Yonago Acta medica 1998;41:105–112

Cytokeratin-Positive Cells in Lymph Nodes in Which Metastases Are
Undetectable by Conventional Histological Staining in Advanced Gastric
CancerMichio Maeta, Jianhui Cai, Shinichi Oka, Hiroaki Saito, Akira Kondo, Shunichi
Tsujitani, Masahide Ikeguchi and Nobuaki Kaibara
First Department of Surgery, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
Detection of occult metastases in lymph nodes by immunostaining is becoming of increasing interest as a way to improve the accuracy of predicting the prognosis for patientswith gastric cancer. Immunohistochemical detection of cytokeratin (CK) is recognized
as the most sensitive method for identification of cancerous epithelial cells. In thisstudy, lymph nodes were stained for CK in an effort to detect micrometastases and theclinical implications of the results were examined. We immunostained sections from a
total of 1,198 lymph nodes from 25 totally gastrectomized patients with T3 or T4 gastriccancer who had been diagnosed as having no nodal involvement by conventional
hematoxylin-eosin (HE) staining. Eighty (6.7%) of 1,198 lymph nodes from 15 (60%)
of the 25 patients were immunostained with a CK-specific monoclonal antibody. CKpositive cells were more frequent in patients with macroscopic types of 3,4 and 5 gastriccancer. Patients with nodes that were both HE-negative and CK-negative had the best
postoperative survival, followed by patients with HE-negative and CK-positive nodes
and, finally, by patients with HE-positive nodes. Our results indicate that the presenceof micrometastases in lymph nodes is a reliable indicator of the prognosis of patientswith advanced gastric cancer.
Key words: cytokeratin; gastric cancer; lymph nodes; micrometastases; prognosis

In patients with gastric cancer, metastasis to thelymph nodes is considered to be one of the mostimportant predictive factors in the prognosis(Kaibara et al., 1988; Maruyama et al., 1989).
Histological nodal metastasis is examined routinely by conventional hematoxylin-eosin (HE)staining. However, discrete cancer cells or
small clusters of cancer cells within the nodes,
namely, micrometastases, are easily overlooked
after HE staining (Ishida et al., 1997; Sasaki etal., 1997). Immunostaining for cytokeratin(CK), using the monoclonal antibody CAM 5.2,
is a very sensitive method for the detection ofsuch micrometastases when cancers have developed from epithelial tissues (Makin andBodmer, 1984; Sedmak et al., 1989; Maehara etal., 1996a, 1996b; Ishida et al., 1997; Sasaki etal., 1997).

Lymph nodes around the abdominal aorta
are the furthest from the primary gastric lesionin the abdominal cavity. These paraaortic
nodes are classified as most-distant group 4(N4) in the Japanese system for classification ofgastric cancer (Japanese Research Society for
Gastric Cancer, 1993). We previously conducted a prospective study to compare morbidity,
mortality and survival, from May 1990 to April
1997, between conventional extended D3 lymphadenectomy and super-extended D3 plus
paraaortic lymphadenectomy (PAL; Yonemura
et al., 1991). In the current study, we examined
all lymph nodes by immunostaining for CK inpatients who had been diagnosed as having nonodal metastasis by HE staining in our earlier
prospective study. The purpose of this study
was to evaluate the clinical implications of CK-

Abbreviations: CK, cytokeratin; HE, hematoxylin-eosin; PAL, paraaortic lymphadenectomy105

M. Maeta et al.

positive cells in apparently metastasis-negative
nodes.

