Yonago Acta medica 2003;46:125–131

Immunohistochemical Detection of Occult Serosal Microinvasion in Primary Lesions of Gastric Cancer with Subserosal Invasion
Zheng Qiang Wei, Michio Maeta*, Haruhiko Yoshida**, Masahide Ikeguchi, Shunichi
Tsujitani and Nobuaki Kaibara
Division of Surgical Oncology, Department of Surgery, School of Medicine, *Department of Fundamental Nursing and **Department of Pathobiological Science and Technology, School of
Health Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan
In gastric cancer, the presence or absence of serosal invasion by cancer in the primarylesion is an important prognostic factor. Pathological findings are routinely determinedby hematoxylin-eosin (H&E) staining, but it is well known that micrometastasis or microinvasion are easily overlooked by H&E staining. Cytokeratin (CK) proteins serve as reliable
markers for cells from epithelial origins. The purpose of this study was to clarify theusefulness of CK immunohistochemical staining in the detection of serosal microinvasionin gastric cancer with subserosal invasion. We examined 50 primary lesions from 50 gastriccancer patients with subserosal invasion. Two consecutive sections were prepared for
simultaneous staining with ordinary H&E and CK immunostaining with anticytokeratin
antibody (CAM 5.2), respectively. Although there were no differences in the postoperative
survival rates between patients with or without microinvasion, serosal microinvasion was
detected in 8 (16%) of 50 patients by CK staining, including 1 patient whose invasion was
detected by both H&E and CK stainings. CK immunostaining enabled us to make anaccurate and detailed diagnosis which we believe to be useful for detecting serosal microinvasion in the primary lesion in gastric cancer with subserosal invasion.
Key words: cytokeratin; gastric cancer with subserosal invasion; hematoxylin-eosin staining; immunohistochemical staining; serosal microinvasion

The presence of serosal invasion by cancer within
the gastric wall is the most important prognostic
factor for patients with gastric cancer (Koga et al.,
1984; Kaibara et al., 1986; Baba et al., 1989), as wellas the extent of lymph node metastasis (Maruyama
et al., 1989; Siewert et al., 1993; Matsushita et al.,1995). Even when curative gastrectomy is performed on patients with gastric cancer with serosal invasion, many patients die from recurrence of thecancer and the prognosis of such patients remains

poor. The development of peritoneal metastasis accounts for more than half of all recurrences after
curative resection for gastric cancer with serosal
invasion (Kaibara et al., 1989). When cancer cells
penetrate the tunica serosa of the stomach, exfoliation of these cells from the surface of the primarycancerous lesion into the peritoneal cavity is induced. Many investigators have reported that patientswith gastric cancer with serosal invasion have freecancer cells in the peritoneal cavity and these cancer cells are viable by enzymological and autoradi-

Abbreviations: CK, cytokeratin; H&E, hematoxylin-eosin

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ographycal examination (Nakajima et al., 1978;
Iitsuka et al., 1979; Tanida et al., 1982). Additionally,
these cancer cells may be able to implant themselves and proliferate in the peritoneum causing
postoperative peritoneal metastasis. Therefore,
precise diagnosis of the presence or absence ofserosal invasion by the primary gastric lesion isvery important for appropriate postoperative chemotherapy in order to prevent the development ofperitoneal metastasis.
Clinicopathologic diagnosis relies on hematoxylin-eosin (H&E) staining in a respective section oflymph nodes and primary lesions to ascertain thepresence of metastases and the depth of tumor invasion. However, it has been reported that discrete
cancer cells, namely occult micrometastases or
microinvasions are sometimes overlooked by routine examination with H&E staining (Trojani et al.,
1987; Ishida et al., 1997; Sasaki et al., 1997).
Immunohistochemical techniques with antibodies against cytokeratin (CK) can identify lymph
node micrometastasis overlooked by H&E staining
in various solid malignancies (Sedmak et al., 1989;
Greenson et al., 1994; Maehara et al., 1996), because
CK proteins serve as reliable markers for cells ofepithelial origin. In 1999, we reported that in gastric cancer with submucosal invasion, CK immunohistochemical analysis can detect not only micrometastases in lymph nodes but also microinvasionof the muscularis propria in primary lesions (Cai etal., 1999). Therefore, CK staining also seemed to be
useful in the detection of the serosal microinvasionof gastric cancer with subserosal invasion. The purpose of the current study was to evaluate the usefulness of CK staining in the detection of microinvasion (serosal invasion) in gastric cancer with subserosal invasion in which serosal invasion by thecancer was undetectable by routine H&E staining.

