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Vol. 4, 2129-2134, Septenther 1998 Clinical Cancer Research 2129
Tumor Angiogenesis and Micrometastasis in Bone Marrow of
Patients with Early Gastric Cancer’
Yoshihiko Maehara,2 Shota Hasuda, Toru Abe,
Eiji Oki, Yosbihiro Kakeji, Shinji Ohno, and
Keizo Sugimachi
Department of Surgery II, Faculty of Medicine, Kyushu University,
Fukuoka 812-8582, Japan
ABSTRACT
In a subset of patients with early gastric cancer, therewere recurrences of the disease after a curative resection
had been done. Direct evidence of tumor seeding in distantorgans at the time of surgery for gastric cancer is notavailable. An immunocytochemical assay for epithelial cy-tokeratin protein may fill this gap because it is a feature ofepithelial cells that would not normally be present in bonemarrow. From 1994-1997, the bone marrow of 45 patientswith early gastric cancer was examined for tumor cells,using immunocytochemical techniques and an antibody re-
acting with cytokeratin, a component of the intracytoplas-talc network of intermediate filaments. Intratumoral mi-
crovessels were stained with anti-CD31 monoclonalantibody. Clinicopathological characteristics were deter-mined for subjects with cytokeratin-positive cells in the bonemarrow. Of these 45 patients, 9 (20.0%) had cytokeratin-positive cells in the bone marrow at the time of primarysurgery. These positive findings were not related to tumoradvance-related factors of lymph node metastasis and dis-tinct lymphatic and vascular invasion. Microvessel densityin the primary tumor exceeded 2-fold in cytokeratin-positivecells, compared with findings in negative cells (P < 0.05).
Tumor cells in bone marrow are indicative of the general
disseminative metastasis in patients with early gastric can-
cer, and the metastatic potential was closely related to an-giogenesis in the primary tumor.
INTRODUCTION
Early gastric cancer is defined as a lesion in which the
depth of invasion is limited to the mucosa or mucosa and
submucosa, regardless of whether a regional lymph node me-
tastasis is evident on histological examination, and the postop-
erative prognosis is usually favorable (1, 2). The diagnosis of
Received 12/16/97; revised 3/27/98; accepted 6/5/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.I This work was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science, Sports and Culture of Japan.2 To whom requests for reprints should be addressed. Fax: 81 92 6425482.
early gastric cancer is much more frequent in Japan because
sophisticated diagnostic techniques are now available (3).
However, even after “curative” resection of early gastric
cancer, there are recurrences in a subset of patients (4-8).
Hematogeneous metastasis is the predominant route in such
cases. It is difficult to control tumor growth in cases of a distant
metastasis in these patients, and knowledge of the stage of
subclinical tumor cell dissemination is needed to design adju-
vast treatment. Various parameters, including DNA pboidy and
oncogenes, aid in predicting the recurrence and the prognosis of
early gastric cancer (9-12). A feature common to all of these
prognostic factors is that, from the excised tumor tissues, one
attempts to extrapolate to the malignant potential of occult cells
that may possibly be present. Diagnostic techniques presently
available are not sufficiently sensitive to detect unicellular or
oligoceblular micrometastasis.
Because cytokeratin proteins are essential constituents of
the cytoskebeton of both normal and malignant epithelial cells,
they can serve as reliable markers for the epithelial origin of
cells (13). The use of a monoclonal antibody against the
cytokeratin component expressed by all tumor cells derived
from simple epithebia facilitates identification of one of l0�
epitheliab tumor cells in bone marrow. Evidence of microme-
tastasis in the bone marrow means an early relapse and a poorer
clinical outcome for patients with gastric, coborectab, mammary,
and lung cancers (14-19). Schbimok et a!. (20) found no posi-
tive cells in a large series of bone marrow aspirates from 102
patients with no evidence of malignant epitheliab disease. We
reported the presence of micrometastasis in bone marrow for
32.6% patients with gastric cancer (21). However, the presence
of micrometastasis was not focused on patients with early gas-
tric cancer.
