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Annals of Surgical Oncology 9:771-774 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Prognostic Effect of Lymph Node Micrometastasis in Patients With Histologically Node-Negative Gastric Cancer

Kazuhiro Yasuda, MD, Yosuke Adachi, MD, Norio Shiraishi, MD, Masafumi Inomata, MD, Hiroaki Takeuchi, MD and Seigo Kitano, MD, FACS

From the Department of Surgery I, Oita Medical University, Oita, Japan.

Correspondence: Address correspondence and reprint requests to: Kazuhiro Yasuda, MD, Department of Surgery I, Oita Medical University, 1-1 Idaigaoka, Hasama-machi, Oita 879-5593, Japan; Fax: 81-97-549-6039; E-mail: kyasuda{at}oita-med.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: There is no consensus as to the impact of lymph node micrometastasis on survival of patients with gastric cancer. The aim of this study was to clarify the prognostic significance of lymph node micrometastasis in patients with histologically node-negative gastric cancer.

Methods: Lymph nodes (n = 2039) from 64 patients with histologically node-negative gastric cancer (T2, T3) were evaluated for micrometastasis. Three serial 5-µm sections of the resected lymph nodes were prepared for immunohistochemical staining with the anti-cytokeratin antibody CAM 5.2.

Results: Micrometastasis was found in 73 of 2039 nodes (4%) and 20 of 64 patients (32%). The 5-year survival rate was significantly lower for patients with lymph node micrometastasis than for those without lymph node micrometastasis (66% vs. 95%, P < .01). The 5-year survival rate was significantly lower when there were four or more positive micrometastatic nodes (94% vs. 29%, P < .01) and when there were extragastric micrometastatic nodes (89% vs. 53%, P < .01).

Conclusions: Lymph node micrometastasis was associated with poor outcome in patients with histologically node-negative gastric cancer. The number and the level of lymph node micrometastases are useful prognostic markers for deciding treatment strategies for additional therapy and follow-up.

Key Words: Gastric cancer • Lymph node micrometastasis • Immunohistochemistry • Cytokeratin • Prognosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regional lymph node metastasis is well established as the most important prognostic factor in patients with gastric cancer.1–3 Despite curative resection of their primary tumor, some patients with histologically node-negative gastric cancer will die as a result of local or distant tumor recurrence. Therefore, additional markers would be helpful for predicting patients at risk for recurrence.

During the mid 1990s, progress in methods of immunohistochemical and molecular genetic analysis made it possible to identify micrometastatic foci not detected by ordinary hematoxylin and eosin examination.4–6 Some studies examined the frequency of lymph node micrometastasis in histologically node-negative gastric cancer limited to the mucosa or submucosa (T1).7,8 However, T1 tumors have a good prognosis, and the prognostic significance of lymph node micrometastasis from gastric cancer remains unclear. In colorectal cancer, several studies showed that lymph node micrometastasis was correlated with a poor prognosis for patients with Dukes’ B tumors.9–11

To clarify the prognostic importance of lymph node micrometastasis in patients with gastric cancer, histologically node-negative gastric cancers invading the muscularis propria or deeper (T2 and T3) were selected for this study. We examined the relationship between micrometastasis, clinicopathologic characteristics, and survival and, in particular, assessed the prognostic significance of the number and level of lymph node micrometastases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Between January 1982 and December 1996, 565 consecutive patients with primary adenocarcinoma of the stomach underwent gastrectomy at the Department of Surgery I, Oita Medical University, Oita, Japan. Of these patients, 69, who had tumor invasion of the muscularis propria or deeper (T2 and T3) and no lymph node metastasis detected by routine histopathology were enrolled as the subjects of this study. Patients with tumors limited to the mucosa or submucosa and those with liver metastasis, peritoneal dissemination, or invasion to an adjacent organ were excluded.

The age and sex of patients, location of tumors, and type of operation were reviewed from medical charts and operative records. The histological type, depth of wall invasion, gross type, size, and lymphatic and venous invasions were examined through pathologic reports and photographs of the resected specimens. Histological type was classified into two categories: well differentiated type and poorly differentiated type.12 The level of regional lymph node metastasis was classified as perigastric (level 1) and extragastric (level 2) lymph nodes, according to the anatomical distribution. These clinicopathologic findings were based on the Japanese classification of gastric carcinoma outlined by the Japanese Gastric Cancer Association.13

Routine Histopathology and Immunohistochemical Staining
Primary tumors and lymph nodes were fixed in 10% formalin solution and embedded in paraffin. Routine histopathologic examination was performed on several tissue sections containing the deepest invasive margin of the tumor and stained with hematoxylin and eosin. The presence or absence of lymph node metastasis was examined routinely by hematoxylin and eosin by using a representative cut section including the largest diameter of the lymph nodes. From each node, one 3-µm-thick section was prepared for hematoxylin and eosin staining, and another three serial 5-µm sections were prepared for immunohistochemical staining with CAM 5.2 (Becton Dickinson, San Jose, CA), a mouse monoclonal antibody reactive with human cytokeratin numbers 8 and 18.14 The streptavidin-biotin immunoperoxidase technique (Histofine SAB-PO kit; Nichirei Corp., Tokyo, Japan) was used. Deparaffinized and rehydrated sections were trypsinized with .1% calcium chloride solution at 37°C for 20 minutes. After nonspecific reactions were blocked with 10% normal rabbit serum, the sections were incubated with CAM 5.2 diluted 1/5, biotinylated rabbit anti-mouse immunoglobulin, and streptavidin-peroxidase. Between each two incubation steps, sections were washed carefully in phosphate-buffered saline. Micrometastasis was defined as the presence of tumor cells—single or in small clusters—detected only by immunohistochemistry, that is, unidentifiable by hematoxylin and eosin staining.

