International Journal of Clinical Medicine, 2013, 4, 428-431
http://dx.doi.org/10.4236/ijcm.2013.410077 Published Online October 2013 (http://www.scirp.org/journal/ijcm)
Amplification of Papillomavirus Oncogene Transcripts
Assay for Bowenoid Papulosis
Takahide Kaneko1*, Gen Nakahishi2, Koji Nakajima1, Takayuki Aizu1, Kayo Jin1, Daiki Rokunohe1,
Yasushi Matsuzaki1, Nakano Hajime1, Toshihiro Tanaka2, Daisuke Sawamura1
1Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan; 2Department of Dermatology,
Shiga University of Medical Science, Otsu, Japan.
Received July 31st, 2013; revised August 20th, 2013; accepted September 23rd, 2013
Copyright © 2013 Takahide Kaneko et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Human papillomavirus (HPV) is an important causative agent of cervical carcinoma and some malignant cutaneous
tumors. The integration of HPV DNA into the host genome is one of the most important risk factors for malignant
transformation. Here, we report the case of a 53-year-old man with multiple, black-b rown genital macules and nodules.
Histological findings resembled those for Bowen’s disease, and we made a diagnosis of bowenoid papulosis. This con-
dition is generally benign, but invasive squamous cell carcinoma (SCC) arising from bowenoid papulosis has been pre-
viously reported. Therefore, we took biopsy specimens from 10 lesions and tested them for the presence of HPV DNA.
DNA sequencing identified HPV type 16 (HPV-16) DNA in all samples. Next, we excised 4 different relatively large
lesions and performed an amplification of papillomavirus oncogene transcripts (APOT) assay. This assay showed that
all samples had only episome-derived viral transcripts, indicating no integration of HPV DNA into the host genome. We
have followed this case for 3 years, and no progression to Bowen’s disease or SCC has so far been observed. We con-
clude that the APOT assay is a feasible method for evaluating the malignant potential of bowenoid papulosis.
Keywords: HPV 16; Integration; Malignant Transformation
1. Introduction
Human papillomavirus (HPV) infection of the genital
skin shows a variety of presentations ranging from be-
nign condyloma acuminata to squamous cell carcinoma
(SCC) [1,2]. Bowenoid papulosis is a condition that his-
tologically resembles Bowen’s disease and manifests
small verrucous papules and nodules on the genitalia [3].
The condition is benign and has a tendency towards
spontaneous resolution. The high-risk, oncogenic HPV
type 16 (HPV-16) has been detected in most cases, and
invasive SCC arising from bowenoid papulosis has been
described in some cases [4]. Here we report the case of a
53-year-old man with bowenoid papulosis in which the
amplification of papillomavirus oncogene transcripts
(APOT) assay served as a feasible method for evaluating
prognosis.
2. Materials and Methods
2.1. Patient
A 53-year-old man presented with multiple skin lesions
on the genital area. The lesions were first noticed 3 years
earlier. His medical history included diabetes mellitus for
3 years and hypertension for 5 years. His family history
was unremarkable. Physical examination revealed multi-
ple, keratotic, black-brown macules and nodules 0.5 - 3.0
cm in diameter on his genitalia (Figure 1). Inguinal
lymph nodes were not palpable. Skin biopsies were taken
from 2 lesions, including both nodule and plaque. Histo-
logical findings from the nodule and plaque showed a
variable extent of hyperkeratosis, irregular acanthosis,
papillomatosis, and cytological atypia with few dyskera-
totic cells in the entire upper epidermis (Figure 2).
2.2. Immunohistochemical Study
*The authors have no conflict of interest to declare.
#Corresponding author. Skin sections were fixed in 10% formaldehyde and

Amplification of Papillomavirus Oncogene Transcripts Assay for Bowenoid Papulosis 429
Figure 1. Clinical findings. Mu ltiple, keratotic, black-brow n
macules and nodules 0.5 - 3.0 cm in diameter on the pa-
tient’s genitalia.
Figure 2. Histological findings. Variable extent of hyperk-
eratosis, irregular acanthosis, papillomatosis, and cytologi-
cal atypia with few dyskeratotic cells in the entire upper
epidermis.
embedded in paraffin. The sections were routinely passed
through xylene and a graded alcohol series, and stained
with several antibodies using the avidin-biotin-peroxi-
dase complex method. Peroxidase binding was detected
using the diaminobenzidine method, and the sections
were then lightly counterstained with hematoxylin for
microscopic examination.
