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C L I N I C A L A N D LA B O R A T O R Y I N V E S T I G A T I O N S
Department of Dermatology, 2Diagnostic Pathology and 3Oncology Clinical Development, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi,
Maebashi, Gunma 371-8511, Japan
4
Department of Dermatology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan
Summary
Correspondence
Akira Shimizu.
E-mail: shimizuakira@gunma-u.ac.jp
Funding sources
None.
Conflicts of interest
None declared.
DOI 10.1111/bjd.13234
The prevalence of HPV is not high, and bowenoid papulosis is an important HPVassociated precancerous lesion in Japanese patients.
Ethnic predispositions may exist; therefore, further investigation is necessary in
order to assess the relationship between ethnicity and the prevalence of HPV infection.
Squamous cell carcinoma (SCC) usually develops from preceding lesions, including actinic keratosis, Bowen disease, burns
and chronic radiation dermatitis. In addition, an association
between SCC and high-risk human papillomavirus (HPV) has
been reported.13 The prevalence of HPV infection and
2014 British Association of Dermatologists
779
trols. The positive controls were HPV types 16 and 56, which
had been extracted by the same method as was used in this
study, and which were reported previously (Fig. S1; see Supporting Information).10 A pUCHPV16 plasmid containing HPV
type 16 DNA was used as a positive control. We evaluated the
sensitivity of PCR using the pUCHPV16 plasmid and found
the detection limit to be approximately 100 copies (data not
shown).
Immunohistochemical analysis
Immunohistochemical analysis was performed on paraffin sections using the polymer peroxidase method [Histofine Simple
StainTM MAX PO (MULTI) kit (Nichirei Corp., Tokyo, Japan)].
Briefly, deparaffinized and rehydrated sections were treated
with 03% H2O2 in methanol for 30 min to block endogenous
peroxidase activity. To expose the antigens, sections were
autoclaved in citrate buffer (pH 60) for 5 min at 121 C and
cooled for 30 min. After rinsing in phosphate-buffered saline,
the sections were incubated with affinity-purified anti-HPV
antibodies (K1H8, dilution 1 : 1000; Dako, Kyoto, Japan)
overnight followed by immunohistochemical staining with a
Histofine Simple StainTM MAX PO (MULTI) kit. The peroxidase
reaction was carried out using 002% 3,30 -diaminobenzidine
(DAB) tetrahydrochloride and 001% H2O2 in 005 mol L1
Tris-HCl (pH 74). p16INK4a staining was performed using the
CINtecTM p16INK4a Histology Kit (Dako) according to the
manufacturers instructions. The overexpression of p16INK4a
proteins is a marker for high-risk types of HPV infection.10 All
sections were separately evaluated for patterns of immunohistochemical labelling. For p16INK4a, the presence of nuclear
staining with or without cytoplasmic staining was considered
a positive result.
In situ hybridization
We next performed in situ hybridization to detect HPV DNA
and proteins in the specimens. The catalysed signal amplification method (GenPoint System; Dako) was used4 with formalin-fixed, paraffin-embedded specimens. Briefly, formalinfixed, paraffin-embedded 4-lm tissue sections mounted on silanized slides were deparaffinized. Each sample was pretreated
in buffer (Target Retrieval Solution; Dako) at 95 C for
40 min, digested with 4 lg mL1 proteinase K at room temperature for 10 min, incubated in 03% H2O2 in methanol for
20 min, then air dried. After heat denaturation at 90 C for
5 min in hybridization solution mixed with biotinylated highrisk HPV probe cocktail (GenPoint HPV; Dako), which contains DNA of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59 and 68, the slides were incubated at 37 C overnight. Then, the slides were washed with 01 9 saline sodium
citrate at 50 C for 20 min. Following treatment with horseradish peroxidase-conjugated streptavidin (Dako), the biotinyl
tyramide amplification reaction was conducted. Then, the substrates were exposed to streptavidin conjugated with horseradish peroxidase. The sections were incubated with DAB
2014 British Association of Dermatologists
Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publishers website:
Fig S1. Detection of human papillomavirus DNAs using different consensus primer pairs.
Fig S2. Detection of human papillomavirus DNA and b-globin using different consensus primer pairs.
Results
Prevalence of human papillomavirus infection among
patients with squamous cell carcinoma
The clinical and virological data of the 38 patients studied are
summarized in Table 1. The subjects included 24 patients
with facial SCC, four patients with hand SCC, two patients
(a)
each with finger, leg and foot SCC and one patient each with
trunk, labia majora, scrotum and scalp SCC. Actinic keratosis
was histopathologically and clinically considered to be a precancerous lesion in 27 of the 38 patients, and bowenoid papulosis (BP) and Bowen disease in two patients each as
precancerous lesions. No obvious precancerous lesions were
identified in seven patients. Using three different primer pairs,
we detected HPV DNA in two patients (5%) (Fig. S2; see Supporting Information; and Table 1), whereas all of the 18 control subjects were found to be HPV DNA negative (data not
shown). The internal control DNA, b-globin, was detected in
all 38 cases (Fig. S2 and Table 1). Direct sequencing confirmed HPV type 16 in two cases (cases 1 and 35). The copy
(b)
(c)
(e)
(d)
(f)
The other patient positive for HPV type 16 (case 35) was
an 81-year-old woman with a 5-month history of a nodule
on the labia majora. Physical examination revealed a pedunculated 80 9 55-mm reddish elastic nodule on the labia majora.
There were also multiple black pigmented nodules around the
reddish nodule (Fig. 2a). Inguinal lymph node swelling was
observed. A histopathological examination revealed hyperkeratosis, parakeratosis, papillomatosis and acanthosis. Tumour cell
proliferation into the dermis with inflammatory-cell infiltration was remarkable (Fig. 2b). Atypical keratinocytes proliferated throughout the epidermis, and dyskeratotic cells were
observed in some areas (Fig. 2c). Immunostaining of p16INK4a
was positive (Fig. 2d), while in situ hybridization and HPV
immunohistochemistry were negative. A computed tomography scan showed left inguinal lymph node enlargement.
Therefore, a diagnosis of SCC with lymph node metastasis was
established. The clinical and histopathological findings strongly
suggested the diagnosis of SCC arising from BP. After receiving radiation therapy, the patient refused any further
treatments.
Discussion
A relationship between cutaneous SCC and HPV infection has
been reported.13,1113 As for mucous membrane type HPV,
such as HPV type 16, Hama et al.1 reported that the prevalence
of HPV infection in patients with SCC of the hands and fingers
was 23%. On the other hand, epidermodysplasia verruciformis
type HPV was detected in 27% of patients with SCC.2 Shamanin et al.3 reported that 31% of patients with SCC were HPV
positive, including infection with HPV types 4, 8, 9, 23, 32,
42 and 51. However, the methods used in these studies were
all PCR-based typing methods employing different primers,
suggesting that the different prevalence of HPV between the
studies may be due to the methodological conditions in each
(a)
(b)
(c)
(d)
Acknowledgments
We thank Tohru Kiyono for kindly supplying us with the
pUCHPV16 plasmid.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publishers website:
Fig S1. Detection of human papillomavirus DNAs using different consensus primer pairs.
Fig S2. Detection of human papillomavirus DNA and b-globin using different consensus primer pairs.