rectomy in the First Department of Surgery, Tottori
University Hospital. We excluded 14 patients who
had metachronous or simultaneous primary malignancies in organs other than the stomach and/or
patients whose primary lesions were limited to themuscularis propria but classified as gastric cancer
with subserosal invasion by the presence of lymphatic or venous invasions by the cancer in the telasubserosa apart from the primary lesion. The subjects of this study were 50 patients whose depth ofcancer invasion was limited to the tela subserosa of
the gastric wall. There were 30 men and 20 women,
ranging in age from 31 to 80 years, with a mean ageof 63 years. Clinicopathologic data were evaluatedaccording to the General Rules for Gastric Cancer
Study in Surgery and Pathology (Japanese ResearchSociety for Gastric Cancer, 1981).

CK-specific immunostaining of the primarylesions
One to 4 paraffin-embedded blocks of the primarylesions were prepared in each patient where the subserosal invasion by the cancer had been identified
by the previous H&E-stained sections. Serial sections of 4 µm in thickness from each block were
subjected to conventional H&E staining and CKspecific immunostaining to allow comparison of results by the 2 methods in adjacent sections of eachprimary lesion. For controls, sections from 5 patients with mucosal cancer and 5 patients with serosal invasion in the primary lesion were also stainedwith the 2 methods in the same manner.
Immunohistochemical staining was performed by the streptavidin-biotin immunoperoxidase
method with a murine monoclonal antibody, CAM
5.2 (Becton Dickinson, San Jose, CA) against lowmolecular weight CK. CAM 5.2 especially recognizes intracellular CK component numbers 8 and
18, an intermediate filament representing the intracellular network of the cytoskeleton that is expressed in simple epithelia. In brief, dewaxed and dehydrated sections were heated in a microwave oven(700 W) for 10 min for retrieval of antigens in thespecimens. Endogeneous peroxidase was blocked
by incubation of samples with 3% hydrogen peroxi-

Patients and Methods

Patients
Between 1985 and 1995, 64 patients with gastriccancer with subserosal invasion underwent gast126

Detection of serosal microinvasion

ered to be significant. None of the patients was lost
during follow-up.

dase in 100% methanol. The tissue sections werethen incubated with primary antibody CAM 5.2 at
25 µg/mL overnight at 4˚C. The 2nd set of antibodies, biotinylated against mouse immunoglobulin, were applied with subsequent application ofperoxidase-labeled streptavidin. Reaction products
were visualized with diaminobenzidine as the chromogen and sections were counterstained with methyl green. Tris-buffered saline was used instead of
the primary antibody for negative controls.
The H&E-stained slides were first assessed forthe determination of the depth of invasion in the primary lesion, followed by the assessment of immunostained slides. Microinvasion in the primary lesions
was defined as the presence of isolated or clusteredcancer cells exposed to the tunica serosa of thestomach wall.

Results
The controls used were 5 CK-stained specimens ofmucosal cancer whose cancerous lesions were confirmed to be limited to the mucosal layer without
microinvasion of the submucosal layer. Moreover,the non-cancerous normal epithelium was also CKpositive in all specimens. Five specimens stainedwith CK of serosally exposed cancer confirmed
complete exposure of cancer cells on the tunica serosa of the stomach. Further, some mesothelial
cells and/or smooth muscle were stained with CK
unexpectedly in some specimens.
Table 1 summarizes the results of the current
study in 50 gastric cancer patients with subserosal
invasion in which serosal invasion was undetectable by H&E staining. Eight patients (16%) were
determined to have serosal microinvasion, including 1 patient (Patient 1) whose serosal invasion was
detected by both H&E and CK stainings (Figs. 1aand b). In 7 patients (Patients 2 to 8), serosal microinvasion was detected by CK staining, but H&E
staining failed to detect it, as shown in Figs. 1c and d.

