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Annals of Surgical Oncology 8:741-745 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

A Prospective Analysis of Plasma Endostatin Levels in Colorectal Cancer Patients With Liver Metastases

Andrew L. Feldman, MD, H. Richard Alexander, Jr., MD, David L. Bartlett, MD, Karen C. Kranda, RN, Marshall S. Miller, BS, Nick G. Costouros, BS, Peter L. Choyke, MD and Steven K. Libutti, MD

From the Surgery Branch, National Cancer Institute (ALF, HRA, DLB, KCK, MSM, NGC, SKL) and the Diagnostic Radiology Department (PLC), Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland.

Correspondence: Address correspondence and reprint requests to: Steven K. Libutti, MD, Surgery Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 2B07, 9000 Rockville Pike, Bethesda, MD 20892; Fax: 301-402-1788; E-mail: libuttis{at}mail.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Circulating inhibitors of angiogenesis have been suggested to affect the growth of distant micrometastatic disease in patients with cancer. This study was designed to evaluate circulating endostatin levels in colorectal cancer patients with liver metastases.

Methods: Plasma samples from 30 colorectal cancer patients with liver metastases were analyzed for endostatin and vascular endothelial growth factor (VEGF) by using competitive enzyme immunoassays. Samples were compared with plasma from age- and sex-matched healthy controls; values >2 SD above the control mean were considered elevated.

Results: Plasma endostatin levels were significantly higher in the 30 cancer patients than controls (P < .0001) and correlated with preoperative VEGF levels (P = .0008). Eighteen patients underwent surgical treatment (liver resection, n = 10; or isolated hepatic perfusion with melphalan, n = 8). Seventeen treated patients were available for follow-up. Eight of 11 patients who progressed had elevated plasma endostatin levels at the time of progression. None of six patients who remained progression free had elevated endostatin levels at last follow-up (P = .02).

Conclusions: Plasma endostatin levels are elevated in colorectal cancer patients with liver metastases and correlate with VEGF levels. Elevated endostatin levels during follow-up are associated with disease progression. Understanding the role of endogenous endostatin in cancer patients may lead to novel strategies to inhibit tumor angiogenesis.

Key Words: Angiogenesis • Endostatin • Colorectal cancer • Liver metastasis • Vascular endothelial growth factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nearly 130,000 new cases of colorectal cancer are diagnosed annually in the United States, and mortality from the disease approaches 50%.1 Liver metastases occur in approximately 45% of patients at some point in their disease course. Although a select group of patients have favorable outcomes after liver resection, alternative therapeutic options are needed to treat patients whose tumors recur after resection, as well as those with unresectable disease.2 Because solid tumors have been demonstrated to be dependent on angiogenesis for sustained growth,3 inhibiting tumor angiogenesis has emerged as an attractive treatment strategy for patients with cancer.

We previously have shown that endostatin,4 an endogenous antiangiogenic agent now in clinical trials, is present in the circulation of normal subjects and elevated in patients with certain cancers.5,6 However, endostatin levels in patients with colorectal cancer have not been reported. Because the liver is the major site of morbidity and mortality in these patients, as well as the major site of production of the endostatin precursor, collagen XVIII,7 we prospectively analyzed circulating endostatin levels in colorectal cancer patients with liver metastases.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The study population consisted of 30 colorectal cancer patients with liver metastases enrolled onto institutional review board–approved clinical research protocols in the Surgery Branch, National Cancer Institute, for possible surgical treatment. Informed consent was obtained for all patients. A prospective database was maintained, cataloging demographic data, treatment, and response.

Plasma samples were obtained before surgery and during follow-up visits. In addition, we obtained plasma samples from age- and sex-matched volunteer blood donors (controls) from the Department of Transfusion Medicine, National Institutes of Health. Samples were stored at <-30°C until ready for analysis. Plasma endostatin and vascular endothelial growth factor (VEGF) levels were determined in duplicate by using competitive enzyme immunoassays (Accucyte; Cytimmune Sciences, College Park, MD) as previously described.5

Preoperative helical computed tomography images (HiSpeed Advantage or Light Speed; GE Medical Systems, Milwaukee, WI) were analyzed, and the greatest perpendicular diameters of each hepatic lesion were measured. Measurements were independently audited by a radiologist (P.L.C.) blinded to plasma cytokine levels. Tumor volumes were calculated with the formula 4/3{pi}r3, where r represents the mean radius of the two measurements taken. Hepatic tumor burden in each patient was calculated by adding the volumes of individual hepatic tumors.

