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Prospective analysis of circulating endostatin levels in patients with renal cell carcinoma†
Article first published online: 3 OCT 2002
Copyright © 2002 American Cancer Society
Volume 95, Issue 8, pages 1637–1643, 15 October 2002
How to Cite
Feldman, A. L., Alexander, H. R., Yang, J. C., Linehan, W. M., Eyler, R. A., Miller, M. S., Steinberg, S. M. and Libutti, S. K. (2002), Prospective analysis of circulating endostatin levels in patients with renal cell carcinoma. Cancer, 95: 1637–1643. doi: 10.1002/cncr.10845
This article is a US Government work and, as such, is in the public domain in the United States of America.
- Issue published online: 3 OCT 2002
- Article first published online: 3 OCT 2002
- Manuscript Accepted: 20 MAY 2002
- Manuscript Revised: 26 APR 2002
- Manuscript Received: 8 MAR 2002
- renal cell carcinoma;
- vascular endothelial growth factor;
The aim of the current study was to assess circulating levels of endogenous endostatin in patients with renal carinoma and to determine the relationship of these levels to circulating levels of vascular endothelial growth factor (VEGF) and prognosis.
The authors prospectively studied 66 patients (48 male, 18 female; mean age, 50 years) undergoing nephrectomy for renal carcinoma on clinical trials at the National Cancer Institute. Metastases were present in 51 of 66 patients (77%) at the time of nephrectomy. Preoperative and followup serum endostatin and VEGF levels were determined using competitive enzyme immunoassays and compared to a group of 32 age- and gender-matched healthy controls. Associations between circulating endostatin levels and clinicopathologic variables, including survival, were determined.
Preoperative endostatin levels were higher in renal carcinoma patients than in healthy controls (P = 0.05). There was a weak to moderate correlation between pretreatment serum endostatin levels and serum VEGF levels (r = 0.47; P = 0.001), and levels of both proteins increased significantly following nephrectomy (P < 0.0001 and P < 0.0001, respectively; n = 41). In addition, patients whose endostatin levels increased more than twofold after nephrectomy had significantly poorer prognoses than patients without such an increase (P = 0.018). This association was more pronounced when patients without metastases were excluded (P = 0.0037).
Circulating endostatin levels are elevated in patients with renal carcinoma and correlate with circulating VEGF levels. Endostatin levels increase after nephrectomy, and patients with the greatest increases experience shortened survival times. These findings suggest an association between tumor aggressiveness and the production of endogenous endostatin in patients with renal carcinoma. Cancer 2002;95:1637–43. Published 2002 by the American Cancer Society.
Because solid neoplasms rely on angiogenesis for sustained growth,1 the inhibition of tumor angiogenesis is an attractive new strategy for treating cancer patients. Renal cell carcinoma (RCC) has been an attractive target for clinical trials of angiogenesis inhibitors because it is a highly vascular tumor and is known to produce the proangiogenic cytokine vascular endothelial growth factor (VEGF).2
Numerous endogenous inhibitors of angiogenesis have been identified,3 mostly in mice. Interestingly, some of these inhibitors, including angiostatin and endostatin, are produced by tumors themselves.4, 5 In mice, angiostatin production by a subcutaneous Lewis lung carcinoma tumor inhibits the growth of lung metastases, which grow rapidly when the primary tumor is resected.4, 6 It has been suggested that this phenomenon exists in humans with cancer;7 however, this hypothesis has not been rigorously proven, nor are the human inhibitors that might be responsible known.
To investigate this phenomenon further, we previously undertook a small, retrospective study of circulating endostatin levels in patients with RCC and found that endostatin levels were elevated compared to those of healthy controls.8 Curiously, endostatin levels correlated with levels of VEGF, which have been reported to be associated with aggressive disease and poor outcome.9 Furthermore, subsequent studies of endostatin levels in patients with soft tissue sarcoma and metastatic colon carcinoma revealed an association between elevated endostatin levels and poor outcome.10, 11
Because our previous study was too small to adequately study disease outcome, we undertook a larger, prospective analysis to confirm our previous findings and determine whether endostatin levels were associated with outcome in patients with RCC.