Angiogenesis is closely involved in tumor progression and
metastasis (22, 23). Weidner et a!. (24) reported that microves-
sd density evaluated by immunostaining for endotheliab cells
was an independent prognostic indicator in breast cancer. The
level of microvessel density particularly correlated with hemato-
geneous metastasis of gastric and coborectal cancers (25, 26).
CD-3 1 is a pbatebet/endothelial cell adhesion molecule and is a
more sensitive marker for endotheliab cells than is factor VIII
antigen (27, 28). We examined micrometastasis in bone marrow
from patients with early gastric cancer, using an anti-cytokeratin
monoclonal antibody, and we determined the microvessel den-
sity in the primary tumor, using an anti-CD31 monocbonal
antibody. Bone marrow aspirates were taken immediately prior
to the initial surgery done from 1994 to 1997.
PATIENTS AND METHODS
Patients. This study included 45 unselected Japanese
patients with primary early gastric cancer, all of whom under-
went curative gastric resection in the Department of Surgery II,
Kyushu University, from 1994 to 1997. The depth of invasion
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2130 Gastric Cancer and Bone Marrow Micrometastasis
Table I Clinicopathological characteristics of patients with earlygastric cancer with and without cytokeratin-positive cells in the
bone marrow
Cytokeratin- Cytokeratin-negative cases positive cases
(n36) (n9) P
GenderMale 25 6 NS�Female I I 3
Age (yr) 60.9 ± 1 1 8b 56.4 ± 6#{149}8b NSTumor maximal diameter (cm) 2.81 ± 2.14” 2.52 ± 2.26” NS
Location of tumorUpper 9 2 NSMiddle 17 3Lower 10 4
HistologyDifferentiated 22 4 NSUndifferentiated 14 5
Depth of invasionMucosa 21 6 NSSubmucosa 15 3
Lymph node metastasisNegative 30 6 NSPositive 6 3
Lymphatic invasionNegative 3 1 8 NSPositive 5 1
Vascular invasionNegative 3 1 8 NS
Positive 5 1
Gastric resectionPartial 25 7 NSTotal 11 2
Lymph node dissection’Dl 2 1 NSD2andD3 34 8
a NS, not significant.
,) Mean ± SD.C Dl , complete removal of group 1 lymph node alone; D2, corn-
plete removal of groups 1 and 2 lymph nodes; and D3, completeremoval of groups 1, 2, and 3 lymph nodes.
was mucosa in 27 patients and mucosa and submucosa in 18
subjects. Standardized procedure was that a gastric resection
was done, after determining the resection line 3 cm apart from
the macroscopic edge of the localized tumor, and 6 cm for the
infiltrative tumor (29, 30). Prophylactic lymph node dissection
of more than D2 resection was carried out (3 1). Complete
excision of invaded organs was done, irrespective of the number
of sites on the organs, when there was no evidence of peritoneab
dissemination, liver metastasis, and widespread nodal involve-
ment (32). All patients were examined clinically and patholog-
ically with respect to the factors given in Table 1. Pathological
diagnosis and classification of the resected gastric cancer tissues
were made according to the General Rules for the Gastric
Cancer Study in Surgery and Pathology in Japan (33, 34).
Informed consent to participate in this study was obtained from
all patients prior to their surgery.
