Oral Oncology 50 (2014) 840–847

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

Detection and typing of Human Papillomaviruses (HPV) in malignant,

dysplastic, nondysplastic and normal oral epithelium by nested
Polymerase Chain Reaction, immunohistochemistry and transitional
electron microscopy in patients of Northern Greece
E. Blioumi a,⇑, D. Chatzidimitriou b, Ch. Pazartzi c, Th. Katopodi d, G. Tzimagiorgis e,
E.-N. Emmanouil-Nikoloussi f, A. Markoloulos a, C. Kalekou a, N. Lazaridis g, E. Diza h, D. Antoniades a
a
Department of Stomatology, School of Dentistry, Aristotle University of Thessaloniki, Greece
b
B’ Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki, Greece
c
Department of Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Greece
d
Department of Biology, School of Medicine, Aristotle University of Thessaloniki, Greece
e
Department of Biochemistry, School of Medicine, Aristotle University of Thessaloniki, Greece
f
Department of Histology, Embryology and Anthropology, School of Medicine, Aristotle University of Thessaloniki, Greece
g
Department of Orofacial Surgery, School of Dentistry, Aristotle University of Thessaloniki, Greece
h
Department of Microbiology, AHEPA University General Hospital of Thessaloniki, Greece

a r t i c l e i n f o s u m m a r y

Article history: Objectives: To evaluate the role of HPV in oral carcinogenesis, we examined the prevalence of HPV in
Received 24 February 2014 malignant, potentially malignant and normal oral epithelium and studied the relation of HPV prevalence
Received in revised form 24 May 2014 with other factors obtained from the patient’s records.
Accepted 12 June 2014
Materials and methods: Our material consisted of 291 tissue specimens from 258 individuals. From every
Available online 17 July 2014
individual formalin fixed and paraffin embedded tissues were examined by nested Polymerase Chain
Reaction (NPCR) for the detection of HPV DNA and by immunohistochemistry (IHC) for the in situ detec-
Keywords:
tion of HPV L1 protein. Positive PCR products were sequenced in order to type HPVs. Also 33 fresh tissues
Oral cancer
Human Papillomavirus (HPV)
were obtained, fixed and used to detect HPV particles by transitional electron microscopy (TEM).
Oral dysplasia Results: HPV was detected in 32.9% of the tissue specimens by NPCR, in 4.7% by immunohistochemistry
Polymerase Chain Reaction and in 28.1% by TEM. In detail, by nested PCR HPV L1 DNA was detected in 40% of normal tissues, 40% of
Immunohistochemistry fibromas, 35.8% of non-dysplastic leukoplakias, 31.6% of dysplastic leukoplakias and 22.2% of oral squa-
Electron microscopy mous cell carcinomas. The HPV viral load of 96.5% of the samples was very low (1 viral copy per 102–104
cells). HPV16 prevails in all histological groups in 89–100%.
Conclusion: We conclude that HPV does not seem, from the specific sample examined, to play a substan-
tial role in oral carcinogenesis. However, it cannot be excluded that HPV could be involved in oral
carcinogenesis only in cases with high viral load or at early stages of carcinogenesis possibly through
the hit-and-run mechanism.
Ó 2014 Elsevier Ltd. All rights reserved.

Introduction
Oral cancer is the twelfth most frequent malignancy in the
world, representing almost 2.5% of malignant tumors [1]. The prev-
alence and incidence of oral cancer in 2002 worldwide was esti-
mated about 741,000 and 274,289 cases, respectively [1].
⇑ Corresponding author. Present address: Theophrastou 3, P.S. 55132, Thessalo-
niki, Greece. Tel.: +30 2310 452576; fax: +30 2313 304467, mobile: +30
6942577911.
E-mail address: eblioumi@yahoo.com (E. Blioumi).
