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Gynecologic Oncology 93 (2004) 435 – 440
www.elsevier.com/locate/ygyno
Detection of hypermethylated genes in tumor and plasma of cervical
cancer patients
H.J. Yang, a V.W.S. Liu, a,* Y. Wang, a K.Y.K. Chan, a,b P.C.K. Tsang, a U.S. Khoo, b
A.N.Y. Cheung, b and H.Y.S. Ngan a
a
Department of Obstetrics and Gynaecology, Faculty of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China
b
Department of Pathology, Faculty of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China
Received 22 September 2003
Abstract
Objective. The aim of this study is to investigate the prevalence of promotor CpG island methylation of the death-associated protein
kinase (DAPK), p16, and O6-methylguanine-DNA methyltransferase (MGMT) genes in both tumor and plasma samples of cervical cancers.
Methods. Methylation-specific PCR (MSP) was employed to detect promotor CpG island methylation of the DAPK, p16, and MGMT
genes in 85 surgical tumor tissue samples and 40 pretreatment plasma samples from cervical cancers.
Results. Promotor CpG island methylation of DAPK, p16, and MGMT was detectable, respectively, in 60%, 28.2%, and 18.8% of cases of
cervical tumor DNA; and in 40%, 10%, and 7.5% of cases of patients’ plasma DNA. Moreover, at least one of the three methylated genes was
detected in 75.3% (64/85) of cases of tumor and in 55% (22/40) of cases of plasma. Higher prevalence of methylation of DAPK was found in
squamous cell carcinoma than in adenocarcinoma in both univariate and multivariate analysis. Methylation of p16 was significantly
associated with that of MGMT in both univariate and multivariate analysis. The methylation pattern in primary tumor and plasma was found
to be concordant in 23 patients with matched tissue and plasma samples. In cases positive for DAPK and p16 methylation in tumor, detection
in the paired plasma sample was 64.3% (9/14) and 33.3% (3/9), respectively.
Conclusions. Promotor CpG island methylation is a frequent event in cervical carcinogenesis. Detection of the methylated sequences in
the circulation suggests that plasma DNA methylation warrants further study to determine its potential role in cancer management.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Cervical cancer; Promoter CpG island hypermethylation; DAPK; p16; MGMT
Introduction
Cervical cancer is the second most common cancer in
women worldwide [1]. Human papillomavirus (HPV) is the
major causative agent [2– 5]. However, majority of patients
with HPV-associated lesions such as cervical intraepithelial
neoplasm (CIN) will remain stable or spontaneously regress
over time [6]. Therefore, other genetic and epigenetic events
are likely to be involved in cervical carcinogenesis.
Promoter CpG island hypermethylation is a frequent
epigenetic event in many human cancers [7,8]. It is a
potential pathway for tumor suppressor gene inactivation.
* Corresponding author. Department of Obstetrics and Gynaecology,
The University of Hong Kong, Room 747, Faculty of Medicine Building,
21 Sassoon Road, Hong Kong SAR, China. Fax: +852-2816-1947.
E-mail address: vwsliu@hkusua.hku.hk (V.W.S. Liu).
0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2004.01.039
The profile of gene hypermethylation is different for each
cancer type. Some genes are hypermethylated in multiple
tumor types, e.g., p16; while other changes are tumor-type
specific, e.g., hypermethylation of p73 is only observed in
hematological cancers [9]. Promoter hypermethylation of
the death-associated protein kinase (DAPK ), p16, and O6methylguanine-DNA methyltransferase (MGMT) genes has
been reported in 30– 60% of cervical cancers, but found to
be absent in normal tissues [10 – 13]. Furthermore, hypermethylation of p16 and MGMT seems to be an early event in
cervical tumorigenesis [11,12].
