Clinical Oral Investigations

https://doi.org/10.1007/s00784-019-03048-y

ORIGINAL ARTICLE

Detection of HPV in oral leukoplakia by brushing and biopsy:

prospective study in an Italian cohort
Fedora Della Vella 1 & Giuseppe Pannone 2 & Assunta Patano 1 & Rossella Ninivaggi 1 & Raffaele Del Prete 1 &
Dorina Lauritano 3 & Massimo Petruzzi 1

Received: 11 March 2019 / Accepted: 5 August 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Aim The aim of the present study was to evaluate the prevalence of HPV infection in oral leukoplakia, specifying the HPV
genotypes eventually involved. We also compared the micro-biopsy and brushing HPV detecting efficacy.
Materials and methods Consecutive patients with a presumptive diagnosis of oral leukoplakia were enrolled. Demographical,
behavioral data (smoking, alcohol) and lesion features were recorded. Each patient underwent a brushing procedure, performed
with a cytobrush rubbed on the lesion, and then a biopsy was performed. The brushing and micro-biopsy specimens were both
analyzed with the HPV 28 Anyplex II Seegene RT-PCR. The prevalence of HPV infection was calculated considering the two
methods’ outcomes separately and then combining both. Cohen’s k coefficient was used to assess the agreement between the two
methods.
Results Sixty-five patients were enrolled with a mean age of 60 years. The HPV infection prevalence was 17%, decreasing to 5%
considering the brushing outcomes alone. The most frequently detected genotypes were 6 (12%), 11 (3%), 42 (3%), and 16 (3%).
No statistically significant correlation was found between HPV infection and the variables analyzed, except for smoking and the
type of mucosa (p < 0.05). The strength of agreement between cytobrush and micro-biopsy was “fair” (k = 0.384).
Conclusions The present study showed a low prevalence of HPV infection in oral leukoplakia. The micro-biopsy appeared to be
more reliable than brushing in detecting HPV DNA in oral leukoplakia, but the method invasiveness discourages its employ as a
screening tool. The importance of HPV in the etiopathogenesis of oral potentially malignant lesions remains unclear; further
studies are needed to establish the HPV role in oral leukoplakia.
Clinical relevance HPV involvement in oral leukoplakia and an effective and appropriate detecting technique are still a debated
issue. From this study, the restricted use of brushing did not appear sufficient to assess the presence of HPV infection with PCR
techniques in samples obtained from oral leukoplakia.

Keywords Leukoplakia . Papilloma virus . HPV . Oral premalignant disorder . RT PCR . Brushing

Fedora della Vella and Massimo Petruzzi contributed equally to this work
and are co-first authors.

* Fedora Della Vella Raffaele Del Prete

dellavellaf@gmail.com raffaele.delprete@uniba.it
* Massimo Petruzzi Dorina Lauritano
massimo.petruzzi@uniba.it dorina.lauritano@unimib.it
Giuseppe Pannone
giuseppe.pannone@unifg.it
1
Interdisciplinary Department of Medicine, University of Bari “Aldo
Moro”, Bari, Italy
Assunta Patano 2
Department of Clinical and Experimental Medicine, University of
assu.patano@hotmail.it
Foggia, Foggia, Italy
3
Rossella Ninivaggi Department of Medicine and Surgery, |University of Milan-Bicocca,
rossella.ninivaggi@hotmail.it Monza, Italy

