Copyright � The Korean Academy
of Medical Sciences
J Korean Med Sci 2007; 22 (Suppl): S47-51
ISSN 1011-8934
High Frequency of Genetic Alterations in Non-small Cell Lung Cancer
Detected by Multi-target Fluorescence In Situ Hybridization
Detection of genetic alterations could provide a tool as an adjuvant for the diagnosis of non-small cell lung cancer (NSCLC) and to define patients at risk for early
relapse. In this study, a multi-target fluorescence in situ hybridization (FISH) assay
was conducted to investigate the correlation between the alterations of chromosomes, including 5p15.2, 6p11.1-q11, 7p12, and 8q24.12-q24.13 (LaVysion Test),
and clinicopathological variables, and to clarify the potential of the multi-target FISH
assay in 37 NSCLC. The most notable finding was the higher frequency of a gain
in chromosome 5p15.2 in early-stage (I+IIa) lung cancers. The frequency of the
gain was 81.3% (16/22) in stage I tumors. The frequencies of gains in 6p11.1-q11
and 8q24.12-q24.13 were 61.5% (8/13) and 84.6% (11/13) in stage IIIa cancers,
as compared with lower frequencies in stage I tumors at 25.0% and 31.3%, respectively. There was also a significant difference in the histological type. Our results
suggest that a gain in 6p11.1-q11 and 8q24.12-q24.13 plays an important role in
tumor progression and is associated with histological differentiation. On the other
hand, gene amplification in the 5p region was one of the most consistent alterations
in early-stage lung cancer, and thus a series of genes in the critical 5p15.2 region
might potentially associated with the development of lung cancer.
Key Words : NSCLC; Genetic Alteration; Multi-target FISH; 5p15.2; 6p11.1-q11; 7p12; 8q24.12-q24.13
INTRODUCTION
Ji Un Kang, Sun Hoe Koo,
Kye Chul Kwon, Jong Woo Park,
So Youn Shin, Jin Man Kim*,
Sung Su Jung�
Departments of Laboratory Medicine, Pathology* and
�
Internal Medicine , Chungnam National University
Hospital, Daejeon, Korea
Received : 22 November 2006
Accepted : 22 January 2007
Address for correspondence
Sun Hoe Koo, M.D.
Department of Laboratory Medicine, Chungnam
National University Hospital, 640 Daesa-dong,
Jung-gu, Daejeon 301-721, Korea
Tel : +82.42-220-7798, Fax : +82.42-257-5365
E-mail : shkoo@cnu.ac.kr
*This work was supported by grant No. R11-2002-10000000-0 from the ERC program of the Korea Science
& Engineering Foundation.
Genomic alterations of regions on chromosome arm 5p
have been observed frequently in lung cancers (4); however,
copy number alteration has rarely been described in earlystage lung cancer. Understanding chromosomal alterations
in early-stage lung cancer led to the discovery of candidate
genes and will also offer an insight into its pathogenesis.
Moreover, classical cytogenetics (5), interphase FISH (6-8),
and comparative genomic hybridization (9, 10), have shown
gains in chromosomes 6, 7, and 8 in approximately 50% of
the NSCLC, which suggests that chromosomes 5, 6, 7, and
8 might be suitable targets for diagnostic FISH probes.
In this study, a multi-target FISH assay was performed to
investigate the correlation between chromosomal alterations
in each probe and clinicopathological variables, and to clarify the potential of the multi-target FISH assay in patients
with stage Ia-IIIa NSCLC.
Chromosomal aberrations can be a hallmark of cancer, and
molecular cytogenetic analyses have a considerable potential
as diagnostic adjuncts. Several chromosomal and molecular
abnormalities have been identified in non-small cell lung carcinomas (NSCLC) (1, 2). Although early-stage carcinomas
have been found to have fewer molecular alterations than advanced-stage carcinomas, such early molecular changes might
be used as molecular screening tools for the early detection
of NSCLC in high-risk patient populations.
