Oral Oncology 38 (2002) 227–234

www.elsevier.com/locate/oraloncology

Review

Oral squamous cell carcinoma; from an hypothesis about a virus, to

concern about possible sexual transmission
Crispian Scully*
International Centres for Excellence in Dentistry, and Eastman Dental Institute for Oral Health Care Sciences UCL, University of London,
256 Gray’s Inn Road, London WC1X 8LD UK

Received 25 July 2001; accepted 1 August 2001

Abstract
Nearly two decades ago, we produced the first evidence for the presence of viral nucleic acids in oral squamous cell carcinoma
(OSCC) tissues, hypothesising that there may be a viral involvement in at least some OSCC. Subsequently, human papilloma-
viruses (HPV) in particular have been implicated in OSCC. Antibody responses to HPV are seen and HPV-DNA detected in
tumors by us and many others, the virus being mainly HPV-16, the genotype associated with ano-genital cancer. HPV are seen by
in situ hybridisation only in tumour and premalignant tissue but not in surrounding normal mucosa suggesting HPV has a causal
relationship. HPV may also be integrated in the host genome, further suggesting a causal role. Studies of patients with OSCC
have suggested possible sexual transmission of HPV. Recent studies have indicated that HPV may be aetiologically important
particularly in some types of oropharyngeal cancer, at least in tonsillar carcinogenesis, and may represent an alternative pathway
in carcinogenesis to the established factors of tobacco and alcohol. We have come a very long way in the two decades since our
first suggestion of a viral aetiopathogenesis was greeted with incredulity, and data from on-going studies by the International
Agency for Research on Cancer, Johns Hopkins Oncology Center and others are eagerly awaited. # 2002 Elsevier Science Ltd.
All rights reserved.
Keywords: Oral cancer; Viruses; Papillomavirus

1. Introduction mucosa suggested an association with HSV [5,6]. Others

There is substantial evidence that oral squamous cell
carcinoma (OSCC) is usually aetiologically linked with
tobacco and/or alcohol or betel use, but there are clearly
patients who develop OSCC in the absence of exposure
to these, and in the absence of any obvious predisposing
genetic defect [1–3]. Microbial agents are possible
aetiological agents [4] and nearly two decades ago, we
produced the first evidence for the presence of viral
nucleic acids in OSCC tissues, hypothesising that there
may be a viral involvement in at least some OSCC [5–7]
as in ano-genital cancer [8,9]. This has been the subject
of a number of subsequent reviews [7,10–22].
This first study in the field examined OSCC for herpes
simplex virus (HSV). Our demonstration of in situ
hybridisation of RNA complementary to HSV-DNA in
OSCC but not to autologous, clinically normal oral
* Tel.: +44-2079-151-038; fax: +44-2079-151-039.
E-mail address: c.scully@eastman.ucl.ac.uk (C. Scully).
1368-8375/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S1368-8375(01)00098-7
have since demonstrated HSV-1 DNA in OSCC [23]. A
number of studies have shown changes in levels of
serum antibodies to HSV patients with OSCC [23–27].
There is a higher reactivity to the HSV immediate early
protein ICP4 in patients with OSCC, suggesting a dif-
ferent course of an earlier herpetic infection, with a
prolonged exposure to early immediate proteins of
HSV as a consequence of smoking [24]. Evidence
of HSV viral ‘‘footprints’’ has thus given interesting
results, though this is not proof of a causal relationship
since HSV antibodies are common in the apparently
healthy general population, and hybridisation could also
be revealing segments of normal host nucleic acid with
homology to part of the HSV genome. In contrast, fail-
ure to demonstrate HSV products would not, of course,
exclude a hit and run mechanism of carcinogenesis. In
one US epidemiological study, the relative risks for
OSCC associated with serologically detected HSV-1 and
HSV-2 infections were 0.8 (95% CI 0.3–1.7) and 1.8 (CI
0.7–4.6), respectively [28] but, although those infected
228 C. Scully / Oral Oncology 38 (2002) 227–234
with the mainly genital virus HSV-2 were at elevated
risk of OSCC, these associations may have been due to
chance.