Recently, hypermethylated genes have also been detected
in DNA from plasma, serum, urine, and sputum by methylation-specific PCR (MSP) [14 –19]. As this method can
detect as little as one methylated gene per 1000 unmethylated copies, it has sufficient sensitivity to detect low
concentrations of tumor DNA in plasma. Compared to the
large number of different mutations, promoter hypermethy-
436
H.J. Yang et al. / Gynecologic Oncology 93 (2004) 435–440
lation occurs over the same regions of a given gene in each
form of cancer. Unlike allelic loss, detection of methylation
constitutes a positive signal, which is easier to be detected
against a background of normal DNA. Identification of the
occurrence of methylation for a relatively small subset of
genes in plasma might provide a potentially powerful
system of biomarkers for cancer detection. Therefore, in
this study, we examined the prevalence of promoter CpG
island methylation of DAPK, p16, and MGMT in tissues and
plasma of cervical cancer patients.
Materials and methods
Patients and tissue samples
This study was approved by the Ethics Committee of the
University of Hong Kong (EC 2011-03). One hundred and
two cervical cancer patients, who had been diagnosed and
treated in Queen Mary Hospital, Hong Kong, from January
1990 to February 2003, were recruited for this study. Tumor
tissues were obtained from 62 patients. Pretreatment peripheral blood samples were obtained from 17 patients. Paired
tumor tissue and blood samples were available in another 23
patients. In addition, 100 normal tissue samples including
white blood cells, cervix, and ovary from these patients
were used as controls. Another 30 plasma samples taken
from normal individuals were also used as controls for
plasma DNA analysis. Informed consent was obtained from
all individuals for the collection of tissue or blood. The
histological type and grade of tumor were classified according to WHO criteria. The stage of each cancer was established according to the International Federation of Gynaecology and Obstetrics (FIGO) criteria.
DNA isolation
Nine-milliliter pretreatment peripheral blood, collected in
a tube containing 1 ml of 3 M sodium citrate, was fraction-
ated by 1/1 volume Ficoll and centrifuged at 2000 g for
20 min at room temperature. The top plasma layer was
transferred to a microcentrifuge tube and centrifuged at
8000 g for 3 min to spin down all suspended cells and/
or cell debris. Then plasma was collected and stored at
20jC until further analysis. Plasma DNA was extracted
using the High Pure PCR Product Purification Kit (Roche
Applied Science, Mannheim, Germany) following procedures according to the manufacturer. Briefly, 200 Al plasma
was mixed thoroughly with 1 ml binding solution, and then
centrifuged at 10,000 g for 1 min. After discarding the
flow through, DNA was rinsed in washing solution twice,
then finally eluted by 50 Al sterile water.
Genomic DNA was extracted from tissues using the
standard Proteinase K treatment followed by phenol/chloroform extraction.
Methylation-specific PCR (MSP)
Sodium bisulfite treatment of genomic and plasma DNA
was performed before MSP. Genomic DNA (1 Ag) or 50
Al plasma DNA was denatured with 0.3 M NaOH for 10 min
at 37jC, and then bisulfite-treated as described by Herman
et al. [20]. DNA was then purified by Wizard DNA Cleanup
System (Promega, Madison, WI). DNA was treated again
with NaOH at room temperature, neutralized by ammonium
acetate, and finally ethanol-precipitated. After treatment, the
unmethylated cytosine will be converted to uracil, whereas
the methylated cytosine is unchanged. Uracil is recognized
as thymine by Taq polymerase.
The treated DNA was suspended in 30 Al of Tris buffer
and subjected to MSP using unmethylation-specific or
methylation-specific primers. The three genes were analyzed individually. Primer sequence, annealing temperature,
and the expected product size are listed in Table 1. Each 20
Al PCR contained 2 Al of bisulfite-treated genomic DNA or
5 Al of bisulfite-treated plasma DNA, 1 PCR buffer, 0.25
AM of each primer, 250 AM (each) dNTP mix, and 0.75
Unit of FastStart Taq DNA polymerase (Roche Applied
Table 1
Primer sequences, annealing temperatures, and PCR product sizes
Gene
p16
MGMT
DAPK
Uf
Ur
Mf
Mr
Uf
Ur
Mf
Mr
Uf
Ur
Mf
Mr
Sequence
Product
size (bp)
Annealing
temperature (jC)
5V-TAA TTA GAG GGT GGG GTG GAT TGT-3V
5V-CAA CCC CAA ACC ACA ACC ATA A-3V
5V-TTA TTA GAG GGT GGG GCG GAT CGC-3V
5V-GAC CCC GAA CCG CGA CCG TAA-3V
5V-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3V
5V-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3V
5V-TTT CGA CGT TCG TAG GTT TTC GC-3V
5V-GCA CTC TTC CGA AAA CGA AAC G-3V
5V-GGA GGA TAG TTG GAT TGA GTT AAT GTT-3V
5V-CAA ATC CCT CCC AAA CAC CAA-3V
5V-GGA TAG TCG GAT CGA GTT AAC GTC-3V
5V-CCC TCC CAA ACG CCG-3V
151
60
150
60
93
59
81
59
108
60
98
60
Uf : unmethylation forward primer; Ur: unmethylation reverse primer; Mf: methylation forward primer; Mr: methylation reverse primer.