Bone Marrow Specimens. Details are given in our study
published previously (21). Preoperativeby, 1-2 ml of bone mar-
row aspirates from the sternum were taken in syringes contain-
ing 100 units of heparinlmb marrow, and bone marrow cells
were prepared. After density centrifugation through Ficoll-
Hypaque (400 X g for 30 mm), mononuclear cells were cob-
bected from the interphase. The cells were suspended with 0.5
ml RPMI 1640 containing 10% FCS, yielding a concentration of2 X l06/ml; the cells were then smeared on glass slides and
fixed with acetone (30 mm at 4#{176}C).For immunostaining, the
monocbonal antibody CK2 (IgGl; Boehringer Marntheim,
Mannheim, Germany) or CAM 5.2 (IgG2�, Becton Dickinson,
CA) was used at a concentration of 0.2 p�g/ml (17). This anti-
body recognizes intracellular cytokeratin component no. 1 8, an
intermediate filament representing the intracellular network of
the cytoskeleton that is expressed in simple epithelia and no-
where else. The antibody reaction was developed using the
labeled avidin-biotin technique (35), and biotin-labeled anti-
body and alkaline-phosphatase-labeled avidin were used se-
quentially. Naphthol-AS-BI-phosphate was used as a substrate
of alkaline phosphatase, and the released naphthol-AS-BI was
coupled with hexazotized new fuchsin. Endogenous phospha-
tase was inhibited by preincubation with levamisobe. Cells con-
taming cytokeratin were stained bright red. Two observers
(Y. M. and Y. K.) examined the positivity of micrometastasis of
bone marrow independently.
Immunohistochemistry of CD31. Tumors were cob-
lected and fixed in 10% formalin. Sections of 5-p.m thickness
from paraffin-embedded blocks were deparaffinized in xybene
and rehydrated in a graded series ofethanol. After quenching the
endogenous peroxidase activity in methanol containing 0.3%
(v/v) hydrogen peroxidase for 30 mm, sections were pretreated
with 12.5 mg of protease type XXIV (Sigma Chemical Co., St.
Louis, MO)/100 ml PBS (pH 7.4) for 15 mm at 37#{176}C.Nonspe-
cific binding was blocked by treatment with 10% (v/v) normal
goat serum for in PBS for 15 mm. Primary mouse anti-CD31
antibody (1:50 at 4#{176}C;DAKO Corp., Carpinteria, CA) was
applied to the sections, and the preparations were incubated
overnight in a moist chamber (36, 37). After washing in PBS,
biotinylated goat anti-mouse immunogbobulin G (Vector Labo-
ratories, Burlingame, CA) diluted 1:200 was applied, followed
by incubation for 30 mm at room temperature. After a thorough
washing in PBS, peroxidase-conjugated streptoavidin (DAKO
LSAB kit; DAKO) was applied, and the preparations were
incubated for 30 mm. Peroxidase labeling was developed with
diaminobenzidine and hydrogen peroxidase, and nuclear coun-
terstaining was performed with Mayer’s hernatoxylin solution
(Fig. 1). Specificity of binding for all antibodies was examined
by applying nonimmune sera instead of specific antibodies.
Microvessel Counting. Evaluation of microvesseb den-
sity (as a continuous variable) was determined, using the mod-
ified technique of Weidner et a!. (24). The entire tumor section
was systematically scanned at X 100 to search for areas of the
most intense neovascularization; these were identified as having
the highest density of red-brown staining, CD31-positive cells
or cell clusters. These neovascular “hotspots” were included into
the counts only if they were adjacent to tumor tissue. Whenever
a highly vascubarized area was evident at X 100, individual
microvessebs were counted on a single X 200 field (1 mm2) in
this area. Any red-brown-staining endothelial cell or endothelial
cell clusters, clearly separated from adjacent microvessels, were
regarded as a single, countable microvessel. Neither vessel
lumens nor RBCs were used to define a microvesseb. Immu-
nopositive macrophages and plasma cells were excluded on
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Clinical Cancer Research 2131
Fig. 1 Immunohistochemicalstaining of endothelial cellswith antibody against CD-31.X200.
morphological grounds. Results were expressed as the mean of
values in five fields.
Postoperative Chemotherapy. All of the patients with
cytokeratin-positive cells were treated with postoperative adju-
vant chemotherapy. An iv. injection of 10 mg of mitomycin C
(Kyowa Hakko Co., Japan) was given on the day of operation,
and fluorinated pyrimidine UFT (Taiho Pharmaceutical Co.,
Japan; Ref. 38) p.o. at a daily dose of 400 mg was started 2
weeks after the operation and was continued for 1 year.
