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BJD

British Journal of Dermatology

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

Detection of human papillomavirus (HPV) in patients with


squamous cell carcinoma and the clinical characteristics of
HPV-positive cases
A. Shimizu,1 M. Kato,1 Y. Takeuchi,1 T. Sano,2 K. Kaira,3 H. Uezato4 and O. Ishikawa1
1

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

Accepted for publication


25 June 2014

Funding sources
None.

Conflicts of interest
None declared.
DOI 10.1111/bjd.13234

Background The association between human papillomavirus (HPV) and squamous


cell carcinoma (SCC) has been reported; however, the prevalence of HPV infection varies, and the clinical characteristics of HPV-positive cases remain
unknown.
Objectives To elucidate the frequency of HPV infection in a series of Japanese
patients with SCC and to identify the characteristics of HPV-positive cases.
Methods We evaluated 38 patients with SCC treated at our department. HPV typing
was performed using SCC samples from different body sites. Immunohistochemical staining for HPV proteins and p16INK4a was performed, in addition to polymerase chain reaction and in situ hybridization. The clinical characteristics of the
HPV-positive cases were clarified.
Results Two genital lesions were positive for HPV type 16. Both cases showed basaloid features histopathologically, and were considered to have SCC that had
arisen from bowenoid papulosis. p16INK4a expression was observed in 11 cases,
including the two HPV-positive cases.
Conclusions The present study indicates that the prevalence of HPV is not high and
that bowenoid papulosis is an HPV-associated precancerous lesion. Further investigation is necessary to assess the relationship between HPV infection and SCC.

Whats already known about this topic?

The prevalence of human papillomavirus (HPV) infection varies in squamous cell


carcinoma (SCC), possibly because of the different detection methods used.
The clinical characteristics of HPV-positive SCC remain unknown.

What does this study add?

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

whether HPV is really a causative agent or simply a bystander


remains unclear. Furthermore, the clinical characteristics of
HPV-positive cases remain unknown. We herein examined the
presence of HPV DNA in 38 cases of SCC, using polymerase
chain reaction (PCR) with three different consensus primer
British Journal of Dermatology (2014) 171, pp779785

779

780 Detection of HPV in SCC and its characteristics, A. Shimizu et al.

pairs. We further carried out immunohistochemistry for HPV


proteins and p16INK4a, as well as in situ hybridization. We clinically characterized the HPV-positive lesions and discuss them
in context with previous studies.

