Experimental and Molecular Pathology 98 (2015) 119123

Contents lists available at ScienceDirect

Experimental and Molecular Pathology

journal homepage: www.elsevier.com/locate/yexmp

TIMP-2 gene methylation in cervical precursor and invasive lesions

Yara Furtado a,b,c,, Gutemberg Almeida b,c, Filomena Aste Silveira b,c, Ktia S. Silva d, Paula Maldonado c,
Isabel Cristina do Val e, Silvia Cavalcanti e, Miranda-Alves L f, Maria da Gloria da Costa Carvalho g
a

Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Brazil

Postgraduate Program in Surgical Sciences, Universidade Federal do Rio de Janeiro UFRJ, Brazil
Institute of Gynecology, Universidade Federal do Rio de Janeiro (UFRJ), Brazil
d
National Institute for Women, Children and Adolescents, Fernandes Figueira Institute, Fundao Oswaldo Cruz (FIOCRUZ), Brazil
e
Universidade Federal Fluminense (UFF), Brazil
f
Biomedical Sciences Institute, Universidade Federal do Rio de Janeiro (UFRJ), Brazil
g
Laboratory of Molecular Pathology, Department of Pathology, Clementino Fraga Filho University Hospital, Universidade Federal do Rio de Janeiro (UFRJ), Brazil
b
c

a r t i c l e

i n f o

Article history:
Received 31 December 2014
Accepted 7 January 2015
Available online 9 January 2015
Keywords:
Methylation
TIMP-2
Papillomavirus infection
Cervix
Neoplasia

a b s t r a c t
Objective: To analyze the presence of HPV-DNA and TIMP-2 gene methylation in cervical precursor and invasive
lesions, as well as to study the associations among the latter, the presence of HPV-DNA, and the clinical evolution
of such lesions.
Methods: Cross-sectional study that includes 49 biopsy or brush smear samples from women with a normal cervix, LSIL, HSIL, microinvasive carcinoma and invasive carcinoma. The presence of HPV-DNA and specic methylation was analyzed using PCR. Thirty-eight biopsy samples for HSIL, microinvasive carcinoma and frank invasive
carcinoma as well as 11 brush smear samples for LSIL and normal cervices were analyzed.
Results: TIMP-2 gene methylation was detected in 86.8% (33/38) of the samples from the group with lesions and
50% (4/8) of the normal samples (p = 0.03). HPV-DNA was detected in 81.6% (31/38) of the samples from the
group with lesions and 25% (2/8) of the normal samples (p = 0.003). HPV-DNA was more frequent in the methylated samples (50%), and the group with methylation had a higher risk of unfavorable evolution than the group
without methylation; however, such observations were not statistically signicant (p = 0.19).
Conclusion: TIMP-2 gene methylation and the presence of HPV-DNA were characteristic of the group with cervical
lesions. Methylation was not associated with the presence of HPV-DNA or an unfavorable clinical evolution.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Cervical cancer is the second leading cause of death by cancer among
women throughout the world (Ferlay et al., 2002). In Brazil, cervical
cancer is the third most common tumor in the female population and
the fourth leading cause of death in women by cancer (INCA
Instituto Nacional do Cncer, 2013). Diagnosing the tumor at advanced
stages is one of the primary causes of high mortality and only allows for
palliative radiotherapy and chemotherapy treatment with a great recurrence risk in a short period of time (Thuler, 2008).
Human papillomavirus (HPV) is necessary for the development of
precursor and invasive lesions, as has been demonstrated through epidemiologic and molecular studies (Bosch and de Sanjos, 2003). A series
of molecular genetic and epigenetic events transpire during the initial
progression of HPV-induced lesions through cervical cancer development, which ultimately prevent the cell from generating an adequate

Corresponding author at: Instituto de Ginecologia, Rua Moncorvo Filho, No. 90, Centro,
Rio de Janeiro, RJ CEP 20211-340, Brazil.
E-mail address: yarafurtadorj@terra.com.br (Y. Furtado).

http://dx.doi.org/10.1016/j.yexmp.2015.01.008
0014-4800/ 2015 Elsevier Inc. All rights reserved.