Specifically in Greece, 399 new oral cancers and 125 deaths come
up every year [2]. Although more than 40 different types of neo-
plasms grow up in the oral cavity, squamous cell carcinomas
account for 85–94% of all oral malignancies [3–6]. The etiology of
oral cancer seems to be multifactorial. A number of extrinsic
factors, such as smoking, alcohol consumption, tobacco chewing,
ultraviolet radiation and several viruses, seem to play role in oral
carcinogenesis. Intrinsic agents, including oncogenes, tumor
suppression genes, hormones, immunosuppresion and enzymatic
mechanisms also seem to be involved in the initiation and

http://dx.doi.org/10.1016/j.oraloncology.2014.06.011
1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
 E. Blioumi et al. / Oral Oncology 50 (2014) 840–847
progression of oral carcinomas [3,7]. Moreover, many of the squa-
mous cell carcinomas develop on the ground of potentially malig-
nant disorders (PMDs) [7]. Oral leukoplakia is the most frequent
PMD, representing 85% of these lesions [4].
HPV is a small, epitheliotropic, DNA virus, which belongs to a
large family of viruses, the Papillomaviridae, containing more than
100 different human types [8–11]. PV genome consists of 7900
base pairs of closed-circular double-stranded DNA, containing 8
open reading frames. Some of HPV types, such as 16, 18, 31, 33,
35, 39, 45, 51, 52, 56, 58, 59, 66, are carcinogenic to humans and
are characterized as ‘high risk types’. Moreover high risk types
are 26, 30, 34, 53, 67, 68, 69, 70, 73, 82 [8].
The first time that a relationship between HPV and head and
neck cancer was mentioned was in 1980 [12–15]. Since then, the
findings of HPV participation in the onset and the progression of
oral carcinomas differ among the researchers. Specifically, the
prevalence of HPV in oral carcinomas varies between 0% and
100% [16–26], and in the normal oral epithelium varies between
0% and 81% [27–35]. Most of the reports have selectively studied
a certain type of oral lesions. To evaluate the role of HPV in oral
carcinogenesis, we comprehensively investigated HPV infection
in 258 specimen of oral mucosa, representing the whole spectrum
of normal, hyperplastic, potentially malignant and malignant
lesions, using a combination of three different techniques:
nested-PCR, immunohistochemistry and transitional electronic
microscopy, and studied the relation of HPV prevalence with other
factors obtained from the patient’s records, such as histologic
diagnosis, gender, age, anatomic location, smoking and alcohol
drinking.
Material
Our material consisted of 291 tissue specimens from 258 indi-
viduals. From every individual, formalin fixed and paraffin embed-
ded tissues (FFPE) were examined by nested Polymerase Chain
Reaction and immunohistochemistry. Fresh tissues were obtained
and examined by transitional electron microscopy from 33 of the
above patients.
The FFPE tissues consisted of 63 oral squamous cell carcinomas
(OSSC), 57 oral leukoplakias with dysplasia (OLD), 53 leukoplakias
without dysplasia (OL), 50 fibromas with normal epithelium (F)
and 35 normal mucosas (N). The pathologic tissues were taken
from the routine files (1970–2009) of the Department of Stomatol-
ogy, School of Dentistry, Aristotle University of Thessaloniki,
Greece, whereas the normal tissues were taken from the normal
excision borders of healthy subjects during fibroma, epulis, muco-
cele biopsies or third molar extractions in the Department of Sto-
matology or Maxillofacial Surgery (2008–2009).
The 33 fresh oral specimens were collected during the biopsy of
squamous cell carcinomas or hyperplastic or dysplastic lesions.
They consisted of 15 OSSCs, 8 OLDs, 9 OLs and 1 fibroma. For each
case, patient’s gender and age, smoking or alcohol drinking, lesion’s
location, histological diagnosis and histological grading of dyspla-
sia or cancer differentiation, according to Smith and Pindborg’s cri-
teria [36], were recorded.
