Ann Thorac Surg 2002;73:1060-1064
© 2002 The Society of Thoracic Surgeons
Original article: general thoracic
Clinical significance of nm23 expression in resected pathologic-stage I, non-small cell lung cancer
Hiromichi Katakura, MDa,
Fumihiro Tanaka, MDa,
Hiroki Oyanagi, MDa,
Ryo Miyahara, MDa,
Kazuhiro Yanagihara, MDa,
Yosuke Otake, MDa,
Hiromi Wada, MD*a
a Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan
Accepted for publication December 2, 2001.
* Address reprint requests to Dr Wada, Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Shogoin-kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan
e-mail: wadah{at}kuhp.kyoto-u.ac.jp
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Abstract
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Background. Clinical significance of the status of nm23 gene, originally identified as an antimetastatic gene, in non-small cell lung cancer remains unestablished, whereas many clinical studies have demonstrated that reduced nm23 expression is correlated with tumor progression and poor prognosis in a variety of malignant tumors such as breast carcinoma.
Methods. nm23 expression was examined immunohistochemically in a total of 117 patients with completely resected pathologic stage I non-small cell lung cancer.
Results. nm23 expression was positive in 73 (62.4%) patients, and there was no correlation between nm23 expression and age, sex, performance status, pathologic T factor, histologic type, or degree of cancer cell differentiation. The 5-year survival rates of nm23-positive and nm23-negative patients were 79.7% and 57.8%, respectively, demonstrating a significantly poorer prognosis in nm23-negative patients (p = 0.013), which was confirmed by a multivariate analysis. nm23 was not correlated with the incidence of apoptosis, proliferative activity, or p53 status.
Conclusions. nm23 expression was a significant factor for predicting a favorable prognosis, suggesting antimetastatic potential of the nm23 gene in non-small cell lung cancer.
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Introduction
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The nm23 gene was originally identified as an antimetastatic gene by means of differential hybridization screening of murine melanoma K-1735 sublines showing different metastatic potential [1]. Following identification of the first nm23 gene, which was named the nm23-H1 gene, a second nm23 gene (nm23-H2 gene) was identified [2]. In addition to several experimental studies demonstrating that the nm23-H1 gene is a direct antimetastasis suppressor [3–5], many clinical studies have revealed that reduced nm23 expression is correlated with the metastatic potential of a variety of malignant tumors such as breast carcinoma [6].
Clinical studies on nm23 expression in non-small cell lung cancer (NSCLC) have demonstrated conflicting results, however; some studies suggested that nm23 was associated to antimetastatic potential [7–9], and others did not [10, 11]. Surprisingly, some studies suggested that nm23 was correlated with tumor progression [12–14]. These conflicting results may be partly due to small numbers of patients as well as heterogeneity of the pathologic (p-) stages documented in these studies. In the present study, therefore, a retrospective analysis was conducted in a homogenous patient population (only p-stage I patients) to clarify the clinical significance of nm23 in resected NSCLC. In addition, correlation between nm23 status and proliferative activity were examined to investigate the role of nm23 in tumor cell proliferation and induction of apoptosis. Correlation between nm23 status and p53 status, which had been reported to be a significant prognostic factor in NSCLC, was also examined.
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Material and methods
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A total of 117 consecutive patients with p-stage I NSCLC of which histologic sections were available and who underwent complete tumor resection and mediastinal lymph node dissection without any preoperative therapy at the Department of Thoracic Surgery, Kyoto University, between January 1, 1987, and December 31, 1992, were retrospectively reviewed (Table 1).
Pathologic stage was reevaluated and determined according to the tumor-node-metastasis (TNM) classification as revised in 1997. Histologic type was also reevaluated and determined according to the classification by the World Health Organization as revised in 1999. There was no operation-related death. For all patients, inpatient medical records, chest roentgenograms, whole-body computed tomography (CT) films, bone and gallium scanning data, and records of surgery were reviewed. Follow-up of the postoperative clinical course was conducted by outpatient medical records and by inquiries by telephone or letter. The day of thoracotomy was considered the starting day for counting postoperative survival days, and the mean follow-up duration was 1,581 days and ranged from 134 to 5,660 days.
Tissue preparation
Serial 4-µm sections were cut from each formalin-fixed and paraffin-embedded tumor specimen and served for routine hematoxylin and eosin (HE) staining, immunohistochemical staining (IHS), and the terminal deoxynucleotidyl tranferase-mediated dUTP (deoxyuridine triphosphate)-biotin nick end-labeling (TUNEL) staining to detect apoptotic cells. Before IHS to detect expression of nm23, proliferative cell nuclear antigen (PCNA) and p53, sections were heated in a microwave oven for 5 minutes, three times each to retrieve their antigeneities. Before the TUNEL staining, sections were digested with 20 ug/mL proteinase K (Boehringer Manheim, Manheim, Germany) for 20 minutes.