Patients and Methods

Patients
Between May 1990 and April 1997, 70 patientswere entered into a prospective study after preoperative informed consent had been obtained.
The eligibility requirements for inclusion in the
study were as follows. Patients had to be under
75 years of age without other malignancies inorgans other than the stomach.
After laparotomy, we also excluded patientswith peritoneal seeding or liver metastasis, aswell as those who had macroscopic metastasesin the paraaortic lymph nodes. Patients who
had T3 (serosal invasion) or T4 (invasion of
adjacent structures) gastric cancer and who had
undergone potentially curative total gastrectomy were entered into the study after extended
dissection of group 1 (N1), group 2 (N2) andgroup 3 (N3) lymph nodes, namely, the D3 procedure. The extent of lymphadenectomy was
expressed as follows: D1, complete removal of
N1 nodes; D2, complete removal of N1 and N2
nodes; D3, complete removal of N1, N2 and N3
nodes; and D3 plus PAL, complete removal of
N1, N2 and N3 nodes, as well as paraaortic lymphadenectomy. The 70 patients were divided,by alternate designation at the time of operation, into the D3 (n = 35) and D3 plus PAL (n =35) groups.

Methods
All dissected lymph nodes were prepared forhistologic analysis by one central crosssectional passage through the hilus for microscopic examination of metastasis. Among 70
patients, 25 were diagnosed, after HE staining,as having no metastasis in all dissected nodes(12 of the 35 patients in the D3 group and 13 of
the 35 patients in the D3 plus PAL group). In
the present study, we examined a total of 1,198
nodes, consisting of 1,024 nodes (mean 41
nodes per patient) in groups N1, N2 and N3 and174 nodes (mean 13 nodes per patient) in group
N4 (paraaortic nodes) from these 25 nodenegative patients.
Paraffin-embedded blocks of the 1,198
lymph nodes and the 25 primary gastric tumors
were prepared from the above mentioned 25 patients. Serial sections of 4 µm in thickness wereprepared from the blocks and subjected to HE
staining and CK-specific immunostaining toallow comparison of results obtained by the 2
methods in adjacent sections of each lymph
node and in the primary gastric tumor.
Immunohistochemical staining was performed by the streptavidin-biotin method with amurine monoclonal antibody, CAM 5.2 (Becton
Dickinson, San Jose, CA) against low-molecularweight CK. In brief, dewaxed and dehydrated
sections were heated in a microwave oven (700W) for 10 min for retrieval of antigens in the specimens. Endogenous peroxidase was blocked byincubation of samples with 3% hydrogen peroxide in 100% methanol. The tissue sections werethen incubated with the primary antibody CAM
5.2 at 25 µg per mL overnight at 4˚C. The
second antibodies, biotinylated antibodies
against mouse immunoglobulin, were applied,and sections were then incubated with peroxidase-labeled streptavidin. Reaction products
were visualized with diaminobenzidine as thechromogen, and sections were counterstainedwith methyl green for visualization of lymphocytes. Tris-buffered saline was used instead of
the primary antibody for negative controls.
The lymph nodes were divided into 5 CKgroups according to the criteria defined by
Ishida and colleagues (1997): CK-group 0 (CG0), no cancer cells in the section of the node;
CK-group 1 (CG 1), only 1 discrete cancer cellin the section of the node (Figs. 1a and b); CKgroup 2 (CG 2), 2 or more discrete but not
aggregated cancer cells in the section of thenode; CK-group 3 (CG 3), in addition to discrete cancer cells, 1 or more aggregates of 2 ormore cancer cells in the section; and CK-group
4 (CG 4), metastasized cells detectable bystandard HE staining (Figs. 1c and 1d). The
determination of the CK-group for each patient
was based on the highest group recorded for anyof that patient’s lymph nodes.

106Cytokeratin-positive nodes in gastric cancer

a

b

c

d

Fig. 1. Examples of stained lymph nodes after staining with hematoxylin-eosin (HE) and after immunostaining specific for cytokeratin (CK). a and b: a discrete cancer cell is hard to identify after HE staining (a)but is readily identifiable after CK-specific immunostaining (b) (× 95). c and d: aggregated cancer cells arerecognized after both HE staining (c) and immunostaining with CK (d) (× 48).