immune response and support tissue invasion and metastasis (StetlerStevenson et al., 1991).
In mammals, methylation is the most common epigenetic modication and functions as a regulatory mechanism for gene integrity and
expression (Furtado et al., 2010). Abnormal CpG island methylation in
promoter regions for tumor suppressor genes is related to gene
transcription inactivation in cancer and is a molecular biomarker
(Strathdee and Brown, 2002). Methylation of genes that express proteolytic enzyme inhibitors, such as matrix metalloproteinases (MMPs), can
be an important event in invasion (Ivanova et al., 2004).
Matrix metalloproteinases (MMPs) are zinc- and calciumdependent lytic enzymes and are secreted as proenzymes (Davidson
et al., 1999; Ivanova et al., 2004; Strathdee and Brown, 2002). One
specic MMP group that belongs to the Matrixines family has been implicated in the proteolytic system of carcinogenesis because it participates in stroma, lymphatic and blood vessel invasion (Brummer et al.,
2002; Ivanova et al., 2004).
MMP activity is controlled by tissue inhibitors of metalloproteinases
(TIMPs), which are 22 kDa proteins that form a complex with the enzymes by binding at their active site (Benassi et al., 2001; Branca et al.,
2006). Tumor invasion and metastases can be inhibited by TIMP

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Y. Furtado et al. / Experimental and Molecular Pathology 98 (2015) 119123

Table 1
Distribution of TIMP-2 gene methylation in accordance with cyto/histological diagnosis.
LSIL n(%) HSIL n(%) Microinvasive
Invasive
Control
carcinoma n(%) carcinoma n(%) n(%)
Methylated
6(100)
Unmethylated 0
Total
6(100)

11(91.6)
1(8.3)
12(100)

11(84.6)
2(15.3)
13(100)

5(71.4)
2(28.5)
7(100)

4(50)
4(50)
8(100)

p = 0.03.

overexpression in tumor cells (Branca et al., 2006). Thus, gene inactivation through methylation of the promoter region may support a loss of
control over tissue proliferation, which facilitates invasion and metastasis (Benassi et al., 2001; Nuovo et al., 1995).
The objective for the study herein was to analyze the presence of
HPV-DNA and TIMP-2 gene methylation in cervical precursor and invasive lesions as well as to study the associations among the latter, the
presence of HPV-DNA and clinical evolution of such lesions.

A small fragment of the surgical specimen was removed for molecular analysis and frozen at 20 C in Eppendorf tubes for DNA extraction.
Subsequently, the presence of HPV-DNA was analyzed through polymerase chain reaction (PCR), and the presence of TIMP-2 gene promoter
region methylation was analyzed through methylation-specic PCR
(MSP). The remaining surgical specimen was xed in 10% buffered formalin, dehydrated, embedded in parafn, cut into 5 m sections and
stained with HematoxylinEosin for histological analyses at the Laboratory of Pathology in the Institute of Gynecology at the UFRJ.
The specimens collected through brush smears were also stored in
Eppendorf tubes at 20 C.
These methods were performed at the Laboratory of Control of Gene
Expression in the Institute of Biophysics Carlos Chagas Filho for the UFRJ
and the Laboratory of Virology at the Department of Microbiology and
Parasitology for the Fluminense Federal University (Universidade
Federal Fluminense UFF).
The project was approved by the Research Ethics Committee at the
UFRJ Maternity-School using the protocol number 25/2009 on December
11th, 2009.

2. Methods
2.2. DNA extraction
2.1. The population used for the study
The work described herein is a cross-sectional study evaluating
TIMP-2 gene methylation and the presence of HPV-DNA in cervix samples from 49 women at the Cervical Pathology Outpatient Clinic and
General Outpatient Clinic in the Institute of Gynecology for the Federal
University of Rio de Janeiro (Universidade Federal do Rio de Janeiro
UFRJ) from July 2005 to July 2011.
The samples were collected from outpatient cervical biopsies generated using a surgical high-frequency ultrasound device (Wavetronic
5000 Loktal Brazil) or through conventional conization at a surgical
center. The negative controls were cervix fragments from hysterectomized specimens (derived from surgeries for benign uterine corpus pathologies) or cervical brush smears collected at the General Outpatient
Clinic in the above institution. Samples from women with a low-grade
squamous intraepithelial lesion (LSIL) diagnosed through cytology and
colposcopy were also collected using a cervical brush smear from the
Cervical Pathology Outpatient Clinic.
The population used for this study included seven women with LSIL,
13 with high-grade squamous intraepithelial lesions (HSIL), 13 with
microinvasive carcinoma (FIGO IA1) (Pecorelli et al., 2009) and seven
with frank invasive squamous carcinoma. The negative control group
included nine women; four samples were collected from biopsies and
ve from brush smears.
Clinical evolution was favorable for women with no recurrence or
cervical lesion persistency upon treatment, whereas the evolution was
unfavorable for women with disease recurrence or persistency upon
treatment. For the LSIL cases that only included a follow-up without
treatment for two years, evolution was unfavorable when the
woman exhibited lesion persistency or cytological progression.

