Ann Thorac Surg 1996;62:1500-1504
© 1996 The Society of Thoracic Surgeons
Original Article: General Thoracic
Immunohistochemical Analysis of nm23-H1 in Stage I Non–Small Cell Lung Cancer: A Useful Marker in Prediction of Metastases
Wu-Wei Lai, MD,
Ming-Ho Wu, MD,
Jing-Jou Yan, MD,
Feng-Feng Chen, MD
Division of Thoracic Surgery, Departments of Surgery and Pathology, National Cheng Kung University Hospital, Taiwan, Republic of China
Accepted for publication June 15, 1996.
 |
Abstract
|
|---|
Background. About 30% to 40% of patients with pathologic stage I non–small cell lung cancer die within 5 years after complete resection. The identification of subgroups with high risk of recurrence is important. The level of nm23-H1/nucleoside diphosphate kinase expression has been reported to correlate inversely with the metastatic potential of some tumors.
Methods. We examined immunohistochemically the expression of nm23-H1 in 32 stage I non–small cell lung cancers to determine its value in predicting the outcome of the operation.
Results. Twelve (37.5%) recurrences and ten deaths occurred in this series. There were eight (25%) distant and four (12.5%) locoregional recurrences. Of the 32 pathologic stage I non–small cell lung cancers, 10 (31.3%) were positive for nm23-H1 gene product. None of the patients with a higher nm23-H1 protein level had detectable distant metastases at the end of this study (
2 test, p < 0.05). The median follow-up time to date is only 35 months (range, 18 to 83 months), and so far no significant difference in overall survival rate has been observed between the groups with high and low nm23-H1 protein levels.
Conclusions. The level of nm23-H1 protein is more useful than the T status or histologic type for the prediction of distant metastases, whereby cases may be selected for postoperative adjuvant chemotherapy.
|
Introduction
|
|---|
The average 5-year survival rate for patients with stage I non–small cell lung cancer (NSCLC) is approximately 64.6% (range, 55% to 72%) [1]. This means that about 30% to 40% of stage I patients will have recurrence of the tumor and die despite complete resection [2]. At present, postoperative adjuvant chemotherapy is not a routine standard procedure for these patients. We think that methods to identify the group at high risk of recurrence may lead to selective and effective chemotherapy of these patients.
Nm23 was originally identified as the antimetastatic gene [3]. The level of nm23-H1/nucleoside diphosphate kinase expression has been reported to correlate inversely with distant metastatic potential in some tumors [4, 5]. Whether nm23-H1 gene product is associated with distant metastatic potential in early stage I NSCLC has not been evaluated. This study represents an attempt to determine whether immunohistochemical determination of nm23-H1 gene product could be of value in prognostication.
|
Material and Methods
|
|---|
Patients
Between July 1988 and October 1994, 111 patients underwent resection of lung malignancy in the National Cheng Kung University Hospital, Tainan, Taiwan. Of these, 38 were staged pathologically as stage I NSCLC (squamous cell carcinoma, 25; adenocarcinoma, 13), and as of November 1995, these had been followed up for more than 18 months. There were 25 men and 7 women in this series, with a mean age of 61.5 years (squamous, 15 men and 4 women; mean age, 63 years; adenocarcinoma, 10 men and 3 women; mean age, 59.3 years). The median follow-up period was 35 months (range, 18 to 83 months). The medical charts, operative records, and pathologic reports were reviewed. Six of the patients with squamous cell carcinoma were excluded: 3 had incomplete follow-up; 1 died after 4 months of respiratory failure due to bronchopleural fistula; 1 died after 8 months of cerebrovascular disease; and 1 had local recurrence 2 months later because of microscopic incomplete bronchial resection.