Statistical analysis
Statistical analysis was performed by the chi-square
test to examine the relationships between microinvasion and the clinicopathologic characteristics ofthe primary lesion. For analysis of survival after
gastrectomy, we used the Kaplan-Meier method
and the generalized Wilcoxon test for the statisticalsignificance. A P value less than 0.05 was consid-

Table 1. Clinicopathologic findings of 8 patients with serosal microinvasion
Patient
Depth of invasion
Num- Age (year)/
H&E
CK
ber
genderGrossfinding

Tumor
size
(cm)

Histologic
type

Nodal
metastasis

1
2
3
4
5
6
7
8

Type 2Type 2Type 2Type 2Type 3
Type 3
Type 2Type 5

3.9
8.6
8.0
7.0
2.2
11.0
3.5
4.6

Undiff
Diff
UndiffUndiff
Diff
UndiffUndiffUndiff

+
+
+
–
–
+
+
–

78/F
73/M
70/F
76/M
37/F
80/M
42/M
75/F

se
ss
ss
ss
ss
ss
ss
ss

se
se
se
se
se
se
se
se

Vascular invasion
Lymphatic Venous
+
+
+
+
–
+
+
+

–
+
+
+
–
+
–
+

CK, cytokeratin; Diff, differentiated adenocarcinoma; F, female; H&E, hematoxylin and eosin; M, male; se, tunica
serosa; ss, tela subserosa; Undiff, undifferentiated adenocarcinoma.
Type 2, ulcerated carcinomas with sharply demarcated and raised margins; Type 3, ulcerated carcinomas without
definite limits, infiltrating into the surrounding wall; Type 5, non-classifiable carcinomas that cannot be classified into
any of the other types.

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Z. Q. Wei et al.

a

b

c

d

e

f

Fig. 1. Comparison of the results between H&E and CK stainings.
a and b: Example of stained primary lesion after staining with H&E (a) and CK (b) (2.5 × 10 each). Serosal
microinvasion was detected by both H&E and CK stainings (Patient 1).
c and d: Example of stained primary lesion after staining with H&E (c) and CK (d) (2.5 × 10 each). Serosal
microinvasion was not detected by H&E staining but by CK immunostaining.
e and f: Example of stained primary lesion after staining with H&E (e) and after staining with CK (f) (2.5 × 10each). Serosal microinvasion was not detected by either H&E or CK staining.

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Detection of serosal microinvasion

Table 2. Serosal microinvasion in the primary lesion and clinicopathologic characteristics
Variable

Numberofpatients

Microinvasion of the serosa
Positive (%)
Negative (%)[n = 7][n = 42]Age (year)
Gender (male/female)
Gross finding

49
30/19
8
24
13
1
3
19
30
26
23
19
30
15
34
17
32

64.7 ± 17.5
4/3
0
4 (17)
2 (15)0
1 (33)
2 (11)
5 (17)
3 (12)
4 (17)
3 (16)
4 (13)
1 ( 7)
6 (18)
2 (12)
5 (16)

Histologic type
Depth of invasion*
Nodal metastasis
Lymphatic invasion
Venous invasion

Type 0, 1
Type 2Type 3
Type 4
Type 5DifferentiatedUndifferentiatedssα, βssγ
Negative
Positive
Negative
Positive
Negative
Positive

P value

62.2 ± 10.9
26/16
8
20 (83)
11 (85)1
2 (67)
17 (89)25 (83)23 (88)19 (83)16 (84)26 (87)14 (93)28 (82)15 (88)27 (84)0.64230.54950.55900.81080.31140.7132

Types 0 and 1, superficial, flat tumors with or without minimal elevation or depression and polypoid tumors,
sharply demarcated from the surrounding mucosa, usually attached on a wide base, respectively; Type 4, diffusely
infiltrating carcinomas in which ulceration is usually not a marked feature; Type 2, 3 and 5, see footnote to Table 1.
* When cancerous invasion extends to the tela subserosa (ss), the growth patterns can be divided into 3 categories;ssα, expansive growth pattern with a distinct border; ssβ, intermediate pattern between ssα and ssγ; ssγ, infiltrative
growth pattern with an ill-defined border.

Six of these 8 patients with serosal microinvasion
had undifferentiated adenocarcinoma as a histologic type of gastric cancer. The remaining 42 patients were determined to have no serosal microinvasion by either H&E or CK-staining (Figs. 1eand f).
Clinicopathologic characteristics of the 7 patients with serosal microinvasion in the primary lesions (Patient 1, whose serosal invasion was detected by H&E and CK stainings, was excluded)
were compared with those of 42 patients without it(Table 2). Microinvasion was found to be more frequent in undifferentiated types of cancer and in patients with lymphatic invasion by cancer. However,there were no statistical differences between the 2groups among any variables.
Figure 2 compares the 5-year Kaplan-Meiersurvival curves for 7 patients with and 42 patients

without microinvasion. There was no difference insurvivals between the 2 groups.