Methods
Three different techniques were applied on the specimens
collected: nested Polymerase Chain Reaction was applied on 258
tissue specimens for the detection of HPV DNA, immunohisto-
chemistry was applied on 258 tissue specimens for the in situ
detection of HPV L1 protein and transitional electron microscopy
was applied on the 33 fresh tissue specimens for detection of viral
particles. Nested-PCR, the most sensitive and specific technique,
841
was used mostly for the evaluation of HPV prevalence and the sta-
tistic analysis, whereas the other two techniques offered informa-
tion about the inter-epithelial and inter-cellular location of HPVs.
Nested Polymerase Chain Reaction
Three to five adjacent 10 lm sections of each paraffin embed-
ded tissue were collected in sterile 1.5 mL microcentrifuge tube.
DNA was extracted with the Qiagen QIAamp DNA extraction
method. To avoid cross-contamination at any step of the proce-
dure, paraffin-embedded compounds were cut between samples
and subsequently subjected to DNA extraction and PCR analysis.
DNA’s concentration and purity were estimated using 260 nm
and 280 nm spectrophotometry (Eppendorf Centrifuge).
Strict procedures were followed to avoid false-positive reac-
tions due to contamination. DNA extraction and PCR reactions
were carried out in separate rooms, sterilized with UV radiation
both before and after reactions for 10 min. All surfaces were decon-
taminated with a special solution (DNA AWAY, Molecular Bioprod-
ucts). Reagents were prepared and aliquoted, filtered pipette tips
and one-use gloves were used. Every PCR reaction was applied to
10 samples, one positive control (DNA extracted from cervical can-
cer) and one or more negative controls (distilled sterile water).
To check the integrity of the extracted DNA and the absence of
PCR inhibitors a 110 bp segment of human b-globin gene was
amplified by PCO3/PCO4 primers [37,38].
Detection of HPV sequences was performed by two sets of con-
sensus primers, MY09/MY11 and GP5+/GP6+, which amplify a
450 bp and an internal 150 bp region, respectively, in the highly
conserved L1 HPV gene. In the first amplification step, each reac-
tion of 50 ll contained in final concentrations: 1X PCR buffer,
2 mM MgCl2, 200 lM of each dNTP, 1 lM of each primer MY09
(50 -CGTCCMARRGGAWACTGATC-30 ) and MY11 (50 -GCMCAGGGW-
CATAAYAATGG-30 , M: A + C, R: A + G, W: A + T, Y: C + T), 1.5 units
Taq DNA polymerase and 20 ll of DNA (600–1000 mg). The mix-
ture was subjected to a 5 min denaturation at 94 °C, then 40 cycles
of amplification at 94 °C for 1 min, 55 °C for 1 min and 72 °C for
1 min, in addition to a 5-min extension at 72 °C [12].
2 ll of the above PCR product were used in the second amplifi-
cation step of the PCR reaction. Each reaction of 50 ll contained in
final concentrations: 1X PCR buffer, 3.5 mM MgCl2, 200 lM of each
dNTP, 0,5 lM of each primer GP5+ (50 -TTTGTTACTGTGGTAGATAC-
TAC-30 ) and GP6+ (50 -GAAAAATAAACTGTAAATCATATTC-30 ), 1.25
units Taq DNA polymerase and 2 ll of the first PCR product. The
mixture was subjected to a 5 min denaturation at 95 °C, then
40 cycles of amplification at 94 °C for 1 min, 40 °C for 2 min and
72 °C for 2 min, in addition to a 3-min extension at 72 °C [11,37].
After amplification, the reaction products were electrophoresed
on 2% agarose and visualized by ethidium bromide.
To type the HPV, the final nested PCR product was further puri-
fied with PureLink™ purification procedure (Invitrogen Corpora-
tion, UK) and got sequenced with the Sanger method in
automatic analyzer (CEQ 8000, Beckman Coulter, GENOTYPOS,
Athens, Greece). Chromograms were analyzed with Chromas pro-
gram. The DNA sequences were aligned and compared to
sequences of known HPV types, found in NCBI Genbank (NCBI,
National Institute of Health, Bethesda, MD, USA), with the use of
NCBI Blast program (http://blast.ncbi.hlm.nih.gov/Blast.cgi).
