Submission: November 13, 2018; Published: November 29, 2018
*Corresponding author: Joharia Azhar, Associate Professor and Consultant Oral Pathologist, Chair PBL unit, Department of Diagnostic Oral Sciences, Princess Nourah Bint Abdulrehman University, Riyadh, KSA, Saudi Arabia.
How to cite this article: Joharia A, Sadia S, Ishtiaq Q. The Role of Human Papilloma Virus in the Development of Oral Squamous Cell Carcinoma in
Pakistani Population. Adv Dent & Oral Health. 2018; 10(1): 555778. DOI: 10.19080/ADOH.2018.10.555778
Oral cancer is one of the most prevalent cancers in Pakistan and the Pakistani population seems to be especially susceptible to the development of oral cancer. Human Papilloma virus has already been implicated in the etiology of cervical cancer, and strong evidence suggests its close association with the oral cancer. The aim of our study was to define the role of Human Papilloma virus in the development of oral squamous cell carcinoma patients in Pakistani population. Ninety-five patients with oral squamous cell carcinoma samples were recruited for this study. HPV infectivity was determined histopathological by observing Koilocytosis and molecularly by conducting PCR using HPV consensus primers. HPV genotyping was then carried out using the HPV genotyping PCR assay technique. The results of HPV prevalence and genotyping were evaluated in relation to various clinico-pathalogical parameters Statistical Package for the utilizing social sciences version 12.0 for windows (SPSS Inc, Chicago, IL). HPV was detected in 78% cases, out of whom 74% were associated with HPV16, 4% with HPV 18, 15% were co infected with HPV 16 and 18, and 7% were positive for HPV by the general primer and could not be type specified. This study finds a significant association between type 16 HPV prevalence and oral squamous cell carcinoma with male patients showing high co-relations.
Pakistan is one of the high-risk countries for oral cancer development, where oral cancer is the most prevalent cancer in men and may account for up to 30% of all new cases of cancer per year, compared to 3% in the UK and 6% in France . Many known etiological factors for oral cancer development, like Pan, Betel Nut, Areca nuts and smoking are very commonly used in Pakistan, however not all the people exposed to these factors develop cancer. Furthermore, many patients not exposed to any of these factors develop cancer. This indicates the presence of additional risk factors, including Human Papilloma Virus that can make these people more susceptible to cancer.
More recently research has associated certain genotypes of HPV to the Papillary SCC of the Head and Neck , to OSCC , to the upper aerodigestive tract tumours [4,5], not to mention malignant and pre-malignant lesions of the uterine cervix , ulva , penis , and conjunctiva . However, the exact mechanism of the interaction of HPV and the significance of its presence in the development of carcinogenesis is still not clear4. Discrepancies in the prevalence of HPV in OSCC have been observed, geographically, in the more recent studies, creating further confusion4. Certain
studies investigating role of HPV in PSCC, a subset of SCC have reported prevalence of transcriptionally active HPV in almost 54% of cases2, while some others report a failure of PCR studies to show transcriptionally active HPV .
The aim of this study was to detect the prevalence of HPV in the OSCC patients of the Pakistani origin and to determine the most prevalent genotype of HPV associated with OSCC in Pakistani patients.
This study is based on samples retrospectively collected from 72 different oral cancer patients diagnosed with oral squamous cell carcinoma OSCC during the period of 2005-2010. Inclusion criteria were complete clinicopathologic data and availability of enough paraffin- embedded tumour material. The clinico- pathological data consisted of age, gender, geographical location of the patient, site of lesion and histopathological grade. OSCC samples were formalin fixed paraffin embedded. A total of 72 blocks of paraffin-embedded tissue including samples diagnosed as oral squamous cell carcinoma stages I, II and III, verrocus
carcinoma, focal dysplasias, and fibroepithelial hyperplasias
were retrieved from the archive of Pakistan Institute of Medical
sciences (PIMS), and KRL hospital Islamabad, from year 2005 to
2010. Samples were taken from different lesion sites including
maxilla, mandible, tongue, floor of the mouth, labial and buccal
mucosa, naso-pharynx and vocal cords.