Materials and methods


Patients and specimens
Thirty-eight patients with SCC were recruited in this study.
Their mean age was 77 years, ranging from 49 to 98 years.
As negative controls, normal skin samples from 18 healthy
subjects, obtained by surgical operation, were also examined.
This study was approved by the Genome Ethics Committee at
Gunma University Graduate School of Medicine.
Polymerase chain reaction with consensus sequence
primers and sequence analysis
Formalin-fixed, paraffin-embedded samples were cut, and
DNA was extracted using Dexpat (Takara, Kyoto, Japan).
HPV PCR was performed using L1C1/L1C2,4,5 GP5+/GP6+4,6
and CPI/CPIIS7,8 consensus primers. To detect viral DNA by
PCR, an 18-lL reaction mixture, containing 1 9 Ex Taq buffer, 2 mmol L1 MgCl2, each deoxyribonucleotide triphosphate at 200 nmol L1, 60 ng sense and antisense PCR
primers and 5 U TaKaRa Ex Taq (Takara) (final concentrations) was added to 2-lL DNA extracts. The PCR protocol
employed for the primer pair L1C1/L1C2 was 40 cycles under
the following conditions: denaturation at 94 C for 1 min,
annealing at 48 C for 15 min and extension at 72 C for
2 min. For the primer pair GP5+/GP6+, the PCR protocol was
40 cycles under the following conditions: denaturation at
94 C for 1 min, annealing at 40 C for 1 min and extension
at 72 C for 15 min. For the primer pair CPI/CPIIS, the conditions were as follows: 40 cycles of denaturation at 94 C for
1 min, annealing at 52 C for 1 min and extension at 72 C
for 1 min. In addition, PC03/PC04 (Takara), a ready-made
primer pair, was used for the amplification of b-globin. For
this primer pair, the PCR protocol was 40 cycles under the
following conditions: denaturation at 94 C for 1 min,
annealing at 55 C for 45 s and extension at 72 C for 1 min.
Each PCR condition was modified by us. The L1C1/L1C2 consensus primers can be used to detect HPV types 6, 16, 18, 31,
32, 33, 35, 39, 42, 45, 51, 52, 53, 54, 56, 58, 59, 61, 66,
67, 68, 70, 71, 72, 73, 82, 84, 86, 90, 91 and ME180.9
GP5+/GP6+ consensus primers can be used to detect HPV
types 6, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39, 40, 43, 45,
51, 52, 54, 55, 56, 58, 59 and 66.6 In addition, CPI/CPIIS
consensus primers cover HPV types 1, 2, 3, 4, 5, 6b, 7,
8,10a, 11, 14a, 16, 18, 19, 20, 21, 24, 25, 31, 33, 36 and
46.7,8 The amplified PCR products were electrophoresed on
2% agarose gel, and the PCR products were sequenced with
an automated sequencer. Before testing the samples, we confirmed that each primer pair successfully amplified positive
controls and HPV DNAs, and we also included negative conBritish Journal of Dermatology (2014) 171, pp779785

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

Detection of HPV in SCC and its characteristics, A. Shimizu et al. 785


14 Witkiewicz AK, Knudsen KE, Dicker AP, Knudsen ES. The meaning
of p16ink4a expression in tumors: functional significance, clinical
associations and future developments. Cell Cycle 2011; 10:2497
503.
15 Ikenberg H, Gissmann L, Gross G et al. Human papillomavirus
type-16-related DNA in genital Bowens disease and in Bowenoid
papulosis. Int J Cancer 1983; 32:5635.
16 Yoneta A, Yamashita T, Jin HY et al. Development of squamous
cell carcinoma by two high-risk human papillomaviruses (HPVs),
a novel HPV-67 and HPV-31 from bowenoid papulosis. Br J Dermatol 2000; 143:6048.
17 Park KC, Kim KH, Youn SW et al. Heterogeneity of human papillomavirus DNA in a patient with Bowenoid papulosis that progressed to squamous cell carcinoma. Br J Dermatol 1998; 139:1087
91.
18 Kato T, Saijyo S, Hatchome N et al. Detection of human papillomavirus type 16 in bowenoid papulosis and invasive carcinoma
occurring in the same patient with a history of cervical carcinoma.
Arch Dermatol 1988; 124:8512.

2014 British Association of Dermatologists

19 Gillison ML, Koch WM, Capone RB et al. Evidence for a causal


association between human papillomavirus and a subset of head
and neck cancers. J Natl Cancer Inst 2000; 92:70920.
20 Cubilla AL, Reuter VE, Gregoire L et al. Basaloid squamous cell carcinoma: a distinctive human papilloma virus-related penile neoplasm: a report of 20 cases. Am J Surg Pathol 1998; 22:75561.
21 Holzinger D, Schmitt M, Dyckhoff G et al. Viral RNA patterns and
high viral load reliably define oropharynx carcinomas with active
HPV16 involvement. Cancer Res 2012; 72:49935003.

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.

British Journal of Dermatology (2014) 171, pp779785

782 Detection of HPV in SCC and its characteristics, A. Shimizu et al.

chromogen substrate and counterstained with haematoxylin.


The probe was a biotinylated high-risk HPV probe cocktail
(GenPoint HPV; Dako) that contained HPV DNA types 16, 18,
31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.

