Human papillomavirus infection and immunohistochemical expression of cell cycle proteins pRb, p53, and p16 INK4a in sinonasal diseases
Yamashita et al. Infectious Agents and Cancer
Human papillomavirus infection and immunohistochemical expression of cell INK4a cycle proteins pRb, p53, and p16 in sinonasal diseases
Yukashi Yamashita 0 1
Masahiro Hasegawa 0 1
Zeyi Deng 1 3
Hiroyuki Maeda 1
Shunsuke Kondo 1
Asanori Kyuna 1
Sen Matayoshi 1
Shinya Agena 1
Takayuki Uehara 1
Hideaki Kouzaki 2
Takeshi Shimizu 2
Taro Ikegami 1
Akira Ganaha 1
Mikio Suzuki 1
0 Equal contributors
1 Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus , Okinawa 903-0215 , Japan
2 Department of Otolaryngology, Head and Neck Surgery, Shiga University of Medical Science , Otsu 520-2192 , Japan
3 Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University , Guangzhou , China
Background: We aimed to clarify the possible role of human papillomavirus (HPV) infection in the malignant transformation of sinonasal inverted papilloma (IP). Methods: Subjects comprised 32 patients with chronic rhinosinusitis (CRS), 17 with IP, 5 with IP and squamous cell carcinoma (IP + SCC), and 16 with primary sinonasal SCC. HPV presence, viral loads, and physical status were investigated using polymerase chain reaction. Retinoblastoma (pRb), p53, and p16INK4a gene products were investigated by immunohistochemistry. Results: HPV DNA was detected in 6.3 % of cases with CRS, 29.4 % with IP, 40 % with IP + SCC, and 25 % with SCC. IP cases had significantly higher HPV presence than CRS cases (p = 0.04). High-risk HPV-16 was the most frequently encountered subtype (10/13, 76.9 %). HPV-16 viral loads varied from 2.5 to 7953 E6 copies/50 ng genomic DNA. Patients in the SCC and IP + SCC groups had significantly higher viral loads than those in the IP and CRS groups (p < 0.01). All SCC and IP + SCC patients with HPV-16 demonstrated mixed-type integration, whereas 4 of 5 HPV-16 patients in the IP and CRS groups showed episomal type infection (p = 0.04). Positivity to pRb was found in 78.1 % of CRS, 35.3 % of IP, and 68.8 % of SCC cases. The presence of HPV DNA negatively correlated with pRb expression in SCC (p = 0.029) and IP (P = 0.049) groups. Although 62.5 % of SCC cases exhibited p53 positivity, only 5.9 % of IP, and no CRS cases were positive. Regardless of HPV status, p16INK4a positivity was frequently detected in IP cases (82.4 %), less in SCC (12.5 %) cases, and was not detected in the CRS group. Neither the IP nor SCC cohorts showed any correlation between HPV presence and the expression of either p53 or p16INK4a. Conclusions: HPV infection was more frequent in the IP, IP + SCC, and SCC groups than the CRS group. Higher viral loads and integration observed in the IP + SCC and SCC groups, and an inverse correlation between HPV presence and positive pRb indicated that persistent infection and integration play a part in tumorigenesis and malignant transformation in certain IP cases. However, p16INK4a is not a reliable surrogate marker for HPV infection in IP.
Human papillomavirus; Cell cycle protein; Inverted papilloma; Malignant transformation; Integration; Viral load; Sinonasal disease
A variety of benign and malignant tumors may arise in
the sinonasal cavity. Of these, inverted papilloma (IP), a
benign neoplasm, has unique clinical characteristics; of
note are its high rates of recurrence and malignant
transformation. According to several systematic reviews
and meta-analyses, the malignant transformation rate of
IP is estimated to be approximately 10 % [3, 17, 20].
Human papillomavirus (HPV) infection in the
sinonasal tract is estimated to be present in 37.8 % of IPs 
and 27 % of squamous cell carcinomas (SCCs) .
