Download Relationship between Chromosomal Instability and Intratumoral

Vol. 6, 2815–2820, July 2000
Clinical Cancer Research 2815
Relationship between Chromosomal Instability and Intratumoral
Regional DNA Ploidy Heterogeneity in Primary
Gastric Cancers1
Tomoko Furuya, Tetsuji Uchiyama,
Takuo Murakami, Atsushi Adachi,
Shigeto Kawauchi, Atsunori Oga, Takashi Hirano,
and Kohsuke Sasaki2
cers contained only diploid population. In contrast, all tumors without (near) diploid regions were advanced cancers.
These observations suggest that CIN is a necessary prerequisite for developing intratumoral DNA ploidy heterogeneity
with DNA aneuploidy.
Department of Pathology, Yamaguchi University School of Medicine,
Ube 755-8505 [T. F., S. K., A. O., K. S.]; Department of Surgery,
Iwakuni Medical Center, Iwakuni 740-0021 [T. U., T. M., A. A.]; and
Biopolymers Laboratory, National Institute of Bioscience and HumanTechnology, Tsukuba 305-8566 [T. H.], Japan
INTRODUCTION
ABSTRACT
The purpose of this study was to elucidate the relationship between intratumoral regional heterogeneity in DNA
ploidy and chromosomal instability (CIN) in primary gastric
adenocarcinomas. In 45 sporadic gastric adenocarcinomas,
we measured DNA ploidy and numerical aberrations for
chromosomes 7, 11, 17, and 18 by laser scanning cytometry
and fluorescence in situ hybridization, respectively, in small
tissue specimens taken from 2 to 6 (on the average 4) different portions of the same tumor. A total of 231 specimens
including 45 normal control specimens were examined. All
98 tumor specimens with DNA aneuploidy (DNA index >
1.2) showed large intercellular variations in chromosome
copy number, indicating CIN. In contrast, 85 tumor specimens with (near) diploidy (1.0 < DNA index <1.2) exhibited
much small intercellular variations in chromosome copy
number as compared with aneuploid specimens (P <
0.0001). The relationship between DNA ploidy and intercellular variation in chromosome copy number was true for
tumors consisting of a mixture of (near) diploid and aneuploid subpopulations. These data indicate that DNA aneuploidy is associated with CIN but that (near) diploidy is not.
Intratumoral regional DNA ploidy heterogeneity was conspicuous in 33 (92%) of 36 tumors with regions of DNA
aneuploidy, and all aneuploid specimens showed great intercellular variation in chromosome copy number. Diploid
regions were predominant in early stage cancers (intramucosal and submucosal cancers), and five of eight early can-
Received 9/3/99; revised 4/12/00; accepted 4/12/00.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
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indicate this fact.
1
This work was supported in part by The New Energy and Industrial
Technology Development Organization of Japan.
2
To whom requests for reprints should be addressed, at Department of
Pathology, Yamaguchi University School of Medicine, Ube 755-8505,
Japan. Phone: ⫹81-836-222222; Fax: ⫹81-836-222223; E-mail:
[email protected].
Although the mortality rate of patients with gastric cancer
has decreased due to early detection coupled with improvements
in therapeutic procedures, many patients still die of the disease.
In general, the prognosis for patients with early cancer is excellent, and prognosis for patients with advanced cancers is poor.
However, there are exceptions; some early cancers progress
rapidly, and the prognosis in advanced cancer patients is not
always grave. The biological characteristics of the cancer depend primarily on genetic alterations in cancer cells. In this
context, there is an important issue which we have to face
concerning a common genetic feature for all malignancies.