HPV positive specimens were subjected to a type-specific PCR
for the amplification of a 120 bp segment of the HPV16 E6 gene.
Each reaction of 50 ll contained in final concentrations: 1X PCR
buffer, 1.5 mM MgCl2, 200 lM of each dNTP, 1 lM of each primer
F (50 -TCAAAAGCCACTGTGTCCTG-30 ) and R (50 -CGTGTTCTTGAT-
GATCTGCA-30 ), 2 units Taq DNA polymerase and 10 ll of sample
DNA (300–800 mg). The mixture was subjected to a 2 min denatur-
ation at 95 °C, then 40 cycles of amplification at 94 °C for 1 min,
842 E. Blioumi et al. / Oral Oncology 50 (2014) 840–847
53 °C for 1 min and 72 °C for 2 min, in addition to a 7-min exten-
sion at 72 °C [34].
The sensitivity of the above PCR protocols was determined
using serially diluted DNA extracted from cervical cancer.
Immunohistochemistry
Three adjacent histological sections of each sample were incu-
bated in 60 °C for 1–2 h, dewaxed in xelene and rehydrated
through alcohol to water. The antigens were retrieved by dipping
the sections in citrate buffer solution and warming them in micro-
waves for 10 min at the highest temperature. The sections were
washed with Tris Buffer Solution (TBS) and incubated with 0.3%
hydrogen peroxide in methanol for 30 min in dark to quench the
endoxegous peroxidase activity. Non-specific background staining
was reduced by incubating the sections with fetal bovine serum
(FBS) for 1 h. The sections were then incubated overnight at 4 °C
with the antibodies; the first section, with an antibody against
HPV L1 capsid protein (Polyclonal rabbit Anti-papillomavirus anti-
body (Predilute Antibody, Ready-to-use, 08-0075, Zymed Laborato-
ries, Invitrogen Immunodetection, USA) in 7 mg/ml concentration,
the second section with a negative antibody (N-universal Negative
control rabbit, code N1699, DakoCytomation, Denmark) for nega-
tive control and the third section with monoclonal mouse antibody
against cytokeratin (Monoclonal mouse anti-human cytokeratin,
MNF clone, code M 0821, DakoCytomation, Denmark) for positive
control of the quality and the antigenicity of the tissue. The sec-
tions were then washed with TBS and incubated with for 60 min
with the secondary antibody and peroxidase (ChemMate™ Dako
Envision/HRP, Rabbit/Mouse, DakoCytomation, Denmark) in room
temperature. Finally, the sections were incubated with DAB (diam-
inobenzidine) diluted 1:50 in hydrogen peroxide for 1.5–2 min,
stained with haematoxylin, dehydrated and mounted. The tech-
nique was further controlled by staining specimen with known
HPV (oral wart, cervical epithelial dysplasia) and by staining a
specimen without any history of HPV infection (normal minor sal-
ivary gland). All specimens were read by a trained pathologist
without information on the origin of the specimen so as to avoid
bias results.
Positive staining was registered the localized dark brown inter-
nuclear or para-nuclear staining of the epithelium, whereas dis-
perse cytoplasmic staining was considered as non-specific.
Transitional electron microscopy
Immediately after excision, a 3 mm-diameter specimen was cut
into slices of less than 1 mm thickness and fixed by immersion in
3% gluteraldehyde in phosphate buffer (PH:7.4) for 1 h at 4 °C.
After brief washing with phosphate buffer solution, the specimens
were postfixed with 2% osmium tetroxide for 1–1.5 h at 4 °C. They
were then blocked with 1% uranyl acetate for 12–14 h, dehydrated
through a graded ethanol series, and embedded in EPON 812 (Ser-
na Electrophoresis GmbH, Heidelberg). Ultra thin sections were cut
using a diamond knife on a Leica EM UCG, stained with Reynold’s
stain and examined with a JEOL Electron Microscope, JEM-2000
FX, at 80 kV.