Fluorescence in situ hybridization (FISH) has been used
successfully for targeted analysis of numerous chromosomal
abnormalities in interphase nuclei of various types of solid
tumors (3). Despite the high frequency of aneusomy in lung
cancer, FISH has not yet been fully exploited for early detection and monitoring of this tumor type, in part because of
the unavailability of validated probes specifically for lung carcinoma. We used the recently developed multi-target FISH
probes, LaVysion (Vysis, Downers Grove, IL, U.S.A.), for
simultaneous analysis of chromosome 6 and the 5p15.2,
7p12 (EGFR gene), and 8q24.12-q24.13 (MYC gene) loci.
This made it possible to simultaneously screen copy number
changes for four distinct DNA sequences.
MATERIALS AND METHODS
Patients and sample preparation
Touch imprints were prepared from 37 NSCLC at the time
of surgical resection. At the same time, fifteen imprints of
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J.U. Kang, S.H. Koo, K.C. Kwon, et al.
S48
normal areas were obtained from the lobected tissue of the
controls under the guidance of a pathologist. All subjects had
a lung mass that was operable. In addition, none of them had
undergone chemotherapy or irradiation. Imprints were obtained according to a slightly modified version of the protocol described by Varella-Garcia et al. (11). The freshly resected
tumor was imprinted on silanized slides. After washing, the
specimens were fixed in a 3:1 solution of methanol:glacial
acetic acid and were stored at -20℃ until analysis. The charts
of all patients were reviewed for any significant medical history. Permission to perform the study was obtained by the
institutional review board (IRB) at Chungnam University
Hospital, Daejeon, Korea.
morphologically abnormal cells were assessed, or the entire
sample was screened. Slides that contained fewer than six cells
were considered to be inadequate for evaluation. Overlapping
cells or cells with blurred signals were not assessed. Signals
that were located very close to each other were interpreted as
split signals and were counted as one signal. Slides showing
suspicious signals were re-examined by another technician
to verify the results. The test results were defined based on
previous studies (7, 14, 15). The identification of 6 or more
cells with polysomy, defined as a gain of 2 or more chromosomes in a cell, defines cancer positivity in this study.
Fluorescence in situ hybridization
We utilized several techniques for statistical analysis to
verify our results. Possible correlations between clinicopathological variables (cancer type and lymph node metastasis) and
the mean chromosome copy number were evaluated via independent t-tests. We employed one-way ANOVA (analysis of variance) in our comparisons of the differences in the
mean chromosome copy number between TNM (tumor-nodemetastasis) tumor stages. p values <0.05 were considered
significant.
A LaVysion kit (Vysis, Downers Grove) was used for FISH.
The probe set included one centromeric sequence (6p11.1q11) labeled with SpectrumAqua and three single copy DNA
sequences. These sequences recognize 450-kb sequences mapped at 5p15.2 (SpectrumGreen), 7p12 (including the EGFR
gene, SpectrumRed), and 8q24.12-q24.13 (including the
MYC gene, SpectrumGold).
The laboratory process was performed according to the
recommendations of the manufacturer with minor modifications. In brief, the slides were denatured by incubation
with formamide (70% in 2×SSC) at 73℃ for 5 min in a
water bath. The slides were then dehydrated through a graded ethanol system (70% for 1 min, 85% for 1 min, and 100%
for 1 min). A hybridization solution (10 L) was applied to
each slide, which was then cover-slipped and sealed with
rubber cement. After incubation for over 16 hrs at 37℃ in
a humidified chamber, the slides were washed with 0.4×
SSC/0.3% NP-40 for 2 min at 73℃. The slides were placed
in 2×SSC/0.1% NP-40 for 1 min at room temperature. A
4, 6-diamidino-2-phenylindole-antifade solution (10 L) was
then applied to each spot, which was subsequently coverslipped.
The slides were observed under a fluorescence microscope
connected to a cooled, charge-coupled device camera and an
image analyzer system, CytoVision (Applied Imaging, Ltd.,
Newcastle, United Kingdom). Two observers were independently involved in the FISH analysis, and investigators responsible for the FISH analysis were blinded to the status of
specimens, regarding all variables, except for the date of specimen collection. The score was calculated as an average between two observers’ scores.
