Detection of human papilomavirus types 16 and 18 in pathologic samples from patients with cervical cancer by PCR and RFLP methods

Abstract

Infection with human papiloma virus (HPV) is the most frequent sexually transmitted disease worldwide. HPV types 16, 18, 31 and 33 are considered as the most important types in the cervical cancer.This study was undertaken on 64 samples of archival cervical carcinoma pathologic to assess the rate of HPV infection (HPV16,18) in cervical carcinoma among Iranian patients. HPV DNA was detected by polymerase chain reaction (PCR) and typing by restriction fragment length polymorphism (RFLP) analysis.The total prevalence of HPV in this study (HPV16,18) for all cases was 59.4% (38/64). HPV type 16 was the most common one (22/64, 34%) followed by HPV type 18 (16/64, 25%). On the basis of the rate of HPV (16,18) which were detected in squamous cell carcinoma and adenocarcinoma,only women with HPV18 infection showed a statistically significant risk for development of cervical cancer (P=0.019) while P value for HPV16 was 0.47 . INTRODUCTION In developing countries, cervical cancer is the most frequent female malignancy after the breast cancer (1). However, the prevalence of cervical cancer in Iran is much lower than other parts of the world (2). Clinical and epidemiological studies have shown that papiloma viruses (HPV) play a major role in the development of different types of cervical lesions, and are therefore considered as the major infectious aetiological agents of genital lesions as well as cancers (1). The other known risk factors for cervical cancer are high parity, smoking, sexual behavior and hygiene (1,3,4,5). To date, more than 85 HPV types have been characterized (6). HPV types 16, 11, 42, 43 and 44 are associated with benign lesions (condylomas), whereas HPV 31, 33, 35, 51, 52 and 58 are detected more frequently in low grade squamous intraepithelial lesions (LSIL) and HPV 16, 18 are predominant in high grade LSIL and invasive carcinomas (7). Human papiloma viruses (HPV) are causative agents of cervical cancer in women and some other genital mucosal and skin lesions. Cervical cancer takes many years to develop, and changes can be detected in the cervix for some time before the appearance of cancer. In principle, screening of women for these changes may allow treatment of those with early signs of developing disease, like cancer (8). The lack of standardized detection methods represents a major problem for monitoring HPV in developing countries. However, sensitive and special methods, based on the detection of HPV DNA, are available, of which, the polymerase chain reaction (PCR) technique is the method of choice for epidemiological studies (2,8,9,11). In this study the presence of HPV nucleic acid sequences in samples was investigated by PCR method. The identification of the HPV types was performed using restriction fragment length polymorphism (RFLP) analysis.

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DARU Volume 14, No. 2, 2006
78
Corresponence: Parviz Maleknejad, Department of Microbiology , School of Medicine, Tehran University of
Medical Sciences, Tehran, Iran, E-mail: maleknej@sina.tums.ac.ir


DETECTION OF HUMAN PAPILOMAVIRUS TYPES 16 AND 18 IN
PATHOLOGIC SAMPLES FROM PATIENTS WITH CERVICAL
CANCER BY PCR AND RFLP METHODS

1PARVIZ MALEKNEJAD, 2MOHAMMAD RAKHSHAN, 3BAHRAM KAZEMI,
1FARNAZ FAROKH, 1SHADI SHAHSAVAN

1Department of Microbiology, School of Medicine, Tehran University of Medical
Sciences, 2Department of Pathology, 3Cellular and Molecular Biology Research Center,
School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

ABSTRACT
Infection with human papiloma virus (HPV) is the most frequent sexually transmitted disease worldwide.
HPV types 16, 18, 31 and 33 are considered as the most important types in the cervical cancer.This study
was undertaken on 64 samples of archival cervical carcinoma pathologic to assess the rate of HPV
infection (HPV16,18) in cervical carcinoma among Iranian patients. HPV DNA was detected by
polymerase chain reaction (PCR) and typing by restriction fragment length polymorphism (RFLP)
analysis.The total prevalence of HPV in this study (HPV16,18) for all cases was 59.4% (38/64). HPV
type 16 was the most common one (22/64, 34%) followed by HPV type 18 (16/64, 25%). On the basis of
the rate of HPV (16,18) which were detected in squamous cell carcinoma and adenocarcinoma,only
women with HPV18 infection showed a statistically significant risk for development of cervical cancer
(P=0.019) while P value for HPV16 was 0.47 .
Keywords: HPV, Cervical cancer, PCR, Iran