The second group of samples consisted of 23 fresh samples
in which around 6 were fresh unfixed biopsy specimen while 17
were fresh endoscopic samples obtained from ENT department
CMH, Rawalpindi. The samples collected were stored at 4 °C in
Diagnostic Laboratory, NCVI, NUST, Islamabad until they were
ready for further processing.
Representative tissue sections of 3 μm thickness were cut
on a microtome and the slides were kept at 60°C for 30 minutes.
Sections were stained with Harri’s Haematoxylin and observed
under the microscope. Histopathological diagnosis was confirmed
and histopathological evaluation for staging was carried out.
Genomic DNA Extraction from Formalin Fixed Paraffin
Embedded (FFPE) Tissue Biopsies: Using a standard microtome
with disposable blades, sections of 20μm thicknesses was
obtained from the block with the microtome blade properly
cleaned in between every sample to avoid contamination. Each of
the sections was placed in a 1.5ml eppendorf tube. Blocks with the
similar amount of tissue and the same section thickness were used
for all DNA isolation methods. DNA was extracted from formalinfixed
paraffin blocks by Xylene-ethanol method. Cut section from
each case was deparaffinized and DNA was extracted by adding
one ml of xylene to each 20μm section. Tubes were agitated for
30 min at room temperature and spun at 13000rpm for 10min.
The supernatant was removed carefully, one ml fresher xylene was
added, and the step repeated. Two identical washes with 100%
ethanol were performed followed by spinning at 13000rpm for
10 minutes. Tissue pellets were air dried at 37°C. Tissue digestion
was done by adding 400μl of the tissue digestion buffer (Tris-Cl
(100mM, pH = 7.5), Tween-20 (0.05%)) to each tube containing
tissue pellet. Then, 20μl of 500μg/ml of Proteinase K (Invitrogen)
was added, agitated, and tubes were left overnight at 56 °C in
water-bath. Digestion buffer was inactivated by heating for 10
minutes in boiling water and the cell debri was spun down. The
supernatant containing the DNA was transferred to a new DNA
tube and was used directly in PCR. The extracted DNA was frozen
at -20 °C until use.
Genomic DNA extraction from fresh biopsy/ endoscopy
tissue samples: Fresh biopsy specimen was homogenized in
glass homogenizer. Homogenized tissue was then placed in 1.5ml
eppendorf tube to which 400μl of digestion buffer [Tris- Cl (100
mM, pH = 7.5), Tween-20 (0.05%)] was added. Then 5μl of 20μg/
μl Proteinase K (Invitrogen) was added to it, and tubes left to
agitate overnight at 56 °C in a water-bath. Digestion buffer was
inactivated by heating for 10 minutes in boiling water and the cell
debri was spun down. The supernatant containing the DNA was
transferred to a new DNA tube and was used directly in PCR. The
extracted DNA was frozen at -20 °C until use.
DNA concentration was measured by spectrophotometer
(Eppendorf Biophotometer plus, Germany). Optical density
was measured at 260 and 280nm. The ratio of absorbance at
260/280 was used to determine quality of DNA. A ratio above 1.0
was inacceptable range. Successful DNA extraction was assessed
by PCR amplification of a 130bp fragment of the β-globin gene.
Sequences of primers are given in Table 1.
HPV detection PCR was performed using primers published
elsewhere (Baay MF, et al, 1996). The sequence for the primers
GP5 and GP 6 is given in the Table 1 along with the length of
respective amplimer and target gene. Polymerase chain reaction
round was performed in 0.2ml tubes (Axygen , USA) with 5μl of
sample DNA dilution in 25μl of reaction mixture, using 0.4μl of
MBI Fermentas, UK Taq polymerase (2U/μl), 2.4μl MgCl2 (25
mM), 3.0μl of (10X) PCR Buffer, 1.0μl DNTPs (2mM), 2.0μl each of
both Forward and reverse primers (10 pmol/μl) and 9.2 μl dH2O
(nuclease free). The PCR was performed on Applied Biosystem
(ABI Veriti, Singapore) 96 well thermocycler. The PCR thermal
profile was 94 °C for 4 minutes, and 35 cycles of 94 °C for 30 seconds, 45 °C for 30 seconds, 72 °C for 30 seconds, and 7 minutes
final extension at 72 °C.