The signals were scored on a cell-by-cell basis after hybridization and washing. The slides were assessed by scanning
for morphologically abnormal cells with nuclear enlargement,
irregular nuclear contour, patchy or lighter nuclear DAPI
staining, and cell clusters, all of which were indicative of
malignancy (12, 13). A normal cell contains two red, two
gold, two green, and two aqua signals. A minimum of 25
Statistical analysis
RESULTS
The overall sensitivity of FISH in lung cancer
FISH was performed to detect genetic alterations of chromosomes 5p15.2, 6p11.1-q11, 7p12, and 8q24.12-q24.13
(LaVysion Test), which showed very high sensitivity in detecting stage Ia to IIIa non-small cell lung cancers. A summary
of clinicopathological data of 37 non-small cell lung cancer
patients are shown in Table 1. When the aneusomy rate was
defined as the percentage of carcinomas with aneusomy for
at least two DNA targets, the aneusomy rate with the LaVyTable 1. Demographic data for patients with lung cancer
Number (%)
Sex
Male
Female
Age (M±SD)
< 59
60-69
>70
Histological type
Squamous cell carcinoma
Adenocarcinoma
TNM stage
I (a+b)
II (a+b)
IIIa
TNM stage, tumor-node-metastasis stage.
26 (70.3)
11 (29.7)
8 (21.6)
20 (54.1)
9 (24.3)
23 (62.2)
14 (37.8)
16 (43.2)
8 (21.6)
13 (35.1)
Genetic Alterations in Non-small Cell Lung Cancer
S49
A
B
C
Fig. 1. Representative examples of a multi-color, multi-target FISH assay from patients with lung cancer. (A) Normal control (6p11.1-q11;
aqua, 5p15.2; green, 7p12 red, 8q23.12-q24.13; yellow). (B) FISH-positive cells with polysomy of adenocarcinoma (5 red, 4 green, 4 aqua,
and 2 yellow signals). (C) FISH-positive cells with polysomy of squamous cell carcinoma (3 red, 1 green, 9 aqua, and 7 yellow signals).
Table 2. Relationship between TNM tumor stage and mean chromosome copy numbers in non-small cell lung cancer (mean±SD)
Chromosome
No.
5p15.2
6p11.1-q11
7p12
8q24.12-q24.13
TNM stage
Control
2.00±0.48
2.00±0.38
1.90±0.26
2.10±0.26
p value*
I (a+b)
II (a+b)
IIIa
Total
4.63±1.67
2.50±1.79
3.94±1.53
3.13±1.93
4.50±2.33
3.38±1.41
3.88±1.25
4.38±2.88
4.69±2.02
4.54±2.22
4.38±2.02
5.77±2.42
4.62±1.89
3.41±2.05
4.08±1.64
4.32±2.55
0.976
0.024
0.718
0.017
TNM stage, tumor-node-metastasis; SD, standard devication. *One-way ANOVA test.
Table 3. Relationship between lymph node metastasis and mean
chromosome copy numbers in each probe (mean±SD)
Chromosome
No.
5p15.2
6p11.1-q11
7p12
8q24.12-q24.13
Lymph node
Absent
Present
4.61±1.90
2.78±1.73
4.00±1.38
3.52±2.35
4.64±1.95
4.43±2.17
4.21±2.05
5.64±2.37
p value*
0.959
0.025
0.732
0.013
*SD, standard deviation. *Independent t-test.
Table 4. Relationship between histologic subtype and mean
chromosome copy numbers in each probe (mean±SD)
Chromosome
No.
5p15.2
6p11.1-q11
7p12
8q24.12-q24.13
Cancer type
Squamous cell
carcinoma
Adenocarcinoma
p value*
4.61±1.99
2.74±1.29
3.83±1.19
3.48±1.88
4.64±1.78
4.50±2.59
4.50±2.16
5.71±2.95
0.957
0.030
0.301
0.020
*SD, standard deviation. *Independent t-test.
sion probe was 92.6% (35/37). Abnormalities of at least one
of the chromosomes were observed in all cases (37/37), and 12
tumors (32.4%) showed polysomy of all four chromosomes.