Certainly, HSV can be an oncogenic virus. HSV-1 is
capable of transforming cells in vitro [29] provided
cytolysis is inhibited by factors such as ultraviolet light
[29] or certain chemicals [30]. In some in vitro systems
such as SV40-transformed hamster embryo cells, HSV is
more effective than are some chemical carcinogens in
amplifying SV40 DNA sequences [31,32], acting via
HSV-encoded DNA polymerase [32,33]. Several reports
indicate that HSV can act synergistically with chemical
carcinogens in causing oncogenic transformation [34–
36]. In vitro HSV induces chromosomal aberration,
mutations, and gene amplification, and in the hamster
cheek pouch model of dimethylbenzanthracene-induced
carcinogenesis enhances erb-B1 oncogene amplification
and overexpression [37], a feature that coincides with
the appearance of malignancy. Animal studies also sug-
gest that HSV may be carcinogenic, acting as a co-
carcinogen with tobacco or other chemicals [37–40]
and that immunisation against HSV prevents the co-
carcinogenic activity of HSV with dimethylbenzan-
thracene [41].
Substantial evidence suggests therefore, that HSV
might, under particular circumstances, be oncogenic
[7,12,24,42–44] but further studies are needed to clarify
any causal role of HSV in oral carcinogenesis. More-
over, carcinogenesis is not a single step procedure with a
single aetiology and it has been suggested that HSV may
act synergistically in carcinogenesis with other factors,
including with human papillomaviruses (HPV). With
regards to cervical carcinoma, epidemiological evidence
indicates that this may be possible and, in experimental
situations, it has been demonstrated that keratinocytes
immortalised by HPV genotype 16 DNA are tumori-
genic in nude mice following transfection with HSV-
DNA [45,46].
HPV are small epitheliotropic DNA viruses that can
induce hyperplastic, papillomatous, and verrucous
squamous cell lesions in the stratified squamous epi-
thelia of skin and mucosae, including the oral mucosa
[14–16,47,48]. Nearly 100 HPV genotypes have been
identified thus far. Some mucosotropic genotypes, such
as HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-
35 and others, are associated with mucosal warts or
carcinomas, especially in the ano-genital region [9]. Of
these, HPV-6 and HPV-11 have only a low risk of
association with malignancy and are termed benign,
while others such as HPV-16 and HPV-18 are strongly
associated with malignancy and termed malignant or
oncogenic or high-risk genotypes.
HPV have been implicated in oral and head and
neck squamous carcinoma [15,16,19] but the field has
been controversial. However, the mystery around the
role HPV may play in head and neck squamous cell
carcinoma (HNSCC) and OSCC, though remaining, is
now fast unravelling.
Many groups have identified HPV in patients with
cancer but the results of case series and case control
studies have not been consistent and the identification
of a virus does not prove a causal relationship, since it
may have been activated by the disease rather than the
converse. Thus, HPV-DNA detection is insufficient evi-
dence for a causal role. Furthermore, HPV infection
tends to be focal and may be transient, so the lack of
detection does not rule out virus involvement. Anti-
bodies to HPV capsid antigens however, are reliable
markers of past or present infection [49] and their pres-
ence indicative of HPV. Finally, should there be evi-
dence of HPV infection, a clonal relationship between
HPV and the tumor, as proved by virus integration into
the host cell genome, would argue against HPV being
merely a secondary invader, and would strongly suggest
a causal role in carcinogenesis. HPV genomes E6 and
E7 are the two major viral oncogenes expressed in
tumor tissue and both stimulate cell proliferation and
interfere with tumor suppressor proteins: their identifi-
cation in the tumors would further substantiate an
oncogenic role for HPV.