H.J. Yang et al. / Gynecologic Oncology 93 (2004) 435–440
Table 2
Prevalence of methylation of genes in tissue and plasma samples of cervical
cancer patients
Direct sequencing
Methylated and unmethylated PCR products were confirmed by direct sequencing. The sequencing primers used
were forward primers. Sequencing reactions were carried
out using the BigDyek Terminator Cycle Sequencing V2.0
Ready Reaction Kit (Applied Biosystems, Foster City, CA)
and analyzed automatically by ABI 310 DNA sequencer
(Applied Biosystems). The cytosine in the unmethylated
PCR products converts to uracil, whereas the cytosine in the
methylated PCR products is unchanged.
Methylated Methylated Methylated Overall
DAPK
p16
MGMT
methylated
Tumor
(n = 85)
Nontumor
(n = 100)
Plasma
(n = 40)
Normal plasma
(n = 30)
Paired cases
Tissue
(n = 23)
Plasma
(n = 23)
51
(60%)
0
24
(28.2%)
0
16
(18.8%)
0
64
(75.3%)
0
16
(40%)
0
4
(10%)
0
3
(7.5%)
0
22
(55%)
0
14
(60.8%)
9
(39.1%)
9
(39%)
3
(13%)
4
(17.4%)
1
(4.0%)
18
(78.3%)
12
(52.2%)
33.3%
25%
437
Statistical analysis
Percentage of patients 64.3%
with positive
methylation in both
plasma and tissues
All the statistical analyses were performed with the
software SPSS Windows version 10.0 (SPSS Inc., Chicago,
IL). Numerical and categorical data were analyzed for
statistical significance using t test and chi-square analysis,
respectively. Parameters including age, histology type
[squamous cell carcinoma (SCC) vs. adenocarcinoma
(AC)], stage (early vs. late), grade of differentiation (G1
vs. G2 –3), and methylation status of the three genes were
put to multivariate analyses using logistic regression. A P
value of <0.05 was taken as significant.
Science). Thermal cycling was initiated at 95jC for 5 min,
followed by 45 cycles of 95jC for 30 s, the specific
annealing temperature for 30 s, and extension temperature
at 72jC for 30 s; and a final extension at 72jC for 2 min.
A methylation-positive DNA control was made in vitro
using SssI methylase (New England Biolabs, Beverly, MA)
which methylated every cytosine of CpG dinucleotide in
the DNA. An untreated blood DNA from a normal individual was used as negative control. The same PCR
conditions were used for tumor, normal, and plasma
DNA. PCR products were separated by 8% nondenaturing
polyacrylamide gel.
Results
Prevalence of promoter CpG island hypermethylation in
tissue samples of cervical cancer
Methylation of the three genes were detected in cervical
cancer samples but not in any of the normal tissue samples.
The prevalence of methylation of the three genes in 85
Table 3
Prevalence of methylation of different genes in tissues of cervical cancer patients and relation to clinical data
All
subjects
Mean age (years)
P valuea
Histological type
Squamous
Adenocarcinoma
P valueb
Stage
Early stage
Late stage
P valueb
Grade
G1
G2 – G3
P valueb
85
50.2
85
61
24
85
41
44
83c
30
53
DAPK
P16
MGMT
Any gene
+
+
+
+
51
51.0
0.457
34
48.6
24
53.4
0.183
61
48.7
16
49.0
0.756
69
50.3
64
52.1
0.024
21
43.9
42
9
0.008
19
15
18
6
0.678
43
18
11
5
0.766
50
19
50
14
0.023
11
10
22
29
0.249
19
15
13
11
0.492
28
33
11
5
0.068
30
39
28
36
0.149
13
8
18
32
0.973
12
21
12
11
0.060
18
42
7
8
0.349
23
45
24
39
0.511
6
14
+: positive for methylation; : negative for methylation.
a
P value was obtained by t test.
b
P value was obtained by chi-square test.
c
Two patients were uninformative on the WHO grading.