Detection of the Recurrence of Cancer
Locoregional recurrence was defined as clinically or radiologically demonstrable evidence of tumor at the bronchial surgical stump or within the same lung, pleural space, mediastinum, supraclavicular nodes, or ipsilateral chest wall [6]. Distant recurrence was diagnosed when there were multiple nodules on the contralateral lung or a solitary nodule on the contralateral lung with extrapulmonary metastases. Diagnosis of a second primary lung cancer was based on the presence of a solitary lung nodule in the absence of other sites of recurrence and an interval between the first operation and recurrence of longer than 2 years [7, 8].
Immunohistochemical Determination of nm23-H1 Protein
The nm23-H1 protein was detected with a specific monoclonal antibody against nm23-H1 protein, nucleoside diphosphate kinase A (Oncogene Science, Cambridge, MA).
Formalin-fixed, paraffin-embedded, 4-µm sections were obtained from the department of pathology. They were deparaffinized with xylene and progressively dehydrated in decreasing concentrations of alcohol. Endogenous peroxide activity was blocked by incubating the sections in a 3% solution of hydrogen peroxide in methanol for 5 minutes. The sections were immersed in normal goat serum for 15 minutes to reduce nonspecific staining and then incubated overnight at 4°C with 1:100 monoclonal antibody. They were washed with phosphate-buffered saline solution and incubated with a 1:30 dilution of biotinylated goat anti-mouse immunoglobulin G (DAKO, Kyoto, Japan) at room temperature for 30 minutes. The sections were washed with phosphate-buffered saline and covered with a 1:100 dilution of streptavidin-biotin-peroxidase complex (DAKO) at room temperature for 30 minutes. The antibody was visualized with 3,3`-diaminobenzidine tetrahydrochloride and 0.3% H2O2 in phosphate-buffered saline solution. The negative control consisted of sections not incubated with primary antibodies to nm23-H1, whereas the positive control was prepared with breast cancer tissues, as recommended by the manufacturer (Oncogene Science). The slides were lightly counterstained with hematoxylin. The results, based on the intensity of the staining reaction with monoclonal antibody, were classified into two grades. Negative means that the staining reaction of the tumor section was similar to that of the negative control; positive denotes that greater than 10% of the cancer cells had a staining reaction similar to or more intense than that of the positive control [9].
Statistical Analysis
Statistical significance was evaluated using the 2 test, with p < 0.05 as the criterion. Patients who had incomplete follow-up or died of postoperative complications or of nontumor-related disease were excluded. Survival time and disease-free interval were computed from the date of operation. Survival curves were plotted according to the Kaplan-Meier method, and differences between the curves were analyzed by the log rank test [10]. These analyses were performed with the SPSS 6.0 software package for Windows (SPSS Inc, Chicago, IL).
|
Results
|
|---|
The operative procedures and conditions of recurrence in the 32 stage I NSCLC cases are shown in Table 1
. According to the Kaplan-Meier survival curves of these patients (Fig 1), the 5-year survival rate was 60.5% (squamous, 62.3%; adenocarcinoma, 57.7%). Differences in survival of the patients were not significant in terms of T status (T1 or T2; data not shown) or histologic type (squamous, adenocarcinoma) of the tumor.
View larger version (31K):
[in this window]
[in a new window]
|
Fig 1. . Cumulative (cum) survival by histologic type (p > 0.05), or time to death after operative treatment. (Adeno. = adenocarcinoma.)
|
|
During follow-up, there were 12 (37.5%) recurrences (squamous, 36.8%, 7/19; adenocarcinoma, 38.4%, 5/13) and ten deaths. There were eight (25%) distant recurrences (squamous, 26.3%, 5/19; adenocarcinoma, 23%, 3/13) and four (12.5%) locoregional recurrences (squamous, 10.5%, 2/19; adenocarcinoma, 15.4%, 2/13; Table 2). Two of the four patients with locoregional recurrence are alive, and 1 of these has had control of his recurrence by the second operation. There was no significant correlation between the incidence of recurrence (locoregional plus distant) and the T status (data not shown) or histologic type of the tumor (Fig 2).