Evidence for HPV presence in HNSCC has been
detected in numerous studies. For example, in one sero-
logical study, the odds ratio for HNSCC in subjects
positive for HPV-16 antibodies was 2.2 (CI 1.4–3.4)
suggesting HPV-16 to be a risk factor [49]. Based upon
the most sensitive method of detection, polymerase
chain reaction (PCR), the overall prevalence of HPV-
DNA in HNSCC tumor tissue was 34.5% (416 of 1205
tumors), the majority of HPV-positive tumors con-
tained the ‘‘high risk’’ HPV genotypes 16 (40.0%) and
18 (11.9%) [20]. Several of these and other subsequent
studies [50–52] have contained OSCCs as well as other
oropharyngeal carcinomas. For example, in an Italian
study, 36% HNSCC contained HPV and oncogenic
HPV (HPV-16, 18, 31, 45, 56, 57) were identified
in 26% HNSCC [52]. Failure to detect the HPV
E2 genome suggested viral integration into the tumor
genome [52].
Recent studies from the MD Anderson Center sup-
port an aetiologic role for HPVs in at least a sub-set of
HNSCC, especially in tonsillar carcinoma. These studies
have implicated HPV particularly in a sub-type of
poorly differentiated tumor with basaloid histological
features arising from the area of Waldeyer’s tonsillar
ring in the posterior tongue and fauces [50]. In the MD
Anderson study, 25% of HNSCC contained HPV, and
90% were HPV-16. In that study, around 57% of ton-
sillar tumors were HPV-positive compared with 12%
OSCC. In a review of other studies on various head and
neck sites, HPV was most often detected in tumors of
the oral cavity (59%), followed by the pharynx (43%),
and larynx (33%) [20].
 C. Scully / Oral Oncology 38 (2002) 227–234
HPV early-region transcripts have been found in ton-
sillar cancer [53,54] and there is reduced cyclin D1
synthesis and overexpressed p16 [55] suggesting an effect
on retinoblastoma protein pRb. HPV-containing
tumors appear to contain less p53 mutations and to be
seen more frequently in non-smokers/non-drinkers [50].
HPV was seen by in situ hybridisation only in tumour
and premalignant tissue but not in surrounding normal
mucosa [50] suggesting a causal relationship. Tonsillar
tumors containing HPV appear to have a better prog-
nosis [50,56] as do OSCC in some but not all [57] stud-
ies. Other studies, such as one Italian study, have
confirmed a more frequent association of HPV with
tonsillar carcinomas (50%) compared with carcinomas
of the tongue (38%) or buccal mucosa (12%) [52]. HPV-
16 DNA was also found by PCR in 50% of orophar-
yngeal and 14% of tongue cancers in a Scandinavian
study [49].
There have been numerous studies on oral squamous
cell carcinoma over the past 15 years. DNA technology
has shown that a substantial portion of OSCC and pre-
malignant lesions contain HPV sequences, often of the
‘‘high risk’’ genotypes (mainly HPV-16 and HPV-18).
The rate of HPV detection in OSCC has varied widely
in reports based on clinical material from OSCC from 0
to 94% [58–63] but the ability to detect HPV is of
course strongly dependent on the sensitivity of the
method used, as well as the representativeness of
the sample analysed. HPV detection is higher when
analyzed by in situ hybridization and polymerase chain
reaction (PCR), and studies with these techniques have
disclosed HPV 11, 16 or 18 DNA sequences in up to
60% of OSCC [60,62–68] and in up to 28% of oral
potentially malignant lesions [68,69]. Nested PCR stud-
ies have found HPV in even more lesions; for example,
in one Greek study in up to 86% of hyperplasias, 100%
of dysplastic lesions and 95% OSCC [51]. When data
from 94 reports that analyzed 4680 OSCC samples were
included in a recent meta-analysis [70], HPV were found
to be between two and three times more likely to be
detected in precancerous oral mucosa and 4.7 times
more likely to be detected in OSCC than in normal
mucosa. The probability of detecting high-risk HPVs in
OSCC was 2.8 times greater than that of low-risk HPVs,
providing further quantitative evidence that oral infec-
tion with HPV, particularly with oncogenic genotypes,
is a significant independent risk factor for OSCC [70].
