Oral Oncology 50 (2014) 241–246

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Patients with HPV-related tonsil squamous cell carcinoma rarely harbour oncogenic HPV infection at other pharyngeal sites q Selvam Thavaraj a,⇑, Angela Stokes a, Kazuya Mazuno b, Rhonda Henley-Smith c, Yae-eun Suh c, Vinidh Paleri d, Mahvash Tavassoli e, Edward Odell a, Max Robinson f a

Oral Pathology, King’s College London Dental Institute, London, United Kingdom Department of Oral Pathology, Osaka Dental University, Osaka, Japan Oral Pathology, Guy’s and St. Thomas’ NHS Foundation Trust, London, United Kingdom d Otolaryngology-Head and Neck Surgery, Newcastle upon Tyne Hospitals NHS Trust, United Kingdom e Molecular Oncology, King’s College London Dental Institute, London, United Kingdom f Centre for Oral Health Research, School of Dental Sciences, Newcastle University, Newcastle Upon Tyne, United Kingdom b c

a r t i c l e

i n f o

Article history: Received 15 August 2013 Received in revised form 27 November 2013 Accepted 14 December 2013 Available online 13 January 2014 Keywords: Oropharynx Tonsil Squamous cell carcinoma Human papillomavirus Field cancerisation

s u m m a r y Objectives: Patients with human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) have a reduced risk of developing second primary upper aerodigestive tract (UADT) tumours compared to patients with HPV-negative primary tumours at the same site. To determine whether this finding might be explained by a lack of viral-induced field cancerisation or multifocal infection, we investigated whether there was epithelial dysplasia and/or evidence of HPV infection at other pharyngeal mucosal sites in patients presenting with the disease. Materials and methods: Sixty-three patients with primary tonsil SCC and 108 pharyngeal endoscopic biopsies, representing at least one pharyngeal subsite from each patient, were included in this study. Tissue samples were tested using HPV PCR (GP5+/6+), p16 immunohistochemistry (IHC) and high risk HPV DNA in situ hybridisation (ISH). Results: There were 46 patients with HPV-related SCC and 17 patients with HPV-negative disease. PCR detected HPV DNA in a fifth of pharyngeal endoscopic biopsies and was equally likely to be from a patient with HPV-related SCC as from a patient with HPV negative disease. All PCR positive cases were tested using p16 IHC and high risk HPV ISH and only three biopsies were positive. Significantly, these three biopsies all showed evidence of epithelial dysplasia and were from patients with an HPV positive index tumour. Conclusion: Our data suggest that virus-induced field cancerisation and/or multifocal oncogenic HPV infection of the pharynx is uncommon in OPSCC and supports the concept that these patients have a lower risk of developing second primary tumours of the UADT. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Introduction The majority of head and neck squamous cell carcinomas (HNSCC) are associated with tobacco and alcohol use and result from cumulative genetic damage and epigenetic changes in mucosal keratinocytes. These molecular events are known to develop across wide areas of the upper aerodigestive tract (UADT) mucosa

q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Address: Oral Pathology, Floor 28, Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, United Kingdom. Tel.: +44 207 188 8729. E-mail address: [email protected] (S. Thavaraj).

and underpin the concept of ‘field cancerisation’ [1]. Field cancerisation is thought to contribute to poor outcome in patients with HNSCC because patches of altered UADT mucosa persist following ablative treatment and give rise to second primary tumours. It is estimated that up to 27% of patients with HNSCC develop second primary tumours [2]. However, there is accumulating evidence that patients with a subtype of HNSCC, namely HPV-related oropharyngeal squamous cell carcinoma (OPSCC), have a reduced risk of developing second primary tumours [3–9], a factor that is likely to contribute to the improved overall and disease-specific survival in these patients. The natural history of oncogenic HPV infection in the head and neck region is unknown. Head and neck mucosal HPV carriage rates of between 7% and 31% have been reported in normal individuals [10–12]. Whilst the majority of infected individuals clear the

1368-8375/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.oraloncology.2013.12.012

