Download Chapter 6 Comprehensive CADM1 promoter methylation

Chapter 6
Comprehensive CADM1 promoter methylation
analysis in NSCLC and normal lung specimens
Remco M. van den Berg, Peter J. F. Snijders, Katrien Grünberg, Clarissa Kooi, Marieke
D. Spreeuwenberg, Chris J.L.M. Meijer, Pieter E. Postmus, Egbert F. Smit, Renske D.M.
Steenbergen
In press, published online Lung Cancer 2010.
Abstract
Methylation-mediated silencing of the tumour suppressor CADM1 has
been functionally linked to lung cancer development. We aimed to determine
whether CADM1 promoter methylation is a candidate early detection marker
for lung cancer. To this end frozen tissue samples of 36 non-small cell lung
cancers, 26 corresponding tumour distant normal tissue samples as well as 6
samples of normal lung from non-lung cancer patients were tested for DNA
methylation at three different regions within the CADM1 promoter (M1, M5
and M9) using methylation specific PCR followed by methylation specific
reverse line blot analysis.
Sixty-four % of tumour samples tested positive at the M1 region, 47% at
M5 and 74% at the M9 region, compared with 65% (M1), 23% (M5) and 46%
(M9) of paired normal tissue samples. Methylation of each of these promoter
regions was also detected in the majority of non-lung cancer control samples.
Dense methylation, defined as methylation at ≥2 promoter regions, was detected
in 66% of tumour samples compared with 38% of paired normal tissues and
67% of non-lung cancer control samples. Within the small subgroup of female
patients dense methylation was found in all tumour samples but only 22%
of paired normal samples. Neither methylation of individual sites nor dense
methylation was correlated with disease free survival. In conclusion, CADM1
promoter methylation is a frequent event in NSCLC as well as normal lung,
both of lung cancer and non-lung cancer patients. Hence, CADM1 methylation
analysis is unlikely to have diagnostic value for the early detection of lung
cancer in an unselected population. However, a diagnostic value for selected
subjects, such as females, cannot be excluded.
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Introduction
Lung cancer claims more lives than any other malignancy. Two main
categories are distinguished: small cell lung cancer (SCLC) and non-small cell
lung cancer (NSCLC). The latter, an umbrella term for a number of histological
subtypes that comprise about 80% of lung cancers, can be cured by surgery
in its earlier stages. However, this disease often becomes symptomatic only
after the tumour has metastasised. Efforts to find screening tests that enable
early diagnosis, when curative treatment is still an option, should be pursued.
Malignant transformation is caused by a stepwise accumulation of molecular
changes. Theoretically, detection of these changes by sensitive nucleic acid
amplification assays that are available nowadays could serve as a molecular
marker for pre-symptomatic lung cancer detection, e.g. by sputum analysis of
risk groups. Hypermethylation of tumour suppressor gene promoter regions
has generated attention since this is a frequent event in the onset of many
cancers 1, 2. Different methods to determine DNA methylation have been
developed 3. Of these, methylation specific PCR (MSP) is one of the most
sensitive methods and consequently well suited to detect low target frequencies
of tumour DNA in sputum samples.
Support for a functional involvement of methylation events in lung cancer
development has come from a recent study of Damiani et al., who demonstrated
that transformation of immortalized bronchial epithelial cells upon exposure to
carcinogens was causally related to an increase of DNA Methyl Transferase 1
(DNMT1) protein and concomitant de novo methylation of tumour suppressor
genes 4. Indeed, a large number of genes has been found to be methylated
in lung cancer, of which p16, CDH13 and RASSF1A are amongst the most
commonly methylated genes being described in multiple studies 5. Analyses
on sputum samples revealed that a population with increased risk of lung
cancer might be discerned using methylation markers 6-11. One of the promising
methylation marker panels described for sputum consists of APC, p16, 3OST2
and RASSF1A 11. However, combinations of markers with better sensitivity
and specificity than those that have been studied so far are required to allow
implementation in screening tests 5. Thus, there is a need for additional markers
in lung cancer that can complement currently established panels to increase
their efficacy.