Viral particles were detected as spherical particles 50–60 nm in
diameter, sparsely distributed or forming clusters, localized in the
nucleus or in the cytoplasm or in the narrow extracellular space
between the epithelial cells of oral mucosa.
Statistical analysis
Statistical analysis was performed by X2 test for the study of
correlation between categorical quality parameters using SPSS
15.0 software package. P-value was calculated either with Exact
method or Monte Carlo method [39]. To compare percentages,
we used Fisher’s Exact Test [40]. Pearson’s linear correlation coef-
ficient was used to correlate quantity parameters. P values lower
than 0.05 were considered significant.
Results
258 Samples were derived from the buccal mucosa (30.2%), the
tongue (24.0%), the gingival and alveolar mucosa (19.4%), the lips
(14%), the floor of the mouth (8.5%) and the palate (3.5%). No sam-
ples were selected from the posterior oropharyngeal sites since
patients with such lesions are mostly referred to ENT or oral and
maxillofacial surgeons. 127 samples were derived from male
patients and 131 from female patients. The age of patients ranged
from 12 to 94 years, with a mean age of 53.1 years (mean devia-
tion: 16.6 years). 57.8% of samples were derived from smokers or
ex-smokers (>1 cigarette per day), and 15.5% of samples were
derived from alcohol consumers (>1 portion per day). Cancerous
lesions were classified into well-differentiated (37), moderately
differentiated (21) and poorly differentiated (5), according to Smith
and Pindborg’s criteria. Dysplastic lesions were classified into mild
epithelial dysplasia (34 patients), moderate dysplasia (6), severe
dysplasia (10) and in situ carcinoma (7).
The results of the different techniques to demonstrate HPV
revealed that 90 out of 258 patients were HPV positive (35.9%)
by at least one technique. All techniques proved to be reliable as
all control specimens gave consistent results irrespective of the
technique employed.
B-globin was detected in all tissues studied. NPCR analysis
detected the presence of HPV genome in 85/258 specimen
(32.9%). Of the positive specimens, 3.5% were identified by the first
step and 96.5% by the second step of NPCR assay (Fig. 1). By the
first step, HPV was detected in 2 carcinomas with moderate differ-
entiation and 1 in leukoplakia with mild dysplasia. Two samples
were derived from the tongue and one from the buccal mucosa.
HPV DNA was detected in 22.2% of OSCCs, 31.6% of dysplastic
leukoplakias, 35.8% of non-dysplastic leukoplakias, 40% of fibromas
and 40% of normal oral epithelia. Statistical analysis with X2 test in
the whole sample revealed a negative, weak and statistically signif-
icant relation between age and HPV DNA detection (p = 0.017). HPV
DNA was detected more frequently in younger individuals
(653 years old). No significant relation was detected between
HPV prevalence and gender (p = 1.000), histological diagnosis
(p = 0.247), histological grading (p = 0.469), location (p = 0.317),
smoking (p = 0.182) or alcohol drinking p = 0.133). In oral carcino-
mas, a statistically significant relation was detected between HPV
L1 DNA positivity and male gender (p = 0.023) and location on
the tongue (p = 0.059, a = 0.1). In oral leukoplakias with dysplasia,
HPV detection was significantly higher in female patients
(p = 0.012) and non-alcohol drinkers (p = 0.022). In normal sam-
ples, fibromas and benign leukoplakias, no significant relation
was found between HPV and the above parameters (Table 1).
The typing of the HPV positive samples indicated HPV16 in
88.6%, HPV13 in 3%, HPV56 in 3%, double infection of HPV13/16
in 3%, double infection of HPV16/56 in 1% and HPV66 in 1.5%.
Totally, 97.1% of positive samples contained high risk HPV types
(HPV16, 56, 66). The distribution of HPV types in every patient
group is shown in table 2. Double infections were detected by
the combination of the results of sequencing and the results of
HPV16 E6 PCR. E6 was detected in 69.7% of HPV16 L1 positive spec-
imens. No significant relation was found between histological diag-
nosis and HPV16 E6 detection (p = 0.988).