INTRODUCTION
In developing countries, cervical cancer is the
most frequent female malignancy after the breast
cancer (1). However, the prevalence of cervical
cancer in Iran is much lower than other parts of
the world (2).
Clinical and epidemiological studies have shown
that papiloma viruses (HPV) play a major role in
the development of different types of cervical
lesions, and are therefore considered as the major
infectious aetiological agents of genital lesions as
well as cancers (1). The other known risk factors
for cervical cancer are high parity, smoking,
sexual behavior and hygiene (1,3,4,5).
To date, more than 85 HPV types have been
characterized (6). HPV types 16, 11, 42, 43 and 44
are associated with benign lesions (condylomas),
whereas HPV 31, 33, 35, 51, 52 and 58 are
detected more frequently in low grade squamous
intraepithelial lesions (LSIL) and HPV 16, 18 are
predominant in high grade LSIL and invasive
carcinomas (7).
Human papiloma viruses (HPV) are causative
agents of cervical cancer in women and some
other genital mucosal and skin lesions. Cervical
cancer takes many years to develop, and changes
can be detected in the cervix for some time before
the appearance of cancer. In principle, screening

of women for these changes may allow treatment
of those with early signs of developing disease,
like cancer (8).
The lack of standardized detection methods
represents a major problem for monitoring HPV in
developing countries. However, sensitive and
special methods, based on the detection of HPV
DNA, are available, of which, the polymerase
chain reaction (PCR) technique is the method of
choice for epidemiological studies (2,8,9,11). In
this study the presence of HPV nucleic acid
sequences in samples was investigated by PCR
method. The identification of the HPV types was
performed using restriction fragment length
polymorphism (RFLP) analysis.

MATERIALS AND METHODS

Tissue samples
Formalin fixed paraffin-embedded samples from
patients with cervical cancer (n=64) were
processed (archival pathologic specimen of
Mahdie and Loghaman Hospitals).
The mean age of patients was found to be 52.5
(32-73) years.

Processing of samples
In the first step a thick section from samples was
provided and placed in sterile 2 ml eppendorf

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Maleknejad et al
tubes. Paraffin was removed with warm xylene
extraction (60°C) followed by washing with 90%
ethanol two times. Samples were dried in
thermomixer for 30 minutes and were then
incubated in digestion buffer (10 mM Tris-HCl, ,
25 mM EDTA, 0.5% SDS, 2 mg/mol proteinase
K) pH= 8.5 at 37°C.
After centrifugation at 5000 rpm, light phase was
used for DNA extraction.

DNA extraction
An equal volume of phenol and chloroform were
then added to samples, mixed and centrifuged.
Light phase was transferred to a microtube and
mixed with an equal volume of chloroform and
centrifuged. It was then mixed with two volume of
ethanol and freezed at -20°C over night.
The formalin was removed from the pelletted
DNA by drying. The amplification reaction was
performed in a final volume of 50µl containing 40
pico mol of each
(5'GAATATGATTTRCAGTTTATTT3')
HP168 R (5'TCTYKGAAACTTTCCTT3'), 0.25
unit Taq DNA polymerase, 10mM dNTP, 50 mM
MgCl2, 5µl 10x PCR buffer and the mixture
denatured at 94°C for 5 minutes.
These sequence primers was designed by oligos
program from sequence of strains HPV 16 (Gene
bank Accession number S71514 )and HPV 18
(Gene bank Accession number U 45893 ).
These primers were amplified a 269 bp fragment.
Depending on the type of virus, the amplicons had
either Bam H1 restriction site for HPV 18 and Eco
R1 restriction site for HPV 16 .
Then, 35 cycles of PCR were performed by
denaturation at 94°C for 30s, annealing at 52°C
for 30s, and extension at 72°C for 30s.
At the end of last cycle, mixture was incubated at
72 °C for 5 min.
The PCR was performed in a eppendorf
thermocycler. For every reaction, negative control
(master mix and sterile distilled water), a positive
control (HPV18 DNA, HPV 16 DNA) were
employed. PCR product (269 bp) were analysed
by electrophoresis through a 1.5% agarose gel and
stained with ethidium bromide and visualized on a
UV transilluminator (Fig 1, 2).
RFLP method( Restriction Fragment Length
Polymorphism Analysis)
identification of HPV types. The PCR products
and DNA from positive control (HPV16,18 ) were
digested with 20 units of endonuclases EcoR I for
HPV16 and Bam H I for HPV18 . The fragments
subsequently were separated in polyacrylamide gel
electrophoresis and DNA patterns were visualized
by UV transillumintor after staining the gel with
ethidium bromide. Restriction fragments were
seperated on polyacrylamide gel and were
primer, HP 168F
and
permits reliable
visualized by UV transilluminator after ethidium
bromide staining.

Statistical analyses
Te relationship between HPV (16, 18) infection,
age ofppatient,aandttwottypes ofccervical cancers
(adenocarcinoma, squamous cell carcinoma) were
assessed by Fisher's test.
All data were analysed using [1] SAS version 8.00
and were two-sided where P< 0.05 was regarded
as significant.