Malignant tumors intrinsically manifest genetic instability, and
consequently genetic aberrations successively accumulate in
tumor cells as the tumor progresses. Recently, genetic instability
in colon cancer cell lines was conceptually classified into two
distinct types: MIN3 and CIN (1– 4). MIN, which is a replication
error phenotype found in hereditary nonpolyposis colorectal
cancer, results from abnormalities in the DNA mismatch repair
pathway. The mechanism of MIN has been partially elucidated
(5, 6). Mutations in mismatch repair genes such as hMSH2 and
hMLH1 have been found in some colorectal cancers and are
though to be a cause of MIN. In contrast, defects in chromosome
segregation induced by aberrations of mitosis-regulating genes
results in a gain and/or loss of chromosomes in tumor cells (4,
7, 8), which gives rise to aneuploidy (3, 4). MIN has been
studied intensively in various malignant tumors. The incidence
of MIN was reported to be 16 –39% in gastric cancer (9 –11).
However, reports concerning CIN in primary cancers including
gastric cancers is scarce (12). This is due in part to methodological difficulties, because CIN, which is defined as a rate, cannot
be assessed from a single experiment. Recent investigations
have demonstrated that the extent of chromosome copy number
variations can be used as a surrogate marker for CIN (1– 4). In
vitro studies of colon cancer cell lines suggested that whereas
MIN generates subtle changes in nuclear DNA content, CIN
causes aneuploidy that is detectable by cytometry (1–3).
To examine whether the relationship between CIN and
DNA aneuploidy found in colon cancer cell lines holds for
3
The abbreviations used are: MIN, microsatellite instability; CIN, chromosomal instability; FISH, fluorescence in situ hybridization; LSC,
laser scanning cytometry; PI, propidium iodide; DI, DNA index.
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2816 Chromosomal Instability in Gastric Cancer
primary gastric cancers, we measured variations in chromosome
copy number and DNA ploidy in tissue specimens of surgically
removed gastric carcinomas using FISH and LSC, respectively.
This study reveals close relationships between intercellular variation in chromosome copy number and DNA ploidy and provides insight into the evolution of intratumoral heterogeneity in
ploidy in primary gastric cancers. This is the first report that
CIN causes intratumoral regional heterogeneity in DNA ploidy
in primary gastric cancers.
MATERIALS AND METHODS
Specimens. We used 45 consecutive surgically removed
gastric cancers: 8 early (3 mucosal and 5 submucosal tumors)
and 37 advanced cancers (Table 1). There were 32 male patients
and 13 female patients with an average age of 67.4 years (range,
44 – 86 years). Family histories were noncontributory for all
patients. Usually, tumor tissue specimens were taken from five
different parts of the same tumor, and as a control, an additional
specimen was taken from the mucosa distal to the tumor. A total
of 231 tissue specimens were examined, 186 tumor specimens
and 45 normal mucosal tissue specimens. Tissue specimens
were stored at ⫺80°C until use.
Touch Smear Preparations for FISH and LSC. At
least five touch smears were prepared by touching thawed tissue
specimens to glass slides after wiping blood from the cut surface
of the specimens with a paper towel. One touch sample was
dipped in 70% ethanol for fixation for DNA measurement by
LSC (13). The others were dried well and fixed with 100%
ethanol for FISH analysis (14).
FISH. Touch smears fixed in 100% ethanol were refixed
in 0.2% paraformaldehyde-PBS at 4°C for 10 min as previously
described (14). We examined numerical aberrations in chromosomes 7, 11, 17, and 18 using biotinylated alphoid satellite DNA
probes specific for the pericentromeric region of each chromosome (D7Z1, D11Z1, D17Z1, and D18Z1, respectively; Oncor,
Inc., Gaithersburg, MD), as described elsewhere (13). Briefly,
10 ␮l of a hybridization mixture containing 1 ␮g/ml salmon
sperm DNA (Sigma Chemical Co., St. Louis, MO), 55% formamide, 2 ⫻ SSC (1 ⫻ SSC is 0.15 M NaCl and 15 M sodium
citrate), and 10% dextran sulfate was heated in a water bath at
70°C for 5 min. The DNA mixture was applied to the slides,
which were denatured at 70°C for 2 min. Incubation for hybridization was performed overnight at 37°C in a moist chamber.