Introduction
Human papillomavirus (HPV) infections are responsible for
the most common sexually transmitted diseases, with a report-
ed infection prevalence of 30–50% in sexually active women
and of 5–40% in men [1, 2]. Most part of HPV infections is not
clinically manifested, because of the tendency of the virus to
keep its genome into an episomal form, with minimal tran-
scriptional activity [3]. This is especially true for the so-called
low-risk (LR) genotypes, while the high-risk (HR) ones, in-
cluding groups 1 and 2 according to International Agency for
Research on Cancer (IARC) classification [4], tend to incor-
porate their DNA into the host cell genome [5]. This condition
allows viral protein synthesis and promotes replication, possi-
bly leading to clinical manifestations, like squamous papillo-
ma, condyloma, and warts [6]. Due to its predominantly sex-
ual transmission, HPV tends to infect basal epithelial cells of
the genital tract. Nevertheless, other body epithelial mucosae
can be infected, like the oropharynx, both through horizontal
transmission and auto-inoculation [7]. The oral HPV preva-
lence reported ranges between 2.6 and 26%, significantly var-
iable according to the specimen collection method and ana-
lyzed population [8, 9]. The oncogenic activity of some HPV
genotypes is well established, just like their role in uterine
cancer etiopathogenesis, with correlation percentages reported
from 85 to 100% [10]. This is not the case for oral cancer and
oral potentially malignant disorders (OPMDs), whose corre-
lation with HPV infection is still debated. The oral leukoplakia
is one of the most frequent OPMDs in general population [11],
and it is defined as “a white patch or plaque that cannot be
characterized, clinically, or pathologically, as any other dis-
ease” [12]. Depending on clinical and histological criteria,
percentages of leukoplakia malignant transformation may
range from 0.13 to 17.5% [12, 13]. Two main clinical sub-
types of leukoplakia are recognized: homogeneous and non-
homogeneous, which includes the nodular, speckled, and
verrucous type. The non-homogeneous leukoplakias are
deemed to have a greater risk of malignant transformation.
Other prognostic features include size, affected site, sex, age,
alcohol and tobacco consumption, and presence of dysplasia.
The positivity for HR HPV infection has also been proposed
as a malignancy prognostic factor for leukoplakia [14].
Actually, most of the studies available on this subject ana-
lyzed HPV as a tumor risk factor for the oropharyngeal area,
not considering the relevant differences between the properly
oral cavity and the pharynx, such as the local microbiota, the
organization of lymphatic tissue, and the presence of saliva. In
fact, the limited resources referring purely to the mouth are
discording about the implication of HPV in OPMDs [15, 16].
Furthermore, it is not currently recognized a gold standard
method for HPV detection, that is generally performed on a
cytological smear collected using a swab or a brush [17]. This
is a minimally invasive technique, but its effectiveness is not
Clin Oral Invest
totally accepted, especially in the oral cavity, where there is a
high risk of contamination. Other possibilities can be posed by
oral rinses, unfortunately lacking in site specificity, and micro-
biopsy that requires a higher ability of the operator and that is
more invasive [18, 19].
The aim of the present study was to evaluate the prevalence
of HPV infection in a group of patients affected by oral leu-
koplakia, using and comparing two different chair-side spec-
imen collection methods: brushing and micro-biopsy.
Materials and methods
Study design
This single-center, prospective, observational study was car-
ried out at the University of Bari - Oral Medicine Section of
Dental Clinic (Bari, Italy), from October 2017 to December
2018. The study was conducted according to the Declaration
of Helsinki and independently approved and reviewed by the
Institution’s Ethical Committee.
Patients
Consecutive patients referred to the Dental Clinic of
University of Bari “Aldo Moro” with a presumptive diagnosis
of leukoplakia were enrolled. Oral leukoplakia was defined
according to 2017 World Health Organization (WHO) criteria
[12]. All patients released a written informed consent. Patients
who had received an anti-HPV vaccination, with an incom-
patible histopathological diagnosis, or who refused to give
consent to the study participation were excluded. For each
patient, demographical data and smoking and daily alcohol
units assumed were recorded. During the oral clinical exam,
the lesions occurring on the gingival mucosae, hard palate,
and dorsal tongue were reported as involving keratinized sites,
while the ones occurring on the buccal and labial mucosae,
ventral tongue, mouth floor, and soft palate were reported as
involving non-keratinized sites.
Brushing
The brushing sampling was performed using the cytobrush
(Nuova APTACA S.r.l., Canelli -AT-, Italy), a single-use, ster-
ile, medical tool, consisting of a nylon conoid brush mounted
on a rigid stick. The brushing procedure was performed rub-
bing the cytobrush with a rotation-translation movement on
the lesion, avoiding the contact with any other surface that