One candidate marker is methylation of the CADM1 gene. CADM1 (Cell
adhesion molecule 1, originally called TSLC1 for tumour suppressor in lung
cancer 1, and also referred to as IGSF4) is located in a region of chromosome
11q, which is commonly affected by deletions in all types of lung cancer 12. It
encodes a transmembrane protein of the immunoglobulin superfamily involved
89
in cell-cell interactions between epithelial cells. The CADM1 gene has been
shown by functional complementation studies to inhibit tumorigenesis of
lung- and cervical cancer cell lines 13, 14. Reduced expression and/or promoter
methylation of CADM1 has been found in carcinoma of the lung, cervix,
nasopharynx, oesophagus and breast 14-18. The gene is silenced in most cases
through methylation of its promoter, either or not in combination with allelic
loss 15, 18. In a series of 48 NSCLCs examined using methylation-specific single
strand conformation polymorphism (MS-SSCP) and bisulfite sequencing, 21
tumours showed CADM1 promoter hypermethylation, whereas no abnormal
methylation in the adjacent normal lung tissue could be detected15. CADM1
methylation was more frequently detected in high-grade lung tumours 14, 15
and also found to be associated with tobacco smoking and poor prognosis19.
A recent study investigated CADM1 as part of a panel of 27 methylation
markers in NSCLC using semi-quantitative real-time ms-PCR (methylight).
Although CADM1 was more frequently methylated in tumour tissue than in
normal tissue (18% vs. 4% respectively), it was not included in the panel of 8
markers that the authors deemed most relevant because any added sensitivity
did not outweigh the decrease in specificity 20. Although CADM1 has proven
tumour suppressive activity in a lung cancer cell line13, and expression in lung
cancers is often reduced 21, 22, the reported frequencies of promoter methylation
of this gene are highly variably amongst different studies. Therefore, no
firm conclusions can be drawn yet about the potential value of CADM1 as
biomarker for early lung cancer detection. For this a more comprehensive
analysis is warranted.
Towards this aim we examined methylation of three CADM1 promoter
regions in lung cancer tissue specimens and adjacent normal lung tissue samples,
as well as normal lung tissue of non-lung cancer patients using methylation
specific PCR (MSP) and a methylation specific reverse line blot hybridisation
read-out for PCR products23. Previous application of this methodology to
cervical carcinomas and precursor lesions, revealed an association between
methylation at ≥2 of the CADM1 promoter regions (i.e. dense methylation)
and both reduced CADM1 expression and increased severity of the lesion23.
90
Materials and methods
Tissue samples
Tissue samples from 36 patients who had surgery for NSCLC between
July 1987 and September 2004 as well as lung samples from 6 patients who
underwent surgery for lung diseases other than primary lung cancer were
obtained from the lung tissue bank of the department of Pathology of the VU
University Medical Center (table 1). Specimens had been collected within an
hour after surgical resection and tissue of the tumour had been snap-frozen and
stored in liquid nitrogen until use. For 26 patients normal lung tissue taken
from the surgical specimen at maximum distance from the tumour had been
collected and snap-frozen as well. Normal lung tissue samples from non-lung
cancer controls were obtained from patients undergoing surgery for lesions
that turned out to be pulmonary metastases of colorectal carcinoma (n=3) or
non-malignant disease (fibrous scar tissue n=3). Diagnosis of the lesion was
obtained from the pathology report. A board certified pathologist confirmed the
histology of the frozen material. Data on staging and follow up were obtained
from patient files. This study followed the ethical guidelines of the Internal
Review Board of the VU University Medical Center.
Table 1
Patient
characteristics
according to
histology
AC
n=11
SCC
n=19
LC-other
n=6
Non-LC
n=6
Age
60±11
69.6±7.9
59.2±21.6
67.0±7.7
Follow-up time (yrs)
4.2±3.1
3.9±4.5
1.3±1.9
1.3±1.3
Male
Disease free survival (yrs)*
5 (45)
5.0±1.2
14 (74)
9.9±2.2
5 (83)
1.9±1.0
5 (83)
Recurrence
6 (55)
6 (32)
3 (50)
Data presented as mean ± standard deviation or n (%) unless otherwise indicated.
