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Oral Oncology 50 (2014) 241246

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Patients with HPV-related tonsil squamous cell carcinoma rarely


harbour oncogenic HPV infection at other pharyngeal sites
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, Kings College London Dental Institute, London, United Kingdom
Department of Oral Pathology, Osaka Dental University, Osaka, Japan
Oral Pathology, Guys 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, Kings 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
nding might be explained by a lack of viral-induced eld 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 fth 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. Signicantly, 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 eld 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.
2013 The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.

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

Corresponding author. Address: Oral Pathology, Floor 28, Tower Wing, Guys
Hospital, Great Maze Pond, London SE1 9RT, United Kingdom. Tel.: +44 207 188
8729.
E-mail address: selvam.thavaraj@kcl.ac.uk (S. Thavaraj).

and underpin the concept of eld 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 [39], a factor that is likely
to contribute to the improved overall and disease-specic 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 [1012]. Whilst the majority of infected individuals clear the

1368-8375 2013 The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.
http://dx.doi.org/10.1016/j.oraloncology.2013.12.012

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S. Thavaraj et al. / Oral Oncology 50 (2014) 241246

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 eld cancerisation. In this context, the concept of eld cancerisation requires a broader denition. Unlike elds 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 eld, 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 elds 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 identied 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 sufcient
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-xed parafn embedded (FFPE) tissue using the QIAamp DNA (FFPE) tissue
kit (Qiagen, UK) according to the manufacturers 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 ampliable DNA. HPV DNA was amplied by PCR using the improved
general primer set GP5+/6+, which amplies 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 microuidics-based platform (Bioanalyzer 2100, Agilent
Technologies, UK) according to manufacturers 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
microuidics-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 specic 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 manufacturers instructions.
Statistical analysis
Fishers exact test was used to evaluate correlation between different groups. P values below 0.05 were regarded as signicant. All
data was analysed using SPSS software version 20.
Results
Patients and specimens
Sixty-three patients fullled 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

S. Thavaraj et al. / Oral Oncology 50 (2014) 241246

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 signicant 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
17108 months; Supplementary Table 1).
HPV testing of pharyngeal endoscopic biopsies
The majority (78 of 108; 72%) of pharyngeal endoscopic biopsies yielded PCR ampliable 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 fth 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, Fishers 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, Fishers 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

243

thermal damage that precluded accurate microscopic analysis


(data not shown).
Discussion
The majority of HNSCCs arise in a background of altered mucosa
(eld cancerisation) [1]. These pre-malignant elds of altered mucosa may be identied morphologically as epithelial dysplasia, but
phenotypic changes may be preceded by genetic or epigenetic
aberrations that are not identiable 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 signicantly
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 eld 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 signicantly
lower than those with HPV-negative disease [39]. Nevertheless,
the occurrence of synchronous and/or metachronous HPV-positive
OPSCC, albeit at a lower rate, raises the possibility of viral-induced
eld cancerisation [1921]. 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).

244

S. Thavaraj et al. / Oral Oncology 50 (2014) 241246

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 signicant 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 Waldeyers 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 eld
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 identied p16 overexpression and ISH positivity in one of eight cases. Interestingly,
and in agreement with our ndings, 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 sufcient to screen for


multifocal HPV infection, which can then be conrmed 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

S. Thavaraj et al. / Oral Oncology 50 (2014) 241246

245

Figure 3. DNA extracted from pharyngeal endoscopic biopsies from patients with an index tonsil squamous cell carcinoma were tested for the presence of ampliable 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 fth 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 eld 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 [39].
Conict 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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