Statistical Analysis. The BMDP Statistical Package pro-
gram (BMDP, Los Angeles, CA) for the IBM 3090 mainframe
computer was used for all analyses (39). The BMDP P4F and
P3S programs were used for the x2 test and the Mann-Whitney
test to compare data on patients with and without cytokeratin-
positive cells in their bone marrow. The bevel of significance
was P < 0.05.
RESULTS
Cytokeratin-positive cells were present in 9 of 45 (20.0%)
patients with early gastric cancer, the depth of penetration being
the mucosa or mucosa and submucosa. Alkaline phosphatase-
stained cells in cytocentrifuge preparations varied from a single
cell to a cluster of 10 cells, as determined histologically.
Cytokeratin-positive cells were confirmed to be cancer cells,
determined using Papanicolaou staining. Seeding of cancer cells
had already occurred in these cases, even though extensive
lymph node dissection had been done and a surgically “cura-
tive” operation was carried out.
In a patient with mucosal gastric cancer, the tumor meas-
ured only 0.9 X 0.6 cm, was a macroscopically depressed type
(lIc), localized in the angle of the stomach, and was limited to
the mucosa (Fig. 2, A and B). In this patient, a small cluster of
cytokeratin-positive cells was present in bone marrow aspirates
(Fig. 2C).
Micrometasta.sis in the Bone Marrow and Clinicopath-
obogical Factors. Positive findings of micrometastasis in the
bone marrow of patients with early gastric cancer did not
depend on gender, age, tumor size, tissue differentiation or
location ofthe tumor (Table 1). The depth of penetration was the
mucosa and mucosa and submucosa, and the tumor size was
smaller. The rates of lymph node metastasis and distinct lym-
phatic and vascular involvements were low, and these events
were not rebated to the presence of micrometastasis. There were
evidently no cytokeratin-positive micrometastatic cells in the
lymph nodes.
Micrometastasis in the Bone Marrow and MicrovesselDensity. Microvesseb counts determined by anti-CD3 1 anti-
body in primary gastric cancer tissues were 13.5 ± 6.0 for the
cytokeratin-negative patients and 27.9 ± 8.3 for the cytokeratin-
positive ones, with a statistical significance of P < 0.05
(Table 2). Thus, the presence of micrometastasis in the bone
marrow showed a close relation to tumor angiogenesis.
DISCUSSION
Even after curative resection, there can be recurrences of
the cancer in patients with early gastric malignancy (4-8). The
most frequent mode of recurrence is by a hematogeneous
spread, and tumor cells have already disseminated to distant
organs at the time of surgery. Availability of a highly sensitive
method would aid in predicting metastatic potential and clinical
outcome, and more effective treatments could be designed.
Clinicopathological factors of age of the patient, tissue differ-
entiation, growth pattern of pen A type, and nodal involvement
were reported to be prognostic for the occurrence of recurrences
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2132 Gastric Cancer and Bone Marrow Micrometastasis
Fig. 2 Resected tissue specimen and microphotographs from a patientwith mucosal gastric cancer. Macroscopic size of the tumor was 0.6 X0.9 cm and the depressed type (A). Cancer cells were limited in the depthof the gastric mucosa (B; X 18). High-power photomicrograph of bonemarrow specimen reveals a small cluster of malignant cells (C; X 1000).
in early gastric cancer ( 1 , 7, 40). Gastric cancer markers may
provide prognostic information independent of and complemen-
tary to conventional parameters, including growth potential,
oncogenes, tumor-suppressor genes, and DNA flow cytometry,
as well as other growth factors (9 -1 2, 4 1 ). The common char-
acteristic of these prognostic factors is that they correlate a
property of the primary tumor with the subsequent outcome.