AC: tumours with adenocarcinoma features (adenocarcinoma (n=8), adenosquamous
carcinoma (n=2), bronchioloalveolar carcinoma (n=1)), SCC: squamous cell
carcinoma, LC-other: lung cancer-other: i.e. LCC (n-4), MEC (n=1) and carcinoid
(n=1)
* Mean ± standard error
91
Laboratory procedures
DNA extracted from frozen tissue was subjected to bisulfite modification
using the EZ DNA methylation kit™ (Zymo Research, Orange, Ca, USA).
500 ng of DNA was modified and 25 ng of modified DNA was used for
MSP. Laboratory procedures and MSP primers and probes for three regions
in the CADM1 promoter (indicated as M1, M5 and M9, respectively) were
as described previously 23. In short, PCR mixtures contained 25ng modified
DNA, 0.5μM primers, each dNTP at 200μM, FastStart Taq PCR buffer (Roche
Diagnostics), 1.5mM MgCl2, and 1.25U FastStart Taq DNA polymerase (Roche
Diagnostics). All U-targeted primers were specific for unmethylated DNA,
while M-targeted primers only detected methylated DNA. Antisense primers
were biotinylated for reverse line blot (RLB) detection, in which denatured
PCR-products were hybridized to oligoprobes specific for unmethylatedand methylated-DNA, respectively. In each run H2O, unmodified DNA and
unmethylated DNA (primary keratinocytes) were included as negative controls
and DNA isolated from the cervical cancer cell line SiHa was included as a
positive control. Analytical sensitivity of each MSP-RLB assay, as determined
on a dilution series of methylated DNA in unmethylated DNA, was 1% for M1,
5 % for M5 and 0.5% for M9 23.
Statistics
Proportions of methylated sites in tumour and tumour-distant normal
samples of NSCLC patients were compared using the McNemar exact test and
the relation of ‘dense methylation’ (i.e. MSP positivity at ≥2 sites) with clinical
characteristics was analysed using the Fisher exact test. The presence of a
relation between dense methylation in tumour samples and different patient
characteristics was tested using stepwise binary logistic regression analysis
with forward selection. Relation between clinical characteristics of NSCLC
patients, dense methylation or methylation of individual sites and disease free
survival was analysed using Kaplan Meier survival analysis with a log rank
test. A p-value of ≤0.05 was considered statistically significant.
92
Results
Patient characteristics according to histology are summarized in table 1.
Eleven patients had tumours with adenocarcinoma features; adenocarcinoma
(AC; n=8), adenosquamous carcinoma (ASC; n=2) and bronchioloalveolar
carcinoma (BAC; n=1). Other histological subtypes were squamous cell
carcinoma (SCC, n=19); large cell carcinoma (LCC, n=4); mucoepidermoid
carcinoma (MEC, n=1) and carcinoid tumour (n=1). Methylation results of
individual samples are shown in table 2.
Sixty-four percent of tumour samples (23/36) showed CADM1 promoter
methylation at the M1 site, 47% (17/36) at the M5 site and 74% (26/35) at the
M9 site. Dense promoter methylation (defined as methylation of 2 sites or more,
and previously associated with reduction of CADM1 expression in cervical
lesions23) was present in 66% (23/35) of tumour samples. Dense methylation
was significantly more frequent in female patients tumour specimens (6AC, 5
SCC and 1 carcinoid); all (12/12) specimens of female patients revealed dense
methylation versus 46% (11/24) of the male patients tumours (p=0.002) (table
3).
Normal lung tissue taken at a maximum distance from the tumour was
available for 26 of 36 NSCLC patients. The M1 site was methylated in 65%
(17/26), M5 in 23% (6/26) and M9 in 46% (12/26) of these samples. Dense
methylation was found in 38% (10/26) of adjacent normal lung tissue samples.