Both MY09/MY11, GP5+/GP6+ and HPV16 E6 protocols detected
a minimum of 0.78 ng of total (human and HPV) DNA in a 50 ll
reaction, having a sensitivity of 1 HPV copy per 102 cells. Nested
 E. Blioumi et al. / Oral Oncology 50 (2014) 840–847 843

Figure 1. PCR products of biopsy specimens. (A) PCR products with external MY09/MY11 primers of HPV L1 DNA after staining with ethidium bromide and photography
under UV illumination. DNA band of 450 bp indicated the presence of HPV DNA. Lanes 1–6: oral carcinoma tissues, NCTL: negative control, POS: positive control, Lad: 100 bp
DNA Ladder (molecular weight marker). (B) PCR products with internal GP5+/GP6+ primers of HPV L1 DNA. DNA band of 150 bp indicated the presence of HPV DNA. Lanes 1–
10: oral carcinoma tissues, NCTL: negative control, POS: positive control, Lad: 100 bp DNA Ladder (molecular weight marker).

Table 1
HPV prevalence with nested PCR and study of the associations of HPV DNA with histological diagnosis, gender, age, location, smoking and alcohol drinking in the total sample
(n = 258).

Parameters Categories HPV () HPV (+) Total Association p

Diagnosis Normal 21 14 (40.0%) 35
Fibromas 30 20 (40.0%) 50
Leukoplakias 34 19 (35.8%) 53
Dysplasias 39 18 (31.6%) 57
Carcinomas 49 14 (22.2%) 63
173 85 258 Nonsignificant p = 0.247
Gender Male 85 41 (32.3%) 127
Female 88 43 (32.8%) 131
173 85 258 Nonsignificant p = 1.000
Age 653 73 50 (40.7%) 123
>53 100 35 (25.9%) 135
173 85 258 Significant p = 0.017
Location Tongue 39 23 (37.1%) 62
Floor of mouth 17 5 (22.7%) 22
Buccal 52 26 (33.3%) 78
Alveolar mucosa. 30 20 (40.0%) 50
Lips 28 8 (22.2%) 36
Palate 7 2 (22.2%) 9
Oropharyngeal 0 1 (100%) 1
173 85 258 Nonsignificant p = 0.317
Smoking No 105 44 (29.5%) 149
Yes 68 41 (37.6%) 109
173 85 258 Nonsignificant p = 0.182
Alcohol No 144 77 (34.8%) 221
Yes 29 8 (21.6%) 37
173 85 258 Nonsignificant p = 0.133
Histological grading No dysplasia 85 53 (38.4%) 138
Mild dysplasia 23 11 (32.4%) 34
Moderate dysplasia 3 3 (50.0%) 6
Severe Dysplasia 8 2 (20.0%) 10
In situ carcinoma 5 2 (28.6%) 7
Well-differentiated 29 8 (21.6%) 37
Moderately-differentiated 16 5 (23.8%) 21
Poorly-differentiated 4 1 (20.0%) 5
173 85 258 Nonsignificant p = 0.469

PCR protocol detected a minimum of 0.78 pg of total DNA, namely HPV L1 antigen was detected in 12 out of 258 samples (4.7%) by
NPCR was 100 times more sensitive than the above three PCR pro- immunohistochemistry. Positive stain was localized inter- or para-
tocols with a sensitivity of 1 viral copy per 104 cells. nuclearly, in few epithelial neoplastic or dysplastic cells, often
844 E. Blioumi et al. / Oral Oncology 50 (2014) 840–847
Table 2
Distribution of HPV types in patients’ groups after sequencing.
Group HPV types
HPV16 HPV13 HPV56
Normal 11
Fibromas 15 1 1
Benign leukoplakias 17 1 18
Dysplasias 8 1
Carcinomas 11
Total 62 2 1
separate in intermediate (spinous) layer and seldom in clusters,
rete ridges, or in cells with koilocytic appearance (Fig. 2). HPV L1
antigen was detected in 0% of normal mucosa, fibromas and leuko-
plakias without dysplasia, and 1.8% of dysplastic leukoplakias and
17.5% of OSCCs.
