Prevalence of HPV infection in hypopharyngeal and laryngeal squamous cell carcinoma at Thailand's largest tertiary referral center
Pongsapich et al. Infectious Agents and Cancer
Prevalence of HPV infection in hypopharyngeal and laryngeal squamous cell carcinoma at Thailand's largest tertiary referral center
Warut Pongsapich 0
Nitathip Eakkasem 0
Sontana Siritantikorn 1
Paveena Pithuksurachai 0
Kshidej Bongsabhikul 0
Cheerasook Chongkolwatana 0
0 Department of Otorhinolaryngology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok 10700 , Thailand
1 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok 10700 , Thailand
Background: Following the well-established relationship between human papillomavirus (HPV) and cervical carcinoma, the carcinogenicity of this virus has also been confirmed in subsets of head and neck carcinoma (HNCA), but mainly in the oropharynx. Other subsites of HNCA with less known association to HPV have never been studied in Thailand. Accordingly, the aim of this study was to investigate the prevalence of HPV DNA in hypopharyngeal and laryngeal squamous cell carcinoma in Thai population. Methods: This cross-sectional study included hypopharyngeal and laryngeal squamous cell carcinoma patients diagnosed and treated at the Department of Otorhinolaryngology, Siriraj Hospital during the September 2011December 2013 study period. Presence of HPV genome was confirmed by polymerase chain reaction from pathologically-confirmed fresh specimens. Demographic data and risk factors of HPV infection were evaluated. Results: Eighty patients were included, and 95% of those were male. Only one patient was noted with positive HPV62 serotype. Most patients consumed tobacco and/or alcohol. Five patients had no risk factors for cancer development. Risk of HPV infection was evaluated by self-reporting questionnaire. The mean age of sexual debut was 20.17 years. Forty-eight patients had multiple sexual partners. Sixteen and seven patients had history of sexually transmitted disease infection and habitual oral sex contact, respectively. Conclusion: There was no oncogenic HPV DNA detected within pathologic specimens of laryngeal and hypopharyngeal cancers in this study. Compared to rates reported from developed countries, the prevalence of HPV-related HNCA in Thailand is very low.
Thailand; Prevalence; Human papillomavirus; HPV infection; Laryngeal and hypopharyngeal squamous cell carcinoma
The incidence of head and neck cancer (HNCA) is
currently declining. In 2012, the World Health Organization
(WHO) estimated 686,000 new cases of HNCA [
comprising 300,000 cases of oral cavity cancer, 142,000 cases
of pharyngeal carcinoma, and 157,000 cases of laryngeal
carcinoma. HNCA-related deaths were estimated to be
375,000 cases per year. Moreover, the burden of cancer
has gradually shifted to less developed countries which
now account for 65% of cancer deaths worldwide [
Certain lifestyle behaviors, such as smoking, poor diet,
and physical inactivity, are known major risk factors for
carcinogenesis. Other possible risk factors for the upper
aerodigestive malignancies include chronic mucosal
irritation and gastroesophageal reflux disease. Infection has
been acknowledged as an etiological factor in certain types
of cancer, including stomach, liver, and cervical cancer. In
HNCA, human papillomavirus (HPV) infection has been
proven to be a major cause of cancer development only at
the base of tongue and in the tonsillar regions.
Nonetheless, due to the heterogeneity of HNCA, the relationship
between HPV infection and other subsites of the head and
neck has not been conclusively established.
The global impact of HPV-related malignancies has
been analyzed in accordance with criteria set forth by the
International Agency for Research on Cancer. A strong
association has been identified between HPV and HNCA in
high Human Development Index (HDI) regions and
countries, including Europe, North America, Australia, New
Zealand, and Japan [
]. The incidence of HNCA is
disproportionate among ethnic groups, with the highest
prevalence in non-Hispanic white men [
]. HPV related cancer
is also more prevalent in people with high socioeconomic
status, high education and young patients [
typical cervical node involvement, the prognosis in these
demographic subsets is excellent.
Even though HPV infection can develop in almost all
parts of the upper aerodigestive tract, the classical
manifestation is often located at the larynx. Laryngeal
papillomatosis is a benign pathologic lesion that has been
recognized for almost a century. The larynx is usually
infected by low oncogenicity HPV subtypes that have a
malignant transformation rate of less than 2% [
systematic review by Kreimer, et al. found 24% prevalence
of HPV in more than 1400 laryngeal or hypopharyngeal
cancer patients using the polymerase chain reaction
(PCR)-based method [
Subsites of HNCA with less known association to
HPV than the oropharynx have never been studied in
Thailand. Accordingly, the aim of this study was to
investigate the prevalence of HPV DNA in hypopharyngeal
and laryngeal squamous cell carcinoma at Thailand’s
largest national tertiary referral center.
This cross-sectional study included hypopharyngeal
and laryngeal squamous cell carcinoma patients
diagnosed and treated at the Department of
Otorhinolaryngology, Siriraj Hospital, Thailand’s largest national
tertiary referral center, during the September 2011 to
December 2013 study period. The protocol for this
study was approved by the Siriraj Institutional Review
Board (SIRB), Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, Thailand.