Surgical treatment of liver metastases was performed after verifying the absence of significant extrahepatic disease and consisted of liver resection or isolated hepatic perfusion with melphalan as previously described.8 Postoperative follow-up and determination of disease status in treated patients were performed according to clinical protocols.

Data are presented as mean ± SD. Statistical analyses were performed with the Mann-Whitney U-test, Wilcoxon signed rank test, Kruskal-Wallis test, Spearman rank correlation, and Fisher’s exact test, where appropriate. Two-tailed P values <.05 were considered statistically significant. Cytokine levels >2 SD above the control mean were considered elevated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics
Patients included 21 men and 9 women, with a mean age of 60 years (Table 1). Controls included 20 men and 8 women, with a mean age of 56 years. Original stage of disease was stage 2 in 6 patients, stage 3 in 12 patients, and stage 4 in 12 patients. Median time between colon resection and the development of liver disease in 18 patients with metachronous metastases was 12 months (range, 1–60 months). The mean number of hepatic metastases as determined by computed tomography analysis was 10.4 ± 18.7, and the mean total volume of metastases was 409.1 ± 514.7 ml. Eighteen patients underwent surgical treatment, consisting of liver resection in 10 patients and isolated hepatic perfusion in 8 patients. The remaining 12 patients underwent biopsy only, placement of a hepatic arterial infusion pump, or no procedure.


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  		TABLE 1. Clinical characteristics of 30 colorectal cancer patients with liver metastases
 

Preoperative Plasma Endostatin and VEGF Levels
Preoperative plasma endostatin levels were significantly higher in the cancer patients than in controls (71.6 ± 28.6 and 43.2 ± 15.1 ng/ml, respectively; P < .0001)(Fig. 1). VEGF levels did not differ significantly between the two groups (2.7 ± 2.7 ng/ml and 2.3 ± 1.6 ng/ml, respectively; P = .83). As defined previously, endostatin levels >73.4 ng/ml were considered elevated. Using this criterion, preoperative endostatin levels were elevated in 12 (40%) of the 30 patients.



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  		FIG. 1. Preoperative plasma endostatin and vascular endothelial growth factor (VEGF) levels in colorectal cancer patients with liver metastases. (A) Plasma endostatin levels were significantly higher in the cancer patients than in controls (71.6 ± 28.6 and 43.2 ± 15.1 ng/ml, respectively; P < .0001, Mann-Whitney U-test). (B) VEGF levels did not differ significantly between the two groups (2.7 ± 2.7 and 2.3 ± 1.6 ng/ml, respectively; P = .83). Dashed lines indicate median values.

 

Correlation Between Preoperative Endostatin and VEGF Levels
There was a highly significant correlation between preoperative endostatin and VEGF levels in the cancer patients (r = .63, P = .0008) (Fig. 2). This correlation was absent in controls (r = .06, P = .75). There was no significant correlation between preoperative endostatin levels and age (r = -.11, P = .56) and no association with sex (P = .10).



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  		FIG. 2. Correlation between preoperative endostatin and vascular endothelial growth factor (VEGF) levels. (A) There was a highly significant correlation between preoperative endostatin and VEGF levels in the cancer patients (r = .63, P = .0008; Spearman rank correlation). (B) This correlation was absent in controls (r = .06, P = .75).

 

Association Between Preoperative Endostatin Levels and Burden of Disease
The mean number of hepatic metastases among patients with elevated preoperative endostatin levels (19.4 ± 27.6) was significantly higher than among those without elevated levels (4.5 ± 4.6; P = .04). Similarly, the mean total volume of hepatic metastases among patients with elevated preoperative endostatin levels (670.4 ± 643.4 ml) was significantly higher than among those without elevated levels (240.0 ± 333.3 ml; P = .03). Finally, only 5 (42%) of 12 patients with elevated preoperative endostatin levels were deemed eligible for surgical treatment (liver resection or isolated hepatic perfusion, as described previously), compared with 13 (72%) of 18 patients without elevated levels (P = .01). Preoperative endostatin levels were not associated with either tumor stage at initial presentation or histological grade (P = .73 and P = .15, respectively).