When dichotomised into patients older or younger than the median of 68 years,
for the purpose of creating table III, significantly more patients with dense
methylation of normal tissue were older than 68 (table 3). However, a T-test
of mean ages showed no significant difference between patients with and
without dense methylation of adjacent normal lung tissue. When comparing
methylation at individual sites of paired tumour and normal samples using the
McNemar test (table 4), only the proportion of tumour samples methylated at
the M9 site was significantly higher than that of corresponding normal samples
(p value 0.012). The frequency of dense methylation was also higher in tumour
than in corresponding normal samples (p=0.039). This, however, could mainly
be attributed to the high frequency of dense methylation in female subjects; all
twelve females had densely methylated tumour tissue, whereas in only two out
of nine matched normal tissue samples of female patients the corresponding
tumour distant normal tissue showed dense methylation as well. In male
subjects there was no significant difference between dense methylation in
tumour and corresponding normal tissue. The relation between sex and dense
93
94
Sex
Age
Histology
pTNM
m
m
f
f
m
f
f
f
m
f
m
m
m
m
m
m
m
m
f
f
m
m
m
f
f
m
m
m
f
m
f
m
m
m
m
m
72
45
67
47
77
52
51
49
71
63
67
70
59
87
74
75
80
80
62
65
69
70
55
62
67
73
62
75
69
70
45
71
77
74
67
22
AC
AC
AC
AC
AC
AC
AC
AC
ASC
ASC
BAC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
atypical carcinoid
LCC
LCC
LCC
LCC
MEC
T1N0
T1N0
T1N0
T1N2
T2N0
T3N0
T3N0
T3N2
T1N0
T2N0
T1N0
T1N0
T1N0
T1N0
T1N0
T1N0
T1N0
T1N0
T1N0
T1N0
T2N0
T2N0
T2N0
T2N0
T2N0
T2N2
T3N0
T3N0
T3N0
T3N1
T1N0
T2N0
T3N0
T3N0
T3N1
T1N0
m
m
m
m
m
f
77
70
69
70
61
55
control*
control*
control*
control#
control#
control#
M1
Tumor
M5 M9
M1
Normal
M5 M9
Not available
Not available
Not available
ND
Not available
Not available
Not available
Not available
Not available
Not available
Not available
methylated
unmethylated
AC: adenocarcinoma, ASC: adenosquamous carcinoma, BAC: bronchioloalveolar carcinoma,
SCC: squamous cell carcinoma, LCC: large cell carcinoma, MEC: mucoepidermoid carcinoma, *
pulmonary metastasis, # non-malignant, ND: not done because of insufficiënt material available
Table 2
Summary of
methylation results
on tumour samples,
adjacent normal
samples and controls
18
I
24
2
4
19
11
67 (16/24)
100 (2/2)
25 (1/4)
58 (11/19)
82 (9/11)
10 (10/18)
72 (13/18)
50 (12/24)
92 (11/12)
64 (23/36)
M1
Tumour
68 (13/19)
100 (2/2)
0 (0/4)
85 (11/13)
40 (4/10)
72 (8/11)
60 (9/15)
63 (10/16)
67 (6/9)
65 (17/26)
Normal
54 (13/24)
100 (2/2)
25 (1/4)
53 (10/19)
36 (4/11)
33 (6/18)
61 (11/18)
67 (9/24)
67 (8/12)
47 (17/36)
M5
Tumour
26 (5/19)
50 (1/2)
0 (0/1)
23 (3/13)
20 (2/10)
36 (4/11)
13 (2/15)
29 (5/17)
11 (1/9)
23 (6/26)
Normal
83 (19/24)
100 (2/2)
25 (1/4)
78 (14/19)
91 (10/11)
67 (12/18)
88 (15/18)
56 (15/24)
100 (12/12)
74 (26/35)
M9
Tumour
47 (9/19)
0 (0/2)
0 (0/1)
46 (6/13)
60 (6/10)
63 (7/11)
33 (5/15)
47 (8/17)
44 (4/9)
46 (12/26)
Normal
67 (16/24)
100 (2/2)
25 (1/4)
63 (12/19)
73 (8/11)
50 (9/18)
77 (14/18)
46 (11/24)
100 (12/12)
66 (23/36)
≥2 sites
Tumour
0.72
0.702
0.16
0.002
P1
73 (8/19)
50 (1/2)
0 (0/1)
46 (6/13)
30 (3/10)
64 (7/11)
20 (3/15)
47 (8/17)
22 (2/9)
38 (10/26)
Normal
0.66
0.672
0.04
0.39
P1
II and III
12
7 (58)
57 (4/7)
33 (4/12)
14 (1/7)
58 (7/12)
43 (3/7)
58 (7/12)
28 (2/7)
Data presented as n or % (n/total) unless otherwise indicated, 1Fisher exact test, 2only using AC and SCC, AC: tumours with adenocarcinoma features,
SCC: squamous cell carcinoma, LCC: large cell carcinoma
Stage
Other
LCC
SCC
AC
18
>68
<68
68
65±12.9
24
12
Median
Mean
Male
Female
Histology
Age
Sex
Total
Total
Table 3
Percentage
methylation per site
in tissue samples from
NSCLC patients
95
Normal
Tumour
U
M
total
P*
6
13
19
0.75
U
13
1
14
Total
20
M
10
Dense U
6
M1
M5
M9
U
M
Total
M
U
Total
M
Total
3
9
7
4
14
10
16
4
17
5
6
1
7
26
12
26
5
11
21
2
8
12
8
10
26
18
26
0.07
0.012
0.039
U: unmethylated, M: methylated, * 2-sided McNemar test
methylation in tumour samples was confirmed by stepwise binary logistic
regression analysis with forward selection; no other variables (tested were age,
histology and disease stage) were a significant improvement of the model.