All samples were further genotyped for type 16 and type 18, by
carrying out the type specific PCR published elsewhere . The
sequence for the primers for type 16 and 18 are given in the Table
1, along with the length of respective amplimer and target gene.
Polymerase chain reaction round was performed in 0.2ml tubes
(Axygen , USA) with 5μl of sample DNA dilution in 20μl of reaction
mixture for both reactions, using 0.4μl of MBI Fermentas, UK Taq
polymerase, 2.3μl MgCl2 (25 mM), 2.0μl of (10X) PCR Buffer, 1.0μl
DNTPs (2mM), 1.0μl each of both Forward and reverse primers
(10pmol/μl) and 7.3μl dH2O (nuclease free). The PCR was
performed on Applied Biosystem (ABI Veriti, Singapore) 96 well
thermocycler. The PCR thermal profile for both reactions was 94
°C for 4 minutes, and 35 cycles of 94°C for 30 seconds, annealing
temperature for type 16/18 was 54/61 °C for 30 seconds, 72 °C for
30 seconds, and 7 minutes final extension at 72 °C.
Data were processed by SPSS statistical software program
version 12.0. The correlations were analysed by running cross
tabulation tests. Cross tabulations were analysed using chi-square
test or fisher’s exact test and Pearson co-relation test where
applicable. P value measures the significance of an association and
is significant when it is less than or equal to 5% (0.05).
The study population consisted of a total of 95 patients
admitted to the dental ward of PIMS, Islamabad, Maxillo-facial
Surgery ward of KRL hospital, Islamabad and ENT ward of CMH,
Rawalpindi. The demographic parameters included in our study
were age, gender, geographic location and specimen site. Patients
ages ranged from 16 years to 86 (mean age=51 years; ±SD=14).
87% of the patients were above 35 years of age (n=83). 55% of
the patients were males (n=52; ±SD=0.500) while around 45%
(n=43; ±SD=0.500) were females. A total of 86% of males were
>35 years of age (n=45) and about 89% of the female patient
population was >35 years (n=38). There were 80 (83%) cases
of squamous cell carcinoma, 3 (3.2%) cases of oral submucous
fibrosis which is a pre-malignant condition and 12 (13%) cases
of fibroepithelial polyps. The most common anatomic site of
involvement was mandible including the tongue area with 40%
(n= 38) of the cases occurring here. The next most common site
was the nasopharynx and vocal cords with 23.2% (n=22) of the
patients having carcinoma of the oral cavity that had metastasized
to the neck. Around 20% (n=19) of the cases involved either
the buccal or the labial mucosa with 4% (n=4) showing gingival
involvement, while maxillary and palatal involvement was found
in 12.4% (n=12) of the cases.
The histopathological evaluation was carried out and the
squamous cell carcinomas were graded. We had a total of 49 (52%)
cases of well differentiated squamous cell carcinomas designated
as histopathological stage I cancer. 25 (26%) cases were of
moderately differentiated squamous cell carcinoma graded as
stage II cancer while 4 (4%) cases were of the poorly differentiated
variety graded as stage III cancer. All the samples were also
observed for the presence of koilocytes (Figure 1) to determine
the efficacy of morphological assays in the identification of HPV
infection. Out of the 95 samples about 50 (53%) were positive for
the presence of koilocytes.
Out of a total of 95 oral squamous cell carcinoma samples 72
were formalin fixed and paraffin embedded, 6 were fresh biopsy
specimen unfixed and 17 were endoscopic specimen. Out of these
95, 51 (54%) were positive for HPV when GP PCR was done
(Figure 2 & 3). However, it was observed during genotyping PCR’s
that some of the samples previously determined to be negative
by the GP PCR were positive for type 16 HPV. Therefore, the total
infected samples were calculated to be 74 (78%) (Figure 4). The
sensitivity and specificity of the GP PCR for HPV detection were
also determined and was found to be 74% and 81% respectively.