PATIENTS AND METHODS
Patients and Collection of Specimens
The subjects for the current study were 66 consecutive patients with sporadic renal cell carcinoma who underwent nephrectomy on institutional review board-approved protocols at the National Cancer Institute, Bethesda, Maryland from July 1999 through March 2001. Informed consent was obtained from all patients. There were 48 men and 18 women, with a mean age of 50 years (range, 22–76 years). Serum samples were collected preoperatively and at the first postoperative followup visit (mean, 8 weeks postoperatively; range, 4–16 weeks). Age- and gender-matched control serum samples were acquired from healthy volunteer blood donors at the Department of Transfusion Medicine, Warren G. Magnusson Clinical Center, National Institutes of Health, Bethesda, Maryland. Control samples were selected to maintain similar gender ratios and mean ages between the cancer patients and controls; matching was performed prior to determination of serum cytokine concentrations. There were 32 controls (23 men and 9 women) with a mean age of 48.5 years (range, 30–71 years). Samples were aliquoted and stored at −30 °C until ready for analysis.
Determination of Circulating Endostatin and VEGF Levels
Serum endostatin and VEGF concentrations were determined using competitive enzyme immunoassays (Cytimmune Sciences, College Park, MD) as previously described.8 Briefly, rabbit-anti-human endostatin or VEGF antibodies were allowed to bind to 96-well plates precoated with goat anti-rabbit antibodies in the presence of serum samples or recombinant protein standards and a biotinylated protein conjugate. After washing, streptavidin conjugated alkaline phosphatase was allowed to bind to the immobilized biotinylated protein. Plates were rewashed, color reagent was added, and the quantity of formazan product was determined by measuring the 450 nm absorbance using a microplate reader (Titertek, Huntsville, AL). Sample concentrations were calculated by comparison to standard curves. Two aliquots of each sample were measured in duplicate, and the mean value from these four determinations was used as the final concentration. Samples with undetectable endostatin or VEGF concentrations were defined as having concentrations midway between 0 and the lower limit of detection (resulting in values of 1.0 ng/mL for endostatin and 0.1 ng/mL for VEGF).
Data are presented as the mean ± the standard error. Elevated serum levels of endostatin or VEGF were defined as levels above the 95th percentile of the distribution for the control subjects: 43.1 ng/mL for endostatin and 4.9 ng/mL for VEGF. Differences between groups were evaluated using the Wilcoxon rank sum test. The statistical significance of paired differences between pre-treatment and followup was assessed using the Wilcoxon signed rank test. The correlation between continuous variables was assessed using the nonparametric Spearman rank correlation method. The association between age and group of subjects was determined using the chi-squared test.
For evaluations stratified by pre-treatment protein levels, survival duration was computed from the date the pre-nephrectomy laboratory values were obtained until the date of death or last followup. Survival curves were constructed using the Kaplan-Meier method and compared using log-rank analysis. For evaluations using followup laboratory values as grouping variables, a landmark method of analysis was used to determine survival probabilities.12 To do so, survival analyses began followup from a date 112 days after nephrectomy to provide an unbiased, conditional estimate of the difference in survival between groups of patients determined according to the protein levels they attained at that followup visit. The period of one hundred twelve days was selected because it corresponded to the latest date at which a followup value was determined. By so doing, 8 of 41 patients with followup data were eliminated, since they had followup of less than 112 days. All P values are two-tailed and were not adjusted for other comparisons.
Clinical characteristics of the 66 patients are summarized in Table 1. The majority (80%) of patients had clear cell histology, and 51 of 66 (77%) had metastases, most commonly involving the lung. Followup blood samples were obtained in 41 patients (62%). Of the remaining patients, 1 patient had operative mortality, 2 underwent operation at the time of writing, and 22 did not return for geographic reasons and/or because of rapidly progressive disease. Forty-eight of 66 patients were male (73%) as were 23 of 31 controls (74%; P = 0.93). The median age of the patients was 49.5 years, compared to 48.5 for controls (P = 0.62).