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)

British Journal of Dermatology (2014) 171, pp779785

Fig 1. Clinical appearance and histopathology


of the human papillomavirus (HPV) type 16positive tissue in case 1. (a) A mulberry-like,
reddish, elastic hard nodule surrounded by
multiple black pigmented nodules. (b)
Atypical keratinocytes were observed
throughout the epidermis. The white arrows
indicate clumping cells. Several dyskeratotic
cells were also observed. Koilocytes were
noted beneath the cornified layer. Original
magnification 9 400. (c) Atypical tumour
cells had invaded the dermis. Inflammatorycell infiltration was seen around the tumour
cells. Original magnification 9 100. (d) HPV
DNA was detected in the nuclei of the
epithelium of the squamous cell carcinoma
(SCC) lesion using in situ hybridization (black
arrow). Original magnification 9 400. (e)
Immunohistochemistry using anti-HPV
antibodies (K1H8; Dako) demonstrated HPV
capsid proteins in the epithelium of the SCC
lesion (black arrows). Original magnification
9 400. (f) Immunohistochemistry using antip16INK4a antibodies demonstrated the
overexpression of p16INK4a in the epithelium
of the SCC lesions. Original magnification
9 40.
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Detection of HPV in SCC and its characteristics, A. Shimizu et al. 783

numbers of the two HPV-positive cases were estimated to be


approximately 104 copies lL1 using serially diluted pUCHPV16 plasmids (data not shown). Eleven patients, including
the two HPV type 16-positive patients, were positive for
p16INK4a. An immunohistochemical analysis of the HPV L1
protein and in situ hybridization detected only one patient, in
whom HPV type 16 was detected, with positive staining.
Clinical features of human papillomavirus type 16positive squamous cell carcinoma
One HPV type 16-positive patient (case 1) was a 71-year-old
man with a 3-year history of a small nodule on the scrotum.
A physical examination revealed a 40 9 29-mm mulberry-like
reddish elastic nodule. There were also multiple black pigmented nodules on the large pigmented flecks around the reddish nodule (Fig. 1a). No inguinal lymph node swelling was
noted. A histopathological examination revealed hyperkeratosis, parakeratosis, papillomatosis and acanthosis. Atypical keratinocytes proliferated throughout the epidermis, and clumping
cells, koilocytes and dyskeratotic cells were noted in some
areas (Fig. 1b). Atypical tumour cell proliferation into the dermis with inflammatory-cell infiltration was apparent (Fig. 1c).
On the other hand, in the left portion of the specimen, atypical cell growth was observed throughout the epidermis, and
koilocytosis was noted under the stratum corneum, consistent
with the findings of BP. A diagnosis of SCC arising from BP
was established. The lesion was resected with a 1-cm margin
from the pigmented fleck, and full-thickness skin grafting was
performed. There has been no recurrence for 5 years. HPV
type 16-positive cells with nuclear staining were observed in
the upper epidermis and stratum corneum of the SCC lesion
on in situ hybridization (Fig. 1d) and HPV immunohistochemistry (Fig. 1e). Immunostaining of p16INK4a was positive
(Fig. 1f).

Fig 2. Clinical appearance and histopathology


of the HPV type 16-positive tissue in case 35.
(a) A pedunculated 80 9 55-mm reddish,
elastic tumour on the labia majora. Multiple
black pigmented nodules were seen (black
arrows) around the reddish tumour. (b)
Downward proliferation of rete ridges with
surrounding cell infiltration was observed.
Invasion of tumour cells was noted. Original
magnification 9 40. (c) Atypical keratinocytes
were observed throughout the epidermis.
Several dyskeratotic cells were also observed.
Original magnification 9 100. (d) Staining of
p16INK4a in the epithelium. Original
magnification 9 40.
2014 British Association of Dermatologists

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)