The following formulas were utilized to determine the sensitivity
SENSITIVITY = Those that are test positive / All with disease= 74%
SPECIFICITY= Those that are test negative / All without disease= 81%
Cross tabulation analysis showed that out of 43 females 29
(67%) were infected. Similarly, out of 52 males 45 (87%) were
infected. Sex difference among the patients was significantly corelated
to HPV infectivity as determined by the Pearson chi square
test (P=0.02). HPV was detected in 37(82%) out of 45 stage I
well differentiated squamous cell carcinomas, 21 (84%) out of
25 stage II moderately differentiated carcinomas, 3 (75%) out of
4 stage III poorly differentiated carcinomas. The single sample of verrocus carcinoma was positive for HPV as well as 12 (75%)
out of 16 fibro-epithelial proliferations. All in all, no significant
association was found between age groups, site of involvement
and histological differentiation.
Out of the total 74 infected patients about 55 (74%) were
infected with HPV type 16 (Figure 5), 3 (4%) were infected with
HPV type 18 (Figure 6), 11 (15%) had HPV type 16 and 18 coinfection
and 5 (7%) were positive for HPV by GP PCR but could
not be type specified (Figure 7).
OSCC is normally considered to be linked with tobacco, alcohol
and betel/areca nut chewing, however a percentage of patients not
genetically predisposed to developing cancer, manage to develop
oral carcinogenesis in the absence of all these agents [12-14].
This fact indicates the presence of another etiological agent which
can cause cancer independent of these factors. The evidence of
presence of viral nucleic acids in OSCC tissue was presented for
the first time in 1982, 1983 by Scully C et al. . A meta-analysis
by Miller CS & Johnstone BM performed in 2001 revealed HPV
to have been significantly associated with oral cancer as well as pre-malignant lesions . The role of HPV in the development of
squamous cell carcinoma of the cervix is a well-established fact.
Although anatomically distantly located the squamous epithelia of
both the cervix and oral cavity have remarkable similarities. The
HPV high risk types found in association with cervical cancer like
type 16, 18, 31 and 33 have also found to be associated with the
cancerous lesions of the oral cavity as well as the naso-pharynx
In our study, two methods were employed to determine
the presence of HPV. The first one was the histopathological
evaluation, in which the morphological changes occurring in the
HPV infected squamous cell were observed. This was especially
emphasized as more than one third of Pakistani population lives
below the poverty line , and most of them cannot afford to
have PCR analysis done to determine HPV infection. We had a
total of 95 samples which were histopathological analysed for the
presence of koilocytes, which are HPV infected squamous cells
with pyknotic nuclei and a clear halo around the cytoplasm (Figure
1). 50 samples tested positive for the presence of koilocytes out of
95, which constitutes about 53% of the total samples. All these 50
samples were later tested positive for HPV infection using GP PCR
assays (Table 2).
Histopathological grading of the OSCC samples was also carried
out in order to determine the severity of the cancerous process.
The histopathological parameters that were observed were the
degree of differentiation, cellular atypia, presence of keratin pearls
and individual cell keratinizations and degree of dysplasiam. The
lesions were classified as well differentiated or stage I, moderately
differentiated or stage II and poorly differentiated or stage III. In
our study we had a total of 49 (52%) cases of well differentiated
squamous cell carcinomas designated as histopathological stage
I cancer. 25 (26%) cases were of moderately differentiated
squamous cell carcinoma graded as stage II cancer while 4 (4%)
cases were of the poorly differentiated variety graded as stage III
cancer. The most prevalent type of OSCC in our study group was
the well differentiated squamous cell carcinoma or stage I cancer,
which may represent a trend in patients of Rawalpindi/ Islamabad
origin to consult the dentists early on in the cancerous process,
which is contrary to the studies done in the rural areas of Pakistan
where stage II and stage III cancers are more prevalent, due to lack
of easy accessibility to a dentist as well as awareness dearth .