The possible aetiological role of HPV infection in the
pathogenesis of oral potentially malignant lesions and
OSCC has been supported by the discovery of HPV-
suggestive lesions in oral potentially malignant lesion
specimens as well as by DNA-hybridization studies dis-
closing HPV 11, 16 and 18 DNA [16,47,63,64,71] and
demonstration of HPV-DNA in tissue [50,51]. HPV is
identified in oral squames in significantly more patients
with oral cancer than controls [72]. A particularly high
229
prevalence of HPV has been found in betel-quid-
associated OSCC in India [73], the HPV association
with OSCC was independent of tobacco and alcohol
use, but current tobacco and current alcohol users were
at risk of OSCC [72].
Most studies have identified high-risk HPV-16 geno-
types in OSCC but a novel HPV 16-related virus was
found by us in a UK study in about 40% OSCC [74]
and others have found a number of oncogenic HPV,
predominantly HPV-16 and HPV-18 in OSCC
[51,68,75]. Interestingly, HPV-16 subtypes associated
with HNSCC have changes in the promoter-enhancer
region that make them especially active in oral kera-
tinocytes [76]. It would appear that HPV-16, the virus
strongly implicated in cervical and other ano-genital
cancer, is the main but not necessarily sole, virus impli-
cated in OSCC.
Southern blot hybridisation results are consistent with
HPV existing in HNSCC in an episomal or integrated
way [50] and the absence of HPV E2 region of the viral
genome is consistent with viral integration into the
tumor genome [52]. This is interesting, since certain
HPV also clearly have oncogenic properties. HPV can
transform oral keratinocytes [77], especially with che-
mical carcinogens [78] but only the high risk HPV types
such as HPV-16 [77] have immortalizing activities [79].
Transformation studies with oncogenic HPV-DNAs
has localised the transforming activity to certain early
genes, mainly to the E6 and E7 oncogenes. Proteins
(oncoproteins) encoded by the E6 and E7 genes from
‘‘high cancer risk’’ HPV genotypes are able to interfere
with cellular growth regulatory proteins [80]. The E6
oncoprotein can bind to and interfere with the p53
tumour suppressor gene product [80]. The importance
of this E6/p53 interaction is substantiated by the obser-
vation that there is a correlation between the ability of
the HPV genes to bind these cell growth regulatory
proteins and the oncogenicity of the HPV type. E6 pro-
teins from high risk type viruses HPV 16 and 18 can
associate with p53, whereas no complex can be detected
with E6 proteins from HPV 6 or 11 (‘‘low risk’’) [80]. E6
from high risk HPV can also activate telomerase [81],
another factor involved in carcinogenesis [1–3].
The E7 oncoprotein can bind to and interfere with the
retinoblastoma tumour suppressor gene product pRB
[82]. The importance of the E7/pRB interaction is sub-
stantiated by the observation that there is a correlation
between the ability of the HPV proteins to bind cell
growth regulatory proteins and the oncogenicity of the
HPV type. E7 proteins from the oncogenic HPV types
16 and 18 bind pRB more strongly than does E7 from
the benign HPV types 6 and 11 [82]. Analysis of HPV E7
mRNA in OSCC and cell lines by reverse transcriptase–
polymerase chain reaction (RT–PCR) showed that HPV
E7 mRNA was present in 90% of patients with OSCC
[83]. Quantitative RT–PCR and western blot analysis
230 C. Scully / Oral Oncology 38 (2002) 227–234
on transformed oral epithelial cell lines demonstrated
that the mRNA level of HPV-16 E7 corresponded to E7
protein level, suggesting that HPV oncogene expression
is primarily regulated at the transcriptional or post-
transcription level [83]. Although the E7 protein alone
has transforming and immortalizing activities in rodent
cells, cooperation between E6 and E7 appears to be
both necessary and sufficient for the efficient immort-
alization of primary human genital keratinocytes.
Sequence rearrangements in the upstream regulatory
region (URR) may also be responsible for the onco-
genicity of specific HPV types [84].