The slides were rinsed in washing solution containing 50%
formamide and 2 ⫻ SSC at 45°C and processed to stain the
hybridized probe using FITC-avidin (Vector Laboratory, Burlingame, CA). Nuclei were counterstained by adding glycerol
with PI (2 ␮g/ml, Sigma) and p-phenylenediamine dihydrochloride (1 ␮g/␮l, Sigma).
Scoring of Hybridization Signals. The number of hybridization signals in each nucleus was determined by observing
more than 200 nuclei on each slide with an epifluorescence
microscope equipped with a ⫻100 oil immersion objective
(Olympus Co., Tokyo, Japan).
DNA Measurement by LSC. DNA ploidy was determined as described previously (13, 15–17). Touch preparations
fixed in 70% ethanol were dipped in PI solution (25 ␮g/ml in
PBS) containing 0.1% RNase (Sigma). A coverslip was placed
Table 1
Pathological data and DNA ploidy in gastric cancers
Case Age Sex Histologya Depthb LNMc LMd Ploidye DPHf
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
59
63
61
51
68
86
71
64
53
78
48
44
73
77
80
70
68
48
68
82
75
54
80
82
59
57
78
66
64
72
67
74
57
85
73
71
70
66
77
65
49
63
83
63
71
M
M
M
F
M
M
M
M
M
F
F
F
M
F
F
M
M
F
M
M
F
M
F
F
F
M
F
F
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
por2
tub1
por2
por2
por2
tub2
tub2
por2
por2g
por2
tub2
por2
muc
tub2
sig
tub2
por2
sig
por1
tub2
pap
tub2
por2
tub2
sig
por1
por1
por1
por1
por1
muc
tub2
por1
pap
pap
sig
tub2
por2
tub2
tub2
por2
sig
tub2
por2
por2
ss
sm
se
si
si
si
ss
sm
si
se
m
ss
se
ss
se
se
si
m
se
se
ss
se
se
pm
ss
ss
pm
sm
se
ss
se
se
ss
sm
ss
sm
se
si
pm
ss
se
m
pm
se
si
⫺
⫺
2
⫺
⫺
1
⫺
⫺
4
2
⫺
⫺
2
⫺
1
3
1
1
1
2
⫺
1
⫺
⫺
1
⫺
1
1
1
1
3
3
6
⫺
2
1
1
2
⫺
2
2
⫺
⫺
2
2
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
D
M
A
D
M
A
M
M
D
M
M
D
A
A
M
A
D
D
A
D
A
A
M
A
M
A
M
D
M
A
D
M
M
D
A
D
M
M
A
A
M
D
D
M
M
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫹
⫹
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫹
⫺
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫹
a
Histological type: por1, poorly differentiated adenocarcinoma,
solid type; por2: poorly differentiated adenocarcinoma, nonsolid type;
tub1, well differentiated tubular adenocarcinoma; tub2, moderately differentiated tubular adenocarcinoma; sig, signet ring cell carcinoma;
muc, mucinous carcinoma.
b
Depth of tumor invasion: m, mucosa; sm, submucosa; ss, subserosa; se, serosal exposure; si, invasion to the pancreas.
c
LNM, lymph node metastasis. Figures indicate the number of
lymph nodes involved in carcinoma.
d
LM, liver metastasis: ⫹, positive; ⫺, negative.
e
D, a tumor in which every specimen shows (near) diploidy; M, a
tumor in which (near) diploid and aneuploid subpopulations coexist; A,
a tumor in which every specimen shows aneuploidy.
f
DNA ploidy heterogeneity: ⫹, present; ⫺, absent.
g
Scirrhous carcinoma.
on the slide and sealed with nail polish. DNA content was
measured with a laser scanning cytometer (LSC 101; Olympus
Co.). Usually, at least 5000 cells were examined for each sample. DNA histograms were generated, and DNA ploidy was
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Clinical Cancer Research 2817
Table 2 Classification of gastric adenocarcinomas based on DNA
ploidy and intratumoral regional DNA
Diploid tumors (9)a
WHb (3)
WOH (6)
a
b
Mixed tumors (22)
WH (22)
WOH (0)
Aneuploid tumors (14)
WH (11)
WOH (3)
Numbers in parentheses, number of cases.