could contaminate the bristles. The collected specimens were
put in a sterile propylene cup.
Clin Oral Invest
Micro-biopsy
An excisional biopsy was performed for each lesion using a
blade no. 15 scalpel. Every tissue specimen was further sec-
tioned, obtaining a sample immediately fixed in 10% buffered
formalin to be analyzed by an expert pathologist and a smaller
representative sample (about 4 mm) that was put in saline
solution NaCl 0.9% and designed to the virologic molecular
analysis.
Molecular analysis
The detection of HPV DNA was performed by the real-time
polymerase chain reaction (RT-PCR) HPV 28 Anyplex II
Seegene (Seegene, Seoul, Korea), a multiplex PCR allowing
the simultaneous identification of 28 HPV genotypes (6, 8, 11,
16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56,
58, 59, 61, 66, 68, 69, 70, and 73), precisely 19 HR (16, 18,
26, 31, 33, 35, 39, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 70,
and 73) and 9 LR (6, 8, 11, 40, 42, 43, 44, 61, and 69). The
process, including DNA extraction, HPV testing, and data
analysis, was performed according to the manufacturer’s in-
structions. The HPV 28 Anyplex II Seegene employs two
melting temperature analysis systems (TOMs) that enable a
semi-quantitative dosage of more analytes at the same time.
Data analysis and statistics
The sample size calculation was done referring to an estimate
population of Italians aged between 18 to 80 years, 2.3% of
leukoplakia prevalence, using n = Nx/[(N − 1)E2 + x], with
95% confidence level and 5% margin of error. The prevalence
of HPV infection and the percentages of the found genotypes
were calculated in the analyzed population, considering the
brushing and biopsy outcomes separately and then combining
both. The chi-square test was performed in order to evaluate
the correlation between HPV infection and age (cut-off: sam-
ple mean age), sex, smoking habit, alcohol consumption, pres-
ence of dysplasia according to 2017 WHO guidelines [12],
and type of oral mucosa involved (keratinized or non-
keratinized), setting a significance for p < 0.05. The agreement
degree between brushing and micro-biopsy was calculated
using Cohen’s k coefficient.
Results
Sixty-five patients were enrolled, 35 males and 30 females,
with a mean age of 60 years (SD ± 14.8).
Fourteen patients declared to smoke an average number of
10 cigarettes per day and 7 were ex-smokers, while the re-
maining 44 declared not to smoke. Twenty-three patients con-
sumed at least 1 alcoholic unit a day, while the other 42 did not
report to assume alcohol. Thirty-one lesions occurred on non-
keratinized mucosal sites (19 on buccal and labial mucosae, 5
on mouth floor, 5 on ventral tongue, and 2 on soft palate) and
34 on keratinized mucosa (21 on dorsal tongue, 10 on gingival
mucosae, and 3 on hard palate). Thirty-seven out of the
leukoplakias analyzed were clinically homogeneous, while
28 had a non-homogeneous aspect. Twenty-two leukoplakias
(34%) showed dysplasia at the pathological exam. The aver-
age follow-up time was 8 months.
The total prevalence of HPV infection was 17%, but only 3
lesions (5%) presented with HR HPV (HPV 16 and HPV 53).
The HPV prevalence decreased to 5% (0% HR HPV) when
considering the brushing outcomes alone, while it remained
17% when considering only the micro-biopsy.
No significant correlation was found between HPV infec-
tion and age, sex, alcohol consumption, and presence of dys-
plasia (p > 0.05), while smoking and the type of oral mucosa
were statistically associated with the infection (p < 0.05)
(Table 1).
All the patients who presented with HPV infection had a
positive micro-biopsy, three of them showed a positive
cytobrush test too. Differently, none of the positive cytobrush
patients had a negative micro-biopsy outcome. The HPV ge-
notypes detected were as follows: 6 (8 patients, 12%), 42 (3
patients, 5%), 11 (2 patients, 3%), 16 (2 patients, 3%), 35 (1
patient, 2%), 43 (1 patient, 2%), and 53 (1 patient, 2%). Four
biopsy specimens presented multiple HPV genotype infec-
tions: 6–35, 16–42, 6–11, and 6–16-43, respectively. None
of the HR HPV was detected from the brushing specimens.
Lack of coincidence among the detected genotypes was ob-
served between cytobrush and micro-biopsy specimens. The
strength of agreement between the cytobrush and micro-
biopsy methods was “fair” (k = 0.384). Data about the
collecting methods’ outcomes are resumed in Table 2.
Discussion
This study evidenced a low prevalence of HPV infection in
oral leukoplakia. Only a small percentage of patients resulted
in positive HPV, and even less were affected by an HR HPV
infection. None of the specimens was positive for HPV 18, in
contrast to data pointing HPV 18 and 16 as the most frequent-
ly involved in OPMDs [16, 20].
The micro-biopsy was better than the brushing in HPV
detection, although HPV genotypes found with the two
methods were never coincident. Also, all the considered var-
iables were not significantly associated with the HPV infec-
tion, exception for smoking and the type of oral mucosa in-
volved. The association between smoking cigarettes and HPV
presence occurred to be statistically significant, probably due
to the smoking affecting the host immune response that facil-
itates the HPV infection [21]. Moreover, the higher frequency
 Clin Oral Invest