Follow-Up and Statistical Analysis
Follow-up data were obtained from census registry certificates or outpatient records. All patients were followed up for at least 5 years, and median follow-up was 101 months. The {chi}2 test and Fisher’s exact test were performed to determine the relationship between lymph node micrometastasis and clinicopathologic variables. Five-year survival rates were calculated with the Kaplan-Meier method, and survival curve differences were measured by the log-rank test. In multivariate analysis, independent prognostic factors were determined by a Cox proportional hazards model. A P value <.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Re-evaluation of the original hematoxylin and eosin slides showed that five patients had lymph node metastasis, and they were excluded from this study. Overall, 2039 lymph nodes from the remaining 64 patients with histologically node-negative gastric cancer were examined. The number of lymph nodes per patient ranged from 5 to 83, with a median of 32 and a mean of 33. Lymph node micrometastasis was detected in 73 nodes (4%) and 20 patients (31%), and the number of lymph nodes involved ranged from 1 to 12, with a median of 2 and a mean of 3.6.

Table 1 shows the clinicopathologic factors of patients with or without lymph node micrometastasis. There was no significant relationship between the presence or absence of lymph node micrometastasis and clinicopathological variables including patient age or sex, tumor location, histological type, depth of wall invasion, gross type, tumor size, and lymphatic or venous invasion.


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  		TABLE 1. Clinicopathologic factorsa
 
The 5-year survival rate for the group of patients with lymph node micrometastasis was significantly lower than that for the group without lymph node micrometastasis (66% vs. 95%, P < .01). Furthermore, the 5-year survival rate was significantly lower for the group of patients with four or more positive micrometastatic nodes than for the group with zero to three positive micrometastatic nodes (29% vs. 94%, P < .01) (Fig. 1), and the 5-year survival rate was significantly lower for the group with extragastric (level 2) micrometastasis than for the group with no or perigastric (level 0 or 1) micrometastasis (53% vs. 89%, P < .01) (Fig. 2). The mode of the recurrence was peritoneal metastasis in seven patients, lung metastasis in two, and others in one. Multivariate analysis showed that among 11 clinicopathologic factors, the age, lymph node micrometastasis, and depth of wall invasion were independent predictors of a poor survival (Table 2).



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  		FIG. 1. The 5-year survival rate for the group of patients with four or more micrometastases was significantly lower than that for the group with zero to three micrometastases (29% vs. 94%, P < .01).

 


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  		FIG. 2. The 5-year survival rate for the group with extragastric (level 2) micrometastases was significantly lower than that for the group with no or perigastric (level 0 or 1) micrometastases (53% vs. 89%, P < .01).

 

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  		TABLE 2. Independent prognostic factors
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that lymph node micrometastasis is an independent prognostic indicator for patients with histologically node-negative gastric cancer invading the muscularis propria or deeper (T2 or T3). In addition to the presence of lymph node micrometastasis, the number and level of micrometastases in the lymph nodes were strongly associated with the survival time of patients.

It is controversial whether lymph node micrometastasis detected by immunohistochemistry predicts the clinical outcome of patients with histologically node-negative gastric cancer.15–19 Nakajo et al.18 reported that lymph node micrometastasis was correlated with a significantly worse survival rate in patients with T1 or T2 tumors. Cai et al.19 also found a significant relationship between lymph node micrometastasis and poor prognosis in patients with T3 gastric cancer. However, Fukagawa et al.16 showed that the presence of lymph node micrometastasis did not affect the survival in large numbers of patients with T2 gastric cancer.

Our results indicate that the prognostic significance of lymph node micrometastasis is found in the metastatic pattern, rather than just in the presence of micrometastatic tumor cells. The number and the level of lymph node metastases histologically determined by hematoxylin and eosin staining are generally accepted as important indicators associated with patient survival in gastric cancer.2,3 Previous studies also indicated that the status of lymph node micrometastasis was linked to patient prognosis. Siewert et al.15 observed that the presence of three of more tumor cells in more than 10% of the dissected lymph nodes was the only significant prognostic factor. In a similar finding from another study, the patient group with micrometastasis in level 2 nodes had a significantly poorer survival rate than that of the group with micrometastasis limited to level 1 nodes.19 In our study, micrometastasis of four or more lymph nodes or micrometastasis of level 2 nodes was significantly associated with a poor outcome.