The recurrence-free survival curves (interval between operation and recurrence) of these patients obtained by the method of Kaplan and Meier are also shown in Figure 2
. The estimated rate of recurrence was on average 10.4% per year of observation. The median duration from operation to the recurrence in 12 patients was 26 months (range, 4 to 58 months). In 75% of the patients with recurrence, it developed within the first 2.5 years after the operation.
Of the 32 pathologic stage I NSCLC cases, 10 (31.3%) were positive for nm23-H1 gene product (squamous, 26.3%, 5/19; adenocarcinoma, 38.5%, 5/13). Positive cells showed diffuse cytoplasmic staining (Fig 3). Little or no nm23-H1 expression was observed in noncancerous parts of the lung.
View larger version (132K):
[in this window]
[in a new window]
|
Fig 3. . Immunohistochemical staining of nm23-H1 protein in stage I non–small cell lung cancer (adenocarcinoma). Positive cells showed diffuse cytoplasmic staining. No immunoreaction was observed in noncancerous tissue. (Original magnification x200.)
|
|
There appeared to be no significant correlation between distant metastases and the T status or histologic type in these stage I NSCLC patients. The incidence of distant metastases was significantly higher in patients with a lower level of nm23-H1 protein (see Table 2, Fig 4; p < 0.05, 2 test, log rank test), although the level of nm23-H1 protein had no significant bearing on the overall survival (Fig 5). Table 3 shows the relation between the level of nm23-H1 protein and the degree of histologic differentiation.
View larger version (26K):
[in this window]
[in a new window]
|
Fig 4. . Kaplan-Meier distant recurrence-free curve of stage I non–small cell lung cancer with regard to nm23-H1 protein expression. There was a significant difference between low expression (n = 22), with eight distant metastases, and high (+) expression (n = 10), with no detectable distant metastases yet (p < 0.05). The line of high expression was not plotted because of the design of SPSS 6.0 software. The median follow-up period for these 10 patients with high expression of nm23-H1 was 40.5 months (range, 19 to 79 months).
|
|
View larger version (31K):
[in this window]
[in a new window]
|
Fig 5. . Kaplan-Meier cumulative (cum) survival curve of stage I non–small cell lung cancer patients with regard to nm23-H1 protein expression. There was no statistical significance (p > 0.05) between high (+) expression (n = 10) and low (-) expression (n = 22).
|
|
|
Comment
|
|---|
Patients with pathologic stage I NSCLC are generally considered to have neither distant nor mediastinal lymph node metastases beyond the resected specimen. Yet the average 5-year survival rate for these patients is only 64.6% (range, 55% to 72%). Thus, approximately 35% (range, 30% to 40%) of these patients have recurrent lung cancer and die within 5 years after complete resection. The existence of heterogeneity among these patients should be recognized [1]. The 5-year survival rate in our series was 60.5% (squamous, 62.3%; adenocarcinoma, 57.7%), which places it at the lower end of the range reported in the literature. The general incidences of recurrence reported in the literature among patients with stage I NSCLC are 7% to 19% for locoregional recurrence and 18.2% to 20.2% for distant recurrence [2, 6, 11]. Our experience with locoregional recurrence was quite consistent, at 12.5% (4/32). In contrast, the incidence of distant metastases, 25% (8/32) in our series, was slightly higher than what is generally reported.
A variety of clinical and pathologic features have been recognized as important determinants of outcome of operative treatment of stage I NSCLC. The tumor size (T1 versus T2) and histologic subtype (squamous vs nonsquamous) have been the most widely studied. Patients with T1 N0 tumor have had better prognosis. The correlation between postoperative survival and the histologic subtype as observed by different groups has not been consistent [1, 2, 6]. To predict the probability of distant metastases, neither T status nor histologic types is useful. The level of nm23-H1 protein, on the other hand, seems to be a practical index (2 test, p < 0.05) (see Table 2).