By transitional electron microscopy, viral particles were
observed in 9 out of 33 specimens. Specifically, no particles were
found in fibromas and non-dysplastic leukoplakias, whereas they
were detected in 37.5% of dysplastic leukoplakias and 40% in
OSCCs. HPV particles were spherical, 50 nm in diameter and were
localized mainly in the nucleus of epithelial cells, or in the cyto-
plasm next to the nuclear membrane. Particles were detected more
frequently in the spinous layer and more seldom in the basal and
prickle cell layer, forming clusters in isolated cells (Fig. 3).
Discussion
Reports on HPV involvement in oral carcinogenesis are conflict-
ing, with percentages ranging from 0% [18,41–43] to 100% [17,20].
The observed discrepancies can be attributed to differences in epi-
demiologic data, sample collection techniques, sample preserva-
tion techniques, HPV detection methods and protocols. In our
study, three different techniques were used to demonstrate HPV
in oral specimens. All techniques proved to be reliable as all control
specimens gave consistent results irrespective of the technique
employed.
The study revealed that HPV was detected in 34.9% of the tissue
specimens by at least one of the three methods used. The majority
of the positive samples were found with the NPCR technique
(32.9%), less were observed by electron microscopy (28.1%) and
very few by immunohistochemistry (4.7%). The sensitivity of NPCR
was roughly estimated to reach 1 viral copy per 104 epithelial cells,
about 100 times more than MY, GP+ and TS-PCR’s sensitivity
(1/102). These findings agree with the results of Gillison and Keert
[44] and Remmerbach [45]. The fact that 96.5% of positive NPCR
samples were detected by the second step, means that viral load
ranged between 1 copy per 102 and 104 epithelial cells. According
to the clonal expansion theory [46,47], if viral load is less than 1
copy per cell – as it is for the majority of our positive specimens
– HPV does not seem to play any role in oral carcinogenesis. Higher
viral load (>1 copy per 102 cells) was detected only in 1.8% of dys-
plastic and 3.2% of cancerous lesions by the use of MY-PCR.
An unusual finding of our study was the decreasing percentage
of HPV DNA positive samples during the transition of normal epi-
thelium to malignancy. In detail, by NPCR HPV L1 DNA was
detected in 40% of normal tissues, 40% of fibromas, 35.8% of non-
dysplastic leukoplakias, 31.6% of dysplastic leukoplakias and
22.2% of oral carcinomas. Our results agree partly with some stud-
ies [30,31,48,49], although there are many studies reporting con-
tradictory results [11,50,51]. Our findings can be explained by
the ‘hit-and-run mechanism’. According to that theory, HPV may
play a role in the initial stages of carcinogenesis, inactivating p53
or pRb and predisposing the cells to accumulate additional genetic
HPV66 HPV13/16 HPV16/56 Total
1 12
2 1 20
9
11
1 2 2 70
lesions. The disturbed tissue has higher risk of progression into a
malignant phenotype, with the later stages being independent of
HPV [47,48,52,53].
HPV antigens or particles were detected, disperse or in clusters,
in few and isolated oral epithelial cells, which may indicate a local-
ized colonization of HPV in oral epithelium, without any clinical
significance for malignant transformation [54]. Also, para-nuclear
detection of HPV antigens or particles could be attributed to frag-
mentation of infected nuclei and release of virus particles and
virus- associated proteins, whereas cytoplasmic stain may indicate
non-specific reactivity of cytoplasmic substances with HPV anti-
bodies [14].
