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Does Transcriptionally Active Papillomavirus
Associate with Glottic Laryngeal Cancer?
Gustavo Barreto da Cunha1,2, Leonardo Haddad1*, José Eduardo de Sá Pedroso1, Ricardo Artigiani Neto3 and
Noemi Grigoletto De Biase1
1 Otolaryngology and Head and Neck Surgery, Universidade Federal de São Paulo, Brazil
2 Otolaryngology and Head and Neck Surgery, Universidade Federal de São Paulo, Brazil
3Pathology Department, Universidade Federal de São Paulo, Brazil.
Submission: February 25, 2020; Published: April 24, 2020
*Corresponding author:Leonardo Haddad, Otolaryngology and Head and Neck Surgery, Escola Paulista de Medicina - Universidade Federal de São
Paulo, São Paulo, 40038-002, Brazil
How to cite this article: Gustavo Barreto da Cunha, Leonardo Haddad, José Eduardo de Sá Pedroso, Ricardo Artigiani Neto, Noemi Grigoletto De
Biase. Does Transcriptionally Active Papillomavirus Associate with Glottic Laryngeal Cancer?. Glob J Oto, 2020; 22(1): 556076. DOI: 10.19080/GJO.2020.22.556083
Purpose: To evaluate the association of human papillomavirus in its active form and invasive glottic laryngeal cancer
Methods: A case-control study was conducted to evaluate the association of transcriptionally active human papillomavirus in patients
diagnosed with glottic laryngeal squamous cell carcinoma and vocal cord polyps as cancer-free controls. p16INK4a immunohistochemistry and
DNA in situ hybridization were used to identify positive cases for the virus.
Results: 132 subjects were included in the study, 66 patients diagnosed with laryngeal glottic squamous cell carcinoma, and the remaining
66 participants formed the control group. Among the individuals in the study group, only 8 were women with a mean age of 62.3 years. Smoking
and tobacco exposure (pack years) were positively associated with laryngeal cancer. p16INK4a immunohistochemistry was positive in 5 out of
the 66 (7.6%) patients with squamous cell carcinoma, whereas no case among the control group was positive. However, all the 5 cases presented
negative in situ hybridization for human papillomavirus DNA and were therefore classified as negative human papillomavirus status. p16INK4a
immunohistochemistry was not significantly associated with glottic laryngeal squamous cell carcinoma or human papillomavirus status, nor
with any specific group. There was, however, a tendency to associate it with the laryngeal cancer group and patients who never smoked.
Conclusion: Transcriptionally active papillomavirus did not associate with glottic laryngeal cancer.
Keywords:Laryngeal Neoplasms; Glottis; Vocal Cords; Papillomavirus Infections; In Situ Hybridization
The most important risk factors related to laryngeal cancer,
as well as to other head and neck neoplasms, are smoking and
alcoholism, which become even more important when associated
. However, during the last few decades, the studies involving the
relationship among human papillomavirus (HPV) infection and
the development of these neoplasms have increased . Several
studies have demonstrated a strong association among active HPV
infection and oropharyngeal carcinoma [3,4]. On the other hand,
the participation of HPV in the etiology of laryngeal squamous cell
carcinoma (SCC) and other sites of the head and neck SCC has not
been definitively established.
HPV-related SCC has an increased expression of p16INK4a,
whereas in non-HPV-related cases chronic exposure to tobacco
and alcohol can generate mutational losses of p16INK4a and p53
protein genes, resulting in a low expression of this protein.
Therefore, the immunohistochemical panel of p16INK4a has been
described as a surrogate biomarker for active HPV infection in
head and neck carcinomas , mainly in oropharynx cancer, but
in laryngeal cancer as well .
However, detection of low levels of HPV-DNA and absence of
viral transcription has a limited biological value and may indicate
that HPV does not play a role in malignant transformation of
laryngeal cancer [7,8]. HPV-DNA has also been found in normal
tissues and in benign lesions of the vocal folds , which supports
the idea that that the presence of HPV alone may not be related
to a causal factor. Thus, detection methods that reflect active HPV transcription, including p16INK4a immunohistochemistry (p16
IHC), reverse transcriptase polymerase chain reaction (RT-PCR)
or in situ hybridization (ISH) for HPV-DNA or E6/E7 mRNA are
required to demonstrate biologically significant HPV . This
study aims to evaluate the association of active HPV infection and
the glottic laryngeal SCC.
We conducted a case-control study in which the association
of transcriptionally active HPV was studied in tumors of patients
previously diagnosed with invasive glottic laryngeal SCC. 66
consecutive adult patients (>18 years old) recently diagnosed
with glottic laryngeal SCC through biopsy of the lesion under
suspension laryngoscopy and anatomopathological analysis were
recruited from July 2014 to June 2017. All patients previously
treated with chemotherapy or radiotherapy were excluded.
