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Annals of Surgical Oncology 8:158-162 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Detection and Prediction of Micrometastasis in the Lymph Nodes of Patients With pN0 Gastric Cancer

Akihiro Nakajo, MD, Shoji Natsugoe, MD, Sumiya Ishigami, MD, Masataka Matsumoto, MD, Saburo Nakashima, MD, Shuichi Hokita, MD, Masamichi Baba, MD, Sonshin Takao, MD and Takashi Aikou, MD

From the First Department of Surgery, Kagoshima University School of Medicine, Kagoshima, Japan.

Correspondence: Address correspondence to: Akihiro Nakajo, MD, First Department of Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 891-0075, Japan; Fax: 81-99-265-7426; E-mail: jo3{at}sa2.so-net.ne.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The clinicopathologic significance of micrometastasis (MM) and tumor cell microinvolvement (TCM) in regional lymph nodes as identified by immunohistochemical staining for cytokeratin expression was evaluated in patients with node-negative gastric cancer.

Methods: MM was defined as tumor cells with stromal reaction, and TCM was defined as individual tumor cells without stromal reaction. We investigated 1761 lymph nodes obtained from 67 gastric cancer patients whose diagnosis showed no lymph node metastasis by routine histological examination. The depth of tumor invasion was T1 (submucosa) in 33 patients and T2 (muscularis propria and subserosa) in 34 patients. The lymph nodes were examined immunohistochemically for the presence of tumor cells using anti-cytokeratin AE1/AE3 monoclonal antibody. Both the biopsy tumor specimens obtained prior to surgery and the resected primary tumors were immunostained with E-cadherin (E-cad) monoclonal antibody.

Results: Thirty (1.5%) of the 1761 lymph nodes showed MM and/or TCM. MM with or without TCM was found in 10 patients, and TCM alone was found in 4 patients; 6 (18.2%) of the 33 patients with T1 tumor and 8 (23.5%) of the 34 patients with T2 tumor had occult lymph node metastasis. The 5-year survival rate was worse among those with MM with or without TCM, than among those without MM. Nearly all of the patients with MM and/or TCM had reduced or negative E-cad expression in the primary tumor.

Conclusions: We demonstrated that the incidence of MM and/or TCM in the lymph nodes of patients with gastric cancer is quite high, and that such metastasis is associated with the prognosis of patients with pN0. Examination of E-cad expression in biopsy tumor specimens may be useful for predicting MM and/or TCM.

Key Words: Micrometastasis • Lymph node • Cytokeratin • E-cadherin • Prognosis • Gastric cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Some gastric cancer patients who undergo curative resection subsequently develop recurrent disease, and this occurs even in patients who are diagnosed as free of lymph node metastasis by routine histological examination. It is suggested that in gastric cancer patients, the tumor spread is more advanced than indicated by the results of routine histological diagnosis. The development of sensitive immunohistochemical stains using specific monoclonal antibodies has made it possible to detect a small tumor cell cluster or even single tumor cells in histological tissue sections. Cytokeratin (CK) is an epithelial marker and is a type of intermediate filament. Immunohistochemical detection of occult lymph node metastasis using an epithelial marker has been reported in various carcinoma types, including lung,1,2 breast,3,4 gallbladder,5 esophagus,6 stomach,7,8 and colon cancers.9,10,11

Recently, the malignant property of primary tumors has been elucidated using biological methods. The relation between cell adhesion molecules and lymph node metastasis in primary tumors has also been reported. E-cadherin (E-cad) is a member of the cadherin family and plays an important role in regulating intercellular adhesion in epithelial tissues. Reduced E-cad expression is closely associated with lymph node metastasis.12,13

The purposes of this study were to investigate the incidence and clinical significance of occult lymph node metastasis in gastric cancer patients who were diagnosed as pN0 by routine histological examination, and to examine whether the level of E-cad expression in preoperative biopsy specimens can predict the occurrence of such metastasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Sixty-seven consecutive patients with primary gastric cancer, who were diagnosed as free of lymph node metastasis by routine histological examination using hematoxylin and eosin staining, were enrolled in this study. All of the patients underwent curative gastric resection with lymphadenectomy at the First Department of Surgery, Kagoshima University Hospital, between 1985 and 1997. None of the patients had received preoperative chemotherapy or radiotherapy. All patients were followed after the surgery. The median length of the follow-up period was 41 months (range, 4–136 months).

