1. Detection of aberrant methylation
of 10 genes in genomic DNA of
lung tumors
Lung cancer is the leading cause of cancer-related death in most
of the countries [1]. Two-thirds of patients have advanced
stages of tumors at the time of diagnosis, which results high
mortality among lung cancer patients [2]. Identification of
cancer-specific markers could help to develop early diagnostic
tests and to improve the survival rates substantially. Epigenetic
changes, such as DNA methylation of CpG islands in the
promoter of some tumor suppressor genes (TSGs), are
frequently associated with ‘gene silencing’ [3]. Thus far, TSGs
such as APC, p16, CDH1, RARβ-2, and RASSF1A have been
found to have hypermethylated promoters in 30% of lung
tumors [4, 5]. We have determined the frequency of promoter
methylation of 10 TSGs (APC, SHP1, CDH1, SFRP1, Socs,
GSTP1, P16, DLC1, DAPK, and RarB2) in surgically removed
lung tumors. The tumor tissues were obtained from 30 lung
cancer patients, who underwent curative surgery from 2009 to
2011 at Masih Daneshvari Hospital. The histological
Table 1. The percent methylation rates were determine by quantitative methylation-specific PCR for seven selected tumor suppressor genes in 30 surgically
removed lung cancer tumors
Tumor APC CDH1 SFRP1 P16 DLC1 DAPK RARB-2 Type Stage Sex Age
TT-01 52 12 26 7 47 36 22 NSCLC IIIB M 46
TT-02 46 10 0 0 0 1 0 NSCLC IB M 67
TT-03 91 0 25 0 0 5 0 NSCLC IIIB F 75
TT-04 38 8 0 0 0 4 0 NSCLC IIB F 54
TT-05 25 11 0 0 0 9 1 NSCLC IIB M 53
TT-06 36 8 0 0 0 0 0 SCLC Extensive M 24
TT-07 45 13 0 0 0 0 0 SCLC Limited M 66
TT-08 80 13 4 29 0 20 0 NSCLC IIB M 46
TT-09 73 24 16 10 15 25 0 Other Limited M 62
TT-10 0 14 0 0 0 0 0 NSCLC IB M 31
TT-11 0 13 0 0 0 12 0 NSCLC IIB F 54
TT-12 100 0 0 0 0 0 0 NSCLC IB M 37
TT-13 36 10 23 9 5 25 0 NSCLC IIB M 61
TT-14 20 0 0 97 0 0 45 NSCLC IIB M 75
TT-15 0 0 0 0 0 0 0 NSCLC IB F 52
TT-16 57 0 0 0 0 0 0 NSCLC IA F 40
TT-17 0 9 0 0 0 0 0 Other Extensive F 54
TT-18 34 0 0 0 80 45 0 NSCLC IB M 66
TT-19 5 17 6 2 7 0 0 Other Extensive M 56
TT-20 0 0 0 0 0 0 0 NSCLC IIA F 68
TT-21 0 0 0 0 0 0 0 NSCLC IIIA F 25
TT-22 40 0 0 0 0 0 0 NSCLC IIIA F 56
TT-23 38 10 0 0 0 6 0 NSCLC IIIA F 37
TT-24 98 0 0 0 0 0 0 Other Extensive F 50
TT-25 32 14 27 1 43 33 12 NSCLC IIIA F 56
TT-26 45 0 0 0 0 1 5 NSCLC IB M 53
TT-27 43 8 0 0 0 13 0 NSCLC IIIA F 63
TT-28 48 32 35 24 38 51 16 NSCLC IIIA M 61
TT-29 39 14 0 0 0 0 0 NSCLC IIB M 58
TT-30 0 0 0 0 0 73 NSCLC IIIA M 53
SSs1 100 100 100 100 100 100 100 END END
The positive control was a DNA fully methylated by bacterial methylase, SssI.