WH, with heterogeneity; WOH, without heterogeneity.
determined. DI was calculated according to accepted principles
(18). A DI of 1.0 indicates DNA diploidy. In this series, tumors
with 1.0 ⬍ DI ⬍ 1.2 were classified as near-diploid cases and
were separated from DNA aneuploid tumors (DI ⱖ 1.2).
Intratumoral Regional DNA Ploidy Heterogeneity. A
tumor consisting of heterogeneous subpopulations with different
DNA ploidies within the tumor was considered to have intratumoral regional DNA ploidy heterogeneity.
Histological Diagnosis. Histological diagnoses were
made via routine 5-␮m sections stained with H&E and categorized according to the Japanese Classification of Gastric Carcinoma System (19).
Statistical Analysis. Student’s t test was used to compare
the population size of tumor cells with signal spots equivalent to
the modal chromosome number between two tumor groups
(diploid versus aneuploid tumor specimens) in gastric cancers
based on DNA indices. Statistical significance was set at P ⱕ
0.05.
RESULTS
Cells isolated from the normal mucosa invariably showed
DNA diploidy (DI ⫽ 1.0). However, DNA indices ranged from
1.0 to 2.64 in gastric cancers. In this series, 88 of 186 tumor
samples were judged to be (near) diploidy (1.0 ⱕ DI ⬍ 1.2), and
the remaining 98 were classified as aneuploidy (DI ⱖ 1.2).
DNA aneuploid (DI ⱖ 1.2) subpopulations were detected in at
least one area of a tumor in 36 (80%) of 45 cases, and (near)
diploid clones (1.0 ⱕ DI ⬍1.2) were found in at least one region
of a tumor in 31 tumors (69%). Gastric cancers were divided
into three groups, diploid, aneuploid, and mixed, based on DNA
ploidy and taking into account chromosomal alterations described later (Table 2). Diploid tumors included tumors consisting of diploid and/or near-diploid cell populations (1.0 ⱕ DI ⬍
1.2). However, there were no cases consisting of only neardiploid subpopulations. Nine cases (20%) were classified as
diploid. Of these, three showed intratumoral regional DNA
ploidy heterogeneity; they contained both diploid and neardiploid subpopulations. Aneuploid tumors (DI ⱖ 1.2) lacked
(near) diploid subpopulations. In this series, 14 tumors (31%)
were classified as aneuploid. Intratumoral regional DNA ploidy
heterogeneity was conspicuous in 11 of these tumors, but in the
remaining aneuploid cases it was difficult to find significant
differences in DI among the specimens. Mixed tumors consisted
of both (near) diploid and aneuploid populations, and 22 (49%)
of 45 tumors were placed within this category. All tumors
included in this group showed DNA ploidy heterogeneity. In
summary, intratumoral regional heterogeneity in DNA ploidy
was detected in 36 (80%) of 45 tumors (Table 2).
Five of eight early gastric cancers were included in the
diploid tumor group. In the remaining three early cancers,
aneuploid regions were found in the limited parts of a tumor. All
cases in which every specimen represented DNA aneuploidy
within a tumor were advanced cancers (Table 1).
Intercellular variation in the chromosome copy number
detected by FISH. In normal mucosa, ⬃90% of cells had two
signal spots for all chromosomes examined, and polysomic
(⬎4 signals) cells were virtually never observed. In diploid
tumors (1.0 ⱕ DI ⬍ 1.2), although there was a small population of polysomic cells, disomic cancer cells were apparently predominant for all chromosomes examined (Fig. 1). In
contrast, DNA aneuploid specimens (DI ⱖ 1.2) exhibited
great intercellular variations in chromosome copy numbers as
compared with diploid tumor specimens (1.0 ⱕ DI ⬍ 1.2)
(Fig. 1). Occasionally, polysomic nuclei were frequent in
aneuploid tumors. In 10 of 14 aneuploid tumors, the modal
chromosome copy number was 3 or 4. The modal chromosome number was 2 for all chromosomes in the remaining
four aneuploid tumors, but the variation in chromosomal
number was great. The intercellular variation in the chromosome copy number was not largely affected by the modal
chromosome copy number in the aneuploid tumor group.