Although a significant number of these tumors are infected
with HPV, especially high-risk HPV types, whether HPV
is involved in the malignant transformation of IP or in
the pathogenesis of SCC has not been confirmed, as its
mechanism and role in malignant transformation remain
obscure. There are several of reports on the clinical
relationship between IP and HPV infection. Two studies by
Beck et al. found that 63 % of IP cases were positive for
HPV DNA, and the presence of HPV sequences
predicted the recurrence of inverted papilloma [4, 5].
Moreover, these studies found that patients with HPV types
16 or 18 have a higher rate of associated malignancy
than patients with HPV 6 or 11 . A systematic review
subsequently confirmed that the presence of HPV was
significantly associated with the likelihood of IP
recurrence . However, there continues to be discussion
over whether there is a significant correlation between
the development of malignant transformation in IP and
HPV types [11, 16, 20].
In our previous study, viral loads and physical status
of HPV were examined using fresh frozen samples of IP,
SCC, and inflammatory mucosa from the sinonasal tract
to clarify the clinical importance of HPV in IP. HPV
genomes were detected in 46.1 % of IPs, 27.3 % of SCCs,
and 7.6 % of the inflammatory group, respectively .
Because the IP group showed significantly higher HPV
positivity rates than the inflammatory group, it was
concluded that HPV infection is involved in the
pathogenesis of IP, and a high viral load and integration of HPV
may play important roles in malignant transformation.
The retinoblastoma (pRb), p16INK4a and cyclin D1
genes are components of the pRb cell cycle control
pathway. The active hypophosphorylated form of pRb binds
and blocks the action of the transcription factor,
inhibiting the transition from G1 to S phase. Cyclin D1
stimulates the phosphorylation of pRb by associating itself
with cyclin-dependent kinases (CDKs). Binding of
p16INK4a to CDK 4 and 6 blocks their association with
the D-type cyclins. Another well-known cellular tumor
suppressor, p53, is involved in processes such as cell
cycle progression, DNA repair, chromatin remodeling,
differentiation, apoptosis and senescence. HPV-mediated
tumorigenesis is mainly due to the activities of two viral
oncoproteins, E6 and E7. HPV E6 can induce the
degradation of p53 by direct binding to the ubiquitin ligase
E6AP, inhibit p53-dependent signaling upon stress
stimuli, and contribute to tumorigenesis [19, 27, 36].
The viral E7 protein binds to and inactivates pRb,
activating E2F independent of cyclin dependent kinase.
The functional inactivation of host pRb by HPV E7
protein results in overexpression of p16, making p16 a
reasonable surrogate marker for the presence of
highrisk human papillomavirus.
Several reports concerning cell cycle protein expression
in IP, IP + SCC, and SCC have been published [1, 6, 14, 19,
21–23, 25, 27, 28, 36, 37]. However, the simultaneous
evaluation of HPV infection and cell cycle protein
expression using IP, IP + SCC, and SCC samples, using chronic
rhinosinusitis (CRS) as a control, has not yet been
reported. Moreover, it is very difficult to compare results
because immunohistochemical methods of evaluation differ
among studies. The aim of this study was to identify the
HPV infection and immunophenotypic features in the
malignant transformation of inverted papilloma. In the
present study, additional cases of IP, IP + SCC, SCC, and
CRS were recruited and HPV status (presence, viral load,
and physical status) and cell cycle proteins related to HPV
infection— retinoblastoma (pRb), p53, and p16INK4a gene
products—were investigated to reinforce our assumptions
from the previous study.
Ethics, consent and permissions
The study protocol was approved in advance by the
Institutional Review Board of the University of the Ryukyus.
This study was conducted to conform to the principles of
the Declaration of Helsinki.