A gain in 5p15.2 (27/37=73.0%) was the most recurrent
finding, followed by gains in 7p12 (24/37=64.9%), 8q24.12q24.13 (21/37=56.8%), and 6p11.1-q11 (15/37=40.5%).
All 37 tumor imprints showed good morphology. We found
no false-positive cases among the 15 normal control specimens (data not shown). Fig. 1 shows representative examples
of the positive FISH results from patients with lung cancer.
The relationship between each probe and the clinicopathological variables
In order to further clarify the chromosomal alterations in
lung cancer, we explored the statistical relationship between
the mean chromosome copy number and clinicopathological
variables in each probe. The mean chromosome copy numbers, in descending order, were 5p15.2 (4.62), 8q24.12-q24.13
(4.32), 7p12 (4.08), and 6p11.1-q11 (3.41) (Table 2). The
5p15.2 region was the most frequently involved, and was
observed in 22 early-stage (I+IIa) lung cancers. The mean
chromosome copy number (4.63) in 16 stage I cancers was
considerably higher than in other regions, followed by 7p12
(3.94), 8q24.12-q24.13 (3.13), and 6p11.1-q11 (2.50). This
region was detected in 81.3% of 16 stage I cancers. Gains of
6p11.1-q11 and 8q24.12-q24.13 were observed in 61.5%
(8 of 13) and 84.6% (11 of 13) of stage IIIa cancers, respectively, while lower frequencies were seen in 25.0% (4 of 16)
J.U. Kang, S.H. Koo, K.C. Kwon, et al.
S50
and 31.3% (5 of 16) of stage I cancers, respectively (Table 2).
Furthermore, the mean chromosome copy number exhibited
a tendency to increase directly with TNM stage, and a significant difference was shown (p=0.024 and p=0.017, respectively) (Table 2).
The frequencies of gene amplification and/or hyperaneusomy at 6p11.1-q11 and 8q24.12-q24.13 were statistically
significantly correlated with specific clinicopathological parameters (p<0.05); TNM tumor stage (I [a+b], II [a+b], IIIa),
lymph node status (absent or present), and histological type
(adenocarcinoma or squamous cell carcinoma (Table 2-4).
Tumors with lymph node metastasis were found to show
higher frequencies than tumors without lymph node metastasis (p=0.025 and p=0.013, respectively) (Table 3). The correlation was significantly higher in adenocarcinoma compared to squamous cell carcinoma (p=0.030 and p=0.020,
respectively), whereas the number of imbalances of 5p15.2
was similar in squamous cell carcinoma and adenocarcinoma (Table 4). Gains of 8q24.12-q24.13 were seen in 71.4%
(10/14) of adenocarcinoma compared to 47.8% (11/23) of
squamous cell carcinoma, while gains of 6p11.1-q11were
detected in 64.3% (9/14) of adenocarcinoma but in only
26.1% (6/23) of squamous cell carcinoma.
DISCUSSION
Earlier identification of cancer cells will lead to a greater
understanding of the chromosomal alterations that occur, and
will allow for a more complete insight into the pathogenesis
and progression of lung cancer. However, no efficient or accurate screening methods have yet been discovered for early
detection of lung carcinoma.
FISH can easily detect the number of specific chromosomes
or certain gene loci in histologic or cytologic tumor specimens, and may be conducted at a very early stage or even at
the precancerous stage. The recent development of multitarget LaVysion FISH probes made it possible to simultaneously screen copy number changes for four distinct DNA
sequences; in each of our samples, an abnormality of at least
one of the chromosomes was observed and 12 tumors (32.4
%) showed polysomy of all four chromosomes.
Classical cytogenetics (5), interphase FISH (6-8) and comparative genomic hybridization (9, 10) show gains of chromosomes 6, 7, and 8 in approximately 50% of NSCLC. A
gain of 5p is also a very common recurrent abnormality in
SCLC (9, 16), which suggests that chromosomes 5, 6, 7, and
8 might be suitable targets for diagnostic FISH probes. This
suggestion was confirmed by the present study, which indicated a high level of sensitivity (35/37=92.6%) of the fourprobe LaVysion assay in lung cancer, and specificity was 100%.