The materials collected from the patients (biopsy fragments and cervical brush smears) were fully digested using proteinase K (10 mg/mL)
for 16 h at 55 C. Subsequently, the mixture was treated twice with
phenol/chloroform (1:1) and precipitated with ethanol p. a. for 16 h at
20 C. The samples were washed with 80% ethanol, resuspended in
20 L water and frozen at 20 C until use. To conrm the extracted
DNA integrity, PCR was performed using primers for p53 exon 5,
which generated a 274-bp product, as described by Pestaner et al.
(1994).
2.3. HPV-DNA detection through PCR
The presence of HPV-DNA was assessed through PCR using the
primers MY09/MY11, which amplify a 450-bp DNA fragment, and the
primers GP5+/GP6+, which amplify a 140-bp DNA fragment. The reaction was performed in a thermocycler using the following protocol:
5 min denaturation at 95 C, 35 cycles of 95 C for 1 min, 60 C for
1 min, 72 C for 1 min and elongation at 72 C for an additional
10 min. The HeLa cell line was used in the positive control reaction to
detect HPV. In the negative control reaction, DNA was substituted
with distilled water. The PCR results were visualized using 10% polyacrylamide gel electrophoresis, followed by silver nitrate staining. The
approximate size of the amplied fragment was calculated using the
molecular weight marker Base Pair Ladder (Pharmacia Biotech).
2.4. Analyzing methylation at the TIMP-2 gene promoter region using MSP
TIMP-2 gene methylation was evaluated in two phases using a
method from Herman et al. (1996) and Rosas et al. (2001). The rst

Fig. 1. Polyacrylamide gel electrophoresis showing PCR products of methylation. The numbers correspond to the identication of patients with cervical lesions, M: methylated,
U: unmethylated, LMW: Base Pair Ladder and bp: base pairs.

Y. Furtado et al. / Experimental and Molecular Pathology 98 (2015) 119123

Table 2
Distribution of HPV DNA detection in accordance with cyto/histological diagnosis.
DNA-HPV

Normal
cervix n(%)

LSIL n(%)

HSIL n(%)

Microinvasive
carcinoma n(%)

Invasive
carcinoma n(%)

Positive
Negative
Total

2(25)
6(75)
8(100)

5(83.3)
1(16.6)
6(100)

9(75)
3(25)
12(100)

11(80)
2(15.3)
13(100)

6(85.7)
1(14.2)
7(100)

p = 0.003.

phase included DNA chemical modication through the sodium bisulte method. This technique transforms unmethylated cytosine into
uracil, but it does not alter methylated cytosine. Thus, approximately
5 L of DNA was diluted with 50 L distilled water and denatured in
0.2 M NaOH for 10 min at 37 C. The denatured DNA was diluted
using 550 L of a solution containing 10 mM hydroquinone (Sigma,
St. Louis, MO) and 3 M sodium bisulte pH 5.0 (Sigma). The solution
was covered with mineral oil and incubated for 16 h at 50 C. Next,
the solution was desalinated using the Wizard DNA Clean-up System
(Promega, Madison, WI), treated with 0.3 M NaOH for 5 min at room
temperature and precipitated with ethanol. The modied DNA was resuspended in 28 L water and stored at 20 C. In the second phase,
PCR was performed using the modied DNA and specic primers for
the methylated and unmethylated TIMP-2 genes in accordance with
Galm et al. (2005). Specically, 23 L of bisulte-modied DNA was
added to a nal volume of 50 L in a PCR mixture with 1 PCR buffer,
deoxyribonucleic triphosphate (1.25 mM each), primers (300 ng for
each reaction), 3 Mm MgCl2 and 1.25 units of Taq polymerase. The
PCR amplications were performed using 40 cycles. The amplication
conditions were as described above. The PCR product was analyzed
using polyacrylamide gel electrophoresis.