British Journal of Dermatology (2014) 171, pp779785

784 Detection of HPV in SCC and its characteristics, A. Shimizu et al.

study. In the present study, we examined 38 patients with


SCC in order to detect HPV and found that two subjects (5%)
were positive for HPV DNA. This is a low rate compared with
previous studies.2,3,12 Differences in the PCR primers and the
method used for DNA extraction, as well as the cohort being
analysed, may account for the differences in the detection
rates. Nine of the 36 HPV DNA-negative tumours in this study
were positive for p16INK4a on immunohistochemistry. Because
most HPV-positive tumours express p16INK4a, it is possible
that some cases of HPV could not be detected. Alternatively,
the expression of p16INK4a in HPV DNA-negative tumours
may reflect disturbances of the retinoblastoma protein signalling pathway, unrelated to HPV infection.14 The reason why
only case 1 showed positive findings for in situ hybridization
and immunohistochemical analysis of the HPV L1 protein is
unknown. There is a possibility that the viral DNA copy number was too low in the sample. Because the patient refused to
undergo surgery, only the biopsied sample was available.
In our patients, HPV type 16-positive cases were considered
to have originated from BP, which is clinically characterized
by the presence of black nodules around the genitals.15 To the
best of our knowledge, five cases of SCC arising from BP have
previously been reported.1,1618 The types of HPV in the
lesions included oncogenic HPV types 16, 31 and 67. BP is
usually a viral-mediated disease that is thought to spontaneously regress; however, physicians should keep in mind the
fact that BP can progress to SCC. The present results suggest
that the detection of HPV depends on the type of precancerous
lesion from which SCC arises. Interestingly, all of the cases of
SCC arising from BP were reported from Asian countries.
Hence, there may be some ethnic predisposition in this
condition.
Histopathologically, the HPV-positive cases in our study
showed basaloid morphology, not conventional keratinizing
SCC. Interestingly, Gillison et al.19 showed that oropharyngeal SCCs that are associated with HPV infection not only
showed poor differentiation and were not keratinizing, but
were also strongly associated with a basaloid morpgology.
In their study, basaloid features were significantly associated
with HPV-positive tumours. In addition, Cubilla et al.20 also
reported that nine of 11 cases (82%) of basaloid penile
SCC were positive for HPV type 16. HPV-positive SCCs may
have an unconventional basaloid histopathological appearance regardless of the part of the body from which they
originate.
Because our case 35, in whom HPV type 16 was detected,
showed lymph node metastasis, HPV-positive SCC might have
a tendency to develop a poor outcome. Croci-Torti et al.
reported that two patients with HPV type 16-positive SCC
showed lymph node metastasis, similar to our case 35.11 Their
two cases were extragenital HPV-positive tumours, and both
of the patients had HPV type 16 detected in the lesions. As
reported by Gillison et al., oropharyngeal SCCs that are associated with HPV infection have a better prognosis than those
that are HPV negative.19 Holzinger et al. reported that a high
viral load in oropharyngeal SCCs is considered to be associated
British Journal of Dermatology (2014) 171, pp779785

with a better prognosis compared with a low viral load.21 In


contrast, Croci-Torti et al.11 reported an aggressive case to have
a lower number of HPV viral copies per cell in the tumoral
tissue. In accordance with this finding, HPV-positive penile
SCCs have been reported to have a worse prognosis.20 There
might also be a difference in the prognosis between cutaneous
HPV-positive SCCs and oropharyngeal HPV-positive SCCs.
Because the number of patients in our study was limited, a
larger study is necessary to address this issue.

Acknowledgments
We thank Tohru Kiyono for kindly supplying us with the
pUCHPV16 plasmid.