1) Patient characteristics (Table 1)
According to Krouse classification  of IP, T2 was
observed in 3 patients, T3 in 13, and T4 in 1. The T4
case had a massive extension to the pterygoid fossa with
no malignant lesions. The observation period after our
surgeries for the IP group ranged from 18 to 98 months
(median 43 months). At the first visit to our clinic, 7
patients had previous history of sinus surgery for IP and 2
cases (11.8 %) had recurrence after surgery.
Metachronous SCC occurred in 3 cases and
synchronous SCC in 2 cases of the IP + SCC group. The periods
between the initial treatments for IP to malignant
transformation in the 3 metachronous SCC cases were
Table 1 Patient profiles
≤50, n (%)
5 months, 6 years, and 9 years. Patients in the IP + SCC
group received surgery and subsequent radiotherapy as
adjuvant treatments for cancerous lesions. Although 1
case recurred after surgery combined with radiotherapy
in the IP + SCC group, salvage endoscopic resection for
the recurrent lesion was performed successfully and the
lesion has not recurred for 9 years. The patient had
HPV-16 infection with mixed integration.
2) HPV detection (Table 2)
HPV genomes were detected in 5 of the 17 patients in
the IP group (29.4 %, Table 2), compared with 4 of the
16 patients (25.0 %) in the SCC group, 2 of the 5
patients (40 %) in the IP + SCC group, and 2 of the 32
patients (6.3 %) in the CRS group. The IP group showed a
significantly higher HPV presence than the CRS group
(p = 0.04, Fisher’s exact test).
The HPV types detected in the present study were
high-risk types only (i.e. HPV-16, HPV-33, and HPV-18)
and multiple HPV infections were not detected. Of the
13 HPV patients, HPV-16 was present in 10, therefore
viral loads and physical status of HPV-16 were
investigated in the positive samples. Viral loads varied from 2.5
to 7953 E6 copies/50 ng genomic DNA. Patients in the
SCC and IP + SCC groups had significantly higher viral
loads than the IP and CRS groups (p < 0.01, Mann–
Whitney U test). In addition, all patients with HPV-16
infection in the SCC and IP + SCC groups demonstrated
mixed type integration, whereas 4 of the 5 patients with
HPV-16 infection in the IP and CRS groups showed
episomal type infection (p = 0.01, chi-square test).
3) Expression of pRb, p53, and p16INK4a by
immunohistochemistry (Table 3)
Figure 1 shows the representative cases and expression
of pRb, p53, and p16INK4a. The expression of pRb was
observed in 78.1 % of the CRS group (Fig. 1d), 35.3 % of
the IP group, none of the IP + SCC group, and 68.8 % of
SCC group. The presence of HPV DNA showed negative
correlation with pRb expression in the SCC group (p =
0.029, chi-square test). A similar relationship between
the presence of HPV DNA and pRb expression was
observed in the IP group (p = 0.049, chi-square test).
The expression of p53 was observed in 10 of the 16
patients (62.5 %) in the SCC group and 1 of the 5
patients (20 %) in the IP + SCC group, whereas it was
expressed rarely in the IP (5.9 %) and CRS (0 %, Fig. 1e)
groups (Table 3). The expression of p53 in the SCC and
IP groups was not significantly related to HPV infection
The expression of p16INK4a was significant in the IP
group at 82.4 % compared with 12.5 % in the SCC group
and no expression (Fig. 1f ) in the CRS group (both p <
0.001, chi-square test). HPV infection in the IP group
was not related to p16INK4a expression (Table 3). The
case with HPV-33 infection in Fig. 2e showed no
expression of p16INK4a, and expression was absent in 2 cases
without HPV infection (Table 3, Fig. 2f ).