|Median; range||49.5; 30–76||–|
|Pathologic tumor type|
|Lymph node only||3||5|
|Lung (± other sites)||45||68|
Circulating Endostatin and VEGF Levels
Preoperative serum endostatin levels were 29.1 ± 1.9 ng/mL in the RCC patients and 22.2 ± 2.1 ng/mL in the healthy controls (P = 0.05; Fig. 1A). Vascular endothelial growth factor levels were 2.1 ± 0.4 ng/mL in the RCC patients and 1.6 ± 0.3 ng/mL in the healthy controls (P = 0.95; Fig. 1B). Among patients for whom followup blood samples were available (n = 41), endostatin levels rose from 31.1 ± 2.6 ng/mL preoperatively to 49.7 ± 3.8 ng/mL postoperatively (P < 0.0001, Fig. 2A), and VEGF levels rose from 2.2 ± 0.6 ng/mL to 4.7 ± 0.5 ng/mL (P < 0.0001, Fig. 2B). The time interval from initial to followup collection was weakly correlated with the followup endostatin levels (r = −0.24, P = 0.13) as well as with the change in endostatin levels (r = −0.16, P = 0.32).
Correlation Between Endostatin and VEGF Levels
There was a weak to moderate correlation between endostatin levels and VEGF levels both preoperatively (r = 0.47, P = 0.001; Fig. 3A) and at followup (r = 0.41, P = 0.0074; Fig. 3B). No correlation between endostatin and VEGF levels was noted among healthy controls (r = −0.04; P = 0.82; Fig. 3C).
Association Between Endostatin/VEGF Levels and Disease Burden
There was no significant difference in preoperative endostatin levels between patients with and without metastatic disease (30.0 ± 2.2 and 26.0 ± 3.8 ng/mL, respectively; P = 0.44); similarly, VEGF levels did not differ significantly between the two groups (2.3 ± 0.5 and 1.4 ± 0.5 ng/mL, respectively; P = 0.72). There was no significant difference in followup endostatin or VEGF levels between patients with and without metastatic disease (data not shown).
Association Between Endostatin/VEGF Levels and Patient Survival
Survival data were available for 62 patients, excluding 1 patient who died intraoperatively, 2 patients who underwent operations recently, and 1 patient who was lost to followup. Median potential followup time for the remaining patients was 14.2 months. Based on an analysis beginning at the on-study date, there were no significant differences in survival between patients with or without elevated preoperative endostatin levels (that is, > 43.1 ng/mL; P = 0.33) or between patients with or without elevated preoperative VEGF levels (that is, > 4.9 ng/mL; P = 0.87). However, using the landmark analysis approach described, there was significantly poorer survival among patients with elevated endostatin levels at followup (P = 0.013). This trend was less pronounced when patients were stratified by followup VEGF level (P = 0.11).
To evaluate the role of elevated followup endostatin levels further, patients were stratified by the response of their endostatin levels to nephrectomy, where a > twofold increase after nephrectomy was considered a significant increase. Patients available for analysis who exhibited such an increase (n = 11) had significantly lower survival probabilities (median survival from landmark date, 8 months) than those without such an increase (n = 22; median survival from landmark not reached; P = 0.01; Fig. 4A). This difference in outcome was not due to an association between increases in endostatin levels and the presence of metastatic disease at the time of nephrectomy (P = 0.90). In fact, the difference in survival was more dramatic when only patients with metastases were considered (P = 0.0037; Fig. 4B). There were too few patients without metastases for statistical analysis. Patients with increased followup VEGF levels, defined by the above criteria, had survival probabilities similar to those of patients without increased levels (P = 0.63).
Recent focus on the development of antiangiogenic strategies for the treatment of cancer patients has led to the discovery of numerous endogenous inhibitors of angiogenesis.3 One of these, endostatin, is a 20-kD, C-terminal cleavage product of the basement membrane constituent, collagen XVIII.5 Endostatin is generated naturally by elastase activity in EOMA murine hemangioendothelioma cells,13 and delivery of recombinant endostatin5 or the endostatin gene14, 15 inhibits tumor growth in mice. Cathepsin L activity also has been associated with the cleavage of murine endostatin from collagen XVIII.16 Recently, a recombinant human form of endostatin has entered clinical trials for patients with advanced forms of cancer.17
Interestingly, endogenous endostatin can be detected in healthy human subjects and patients with a variety of rheumatologic and neoplastic conditions.8, 10, 11, 18–23 In a preliminary retrospective study, we found that preoperative serum endostatin levels in patients with Stage IV clear cell renal carcinoma were elevated when compared to serum from normal volunteer blood donors.8 Subsequent studies showed similar elevations in circulating endostatin levels in patients with soft-tissue sarcoma10 and in patients with liver metastases from colorectal carcinoma.11 In the sarcoma study, elevated endostatin levels were associated with a shorter disease-free survival after resection; in the colorectal carcinoma study, elevated preoperative endostatin levels were associated with increased disease burden, and elevated followup endostatin levels after surgical treatment were associated with poor outcome. Based on these unexpected findings, we conducted the current prospective analysis of circulating endostatin levels in patients with RCC.