107

M. Maeta et al.

Table 1. Incidence of CK-positive lymph nodes in 25 patients with nodes in which metastases
were undetectable after HE staining
Numberofpatients

CK-negative

CK-positive

CG 0

CG 1

CG 2

CG 3

CG4

25
(100)

10
(40)

2
(8)

4
(16)

3
(12)

6
(24)

15 (60)( ), percentage.
CG 0, no cancer cells in the sections of lymph nodes; CG 1, only 1 discrete cancer cell; CG 2, 2 or more discrete
but not aggregated cancer cells; CG 3, in addition to discrete cancer cells, 1 or more aggregates of 2 or more
cancer cells; CG 4, metastasized cells detectable by standard HE staining; CK, cytokeratin; HE, hematoxylineosin.

Statistical analysis was performed by thechi-squared test. The Kaplan-Meier method
was used to calculate cumulative survival
curves and the generalized Wilcoxon test wasused for examination of the statistical significance of differences. A P value of less than
0.05 was considered to be statistically significant.

Results
A total of 1,198 lymph nodes from 25 patientswith advanced gastric cancer were examined inthis study. All 25 primary tumors were consistently immunostained with the monoclonal
antibody against CK. No CK-positive nodes
were found in 10 of the 25 patients, while 15
patients (60%) had CK-positive nodes. Afterthe results of immunostaining had been recorded, all HE-stained samples were reexamined,

and 6 cases (seven lymph nodes) in which metastasized cancer cells had been recognized on
HE-stained slides were classified as CG 4. It
was difficult to identify metastatic cells onother HE-stained slides from 19 patients (a totalof 73 nodes), even when CK-positive cells had
been detected by immunostaining. We divided
our cases into 5 CK-groups, CG 0, CG 1, CG 2,
CG 3 and CG 4, and there were 10, 2, 4, 3 and 6
patients in each group, respectively (Table 1).
Among 1,198 lymph nodes, 80 lymph nodes(6.7%) from a total of 15 patients were immunopositive for CK, as shown in Table 2.
The cytoplasm of immunopositive cells was
diffusely stained and products of the immunoreaction were visualized as brown circles thatwere due to staining by diaminobenzidine. The
nuclei of such cells were barely stained.
Relationships between the presence of CKpositive nodes and the clinicopathological features of primary tumors are shown in Table 3.

Table 2. Number and classification of CK-positive lymph nodes among 1,198 nodes from 25
patients
Number oflymph nodes

CG 0

CG 1

CG 2

CG 3

CG4

1,198
(100)

1118
(93.3)

40
(3.3)

28
(2.3)

5
(0.4)

7
(0.6)

80 (6.7)( ), percentage.
CG 0–4, see footnote to Table 1; CK, cytokeratin.

108

Cytokeratin-positive nodes in gastric cancer

Table 3. Relationships between immunoreactivity in lymph nodes and clinicopathologicalfeatures
Variable

CK-negative[10]

CK-positive[15]

P valueAge (years; mean + SD)
58.9 + 9.8
GenderMale
5
Female5Tumor size
<7 cm
5
>7 cm
5
Histology
Differentiated4
Undifferentiated6
Depth of cancer invasion
Muscularis propria
0
Subserosa
1
Serosa9
Macroscopic type
1, 2
5
3, 4
5
5
0
Lymphatic vessel invasion
Negative
7
Positive
3
Blood vessel invasion
Negative
2
Positive8[ ], number of patients.
CK, cytokeratin; NS, not significant.

53.8 + 10.1

NS

10
5

NS

8
7

NS

3
12

NS

4
0
11

NS

1
10
4

P < 0.05

12
3

NS

8
7

NS

We found no significant relationships between
the presence of CK-positive nodes and the sizeof the tumor, the histological type, the depth oftumor invasion, and lymphatic or blood-vessel
invasion by the primary lesion. However, theincidence of CK-positive nodes was significantly higher in patients with macroscopic
types of 3, 4 and 5 gastric cancer (Table 3).
Table 4 shows the numbers and CG-groups
of CK-positive nodes in terms of the sites of immunopositive lymph nodes. CK-positive nodes
were found most frequently among lymph
nodes along the greater curvature (18 nodes),followed by lymph nodes along the lesser
curvature (17 nodes), infrapyloric nodes (12nodes), and right cardial nodes (10 nodes).
Lymph nodes in group N1 included the largest
number of CK-positive nodes followed bygroup N2. No CK-positive nodes were found inlymph nodes in groups N3 and N4.