Immunohistochemical staining
The procedure of IHS using streptoavidin-biotinylated horseradish peroxidase complex method (LSAB kit; Dako Japan, Kyoto, Japan) was described previously [15]. Sections were incubated at 4°C overnight with an antihuman nm23 polyclonal antibody diluted at 1:50 (Dako Japan) that recognized both nm23-H1 and nm23-H2. Slides were evaluated by two of the authors (HK and FT) independently without knowledge of characteristics of patients or clinical course. Slides incubated without the primary antibody served as negative controls. Tonsil was used as positive control for nm23 staining. A total of 1,000 tumor cells were counted for positive staining and the percentages of positive cells were determined. nm23 expression was judged to be positive when both the authors agreed that the percentage of positive-staining tumor cells exceeded 5%.
An antip53 monoclonal antibody (MoAb) DO-7 (mouse IgG2b, kappa; Dako Japan) and an anti-PCNA, MoAb PC-10 (mouse IgG2a, kappa; Dako Japan) were used as the primary antibodies to determine p53 status and proliferative activity of the tumor, respectively [15]. When the percentage of the cells with nuclear positive staining exceeded 5%, the slide was judged to exhibit aberrant expression of p53. Proliferative index (PI) was defined as the percentage of PCNA-positive staining cancer cells.
TUNEL staining
Detection of apoptotic cells was performed with the TUNEL method as described previously [15]. The TUNEL staining was performed using the In Situ Death Detection Kit, POD (Boehringer Manheim) following the manufactured protocol. Briefly, the sections were incubated for 60 minutes at 37°C with the TUNEL reaction mixture including terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) and fluorescein-labeled deoxyuridine triphosphate. Subsequently, the sections were incubated for 30 minutes at 37°C with antifluorescein antibody, Fab fragment from sheep, conjugated with horseradish peroxidase (POD).
The specificity of the TUNEL staining of apoptotic cells was confirmed by making the negative and the positive control slides at every staining. As negative control slides, sections incubated with the TUNEL reaction mixture without TdT were used. As positive control slides, sections were treated with 0.7 mg/mL DNAse I (Stratagene, La Jolla, CA) for 10 minutes at 25°C before the TUNEL reaction. Apoptotic cells were determined with careful observation of TUNEL-staining sections and serial HE-staining sections. TUNEL-positive staining cells, if they did represent histologic features of necrosis in HE-staining sections, were not considered to be apoptotic cells. In each case a total of 10,000 tumor cells, 1,000 tumor cells each in 10 different fields, were evaluated at high magnification (x400) by two authors independently. The apoptotic index (AI) was defined as the number of apoptotic cells per 1,000 tumor cells.
Statistical methods
Counts were compared by the 
2 test. Continuous data were compared using Student’s t test if the distribution of samples was normal, or using Mann-Whitney U test if the sample distribution was asymmetrical. Postoperative survival rate was analyzed by the Kaplan-Meier method, while differences in survival rates were assessed by the log-rank test. Multivariate analysis of prognostic factors was performed using Cox’s regression model. Differences were considered significant when the p value was less than 0.05. All statistical manipulations were performed using the SPSS for Windows software system (SPSS Inc, Chicago, IL).
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Results
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Expression of nm23 in NSCLC
nm23 expression was positive in 73 (62.4%) among all 117 patients (Table 2).
There was no significant correlation between nm23 expression and age, sex, performance status (PS), pathologic T factor, histologic type, or degree of cancer cell differentiation (Table 2).
Nm23 status and postoperative survival
Five-year survival rates for patients with nm23-positive tumors and nm23-negative tumors were 79.7% and 57.8%, respectively, demonstrating a significantly poorer prognosis for nm23-negative tumors (p = 0.013; Fig 1 and Table 1).
Most treatment-failure patients showed extrathoracic recurrence in both nm23-positive and nm23-negative groups. Multivariate analysis confirmed that positive nm23 expression was an independent and significant factor to predict a favorable prognosis (relative risk 0.491; 95% confidence interval 0.217 to 0.879; p = 0.026).
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Fig 1. Postoperative survival of patients with completely resected p-stage I, non-small cell lung cancer. A comparison is made between tumor with positive nm23 expression and tumor with negative nm23 expression.
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Nm23 expression and PI, AI, p53 status
To investigate how nm23 expression was correlated with postoperative survival, correlation between nm23 expression and PI or AI was examined. Whereas PI for nm23-positive tumor seemed slightly higher than that for nm23-negative tumor (42.7% and 34.9%, respectively), and AI for nm23-positive tumor seemed slightly higher than that for nm23-negative tumor (15.1 and 23.2, respectively), neither of the correlations was statistically significant (Table 3).
