Ann Thorac Surg 2001;71:944-948
© 2001 The Society of Thoracic Surgeons
Original article: general thoracic
Autocrine motility factor receptor expression in patients with stage I non–small cell lung cancer
Murat Kara, MDa,
Yasuhiko Ohta, MDa,
Yoko Tanaka, PhDa,
Makoto Oda, MDa,
Yoh Watanabe, MDa
a First Department of Surgery, Kanazawa University School of Medicine, Kanazawa, Japan
Accepted for publication May 9, 2000.
Address reprint requests to Dr Kara, Guvenlik caddesi, Esenlik sokak 7/10, Asagiayranci, Ankara 06540, Turkey
e-mail: muratkara66{at}hotmail.com
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Abstract
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Background. Expression of autocrine motility factor receptor (AMFR) associates with increased cell migration and poor survival in certain types of human cancers. We assessed the possible correlation between AMFR, clinicopathologic features, and survival in stage I non–small cell lung cancer (NSCLC).
Methods. AMFR expression was analyzed immunohistochemically, using a monoclonal antibody (3F3A) in tumor specimens from 97 patients with curative resection. Vascular endothelial growth factor (VEGF) expression was also examined after accounting for AMFR expression.
Results. Out of 97 tumors, 38 (39.2%) were positively stained with AMFR. The AMFR expression was significantly associated with histologic type of tumor, mainly in adenocarcinoma. Overall survival of patients with AMFR-positive tumors was significantly worse than that of AMFR-negative tumors (p = 0.0050). The AMFR expression appears to be associated with VEGF expression. Patients who were AMFR positive and had high VEGF expression had a worse prognosis compared with the AMFR-negative and low VEGF-expression group (p < 0.0001). Multivariate analysis revealed an independent prognostic impact of AMFR on survival (p = 0.0039).
Conclusions. These results indicate that evaluation of AMFR expression may provide useful guidance in follow-up of patients with NSCLC.
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Introduction
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Stage I non–small cell lung cancer (NSCLC) represents early cancer and is best treated by surgery whenever possible. When only cancer-related deaths are considered 5-year survival for patients with postsurgical stage I lung cancer approaches 75% [1]. Although these groups of patients are presumed to have no lymph node metastases (N0) and no distant metastases (M0), a discouraging ratio from 16% up to 39% have recurrent disease [1–4]. Local recurrence and distant metastases remain the most common cause of death in those patients [5]. It is still unclear as to which factors cause the recurrence and decreased survival in lung cancer. If it becomes possible to define more aggressive lung cancer early in the disease state, extended surgical procedure or adjuvant treatment modalities might be employed before systemic spread is overwhelming, thereby curing more patients.
Autonomous motility of tumor cells plays an important role in recurrent disease [6, 7]. As a specific motility modifier, autocrine motility factor (AMF) was originally identified by its ability to induce migration of cells and has been implicated as playing a role in stimulating motility during invasion and metastasis [8]. AMF stimulates random and directed cell motility via its receptor, AMFR [9]. Recent studies have demonstrated that the increased expression of AMFR is strongly correlated with a high incidence of recurrence and decreased survival of patients with colorectal cancer, bladder cancer, esophageal cancer, and gastric cancer [10–13].
In the study presented here, for the first time we examined the expression of AMFR in specimens from 97 patients with primary stage I NSCLC. We tried to outline the possible correlation between the expression of AMFR, clinicopathologic features, and survival in order to establish the usage of this biomarker for prognostic evaluation.
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Material and methods
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Patients
Surgical specimens were obtained from 97 randomly selected patients with stage I NSCLC. All patients had undergone curative resection of the primary tumor including systematic lymph node dissection at the First Department of Surgery, in Kanazawa University Hospital, between 1988 and 1993. There were 67 male and 30 female patients. Their ages ranged from 46 to 84 years (mean ± standard deviation, 64.9 ± 8.3 years). None of them had received irradiation or chemotherapy prior to surgery. The operative procedures were 85 lobectomies, 9 bilobectomies, and 3 pneumonectomies. Histologically, there were 69 adenocarcinomas and 28 squamous cell carcinomas. Tumors were found to be well differentiated in 52 patients, moderately differentiated in 34 patients, and poorly differentiated in 11 patients. Pathologically, all the patients were proven to have postsurgical stage I NSCLC (T1N0M0 or T2N0M0) disease. According to current staging system for NSCLC [14], there were 62 patients in stage IA and 35 patients in stage IB. Stage I patients were chosen to eliminate the influence of distant metastases and positive lymph nodes on survival. Complete follow-up was obtained by at least 60 months and the mean observation period was 64.1 ± 27.7 months (7 to 105).