NSCLC: non-small-cell lung carcinoma; SCLC: small-cell lung carcinoma.
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2. examination revealed that 24 of the 30 (80%) tumors were non-
small-cell lung cancer (NSCLC), two (6.6%) were small-cell
carcinoma (SCLC), and four (13.3%) other types. The median
age of the patients was 50.66 (range: 25–75) years. The
leukocyte DNA from 25 healthy individuals were used as
control samples. The median age of the control population was
35.7 (range: 25–53) years. All DNAs were extracted using the
QIAamp DNA mini Kit (QIAGENE cat. 51304) and were
subjected to bisulfite treatment, using the Epitect Bisulfite Kit
from Qiagen (Berlin, Germany). The bisulfite-modified DNA
was used as a template for fluorescence-based real-time
polymerase chain reaction (PCR). One of the healthy individual
leukocytes were methylated in vitro with excess SssI
methyltransferase (Zymoresearch, USA) to generate completely
methylated DNA. The TSGs promoter methylation level in each
sample was calculated and normalized with respect to the
internal reference gene, β-actin. The percentage of methylation
ratios (PMRs) represent the relative level of methylation in a
particular sample: 100 × [(GENEX mean value) sample/(ACTB
mean value) sample]/[(GENEX mean value) M.SssI/(ACTB
mean value) M.SssI ] and were used for direct comparison of the
samples. Cutoffs were set by receiver characteristic operator
curves (ROCs). The Fisher’s exact tests (two-sided) were carried
out to detect significant methylation differences between the
two groups. The pathological analysis revealed that most of the
patients (80%) had NSCLC. SOCs, GSTP, and SHP1 genes were
eliminated as possible cancer diagnostic markers, because SOCs
and GSTP1 had no detectable promoter methylation in these
cancer patients and SHP1 showed 100% methylation in both the
experimental and control population. The methylation results of
the remaining seven candidate genes are summarized in Table 1.
Genes such as APC, CDH1, and DAPK were most frequently
methylated when compared with controls, occurring in 23
(77%) versus 8% of control (P = 0.005) for APC, 18 (60%)
versus 8% (P ≤ 0.001) for CDH1, and 12 (40%) versus 0%
(P ≤ 0.0001) for DAPK. The other four genes were also
significantly methylated, 27% (P = 0.005) for SFRP1, 20%
(P = 0.005) for p16, 20% (P = 0.009) for DLC1, and 16%
(P = 0.009) for RARβ2 gene. Only 2 of the 25 control (normal
leukocytes) showed methylation for APC and another 2 for
CDH1 gene. Age was no factor, but gender seems to be a factor
at least in this set of patient population. Our data show that 7 of
13 female patients (54%) had one or no methylated gene, while
14 of 17 male patients (82%) had more than one gene
methylated. Our data suggest that APC, CDH1, and DAPK
could serve as lung cancer-specific panel for methylation
detection and ultimately for diagnostic purposes. At least one
gene from this panel was detected to be methylated at promoter
region in 27 of the 30 lung cancer patients (sensitivity 90%) and
only 4 of 25 control samples (specificity 84%).
R. Sheikhnejad1
*, M. Zohri1
, M. B. Shadmehr2
,
M. Rahmani-Khalili1
, N. Doozande2
, Z. Farsad1
&
K. Sheikhzade3
1
Molecular and Cancer Biology, Tofigh Darau, Research and Drug
Engineering Company, Tehran,
2
Tracheal Diseases Research Center, NRITLD, Shahid Beheshti University of
Medical Sciences, Tehran,
3
Research Center for Modeling in Health, Department of Epidemiology and
Biostatistics, Kerman University of Medical Sciences, Kerman, Iran
(*E-mail: sheikhnejad@msn.com).
funding
This research was supported by Tofigh Daru from the
Department of Molecular Biology Budget.
disclosure
The authors have declared no conflicts of interest.
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doi: 10.1093/annonc/mdt332
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