438
H.J. Yang et al. / Gynecologic Oncology 93 (2004) 435–440
cervical cancers is summarized in Table 2. Methylation was
detectable in at least one gene in 75.3% (64/85) of cervical
cancers. Of these, 24.7% (21/85) of cases were positive for
methylation in two genes, with another 4.7% (4/85) of
patients were positive for methylation in all three genes.
Methylation was more frequently detected in DAPK (60.0%;
51/85) than in p16 (28.2%; 24/85) and MGMT (18.8%; 16/
85). Table 3 illustrates the distribution of methylation of the
three genes according to clinicopathological parameters of
age, histological type, stage, and grade. Patients with methylation were older than patients without methylation ( P =
0.024; Table 3) Methylation of the DAPK gene was found to
be higher in SCC than AC by both univariate and multivariate
analyses ( P = 0.008 and 0.012, respectively) (Table 3), The
prevalence of DAPK methylation shows no significant difference between early stage and late stage ( P = 0.249); or
between less differentiated and well-differentiated cancer ( P
= 0.973). p16 or MGMT methylation was not associated with
any clinical parameters. However, by both univariate and
multivariate analyses, occurrence of methylation of the p16
gene was associated with the occurrence of methylation of the
MGMT gene ( P = 0.011 and 0.047, respectively).
Prevalence of promoter CpG island hypermethylation in
plasma samples of cervical cancer
The prevalence of methylation of DAPK, p16, and
MGMT in plasma was 40.0% (16/40), 10.0% (4/40), and
7.5% (3/40), respectively. Methylated sequence of at least
one gene was detected in a total of 55% (22/40) plasma
samples (Table 2). In contrast, methylated DNA was not
detectable in any of the 30 normal plasma samples. Methylation positivity was found in only one of the three genes
for each case, with the exception of a patient with stage IB2
cancer in which methylation of both DAPK and p16 was
detected in the plasma. A higher percentage of methylation
of DAPK gene in plasma samples was detected than the
other two genes (Table 2).
Of the 40 plasma samples obtained, 23 of them had paired
surgical tumor tissue available. We found a similar pattern of
Fig. 1. Methylation-specific PCR analysis of DAPK hypermethylation. The
control indicated the positions of the PCR products of the unmethylated
allele (U) and the methylated allele (M). In Case 1, promoter methylation
was not detected in tumor tissue or matched plasma sample. In Case 2,
promoter methylation was detected in both tumor and plasma samples.
methylation changes for the three genes in the plasma
samples to their paired tumor tissues (Fig. 1; Table 2).
Methylation of DAPK, p16, and MGMT was, respectively,
detected in 60.8% (14/23), 39% (9/23), and 17.4% (4/23) of
the tissue samples of these paired cases. Of the tissue
methylation positive patients, methylation in plasma for
DAPK, p16, and MGMT was detected in 64.3% (9/14),
33.3% (3/9), and 25% (1/4) respectively. None of the patients
without methylation in cervical cancer tissues was found to
have methylation in their plasma. The occurrence of methylation for the three genes in plasma was associated with that
in their paired tumor tissues ( P = 0.014, Fisher’s Exact Test).
Discussion
Since methylation changes are sometimes associated with
ageing of normal epithelium [21], only those methylation
markers that are always unmethylated in normal cells would
be potentially useful for cancer detection. Methylation of
DAPK, p16, and MGMT has been reported in many cancers
such as esophagus, lung, colon, and cervix. However, they
have not been found methylated in normal tissues including
buffy coat, bronchial brush, and urine samples from healthy
subjects as well as from normal tissues of cancer patients
[8,14,16,22,23]. Hence, in this study, we determined the
prevalence of methylation of these three genes in both tissue
and plasma samples from cervical cancers.