Diminished nm23 expression is often associated with a clinical course of high metastatic potential in several human tumors, including breast carcinoma [12], hepatoma [5, 9], melanoma [13], and colorectal cancer [14]. There exist, however, no reports on the extent of nm23-H1 gene product expression and distant metastases in early-stage (stage I) NSCLC. However, high levels of nm23-H1 protein associated with advanced pathologic stage, positive lymph node status, and poorer prognosis in lung adenocarcinoma of the Clara cell type has been noted [4, 15]. With squamous cell carcinoma of the lung, the increase in nm23 messenger RNA expression is observed in advanced, poorly differentiated tumor, and is inversely correlated with the disease-free survival period [16]. This is in contrast to our observation. None of our patients with a higher nm23-H1 protein level had detectable distant metastases at the end of our study (2 test, p < 0.05; see Table 2, Fig 4).
According to the studies of Ozeki and associates [4] and Higashiyama and colleagues [15], the rate of immunohistochemical positivity of nm23-H1 protein for stage I or stage I plus II adenocarcinoma is 36% and 57.5%, respectively. In our hands, 10 (31.3%) of the 32 cases were positive (squamous, 5/19, 26.3%; adenocarcinoma, 5/13, 38.5%), and little or no nm23-H1 expression has been observed in noncancerous parts of the lung. Engel and associates [16] showed that nm23 messenger RNA was very low or negligible in the normal part of the lung as compared with that containing the lung tumor. In light of our observations, it is interesting to note that, in experimental studies in mice, the nm23 protein level stays low in the primordial tissues of the heart, brain, and most epithelial tissues during their early development and then increases during their functional differentiation. Most tissues retain their high nm23 level in adult life; however, lung and intestine levels are only transient [17]. The mechanisms causing enhanced nm23-H1 expression in NSCLC are unknown. It is also of interest that the nm23-H1 gene has been reported as a late-acting suppresser gene on chromosome 17, on which the p53 and HER2/neu genes are located; these genes are implicated in the development of lung cancer and other cancers [18–21]. Activation of the nm23-H1 gene might be a prerequisite for oncogenesis of NSCLC. It is important to note that the association between nm23-H1 expression and well-differentiated tumors is not absolute [4, 15, 16]. In our study, the correlation between poor histologic differentiation and low nm23-H1 protein level was not established (see Table 3).
To predict distant metastases, the level of nm23-H1 protein was more useful than the T status or histologic type in this study. Patients with a high level of nm23-H1 protein had less incidence of distant metastases but a tendency to have late locoregional recurrence (mean duration from operation to regional recurrence, 56 months; see Table 2). In our opinion, the level of nm23-H1 protein could be a useful tool to identify the subpopulation of patients with early-stage NSCLC who have a higher risk of distant metastases and might benefit from postoperative adjuvant chemotherapy.
|
Acknowledgments
|
|---|
We gratefully acknowledge support by the National Science Council, Taiwan, Republic of China (NSC 83-0412-B-006-081, NSC 84-2331-B-006-032) and Miss Lin-Fang Hou for her help in the statistical analysis of our data.
|
Footnotes
|
|---|
Address reprint requests to Dr Lai, Division of Thoracic Surgery, Department of Surgery, National Cheng Kung University Hospital, 138 Sheng Li Rd, Tainan, Taiwan, Republic of China.