2.5. MSP primers

Specic sequence for unmethylated TIMP-2:
Forward 5-GTA ATA AAA TTG TGG TTT GGT TTA AGT TT-3
Reverse 5-TTC TCT CCT CTT TAT CTC AAA AAC ACA-3.
Specic sequence for methylated TIMP-2:
Forward 5-AAT AAA ATT GCG GTT CGG TTT AAG TTC-3
Reverse 5-CTC TCC TCT TTA TCT CGA AAA CGC G-3.
These primers amplied a DNA fragment approximately 200-bp
long.

2.6. Statistical analysis

A two-tailed Fisher's exact test was performed to assess the association between methylation and the presence of HPV-DNA with clinical
evolution for such lesions. The signicance was 0.05.

3. Results
The average age of the population used for the study was 40.6 years
(ranging between 23 and 67 years).

121

3.1. Detecting TIMP-2 gene methylation

Six percent of cases (3/49; one case of LSIL, one of HSIL and one from
the control group) showed no gene amplication. Methylation was detected in 80.4% (37/46) of the cases analyzed. Where the TIMP-2 gene
was amplied, 86.8% (33/38) of the samples from the women with cervical lesions and 50% (4/8) of the samples from women with a normal
cervix exhibited TIMP-2 methylation. A statistically signicant difference (p = 0.03) was observed for the proportion of methylation in the
group with cervical lesions and the control group (Table 1, Fig. 1).
3.2. HPV-DNA detection
HPV-DNA was detected in 71.7% (33/46) of the samples analyzed.
Specically, 81.6% (31/38) of the samples from women with cervical lesions were positive for HPV-DNA compared with only 25% (2/8) in the
samples from the control group. The difference between these groups
was statistically signicant (p = 0.003) (Table 2, Fig. 2).
3.3. Association between the presence of HPV-DNA and TIMP-2 gene methylation in the group with cyto/histological lesions
The association between the presence of HPV-DNA and TIMP-2 gene
methylation in the group of women with cervical lesions was not statistically signicant (p = 1.00) (Table 3).
3.4. Association between TIMP-2 gene methylation and clinical evolution of
the disease
The group with TIMP-2 gene methylation had a 2.3-fold (CI 95%
0.2322.82) higher risk of unfavorable evolution (recurrence, persistency or progression) than the group without methylation. Where TIMP-2
gene methylation was detected, 36% (12/33) of the women exhibited
unfavorable evolution, whereas this percentage was only 20% (1/5)
without methylation. Although methylation and unfavorable clinical
evolution were associated, the association was not statistically signicant (p = 0.19) (Table 4).
4. Discussion
Detecting methylated genes implicated in tumor suppression in cervix specimens (both biopsy fragments and exfoliated cells) (Furtado
et al., 2010; Ivanova et al., 2004) is technically viable and a signicant
source for potential biomarkers in cervical carcinogenesis. Further, evidence shows that tumor suppressor gene methylation in women infected with HPV may indicate a precursor lesion with a higher risk of
histological progression (Duenas-Gonzalez et al., 2005; Esteller, 2000;
Lattario et al., 2008a).
Only two studies have focused on TIMP-2 gene methylation in the
cervix. In one such study, Ivanova et al. (2004) reported TIMP-2 gene
methylation in 47% of cervical cancer cases and concluded that this epigenetic alteration might be a signicant biomarker for progression of
this cancer and it can be used to evaluate a prognosis during the initial
phase of tumor development. Consistent with such studies, TIMP-2
gene methylation was detected more frequently in LSIL, HSIL and
microinvasive carcinoma herein. Methylation was also more frequent
for the group with unfavorable clinical evolution. Although this analysis
was not statistically signicant (p = 0.19), we can infer that TIMP-2
gene methylation may be a prognostic biomarker for unfavorable lesion

Fig. 2. Polyacrylamide gel electrophoresis showing PCR products of HPV. The numbers correspond to the identication of patients, C+: positive control, C: negative control, bp: base
pairs and LMW: Base Pair Ladder.

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Y. Furtado et al. / Experimental and Molecular Pathology 98 (2015) 119123

Table 3
Association between the presence of HPV DNA and TIMP-2 gene methylation in the cases
of cyto/histological alterations.
DNA-HPV

Methylated n(%)

Unmethylated n(%)

Positive
Negative
Total

27(81.8)
6(18.2)
33(100.0)