The percentage of cells with signal spots equivalent to the
modal chromosome number was significantly smaller in aneuploid tissue specimens (DI ⱖ 1.2) than in (near) diploid ones
(1.0 ⱕ DI ⬍ 1.2) (P ⬍ 0.0001; Table 3). The relationship
between DNA ploidy and intercellular variation in chromosome
copy number was also true in mixed tumors (P ⬍ 0.0001).
Namely, disomic cells were predominant in DNA diploid regions, whereas cells in DNA aneuploid regions exhibited great
intercellular variation in chromosome copy numbers (Fig. 1).
There were foci with the great intercellular variation in the
chromosome copy number in 33 of 36 cases showing intratumoral DNA ploidy heterogeneity (Table 4). These 33 tumors
were aneuploid or mixed tumors, but the remaining 3 cases were
diploid tumors consisting of diploid and near-diploid populations. In contrast, intercellular variations in chromosome copy
numbers were obvious in only three of nine tumors without
DNA ploidy heterogeneity (Table 4). These three cases were
aneuploid tumors, whereas the remaining six were diploid tumors. All tumor specimens with great intercellular variation in
chromosome number showed DNA aneuploidy and vice versa
(Table 5).
DISCUSSION
Tumors are thought to develop along a multistep pathway
in tissues exposed to carcinogens that accumulate genetic alterations in functional targets relevant to tumor evolution. Once a
tumor is established, genetic instability, which is one characteristic of cancer cells, leads to successive genetic abnormalities in
tumor cells. Cells with different genetic alterations successively
emerge in parts of the tumor; some expand within the tumor
after clonal selection, and eventually intratumoral regional heterogeneity becomes conspicuous. Intratumoral regional heterogeneity coupled with genetic instability is an important issue not
only from the standpoint of DNA ploidy measurement but also
with respect to cancer treatment (20 –23). Our results indicate
that analysis of a single tissue specimen may lead to an errone-
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2818 Chromosomal Instability in Gastric Cancer
Fig. 1 Intercellular copy number variation for chromosomes 7,
11, 17, and 18 in diploid (A) and
aneuploid (B) primary gastric
cancers. In a diploid tumor, most
of cells (⬎80%) are disomic for
all chromosomes examined in all
regions, whereas aneusomic cells
are rare. In contrast, the percentage of cells with the modal chromosomal number is lower in an
aneuploid tumor than in a diploid
tumor (P ⬍ 0.0001); furthermore,
intercellular variation in chromosome copy number is conspicuous in aneuploid tumors. These
are seen in all regions within a
tumor. Ordinate, percentage of
cells with different chromosome
copy numbers; abscissa, circled
numbers, part of the tumor from
where a tissue specimen is taken.
N, normal control.
ous interpretation. Fortunately, in this study, the possibility that
we overlooked a major subpopulation is considered low, because most stemlines are detected cytometrically when four
samples taken from different regions of a tumor are examined
(24).
Cytogenetically cancers containing both diploid (DI ⫽ 1.0)
and near-diploid (1.0 ⬍ DI ⬍ 1.2) populations were included in the
diploid tumor group, because tumors in this group were characterized by disomic cells with a small population of aneusomic cells for
all chromosomes examined. Namely, the intercellular variations in
chromosome copy numbers were smaller in DNA (near) diploid
cell populations than in aneuploid cell populations (P ⬍ 0.0001).