In the IP + SCC group, 16INK4a expression was
observed in only patients with HPV infection. However, in
the SCC group, 1 of the 4 patients with HPV infection
showed p16INK4a expression. Cases of p16INK4a positivity
in the SCC (Fig. 1c) and IP + SCC groups (Fig. 2d)
showed strong p16INK4a immunoreactivity compared
with the IP group (Fig. 2a-c). Interestingly, a
metachronous IP + SCC case with HPV infection showed
relatively low expression of p16INK4a when in IP only
(Fig. 2c), whereas intense p16INK4a expression was found
in IP + SCC (Fig. 2d).
We previously reported that HPV infection is involved
in the pathogenesis of IP and that high viral loads and
integration of HPV may play important roles in
malignant transformation . In the present study, HPV
infection was high in IP, IP + SCC, and SCC groups
IP + SCC (n = 5)
IP (n = 17)
HPV16, 3; HPV33, 2
CRS (n = 32)
compared with the CRS group. According to a
metaanalysis of HPV presence in sinonasal lesions, HPV was
detected in 7.0 % of normal sinus mucosa, 4.1 % of nasal
polyps, and 38.8 % of papillomas . Furthermore,
HPV was detected in 65.3 % of exophytic papillomas,
37.8 % of inverted papillomas, and 22.5 % of cylindrical
cell papillomas . According to a systematic review
and meta-analysis of HPV prevalence in head and neck
cancer, HPV was detected in 27.0–29.3 % of SCCs in
sinonasal cancer [13, 33]. In the present study, the prevalence
of HPV in CRS, IP, and SCC groups was consistent with
previous reports [32, 33]. Although high-risk HPV types
were detected in inflammatory diseases as well as in the IP
and SCC groups, significant mixed type integration was
observed in the SCC and IP + SCC groups. In addition,
higher HPV-16 viral loads were detected in the SCC and
IP + SCC groups, compared with the IP and CRS groups.
These findings suggest that persistent infection and the
integration of high-risk HPV types play important roles in
the malignant transformation of IP.
Once HPV integration occurs, it leads to disruption of
the HPV E2 gene, which controls the expression of the
oncogenes E6 and E7 by binding to and repressing their viral
promoter, resulting in their abnormal expression . E6
Table 3 Expression of cell cycle proteins
(n = 16)
(n = 5)
(n = 17)
(n = 32)
IP + SCC 40.0 %
HPV (+), n = 4
HPV(+), n = 2
HPV(−), n = 3
HPV (+), n = 5
HPV (−), n = 12 1
HPV (−), n = 12 10 2
HPV(+), n = 2
HPV(−), n = 30 0
Table 2 HPV presence, HPV type, viral load, and physical status of HPV-16
SCC (n = 16)
High-risk HPV (numbers)
HPV-16, 3; HPV-18, 1
HPV-16 viral load
(E6 copies/50 ng genomic DNA)
binds to the wild-type p53 via an adenosine
triphosphatedependent step, which leads to p53 degradation and
inactivity. The E7 oncoprotein binds to and functionally
inactivates pRB, which controls the crucial G1–S phase
transition. Functional inactivation of pRb by E7 is known
to induce up-regulation of p16INK4a expression [15, 29].
In the present study, pRB expression was frequently
observed in CRS, unlike p53, while expression was
significantly less in the IP and SCC groups. In contrast,
Altavilla et al. reported that all IP cases expressed both
pRb and p16INK4a, regardless of HPV infection .
However, most of the cases in that study carried low-risk
HPV types, whereas in the present study, all HPV cases
carried high-risk types. The inverse correlation between
HPV presence and pRb expression in the IP and SCC
groups indicated that HPV infection in IP and SCC
affects cell cycle protein expression, which suggests that
HPV infection plays a role in tumorigenesis and
Although the wild-type p53 is known to be involved in
the negative regulation of cell growth, the mutant p53
promotes tumor formation through loss of growth
suppression. Since the p53 antibody reacts to both
wild- and mutant-type p53 proteins, it is impossible to
distinguish between them using formalin-fixed,
paraffinembedded (FFPE) samples. However, the accumulation of
p53 to levels detectable by immunohistochemistry is
associated with p53 mutations . In the present study, p53
expression was observed in only 1 patient in the IP and
CRS groups, whereas the SCC group showed high p53
expression regardless of HPV presence. This finding suggests
that the mutant p53 is rarely expressed in IP and CRS.