The control group consisted of patients submitted to laryngeal
microsurgery for vocal polyp, a known benign lesion, during the
same period of the study. Patients under 38 years and women
under 50 were excluded in order to make the sample more like the
study group. It was also excluded those who presented dysplasia
signs in anatomopathological analysis or who had progressed
with head and neck cancer during follow-up. The work was done
in accordance with the appropriate institutional review body and
carried out with the ethical standards. Informed consent was
obtained from all individual participants included in the study.
The immunohistochemical reactions for the p16INK4a antibody
were carried out on paraffin-embedded tissue sections obtained
from biopsies of tumors and polyps, using monoclonal primitive
body GeneAB clone IHC 016 (GenomeMe, Canada).
The immunoblotting pattern of p16INK4a is cytoplasmic and
nuclear and the evaluation was based on the extent of staining,
related to the percentage of stained neoplastic cells. Staining was
scored as positive if it showed nuclear and cytoplasmic staining
in most tumor cells (> 70%), regardless of its intensity. It was
scored as negative if it showed complete absence of p16INK4a or
it appeared only on isolated tumor cells (<40%). Samples with
intermediate staining pattern (between 40 and 70%) were
classified as inconclusive .
ISH was performed by manual technique using the Zytofast
PLUS CISH Implementation kit (Zytovision, Germany) with the
ability to detect high risk genotypes 16, 18, 31, 33, 35, 39, 45, 51,
52, 56, 58, 59, 66, 68, and 82, and low-risk genotypes 6 and 11.
The cases were classified as positive or negative. Negative results
in ISH were considered as negative HPV status.
The Statistical Package for Social Sciences (SPSS Inc., Chicago,
IL, USA), version 17.0 for Windows, was used for database’s
elaboration and descriptive analysis. The results were presented
as tables and graphs. Categorical variables were shown as
frequencies and percentages -n (%). Continuous variables with
normal distribution were expressed as mean and standard
deviation, and those with non-normal distribution were described
as median and interquartile range. The normality of the numerical
variables was verified through descriptive statistics, graphical
analysis and the Shapiro-wilk test.
Chi-square test for categorical variables was used to compare
the clinical and demographic variables between the SCC and control
groups and to compare positive and negative p16 IHC groups
among individuals with laryngeal SCC. Fisher’s exact test was used
when the number of individuals was low. For the comparison of
numerical variables between these groups independent t test was
applied for those with normal distribution and Mann-Whitney
test for those with non-parametric distribution. It was considered
p <0.05 for all analyzes.
132 individuals were included in the study, 66 patients with
glottic laryngeal SCC and 66 participants in the control group
diagnosed with laryngeal polyp. The participants’ clinical and
demographic characteristics are detailed in Table 1. Regarding the
study group, all patients were diagnosed with only one primary
tumor and did not receive treatment prior to surgery. Only 8 were
women and the mean age was of 62.3 years (± 9.2). Smoking
history and tobacco exposure (pack years) were associated with
laryngeal SCC (Figure 1). 5 out of the 66 (7.6%) patients with
SCC presented positive p16 IHC, whereas no control cases were
positive. Nevertheless, all 5 cases presented negative HPV DNA
ISH and were therefore classified as negative HPV status. Cancer
staging of patients with laryngeal SCC is shown in Figure 2. Among
these patients, p16 IHC was positive in 3 T1A (60%), 1 T1B (20%)
and 1 T3 (20%) cases. The p16 IHC distribution among the group
with laryngeal SCC is shown in table 2.
There is a large variation in the proportion of HPV-related
laryngeal SCC in the literature. Besides the demographic and
ethnic variation, the high variation in the diagnostic tests used
may have influenced this result . In the present study, no
cases of glottic laryngeal SCC related to transcriptionally active
HPV infection were identified. In order to confirm the results of
p16 IHC, whilst avoiding false-positive cases, ISH reaction was
performed in all p16 IHC positive cases. Thus, although p16 IHC
was positive in 5 of 66 cases (7.6%), HPV DNA ISH was negative in
all these samples, which demonstrated negative HPV status.
Rodrigo et al also did not find a significant role of HPV
in laryngeal cancer in northern Spain. Only 1 patient out of
62 (1.6%) had positives HPV DNA 16 and E6 mRNA RT-PCR.