Patients aged >20 years that were diagnosed with
laryngeal or hypopharyngeal squamous cell carcinoma
without prior treatment during the study period were
included. Patients were divided into one of two study
groups according to cancer type. Laryngeal cancer was
subclassified into three subsites, including supraglottic,
glottic, and subglottic regions. Hypopharyngeal cancer
was subdivided into pyriform sinus, posterior pharyngeal
wall, and postcricoid regions. Both cancers were staged
according to the seventh edition of American Joint
Committee on Cancer (AJCC) staging criteria [
]. The sample size
of 40 patients per cancer group was calculated based on
the 24% prevalence of HPV-positive laryngeal and
hypopharyngeal cancer reported by Kreimer, et al. [
The case record form used for this study was divided
into two parts, as follows: Part 1) Demographic data and
clinical information; and, Part 2) Data from the
concealed self-reporting Thai-language questionnaire about
behavioral risk factors for HPV infection and cancer
development, including number of lifetime sexual
partners, age of sexual debut, history of habitual oral sex
practice, and history of sexually transmitted diseases.
Patients were notified of their right to decline answering
the sexual behavior questions listed in part 2.
A 1.5–2 mm histologically-confirmed fresh specimen
of hypopharyngeal and laryngeal squamous cell
carcinoma from each included patient was prepared by
pathologist. DNA was extracted, and PCR of linear array
hybridization for specific HPV test was performed in all
80 specimens. Biotinylated DNA was used as a primer
for genotypic testing. The protocol used for HPV
identification was previously described by Steinau et al., with
96% sensitivity and 99% specificity [
]. Oncogenic types
of HPV included type 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 68, and 73 [
Eighty patients were included, and 95% of those were
male. There were 40 patients in the hypopharyngeal
group and 40 patients in the laryngeal group. The mean
age of patients was 61.5 years (range: 34–89). More than
80% of patients presented with an advanced stage, and
almost all of them were smokers (Table 1). Pyriform
sinus was the most common subsite of hypopharyngeal
cancer, while laryngeal cancer predominantly originated
in the glottic region.
More than 70 patients (87.5%) completed the
questionnaire that elicited data about risk behaviors for HPV
infection (Table 2). There was only one positive HPV
DNA finding from all pathologic specimens. That
positive specimen was taken from a 72-year-old male patient
with T2N0M0 pyriform carcinoma. He had no risk
factors for HPV infection, but he reported habitual
smoking and drinking behavior. HPV-62 serotype was
identified in subtype analysis, yet no oncogenic virus
was found within the tumor specimen.
HPV-positive malignancies represented 5–20% of all
]. Data from the US and Europe showed 40–
80% and 20–90% of those HPV infections arising from
the oropharynx, respectively [
]. This prevalence
was relatively high, compared to our previous study [
In our previous study, only 6% of HPV-related cancer
was found in the oropharynx. In this present study, no
oncogenic HPV infection was identified in pathologic
specimens of laryngeal and hypopharyngeal cancer
among Thai study population. This discrepancy in HPV
prevalence might be due to a very high rate of
tobaccoderived carcinogenesis in our population, which is
similar to the result reported from a study in Northern Spain
]. The patients described in low prevalence reports
appear to share similar characteristic. Specifically, most
were non-white men in their seventh decade of life, with
low socioeconomic status and advanced tumors [
HPV is transmitted sexually through micro-abrasions
of skin or mucosa. As a subclinical infection, HPV is the
most common sexually transmitted infection worldwide,
with an estimated lifetime risk of cervical HPV infection
in women of up to 80% [
]. Viral genome is mainly
confined to the basal cell layer of the junction between
squamous and columnar epitheliums. Presence of HPV
infection in head and neck region may be related to
high-risk sexual behaviors, including early age of sexual
activity, number of lifetime sexual partners, habitual oral
sex practice, and history of other sexually transmitted
]. Moreover, the transmission rate might
increase with an inflammation, such as tonsillitis or
pharyngitis due to micro-disruption of mucosal membrane.
The low prevalence of HPV infection in our study
population may reflect a conservative culture of Asian
countries during the past 50 years. With regard to the
sexual behavior questionnaire, it is possible that some of
the reported data was inaccurate. For example, the age
at first sexual activity and the number of life time
partners for some study respondents may have represented
only a ‘best guess’ estimate. It should also be noted that
detection of HPV genome does not conclusively indicate
past high-risk sexual activity. As such, the collection of
previous sexual behavior data may not have been
beneficially informative in this study.
The first reason why HPV may be undetectable in
non-oropharyngeal squamous cell cancer is that different
mucosal linings have different characteristics. While the
palatine and lingual tonsils of the base of the tongue,
which are parts of Waldeyer’s ring, are mostly composed
of lymphoepithelial tissues, the hypopharynx is lined
with columnar epithelium. The other reason is that most
of the human papillomavirus could be cleared by intact
immunity without causing any clinical manifestations.
Only in a small number of people, viral genome can
integrate into host tissue and remain in a latent form, and
then can progress later into an invasive lesion.