In six tumour samples, including 4 SCCs and 2 LCCs no methylation was
detected at any of the three promoter sites examined, whereas two of these
cases (both SCC) had methylation in tumour distant normal samples at one
and two sites, respectively. No clinical or histopathological characteristics
were associated with dense methylation. There was also no relation between
methylation of individual sites or dense methylation in tumour or normal tissue
specimens and disease-free survival. The only clinical characteristic associated
with disease free survival was disease stage (p=0.036, data not shown).
As non-lung cancer controls we used normal tissue samples from patients
who underwent surgery for a solitary pulmonary metastasis of a colon
carcinoma (n=3) or pulmonary lesions that turned out to be fibrous scar tissue
(n=3). Four of these 6 samples (67%) showed dense methylation as well (table
2).
96
Table 4
Comparison of
matched tumour and
adjacent normal
tissue samples
Discussion
CADM1 silencing is functionally involved in pulmonary carcinogenesis13
and promoter methylation of this gene has been proposed as a candidate marker
for early lung cancer detection5. Here, we applied an MSP-based methodology
for assessing CADM1 promoter methylation that was previously validated
on cervical material. By determining the methylation status at three different
regions within the CADM1 promoter, this method predicted gene silencing in
cervical tissue, based on the presence of methylation at ≥2 regions or dense
methylation23. We found that dense methylation was not only frequently present
in lung tumour samples, but also in 38% of corresponding normal samples
taken from the same surgical specimen, as well as four out of six normal lung
samples from patients without primary lung cancer. In the small subgroup of
female patients a clear difference in dense methylation was observed between
tumour and paired normal samples, warranting further studies.
Whereas previous studies limited their analysis to a single region in the
CADM1 promoter, our study included three regions. Methylation of these
regions was heterogeneous; no distinct pattern could be discerned and
methylation frequencies in each region were different. The M9 region was the
only single region where CADM1 methylation was significantly more frequent
in tumour samples than in corresponding normal samples. Nevertheless, 12
out of 26 corresponding normal samples were positive for M9 methylation.
This relatively high frequency of CADM1 promoter methylation in tumour
adjacent normal specimens could be confirmed by application of a newly
developed quantitative MSP assay targeting a region largely overlapping
that of M9 to a subset of cases (n=21). The overall concordance between
both assays was good (86%) (data not shown). Although for the M5 region
the difference between methylated tumour and normal samples was only
borderline significant (p=0.07), it was the region displaying the highest
specificity (the least methylation in corresponding normal samples; 6 out of
26). M5 overlaps the region tested by Fukami et al. and Kikuchi et al.15, 19,
resulting in three CpGs shared between the different analyses. Within this
region similar methylation frequencies in tumour tissue were found; 47% of
primary NSCLCs showed methylation of the M5 region in our study compared
with 44% (21/48 and 45/103, respectively) in both other studies. No CADM1
methylation in matched normal tissue was found in the latter two studies. Other
studies on CADM1 methylation in lung specimens have described methylation
frequencies in matched normal lung samples varying from 4% to 7% 20, 24. It is
likely that these differences in methylation frequency in normal lung samples
reflect differences in the sensitivity and targeted promoter regions between
97
the different assays used. Other studies have described varying degrees of
cancer-related methylation in histologically normal lung tissue of lung cancer
patients, such as adjacent normal tissue of specimens from lung cancer surgery
25
and histologically tumour-free bronchial resection margins 26. Analogous
findings have been reported in other organs, such as for MLH1 in colon27.
