Purpose: The aim of this study was to determine whether collagen XVIII expression is correlated with circulating serum endostatin and whether this has any prognostic value in patients with non–small cell lung cancer (NSCLC).
Experimental Design: Serum endostatin levels were measured quantitatively by a competitive enzyme immunoassay, and collagen XVIII expression in tumor tissue was investigated with an immunohistochemical method in a series of 94 patients who underwent surgery for NSCLC.
Results: Sixty cases (63.8%) had positive immunohistochemical staining with anticollagen XVIII polyclonal antibodies, including strongly positive staining in 11 (11.7%) cases. The mean (± SD) serum endostatin level was 41.6 ± 34.4 ng/ml in the patient group and 16.3 ± 10.3 ng/ml in the control group (P < 0.0001). The 11 cases who were strongly collagen XVIII-positive had significantly higher serum endostatin levels than the cases who were negative or weakly positive (P = 0.0297). The 5-year survival rates of negative, weakly positive, and strongly positive patients were 77.8%, 56.9%, and 43.8%, respectively. The cases with strongly positive collagen XVIII expression had a significantly poorer outcome than cases with negative expression (P = 0.0027). A multivariate analysis with Cox proportional hazards model for disease-specific survival revealed that expression of collagen XVIII (strongly positive versus negative; weakly positive versus negative), tumor classification, and regional lymph node classification were independent prognostic factors.
Conclusions: Our results suggest that expression of collagen XVIII in tumor tissue is strongly associated with a poorer outcome in NSCLC and correlates with elevated levels of circulating serum endostatin.
Distant metastasis is the most common cause of death resulting from solid malignant tumors, including non–small cell lung cancer (NSCLC), even if the tumors have been completely excised. NSCLC frequently gives rise to metastatic lesions in the lungs, brain, bones, adrenal glands, liver, and other organs.
Angiogenesis plays an important role in the development of micrometastases through fine regulation of endogenous angiogenic factors and endogenous negative regulators of angiogenesis in endothelial cells (1) . Endostatin, a specific inhibitor of endothelial cell proliferation and angiogenesis, was initially isolated from the conditioned media of a murine hemangioendothelioma cell line; it has a molecular weight of 20 000 and a structure consistent with the COOH-terminal domain of collagen XVIII (2) . Collagen XVIII is an extracellular matrix protein that is a component of vascular and epithelial basement membranes throughout the body (3) . The long form of collagen XVIII is found exclusively in the liver and is expressed at high levels by hepatocytes and in hepatocellular carcinomas (3 , 4) . The biological function of collagen XVIII remains unclear; however, it has recently received much attention because it is an endostatin precursor.
Many studies have shown that the administration of endostatin suppresses the growth of both primary and metastatic lesions in experimental animal models (2 , 5, 6, 7, 8, 9, 10) . Dhar et al. (11) reported that serum endostatin levels are inversely correlated with angiogenesis. Some reports have also demonstrated a correlation between the development of metastatic lesions and serum endostatin concentration in human malignancies (12, 13, 14) . To date, no reports have demonstrated a link between overexpression of endostatin in tumor tissue and tumor recurrence after surgery.
In this study, we attempted to clarify the significance of endostatin and collagen XVIII expression in angiogenesis in metastatic lesions of NSCLC patients. We investigated the relationships among circulating serum endostatin, expression of the endostatin precursor collagen XVIII in tumor samples, and various clinical features in NSCLC patients. In addition, we assessed whether serum endostatin and collagen XVIII expression influence tumor recurrence and/or prognosis in NSCLC.
MATERIALS AND METHODS
Patient Plasma and Tumor Tissue Samples.
The study group consisted of 94 patients with NSCLC (69 males and 25 females) who underwent surgery at Chiba University Hospital between 1997 and 2000 with suitable preoperative eligible criteria as described previously (14) . The patients ranged in age from 40 to 83 years (mean ± SD, 64.4 ± 10.0 years). NSCLC was diagnosed histologically in surgically excised tissues. Blood samples were drawn before surgery. Histological types included 64 adenocarcinomas, 28 squamous cell carcinomas, and 2 large cell carcinomas. Pathology staging revealed 40 patients with stage IA or IB disease, 14 patients with stage IIA or IIB disease, 34 patients with stage IIIA or stage IIIB disease, and 6 patients with stage IV disease (see Table 1⇓ ). All cases underwent complete resection with 93 lobectomies and 1 segmentectomy. None of the patients received treatment for NSCLC before surgery. For the first year after surgery, patients were followed every month, then every 3 months from 2 to 5 years, and every 6 months thereafter. Informed consent was obtained from all patients, and the study was approved by the institutional ethics board. Classification of histological type, pathological stage, and tumor-node-metastasis (TNM) classification was performed according to the criteria of the American Joint Committee on Cancer, the staging committees of the International Union against Cancer (15) , and the General Rules for Clinical and Pathological Recording of Lung Cancer described by the Japan Lung Cancer Society (16) . As a control, endostatin serum concentrations were measured in healthy volunteers (n = 16).