Lymph node metastasis is linked to tumor progression. Lymph node micrometastasis in gastric cancer is also associated with deep invasion through the gastric wall, large tumor size, and positive lymphatic or venous invasion.7,16,19 In this study, although no relationship was found between the presence of lymph node micrometastasis and clinicopathologic characteristics, a weak association was found between lymph node micrometastasis and depth of wall invasion. Moreover, micrometastasis in four or more lymph nodes was significantly correlated with the depth of wall invasion (T3) and tumor size (>=5 cm).

In this series, the most common pattern of recurrence was peritoneal and lung metastasis. Similar to our results, previous studies reported that lymph node micrometastasis was strongly associated with subsequent development of hematogenous and peritoneal metastases, but not locoregional lymph node recurrence.7,20,21 These past and present data suggest that lymph node micrometastasis reflects the ability of the primary tumor to metastasize or disseminate and represents a marker for the risk of systemic disease.

In conclusion, our results indicate that micrometastasis of four or more lymph nodes or micrometastasis of extragastric (level 2) lymph nodes correlates with tumor progression and with a poor 5-year survival rate among patients with histologically node-negative gastric cancer (T2 and T3). These additional parameters are useful prognostic indicators of the risk for recurrence and may be helpful for deciding treatment strategies for adjuvant chemotherapy.


   Acknowledgments
 
The authors thank M. Hisaka and M. Hayashi for their technical assistance.


   Footnotes
 
The number and level of positive micrometastatic lymph nodes were significantly associated with the prognosis of patients with histologically node-negative gastric cancer. Micrometastases in four or more lymph nodes or in extragastric lymph nodes predict a high risk for death.

Received for publication January 2, 2002. Accepted for publication May 24, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  4. Mori M, Mimori K, Inoue H, et al. Detection of cancer micrometastases in lymph nodes by reverse transcriptase-polymerase chain reaction. Cancer Res 1995; 55: 3417–20.[Abstract/Free Full Text]
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  7. Cai J, Ikeguchi M, Maeta M, Kaibara N. Micrometastasis in lymph nodes and microinvasion of the muscularis propria in primary lesions of submucosal gastric cancer. Surgery 1999; 126: 32–9.
  8. Cai J, Ikeguchi M, Tsujitani S, Maeta M, Kaibara N. Micrometastasis in lymph nodes of mucosal gastric cancer. Gastric Cancer 2000; 3: 91–6.[CrossRef][Medline]
  9. Greenson JK, Isenhart CE, Rice R, Mojzisik C, Houchens D, Martin EW. Identification of occult micrometastases in pericolic lymph nodes of Dukes’ B colorectal cancer patients using monoclonal antibodies against cytokeratin and CC49: correlation with long-term survival. Cancer 1994; 73: 563–9.[CrossRef][Medline]
  10. Sasaki M, Watanabe H, Jass JR, et al. Occult lymph node metastases detected by cytokeratin immunohistochemistry predict recurrence in "node-negative" colorectal cancer. J Gastroenterol 1997; 32: 758–64.[Medline]
  11. Yasuda K, Adachi Y, Shiraishi N, Yamaguchi K, Hirabayashi Y, Kitano S. Pattern of lymph node micrometastasis and prognosis of patients with colorectal cancer. Ann Surg Oncol 2001; 8: 300–4.[Abstract/Free Full Text]
  12. Adachi Y, Yasuda K, Inomata M, Sato K, Shiraishi N, Kitano S. Pathology and prognosis of gastric carcinoma: well versus poorly differentiated type. Cancer 2000; 89: 1418–24.[CrossRef][Medline]
  13. Japanese Gastric Cancer Association. Japanese classification of gastric carcinoma: 2nd English edition. Gastric Cancer 1998; 1: 10–24.[Medline]
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  15. Siewert JR, Kestlmeier R, Busch R, et al. Benefits of D2 lymph node dissection for patients with gastric cancer and pN0 and pN1 lymph node metastases. Br J Surg 1996; 83: 1144–7.[Medline]
  16. Fukagawa T, Sasako M, Mann GB, et al. Immunohistochemically detected micrometastases of the lymph nodes in patients with gastric carcinoma. Cancer 2001; 92: 753–60.[CrossRef][Medline]
  17. Kikuchi Y, Tsuchiya A, Ando Y, Yoshida T, Takenosita S. Immunohistochemical detection of lymph node microinvolvement in node-negative gastric cancer. Gastric Cancer 1999; 2: 173–8.[CrossRef][Medline]
  18. Nakajo A, Natsugoe S, Ishigami S, et al. Detection and prediction of micrometastasis in the lymph nodes of patients with pN0 gastric cancer. Ann Surg Oncol 2001; 8: 158–62.[Abstract/Free Full Text]
  19. Cai J, Ikeguchi M, Maeta M, Kaibara N, Sakatani T. Clinicopathological value of immunohistochemical detection of occult involvement in pT3N0 gastric cancer. Gastric Cancer 1999; 2: 95–100.[CrossRef][Medline]
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