The 67 patients consisted of 49 males and 18 females, who ranged in age from 35 to 85 years (mean age, 64.9 years). According to the Japanese Classification of Gastric Carcinoma,14 the depth of tumor invasion was T1 (submucosa) in 33 patients, and T2 (muscularis propria and subserosa) in 34 patients (Table 1). The number of resected nodes in each patient ranged between 11 and 60, with a median of 26. A total of 1761 regional lymph nodes were examined immunohistochemically.


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  		TABLE 1. Characteristics of the primary gastric cancer patients
 
Immunohistochemical Analyses for CK Expression and E-cad Expression
The dissected lymph nodes were fixed in 10% formalin and embedded in paraffin. Each specimen was sectioned, and the sections were placed on slides. Each slide was placed in an oven at 68°C overnight. Then, the sections were deparaffinized with xylene and dehydrated with 98% ethanol. After incubating the slides in citrate buffer solution (pH 6.0) for 6 minutes in a pressure cooker, the mouse monoclonal antibody against human cytokeratin AE1/AE3 (DAKO CORPORATION, Carpinteria, CA, USA), diluted 150-fold with Tris-buffer, was added on each slide, and the slides were incubated at 37°C for 60 minutes. The reactions for CK were developed using the alkaline phosphatase anti-alkaline phosphatase (APAAP) method.15 The immunohistochemical study for CK expression was limited to one slide per lymph node to expedite routine histological examination. The negative control sections were treated with the same protocol but with the primary antibody omitted. Normal gastric mucosa and the primary tumors of the specimens were used as positive controls and were consistently positive for CK.

The preoperative biopsy tumor specimens and resected primary tumors were subject to immunohistochemical analysis using E-cad monoclonal antibody (Human E-cadherin, TaKaRa, Tokyo, Japan). These specimens were also fixed in 10% formalin and embedded in paraffin. Each specimen was sectioned, and the sections were placed on slides. The sections were deparaffinized with xylene and dehydrated with 98% ethanol. Endogenous peroxidase was blocked by immersing the slides in 0.3% hydrogen peroxide in absolute methanol at room temperature for 30 minutes. After washing with phosphate-buffered saline (PBS) three times for 5 minutes each, the sections were then blocked by treating with a solution of PBS containing 1% bovine serum albumin at room temperature for 30 minutes. The blocked sections were incubated at 4°C overnight with the monoclonal antibody against E-cad diluted 1000-fold with PBS. The reactions for E-cadherin were developed with an avidin-biotin complex immunoperoxidase technique (ABC method, VECTASTAIN ABC kit, Vector Laboratories, Inc, Burlingame, CA, USA).

Evaluation of the Results
Tumor involvement in a lymph node was classified into one of two categories according to the results of immunohistochemical staining for CK: (1) Tumor micrometastasis (MM) — cluster formation of tumor cells with a stromal reaction (presence of granulation tissue or desmoplastic connective tissue). Because a cluster of positively stained tumor cells in the medulla of the lymph node was observed with closely opposed fibroblast, some of which were reactive and were beginning to form granulation tissue or desmoplastic connective tissue, these tumor cells were considered to be micrometastasis (Fig. 1A); or (2). Tumor cell microinvolvement (TCM) — the presence of individual tumor cells without a change in the stroma (Fig. 1B).16 With regard to E-cad expression, if more than 90% of the tumor cells in a tumor positively stained for E-cad, it was classified as having preserved E-cad expression (Fig. 2A). If 90% or less of the tumor cells stained for E-cad, the tumor was classified as having reduced E-cad expression (Fig. 2B).17,18 The stained sections were examined independently by two observers (AN and SN) who had no knowledge of the clinicopathological data.



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  		FIG. 1. A. Micrometastasis (MM) in a lymph node as detected by immunohistochemical staining with anti-cytokeratin AE1/AE3 (x400). Micrometastasis was defined as metastasis consisting of tumor cells or a small cluster of carcinoma cells with surrounding stromal change. B. Tumor cell microinvolvement (TCM) in a lymph node (x400). Microinvolvement was defined as carcinoma cells without surrounding stromal change.

 


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  		FIG. 2. A. Preserved E-cadherin expression in a primary tumor as detected by immunohistochemical staining (x400). A tumor or biopsy specimen in which over 90% of the tumor cells stained for E-cadherin, was considered to have preserved E-cadherin expression. B. Reduced E-cadherin expression in a primary tumor as detected by immunohistochemical staining (x400). A tumor or biopsy specimen in which 90% or less of the tumor cells stained for E-cadherin was considered to have reduced E-cadherin expression.

