Amplification of Papillomavirus Oncogene Transcripts Assay for Bowenoid Papulosis

doi:10.4236/ijcm.2013.410077

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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.

Email: *[email protected]

Received July 31st, 2013; revised August 20th, 2013; accepted September 23rd, 2013

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

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.

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