Change in Endostatin Levels After Surgical Treatment
Of 18 treated patients, 17 patients returned for follow-up. Endostatin levels were determined at the first postoperative follow-up visit (6.6 ± 2.2 weeks after surgery) rather than before hospital discharge to minimize the direct effects of surgery. There was no significant difference between pre- and posttreatment endostatin levels (58.9 ± 24.0 and 70.7 ± 26.5 ng/ml, respectively; P = .33). Only four treated patients had elevated endostatin levels before surgery, precluding statistical analysis; however, levels dropped to below the cutoff value of 73.4 ng/ml after treatment in three of the four patients.

Association Between Endostatin Levels and Disease Progression
Of the 17 patients available for follow-up, median follow-up time was 5.5 months (range, 1.8–16.9 months). Eight (72%) of 11 patients who progressed (median time to progression, 5.5 months) had elevated plasma endostatin levels at the time of progression (mean, 72.7 ± 35.4 ng/ml). None of the six patients who remained progression free had elevated plasma endostatin levels at last follow-up (mean, 42.6 ± 15.4 ng/ml; median follow-up time, 6.2 months; P = .02). There was no significant difference in preoperative endostatin levels between patients who progressed (53.3 ± 28.0 ng/ml) and those who remained progression free at last follow-up (69.2 ± 8.9 ng/ml; P = .13).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Solid tumors rely on angiogenesis for sustained growth3 and produce cytokines that induce neovascularization.9 It is interesting that tumors can also produce antiangiogenic cytokines,4,10,11 an observation hypothesized to be associated with the clinical observation that resection of primary human tumors may lead to growth of distant metastatic disease.12 This phenomenon has been demonstrated in murine models.10,13 Because inhibiting tumor angiogenesis has become an attractive strategy for treating cancer patients, identifying the presence of circulating inhibitors of angiogenesis in humans with cancer might shed light on the role of these endogenous mediators in humans and suggest novel means to enhance their production or antitumor effects.

Endostatin, a 20-kDa, C-terminal fragment of collagen XVIII, is an endogenous inhibitor of angiogenesis first isolated from a murine hemangioendothelioma cell line and found to have potent antitumor activities in mice.4 Recombinant human endostatin now is being investigated in clinical trials in patients with cancer. It is interesting that we have demonstrated the presence of endogenous endostatin in the circulation of healthy humans, and circulating endostatin levels are elevated in patients with clear-cell renal cancer and soft tissue sarcoma.5,6 However, circulating endostatin levels in patients with colorectal cancer have not been reported. Because hepatic metastatic disease represents the major cause of morbidity and mortality in patients with colon cancer, and because the liver is rich in collagen XVIII,7 the parent protein of endostatin, we prospectively analyzed plasma endostatin levels in colorectal cancer patients with liver metastases.

Consistent with previous studies,5,6 detectable circulating endostatin levels were present in a control group of volunteer blood donors. Endostatin levels in the colorectal cancer patients were significantly higher than in controls. Several proteases have been demonstrated to cleave endostatin from collagen XVIII, including elastase14 and cathepsin L.15 With the exception of the original hemangioendothelioma cell line from which murine endostatin was first isolated (which produces cathepsin L), tumor cell lines have not been shown to produce endostatin.4,5 Therefore, it is reasonable to hypothesize that endostatin production in humans occurs as a result of extracellular cleavage of collagen XVIII. Because the liver is particularly rich in collagen XVIII, elaboration of these proteases by tumor would be expected to generate endostatin. It is interesting to note that a previous study of patients with hepatocellular carcinoma found circulating endostatin levels similar to those of controls.16 Therefore, the production of proteases capable of cleaving endostatin from collagen XVIII may vary between tumor histologies and merits further study.