The relationship between CK-positive
nodes and postoperative survival of patients isshown in Fig. 2. To indicate the survival ofcontrol patients, we have included in Fig. 2 thesurvival of 45 of the 70 patients in our prospective study. These 45 patients had been diagnosed as having HE-detectable nodal involvement(therefore, lymph nodes from these 45 patientswere not immunostained in this series). The 5year survival rates for 9 patients (excluding 1
patient who died of complications) with bothHE-negative and CK-negative nodes [HE(–),
CK(–)], for 15 patients with HE-negative but
CK-positive nodes [HE(–), CK(+)], and for 44
patients with HE-positive nodes [HE(+)] were
85.7%, 52.4% and 30.8%, respectively. Although there was no significant difference in survival between patients with HE(–) plus CK(–)
nodes and patients with HE(–) plus CK(+)
nodes, there was a statistically significant dif-109

M. Maeta et al.

Table 4. Number and classification of CK-positive nodes analyzed in terms of sites of lymph
nodes
Site of lymph nodes

CG 1

CG 2

CG 3

CG 4

Total

Group N1 (peri-gastric nodes)
Right cardial
6
2
1
1
10
Left cardial
0
2
0
0
2
Along the lesser curvature
8
4
2
3
17
Along the greater curvature
9
7
1
1
18
Suprapyloric
2
1
0
0
3
Infrapyloric
3
7
1
1
12
Group N2
Along the left gastric artery
4
1
0
0
5
Along the common hepatic artery
4
0
0
0
4
(anterosuperior group)
Around the celiac artery
1
0
0
0
1
At the splenic hilum
2
2
0
1
5
Around the splenic artery
1
2
0
0
3
Group N3*00000Group N4 (paraaortic lymph nodes)
0
0
0
0
0
Total
40
28
5
7
80
* Lymph nodes along the common hepatic artery(posterior group), in the hepato-duodenal ligament, on theposterior surface of the pancreas head, and along the superior mesenteric vein.
CG 0–4, see footnote to Table 1; CK, cytokeratin.

ference in survival between patients with HE(–)
and CK(–) nodes and those with HE(+) nodes(P < 0.05).

Discussion
In the current study, we immunostained sections of 1,198 lymph nodes from 25 patients

who had been diagnosed previously as having
no nodal metastasis by HE staining. Althoughthese 25 patients were selected from our prospective study, our purpose in the present study
was to evaluate clinical implications of CKpositive nodes.
Micrometastases in malignant tumors havebeen a focus of increasing interest in recent
years because of their significance in predicting

100

Percent survival

HE (–), CK (–)[n = 9] 85.7%

HE (–), CK (+) [n = 15] 52.4%

P < 0.05

50

HE (+) [n = 44] 30.8%

1

2

3

4

5

Years after gastrectomy
Fig. 2. Postoperative survival of totally gastrectomized patients with T3 or T4 gastric cancer. HE(–), CK(–):
patients with both HE-negative and CK-negative nodes. HE(–), CK(+): patients with HE-negative but CKpositive nodes. HE (+): patients with HE-positive nodes.