The method we have described here relates to aspects of
the actual behavior of the tumor, microscopic dissemination of
cancer cells in the bone marrow. There are reports of microme-
tastasis in the bone marrow of patients with gastric, coborectal,
Table 2 Microvessel density of early gastric cancer with andcytokeratin-positive cells in the bone marrow
without
Cytokeratin- Cytokeratin-negative cases positive cases
Factor (n = 36) (n 9) P
Microvessel density 13.5 ± 6ff’ 27.9 ± 8.3” 0.05
a Mean ± SD.
breast, prostate, or lung cancers (13-19, 42). Evidence of mi-
crometastasis means an early relapse, and the clinical outcome
for these patients can be predicted. We also found cytokeratin-
positive cells in bone marrow of our patients with gastric cancer,
and the micrometastasis in the bone marrow did not correlate
with p53 overexpression and proliferating activity of the tumor
(21).
Micrometastasis was noted in 20.0% of our patients with
early gastric cancer; thus, seeding of cancer cells can occur even
in the early stages of the cancer. In these cases, small tumors are
just an early manifestation of systemic disease, and metastases
has already occurred. However, the cancer cells were single to
at most 10, and a few cells were present in bone marrow;
however, vascular involvement in primary tumor or lymph node
metastasis is difficult to identify.
Tanigawa et al. (25, 26) reported that tumors that devel-
oped hematogeneous metastasis after surgery had significantly
higher microvessel density than did tumors with rebated perito-
neal metastasis and nonmetastatic tumors. Vascubarization is
usually required for tumor cells to enter the blood circulation
(23). Newly formed tumor vessels are devoid of or backing in
smooth muscle, are tortuous and sinusoidal, have increased
vascular length and diameter, have incomplete endothebial cell
lining and basement membrane, and are prone to spontaneous
hemorrhage and/or thrombosis, thus enabling tumor cells to
enter circulating systems (24, 43). In such cases, cancer cells
released from the primary site would be transported to the bone
marrow. Because the presence of micrometastatic cells in the
bone marrow was closely related to angiogenesis in the primary
tumor, detection of micrometastasis in the bone marrow does
have clinical significance when attempting to evaluate the he-
matogeneous metastatic potential of gastric cancer.
A higher sensitivity test to detect micrometastasis in the
bone marrow can be achieved by making use of the reverse
transcription-PCR (44-46). However, this method needs further
standardization, PCR primers specific for each RNA tool have
to be designed, the optimal number of PCR cycles has to be
defined, as do best cutoff levels, and so on (18, 47).
Overt bone or skeleton metastases are rare in patients with
gastric cancer; however, bone marrow is more often involved
than expected based on clinical findings (17). The apparent
discrepancy between clinically rare bone metastases and the
marrow micrometastases frequently detected by immunocyto-
chemistry can be explained by a reduced proliferative behavior
of the cells and often invoked state of dormancy (20, 42). The
capacity of tumor cells to proliferate in the bone marrow and to
manifest metastasis depends on the microenvironment. Jauch et
a!. ( 1 8) reported that positive bone marrow aspirations are a
surrogate marker of general tumor-cell dissemination or mini-
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Clinical Cancer Research 2133
mal residual disease, rather than the start of metastatic growth in
the skeletal system. The survival time was shorter in the cyto-
keratin-positive group than in negative group in cases of gastric
cancer (16-18). Therefore, these patients may be at a higher risk
for complications arising from peritoneal dissemination or liver
metastasis with no manifest clinical metastasis in the bone
marrow (2, 48, 49).
Because cytokeratin-positive cells were present in the bone
marrow of our patients with early gastric cancer, these cells can
serve as valid indicators of the metastatic activity of these
cancers. Patients presenting with disseminated cytokeratin-
positive cells at the time of primary surgery can be followed to
detect any distant metastasis. Therefore, such patients may be
good candidates for postoperative adjuvant trials, even when a
curative resection is done for patients with early gastric cancer.
ACKNOWLEDGMENTS
We thank M. Ohara for comments and K. Miyammoto and J.
Tsuchihashi for technical assistance.
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1998;4:2129-2134. Clin Cancer Res Y Maehara, S Hasuda, T Abe, et al. patients with early gastric cancer.Tumor angiogenesis and micrometastasis in bone marrow of
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