PREVALENCE
OF HUMAN PAPILLOMAVIRUS IN PENILE CARCINOMA
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DAVID NEVES, GENI N.L. CAMARA, TAINÁ R. ALENCAR, MÁRCIO R. DA CRUZ, CLÁUDIA R.F. MARTINS, LUCIANO G.S. CARVALHO
Division of Cell Biology, Institute of Biology, ICC South, Brasilia University, Federal District Base Hospital, Brasilia, DF, Brazil
ABSTRACT
Introduction:
Squamous cell carcinoma accounts for 95% of penile malignant tumors, and
its association to human papillomavirus (HPV) has been studied by several
authors. Our study describes the prevalence of HPV DNA in penile squamous
cell carcinomas in Brasília, Brazil.
Materials and Methods: Human papillomavirus
DNA was amplified by polymerase chain reaction (PCR) using GP5+/GP6+ and
pU-1M/pU-2R primers, after its extraction from paraffin embedded material
from 59 patients with penile carcinoma. HPV isolates were typed by restriction
enzymes digestion and automated sequencing.
Results: Good quality DNA was obtained from
37 out of the 59 samples previously selected. Prevalence of HPV DNA ranged
from 13.5% (5/37) to 64.8% (19/24) when we considered the amplification
with pU-1M/pU-2R or GP5+/GP6+ primers, respectively. HPV-16 was the only
type detected.
Conclusions: Our data indicate that GP5+/GP6+
PCR allow a good rate of HPV DNA detection and typing in formaldehyde-fixed
and paraffin-embedded tissues. The HPV DNA prevalence can be considered
high when compared to data from other South America studies. The high
frequency of HPV-16 and the low occurrence of HPV-18 are in agreement
with our results from a female population research in the same region
of Brazil. Further investigation should be conducted in order to evaluate
the frequency of HPV types associated to other penile epithelial disorders
and different kinds of neoplasias, as well as in asymptomatic subjects
in the central region of Brazil.
Key words:
penis; tumors; carcinoma; HPV; RT-PCR
Braz J Urol, 28: 221-226, 2002
INTRODUCTION
Penile
cancer is a serious worldwide health problem, despite its scarce occurrence.
The higher rates of the disease are found in developing countries. In
Brazil, the relative frequency of this tumor among all male neoplasias
has been reported as 2.1% (1). By far, the most common histologic type
is the squamous cell carcinoma (SCC), which accounts for more than 95%
of the cases (2). Its etiology appears to be multi-factorial, with a history
of smoking, phimosis and poor hygiene commonly associated (3). Similarly
to cervical SCC, penile SCC has been related to human papillomavirus (HPV)
(4).
Human papillomavirus is a member of the
Papillomaviridae family. The nonenveloped, icosahedral capsid carries
a single molecule of circular double-stranded DNA, ranging from 6800 to
8400 base pairs (bp) (5). More than 40 different genotypes have already
been described in the mucous epithelia of the urogenital surface and aero-respiratory
tract. The mucosotrophic genotypes are classified in two distinct groups:
“low-risk” group, often associated with benign warts and low-grade
intraepithelial lesions, and “high-risk” group, associated with
invasive carcinomas (6,7). The virus targets the basal cell layer of the
epidermis, where a latent infection may be established. Migration of cells
to the skin surface during differentiation allows the progression to active
infection (8).
The studies performed so far have indicated
that HPV has an etiological role in human malignant neoplasias. As early
as 1976, Meisels & Fortin (9) reported findings of HPV infection in
patients with cervical cancer. The frequent association of HPV-16, -18,
-31, and -33 with malignant change is now generally accepted. Other types
such as -35, -39, -41 to -44, -51, -52, and -56 have also been implicated
in cervical, vulvar, or penile cancer. The more common types 6 and 11
are considered to carry a low risk of malignant progression. However,
instances of cancer associated with virtually every HPV type have been
reported (7). These evidences include: viral DNA is found in close to
90 % of anogenital and cervical tumors (6); most of the virus-positive
tumors contain integrated viral DNA (7); and the E6 and E7 viral proteins
of high-risk types are able to immortalize human’s foreskin and cervical
keratinocytes (10).
This transversal study is the first report
on the prevalence of HPV DNA detected by molecular methods using 2 different
sets of primers. We have used paraffin-embedded tissue fragments from
penile squamous cell carcinomas, from patients originally from Brasilia,
central region of Brazil. Moreover, it also describes the frequency of
HPV types detected in the samples.