Immunohistochemical staining procedures
The pathology report with one representative formalin-fixed, paraffin-embedded block of primary tumor and adjacent normal tissue was obtained for each case. The avidin-biotin-peroxidase complex (ABC) technique was used for immunohistochemical staining. Four-micrometer sections were mounted on poly-1-lysine coated glass slides and dried. After deparaffinization with xylene and rehydration with a series of decreasing alcohol concentrations, antigen retrieval was performed by microwaving slides in 5% urea for 20 minutes. Endogenous peroxidase activity was blocked in 0.3% hydrogen peroxide in phosphate-buffered saline (PBS, pH 7.2) for 15 minutes. After rehydration and washing in PBS, sections were incubated with 10% normal goat serum (DAKO, Glostrup, Denmark) for 10 minutes at room temperature to block nonspecific binding of the second antibody. Sections were incubated with anti-AMFR monoclonal antibody, 3F3A [10, 12, 15, 16], at a dilution of 1:200 in PBS overnight at 4°C. After washing in PBS, sections were incubated with biotinylated anti-rat immunoglobulin (Vectastain ABC Kit; Vector, Burlingame, CA) for 20 minutes at room temperature and then washed again in PBS and reacted with streptoavidin-biotin system (DAKO) for 20 minutes at room temperature. Immune conjugate was visualized with PBS containing both 0.02% (w/v) 3,3'-diaminobenzidine tetrahydrocloride and 0.03% (v/v) hydrogen peroxide. All sections were counterstained with Meyer hematoxylin. Negative controls were prepared by substituting PBS for the primary antibody.
Evaluation of AMFR and VEGF expression
All sections were analyzed in a blinded fashion without knowledge of the patient’s clinical information. The degree of monoclonal antibody reactivity was considered positive if unequivocal staining of membrane and cytoplasm was seen in more than 10% of tumor cells as described previously [11–13]. VEGF expression was scored as high if more than 50% of the tumor area was stained, as reported by the authors recently [17].
Statistical analysis
All results were analyzed by a statistical analysis software package (Stat-View version 4.5; SAS Institute, Cary, NC). Statistical comparisons of baseline data between groups were carried out by the 
2 test. The AMFR expression, age, gender, T factor, histology, and differentiation were included in the assessment of prognostic factors. Age was classified as a high or low group relative to the median value. Cancer-specific survival was defined as the time between the operation and the last follow-up or cancer-related death. The cumulative survival rates were calculated by the Kaplan-Meier method and the significance was assessed by the log-rank test. The Cox proportional-hazards model was applied for univariate and multivariate analysis to confirm the prognostic impact of the factors on survival. Significance was defined as p less than 0.05.
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Results
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Among the 97 lung cancer specimens, there were 38 (39.2%) tumors positively stained with AMFR, while the staining in the remaining 59 (60.8%) tumors was negative. The AMFR antigen was mainly identified in both cell membrane and the cytoplasm of cancer cells (Fig 1). Weak staining of bronchial epithelium, vascular endothelium, macrophages, and glandular cells was also noticed in some of the sections.
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Fig 1. Immunohistochemical staining of autocrine motility factor receptor (AMFR) in a well-differentiated adenocarcinoma of the lung. Both cytoplasm and cell membrane staining were confirmed. Scale bar = 50µm.
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There was no significant association between the AMFR expression and age, gender, tumor size, or differentiation. Histologically, out of 69 adenocarcinomas 32 (46.3%) showed positive AMFR expression, which was significantly higher, compared with squamous cell carcinomas of which only 6 (21.4%) showed the same feature (p = 0.0226; Table 1).
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Table 1. Correlation of Clinicopathologic Features and AMFR Expression With Prognostic Value of AMFR Expression in Subgroups of Variables
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Comparison of 5-year survival in each subgroup stratified by AMFR revealed that expression of AMFR had a statistically significant effect in subgroups defined by age (< 65), gender (female), histology (adenocarcinoma), differentiation (moderate/poorly), and tumor size (T1,T2). We also compared the overall survival among subgroups of T-factor. Tumors larger than 3 cm with positive AMFR expression had significantly worse prognosis than tumors smaller than 3 cm without AMFR expression (p = 0.0023; Fig 2). The 5-year survival rate of T1, AMFR-negative tumors was 86.5%, having a significantly higher survival rate than that of T2, AMFR-positive tumors, which was 7.7%.
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Fig 2. Cumulative survival curves of patients with or without autocrine motility factor receptor (AMFR) expression in subgroups of T-factor.
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Significant association was observed between AMFR expression and survival time. Overall survival of patients with AMFR-positive tumors was significantly worse than that of AMFR-negative tumors (p = 0.0050; Fig 3). The 5-year survival rates of the AMFR-positive group and AMFR-negative group were 50.0% and 72.9%, respectively.