Competitive Enzyme Immunoassay and Immunohistochemistry.
Serum endostatin levels were measured with a competitive enzyme immunoassay kit (ACCUCYTE Human Endostatin kit; Cytimmune Science, Inc., College Park, MD) as described previously (14) . The minimum detectable endostatin level was 1.95 ng/ml.
Expression of collagen XVIII was confirmed immunohistochemically in 94 patient tumor samples to clarify the localization of expression as described previously (17) . Formalin-fixed, paraffin-embedded, 4-μm sections on silanized slides (Dako, Glostrup, Denmark) were treated with 3% hydrogen peroxide in PBS [0.01 m sodium phosphate (pH 7.2), 0.15 m NaCl] to block endogenous peroxidase and normal rabbit serum to block nonspecific binding sites and were incubated overnight at 4°C with the primary antibody. Immunohistochemical staining was performed with the streptavidin-biotin technique (Histofine kit; Nichirei, Tokyo, Japan) with anticollagen XVIII polyclonal antibodies that have been established to two polypeptides of collagen XVIII (134–150 and 503–518) according to the manufacturer’s instructions (IBL Co., Ltd., Gunma, Japan). The specificity of these antibodies to NSCLC has been confirmed previously with Western blotting (18) . Peroxidase activity was measured with 3,3′-diaminobenzidine. The primary antibody was absent in negative controls. Sections were counterstained with hematoxylin. Without having any previous knowledge of each patient’s clinical and pathological data, all slides were evaluated independently by two pathologists (A. I., K. H.). A score was established corresponding to the sum of (I) the percentage of positive cells (0, negative; 1, <25% positive cells; 2, 26–50% positive cells; and 3, >50% positive cells) and (II) the staining intensity (0, negative; 1, weak; 2, moderate; and 3, high). The maximum sum of (I) + (II) was 6. Scores between 0 and 2 were regarded as negative, scores of 3 and 4 as positive, and scores between 5 and 6 as strongly positive.
Statistical Analysis and Survival.
We used the Mann–Whitney test to perform statistical analyses of serum endostatin levels in each group. The end point was survival time until death from the primary lung cancer, calculated as the time from the date of surgery to the date of death. The cause of death was confirmed by telephone interview with the doctor who monitored the patient. Cause of death was classified as primary lung cancer-related if the patient had a recurrence of the primary lung cancer at the time of death (distant metastasis, local recurrence, or both), excluding death due to respiratory disease, cardiac disease, brain vascular disease, second malignancy, or others. In this study, we analyzed disease-specific survival from the primary lung cancer by censoring patients with nonprimary lung cancer-related disease at the date of death. Disease-specific survival curves were plotted using the Kaplan–Meier method, and the log-rank test was used to assess the statistical significance of differences between groups. We assessed the simultaneous contribution of each covariate in the multivariate analysis, using Cox’s proportional hazards model for the risk ratio in the SPSS statistical software program package (SPSS version 11.0 for Windows, SPSS Inc., Chicago, IL). We used a probability level of 0.10 as the significance level for adding and deleting a covariable from the model. The correlation between T and N status and staging was evaluated with the Spearman rank correlation coefficient (data not shown); we thus excluded staging, which is well recognized to be strongly associated with survival, as a covariable. P values <0.05 were considered statistically significant.
Immunohistochemical Staining of Tumor Samples with Anticollagen XVIII Antibody and Association between Prognosis and Clinical Variables in 94 NSCLC Patients.
Of 94 patients, 60 (63.8%) had positive immunohistochemical staining for collagen XVIII, including weakly positive staining in 49 (52.1%) cases and strongly positive staining in 11 (11.7%) cases (Fig. 1)⇓ . Tumor classification (P = 0.0015), regional lymph node classification (P < 0.0001), postsurgical stage (P < 0.0001), and immunohistochemical expression of collagen XVIII (P = 0.0108) demonstrated a significant correlation with prognosis, respectively, based on the log-rank test (Table 1)⇓ .
Correlation between Serum Endostatin Levels and Collagen XVIII Expression.
The mean serum endostatin level was 41.6 ± 34.4 ng/ml in the patient group and 16.3 ± 10.3 ng/ml in the control group (P < 0.0001, Mann–Whitney test).
The 11 cases with strongly positive immunohistochemical staining for collagen XVIII had a statistically higher serum endostatin concentration than the cases with negative or weakly positive staining (P = 0.0297, Mann-Whitney test; Fig. 2⇓ ).
Association between Disease-Specific Survival and Collagen XVIII Expression.
Cases with negative, weakly positive, or strongly positive collagen XVIII expression had 5-year survival rates of 77.8, 56.9, and 43.8%, respectively. The cases with strongly or weakly positive collagen XVIII expression had a significantly poorer outcome than the cases with negative expression (P = 0.0027, log-rank test; Fig. 3⇓ ).
Multivariate Analysis for Disease-Specific Survival.