 
Survival data were analyzed by the Kaplan-Meier survival model and expressed as overall survival. Differences in survival were evaluated using the Log rank test. The presence or absence of MM and/or TCM was compared with tumor depth, histology, lymphatic invasion, and venous invasion using the categories shown in Table 1, with Fisher’s exact test. A P value of < .05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Micrometastasis and Tumor Cell Microinvolvement in Lymph Nodes
Tumor cells were found in 30 (1.5%) of the 1761 dissected lymph nodes according to immunohistochemical analysis for CK expression. The 30 CK-positive lymph nodes had been obtained from 14 (20.9%) of the 67 patients. MM with or without TCM was found in the lymph nodes of 10 patients, and TCM alone was found in the lymph nodes of 4 patients. The number of lymph nodes with MM and/or TCM in the 14 patients ranged between 1 and 3 lymph nodes. According to tumor depth, 6 (18.2%) of the 33 patients with T1 tumor and 8 (23.5%) of the 34 patients with T2 tumor had MM and/or TCM. The presence of MM and/or TCM was not significantly related with each of tumor depth, histological type, tumor size, and lymphatic or venous invasion.

E-cadherin Expression
Strong E-cad expression was present in all of the normal gastric epithelium samples. Reduced E-cad expression was found in the resected primary tumors of 12 (85.7%) of the 14 patients with MM and/or TCM, and in 27 (50.9%) of the 53 patients without MM (P = .031). On the other hand, in the preoperative biopsy specimens, E-cad expression was reduced in 13 (92.9%) of the 14 patients with MM and/or TCM, and in 30 (56.6%) of the 53 patients without MM (Table 2). Reduced E-cad expression in the preoperative biopsy specimen was more frequently found among those with MM and/or TCM than among those without such metastasis (P = 0.013). The rate of coincidence between the level of E-cad expression in the preoperative biopsy specimen and that in the primary tumor of a patient was 91.0% (61/67).


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  		TABLE 2. Results of immunohistochemical analysis for E-cad expression in the primary tumors and preoperative biopsy tumor specimens
 
Impact on Survival
The four patients with TCM alone had good prognosis without tumor recurrence. However, the 5-year survival rate of patients with MM with or without TCM was significantly shorter than the 5-year survival rate of those without MM (P < .05) (Fig. 3).



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  		FIG. 3. Survival curve of patients who did or did not have micrometastasis (MM) in one or more lymph nodes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The relationship between lymph node micrometastasis and clinicopathologic findings in gastric cancer has been described in several reports. Siewert et al.19 reported that TCM is an independent prognostic factor in patients with gastric cancer. Ishida et al.8 reported that gastric cancer patients with micrometastasis at Stage II had significantly worse prognosis than those without micrometastasis. In the present study, we evaluated MM and TCM in the lymph nodes of patients with a T1 or T2 gastric cancer, because most of the patients with a T3 or T4 tumor had peritoneal recurrence after the surgery. Furthermore, we selected patients who were diagnosed as node-negative by routine histological examination. Careful review of original sections revealed that tumor cells were indeed present in the original sections of some patients, and such cases were excluded from the present study.

In this series of patients that were histologically diagnosed as pN0, the incidence of MM and/or TCM was 20.9%. It is of interest that the four patients with TCM alone in the lymph nodes did not experience a recurrence. It has been suggested that such single cells can not proliferate in the lymph nodes because they are killed by local and general immunocytes.20 On the other hand, once tumor cells form a cluster, these cells may easily proliferate and have metastatic potential. The prognosis of patients with MM was poorer than that of patients without MM.

The relationship between E-cad expression in the primary tumor and the presence of MM and/or TCM has been reported in esophageal carcinomas.6 It is clinically important that such metastases can be predicted prior to surgery. In the present study, when E-cad expression in the preoperative biopsy specimens and that in the resected primary tumors were compared, the rate of coincidence in a patient was 91.0%, although each tumor had heterogeneous components. Furthermore, 13 of the 14 primary tumors with MM and/or TCM had reduced E-cad expression. These results suggest that testing biopsy tumor specimens for E-cad expression may aid in predicting the occurrence of MM and/or TCM in T1 and T2 gastric cancers. In patients who are preoperatively found to have reduced E-cad expression, extended lymphadenectomy should be performed.

In conclusion, MM and/or TCM was detected at a considerably high rate among patients classified as pN0 by routine histological examination. Although the patients with MM had an especially high risk for recurrence, randomized prospective study is needed to determine if this population actually benefits from postoperative adjuvant therapy. In patients with T1 or T2 gastric cancers, it may be useful to examine E-cad expression in the biopsy tumor specimens for predicting the occurrence of MM and/or TCM, and for performing appropriate lymphadenectomy.