Consistent with our earlier findings,8 serum endostatin levels, but not serum VEGF levels, were elevated in the current study when compared to age- and gender-matched healthy controls. It is interesting that in both studies, some RCC patients had markedly elevated VEGF levels, but overall a significant difference was not detected between cancer patients and controls. Most previous studies have detected elevated circulating VEGF levels in patients with RCC,24–26 although other data suggest this finding is not present universally.27, 28 It is possible that patient selection may influence the degree of elevation detected in VEGF levels. Alternatively, these findings may relate to differences in reactivity between the competitive enzyme immunoassay used in the current study and sandwich-type enzyme-linked immunosorbent assays (e.g., differences in detecting bound vs. free VEGF).28
Surprisingly, levels of both endostatin and VEGF increased dramatically after operation. Thus the pattern of endostatin expression in RCC patients does not appear to be an example of the phenomenon in mice (and hypothesis in humans) that removal of a primary tumor causes a depletion of a circulating angiogenesis inhibitor and subsequent growth of distant disease.4, 6, 7 In fact, a substantial (greater than twofold) postoperative increase in circulating endostatin levels was associated with significantly shorter survival time from the landmark date. This raises the important question of whether circulating, endogenous endostatin in humans has antiangiogenic properties. One study found that endostatin isolated from human circulation had no anti-proliferative effects on endothelial cells in vitro.18 However, studies using recombinant human endostatin suggest that showing functional activity in vitro appears to be more assay-dependent and requires higher doses than recombinant murine endostatin requires.29 It also has been reported that a variety of collagen fragments present in human circulation can be detected with an anti-endostatin antibody.20 In addition, the ability of cathepsin L and elastase, which can generate murine endostatin,13, 16 to contribute to the production of endostatin in cancer patients has not been reported. Taken together, these data indicate that the circulating form of human endostatin remains incompletely characterized. Immunoreactivity in the assay used in the current study might correlate with the presence of fragments of endostatin or alternately glycosylated forms as well as full-length, functional endostatin protein. Thus, the assay does not necessarily predict antiangiogenic activity of the circulating endostatin. The functional status of endostatin-like collagen fragments in human circulation merits further investigation.
It is possible that the increase in followup endostatin levels noted in the current study reflects an effect of surgery. If this were the case, however, we would not expect to find an association between an increase in followup endostatin levels and poor survival. In addition, there was no evidence that the time of collection of the postoperative samples influenced followup endostatin levels. Rather, the current study offers further evidence that high circulating endostatin levels in cancer patients are associated with tumor aggressiveness. The secretion of proteases by tumors is an important step in their ability to invade basement membranes,30 of which collagen XVIII is a constituent.31 Substantial increases in endostatin levels after nephrectomy may represent the activity of residual metastatic disease and thus may be associated with shorter survival times. Interestingly, an association between increased circulating endostatin levels and poor prognosis has recently been reported in patients with acute myeloid leukemia/myelodysplastic syndrome,32 suggesting that this phenomenon is not limited to patients with solid tumors or those undergoing surgery.
In summary, the current prospective study has validated preliminary data showing that circulating endostatin levels are elevated in patients with RCC. Furthermore, as in other histologies, increases in endostatin are associated with poor prognosis. We suspect this reflects an association between tumor invasiveness and the elaboration of tumor-derived proteases that cleave basement membrane collagen XVIII. The antiangiogenic properties of endogenous endostatin released through this process are unclear at present. Further characterization of circulating collagen fragments in humans, as well as the results of ongoing clinical trials using recombinant endostatin, will help elucidate the role of endostatin and related molecules in patients with cancer.
The authors thank Jolynn Procter, B.S., M.Ed. and Susan F. Leitman, M.D., Department of Transfusion Medicine, National Institutes of Health, for help in obtaining volunteer blood samples.