This was also true of DNA (near) diploid regions in tumors con-
sisting of a mixture of (near) diploid and aneuploid populations,
i.e., mixed tumors. These observations indicate that near-diploid
tumors are different from aneuploid tumors and that they should
therefore be included in the diploid group. Gastric cancers confined
to the mucosal layer have been reported to exhibit DNA diploidy
more frequently than advanced cancers (20, 25). The present study
also revealed that diploid cells occupied most of or entire parts of
a tumor in cases of intramucosal cancer and that no early gastric
cancers (mucosal and submucosal tumors) were included in the
aneuploid group. Although aneuploid foci within a tumor were
found in three of eight early gastric cancers, tumors in which every
specimen was aneuploid were always advanced cancers. On the
basis of these observations, we hypothesize that although some
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Clinical Cancer Research 2819
Table 3 Relationship of DNA ploidy to the percentage of cells with
modal number for chromosomes examined in gastric cancers
DNA ploidy of gastric cancerb
Chromosomea
Diploid
1.0 ⱕ DI ⬍ 1.2
Aneuploid
DI ⱖ 1.2
7
11
17
18
77.2 ⫾ 13.2
84.1 ⫾ 10.3
80.2 ⫾ 9.9
80.0 ⫾ 9.4
57.6 ⫾ 16.0
61.8 ⫾ 14.8
63.9 ⫾ 15.5
62.4 ⫾ 18.0
c
a
Approximately 90% of cells from the normal mucosa are disomic
for all chromosomes.
b
Tissue specimens from gastric cancers are classified into two
groups, diploid (1.0 ⱕ DI ⬍ 1.2) and aneuploid (DI ⱖ 1.2) tumors,
based on DNA index. A total of 186 tumor specimens are examined in
this study, 88 samples showing DNA diploidy and 98 samples showing
DNA aneuploidy.
c
Values are mean percentages of cells with modal chromosome
number (mean ⫾ SD). There is significant difference in the percentage
of cells with modal chromosome number between diploid and aneuploid
cancers for all chromosomes examined in this study (P ⬍ 0.0001).
Table 4 Relationship between DNA ploidy heterogeneity and
intercellular variations in chromosome copy numbera
Tumors with DNA ploidy
heterogeneity (36)b
Tumors without DNA ploidy
heterogeneity (9)
Large chromosomal variation(33)c Large chromosomal variation (3)d
No chromosomal variation (3)e
No chromosomal variation (6)f
a
Intercellular variation in the number of chromosomes is large in
33 of 36 tumors with intratumoral regional DNA ploidy heterogeneity
and in 3 of 9 tumors without DNA ploidy heterogeneity. In contrast,
intercellular numerical variation in chromosomes is minimal in the
remaining tumors as well as in normal cell controls.
b
Numbers in parentheses, number of cases.
c
These 33 cases are aneuploid tumors (1.2 ⱕ DI).
d
These 3 cases are aneuploid tumors (1.2 ⱕ DI).
e
These 3 cases are diploid tumors (1.0 ⱕ DI ⬍ 1.2).
f
These 6 cases are diploid tumors (D ⫽ 1.0).
gastric cancers start and develop as DNA aneuploid tumors, a large
proportion of gastric adenocarcinomas arise as diploid tumors.
With tumor progression, cells with different genetic alterations appear successively within the tumor due to genetic
instability, and eventually intratumoral regional heterogeneity
becomes apparent after repeated clonal selection. However, we
must bear in mind that the pattern of intratumoral heterogeneity
may be change over time. This scenario is supported by the
theory that genetic instability is the engine of both tumor progression and tumor heterogeneity (1–3). Because recent investigations indicated a close relationship between DNA ploidy and
CIN in cancer cells (1–3), it is reasonable to assume that CIN,
which leads to asymmetrical cell division, affects the development of intratumoral heterogeneity in DNA ploidy. In contrast,
MIN does not directly lead to DNA aneuploidy because MIN
yields imperceptible alterations in nuclear DNA content (1–3).
During tumor progression, however, mutations in cell division
checkpoint genes may occur in cases with MIN, and eventually,
tumor cells with MIN also have properties of CIN (1– 4). Because CIN is a dominant phenotype (1), genetic instability
results in intratumoral DNA ploidy heterogeneity. CIN is critical
Table 5
Relationship between intercellular variations in chromosome
copy numbers and tumor type
Tumors with large
chromosomal variation (36)a
Tumors without
chromosomal variation (9)
Diploid tumorsb (0)
Aneuploid tumors (14)
Mixed tumors (22)
Diploid tumors (9)
Aneuploid tumors (0)
Mixed tumors (0)
a
Numbers in parentheses, number of cases.