Out of these 95 samples 72 were formalin fixed and paraffin
embedded, 6 were fresh biopsy specimen unfixed and 17 were
endoscopic specimen. In this study group patients ages ranged
from 16 years to 86, 87% of the patients were above 35 years
of age. The most common anatomic site of involvement was
mandible including the tongue area followed by nasopharynx and
vocal cords with 23.2% (n=22) of the patients having carcinoma
of the oral cavity that had metastasized to the neck. Around 20%
(n=19) of the cases involved either the buccal or the labial mucosa
with 4% (n=4) showing gingival involvement, while maxillary and
palatal involvement was found in 12.4% (n=12) of the cases.
GP (general primers) based PCR assay was then performed for
the detection of HPV. This assay uses primers that have already
been reported in the literature . In our study the GP PCR
revealed 51 samples (54%) to be positive for HPV infection while
the remaining 44 were HPV free. However, some of the samples
that were negative for HPV as tested by GP PCR were determined
to be HPV type 16 positive, when genotyped for type specific
HPV. In order to reduce human error, this genotyping PCR was
repeated thrice in duplicates for the samples that were proven
to be negative by GP PCR. The samples positive for type 16 were
HPV positive, so a total percentage of infectivity was derived after
compiling the data derived from both GP PCR based assay and
genotyping assay. The compiled results showed the HPV infected
cases to be a total of 74 (78%) of the 95 OSCC samples (Figure
7), (Tables 2 & 3), which lies within the range of 25%-75% HPV
infectivity levels as have been reported by other studies [21,22].
This discrepancy maybe due to mutational changes in the HPV
genome in the area previously considered to be highly conserved
or the designated area might be comparatively less well conserved
in Pakistani origin HPV, as the studies utilizing them have all been
based on Caucasian population derived HPV.
When HPV infection and its gender distribution cross
tabulations were carried out a very significant association was
observed. Out of the 43 females 29 (67%) were infected with HPV.
Similarly, out of 52 males 45 (87%) were infected. Sex difference
among the patients was significantly co-related to HPV infectivity
as determined by the Pearson chi square test (P=0.02), which showed a male predilection for HPV infection. This finding may
prove to be significant in terms of including anti-HPV vaccination
in vaccination regimens for the males, to protect against oral
Out of the total 74 infected patients about 55 (74%) were
infected with HPV type 16, 3 (4%) were infected with HPV type 18,
11 (15%) had HPV type 16 and 18 co-infection and 5 (7%) were
positive for HPV by GP PCR but could not be type specified (Figure
7). Type 16 emerged as the most prevalent type of HPV in our
OSCC patient population. Type 18 was rare; however, co-infection
was observed in 11 cases. All the cases positive for HPV type 18
were malignant, it was not found in the fibro-epithelial polyps
that were included in our study. The previous studies have also
reported HPV type 16 prevalence over the other types of HPV in
OSCC patients’ population [23-25]. Different HPV types have also
been shown to be variably prevalent because of the geographic
Co-infection was not observed in the HPV infected benign
proliferative lesions, the only lesions co-infected were malignant,
which may indicate a more aggressive pattern of disease in the
case of prevalence of the two high risk types together (Table
3). However, a larger co-infected population group needs to be
studied in order to derive statistically significant association
between co-infection and aggressiveness of the disease. The
5 samples that could not be genotyped for both type 16 and 18
must be infected with some other type of HPV. Cross tabulations
of genotyping and sex, age of patients and primary tumour site did
not show significant association.
a. HPV is very prevalent in OSCC patients of Pakistani
origin, the prevalence being 78%. Therefore, a significant
association exists, and HPV could be the leading cause of oral
b. HPV type 16 is the most prevalent type of HPV found in
association with OSCC patients. Based on this study, serious
efforts must be devoted to containing the viral induced oral
squamous cell carcinoma.