Consistent with others, no methylation of the three genes
was detected in normal tissues in the present study. In
contrast, methylation of at least one of the three genes
studied, DAPK, p16, and MGMT, was detected in about
75.0% of cervical cancers. A similar frequency (79%; 42/
55) was demonstrated for the methylation of at least one of
the p16, APC, HIC-1, DAPK, MGMT, and E-cadherin genes
studied by Dong et al. [10], although additional three genes
were included. In their study, the prevalence of methylation
of DAPK, p16, and MGMT gene was 51%, 30%, and 8%,
respectively. The highest prevalence of methylation among
all genes was also observed in the DAPK, as demonstrated
in this study. The overall findings suggest that methylation
of DAPK, p16, and MGMT occurs frequently and may be
used as markers for cancer detection. Dong et al. [10] found
that promotor methylation of DAPK and p16 was more
common in SCC cases than in AC cases. In this study, we
also found that methylation of DAPK but not p16 was more
common in SCC than AC. This indicates that methylation of
particular genes may preferentially occur in particular cell
types. Recently, Cohen et al. [24] demonstrated that
RASSF1A promoter was hypermethylated in 45% (5/11) of
AC, but not in any 31 cases of SCC. Thus, promoter
hypermethylation may also be a cell-type-specific event.
Great precautions should be taken when selecting methylated DNA markers for cancer detection.
Promoter hypermethylation has been reported in the
plasma/serum of various human cancer patients, including
H.J. Yang et al. / Gynecologic Oncology 93 (2004) 435–440
colorectal cancer [16] and liver cancer [25]. However, methylation in plasma/serum samples from cervical cancers has
not yet been reported. In all 40 plasma samples studied,
methylation of at least one gene was detected in about 55% of
plasma. Furthermore, the occurrence of methylation for the
three genes in plasma was associated with that in their paired
tumor tissues. And no methylated sequence was detected in
plasma from those negative for methylation in paired tissues,
implying an absence of false-positive methylation in this
study. Thus, methylated genes could also be detected in
plasma DNA of cervical cancer patients. The presence of
tumor-specific and methylated gene sequences in peripheral
blood may be developed as a noninvasive approach for cancer
detection. It is possible to detect abnormality using several
frequently methylated markers in majority cases of cancers.
Further analysis in a much larger sample size is warranted.
Recently, Smiraglia et al. [26] demonstrated that DNA
methylation was dynamic using restriction landmark genomic scanning. In paired primary and metastatic tumors
from 13 patients of head and neck squamous cell carcinoma,
the set of methylated loci found in primary tumors was found
to be different from those detected in metastatic tumor. Some
loci were common in both tumor; others were either detected
only in the primary or metastatic tumor. This indicates that
DNA methylation is a reversible process and may change at
different stages of cancer development. Nuovo et al. [12]
used MSP in situ hybridization to demonstrate that hypermethylation of p16 detected in the cancer lesion was also
present in precursor lesions adjacent to cervical cancer.
Virmani et al. [11] also reported the detection of p16 and
MGMT methylation in CIN samples and they suggested that
p16 and MGMT methylation were intermediate events in
cervical carcinogenesis. Though in our study, no significant
difference in methylation of p16 or MGMT was detected
between disease stages or grades, the significant association
between p16 and MGMT methylation suggested that these
two genes are closely related during cervical carcinogenesis.
Other genes such as RARb and GSPT1 have been proposed
to occur early in cervical cancer, while FHIT methylation
was a late, and tumor-associated event [11]. This dynamic
nature of methylation represents a significant difference
between epigenetic and genetic changes. Thorough study
of the prevalence of gene methylation at different stages of
cancer should be carried out before methylated DNA
markers could be used for cancer management.
In conclusion, promotor CpG island methylation is a
frequent event in cervical carcinogenesis. Demonstration of
the methylated sequences in the circulation suggest that
plasma DNA methylation warrants further study to determine its potential role in cancer management.
Acknowledgments
This study was supported by the University of Hong
Kong Conference and Research Grant (10204392/11733/
439
20900/323/01). The authors wish to acknowledge Daniel
Fong for his advice in the statistical analysis of data.
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