Mirza et al. reported that the majority of HPV-positive
cases did not express p53, possibly because of proteolytic
degradation and elimination . On the contrary,
Schwerer et al. reported that the overexpression of p53 in
inverted papilloma compared with normal nasal mucosa
. The range of p53 expression varies from zero to
Fig. 1 Representative immunohistochemistry results of pRb, p53, and p16INK4a. a: pRb immunohistochemistry of SCC without HPV infection.
Bar = 100 μm. b: p53 immunohistochemistry of SCC part of inverted papilloma with SCC. The case showed HPV-16 positive. Bar = 100 μm.
c: p16INK4a expression in SCC with HPV-18 positivity. Tumor cells showed strong immunoreaction to p16INK4a in this case. Bar = 100 μm. d: pRb
expression in chronic rhinosinusitis. This case was HPV negative. Epithelial cells showed diffuse pRb immunoreaction. Bar = 100 μm. e: p53 expression
in chronic rhinosinusitis. This case was also HPV negative. Epithelial cells showed no p53 immunoreaction. Bar = 100 μm. f: p16INK4a expression in
chronic rhinosinusitis. This case was HPV negative. The epithelial cells showed no p16INK4a immunoreaction. Bar = 100 μm
approximately one-third in IP [1, 19, 23, 37], whereas
relatively high rates of expression are found in IP + SCC
[19, 22, 37]. Lin et al. also reported that low expression of
p16INK4a and positive staining for p53 are important
characteristics in IP + SCC compared with IP . Although
the reasons for these discrepancies among the p53
positivity rates between the studies are not clear, different
antibodies and evaluation methods might have affected
Expression of the tumor suppressor p16INK4a has been
proposed as a surrogate marker for HPV infection .
Overexpression of p16 is thought to reflect the presence
of biologically active HPV infection. However, there are
several contradictory reports on the value of p16INK4a as a
Fig. 2 p16INK4a immunohistochemistry in various cases. a: Inverted papilloma with positive p16INK4a reaction. The case had HPV-16 infection with
episomal form. Bar = 200 μm. b: Inverted papilloma with positive p16INK4a expression. Human papillomavirus genome was not identified in this
case. Bar = 200 μm. c: Inverted papilloma with positive p16INK4a reaction. The case became squamous cell carcinoma later (Fig. 2d). The expression
of p16INK4a was more prominent in the basal layer than in the surface layer in inverted papillomas. HPV-16 with mixed integration was detected
in this case. Bar = 200 μm. d: Metachronous squamous cell carcinoma that recurred after primary surgery for inverted papilloma (c). The invasive
carcinoma cells displayed stronger p16INK4 expression compared with the inverted papilloma (c). HPV-16 was also detected with mixed
integration. Bar = 200 μm. e: Inverted papilloma with negative p16INK4a reaction. This case had HPV-33 infection. Bar = 200 μm. f: Inverted
papilloma with negative p16INK4a reaction. The human papillomavirus genome was not identified in this case. Bar = 200 μm
biomarker. Smith et al. found no concordance between
p16INK4a expression and HPV detection in 20 % of head
and neck cancers , possibly due to transcriptionally
inactive infection or an alternate pathway of p16INK4a
activation . In our previous study, the diagnostic sensitivity
of p16INK4a overexpression was 53.2 % for the detection of
HPV DNA in HNSCC, and was considerably better in
oropharyngeal SCC at 80 % . In the present study, p16
INK4a overexpression was identified in only 1 of the 4 SCC
cases with HPV, but in more than 80 % of IP cases. These
findings are consistent with previous reports [1, 19].