RESULTS
The mean age of the patients was 52.5 years,
ranging from 32 to 73 years. The majority of
patients were in the range of 30 to 40 years ( Fig.
3). The total prevalence of HPV in the
study(16,18) for all cases was 59.4% (38/64) (Fig.
3).
The prevalence of HPV type 16 and type 18 were
34.4% (22/64) and 25% (16/64) for all cases
respectively (Fig. 4).
DNA from HPV type16 was detected in 22 cases,
of which 19 cases had squamous cell carcinoma
(86.4%) (19/22) and
adenocarcinoma (14%) (3/22) (Fig. 4).
DNA from HPV type18 was detected in 16 cases
of which 11 cases had squamous cell carcinoma
(68.8%) (11/16) and 5 cases had adeno carcinoma
(31.2%) (5/16) (Fig. 4). On the basis of the rate of
prevalence rates of HPV (16/18) which were
detected in squamous cell carcinoma and
adenocarcinoma only women with HPV18
infection showed a statistically significant risk for
development of cervical cancer (P= 0.019) while p
value for HPV16 was 0.47.

DISCUSSION
Cervical cancer is the second most prevalent
cancer among women worldwide (9).
Currently there is compelling evidence that the
development of human cervical cancer without
involvement of the specific human papiloma virus
(HPV) is exceptional or impossible. In this study
pathologic blocks were used for HPV testing by
PCR and RFLP methods. Today, the diagnosis of
HPV infection is based on the detection of viral
DNA (11,18,19).
Because of the sensitivity of the methods, samples
containing very low levels of HPV DNA, are
becoming viable HPV sample sources and in this
study presence of HPV types 16,18 in samples
(59.4%) were demonstrated.
HPV type 16 was the predominant infection
(34.4%) in this study. In another study in Iran, the
prevalence rate of HPV 16 was reported 6.7% (2).
The prevalence of HPV 16 among Iranian patients
with cervical cancer is lower than those in Croatia
(50%), Australia (53%),
Colombia (69.9%), Spain (66.4%), Italy (32.6%)
remaining (3) had
Thailand (41%)
79

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Figure 1. 1.2% gel electrophoresis of HPV 16 PCR product restriction analysis
M : 100 bp DNA ladder marker; Lane 1 :Postive control; Lane 2 :Negative control; Lane 3 :Digested PCR product
by EcoR1(there is one Restriction site for EcoR1 on HPV 16 sequence); Lane 4 : Digested PCR product by BamHI;
(there is no restriction site for Bam HI on HPV16 sequence; Lane 5 : Uncut PCR product











Figure 2. 1.2% gel electrophoresis of HPV 18 PCR product restriction analysis
Lane M : 100 bp DNA ladder marker; Lane1 : Positive control ; Lane2 : Negative control; Lane3 : Digested PCR
product by EcoRI(there is no restriction site for Eco RI on HPV 18 sequence ); Lane4 : Digested PCR product by
Bam HI; (there is one restriction site for Bam HI on HPV 18sequence ); Lane5 :Uncut PCR product













Figure 3. Prevalence rates of HPV types detection in various age group














Figure 4. Prevalence rates of HPV types detected in adenocarcinoma and squamous cell carcinoma

210 bp
59 bp
300 bp
80 bp
M 1 2 3 4 5 M
191 bp
78 bp
200 bp
80 bp
3
16
1
22
12
11
5
15
2
4
0
5
10
15
20
20-2930-4041-60 > 60
Age
Prevalence rates of
HPV types
HPV 16 HPV 18None
5
3
0
11
19
26
0
10
20
30
HPV18 HPV16 None
HPV types
Prevalence rates of
HPV types
Adenocarcinoma Squamous cell carcinoma
80
Detection of human papilomavirus by PCR and RFLP

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Maleknejad et al
and
prevalence rate of HPV18 was 25% in this study,
other study results of another study in Iran has
been reported 0% (2). This type has been reported
from Australia (17.2%), Thailand (21%) and
South Africa (34%) (13,14,17). The distribution of
HPV 16,18 in squamous cell carcinoma (30/38)
were higher than adenocarcinoma (8/38) (Fig.4).
There was no statistically significant association
between HPV 16 and development of squamous
cell carcinoma and adenocarcinoma (P= 0.47).

China (48.8%) (12-16,19).While the
However a statistically significant relationship
was found for HPV18 (P= 0.019).
In summary, our study suggests that HPV typing
is an important factor for cervical cancer
diagnosis, andmmultiplex PCR, and RFLP are
effective methods for detection of HPV types.

ACKNOWLEDGMENT
This work was mainly supported by Department
of Microbiology, School of Medicine, Tehran
Universityof Medical Sciences.
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