2.3. HPV Typing and APOT Assay
Total DNA was extracted from the lesions and HPV
DNA was amplified using GP5/GP6 primers. The ampli-
fication products were sequenced using an ABI 3100
sequencer.
Total RNA was isolated and reverse transcribed using
a commercial kit (Superscript III First-Strand Synthesis
System; Invitrogen Corp., Carlsbad, CA, USA) and an
oligo-(dT)17 primer coupled to a linker sequence. PCR
amplification for the APOT assay was carried out as pre-
viously described [5,6]. APOT products were visualized
by performing agarose gel electrophoresis, excised from
the gel, and sequenced using an ABI 3100 sequencer.
G3PDH transcript was used as control.
3. Results
3.1. Histologic Detection of HPV
First, we performed an immunohistochemical analysis
using the anti-HPV capsid antigen antibody, K1H8
(DAKO, Tokyo, Japan). The analysis indicated that some
epidermal keratinocytes were HPV-positive (data not
shown). After confirmation of HPV infection, we pro-
ceeded to further HPV analysis.
3.2. Identification of HPV-16
Next, we extracted total DNA from biopsy specimens
taken from 10 pigmented lesions. We performed PCR
amplification using GP5/GP6 primers targeting the HPV
L1 gene and sequenced the PCR products. After align-
ment of our sequences with HPV L1 sequences obtained
in GenBank, conserved sequence regions were selected
and analyzed using a computer software program. We
identified sequences corresponding with HPV-16 in all
10 samples.
3.3. APOT Assay Detected Only
Episome-Derived Viral Transcripts
To know the extent of the integration of HPV into the
patient’s genome, we performed an APOT assay [5-7].
We excised 4, relatively large different lesions. Total
RNA was isolated and reverse transcribed. APOT prod-
ucts were visualized by agarose gel electrophoresis. The
result was shown in Figure 3. PCR amplification de-
tected the bands corresponding to the episomal form of
HPV-16 transcript in samples 1, 2 and 4 while sample 3
showed no clear band. Also we found an addition al short
band in sample 4, the size of which was predicted to be
integrated form. Sequence analysis of those bands re-
vealed that all larger band (black arrow in Figure) had
only episome-derived viral transcripts and the smaller
band (white arrow in Figure 3) was also originated from
the viral transcript, indicating no integration of the HPV
DNA into the host genome (Figure 3). Host DNA could
not be found by sequence analysis.
We have followed this case for 3 years and so far have
observed no progression to Bowen’s disease or SCC.
4. Discussion
Our patient showed multiple pigmented genital macules
and nodules, and our clinical diagnosis was bowenoid
papulosis. Histological findings were compatible to tho se
for Bowen’s disease and indicated bowenoid papulosis.
HPV is an etiological agent in cervical carcinomas and

Amplification of Papillomavirus Oncogene Transcripts Assay for Bowenoid Papulosis 430
Figure 3. Amplification of papillomavirus oncogene tran-
scripts (APOT) assay. Total RNA was isolated from 4 rela-
tively large different lesions and reverse transcribed. APOT
products were visualized by agarose gel. All samples were
numbered from 1 to 4. C: control sample from episomal
transcript. M: DNA maker. G3PDH was used as control.
PCR amplification detected the bands corresponding to the
episomal form of HPV-16 transcript in samples 1, 2 and 4
(black arrow) while sample 3 showed no clear band. Also
we found an additional short band (white arrow) in sample
4.
causes various types of malignant and benign skin tu-
mors, including SCC, Bowen’s disease, and bowenoid
papulosis [1-3]. Bowenoid papulosis is benign, although
HPV-16 has been detected in most cases. In our case, we
took DNA from 10 lesions and detected HPV-16 in all
samples. However, SCC arising from a bowenoid papu-
losis has been described in some cases [4].
Persistent infection with high-risk HPV types, such as
HPV-16, 18, 31, 33, and 45, is necessary but insufficient
for cervical carcinoma development. Integration of the
HPV DNA into the host genome is one of the most im-
portant risk factors for malignant transformation [8]. The
APOT assay can detect transcriptionally activ e virus-host
fusion transcripts and distinguish between episomal and
integrated HPV DNA [6]. In addition, the assay can de-
termine the precise integ ration site of the viral genome in
host chromosomes. The APOT assay requires only a
small tissue sample and allows for the analysis of inte-
gration sites in multiple locations on the lesion.