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virus and do not develop cancer, the factors that contribute to viral-induced carcinogenesis remain to be elucidated. In particular, it is not known whether HPV-related oropharyngeal carcinoma develops in areas of ‘viral-induced’ field cancerisation. In this context, the concept of field cancerisation requires a broader definition. Unlike fields induced by topical carcinogens, HPV-induced carcinomas are thought to arise within reticular crypt epithelia [13] and primarily affect the palatine tonsils, base of tongue and other head and neck mucosal sites with mucosal associated lymphoid tissue. Therefore, in addition to a continuous pre-malignant mucosal field, a risk of second primary tumour could also arise as a result of multifocal infection with the virus. The aim of this study was to determine whether HPV-related OPSCCs arise in fields and/or multifocal areas of dysplastic or HPV infected UADT mucosa. Materials and methods Patients and samples This study had a favourable opinion from the UK National Research Ethics Service (Reference: 10/H0701/27). Patients were retrospectively identified from pathology databases. The inclusion criteria were (1) patients with a primary index tonsil SCC who had UADT endoscopy yielding at least one additional biopsy from another pharyngeal mucosal site (termed ‘pharyngeal endoscopic biopsies’) and (2) the index tumour biopsy and the pharyngeal endoscopic biopsies were harvested at the same operation. Haematoxylin and eosin stained sections from all specimens were reviewed by two pathologists (ST & MR) and assessed for sufficient tissue for HPV testing and histopathological abnormalities. HPV testing Detection of HPV DNA by polymerase chain reaction DNA was extracted from 25 lm sections of formalin-fixed paraffin embedded (FFPE) tissue using the QIAamp DNA (FFPE) tissue kit (Qiagen, UK) according to the manufacturer’s instructions. Contamination of samples with extrinsic DNA was controlled by cleaning the microtome with xylene and discarding the microtome blade between each case. Polymerase chain reactions (PCR) were carried out in a Clinical Pathology Accreditation UK approved diagnostic pathology laboratory using standard operating procedures to prevent sample contamination. All DNA samples were screened for the human b-actin gene by PCR to verify the presence of amplifiable DNA. HPV DNA was amplified by PCR using the improved general primer set GP5+/6+, which amplifies part of the HPV L1 gene encoding the HPV major capsid protein, detecting low-risk genotypes-6, -11, -40, -42, -43 and -44 and high-risk genotypes16, -18, -31,-33, -35, -39, -45, -51, -52, -56, -58, -59, -66 and -68 as previously described [14]. DNA from an HPV16 positive OPSCC-derived cell line (UPCI:SCC090) was used as a positive control. DNA from an HPV negative oropharyngeal SCC-derived cell line (UPCI:SCC089) and omission of template DNA were used as negative controls. Both OPSCC-derived cell lines were a kind gift from Professor Susanne Gollin, University of Pittsburgh. Where there was no detectable b-actin band on DNA resolving gels, the PCR reaction was repeated and the products were assessed on an automated microfluidics-based platform (Bioanalyzer 2100, Agilent Technologies, UK) according to manufacturer’s instruction, which is able to detect amplicon concentrations as low as 1 ng/ml. The PCR DNA resolving gels and digital readout from the automated microfluidics-based platform were examined by two experienced laboratory scientists and a consensus opinion was reached on the presence or absence of an amplicon of appropriate size.

p16 Immunohistochemistry p16 Immunohistochemistry (IHC) was carried out using a proprietary kit (CINtec Histology, mtm Laboratories AG, Germany) on a Ventana Benchmark Autostainer (Ventana Medical Systems Inc., USA), as previously described [15]. A tonsil SCC with high p16 expression was used as a positive control. The primary antibody was omitted from negative controls. p16 IHC was scored using recently described criteria and thresholds; strong nuclear and cytoplasmic expression in greater than 70% of the tumour and an H score greater than 60 [16]. High-risk HPV DNA In-situ hybridisation High-risk HPV DNA in-situ hybridisation (ISH) was carried out using proprietary reagents (Inform HPV III Family 16 Probe (B), Ventana Medical Systems Inc, USA) on a Benchmark Autostainer (Ventana Medical Systems Inc. UK), as previously described [15]. The Inform HPV III Family 16 Probe (B) detects high-risk genotypes HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58 and -66. Three control samples were used: FFPE CaSki cells (HPV-16-positive; 200-400 copies per cell), HeLa cells (HPV-18-positive; 10-50 copies per cell) and C-33A (HPV-negative; Ventana Medical Systems Inc., USA). The high-risk HPV ISH test was scored as positive if there was any blue reaction product that co-localised with cell nuclei. Diffuse nuclear and cytoplasmic (‘episomal’ pattern) staining and punctate nuclear (‘integrated’ pattern) staining were scored as positive. Focal specific staining of only part of the tumour section was regarded as positive. Pale staining limited to the nucleoli of cells and staining of occasional leucocytes and stromal cells were also disregarded, in line with the manufacturer’s instructions. Statistical analysis Fisher’s exact test was used to evaluate correlation between different groups. P values below 0.05 were regarded as significant. All data was analysed using SPSS software version 20. Results Patients and specimens Sixty-three patients fulfilled the inclusion criteria; an index tonsil SCC and at least one pharyngeal endoscopic biopsy. Of the 63 patients presenting with tonsil SCCs, 62 were primary and 1 was a recurrent tumour. There were 48 males and 15 females (M:F ratio 3.2:1). The mean age was 55 years old (Range 38-60 years old). There were 45 patients with an HPV-related SCC (p16 IHC positive, high risk HPV DNA ISH positive) and 18 patients with an HPV negative SCC (Supplementary Table 1). There were a total of 108 pharyngeal endoscopic biopsies; 81 from patients with HPV-related SCC and 27 from patients with HPV negative SCC. The site