110Cytokeratin-positive nodes in gastric cancer

the prognosis of patients. Many human tumors,such as breast (Sedmak et al., 1989), colorectal(Sasaki et al., 1997) and gastric (Maehara et al.,1996a; Ishida et al., 1997) cancers, are associated with such metastases in the lymph nodes orbone marrow (Maehara et al., 1996b), which
probably affect survival.. Various studies have
explored the possible application of epithelial
cell markers in the detection of micrometastases(Trojani et al., 1987; Davison et al., 1990;
Siewert et al., 1996; Broll et al., 1997). In addition, the predictive value of molecular or genetic methods for detection of occult metastases,
which exploit polymerase chain reaction ormutant allele-specific amplification, has beensuggested (Hayashi et al., 1994; Nakamori etal., 1997; Nakanishi et al., 1997; Dorudi et al.,1998). However, it is hard to avoid falsepositive results in many cases, even thoughthese methods reveal occult metastases with socalled supersensitivity. Many investigators
have reported the results of immunostaining for
CK in bone marrow or lymph nodes and havestressed the usefulness of this marker in the detection of micrometastases from gastric cancer(Maehara et al., 1996a, 1996b; Ishida et al.,1997). In the current study, we used monoclonal antibody CAM 5.2, which binds specifically to CK peptide 8.
In our series, all sections of primary tumors
were CK-positive. Moreover, 80 lymph nodes(6.7%) among 1,198 nodes dissected from 15(60%) of 25 patients, who had been diagnosed
as having no metastases by conventional HE
staining, were also CK-positive. However, theCK-positive nodes were confined to groups N1
or N2 and no CK-positive nodes were detected
in groups N3 and N4. The frequency (60%) of
CK-positive nodes in our series of patients withT3 or T4 gastric cancer was higher than thefrequency (40%) reported by Ishida and colleagues (1997) for patients with gastric cancer
that included T1, T2, T3 and T4 cancers, andthan the frequency (23.5%) reported byMaehara and coworkers (1996a) for patientswith early gastric cancer. The deep invasion of
the serosa by the tumor in all of our patientswith T3 or T4 gastric cancer might explain thehigh frequency of CK-positive nodes in ourstudy.
As shown in this study, discrete and scattered cancer cells or small clusters of such cells areeasily overlooked after standard HE staining.
Establishment of an easy method for detecting
such small numbers of cancer cells is clinicallyimportant. In our study, histopathologicalexamination of lymph nodes was made after
only a single central cross-sectional passage.
The sensitivity of detection of metastatic deposits might be expected to improve significantly
by multiple sections of each node (Isozaki et al.,1997). In fact, second sections cut from thelymph nodes examined in the present study
revealed metastatic deposits not only after
immunostaining but also after conventional HE
staining in 7 of the 1,198 lymph nodes. In thesecases, metastatic cells had been undetectable on
the first section that had been stained with HE.
However, this 2-section procedure would not bepractical in routine histopathology. Our results
suggest that immunostaining with CK-specificantibody, in addition to conventional HE staining, would permit a more accurate diagnosis oflymph node metastases.
Metastasis to the lymph nodes is likely tooccur together with the progression of the main
tumor. The major risk factors for lymph nodemetastasis are generally considered to be thelarge size of the primary tumor, infiltrative
growth, diffuse-type gastric cancer, poor differentiation and deep invasion by the primarytumor (Kaibara et al., 1988; Maruyama et al.,1989). In our series, we found that a gross finding of diffuse-type gastric cancer was associated with a high frequency of CK-positive
nodes.
In our patients, the 5-year survival rate forpatients without nodal involvement [HE(–),
CK(–); 85.7%] was the highest, followed byrates for patients with HE(–) plus CK(+) nodes(52.4%) and for patients with HE(+) nodes(30.8%) (Fig. 2). Although there was no significant difference in survival between patientswith or without CK-positive nodes, perhaps
because of the small number of patients, there
was a significant difference between patients111M. Maeta et al.

with HE(–) plus CK(–) nodes and patients with
HE(+) nodes. Our results indicate that the accuracy of predictions of prognosis for patientswith advanced gastric cancer will be enhanced
when CK-immunostaining is performed in
combination with conventional histopathologic
analysis.

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(Received September 29, Accepted October 13, 1998)

112