Survival rate

1.0

7 patients with se microinvasion

0.8
0.6
0.4

42 patients without se microinvasion

0.2
0
0

20

40

60

80

100

Survival time

120

140
(month)Fig. 2. Postoperative survivals (Kaplan-Meier method).
se, tunica serosa.

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Z. Q. Wei et al.

In the current study, the usefulness of CK
staining was examined in order to find out whether
it can detect serosal microinvasion in gastric cancer
with subserosal invasion, because serosal microinvasion may be overlooked by routine H&E staining.
As a result, microinvasion was detected in 8 patients, including 1 whose microinvasion was detected by both CK and H&E stainings. However, serosal microinvasion was not a significant prognostic
factor, and there were no differences in postoperative survivals between patients with or without
microinvasion. The area of microinvasion detected
by CK staining in this series may be too narrow toboth introduce the isolation of free cancer cells into
the peritoneal cavity and affect postoperative survival.
Based on our findings above, it may be suggested that there is no clinical implication in detecting serosal microinvasion using CK staining. However, the important point is how to make an effort todiagnose the presence or absence of serosal invasion and/or microinvasion accurately and precisely. In our series, the number of patients was too
small to answer the question of whether the detection of serosal microinvasion by CK staining is important or not, and thus a multi-institutional study
using a large number of patients is necessary. Nevertheless, induction of the CK staining as in thisseries in the pathohistological diagnosis of serosal
microinvasion may be the 1st trial ever for gastriccancer. We believe that H&E and CK stainings
should be seriously considered for simultaneous use
in the accurate determination of both nodal metastasis and depth of cancer invasion.
Although CK is a reliable marker of cells from
epithelial origins, Makin et al. (1984) reported that
there were some exceptions to negative staining,such as normal stratified squamous esophageal epithelia and of positive staining of cells from nonepithelial origins, such as smooth muscle and mesothelium. As indicated in this series, when CK immunostaining is used for detecting serosal microinvasion, careful attention should be paid to the unexpected exceptional positive staining of mesothelial one-layered cells.

Discussion
Identification of lymph node metastasis is an important factor in predicting postoperative survivalof patients with gastric cancer, and H&E staining
has been widely used for this purpose. However,immunohistochemical techiques with antibodies
against CK can identify micrometastases in lymph
nodes missed during routine H&E staining fromgastric cancer (Maehara et al., 1996; Ishida et al.,
1997; Maeta et al., 1998; Cai et al., 1999), colo-rectal
cancer (Greenson et al., 1994; Adell et al., 1996;
Sasaki et al., 1997) and breast cancer (Sedmak et al.,
1989; Dowlatshahi et al., 1997). Because CK proteins are essential constituents of the cytoskeleton
of both normal and malignant epithelial cells in
lymph nodes, they serve as reliable markers for theepithelial origin of cells. Patients with these CKpositive cells in lymph nodes have a significantly
decreased postoperative survival time.
Since it is known that in patients with gastriccancer, the depth of cancer invasion within thestomach wall is also an important prognostic factor,
as well as nodal metastases, we reported previouslythat immunohistochemical staining with CK was
useful for the detection of microinvasion of themuscularis propria in gastric cancer with submucosal invasion (Cai et al., 1999). In gastric cancer patients with serosal invasion, serosally exposed cancer cells are easily isolated in the peritoneal cavity,
and these cells proliferate as a peritoneal metastasis.
On cytologic examination of peritoneal lavage fluid
immediately after laparotomy at the time of surgery, free intraperitoneal cancer cells are frequently
found in such patients. Kaibara et al. (1986) reported that there were close relationships both between the rate of detection of intraperitoneal freecancer cells and the area of serosal invasion, and between the area of serosal invasion and prognosis inpatients with gastric cancer with serosal invasion.
Therefore, in patients with gastric cancer, the presence of serosal invasion implies a very high risk forthe development of postoperative peritoneal metastasis.

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Received October 15, 2003; accepted November 17, 2003
Corresponding author: Michio Maeta, MD

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