Table 1 Site distribution of pharyngeal endoscopic biopsies from patients with an index tonsil squamous cell carcinoma. HPV positive tonsil SCC 46 patients 81 endoscopic biopsies

HPV negative tonsil SCC 17 patients 27 endoscopic biopsies

Site

Frequency

Site

Frequency

Contra-lateral tonsil Tongue base Soft palate Oropharynx NOS Nasopharynx Hypopharynx Total

17 30 1 4 24 5 81

Contra-lateral tonsil Tongue base Soft palate Oropharynx NOS Nasopharynx Hypopharynx Total

5 11 3 4 2 2 27

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distributions of the pharyngeal endoscopic biopsies are shown in Table 1. Histopathological review of the pharyngeal endoscopic biopsies demonstrated that the majority (105 of 108; 97%) did not show any significant morphological abnormality. Three specimens showed severe epithelial dysplasia (Fig. 1A and B). None of the patients had synchronous UADT tumours. The patients were followed up for a mean period of 48 months (range 17–108 months; Supplementary Table 1). HPV testing of pharyngeal endoscopic biopsies The majority (78 of 108; 72%) of pharyngeal endoscopic biopsies yielded PCR amplifiable DNA; human b-actin positive. The majority (62 of 78; 79%) of samples yielding satisfactory DNA did not show any evidence of HPV DNA following PCR with high sensitivity GP5+/6+ primers (Fig. 2). GP5+/6+ PCR amplicons were detected in around a fifth of samples (16 of 78; 21%) and was equally likely to be from a patient with HPV-related SCC (12 of 59; 20%) as from a patient with HPV negative disease (4 of 19; 21%, p = 1.00, Fisher’s exact test, Fig. 3). All specimens (n = 16) that were positive for HPV by PCR were subject to p16 IHC and highrisk HPV DNA ISH. The majority of specimens (12 of 16) were p16 negative and high risk HPV ISH negative. Four biopsies were p16 positive and three showed evidence of high risk HPV DNA by ISH. The biopsies that were consistently positive across the HPV tests (GP5+/6+ PCR, p16 IHC, high risk HPV ISH) all showed evidence of severe epithelial dysplasia (p = 0.02, Fisher’s exact test) and were derived from three individuals with HPV-related index tumours (Fig. 1). The case that was GP5+/6+ PCR positive, p16 IHC positive, high-risk HPV DNA ISH negative was from a patient with an HPV-related index tumour, but the biopsy showed severe

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thermal damage that precluded accurate microscopic analysis (data not shown). Discussion The majority of HNSCCs arise in a background of altered mucosa (field cancerisation) [1]. These pre-malignant ‘fields’ of altered mucosa may be identified morphologically as epithelial dysplasia, but phenotypic changes may be preceded by genetic or epigenetic aberrations that are not identifiable by conventional transmission light microscopy [17,18]. The persistence of patches of pre-malignant UADT mucosa following ablative therapy are thought to be an important cause for the high rates of recurrence, second primary tumours, and consequently poor overall survival in these patients [17,18]. By contrast, HPV-related OPSCC, a clinically and genetically distinct subtype of HNSCC, demonstrates significantly improved survival compared to HPV-negative tumours at the same site [4]. However, the mechanisms for improved outcome in this subgroup of HNSCC are still largely unknown. Furthermore, despite the rise in the incidence of HPV-related OPSCC, little is known about the natural history of this disease. Therefore, this study sought to determine whether HPV-related OPSCC arise in areas of virally-induced field change within the UADT mucosa and/or as a consequence of multifocal infection with the virus. There is compelling evidence that the incidence of second primary tumours in patients with HPV-related OPSCC is significantly lower than those with HPV-negative disease [3–9]. Nevertheless, the occurrence of synchronous and/or metachronous HPV-positive OPSCC, albeit at a lower rate, raises the possibility of viral-induced field cancerisation [19–21]. HPV-related SCC develops most frequently within the reticular crypt epithelium of mucosal

Figure 1. Photomicrographs of a pharyngeal endoscopic biopsy showing a focus of severe epithelial dysplasia (A and B). There is over-expression of p16 by immunohistochemistry (C) and evidence of high-risk HPV by in-situ hybridisation (D).