Although the mechanisms behind the high prevalence of
p16INK4a in IP are unknown, these results revealed that, in
contrast to OPSCC, p16INK4a immunoreactivity is not a
surrogate marker for HPV infection in IP.
In summary, the abovementioned immunohistochemical
characteristics in specimens from patients with HPV
infection in the IP, IP with SCC, and SCC groups were similar to
other HPV-related tumors. These immunohistochemical
findings and current PCR-based results suggest that HPV
infection is involved in inducing malignant transformation
of IP through alteration of cell cycle protein expression.
HPV infection was more frequently observed in IP, IP +
SCC, and SCC groups than in the CRS group. The
higher viral loads and integration observed in the IP +
SCC and SCC groups and the inverse correlation
between HPV presence and positive pRb in
immunohistochemistry indicated that persistent HPV infection and
integration are involved in tumorigenesis and malignant
transformation in certain IP cases. However, p16INK4a is
not a reliable surrogate marker for HPV infection in IP.
Subjects and methods
The subjects in the present study consisted of 17
patients with IP (IP group), 5 with IP associated with SCC
(IP + SCC group), 16 with primary sinonasal SCC (SCC
group), and 32 with chronic rhinosinusitis (CRS group).
Surgical treatments were performed on all patients in
the IP and IP + SCC groups. Specimens from 17 patients
in the IP group, 1 in the IP + SCC group, 15 in the SCC
group, and 32 in the CRS group were collected and
stored in liquid nitrogen until analysis. FFPE samples
were used for the analysis of only 4 of the 5 cases from
the IP + SCC group, and fresh frozen samples were used
for the analysis of all other cases in the CRS, IP, and
SCC groups, and 1 case in the IP + SCC group.
1) Patient characteristics.
The clinical features in each group, such as age, sex,
and tumor stage, were reviewed from medical records.
2) Detection of HPV genome and identification of
Fresh frozen samples were analyzed for HPV and HPV
types as previously reported . A Gentra Puregene
tissue kit (Qiagen, Maryland) was used to isolate DNA
from the specimens according to the manufacturer’s
specifications. For the extraction of DNA from the
IPSCC and SCC FFPE samples, 3 10 μm-FFPE sections
were placed in a microcentrifuge tube and mixed with
0.5 mL of DEXPAT kit (Takara, Otsu, Japan). After
incubation at 100 °C for 10 min, the tube was immediately
centrifuged at 12,000 rpm for 10 min at 4 °C. The
supernatant was collected carefully, and 10 μL, including the
extracted DNA, was used as a template for a 50 μL
polymerase chain reaction (PCR) reaction according to the
manufacturer’s protocol. The presence and integrity of
the DNA was verified in all samples by PCR β-globin
gene amplification using the primers PC04 and GH20
. Negative controls with water and positive controls
with the DNA of HPV-16-positive CaSki cell line were
included in each amplification series.
The presence of HPV DNA was analyzed by PCR
using the general consensus primer sets GP5+/GP6+
and MY09/11 . DNA samples negative for GP5+/GP6
+ or MY9/11 PCR were re-amplified in a nested PCR
using the GP5+/GP6+ primer pair as previously
described , which can increase the sensitivity of HPV
detection. After purification of positive PCR products,
sequence analysis was performed on an ABI PRISM
3130xl Genetic Analyzer (Applied Biosystems, CA). The
sequences obtained were aligned, and compared with
those of known HPV types available from the GenBank
database using the BLAST program.
To investigate HPV-16 viral loads and physical status,
quantitative real-time PCR using HPV-16 DNA-positive
samples was performed to measure the quantities of the
E6 and E2 genes of HPV-16 with the ABI Prism 7300
Sequence Detection System (Applied Biosystems) and
TaqMan PCR Master Mix II (Roche Molecular Systems,
Foster City, CA). The procedures, including
experimental conditions, primers and TaqMan probes, were
identical to a previous report . Two standard curves for the
E6 and E2 genes were generated by amplification of
serial 10-fold dilutions (101, 102, 103, 104, 105, and 106 viral
copies) of a plasmid pB-actin carrying the complete
HPV-16 early region (Addgene, Inc., Cambridge, MA).