Table 1 HPV infection and

variables analyzed in patients Variables Values HPV+ HPV− p value
affected by leukoplakia (Cytobrush+micro- (Cytobrush+micro-
biopsy) biopsy)

Sex M 5 (8%) 30 (46%) 0.54

F 6 (9%) 24 (37%)
Age < 60 years old 5 (8%) 28 (43%) 0.70
> 60 years old 6 (9%) 26 (40%)
Tobacco consumption Smokers 5 (8%) 9 (14%) 0.02
Not smokers 5 (8%) 39 (60%)
Ex-smokers 1 (2%) 6 (9%)
Alcohol Yes 5 (8%) 30 (46%) 0.54
No 6 (9%) 24 (37%)
Type of oral mucosa Keratinized 3 (5%) 31 (48%) 0.01
Dorsal tongue 3 (5%) 18 (28%)
Gingivae 0 (0%) 10 (15%)
Hard palate 0 (0%) 3 (5%)
Non-keratinized 8 (12%) 23 (35%)
Mouth floor 3 (5%) 2 (3%)
Ventral tongue 1 (2%) 4 (6%)
Soft palate 1 (2%) 1 (2%)
Buccal mucosa 3 (5%) 16 (25%)
Dysplasia Present 4 (6%) 17 (26%) 0.75
Absent 7 (11%) 37 (57%)

Italicized data resulted significantly associated to hpv presence (p<0.05)

of HPV-positive leukoplakias occurring on non-keratinized instance; indeed, the locations where HPV has strongly been
epithelia was consistent with previous studies [22]. A low linked to carcinogenesis, i.e., cervix and tonsils, show non-
keratinization site can ease the access of the HPV in the first keratinized stratified squamous epithelium [22].

Table 2 Cytobrush and micro-

biopsy detection data and HPV+ cases (%) HPV− cases (%) Genotypes (cases)
methods’ agreement degree
scored Cytobrush 3 (5%) 62 (95%) HPV 6 (1)
HPV 42 (2)
Micro-biopsy 11 (17%) 54 (83%) HPV 6 (6)
HPV 11 (2)
HPV 16 (2)
HPV 35 (1)
HPV 42 (1)
HPV 43 (1)
HPV 53 (1)
Cytobrush + micro-biopsy° 11* (17%) 54** (83%) HPV 6 (8)
HPV 11 (2)
HPV 16 (2)
HPV 35 (1)
HPV 42 (3)
HPV 43 (1)
HPV 53 (1)