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bowenoid papulosis. Dermatology 2004; 209:32932.
2 Harwood CA, Surentheran T, McGregor JM et al. Human papillomavirus infection and non-melanoma skin cancer in immunosuppressed and immunocompetent individuals. J Med Virol 2000;
61:28997.
3 Shamanin V, zur Hausen H, Lavergne D et al. Human papillomavirus infections in nonmelanoma skin cancers from renal transplant
recipients and nonimmunosuppressed patients. J Natl Cancer Inst
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4 Shimizu A, Tamura A, Abe M et al. Detection of human papillomavirus type 56 in Bowens disease involving the nail matrix. Br J
Dermatol 2008; 158:12739.
5 Yoshikawa H, Kawana T, Kitagawa K et al. Detection and typing of
multiple genital human papillomaviruses by DNA amplification
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6 de Roda Husman AM, Walboomers JM, van den Brule AJ et al. The
use of general primers GP5 and GP6 elongated at their 3 ends
with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995; 76:105762.
7 Hagiwara K, Uezato H, Arakaki H et al. A genotype distribution of
human papillomaviruses detected by polymerase chain reaction
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in Japan. J Med Virol 2005; 77:10712.
8 Tieben LM, ter Schegget J, Minnaar RP et al. Detection of cutaneous and genital HPV types in clinical samples by PCR using consensus primers. J Virol Methods 1993; 42:26579.
9 Asato T, Maehama T, Nagai Y et al. A large casecontrol study of
cervical cancer risk associated with human papillomavirus infection in Japan, by nucleotide sequencing-based genotyping. J Infect
Dis 2004; 189:182932.
10 Shimizu A, Tamura A, Abe M et al. Human papillomavirus type
56-associated Bowen disease. Br J Dermatol 2012; 167:11614.
11 Croci-Torti A, Tamalet C, Segondy M et al. HPV type 16-induced
skin squamous cell carcinoma located on the groin of immunocompetent patients. Br J Dermatol 2014; 170:7534.
12 Hama N, Ohtsuka T, Yamazaki S. Detection of mucosal human
papilloma virus DNA in bowenoid papulosis, Bowens disease and
squamous cell carcinoma of the skin. J Dermatol 2006; 33:3317.
13 Iannacone MR, Gheit T, Pfister H et al. Casecontrol study of
genus-beta human papillomaviruses in plucked eyebrow hairs and
cutaneous squamous cell carcinoma. Int J Cancer 2014; 134:2231
44.

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Detection of HPV in SCC and its characteristics, A. Shimizu et al. 785


14 Witkiewicz AK, Knudsen KE, Dicker AP, Knudsen ES. The meaning
of p16ink4a expression in tumors: functional significance, clinical
associations and future developments. Cell Cycle 2011; 10:2497
503.
15 Ikenberg H, Gissmann L, Gross G et al. Human papillomavirus
type-16-related DNA in genital Bowens disease and in Bowenoid
papulosis. Int J Cancer 1983; 32:5635.
16 Yoneta A, Yamashita T, Jin HY et al. Development of squamous
cell carcinoma by two high-risk human papillomaviruses (HPVs),
a novel HPV-67 and HPV-31 from bowenoid papulosis. Br J Dermatol 2000; 143:6048.
17 Park KC, Kim KH, Youn SW et al. Heterogeneity of human papillomavirus DNA in a patient with Bowenoid papulosis that progressed to squamous cell carcinoma. Br J Dermatol 1998; 139:1087
91.
18 Kato T, Saijyo S, Hatchome N et al. Detection of human papillomavirus type 16 in bowenoid papulosis and invasive carcinoma
occurring in the same patient with a history of cervical carcinoma.
Arch Dermatol 1988; 124:8512.

2014 British Association of Dermatologists

19 Gillison ML, Koch WM, Capone RB et al. Evidence for a causal


association between human papillomavirus and a subset of head
and neck cancers. J Natl Cancer Inst 2000; 92:70920.
20 Cubilla AL, Reuter VE, Gregoire L et al. Basaloid squamous cell carcinoma: a distinctive human papilloma virus-related penile neoplasm: a report of 20 cases. Am J Surg Pathol 1998; 22:75561.
21 Holzinger D, Schmitt M, Dyckhoff G et al. Viral RNA patterns and
high viral load reliably define oropharynx carcinomas with active
HPV16 involvement. Cancer Res 2012; 72:49935003.

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.

British Journal of Dermatology (2014) 171, pp779785