Only few studies exist on HPV E6 or E7 gene expres-
sion in HNSCC but E7 has been demonstrated in OSCC
[85] and tonsillar carcinomas [55], and tonsillar carci-
nomas express high levels of HPV-16/E6/E7 transcripts
which originate from integrated as well as episomal
HPV-DNA [53]. Interestingly, the tonsillar carcinomas
containing HPV lacked pRb activity, but pRb activity
was present in those that contained no HPV [55]. HPV
E7 DNA is also found in the serum, suggesting either
the presence of tumor cells or nuclear breakdown prod-
ucts in the blood [86]. Antibodies against oncoproteins
E6 and E7 of HPV types 16 and 18 have been demon-
strated in patients with HNSCC, in about 12% and
mainly in persons with tonsillar carcinoma [87] further
supporting a biological role for HPV in these tumors.
The E5 gene may also be involved since the E5 protein
alters responses of the cell receptor tyrosine kinases and
can thus modulate epidermal growth factor receptor
(EGFR) activity.
These interactions provide at least a theoretical model
as to how HPV might be involved in carcinogenesis. The
hypothesis is that the inactivation of normal function of
p53 [88] or pRB (or the related p107) proteins is a criti-
cal step in squamous cell carcinogenesis. p53 may be
mutated by carcinogens or degraded by viruses. Both
p53 mutation-dependent and mutation-independent
pathways may be associated with HPV-mediated carci-
nogenesis, the former mainly in HNSCC and the latter
in cervical tumors. A distinct step in the pathogenesis of
both types of tumors may only be in the mode of p53
inactivation, whereas all other events appear to be
strongly correlated to the presence of HPV [89]. Inacti-
vation of the p53 tumor suppressor protein by the E6
gene product of high-risk HPVs and mutation of the
p53 gene in HNSCC is associated with alterations in
the apoptotic regulatory bcl-2 and bax genes, leading
to downregulation of programmed cell death and
increased cell proliferation [90].
H-ras oncogene mutation also appears correlated
with HPV in oral carcinoma [91], and it is clear that
some HPV can interact with various transcription fac-
tors, especially NF-1 (nuclear factor 1), API (includes
oncogenes jun and fos), protein phosphatase 2A (PP2A),
and others [9]. There are hints for a correlation between
amplification of the Int-2 gene and infection with HPV
in HNSCC [57]. However, despite the central role of
HPV and the early genes, malignant transformation in
vitro appears also to require additional factors like co-
infection with a herpes virus, exposure to tobacco
or alcohol, glucocorticoids or other hormones, or other
co-factors [79].
There is thus abundant epidemiologic and virologic
evidence that high-risk HPVs are tumorigenic in human
epithelia. Molecular studies indicate that tran-
scriptionally active virus is confined to tumor cells and
that HPV has a role in HNSCC development, certainly
in tonsillar and perhaps other oropharyngeal subsets.
Thus HPV may be implicated in some tumors but
clearly not in all OSCC where tobacco and alcohol are
more important aetiological factors [92]. HPV-based
therapeutic vaccines which are currently being devel-
oped for cervical cancer may thus also eventually prove
of benefit in the management of some HNSCC. How-
ever, HPV are clearly neither necessary nor sufficient for
all tumor production and it must be remembered that
much OSCC is induced by the known risk habits
involving tobacco and alcohol.
It is important to consider then the question of HPV
infection in the oral cavity. HPV appear to be common
commensals in the oral mucosa, though their origin
is unclear. HPV DNA was demonstrated in buccal
mucosal cells of 41.6% of the infants born to mothers
in India with HPV-positive cervical smears [93] and, in
USA, HPV is present in the oral cavity in around 5% of
adolescents irrespective of parental race, education,
HPV-related lesions, smoking history, or number of sex
partners; or adolescent’s smoking history or history of
sexual activity [94]. HPV-DNA has been demonstrated
in normal tissues adjacent to HPV-related lesions in the
genital and upper aerodigestive tract (UADT), and in
normal genital mucosa [95], in normal tonsils [96] and,
by us and others, in normal oral mucosa. By Southern
blot hybridization, HPV-DNA has been detected in
from 15% [97,98] to over 40% [74] of biopsies from
clinically normal buccal mucosa from adults, and the
polymerase chain reaction (PCR) technique increased
the detection of HPV-DNA in the former study to
21.8% [97]. HPV-DNA can also be detected by PCR in
exfoliated oral squames from clinically healthy individ-
uals [99,100]. The frequency of HPV positivity in oral
samples from healthy individuals in a number of studies
has ranged from 1 to 60% [20] and it is clear therefore,
that the oral mucosa may act as a reservoir for new
HPV infections and/or as a source of recurring HPV
lesions.