|
References
|
|---|
- Nesbitt JC, Putnam JB, Walsh GL, Roth JA, Mountain CF. Survival in early-stage non-small cell lung cancer. Ann Thorac Surg 1995;60:466–72.[Abstract/Free Full Text]
- Strauss GM, Kwiatkawski DJ, Harpole DH, Lynch TJ, Skarin AT, Sugarbaker DJ. Molecular and pathologic markers in stage I non-small cell carcinoma of the lung. J Clin Oncol 1995;13:1265–79.[Abstract]
- Steeg PS, Bevilacqua G, Kopper L, et al. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 1988;80:200–4.[Abstract/Free Full Text]
- Ozeki Y, Takishima K, Mamiya G. Immunohistochemical analysis of nm23/NDP kinase expression in human lung adenocarcinoma: association with tumor progression in clara cell type. Jpn J Cancer Res 1994;85:840–6.[Medline]
- Nakayama T, Ohtsuru A, Nakao K, et al. Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product. J Natl Cancer Inst 1992;84:1349–54.[Abstract/Free Full Text]
- Martini N, Bain MS, Burt ME, et al. Incidence of local recurrence and second primary tumors in resected stage I lung cancer. J Thorac Cardiovasc Surg 1995;109:120–9.[Abstract/Free Full Text]
- Thomas P, Rubinstein L, and the Lung Cancer Study Group. Cancer recurrence after resection: T1N0 non-small cell lung cancer. Ann Thorac Surg 1990;49:242–7.[Abstract]
- Martini N, Melamed MR. Multiple primary lung cancers. J Thorac Cardiovasc Surg 1975;70:606–12.[Abstract]
- Yamaguchi A, Urano T, Goi T, et al. Expression of human nm23-H1 and nm23-H2 proteins in hepatocellular carcinoma. Cancer 1994;73:2280–4.[Medline]
- Collett D, ed. Modelling survival data in medical research. 1st ed. London: Chapman & Hall, 1994:40–1.
- Pairolero PC, William DE, Bergstralh EJ, Piehler JM, Bernatz PE, Payne WS. Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease. Ann Thorac Surg 1984;38:331–8.[Abstract]
- Bevilacqua G, Sobel ME, Liotta LA, Steeg PS. Association of low nm 23 RNA levels in human primary infiltrating duct breast carcinoma with lymph node involvement and other histopathological indicators of high metastatic potential. Cancer Res 1989;49:5185–90.[Abstract/Free Full Text]
- Florenes VA, Aamdal S, Myklebost O, Madelandsmo GM, Bruland O, Fodstad O. Levels of nm23 messenger RNA in metastatic malignant melanomas: inverse correlation to disease progression. Cancer Res 1992;52:6088–91.[Abstract/Free Full Text]
- Yamaguchi A, Urano T, Fushida S, et al. Inverse association of nm23-H1 expression by colorectal cancer with liver metastasis. Br J Cancer 1993;68:1020–4.[Medline]
- Higashiyama M, Doi O, Yokouchi H, et al. Immunohistochemical analysis of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma: lack of prognostic value. Br J Cancer 1992;66:533–6.[Medline]
- Engel M, Theisinger B, Seib T, et al. High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages. Int J Cancer 1993;55:375–9.[Medline]
- Lakso M, Steeg PS, Westphal H. Embryonic expression of nm23 during mouse organogenesis. Cell Growth Differ 1992;3:873–9.[Abstract]
- Cohn KH, Wang F, Desoto-Lapaix F, et al. Association of nm23-H1 allelic deletions with distinct metastases in colorectal carcinoma. Lancet 1991;338:722–4.[Medline]
- Rosengard AM, Krutzsch HC, Shearn A, et al. Reduced nm23/awd protein in tumor metastasis and aberrant Drosophila development. Nature 1989;342:177–80.[Medline]
- Miller C, Mohandas T, Wolf D, et al. Human p53 gene localized to short arm of chromosome 17. Nature 1984;319:783–4.
- Spurr NK, Solomon E, Jasson M, et al. Chromosomal localization of the human homologues to the oncogenes erbA and B. EMBO J 1984;3:159–63.[Medline]
This article has been cited by other articles:
|
|
|
|
 
H. Katakura, F. Tanaka, H. Oyanagi, R. Miyahara, K. Yanagihara, Y. Otake, and H. Wada
Clinical significance of nm23 expression in resected pathologic-stage I, non-small cell lung cancer
Ann. Thorac. Surg.,
April 1, 2002;
73(4):
1060 - 1064.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A N J Graham, P Maxwell, K Mulholland, A H Patterson, N Anderson, K G McManus, H Bharucha, and J A McGuigan
Increased nm23 immunoreactivity is associated with selective inhibition of systemic tumour cell dissemination
J. Clin. Pathol.,
March 1, 2002;
55(3):
184 - 189.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