Multivariate analysis for disease-specific survival was performed according to the Cox proportional hazards model using four variables (tumor classification, regional lymph node classification, immunohistochemical expression of collagen XVIII, and serum endostatin level) that gave P values < 0.1 in the univariate analysis; postsurgical stage was excluded as a factor from multivariate analysis in this study because of its considerable statistical correlation with regional lymph node classification. The multivariate analysis for disease-specific survival revealed three independent prognostic factors: immunohistochemical expression of collagen XVIII (strongly positive versus negative, weakly positive versus negative), tumor classification (T2, T3, and T4 versus T1), and regional lymph node classification (N1, N2, and N3 versus N0; Table 2⇓ ).
In this study, we investigated the immunohistochemical expression of collagen XVIII in excised tumor tissues and preoperative serum endostatin levels in patients with NSCLC. We have demonstrated that expression of collagen XVIII in tumor tissue is strongly associated with a poorer outcome in NSCLC. In addition, we found that strong collagen XVIII expression correlates with elevated levels of circulating serum endostatin.
Collagen XVIII is a proteoglycan containing heparan sulfate side chains (19) . Although it belongs to the multiplexin subfamily (20) , collagen XVIII is better known as a precursor of endostatin, which has been identified as a 20-kDa COOH-terminal fragment of collagen XVIII, obtained from culture supernatant of a hemangioendothelioma cell line (EOMA), that suppresses endothelial cell proliferation and angiogenesis (2) . The mechanism for converting collagen XVIII to endostatin has not been elucidated, but some reports have noted that the ability of EOMA cells to produce endostatin is linked to their production of the cysteine protease cathepsin L and matrix metalloproteinases (21) . Elastase has also been shown to be capable of cleaving endostatin from collagen XVIII (22) . The generation of endostatin or collagen XVIII by human tumors appears to depend on tumor histology. Some reports have demonstrated that endostatin serum levels are elevated in patients with breast cancer, bladder cancer, renal cell carcinoma, endometrial cancer, and soft tissue sarcoma (12 , 23, 24, 25, 26) , whereas other investigators have reported circulating endostatin levels similar to those in healthy controls in patients with head and neck squamous cell carcinoma (27) , squamous cell vulvar carcinoma (28) , and hepatocellular carcinoma (29) . We have previously demonstrated that serum endostatin levels are elevated in patients with NSCLC (14) . In this study, we report a significant correlation between overexpression of collagen XVIII in tumor tissue from NSCLC patients and elevated serum endostatin levels, which suggests that the source of serum endostatin may be partially derived from NSCLC tumor cells.
We used immunohistochemistry to investigate the expression of collagen XVIII in NSCLC tumor samples in an attempt to determine the site of collagen XVIII production. Collagen XVIII is expressed by a wide variety of cells and tissues, from the basement membrane of epithelial tissue to hepatocytes (3) . Guenther et al. (30) used in situ hybridization to demonstrate that collagen XVIII is expressed in colorectal cancer stroma cells and ovarian cancer cells, and other investigators have reported high expression of collagen XVIII mRNA in tumor tissue from hepatocellular carcinomas (4) . However, no reports of collagen XVIII expression in NSCLC have been published. In this study, we showed that collagen XVIII was expressed in 60 of 94 NSCLC cases, including high expression in 11 cases. We also used univariate analysis to demonstrate that collagen XVIII expression is associated with a poorer outcome and multivariate and univariate analysis to show that collagen XVIII expression is an independent prognostic factor. In some patients in whom collagen XVIII was overexpressed, early-stage distant metastases were found shortly after surgery (a representative case is shown in Fig. 1⇓ ). There have been few reports concerning any correlation between expression of endostatin/collagen XVIII and long-term survival. Hata et al. (31) reported that the prognosis of ovarian cancer patients with high endostatin gene expression was significantly worse than that of patients with low endostatin expression. We have demonstrated that collagen XVIII expression in NSCLC may correspond to a poorer prognosis. This is the first report to clarify clinically the hypothesis of Folkman (1) , which states that production of an inhibitor of angiogenesis (in this case, endostatin/collagen XVIII expression) from malignant tumors correlates with the growth of metastatic lesions after surgery (2) .
In conclusion, serum endostatin detected in NSCLC patients partially originates from tumor tissues. Overexpression of collagen XVIII in NSCLC is associated with an earlier recurrence and poorer outcome compared with tumor tissues that do not express collagen XVIII. Further study on the mechanisms of in vivo production and conversion of collagen XVIII to endostatin could help elucidate this aspect of angiogenesis regulation in NSCLC. Finally, these results suggest that collagen XVIII/endostatin may be a novel therapeutic target for the treatment of NSCLC.
Grant support: Supported in part by Grant-in-Aid 09671362 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Takehiko Fujisawa, MD, Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Chiba, 260-8670 Japan. Phone: 81-43-222-7171, ext. 5464; Fax: 81-43-226-2172; E-mail:
- Received March 5, 2004.
- Revision received April 27, 2004.
- Accepted May 11, 2004.