As shown in Table 1, aberrant expression of p53 was another significant prognostic factor, which was confirmed by a multivariate analysis (relative risk 3.186; 95% confidence interval 1.494 to 7.097; p = 0.003). To examine whether the prognostic significance of nm23 was dependent on p53 status, the correlation was analyzed. As shown in Table 3, there was no correlation between nm23 status and p53 status.
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Comment
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The present study demonstrated that nm23 expression was an independent factor to predict favorable survival after complete resection in early stage, that is, p-stage I, NSCLC, suggesting that nm23 played an important role to prevent distant metastasis in NSCLC. Whereas it has been demonstrated experimentally that the nm23 gene is an antimetastatic gene, the clinical significance of nm23 expression has not been established. In most solid tumors such as breast carcinoma, reduced expression of the nm23 gene and nm23 protein is correlated with metastatic potential as well as poor prognosis [6]. In colon carcinoma, however, two studies demonstrated that nm23 expression was not correlated with metastatic potential; nm23 gene expression was equally elevated in colon carcinomas with low and high metastatic potential as compared with normal coon epithelium [16, 17]. In addition, Hailat and coworkers [18] reported that enhanced expression of nm23 was associated with tumor progression in neuroblastoma. These conflicting results may suggest that the role of the nm23 gene may alter in individual kinds of malignant tumors.
In NSCLC, some studies have suggested that the nm23 gene was an antimetastatic gene [7–9]. Lai and coworkers [7] examined nm23-H1 expression immunohistochemically in 32 patients with resected p-stage I NSCLC, and reported that no patients among 10 patients with positive nm23 expression had postoperative metastatic recurrence whereas 8 patients among 22 patients with negative nm23 expression had distant metastatic recurrence during postoperative follow-up. Kawakubo and coworkers [8] reported that nm23 expression determined immunohistochemically was inversely correlated with nodal metastases and that it was an independent factor to predict a poor prognosis in patients with resected primary lung adenocarcinoma. However, some studies demonstrated that nm23 expression determined immunohistochemically was not a significant prognostic factor in resected NSCLC [10, 11]. In these studies, nm23 expression was examined in 88 patients [10] and 46 patients [11] with resected p-stage I to IV diseases, respectively. Some studies suggested that nm23 expression was correlated with tumor progression or poor prognosis or both [12–14]. Engel and coworkers [12] analyzed nm23 gene expression using Northern blot analysis in 23 patients with squamous cell carcinoma, 5 patients with large cell carcinoma, 3 patients with carcinoid tumors, and 3 patients with sarcomas and reported that nm23 gene expression was correlated with tumor progression and poor postoperative survival. Ozeki and coworkers [13] examined nm23 expression immunohistochemically in 111 patients with p-stage I to IV adenocarcinoma and reported that high levels of nm23 expression were associated with advanced pathologic stage, positive lymph node status, and poor prognosis in Clara cell type adenocarcinoma. Gazzeri and coworkers [14] examined nm23-H1 expression immunohistochemically in 36 patients with squamous cell lung carcinoma and reported that high levels of nm23-H1 expression were associated with tumor stage. These conflicting results documented in clinical studies on nm23 expression in NSCLC are partly due to small numbers of patients as well as to heterogeneous distributions of p-stages. In fact, most studies were conducted with fewer than 100 patients and survival analyses for patients with p-stage IIIB to IV are inappropriate to reveal the prognostic significance of nm23 status. Therefore, the present study may be the first study on nm23 expression with both adequate numbers of patients and homogenous p-stage patients, where the prognostic significance of nm23 expression should be clearly revealed.
In the present study, no correlation between nm23 status and p53 status was documented, which suggested that nm23 might function as a tumor suppressor independent of p53. The present study demonstrated no significant correlation between nm23 status and proliferative activity or incidence of apoptotic cell death, although PI and AI for nm23-positive tumor were slightly higher than those for nm23-negative tumor. It may be possible that the tumor-suppressive function of nm23 is partly explained by inhibition of tumor cell proliferation and that the lack of statistical significance between enhanced nm23 expression and reduced PI is due to the relatively small number of patients reviewed in the present study. A prospective large-scale study should be conducted to clarify the function of nm23. Recently, it has been reported that the nm23-H1 gene may down-regulate the activity of N-actylglucosaminyltransferase V (GnT-V) [19]. GnT-V is a well-known enzyme associated with tumorigenesis and metastasis, as it is a key enzyme in the processing of multiantennary carbohydrate structures closely correlated with cell adhesion [20]. Thus, it can be suggested that the nm23 gene suppresses metastasis by down-regulation of GnT-V activity [19], which should be confirmed in future studies.
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Acknowledgments
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This study was supported in part by the Memorial Foundation by Tahei Ueda. We thank Miss Seiko Sakai for helpful assistance in preparation of the manuscript.
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Abstract
Introduction