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Figure 2. HPV-related squamous cell carcinoma of the right tonsil (A) showing over-expression of p16 by immunohistochemistry (B) and evidence of high-risk HPV by in-situ hybridisation (C). Biopsies from the left tonsil (D), left tongue base (E) and right tongue base (F) do not show any significant abnormality. (G) A resolving gel showing b-actin and GP5+/6+ PCR products from the same case, only the index tumour from the right tonsil and the positive control contain HPV DNA.

associated lymphoid tissue within palatine tonsil and the base of tongue. Whilst the precise mechanism of HPV infection is yet to be established, these sites are thought to be more prone to infection because of increased permeability or active antigen uptake within this specialised mucosa. Mechanical disruption is assumed not to be important as the majority of tumours appear to develop deep in the base of the tonsillar crypts [22]. There are focal, smaller amounts of tonsillar tissue present throughout Waldeyer’s ring and also at ectopic sites within the oral cavity, pharynx and supraglottic larynx, which may represent additional portals of entry for HPV infection. It is also conceivable that ‘non-specialised’ mucosal surfaces could be subject to micro-abrasions thereby facilitating infection of basal keratinocytes as described for the uterine cervix [23]. Whilst keratinocytes with persistent HPV infection may spread laterally from tonsillar crypts to form wider areas of HPV related field change, the risk of second primary HPV-related carcinoma could also be a consequence of independent multifocal infection at other mucosal sites [21]. There is a paucity of studies that have sought to determine whether HPV is present in wider UADT mucosa of patients with OPSCC. Begum et al. [24] undertook p16 IHC and HPV DNA ISH on the contra-lateral uninvolved tonsil in eight patients with primary HPV-related tonsil carcinomas and identified p16 overexpression and ISH positivity in one of eight cases. Interestingly, and in agreement with our findings, p16 over-expression in combination with high-risk HPV DNA by ISH was associated with morphological evidence of epithelial dysplasia [24]. These data suggest that pathologist review of pharyngeal endoscopic biopsies

by conventional microscopy is likely to be sufficient to screen for multifocal HPV infection, which can then be confirmed by conventional laboratory testing (p16 IHC and high risk HPV ISH/HPV PCR). In a recent study, Rietbergen et al. [25] evaluated the tissue margins of HPV-positive OPSCCs treated by surgical resection. They found no evidence of transcriptionally active virus in UADT mucosa surrounding these tumours. Interestingly, in a separate study, Laborde et al. [26], demonstrated HPV DNA in primary OPSCC, corresponding metastatic disease and matched normal tissue. However, in agreement with Rietbergen et al. [25], evidence of viral transcription (E6 and E7) and attendant increased p16 expression was only present in the samples containing tumour. The authors therefore concluded that the HPV DNA in matched normal tissue represents latent infection [26]. We were also able to demonstrate HPV DNA in clinically normal pharyngeal mucosal biopsies; however, rates of HPV detection were similar in patients with HPV-related OPSCC and HPV negative OPSCC. Furthermore, the likelihood of detecting HPV DNA was similar to that reported previously for healthy subjects [10,12], suggesting that clinically healthy mucosa in patients with tonsil cancer do not harbour the virus at rates higher than the normal population. Interestingly, when we tested the PCR-positive pharyngeal biopsies using high-risk HPV DNA ISH and p16 IHC, only three were consistently positive across the laboratory tests employed. This implies that in the majority of cases any detectable viral DNA is likely to be of low-risk genotypes and/or present in very low copy numbers, perhaps as a transient or latent infection [25]. Consequently, detection of HPV DNA alone is unlikely to have clinical utility for identifying individuals at-risk of

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Figure 3. DNA extracted from pharyngeal endoscopic biopsies from patients with an index tonsil squamous cell carcinoma were tested for the presence of amplifiable DNA (b-actin PCR) and screened for HPV DNA using high sensitivity GP5+/6+ PCR. Screen detected HPV positive biopsies were tested using p16 immunohistochemistry and high risk HPV DNA in situ hybridisation.

developing HPV-related progression.

OPSCC

or

in

monitoring

disease

Conclusion The results of this study show that around a fifth of patients with tonsil SCC harbour HPV DNA in the pharynx and the detection rates of HPV DNA were similar between patients with HPV-positive and HPV-negative index tumours. Oncogenic HPV infection, as determined by the presence of high risk HPV DNA and p16 overexpression, was always associated with an HPV-positive index tumour, but was a rare event; less than 3% of biopsies tested, representing less than 5% of patients in this study. Furthermore, oncogenic HPV infection was always accompanied by microscopic evidence of epithelial dysplasia. These data suggest that virus-induced field cancerisation and/or multifocal oncogenic HPV infection of the pharynx may be uncommon in HPV-related OPSCC and it could account for the lower risk of developing a second primary tumour, longer disease free periods and improved overall survival for these patients [3–9]. Conflict of interest statement None declared. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.oraloncology. 2013.12.012.

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