We also found that preoperative endostatin levels correlated with levels of VEGF. We have suggested that a correlation between endostatin and VEGF levels, which we first noted in patients with clear-cell renal cancer, may be associated with the elaboration of proteases that cleave endostatin from collagen XVIII in the peritumoral environment.5 Hypoxia, for example, is often present in tumors and has been shown to induce expression of both VEGF17 and cathepsin L.18 It is also possible that elevated endostatin levels represent a direct response to elevated VEGF levels.5

The ability of endogenous human endostatin to inhibit angiogenesis is unknown at present.19 Thus, it is not clear whether endogenous endostatin represents part of a defense mechanism to protect the host from tumor angiogenesis or simply is a byproduct of proteases secreted by the tumor. In either case, endostatin production might be expected to be increased in patients with more aggressive disease. We found that elevated endostatin levels were associated with greater tumor burden in colorectal cancer patients with liver metastases. Patients with elevated endostatin levels had significantly more metastatic lesions and greater hepatic disease burden and were less likely to be deemed eligible for surgical treatment. Furthermore, elevated follow-up endostatin levels were associated with disease progression. Because of the small number of patients studied and the wide range of circulating endostatin levels observed in healthy controls, a much larger analysis would be required to assess the feasibility of using circulating endostatin levels as a tumor marker of progression or recurrence. In a previous study of patients with soft-tissue sarcomas, we demonstrated that patients with elevated preoperative endostatin levels were more likely to have recurring tumors after resection than patients without elevated endostatin levels.6 Because only four patients with elevated preoperative endostatin levels were eligible for surgical therapy in this study, the potential utility of endostatin levels as a prognostic marker could not be assessed in this population.

In conclusion, circulating endostatin levels are significantly elevated and correlate with circulating VEGF levels in colorectal cancer patients with liver metastases. Elevated preoperative endostatin levels are associated with a greater disease burden, and elevated follow-up endostatin levels are associated with disease progression. Understanding the role of endogenous endostatin in patients with metastatic colorectal cancer may lead to novel treatment strategies to inhibit tumor angiogenesis.


   Acknowledgments
 
The authors thank K. Cipolone, J. Procter, and S. Leitman, Department of Transfusion Medicine, National Institutes of Health, for their help in obtaining volunteer blood samples and T. Moser and C. Helsabeck, Surgery Branch, National Cancer Institute, for their help in obtaining patient samples.


   Footnotes
 
Presented at the 54th Annual Meeting of the Society of Surgical Oncology, Washington, DC, from March 15–18, 2001.

Received for publication March 17, 2001. Accepted for publication July 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  5. Feldman AL, Tamarkin L, Paciotti GF, et al. Serum endostatin levels are elevated and correlate with serum vascular endothelial growth factor levels in patients with stage IV clear cell renal cancer. Clin Cancer Res 2000; 6: 4628–34.[Abstract/Free Full Text]
  6. Feldman AL, Pak H, Yang JC, Alexander HR, Libutti SK. Serum endostatin levels are elevated in patients with soft tissue sarcoma. Cancer 2001; 91: 1525–9.[CrossRef][Medline]
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  8. Bartlett DL, Libutti SK, Figg WD, Fraker DL, Alexander HR. Isolated hepatic perfusion for unresectable hepatic metastases from colorectal cancer. Surgery 2001; 129: 176–87.[CrossRef][Medline]
  9. Desai SB, Libutti SK. Tumor angiogenesis and endothelial cell modulatory factors. J Immunother 1999; 22: 186–211.
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  12. Prehn RT. Two competing influences that may explain concomitant tumor resistance. Cancer Res 1993; 53: 3266–9.[Abstract/Free Full Text]
  13. Holmgren L, O’Reilly MS, Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995; 1: 149–53.[CrossRef][Medline]
  14. Wen W, Moses MA, Wiedershain D, Arbiser JL, Folkman J. The generation of endostatin is mediated by elastase. Cancer Res 1999; 59: 6052–6.[Abstract/Free Full Text]
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  16. Yamagata M, Shiratori Y, Dan Y, et al. Serum endostatin levels in patients with hepatocellular carcinoma. Ann Oncol 2000; 11: 761–3.[Free Full Text]
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