MATERIALS AND METHODS
Samples
Fifty-nine formaldehyde-fixed and paraffin-embedded
tissue fragments from patients attended at Brasília public hospital
from 1994 to 1999 were included in the study. The material, obtained from
males with penile invasive squamous cell carcinoma, was analyzed in order
to detect HPV DNA sequences. Subjects were married, 27 to 82 years old
(mean age of 54 and median of 60), mainly agriculture workers, with history
of penile lesions varying from 2 months to 5 years.
The inclusion criteria was the histopathologic
diagnosis of squamous cell carcinoma. Seventy per cent were histologically
classified as well-differentiated squamous cell carcinoma, whereas 20%
were moderately and 10% poorly-differentiated. Socio-demographic, clinical
and epidemiological data from subjects were recovered from hospital files,
whenever available.
DNA Isolation
from Tissue Sections
Each paraffin block was cut into 15-mm sections
using a new blade for each specimen to minimize block-to-block contamination.
Tissue sections were deparaffinized by treatment with xylem and washed
with ethanol. The proteins were digested by proteinase K [200 µg/ml],
overnight, at 55oC, and the extracted DNA purified by resin from the Nucleon
HT kit (Amershan Pharmacia Int.).
PCR Amplification
To evaluate the quality of the DNA isolation
procedure, samples were submitted to polymerase chain reaction (PCR) (11)
of the b-globin human gene sequence, using a pair of primers named pCO3/pCO4.
DNA amplification for HPV detection was performed in two distinct genomic
regions: E6-E7, using primers pU1M/pU2R, and L1, using primers GP5+/GP6+.
The oligonucleotides sequences used as PCR primers are shown in Table-1
(11-13).
PCR reactions were performed in a final
volume of 50 µl, containing 10 µl of extracted DNA, 20 mM
Tris HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 250 µM dATP, dCTP, dTTP
and dGTP, 2.4 pmol of each primer and 2.5 U of Taq DNA polymerase. Forty
cycles of amplification were conducted in an MJ Research PTC-100 thermocycler
(first cycle: 95ºC-5 min, 40ºC-3 min, 72ºC-3 min; second
to 39th cycle: 95ºC-1 min, 40ºC-3 min, 72ºC-3 min; 40th
cycle: 95ºC-1 min, 40ºC-3 min, 72ºC-3 min). PCR products
were analyzed by electrophoresis on 1.5% agarose gels followed by 10 mg/ml
ethidium bromide staining.
HPV Typing
by RFLP Analysis
Samples with positive HPV DNA were typed
by restriction fragment length polymorphism (RFLP). The products generated
by pU-1M/pU-2R primers were digested by the following enzymes: Acc I,
Ava II, Bgl II and Rsa I (3-4 U) (12). For GP5+/GP6+ (20) PCR products,
we used Rsa I (5 U) digestion (14). The pattern of length polymorphism
for each sample was analyzed by electrophoresis on 8% polyacrylamide gels,
followed by 10 mg/ml ethidium bromide staining.
Automated
Sequence and BLAST Analysis
Automated sequencing followed by Basic Local
Alignment Search Tool (BLAST) (15) was performed, using the amplified
150 bp fragments with GP5+/ GP6+ primers of five HPV DNA samples to confirm
enzyme digestion typing.
RESULTS
Twenty-two
(37.3%) samples proved no significant PCR amplification of the gene fragments
tested, either b-globin gene or HPV DNA fragment, and were excluded of
our series. In the 37 samples suitable for PCR amplification, 24 were
positive for HPV DNA after amplification with GP5+/ GP6+ primers (Figure-1).
The presence of HPV DNA was also detected in 5 out of 37 samples when
pU1M/pU2R primers were used for amplification (Figure-2). The results
of HPV DNA detection are summarized in Table-2. The overall prevalence
of HPV DNA ranged from 13.5% to 64.9%, when we considered the results
of PCR by pU-1M/pU-2R and GP5+/GP6+, respectively.
HPV-16 was found in 22 of the 24 isolates
by digestion with Rsa I. Two samples could not be typed because there
was not enough DNA to repeat the amplification procedure. The five isolates
amplified by pU-1M/pU2R were also typed as HPV-16. Therefore, the prevalence
of HPV-16 in the HPV DNA positive samples submitted to typing was 100%.
