Eur J Cardiothorac Surg 2001;19:904-907
© 2001 Elsevier Science NL
Expression of nm23-H1 gene product in mediastinal lymph nodes from lung cancer patients
Masaki Tomita,
Takanori Ayabe,
Yasunori Matsuzaki,
Toshio Onitsuka
Department of Surgery II, Miyazaki Medical College, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan
Received 2 October 2000;
received in revised form 21 March 2001;
accepted 22 March 2001.
Corresponding author. Tel.: +81-985-85-2291; fax: +81-985-85-5563
e-mail: mtomita{at}post.miyazaki-med.ac.jp
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Abstract
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Objective: Although numerous studies have shown that nm23-H1 gene product expression is inversely related to metastatic potential in some cancers, the expression in lymph nodes has not been studied in detail. An analysis of nm23-H1 gene product expression in mediastinal lymph nodes from lung cancer patients is reported. Methods: One hundred and thirty-four, randomly selected lymph nodes (63 with positive pathological lymph node status) from 39 surgically treated lung cancer patients were examined. Expression of nm23-H1 gene product was determined using specific monoclonal antibodies. Metastatic cancer cells were highlighted using anti-cytokeratin antibody. Results: Expression of nm23-H1 gene product in patients with less and more than 50% nodes-positive was 12/23 (52.2%) and 15/16 (93.8%) cases, respectively. Immunohistochemical studies with cytokeratin revealed micrometastasis in 6/39 (15.4%) patients and 9/71 (12.7%) nodes previously reported as cancer negative. Expression of nm23-H1 gene product in micrometastasis and metastasis-positive nodes was 5/9 (55.6%) and 55/63 (87.3%), respectively. We also found nm23-H1 gene product expression in germinal center cells. However, we found no relationship between expression of nm23-H1 gene product in germinal center cells and extent of metastasis. Conclusions: Our study demonstrates a positive relationship between expression of nm23-H1 gene product and extent of metastasis in mediastinal lymph nodes from lung cancer patients. Our data for normal germinal center cells suggests that nm23-H1 gene product expression does not play a specific biological role in suppressing tumor metastasis in lung cancer.
Key Words: nm23-H1 gene product • Lung cancer • Lymph nodes • Cytokeratin
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1. Introduction
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The nm23-H1 gene was originally identified by differential screening of a murine K1735 melanoma cell line cDNA library using mRNA derived from cell lines with differing metastatic potential [1]. Expression of nm23-H1 gene product was found to be inversely related to metastatic potential, with ten-fold higher mRNA levels in cell clones with low metastatic potential than in those with high metastatic potential [1]. Therefore, it was thought that nm23-H1 gene product may play a specific biological role in suppressing tumor metastasis. In fact, the suppression of metastatic potential was demonstrated experimentally by transfecting nm23 cDNA into high-metastatic tumor cells [2]. It has also been reported that breast cancer with reduced nm23-H1 gene product expression was associated with more frequent lymph node metastasis and a generally poor prognosis [3–5]. A similar relationship between decreased nm23-H1 gene product expression and tumor malignancy has been demonstrated in other cancers [6–9]. With regard to lung cancer, however, previous studies have shown that nm23 gene product expression is unrelated to patient survival [10,11]. The reasons for these differences between lung cancer and other types of cancers remain unclear.
Moreover, the literature contains only a small number of studies on nm23-H1 gene product expression in lymph nodes [6,11–14]. Of these studies, most reported on the expression of nm23-H1 gene product in cancer cells, and only one study examined normal lymph nodes. A study on breast cancer demonstrated significantly lower levels of nm23-H1 gene product expression in normal axillary lymph nodes than in nodes with metastatic deposits [14]. Even though counts were low, it seems likely, based on these results, that expression of nm23-H1 gene product can be observed in normal lymph nodes. The present study investigates nm23-H1 gene product expression in mediastinal lymph nodes from lung cancer patients.
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2. Materials and methods
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2.1. Patients and tissue samples
We randomly selected and examined 134 lymph nodes from 39 primary lung cancer patients (24 adenocarcinomas and 15 squamous cell carcinomas) who were treated surgically at Miyazaki Medical College during 1994–1995 and no patient had pre-operative chemo/radiotherapy. Three to six nodes from each patient were randomly selected. All patients had at least one node-positive. These surgically resected lymph nodes were formalin-fixed and paraffin-embedded. Routine pathological studies revealed 63 nodes to be metastasis positive. Using one paraffin block for each node, studies were conducted to detect the presence of cytokeratin and to ascertain nm23-H1 gene product expression.
2.2. Immunohistochemical studies
Specific monoclonal antibodies against nm23-H1 gene product (Novocastra Laboratories, Newcastle, UK) and cytokeratin 19 (Santa Cruz Biotechnology, California, USA) were used for immunohistochemical studies. Before staining, serialized 4 µm-thick sections were deparaffinized in three changes of Lemosol and rehydrated through a descending series of ethanol. These sections were immersed in 0.6% H2O2 in methanol for 20 min at room temperature to block endogenous peroxidase activity. After blocking non-specific protein bindings with an overnight incubation with Block Ace (Dainippon Inc., Osaka, Japan), the sections were subsequently incubated overnight at 4°C with primary antibodies against human nm23-H1 protein (1:200) and cytokeratin (1:50), respectively. The sections were then incubated with secondary antiserum (1:500) for 1 h, followed by an incubation with peroxidase anti-peroxidase complexes for 30 min at room temperature. Finally, the sections were visualized with the Diaminobenzidine/Metal Concentration (10x) and Stable Peroxide Substrate Buffer (1x) system (Pierce, Rockford, IL, USA). After washing with water, they were counterstained with hematoxylin. Immunohistochemical results were assessed semiquantitatively by two authors.