*In 3 cases, cytobrush and micro-biopsy were both positive in the same lesion
**In 54 cases, cytobrush and micro-biopsy were both negative in the same lesion
°Concordance: k = 0.385 (95% CI from 0.069 to 0.699) (fair agreement)
Clin Oral Invest
Some authors found a stronger correlation between leuko-
plakia and HPV, ranging from 40 to 50%, while other studies
reported prevalences comparable to the healthy mucosa (23–
33%) or even lower (0–18%) [16, 23–27]. This huge variation
is partially due to the different viral detection and identifica-
tion methods employed. From this study, the micro-biopsy
emerged as more reliable for detecting HPV in oral cavity.
The brushing technique is less invasive, and it can be applied
also on healthy tissues; indeed, it is conventionally performed
by the gynecologists to collect vaginal samples, even though
there is no agreement about the device to use: spatula, swab, or
cytobrush [28]. The cytobrush has a greater exfoliative capac-
ity, providing more cells from the superficial epithelium layers
and not only detached ones [28]. The use of this tool in the oral
cavity could be affected by the contamination of saliva, which
easily carries cells and/or viral DNA from other mucosal sites,
like the Waldeyer ring, known to be frequently infected by
HPV [29].
The micro-biopsy resulted in more positive findings. This
is probably due to the chance to access the basal layer, where
the HPV DNA is generally localized in an episomal form,
during silent-latent infections. So far this is the only collecting
method providing deep epithelium cells. Actually, new
collecting brushes specifically dedicated to oral cavity have
been released, made with a customized shape that, according
to manufacturers, provide cells representative of all layers of
the oral epithelium. These devices could represent a good
screening tool and should be subjects of future studies.
Also, micro-biopsy could be partially affected by saliva
contamination, not to mention its invasiveness and inability
to be applied as a screening test. In fact, one of the limitations
of this study is the lack of a healthy control group, because it
was not suitable for the Institute Ethical Committee to perform
adjunctive surgical biopsy on healthy subjects.
Syrjänen and Syrjänen [30] supported a low biopsy efficacy since
they found that HPV DNA was constantly present in the intermediate
and superficial cell layers of the analyzed genital tissues, while rare in
the basal and parabasal layers. The same author attested better adequacy
of biopsy compared with brushing in a successive study conducted on
oral carcinoma and OPMDs [15]. Studies comparing cytobrush and
biopsy already reported a lack of concordance between the two
methods, other than a better biopsy suitability to detect HR HPV,
encouraging its use on OPMDs [31]. Actually, there is still no collecting
method defined as the gold standard. Oral rinses have been proposed,
but their lack of site specificity makes them little reliable as a diagnostic
tool for patients presenting OPMD [32].
About the analysis of the specimens, nowadays molecular
methods are preferred, allowing also the viral genotype detec-
tion. The PCR is considered the quickest and most accurate
assay to detect HPV, based on in vitro genomic amplification
starting at a specific DNA fragment isolated using selective
primers (16–20 nucleotide probes). Generally, these primers
pair with highly conserved regions of the viral DNA, such as
My90/MY011 (L1 region) and CPI and CPII (E1 region). The
HPV 28 Anyplex II Seegene RT-PCR has been validated as a
cervical cancer screening method, with a sensitivity compara-
ble to a pap smear cytologic test [33, 34]. The main limitation
of PCR is that it only gives information about the presence of
the HPV DNA, but not about the viral activity. The p16INK4a
immunohistochemistry (IHC) dosage is recommended to at-
test the ongoing viral replication. This cytogenetic method
uses fluorophores to identify p16 overexpression, expected
in HPV-induced lesions. The HPV E7 protein promotes the
transcription of the CDKN2A/p16INK4a gene through a his-
tone demethylation mechanism. Physiologically, the
p16INK4a increase stops the cell cycle, but the HR HPV E7
also downregulates the tumor suppressor RB proteins, holders
of a pivotal role in the cell cycle negative control [35].
The 8th TNM classification for oropharyngeal squamous
cell carcinoma (OPSCC) released by the WHO includes p16
staining-positivity as a staging parameter, and also the last
American Society of Clinical Oncology (ASCO) guidelines
recommended the p16INK4a dosage as a prognostic test for
OPSCC, even if a p16 activity increase can also exist in non-
HPV etiopathologically driven cancer, showing a p16+/HPV−
pattern. In addition, since a universally recognized cut-off
value to attest the IHC positivity is not available, the
American Joint Committee of Cancer (AJCC) suggested at
least 75% staining with a + 2/3 staining intensity to consider
the test reliable in the oropharynx [36–39]. Multiple studies
agree to recommend the association of PCR and p16 IHC to
guarantee both adequate sensitivity and specificity [29, 30].
The most frequent detected genotypes in the present study
were HPV 6 and 11, considered not cancerogenic by the
IARC (group 3), due to their poor interference on the host cell
life cycle. Nevertheless, HPV 6 and 11 genotypes have been
found in vaginal intra-epithelial neoplasia and larynx and na-
sopharyngeal lesions, with higher prevalence than HPV 16
and 18 (group 1) [4]. The HPV 16 and 53 were the only
cancerogenic genotypes (groups 1 and 2B) found in this study,
both with proven capacity to induce multiple oncogenic ef-
fects and molecular alterations in the host cell [40]. HPV 16,
rather than 18, has been strongly correlated with squamous
cell carcinomas, also in the oral cavity. HPV 53 has been
reported in oral carcinomas with < 1% prevalence [41].
The weak association between HPV DNA presence and
leukoplakia may be explained by the hyperkeratosis which
characterizes this OPMD and could avoid the access to the
viral genome, deeply localized. In fact, the micro-biopsy,
which provides a whole-depth sample, showed higher preva-
lence (17% vs 5%). Another possibility is that the HPV infec-
tion leads a dysplastic transformation via its oncogenic stim-
ulus, and then the virus would be removed thanks to the cel-
lular exfoliation or to the host immune system action. This
mechanism, already proven in some cervical cancers, is called
“hit and run theory” and results in an HPV-induced lesion not

containing the viral DNA anymore, neither in episomal nor
integrated form [42].
Conclusions
The results of this study question the potential role of the HPV
infection in oral leukoplakia. Also, it confirms the fair concor-
dance between brushing and micro-biopsy as collecting
methods in the oral cavity, assessing the higher reliability of
biopsy, especially to detect HR HPV genotypes. Further in-
vestigations are needed to better clarify the effects of the HPV
on the oral mucosa and the use of other potential diagnostic
tools to identify virus presence and activity.
Compliance with ethical standards
Conflict of interests The authors declare that they have no conflict of
interest.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institu-
tional research committee and with the 1964 Helsinki declaration and its
later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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