54 biopsies with immunohistochemistry that stained for p16. 38 PCR analyses succeeded whereas all 54 samples could be tested for p16. Out of 38 PCR samples 2 were positive for HPV 11. In these 38 samples p16 in epithelia was present in all cases and p16 in papilloma in 37 cases. Our results indicate that there is no correlation between p16 and prevalence of HPV in SIP. These findings support the idea that the surrogate marker p16 used for detection of HPV is organ specific [16,17] and that p16 cannot be used in the detection of HPV in SIP. Out of 3 specimens diagnosed with both inverted papilloma and nasal SCC, the first sample was negative for HPV, the second was positive for HPV 18 and the third was not analyzed. During our study period (1984-2014) SIPs incidence rate increased by 7.6%. It has to be noted that research on the causative agents of SIP so far have not been conclusive, though many different potential risk factors (e.g. EBV, chronic inflammation, exposure to occupational smoke) are suspected to play a role. In our SIP study, only 2 out of 38 samples (5%) were HPV positive. These findings diverge from those presented by Syrjanen et al. which observed a prevalence of HPV in SIP of 37.8% [14]. The reason for this disparity might be due to sample sizes, disease classification, or detection methods. Sample sizes of patients with SIP in Syrjanen’s meta-analysis ranged from case reports [19] to larger studies with 101 [20] cases and detection rates varied from 0%-100% [14]. In Syrjanen’s metaanalysis, different types of detection methods were used (PCR, in situ hybridization, southern blot hybridization and dot blot hybridization) which is, along with varying geographic locations, a plausible reason for the large variation in HPV detection outcome. Considering the clinical applicability of mapping HPV status in oropharyngeal SCC with PapilloCheck® , that provides a sensitivity of 89% and 92.5% for CIN2+ (cervical intraepithelial neoplasia) and CIN3+ respectively [21], along with a high level of reliability for detection of 6 low- and 18 high-risk HPV types, we were encouraged to proceed with this analysis. Although this study indicates that SIPs generally express low numbers of HPV, p16 was expressed throughout the epithelia in all samples and in the papilloma parts in all but two samples. p16 is often used as a surrogate marker for high risk HPV, as it is overexpressed by a negative feedback loop when HPV E7 binds to pRb (retinoblastoma protein). However, increased levels of p16 could also originate from p16 pathway changes that are independent of HPV [22]. LaPak et al. discussed infection among other causes as source of p16 elevation [22]. In contrast to previous studies revealing that p16 could be used as a marker for senescence [23], we were unable to correlate p16 expression to age. Lin et al. [24] found that p16 expression usually is preserved in SIP, whereas p16 expression is more likely to be lost when the papilloma lesions undergo malignant change. A ‘hit and run’ hypothesis has previously been postulated. This theory implies that HPV may induce SIP formation, however, the non- keratinizing SIP may not be capable of harbouring an HPV infection [25]. The correlation between the HPV surrogate marker p16 and HPV is clearly site specific in the head and neck region, and in this sense SIP mucosa resembles SCC mucosa in the mobile tongue [17]. Although SIP is a histologically benign disease, it exhibits features similar to mobile tongue cancer, such as properties of local invasion, regional relapse rate, HPV negativity and lack of correlation between p16 and HPV.
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5. Conclusion In summary, the study outcome suggests an incidence increase with 7.6% per year. To assess the etiology of sinonasal inverted papilloma (SIP), we studied the presence of human papilloma virus (HPV) and its surrogate marker p16 in 54 retrospectively-identified SIP patients using PCR and immunohistochemistry. Out of 38 successfully analyzed samples, only 2 samples were positive for HPV 11 whereas p16 was expressed in 37 papilloma parts of the samples and in all epithelial parts of the samples. We conclude that p16 cannot be used as a surrogate marker for high-risk HPV-infection in SIP. Our research group is currently planning more rigorous prospective, multicenter studies in order to further map the observed HPV-negative and p16 positive findings in SIP. We are especially interested in studying p16 levels in healthy sinonasal mucosa. Disclosure of interest The authors declare that they have no competing interest. Acknowledgements This investigation was supported by grants from Lion’s Cancer Research Foundation (KO), Umeå University and Västerbotten County Council (KO), A Allard, I Eriksson, A Holm and K Nylander performed the experiments, K Olofsson, A Holm, G Laurell, K Nylander designed the study; A Holm, A Allard, K Olofsson, K Nylander and G Laurell wrote the paper. Michael Haney assisted with text revision. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.anorl.2017.10.008. References [1] Outzen KE, et al. Inverted papilloma: incidence and late results of surgical treatment. Rhinology 1996;34(2):114–8. [2] Mirza S, et al. Sinonasal inverted papillomas: recurrence, and synchronous and metachronous malignancy. J Laryngol Otol 2007;121(9):857–64. [3] Shanmugaratnam K, Sobin LH. The World Health Organization histological classification of tumours of the upper respiratory tract and ear. A commentary on the second edition. Cancer 1993;71(8):2689–97. [4] van Samkar A, Georgalas C. Long-term quality of life after endoscopic removal of sinonasal inverted papillomas: a 6-year cohort analysis in a tertiary academic hospital. Eur Arch Otorhinolaryngol 2016;273(6):1433–7. [5] Vrabec DP. The inverted Schneiderian papilloma: a clinical and pathological study. Laryngoscope 1975;85(1):186–220. [6] Bewes T, et al. Incidence of neoplasia in patients with unilateral epiphora. J Laryngol Otol 2015;129:S53–7. [7] Bhandary S, et al. Sinonasal inverted papilloma in eastern part of Nepal. Kathmandu Univ Med J (KUMJ) 2006;4(4):431–5. [8] Barnes L. Schneiderian papillomas and nonsalivary glandular neoplasms of the head and neck. Mod Pathol 2002;15(3):279–97. [9] Tritt S, McMains KC, Kountakis SE. Unilateral nasal polyposis: clinical presentation and pathology. Am J Otolaryngol 2008;29(4):230–2. [10] Lombardi D, et al. Limitations and complications of endoscopic surgery for treatment for sinonasal inverted papilloma: a reassessment after 212 cases. Head Neck 2011;33(8):1154–61. [11] Deitmer T, Wiener C. Is there an occupational etiology of inverted papilloma of the nose and sinuses? Acta Otolaryngol 1996;116(5):762–5. [12] Papon JF, et al. Matrix metalloproteinase-2 and -9 expression in sinonasal inverted papilloma. Rhinology 2006;44(3):211–5. [13] Dunn ST, et al. Survey of sinonasal inverted papillomata for Epstein-Barr virus. Head Neck 1997;19(2):98–106. [14] Syrjanen K, Syrjanen S. Detection of human papillomavirus in sinonasal papillomas: systematic review and meta-analysis. Laryngoscope 2013;123(1):181–92. [15] Marur S, et al. HPV-associated head and neck cancer: a virus-related cancer epidemic. Lancet Oncol 2010;11(8):781–9. [16] Loizou C, et al. Incidence of tonsillar cancer in northern Sweden: Impact of human papilloma virus. Oncol Lett 2015;10(6):3565–72.
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Please cite this article in press as: Holm A, et al. Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma. European Annals of Otorhinolaryngology, Head and Neck diseases (2019), https://doi.org/10.1016/j.anorl.2017.10.008