4(80.0)
1(20.0)
5(100.0)

p = 1.00

evolution independent of the evolution and treatment phase. We have

not found published studies demonstrating this type of association to
date.
The other referenced study was by Parashar and Capalash (2012),
which reported no clear relationship between TIMP-2 gene methylation
and proteinase expression in cervical cancer cell lines. Notably, results
from studies using cancer cell lines are inconsistent with studies performed using fresh material removed from a biopsy or cervical brush
smear. The authors hypothesized that only one promoter region was
methylated for this gene or such methylation did not occur in the promoter region proximal to the transcription site, which might promote
partial gene inactivation. We did not assess proteinase expression
because our sole objective was to study TIMP-2 gene methylation as a
biomarker. However, under the hypothesis wherein methylation inactivates only certain regions in the TIMP-2 gene, protein expression may
be reduced, which would impair tumor suppression by TIMP-2.
Interestingly, methylation was also detected in samples from a normal cervix herein. Methylation in the negative controls has been previously described for tumor suppressor genes (Ivanova et al., 2004).
Lattario et al. (2008b) reported DAPK methylation in cervix samples
and concluded that it might be associated with transformation in cervical carcinogenesis. Two HPV-DNA-positive control samples were used
herein. Although the cellular morphology was not altered, TIMP-2
gene methylation was detected in these samples. Thus, this may be a
predictive factor for cervical epithelial lesion progression, which justies the suggestion by certain authors to group such characteristics
and to include a methylation screen in standard cervical cancer detection methods (Sun et al., 2012).
In a study using cell culture, Cardeal et al. (2012) concluded that
combined expression of the HPV 16 oncoproteins E6 and E7 reduces
TIMP-2 gene expression, which promotes increased expression of
MMPs inhibited by TIMP-2. Szalms and Knya (2009) related HPV
oncoprotein E7 expression to host-DNA methylation, and Burgers
et al. (2007) demonstrated that increased DNA methyltransferase 1
(DNMT 1) expression was related to E7 expression both in vitro and
in vivo. Herein, HPV-DNA was more frequently detected in women
with cervical lesions than without such lesions. These observations
were statistically signicant (p = 0.003), whereas the association between methylation and the presence of HPV-DNA was not statistically
signicant (p = 1.00).
Sun et al. 2012 proposed using liquid-based cytology to detect tumor
suppressor gene methylation for early HSIL diagnosis. The authors detected more frequent methylation associated with altered cytologies,
whereas methylation was a rare molecular event in normal cytologies,
which is consistent with the results herein.
In a systematic review of the literature on gene methylation in cervical precursor and invasive lesions, Wentzensen et al. (2009) found 51

Table 4
Association between TIMP-2 gene methylation and unfavorable clinical evolution.

Unfavorable evolution
Favorable evolution
Total
p = 0.19.

Methylated n(%)

Unmethylated n(%)

12(36.3)
21(66.6)
33(100)

1(20)
4(80)
5(100)

published studies. The most frequently cited genes were the following:
death-associated protein kinase (DAPK), cell adhesion molecules
(CADM), retinoic acid receptor, beta (RAR), Ras association domaincontaining protein 1 (RASSF1), cyclin-dependent kinase inhibitor 2A
(p16INK4A), glutathione S-transferase pi (GSTP) and telomerase reverse
transcriptase (TERT). The authors suggest that a panel of two to three
methylated genes could yield sensitivity at 74% and specicity at 95%
in a cervical cancer screening, which demonstrates the signicance of
methylation as a biomarker.
The applied screening methods do not detect lesions with histological evolution towards cancer nor women with a recurrence of precursor
or invasive disease up to 20 years after treatment. These questions must
be answered, which is reected in the published studies that have
attempted to determine the most suitable biomarker for cervical cancer.
DNA methylation is a promising molecular marker, and a panel of methylated tumor suppressor in combination with such screening methods
may improve sensitivity. Using methylation as a screen for precursor lesion histological classication to determine a treatment and monitor
treatment responses may slowly become reality (Harada et al., 2013).
In conclusion, the work herein shows that TIMP-2 gene methylation,
which has been poorly studied until now, was characteristic for women
with cervical lesions and the presence of HPV-DNA. A statistically significant relationship was not detected for TIMP-2 gene methylation and
the presence of HPV-DNA; however, this combination was more frequently observed with an unfavorable evolution.
Further studies are necessary to support the use of TIMP-2 methylation as a cervical lesion biomarker.
Conict of interest statement
The authors declare that there are no conicts of interest.
Acknowledgments
Cancer Foundation, Oncobiology project (Fundao do Cncer,
projeto de Oncobiologia)
National Council for Scientic and Technological Development
(Conselho Nacional de Desenvolvimento Cientco e Tecnolgico
CNPq).
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