For cellular DNA quantification, an external standard
curve was generated using known serial dilutions (0.3, 3,
30, and 300 ng) of human genomic placental DNA
(Sigma-Aldrich, St. Louis, MO), and β-globin was
amplified as described by van Duin et al. . The amount of
DNA (in ng) was calculated by plotting the Ct (threshold
cycle) values against the logarithm of the standard curve.
The relative viral load was identified by calculating the
copy numbers of specimen E6 in 50 ng cellular DNA.
The physical status of HPV-16 was determined
according to a previously described method . The
amount of integrated E6 was calculated by subtracting
the copy numbers of E2 (episomal) from the total copy
numbers of E6 (episomal and integrated). Ratios of E2
copy number/total E6 of <1 indicate the presence of
both integrated and episomal forms (mixed-type
integration). An E2/E6 ratio nearly equal to 1 indicates
predominance of the episomal form, whereas a ratio of 0
indicates the presence of only the integrated form.
3) Immunohistochemistry for pRb, p53, and p16INK4a.
Serial 4 μm-thick sections from FFPE samples were
deparaffinized in xylene and hydrated in a graded series
of alcohol. Epitope retrieval was performed by heating at
95–99 °C for 10 min in Tris/EDTA buffer (pH 9.0).
Endogenous peroxidase activity was quenched by
incubating the sections in 3 % hydrogen peroxide and 15 mM
sodium azide for 5 min. The sections were
subsequently incubated overnight at 4 °C with primary
monoclonal mouse anti-pRb antibody (1:2000;
LifeSpan BioSciences, Seattle WA) for pRb, primary
monoclonal mouse anti-p53 antibody (1:500; Progen
Biotech GmbH, Heidelberg, Germany) for p53
staining, and primary monoclonal mouse anti-p16INK4a
antibody (MTM Laboratories AG, Heidelberg,
Germany). After extensive washing in
phosphatebuffered saline, the slides were incubated for 30 min
at room temperature with a horseradish
peroxidaseconjugated goat anti-mouse secondary antibody
(MTM Laboratories). Immunolabeling was visualized
by incubation in 3-3′-diaminobenzidine and stained
slides were counterstained with hematoxylin.
Cases were considered pRb-, p53-, or p16INK4a-positive
when intense nuclear and/or cytoplasmic reactivity was
present. Positive expression was defined as pRb and p53
staining in more than 25 % , or p16INK4a staining in
40 %, of 2000 tumor cells .
CRS: Chronic rhinosinusitis; FFPE: Formalin-fixed, paraffin-embedded;
HPV: Human papillomavirus; IP: Inverted papilloma; IP + SCC: Inverted
papilloma with squamous cell carcinoma; PCR: Polymerase chain reaction;
pRb: Retinoblastoma gene product; SCC: Squamous cell carcinoma.
YY and MH contributed to the acquisition of samples and clinical data,
supervision of the experiments, and preparation of the manuscript; ZD
contributed to the experimental studies and data acquisition; HM, SK, AK, SM,
SA, and TU contributed to the acquisition of samples, surgical treatments, and
patient follow-ups; HK and TS contributed to the acquisition of samples and
clinical data, patient follow-ups, and study design; TI and AG contributed to the
experimental studies; and MS contributed to the study design, supervision of
experiments, and manuscript review. All authors read and approved the
This study was supported by a KAKENHI grant 15K10785 (C) from the Japan
Society for the Promotion of Science to Dr. Hasegawa. We also thank the Ryukyu
Society for the Promotion of Oto-Rhino-Laryngology for assistance with writing
and technical help. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
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