Received for publication June 8, 2000. Accepted for publication August 18, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chen ZL, Perez S, Holmes EC, Wang HJ, Coulson WF, Wen DR, Cochran AJ. Frequency and distribution of occult micrometastases in lymph nodes of patients with non–small-cell lung carcinoma. J Natl Cancer Inst 1993; 85: 493–8.[Abstract/Free Full Text]
  2. Rasslick VV, Izbicki JR, Kubbuschok B, et al. Immunohistochemical assessment of individual tumor cells in lymph nodes with non–small-cell lung cancer. J Clin Oncol 1994; 12: 1827–32.[Abstract/Free Full Text]
  3. Nasser IA, Lee AK, Bosari S, Saganich R, Heatley G, Silverman ML. Occult axillary lymph node metastases in "node-negative" breast carcinoma. Hum Pathol 1993; 24: 950–7.[CrossRef][Medline]
  4. McGuckin MA, Cummings MC, Walsh MD, Hohn BG, Bennett IC, Wright RG. Occult axillary node metastases in breast cancer: their detection and prognostic significance. Br J Cancer 1996; 73: 88–95.[Medline]
  5. Yokoyama N, Shirai Y, Hatakeyama K. Immunohistochemical detection of lymph node micrometastases from gallbladder carcinoma using monoclonal anticytokeratin antibody. Cancer 1999; 85: 1465–9.[CrossRef][Medline]
  6. Natsugoe S, Mueller J, Stein HJ, Feith M, Hofler H, Siewert JR. Micrometastasis and tumor cell microinvolvement of lymph nodes from esophageal squamous cell carcinoma: frequency, associated tumor characteristics, and impact on prognosis. Cancer 1998; 83: 858–66.[CrossRef][Medline]
  7. Maehara Y, Oshiro T, Endo K, et al. Clinical significance of occult micrometastasis lymph nodes from patients with early gastric cancer who died of recurrence. Surgery 1996; 119: 397–402.[CrossRef][Medline]
  8. Ishida K, Katsuyama T, Sugiyama A, Kawasaki S. Immunohistochemical evaluation of lymph node micrometastases from gastric carcinomas. Cancer 1997; 79: 1069–76.[CrossRef][Medline]
  9. Greenson JK, Isenhart CE, Rice R, Mojzisik C, Houchens D, Martin EWJr. Identification of occult micrometastases in pericolic lymph nodes of Duke’s B colorectal cancer patients using monoclonal antibodies against cytokeratin and CC49. Correlation with long-term survival. Cancer 1994; 73: 563–9.[CrossRef][Medline]
  10. Jeffers MD, O’Dowd GM, Mulchahy H, Stagg M, O’Donoghue DP, Toner M. The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma. J Pathol 1994; 172: 183–7.[CrossRef][Medline]
  11. 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]
  12. Mayer B, Johnson JP, Leitl F, et al. E-cadherin expression in primary and metastatic gastric cancer: down-regulation correlates with cellular dedifferentiation and glandular disintegration. Cancer Res 1993; 53: 1690–5.[Abstract/Free Full Text]
  13. Kinsella AR, Green B, Lepts GC, Hill CL, Bowie G, Taylor BA. The role of the cell-cell adhesion molecule E-cadherin in large bowel tumor cell invasion and metastasis. Br J Cancer 1993; 67: 904–9.[Medline]
  14. Japanese Gastric Cancer Association. Japanese classification of gastric carcinoma. 2nd English ed. Gastric Cancer 1998; 1: 1–15.[CrossRef][Medline]
  15. Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline Phosphatase and monoclonal anti-alkaline Phosphatase (APAAP complexes). J Histochem Cytochem 1984; 32: 219–29.[Abstract]
  16. Natsugoe S, Nakashima S, Matsumoto M, et al. Paraaortic lymph node micrometastasis and tumor cell microinvolvement in advanced gastric carcinoma. Gastric Cancer 1999; 2: 179–85.[CrossRef][Medline]
  17. Shiozaki H, Tahata H, Oka H, et al. Expression of immunoreactive E-cadherin adhesion molecules in human cancers. Am J Pathol 1991; 139: 17–23.[Abstract]
  18. Oka H, Shiozaki H, Kobayashi K, et al. Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis. Cancer Res 1993; 53: 1696–1701.[Abstract/Free Full Text]
  19. Sievert 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]
  20. Ishigami S, Natsugoe S, Tokuda K, et al. Prognostic value of intratumoral natural killer cells in gastric carcinoma. Cancer 2000; 88: 577–83.[CrossRef][Medline]



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