Our findings indicate that HPV is detected in normal and benign
lesions only by PCR, whereas in 16.7% of dysplastic lesions and
47.7% of malignant lesions it is detected by at least two different
techniques. This means that in normal and benign lesions HPV
infection may have a very low concentration and may be latent
(NPCR), while in some dysplastic and malignant lesions HPV can
be found in higher concentration, either as DNA (MY-PCR, IHC or
TEM), or as full viral particle in productive infection (IHC, TEM)
or with both of these forms at the same time (IHC/NPCR, TEM/
NPCR). Transitional electron microscopy seems to be more sensi-
tive than immunohistochemistry perhaps due to its capacity to
detect immature viral particles, indetectable by IHC [55].
In our study, HPV DNA was detected in 40% in both ‘normal epi-
thelium’ groups, which lies in accordance with some studies
[30,34,48,56,57]. However, other groups have not detected any
HPV DNA in normal mucosa samples [11,28,29,58–60]. Very high
sensitivity of NPCR, examination of all epithelial layers and speci-
men collection from every site of oral cavity may explain the high
percentage of positive normal samples.
Our finding of a significant relation between HPV infection of
oral carcinomas and the male gender lies in agreement with many
previous studies [34,51,61–63] and may be explained by the
increased number of sexual partners and orogenital sexual con-
tacts of men. Moreover, our result that there was a significant rela-
tion of HPV detection with tongue carcinomas coincides with the
findings of other studies [15,48,64,65]. The majority of studies
involving oral lesions do not separate oral cavity from oropharynx,
although the relation of HPV to oropharyngeal cancer is more sig-
nificant that to oral cancer [66]. Since our study is focused on oral
cavity, we tried to compare our findings with studies including
similar sample location.
In the total sample HPV DNA was detected more frequently in
younger individuals (653 years old). The higher prevalence of
HPV in the cervix of younger women, the increased sexual activity
of young individuals, the increased number of sexual partners and
the more frequent oral-genital and oral- anal contacts in compari-
son to older people may explain that finding [67]. No significant
relation found between HPV detection and histologic diagnosis
may indicate that in most cases HPV colonizes oral epithelium
without playing a role in malignant progression.
HPV16 was the prevailing viral type, which was detected in
94.3% of positive samples, either in single infections or in coinfec-
 E. Blioumi et al. / Oral Oncology 50 (2014) 840–847
Figure 2. Immunohistochemical stain of HPV antigens in oral squamous cell
carcinoma from the tongue. Papillomavirus structural antigens are identified by
positive brown reaction (arrows) inside the nucleous (A, B), and next to the
nucleous (C, D) in few isolated epithelial cells. These cells may be koilocytes (A, B)
or form clusters (D) (40).
tions. Most studies agree with the high prevalence of HPV16 in
normal tissues [8,27,31,48,57], leukoplakias [29,68] and oral carci-
nomas [31,66,69,70]. High-high risk infection pattern may repre-
sent a synergistic oncogenic action of the coinfecting types.
Conversely, the low–high-risk infections may be indicative of an
845
Figure 3. Transitional Electronic Microscopy in oral specimens. (A) Mild dysplasia
from the floor of the mouth. HPV particles (50–60 nm) are detected inside the
nucleous of oral squamous epithelial cell (TEM, X30.000). (B) Mild dysplasia from
the floor of the mouth. HPV particles are detected in the cytoplasm just next to the
nuclear membrane of oral squamous epithelial cell (TEM, X30.000). (C) Oral
squamous cell carcinoma of the alveolar mucosa, moderately differentiated. HPV
particles are found just out of the nuclear membrane of an epithelial cell of the
spinal layer (TEM, X25.000).
incidental colonization of oral epithelium by the low-risk viral type
and, hence, a solitary tumorigenic effect of the high-risk type [11].
In conclusion, the decreasing HPV prevalence during the transi-
tion from normality to malignancy, the very low viral load of the
majority of the specimens and the sparse detection of viral anti-
gens and particles indicate that HPV does not seem, from the spe-
cific sample examined, to play a substantial role in oral
carcinogenesis. It cannot be excluded that HPV could be involved
in oral carcinogenesis only in cases with high viral load or at early
stages of carcinogenesis through the hit-and-run mechanism.
846 E. Blioumi et al. / Oral Oncology 50 (2014) 840–847

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