These abnormalities are often explained by the concept of ‘field cancerization’,
i.e.(epi)genetically affected tumour adjacent areas that represent a premalignant
condition from which new tumours may emerge. However, given the fact that
(dense) CADM1 methylation was found in a substantial fraction of normal
lung tissue of patients of a similar age who did not suffer from lung cancer,
field cancerization is unlikely to be the only explanation for the observed
methylation background in normal lung tissue. Instead, methylation observed
in normal lung tissue may be the result of environmental insult, aging, or reflect
some degree of physiological, epigenetic regulation of the CADM1 gene in
certain cell types present in lung tissue specimens.
Strikingly, all female patients had dense CADM1 methylation of tumour
samples compared with only 46% of male patients. This finding is contrary
to the findings of Kikuchi et al.19, who found significantly more male patients
with tumour CADM1 promoter methylation. However, they found a relation
with smoking index as well. Since we have no information about the patients’
smoking history it could not be included in our analysis, and it is therefore
uncertain whether different smoking habits could explain the discrepancy
between our results and those of Kikuchi et al. Although this seems irrelevant
for our main conclusion, the lack of specificity of CADM1 methylation testing,
no firm conclusions can be drawn on the potential biomarker value of CADM1
promoter methylation in non-smokers. Moreover, we could not reproduce the
relation between prognosis and CADM1 expression in adenocarcinomas found
in the same study19.
Conclusion
Present study shows that methylation of different sites of the CADM1
promoter is not only common in NSCLC, but can also frequently be detected
in normal lung tissue of both lung cancer patients and patients without lung
cancer. These data indicate that CADM1 methylation analysis has insufficient
specificity to be included in a panel of markers for early lung cancer detection
in an unselected population. We also did not find any evidence for CADM1
methylation analysis being a candidate prognostic marker for patients with
lung cancer. Nevertheless, a diagnostic value for selected subjects, such as
females, cannot be excluded.
98
References
1. F
einberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat
Rev Genet 2006 January;7(1):21-33.
2. Feinberg AP. The epigenetics of cancer etiology. Semin Cancer Biol 2004
December;14(6):427-32.
3. Fraga MF, Esteller M. DNA methylation: a profile of methods and applications.
Biotechniques 2002 September;33(3):632, 634, 636-2, 634, 649.
4. Damiani LA, Yingling CM, Leng S, Romo PE, Nakamura J, Belinsky SA. CarcinogenInduced Gene Promoter Hypermethylation Is Mediated by DNMT1 and Causal for
Transformation of Immortalized Bronchial Epithelial Cells. Cancer Res 2008 November
1;68(21):9005-14.
5. Anglim PP, Alonzo TA, Laird-Offringa IA. DNA methylation-based biomarkers for early
detection of non-small cell lung cancer: an update. Mol Cancer 2008;7:81.
6. Belinsky SA, Liechty KC, Gentry FD et al. Promoter Hypermethylation of Multiple Genes
in Sputum Precedes Lung Cancer Incidence in a High-Risk Cohort. Cancer Res 2006 March
15;66(6):3338-44.
7. Belinsky SA, Palmisano WA, Gilliland FD et al. Aberrant Promoter Methylation in
Bronchial Epithelium and Sputum from Current and Former Smokers. Cancer Res 2002
April 1;62(8):2370-7.
8. Gilliland FD, Harms HJ, Crowell RE, Li YF, Willink R, Belinsky SA. Glutathione
S-Transferase P1 and NADPH Quinone Oxidoreductase Polymorphisms Are Associated
with Aberrant Promoter Methylation of P16INK4a and O6-Methylguanine-DNA
Methyltransferase in Sputum. Cancer Res 2002 April 1;62(8):2248-52.