The source of HPV infection is unclear. HPV can be
spread by saliva or sexually but is not highly transmis-
sible. For example, commercial sex workers in Bangkok
are reservoirs of oncogenic HPV, and cervical cancer in
monogamous Thai women develops in part as a result
 C. Scully / Oral Oncology 38 (2002) 227–234
of transmission of these viruses to them by their hus-
bands from prostitutes [101]. In view of the high infec-
tivity of genital warts, it is interesting to note a
surprisingly low prevalence of oropharyngeal warts in
adults indulging in orogenital contact in a UK study
[102] but this study was based on clinical detection and
not involving stomatologists, and there was no investi-
gation of oral HPV infection. Nevertheless, a study on
oral HPV infection in women with past or present
genital HPV infection [98,103] using dot-blot hybridiza-
tion on exfoliated oral squames showed only a low
(3.8%) HPV-DNA prevalence, but this was almost cer-
tainly an underestimation of the true HPV prevalence
because basal layer epithelial cells cannot be readily
collected in this way.
Epidemiologic studies have shown that exposure to
HPV increases the risk of HNSCC [28,104] suggesting it
may be sexually transferred, and HPV infection may
interact with alcohol and tobacco exposure in tumor
promotion [105,106]. HPV association with OSCC was
greatest in males with a young age of first intercourse,
those with multiple sexual partners, and those with
genital warts [104]. This USA study found that the
associations with a higher lifetime number of sexual
partners and with a total of more than four partners
with whom the subject had engaged in oral sex, was
stronger for patients with HPV-16-positive tumors than
those that were HPV-16-negative [104]. In contrast,
another USA study found that HPV-related genital
lesions, oro-genital sexual behaviour, and number of
sexual partners, did not differ between patients with
OSCC and controls [72].
The Swedish Family Cancer Database was used to
analyse second cancers in the UADT of women first
diagnosed with in-situ or invasive cervical cancer [107].
Tonsillar cancers were increased only among women
aged 50 years or more at diagnosis of in-situ cervical
cancer and this was ascribed to the effects of HPV,
smoking, alcohol or their interaction. Husbands of cervical
cancer patients developed an excess of both tonsillar
cancer and cancer of the tongue. Whether HPV were
transmitted sexually is unknown. Furthermore, patients
with anogenital cancer in another study had a 4.3-fold
increased risk of tonsillar squamous-cell carcinoma [108].
These cancer types also have histopathological and mole-
cular biological similarities. Analysis of second primary
cancers arising after OSCC has shown an excess of oral,
rectal, genital and skin cancer in both males and females
[109]. Cervical cancer was also followed by an excess of
oral, rectal, anal and other genital cancers and some of the
associations were confirmed both ways (oral–oesophagus;
oesophagus–oral; oral–genital; genital–oral; oral–skin;
skin–oral) [109] and though there may be other factors
such as depressed immune function involved, it is difficult
to resist speculation that HPV may have been responsible.
Finally, the Amsterdam group has just provided the
231
‘‘missing link’’ which demonstrates that high risk HPV
are responsible for certain HNSCC by showing that
only HPV E6 mRNA-positive tumors lacked p53
mutation [110].
Thus, HPV may be aetiologically important in some
types of oropharyngeal cancer, and may represent an
alternative pathway in carcinogenesis to the established
factors of tobacco and alcohol. New data from the
current International Agency for Research on Cancer
[111] and Johns Hopkins Oncology Center studies on
HPV and HNSCC may help elucidate further the rela-
tionship. The possibility of sexual or other transmission
also needs to be further explored since in addition to the
above data, associations have been revealed between
skin and oral cancer [112,113].
In any event, we have come a very long way in the
two decades since our first suggestion of a viral aetio-
pathogenesis was greeted with incredulity.
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