2.3. Statistical analysis
Data was analyzed using the Fisher's exact test. Differences were taken as significant when P<0.05.
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3. Results
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3.1. Immunohistochemical staining with cytokeratin
Immunohistochemical staining with cytokeratin revealed micrometastasis in 9/71 (12.7%) nodes previously diagnosed as cancer negative by pathological study. The number of patients who had micrometastasis was 6/39 (15.4%). In the 63 metastasis-positive nodes, all samples showed positive staining with cytokeratin.
3.2. Immunohistochemical staining with nm23-H1 gene product
In most cases, nm23-H1 gene product was observed diffusely in the cytoplasm of the cancer cells. The patients in this series were divided into two groups: Group <50, patients with less than 50% of lymph nodes studied had metastasis; Group >50, those with more than 50% nodes had metastasis. Most of lymph nodes in Group >50 showed extensive metastasis. In the present series, all positive-nodes of an individual patient exhibited a similar pattern of nm23-H1 gene product expression. The expression of nm23-H1 gene product in cancer cells in Group <50 and >50 was observed in 12/23 (52.2%) and 15/16 (93.8%) cases, respectively (Table 1). This result showed a positive relationship between expression of nm23-H1 gene product and extent of metastasis in mediastinal lymph nodes (P=0.023).
Based on the result of cytokeratin staining, the samples were also divided into three groups: Group A, metastasis negative by both routine pathological study and cytokeratin staining; Group B, micrometastasis (cancer negative by pathology, but cancer positive by cytokeratin staining); Group C, metastasis positive. Expression of nm23-H1 gene product in cancer cells in Groups B and C was observed in 5/9 (55.6%) and 55/63 (87.3%) nodes, respectively (Table 2). As shown in Table 2, we noted a positive correlation between expression of nm23-H1 gene product and extent of metastasis in mediastinal lymph nodes (P=0.037). This correlation was also observed in patients with adenocarcinoma as well as in patients with squamous cell carcinoma (data not shown). Furthermore, as shown in Fig. 1, germinal center cells also exhibited positive staining for nm23-H1 gene product. The expression rate of nm23-H1 gene product in germinal center cells, however, did not differ significantly among the three groups as shown in Table 2: Group A, 44/62 (71.0%); Group B, 6/9 (66.7%); and Group C, 38/63 (60.3%).
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Fig. 1. Positive immunohistochemical staining for nm23-H1 gene product in germinal center cells. (Original magnification: 200x).
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4. Discussion
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Metastatic cancer cells were highlighted using anti-cytokeratin antibody [15]. In our study, micrometastasis was detected in 6/39 (15.4%) patients, results consistent with Passlick et al. [16], who observed cytokeratin-positive cells in lymph nodes from lung cancer patients in 11/70 (15.7%). In the present study, the expression of nm23-H1 gene product was significantly higher in Group >50. Furthermore, micrometastatic and metastatic nodes showing positive staining for nm23-H1 gene product expression in our study were 5/9 (55.56%) and 55/63 (87.3%), respectively, suggesting a positive correlation between expression of nm23-H1 gene product and extent of metastasis. Because of these results, we believe expression of nm23-H1 gene product may serve as a marker of lymph node metastasis in lung cancer. Huwer et al. [17], who examined bronchial lavage samples, also reported that the proof of nm23 protein might be to establish the diagnosis of squamous cell lung cancer.
This is not the case, however, in thyroid cancer, where one study reported that metastatic lymph nodes showed no or weak expression of nm23-H1 mRNA, the extent of which was inversely proportional to the degree of cancer cell occupancy [12]. Also, in gastric carcinomas, reduction of nm23 immunoreactivity in the metastatic tumor of regional lymph nodes has also been reported, and the authors concluded that the decrease in nm23 expression was associated with metastasis of human gastric carcinomas [6]. Based on these studies, increased expression of nm23-H1 gene product appears to play a role in suppressing cancer metastasis.
In contrast to these studies, however, we found a positive relationship between expression of nm23-H1 gene product and extent of metastasis in lung cancer patients. Furthermore, in a previous study, we found no significant relationship between nm23-H1 gene product expression in metastatic nodes and patient survival [11]. Other researchers have also documented that expression of nm23-H1 gene product lacks prognostic value in primary lung cancer [10] despite prognostic relationship in other cancers [3–9]. These results suggest that the role of nm23-H1 gene product in lung cancer, both in the primary cancer and in the metastatic nodes, differs from that in other cancers.
In the present study, we also demonstrated nm23-H1 gene product expression in normal germinal center cells. To our knowledge, this is the first study to document nm23-H1 gene product expression in normal lymph node cells. Because the germinal center of lymphoid tissue is well known as a critical microenvironmental site of B cell activation and differentiation in response to antigenic stimuli [18], it is possible to hypothesize that B cells play a role against cancer metastasis through nm23-H1 gene product expression. It is generally accepted, however, that T cell lymphocytes are primarily responsible for host immunity against cancer cells. Furthermore, in our study, nm23-H1 gene product expression in germinal center cells did not differ substantially among the Group A–C. In other words, we found no relationship between expression of nm23-H1 gene product in germinal center cells and extent of metastasis.
Because of the demonstrated positive relationship between expression of nm23-H1 gene product and extent of metastasis in mediastinal lymph nodes, we believe nm23-H1 gene product expression may serve as a marker of lymph node metastasis in lung cancer. Based on our data for normal germinal center cells, we further conclude that nm23-H1 gene product expression does not play a role in suppressing tumor metastasis in lung cancer but has another function. Further studies are needed in this area.
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Acknowledgments
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The authors wish to thank Mrs Yasuko Tobayashi for her skilful technical assistance.
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Abstract
1. Introduction