In order to check the HPV-16 typing using Rsa I analysis, five positive
samples, different from those amplified by pU-1M/pU2R, were automatically
sequenced and analyzed by BLAST, confirming the presence of HPV-16.
DISCUSSION
Data
on cancer incidence show that penile carcinoma is a rare disease in most
developed countries. A high prevalence of this tumor in Brazil associated
to HPV infection has been previously reported (16). Our study is the first
report on HPV detection in penile squamous cell carcinoma in the city
of Brasília, located in the central region of Brazil. The prevalence
of HPV DNA detected in this sampling can be considered high (64.9%) when
compared to data from other regions of Brazil (30%) (2) and Argentina
(71%) (17).
As previously discussed by other authors,
HPV prevalence associated to penile carcinoma can vary significantly in
distinct geographical areas (17). As our results indicate, one shall not
use the information on HPV prevalence from other cities of Brazil (1,16)
to infer the prevalence in one specific region. Variations in HPV prevalence,
as well as in the frequency of HPV types, have implications in: vaccine
testing; choice of diagnostic methods, and epidemiological studies involving
disease control (8).
Despite the preservation problems usually
related to paraffin-embedded tissue fragments (17,18), by using the nucleon
HT kit, we were able to obtain a good quality DNA in 62.7 % (37/59) of
the tested samples. The DNA extraction procedure adopted was the one that
presented the highest percentage of recovery, although four other different
methods had been tested. The percentage of recovery of archival samples
obtained can be considered acceptable, since there is always some percentage
of loss for different reasons (1,2).
The discrepant prevalence of HPV DNA accounted
for pU-1M/pU-2R and GP5+/GP6+ PCRs products may be due to the technical
limitations. Partial degradation of DNA could possibly have affected the
amplification sensibility of pU-1M/pU-2R primers, determining the prevalence
rate 4.8 lower than the rate defined by GP5+/GP6+ amplification. As the
pair of primers for E6/E7 genes (pU1M/2R) amplifies a longer DNA fragment
if compared to GP5+/GP6+, it would be expected that degraded DNA samples
presented a higher efficiency of amplification for lower fragments.
The use of Rsa I digestion for HPV typing
with GP5+/GP6+ amplified products was first reported in 1990 (14). Recently,
we have analyzed all HPV DNA sequences available at the GenBank. We noticed
that, considering the HPV variation that occurs in Brasília (19),
the only other HPV type that might be misinterpreted as HPV-16 is the
HPV-35, when (1,16) Rsa I digestion is used as a typing method. This latter
HPV type can be detected in association with high-risk lesions, but not
in invasive carcinomas (8).
The significant prevalence of HPV-16, a
“high-risk” type (6), is compatible with the histopathologic
diagnosis of squamous cell carcinoma, also referred by other authors (20),
although other reports from Brazil (1,16) and Argentine (17) have found
a predominance of HPV-18. The high prevalence of HPV-18 associated to
cancer is not detected in the central area of Brazil, neither in the male
nor in the female populations studied so far (19).
As only HPV-16 was detected in all analyzed
samples, no correlation could be established considering prognostic factors,
association to clinical or epidemiological data and HPV types.
We consider that other studies should be
conducted in order to evaluate the frequency of HPV types associated to
other penile epithelial disorders and different kinds of neoplasias, as
well as in asymptomatic subjects in the central region of Brazil.
CONCLUSIONS
GP5+/GP6+
PCR allow an acceptable rate of HPV DNA detection and can be used for
formaldehyde-fixed and paraffin-embedded tissues.
HPV prevalence associated to penile carcinoma
seems to be higher in Brasília than in other regions of the South
of Brazil, although compatible to what was found in Argentina.
HPV-16 was the only type detected in penile
cancer in this target population. Further investigation should be conducted
in order to define the distribution of other penile epithelial disorders
and different kinds of neoplasias, as well as in asymptomatic subjects.
______________________________
Financial support: FAP/DF, approved
by CEP/SES/DF on November 3, 1998
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______________________
Received: October 2, 2001
Accepted after revision: April 9, 2002
_______________________
Correspondence address:
Dr. Cláudia Renata F. Martins
Departamento de Biologia Celular,
Instituto de Biologia, ICC Ala Sul,
Universidade de Brasília
Brasília, DF, 70910-900, Brazil
Fax: + + (55) (61) 347-6533
E-mail: cmartins@unb.br