Diploid tumors are seen only as tumors without intercellular
variation in chromosome copy number.
b
for the development of intratumoral heterogeneity. CIN is induced by aberrations in genes relevant to mitosis, but the affected genes are thought to be different among tumors (3).
Accordingly, it is appropriate to assume that the extent of
variations in chromosome copy numbers depends on genes
affected. The present study indicates that the hypothesis proposed by Lengauer et al. (1–3) holds for primary gastric cancers.
DNA ploidy analysis coupled with FISH examination elucidates the relationship between DNA ploidy and genetic instability; furthermore, it provides insight into the evolutionary
mechanisms of intratumoral heterogeneity in gastric adenocarcinomas. To our knowledge, this is the first report that CIN
causes intratumoral regional DNA ploidy heterogeneity in primary gastric cancers.
REFERENCES
1. Lengauer, C., Kinzler, K. W., and Vogelstein, B. Genetic instability
in colorectal cancers. Nature (Lond.), 386: 623– 627, 1997.
2. Lengauer, C., Kinzler, K. W., and Vogelstein, B. DNA methylation
and genetic instability in colorectal cancer cells. Proc. Natl. Acad. Sci.
USA, 94: 2545–2550, 1997.
3. Lengauer, C., Kinzler, K. W., and Vogelstein, B. Genetic instabilities
in human cancers. Nature (Lond.), 396: 643– 649, 1998.
4. Cahill, D. P., Lengauer, C., Yu, J., Riggins, G. J., Willson, J. K. V.,
Markowitz, S. D., Kinzler, K. W., and Vogelstein, B. Mutations of
mitotic checkpoint genes in human cancers. Nature (Lond.), 392: 300 –
303, 1998.
5. Marra, G., and Boland, C. R. Hereditary nonpolyposis colorectal
cancer: the syndrome, the genes, and historical perspectives. J. Natl.
Cancer Inst., 87: 1114 –1125, 1995.
6. Bhattacharyya, N. P., Skandalis, A., Ganesh, A., Groden, J., and
Meuth, M. Mutator phenotypes in human colorectal carcinoma cell
lines. Proc. Natl. Acad. Sci. USA, 87: 7555–7559, 1990.
7. Pihan, G. A., Purohit, A., Wallace, J., Knecht, H., Woda, B., Quesenberry, P., and Doxsey, S. J. Centrosome defects and genetic instability
in malignant tumors. Cancer Res., 58: 3974 –3985, 1998.
8. Zhou, H., Kuang, J., Zhong, L., Kuo, W., Gray, J. W., Sahin, A.,
Brinkley, B. R., and Sen, S. Tumor amplified kinase STK15/BTAK
induces centrosome amplification, aneuploidy and transformation. Nat.
Genet., 20: 189 –193, 1998.
9. Chong, J. M., Fukayama, M., Hayashi, Y., Takizawa, T., Koike, M.,
Konishi, M., Kikuchi-Yanoshita, R., and Miyaki, M. MIN in the progression of gastric carcinoma. Cancer Res., 54: 4595– 4597, 1994.
10. Seruca, R., Santos, N. R., David, L., Constancia, M., Barroca, H.,
Carneiro, F., Seixas, M., Peltomaki, P., Lothe, R., and Sobrinho-Simoes,
M. Sporadic gastric carcinomas with microsatellite instability display a
particular clinicopathologic profile. Int. J. Cancer, 64: 32–36, 1995.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 2000 American Association for Cancer
Research.
2820 Chromosomal Instability in Gastric Cancer
11. Peruncho, M. Cancer of the microsatellite mutator phenotype.
J. Biol. Chem., 377: 675– 684, 1996.
12. Miyazaki, M., Furuya, T., Shiraki, A., Sato, T., Oga, A., and
Sasaki, K. The relationship of DNA ploidy to chromosomal instability in primary human colorectal cancers. Cancer Res., 59: 5283–
5285, 1999.