Recently, we reported the case of a patient with multi-
ple black plaques and an erythematous nodule on the
vulva [7]. On histological exa mination, multifocal vulvar
Bowen’s disease and invasive SCC were found. APOT
assay showed that 2 locations of Bowen’s disease had
only episome-derived HPV-16 transcripts, but another 2
sites of Bowen’s disease and the nodule of invasive SCC
had HPV-16 transcripts derived from integration.
We therefore performed an APOT assay about sample
form 4 lesions in the present case. The result showed that
samples 1 and 2 showed only the bands from episome-
derived HPV-16 tra nscr ipts. No band was obser ved in the
lane of sample 3, but we did not know the reason. Also
sample 4 showed the larger and smaller bands. The for-
mer band was corresponding to that from the episomal
form. The smaller band was thought to be the band from
the integrated form, but sequence analysis could detect
only HPV-16 transcript without host sequence. We con-
cluded that there was no integration of viral genome to
host genome in this case. In fact, a 3-year follow-up did
not show transformation to SCC. We suggest that the
APOT assay is a feasible method for evaluating the
clinical degree of malignancy in bowenoid papulosis.
REFERENCES
[1] S. Majewski and S. Jablonska, “Human Papillomavirus-
Associated Tumors of the Skin and Mucosa,” Journal of
the American Academy of Dermatology, Vol. 36, No. 5,
1997, pp. 659-685.
http://dx.doi.org/10.1016/S0190-9622(97)80315-5
[2] T. M. Darragh, T. J. Colgan, J. Thomas Cox, D. S. Heller,
M. R. Henry, R. D. Luff, T. R. Nayar, J. M. Palefsky, M.
H. Stoler, E. J. Wilkinson, R. J. Zaino and D. C. Wilbur,
“The Lower Anogenital Squamous Terminology Standar-
dization Project for HPV-Associated Lesions: Back-
ground and Consensus Recommendations from the Col-
lege of American Pathologists and the American Society
for Colposcopy and Cervical Pathology,” The Interna-
tional Journal of Gynecological Pathology, Vol. 32, No.
1, 2013, pp. 76-115.
http://dx.doi.org/10.1097/PGP.0b013e31826916c7
[3] C. J. Herquet, “Anogenital Malignancies and Pre-Malig-
nancies,” Journal of the European Academy of Derma-
tology and Venereology, Vol. 25, No. 8, 2011, pp. 885-
895.
[4] A. Yoneta, T. Yamashita, H. Y. Jin, A. Iwasawa, S. Kon-
do and K. Jimbow, “Development of Squamous Cell Car-
cinoma by Two High-Risk Human Papillomaviruses
(HPVs), a Novel HPV-67 and HPV-31 from Bowenoid
Papulosis,” British Journal of Dermatology, Vol. 143, No,
3, 2000, pp. 604-608.
http://dx.doi.org/10.1111/j.1365-2133.2000.03718.x
[5] G. Nakanishi, O. Yamasaki, Y. Nagao and T. Tanaka,
“Detection of Human Papillomavirus Type 67 from Bo-
wenoid Papulosis,” European Journal of Dermatology,
Vol. 20, No. 6, 2010, pp. 819-820.
[6] R. Klaes, S. M. Woerner, R. Ridder, N. Wentzensen, M.
Duerst, A. Schneider, B. Lotz, P. Melsheimer and M. von
Knebel Doeberitz, “Detection of High-Risk Cervical In-
traepithelial Neoplasia and Cervical Cancer by Amplifi-
cation of Transcripts Derived from Integrated Papillo-
mavirus Oncogenes,” Cancer Research, Vol. 59, No. 24,
1999, pp. 6132-6136.
[7] G. Nakanishi, K. Fujii, K. Asagoe, T. Tanaka T and K.
Iwatsuki, “Human Papillomavirus Genome Integration in
Multifocal Vulvar Bowen’s Disease and Squamous Cell
Carcinoma,” Clinical and Experimental Dermatology,
Vol. 34, No. 8, 2009, pp. e965-967.
http://dx.doi.org/10.1111/j.1365-2230.2009.03708.x

Amplification of Papillomavirus Oncogene Transcripts Assay for Bowenoid Papulosis 431
[8] I. Kraus, C. Driesch, S. Vinokurova, E. Hovig, A. Sch-
neider, M. von Knebel Doeberitz and M. Duerst, “The
Majority of Viral-Cellular Fusion Transcripts in Cervical
Carcinomas Cotranscribe Cellular Sequences of Known
or Predicted Genes,” Cancer Research, Vol. 68, No. 7,
2008, pp. 2514-2522.
http://dx.doi.org/10.1158/0008-5472.CAN-07-2776