In the present study, a gain of 5p15.2 was the most frequent alteration (27/37=73.0%), and the mean chromosome
copy number in stage I was considerably higher than in other
regions. Garnis et al. (4) suggested that gains on 5p indicate
early events in lung cancer, and that the amplification of TRIO
and GDNF genes at 5p were some of the most dominant
phenomena in NSCLC. Several molecular cytogenetic studies have shown that chromosomal aberrations occur on the
short arm of chromosome 5 in all major lung tumor types,
and other studies have reported gains on 5p to be among the
most frequent alterations in SCLC (7, 16). These findings
suggest that gene amplification in the 5p region is one of the
most consistent alterations in early-stage lung cancer and the
amplification of this region is a major susceptibility locus in
lung cancer.
Chromosome 7 was shown to be an early marker of tumorigenesis in several types of cancers; polysomies of this chromosome have been demonstrated in hyperplastic and dysplastic epithelium by FISH (17). We detected 64.9% polysomy at 7p12, including the epidermal growth factor receptor, EGFR, and IL-6 genes, which are located on chromosome
7p and are overexpressed in NSCLS (18). Zojer N et al. (6),
demonstrated that aberrations of chromosome 7 occur early
in the development of lung cancer and that the aneuploidy
rate of chromosome 7 was 100% in primary lung cancer.
EGFR overexpression has also been demonstrated in premalignant bronchial epithelium, which suggests that EGFR
plays an important role in lung carcinogenesis (18)
A gain at 8q was the most frequently encountered aberration in 13 stage IIIa cancers (84.6%), with a minimal region
occurring at 8q24.12-q24.13, the region in which the c-MYC
oncogene is located. Several studies suggest that c-MYC amplification or overexpression is associated with a worse prognosis (19). Pei et al. (20) suggested that a gain of 8q was associated with later stage, higher grade tumors. Kubokura et al.
(19) also suggested that the rate of chromosome 8 aberration
is correlated with longevity of survival rate. Amplification
of the MYC family of proto-oncogenes (MYCC, MYCN, and
MYCL1) in human lung cancers has been well characterized
(21). MYCC is amplified in both SCLC and NSCLC, while
MYCN and MYCL1 amplifications are usually limited to
SCLC (21). In this study, the mean chromosome copy number at 8q24.12-q24.13 was significantly correlated with the
stage of TNM tumor (p=0.017). Tumors with lymph node
metastasis were found to occur at a higher frequency than
tumors without lymph node metastasis (p=0.013). We considered the rate of chromosome 8 aberration to be an additional prognostic factor of patients with NSCLC.
In this study, we detected 40.5% polysomy on chromosomes 6p11.1-q11. Juan et al. (22) suggested that amplification in 6p is the most prominent finding in NSCLC. In
the present study, amplification of chromosome 6 was significantly correlated with advanced clinical stage and lymph
node metastasis. These findings suggest that gene amplification of chromosome 6 is strongly associated with the development, progression, and metastasis of tumors.
Squamous cell carcinoma (SqC) and adenocarcinoma (AdC)
Genetic Alterations in Non-small Cell Lung Cancer
are the two most common subtypes of NSCLC. A growing
line of evidence suggests that the SqC and AdC subtypes
progress through different carcinogenic pathways, but the
genetic aberrations promoting such differences remain unclear. In the present study, the frequencies of gene amplification and/or hyperaneusomy at 6p11.1-q11 and 8q24.12q24.13 were significantly higher in AdC. These data suggest that genes located in these chromosomal regions may
be associated with phenotypic properties that differentiate
SqCs from AdCs.
In conclusion, our findings suggest that gains at 5p15.2
might be early events of NSCLC, and gene amplification in
this region might be strongly associated with the development. Additionally, genes located at 6p11.1-q11 and at
8q24.12-q24.13 may be associated with tumor aggressiveness and could represent markers of lung cancer progression.
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