However, this patient was 85 years old, with a smoking and heavy
alcoholism history and presented positive p53 and negative HPV
DNA ISH. Therefore, it was arguable HPV’s participation in this
patient’s carcinogenesis. In this study, 11% of laryngeal tumors
were positive for p16 IHC, which represented a high level of
false-positive results and low correlation with HPV status .
Similarly, Lee et al investigated transcriptionally active HPV only
in glottic laryngeal cancer and suggested that HPV does not appear
to play a causal role in this type of tumor and the detection of this
virus may only be incidental . In the same way, Fakhyr et al
reported that there was no HPV infection in a population of 34
patients with laryngeal cancer, investigated with PCR and ISH .
On the other hand, other studies investigated this relationship
and demonstrated a small but positive association between
active HPV and laryngeal SCC, ranging from 1.6% to 6.7%
[9,14-16]. Chernock et al reported that only 4 out of 60 patients
(6.7%) presented E6/E7 HPV mRNA  and Hernandez et al
demonstrated only 2% p16 IHC and HPV DNA positivity in laryngeal
SCC . Both studies did not find a significant relationship of
p16INK4a expression and HPV DNA status, unlike what is described
in oropharyngeal SCC, which has a well-established etiologic and
prognostic role .
Older studies or those which investigated only the presence
of HPV, instead of its active form, tended to present a higher
prevalence, reaching 16% , 27% , 35%  and up to
75%  of the cases. However, these studies used PCR to detect
HPV DNA as diagnostic method, a method that does not confirm
the integration of HPV DNA into the nucleus of the tumor cell and
does not prove a causal role of the virus in laryngeal carcinogenesis
. The presence of HPV DNA has been demonstrated in a
significant proportion of benign lesions of the larynx. Therefore,
the presence of the virus in many cases of cancer appears to be
only incidental [9,13].
Few studies, including ours, evaluated the participation of
HPV only in glottic laryngeal SCC. Lee et al also found no relation
between HPV infection and carcinogenesis in this population .
There are few studies comparing the prevalence of HPV infection
in glottic and supraglottic cancer, but these are older studies that
do not differentiate the active from inactive HPV forms, and have
very small samples, making it difficult to reach reliable conclusions
[24-27]. Therefore, it is possible that supraglottic laryngeal SCC
behaves more like the pharynx SCC than to the glottic region SCC
itself, which could justify these findings.
In this study, p16 IHC was not significantly associated with
glottic laryngeal cancer or HPV status, nor with any of the specific
groups, although there was a tendency to associate it with
laryngeal SCC and patients who never smoked. HPV-associated
SCCs have an increased expression of p16INK4a, whereas those
related to chronic exposure to tobacco and alcohol tend to
generate mutational losses of p16INK4a and p53 protein genes,
resulting in low expression of these proteins. Thus, p16 IHC has
been used as an HPV-active infection biomarker in head and neck
SCC, especially in oropharynx cancer .
Gheit et al have also suggested the use of this marker in HPVrelated
laryngeal SCC , however most studies published to date
did not find a good correlation between p16INK4a overexpression
and active infection in SCC of this region, suggesting that the
hyperexpression of this protein in laryngeal carcinogenesis may
reflect cellular changes not related to HPV. Some tumors may have
positive p16INK4a due to mutation and inactivation of Rb protein
or E2F amplification . Thus, actual evidence suggests that
p16 IHC alone is not a reliable marker for HPV participation in
carcinogenesis of laryngeal SCC [9,10,17,28,29].
As expected, the group of patients with smoking history,
as well as tobacco exposure (pack years) was associated with
laryngeal cancer. The group of alcoholic patients, however, did not
present the same association. The lack of information regarding
the medical record, especially among patients with laryngeal
polyp seems to have impaired this evaluation. These are the two
main risk factors related to the development of head and neck
SCC . There was also a difference in gender and age between
the study and control groups. Polyps are known benign lesions of
the larynx which affect young patients (less than 40 years old).
Although they are more related to males, they are also highly
prevalent in females (55% x 45%, respectively) , whereas
SCC affects men in a larger proportion and in older patients ,
which explains the difference found.
This study has some limitations. As the patients were
retrospectively evaluated, the anatomopathological analysis were
performed using paraffin-embedded tissue sections obtained from patients’ surgeries. There was loss of data related to medical
records, especially those concerning alcoholism and those of the
control group. There was also a limited number of participants,
which may have contributed to the identification of no HPV
positive cases. Nevertheless, this work presents some other
advantages, such as the use of tests that evaluate transcriptionally
active HPV instead of only detecting the presence of the virus in
the samples, which might not be related to tumor carcinogenesis.
Besides it evaluated a single tumor site, the glottic region, avoiding
generalization of other sites of the larynx’s behavior