9. Chen JT, Chen YC, Wang YC, Tseng RC, Chen CY, Wang YC. Alterations of the p16(ink4a)
gene in resected nonsmall cell lung tumors and exfoliated cells within sputum. Int J Cancer
2002 April 10;98(5):724-31.
10. Palmisano WA, Divine KK, Saccomanno G et al. Predicting Lung Cancer by Detecting
Aberrant Promoter Methylation in Sputum. Cancer Res 2000 November 1;60(21):5954-8.
11. Shivapurkar N, Stastny V, Suzuki M et al. Application of a methylation gene panel by
quantitative PCR for lung cancers. Cancer Lett 2007 March 8;247(1):56-71.
12. Murakami Y, Nobukuni T, Tamura K et al. Localization of tumor suppressor activity
important in nonsmall cell lung carcinoma on chromosome 11q. Proc Natl Acad Sci U S A
1998 July 7;95(14):8153-8.
13. Kuramochi M, Fukuhara H, Nobukuni T et al. TSLC1 is a tumor-suppressor gene in human
non-small-cell lung cancer. Nat Genet 2001 April;27(4):427-30.
14. Steenbergen RDM, Kramer D, Braakhuis BJM et al. TSLC1 Gene Silencing in Cervical
Cancer Cell Lines and Cervical Neoplasia. J Natl Cancer Inst 2004 February 18;96(4):294305.
15. Fukami T, Fukuhara H, Kuramochi M et al. Promoter methylation of the TSLC1 gene
in advanced lung tumors and various cancer cell lines. Int J Cancer 2003 October
20;107(1):53-9.
16. Hui AB, Lo KW, Kwong J et al. Epigenetic inactivation of TSLC1 gene in nasopharyngeal
carcinoma. Mol Carcinog 2003 December;38(4):170-8.
17. Ito T, Shimada Y, Hashimoto Y et al. Involvement of TSLC1 in Progression of Esophageal
Squamous Cell Carcinoma. Cancer Res 2003 October 1;63(19):6320-6.
18. Allinen M, Peri L, Kujala S et al. Analysis of 11q21-24 loss of heterozygosity candidate
target genes in breast cancer: indications of TSLC1 promoter hypermethylation. Genes
Chromosomes Cancer 2002 August;34(4):384-9.
19. Kikuchi S, Yamada D, Fukami T et al. Hypermethylation of the TSLC1/IGSF4 promoter
is associated with tobacco smoking and a poor prognosis in primary nonsmall cell lung
carcinoma. Cancer 2006 March 13.
20. Feng Q, Hawes SE, Stern JE et al. DNA methylation in tumor and matched normal tissues
from non-small cell lung cancer patients. Cancer Epidemiol Biomarkers Prev 2008
March;17(3):645-54.
99
21. Uchino K, Ito A, Wakayama T et al. Clinical implication and prognostic significance of
the tumor suppressor TSLC1 gene detected in adenocarcinoma of the lung. Cancer 2003
September 1;98(5):1002-7.
22. Goto A, Niki T, Chi-Pin L et al. Loss of TSLC1 expression in lung adenocarcinoma:
relationships with histological subtypes, sex and prognostic significance. Cancer Sci 2005
August;96(8):480-6.
23. Overmeer R, Henken F, Snijders P et al. Association between dense CADM1 promoter
methylation and reduced protein expression in high-grade CIN and cervical SCC. J Pathol
2008 August;215(4):388-97.
24. Heller G, Fong KM, Girard L et al. Expression and methylation pattern of TSLC1 cascade
genes in lung carcinomas. Oncogene 2006 February 9;25(6):959-68.
25. Tsou JA, Shen LYC, Siegmund KD et al. Distinct DNA methylation profiles in
malignant mesothelioma, lung adenocarcinoma, and non-tumor lung. Lung Cancer 2005
February;47(2):193-204.
26. Guo M, House MG, Hooker C et al. Promoter hypermethylation of resected bronchial
margins: a field defect of changes? Clin Cancer Res 2004 August 1;10(15):5131-6.
27. Nuovo GJ, Nakagawa H, Sotamaa K, Chapelle AL. Hypermethylation of the MLH1
promoter with concomitant absence of transcript and protein occurs in small patches of crypt
cells in unaffected mucosa from sporadic colorectal carcinoma. Diagn Mol Pathol 2006
March;15(1):17-23.
100