The overall rate of high risk HPV infection detected in
oral rinsed exfoliated cell among sexually active adults in
the US was reported to be 3.7% [
]. There is currently no
consensus regarding which technique for diagnosing HPV
should be used in HNCA. The sensitivity and specificity
of each method are affected by the concentration of the
HPV infection and the quality of diagnostic specimen. In
the current study, real-time polymerase chain reaction
(PCR) was used to detect very small copies of viral DNA.
Moreover, primers were designed to target highly
conserved sequences shared by multiple HPV types, which
facilitated the identification of multiple HPV subtypes.
Despite the high sensitivity of this method, the positive
result did not identify the transcriptional activity of viral
].The HPV type 62 detected in the
hypopharyngeal specimen in this study is categorized as a low-risk
subtype with a non-carcinogenic HPV classification. It is
only a passenger virus of transcriptional silence and plays
no role in the process of tumorigenesis.
In situ hybridization(ISH) is a preferred method for
determining the integration of viral genome into a host cell. To
cover a broader range of viral subtypes, HPV probe
cocktails have recently been invented. This method has higher
specificity, lower sensitivity, is more time consuming, and is
only available in selected centers [
activation of viral oncoproteins E6 and E7 is generally regarded
as the gold standard method for confirmation of HPV
status. The detection of E6/E7 mRNA is required for the
sophisticated technique of RNA extraction followed by PCR
amplification. Given that this method is technically
challenging and has poor reproducibility, it is generally restricted
to the research laboratory [
As a result of its high positive predictive value,
overexpression of p16 protein is proposed as a surrogate marker
of HPV activation in oraopharyngeal cancer. Strong
staining of p16 in classic HPV-related histomorphology is more
practical and cost effective than PCR and ISH [
contrast, without HPV prevalence, the accuracy of p16
immunohistochemistry is low. In cases where the presence
of HPV needs to be conclusive determined, more accurate
HPV testing methods are indicated.
Laryngeal papillomatosis is the most common benign
neoplasm of the larynx. The HPV genome is integrated at
the squamocolumnar junction of the true vocal cord.
Most infections are caused by noncarcinogenic viral
strains type 6 and 11. No strong evidence has yet been
reported that establishes association between HPV infection
and laryngeal cancer. Papilloma viral tropism reflects a
distinct host-viral interplay among different virus
subtypes. A phylogenetic study reported that each variant has
its own distinctive potential to induce lesions among
different types of mucosal epithelium [
]. Consistent with
that reported finding, certain HPV16 subtypes are
specifically associated only with vulvar cancer, while cervical
cancer is associated with other types of high-risk HPV,
such as types 18, 31, and 33 [
In our study, the HPV from the patient who was found
to be positive for HPV type 62 serotype belongs to the
Alphapapillomavirus genus of viruses. HPV-62 is a
lowrisk HPV that was reported as coinfection in 5.1% of the
HPV-positive cervical samples [
]. In India and Egypt,
HPV type 62 is the most prevalent type among low-risk
types of HPV [
]. Similarly, in unvaccinated Thai
women, HPV-62 is the second most frequent HPV
detected in the cervical screening program . Regardless
of its prevalence, HPV type 62 has never been reported
in HNCA worldwide.
The patients enrolled in this study were from a single
center, Thailand’s largest tertiary referral hospital, which
means that we are often referred patients with
complicated and intransigent conditions. As such, it is possible
that our findings may not be generalizable to patients
with the same condition in other settings.
The epidemiological trends of head and neck cancer can
be classified into two groups, HPV-related and
HPVunrelated HNCA. In recent decades, with the increasing
prevalence of HPV-related HNCA, the overall
prevalence of HPV-unrelated HNCA has gradually been
decreasing due to the reduction of tobacco and alcohol
consumption. Hence, HPV status is proved to be an
effective prognostic predictor that can potentially
affect the outcome of treatments. Particularly,
identification of HPV is mandatory in the high prevalence of
In this study, only 1 of 80 cases (0.0125%) had positive
HPV DNA finding. HPV-62 serotype (non-oncogenic)
was identified. There was no oncogenic HPV DNA
detected within pathologic specimens of laryngeal and
Since any subsites other than oropharynx are
considered as HPV-unrelated, the detection is thus not
routinely recommended in our institute. Moreover,
the risks of sexually transmitted diseases in the past
do not determine the possible risk of cancer
development, especially in countries with less strong
association of HPV driven carcinogenesis.
HNCA: Head and neck cancer; HPV: Human papilloma virus
The authors gratefully acknowledge Mrs. Sunattee Kessung and Ms. Jeerapa
Kerdnoppakhun for manuscript assistance.
This study was funded a grant from by the Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, Thailand.
Availability of data and materials
Materials described in the manuscript are available to any physicians intending
to use them for non-commercial purposes.
WP: Author, principle investigator. NE: Co-author, data collection. SS: Co-author,
scientific advisor. PP: Co-author. KB: Co-author, editing proof reader. CC:
Corresponding author. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Siriraj Institutional Review Board (SIRB), Faculty
of Medicine Siriraj Hospital, Mahidol University [COA no.455/2554 (EC2)].
Consent for publication
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