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Fig 3. Cumulative survival curves on the basis of autocrine motility factor receptor (AMFR) expression of tumors in 97 patients with non–small cell lung carcinoma.
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Significant univariate predictors for decreased survival were T-factor or the stage of the tumor (p = 0.0153) and positive AMFR expression (p = 0.0066). Multivariate analysis revealed independent prognostic value of AMFR positivity and T-factor on overall survival (Table 2).
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Table 2. Six Variables as Prognostic Factors in 97 Patients With Non–Small Cell Carcinoma According to Univariate and Multivariate Analysis of Cox Proportional-Hazards Model
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The AMFR positivity rate in the high VEGF-expression subgroup was 60.0%, while that in the low VEGF-expression subgroup was 31.9% (Table 3). The difference was statistically significant (p = 0.0133), suggesting the association between AMFR and VEGF expression. Overall survival of patients with positive AMFR and high VEGF expression was significantly worse than that of patients with negative AMFR and low VEGF expression (p < 0.0001; Fig 4). The 5-year survival rates of the former and the latter group were 13% and 83%, respectively. Expression of AMFR had a significant effect on overall survival in the low VEGF-expression subgroup (p = 0.0497) whereas its expression was not significant in the high VEGF-expression subgroup (p = 0.7721; Table 3).
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Fig 4. Cumulative survival curves of patients according to autocrine motility factor receptor (AMFR) and vascular endothelial growth factor (VEGF) expression levels.
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Comment
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Cell migration appears to be a vital process in tumor cell invasion and metastasis; however, very little information is available as to mechanisms underlying cell motility and motility stimulation. Recently, cell motility has been shown to be affected by various factors including AMF. AMF is a tumor-secreted cytokine that stimulates both random and directed cell migration through binding to its receptor, AMFR/gp78 [15, 16], which has been identified and cloned from murine B16-F1 melanoma cells as a 78-kDa cell surface glycoprotein [18]. It is of interest that the AMFR expression correlates with increased migratory ability in vitro [19] and increased ability in vivo as colonizing lungs in an experimental tumor system [20]. In humans, it was reported that AMFR expression associated with high incidence of recurrence and decreased survival in certain types of cancers [10–13].
We investigated AMFR expression in 97 NSCLC specimens by means of immunohistochemical staining methods and found that AMFR was positively expressed in 39% of NSCLC tumors. When we analyzed the association between AMFR expression and clinicopathologic features, we could only demonstrate significant association of histologic type of tumor with AMFR expression. In adenocarcinomas, 46.3% showed positive AMFR expression, which was statistically significant compared with squamous cell carcinomas, of which only 21.4% showed the same feature (p = 0.0226). However, we could not show any correlation with AMFR expression or any effect of AMFR on survival for squamous cell carcinomas. To our knowledge, this is the first report to clarify the relationship between AMFR expression, clinicopathologic features, and survival in NSCLC.
In our study, T-factor or the stage of the tumor was shown to have influence on the survival either alone (data not shown) or with AMFR expression. Recently, T-factor in NSCLC has been of concern and has also shown to be an independent prognostic factor for stage I NSCLC tumors [21–23]. These findings led the investigators to classify this stage of tumors into two groups as stage IA and stage IB. Although we could not demonstrate any significant association between AMFR expression and resection type (data not shown), we might suggest that regardless of the tumor size, if preoperatively obtained biopsy specimens of NSCLC express high levels of AMFR, performing wider resection, at least not less than standard lobectomy, with lymph node dissection will be of benefit.
Upon analyzing T-factor and AMFR expression by means of multivariate analysis, we found that either positive AMFR expression or T-factor retained its independent prognostic impact.
Interestingly, we found a significant association between AMFR and VEGF expression. As reported previously, VEGF expression has a strong prognostic impact on overall survival in lung cancer [17, 24]. This finding might explain why AMFR expression did not make any significant difference in the high VEGF-expression subgroup.
We conclude that T-factor, particularly T2, is of importance in terms of survival in patients with Stage I NSCLC and that decreased survival is likely when such a tumor expresses high levels of AMFR. AMFR expression is an independent prognostic factor of survival in adenocarcinomas. There is an association between AMFR and VEGF expression that needs further study. AMFR expression may be a marker of metastatic potential, and its use as a guide to adjuvant therapy needs additional confirmation.
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Acknowledgments
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We extend our gratitude to Dr Hideomi Watanabe from Gunma University School of Medicine, Japan, and Dr Avraham Raz from Wayne State University School of Medicine, Detroit, MI, for their kindness in supplying the monoclonal antibody for AMFR.
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Abstract
Introduction