13. Sasaki, K., Kurose, A., Miura, Y., Sato, T., and Ikeda, E. DNA
ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers
and comparison with flow cytometry. Cytometry, 23: 106 –109, 1996.
14. Sasaki, K., Sato, T., Kurose, A., Uesugi, N., and Ikeda, E. Monosomy of chromosome 18 detected by fluorescence in situ hybridization
in colorectal tumors. Cancer (Phila.), 76: 1132–1138, 1995.
15. Kamada, T., Sasaki, K., Tsuji, T., Takahashi, M., and Kurose, A.
Sample preparation from paraffin-embedded tissue specimens for laser
scanning cytometric DNA analysis. Cytometry, 27: 290 –294, 1997.
16. Furuya, T., Kamada, T., Murakami, T., Kurose, A., and Sasaki, K.
Laser scanning cytometry allows detection of cell death with morphological features of apoptosis in cells stained with PI. Cytometry, 29:
173–177, 1997.
17. Kawasaki, M., Sasaki, K., Satoh, T., Kurose, A., Kamada, T.,
Furuya, T., Murakami, T., and Todoroki, T. Laser scanning cytometry
(LSC) allows detailed analysis of the cell cycle in PI stained human
fibroblasts (TIG-7). Cell Prolif., 30: 139 –147, 1997.
18. Hiddemann, W., Schumann, J., Andreeff, M., Barlogie, B., Herman,
C. J., Leif, R. C., Mayall, B. H., Murphy, R. F., and Sandberg, A. A.
Convention on nomenclature for DNA cytometry. Cytometry, 5: 445–
446, 1984.
19. Japanese Research Society for Gastric Cancer. Histological findings. In: Japanese Classification of Gastric Carcinoma, Ed. 12, pp.
60 –107. Tokyo: Kanehara and Co., Ltd., 1995.
20. Fujimaki, E., Sasaki, K., Nakano, O., Chiba, S., Tazawa, H.,
Yamashiki, H., Orii, S., and Sugai, T. DNA ploidy heterogeneity in
early and advanced gastric cancers. Cytometry, 26: 131–136, 1996.
21. Sasaki, K., Hashimoto, T., Kawachino, K., and Takahashi, M.
Intratumoral regional differences in DNA ploidy of gastrointestinal
carcinomas. Cancer (Phila.), 62: 2569 –2575, 1988.
22. Sasaki, K., Takahashi, M., Hashimoto, T., and Kawachino, K.
Flow cytometric DNA measurement of gastric cancers: clinicopathological implication of DNA ploidy. Pathol. Res. Pract., 184:
561–566, 1989.
23. Danova, M., Riccardi, A., Mazzini, G., Wilson, G., Dionigi, P.,
Brugnatelli, S., Fiocca, R., Ucci, G., Jemos, V., and Ascari, E. Flow
cytometric analysis of paraffin-embedded material in human gastric
cancer. Anal. Quant. Cytol. Histol., 10: 200 –206, 1988.
24. Beerman, H., Smith, V. T. H. B. M., Kluin, P. M., Bonsing, B. A.,
Hermans, J., and Cornelisse, C. J. Flow cytometric analysis of DNA
stemline heterogeneity in primary and metastatic breast cancer. Cytometry, 12: 147–154, 1991.
25. Maeda, K., Chung, Y. S., Kubo, T., Nishimura, M., Onoda, N.,
Nitta, A., Arimoto, Y., Kato, Y., and Sowa, M. Nuclear DNA ploidy
pattern and proliferating cell nuclear antigen labeling index in gastric
cancer with mucosal involvement. Gan To Kagaku Ryoho, 21 (Suppl 1):
58 – 61, 1994.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 2000 American Association for Cancer
Research.
Relationship between Chromosomal Instability and
Intratumoral Regional DNA Ploidy Heterogeneity in Primary
Gastric Cancers
Tomoko Furuya, Tetsuji Uchiyama, Takuo Murakami, et al.
Clin Cancer Res 2000;6:2815-2820.
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