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© 2001 American Society for Clinical Oncology Increased Serum Concentration of Angiogenic Factors in Malignant Melanoma Patients Correlates With Tumor Progression and SurvivalFrom the Department of Dermatology, Saarland University Hospital, Homburg/Saar, Germany. Address reprint requests to Uwe Reinhold, MD, Department of Dermatology, The Saarland University Hospital, 66421 Homburg/Saar, Germany; email uwe.reinhold{at}med-rz.uni-sb.de
PURPOSE: To determine the predictive value of the angiogenic serum factors angiogenin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) for the prognosis of patients with malignant melanoma. PATIENTS AND METHODS: Angiogenin, VEGF, bFGF, and IL-8 were measured in sera of 125 melanoma patients with different stages of disease and with or without current therapy including interferon alfa and different cytostatics in comparison with 30 healthy controls using enzyme-linked immunosorbent assay. RESULTS: Serum levels of angiogenin, VEGF, bFGF, and IL-8 were significantly increased in melanoma patients compared with healthy controls. Elevated serum concentrations of VEGF, bFGF, and IL-8 were associated with advanced disease stages and tumor burden. Cytostatic therapy of patients was accompanied by increased serum levels of angiogenin, bFGF, and IL-8. As shown by univariate analysis, elevated serum levels of VEGF (P = .0001 and .0036), bFGF (P < .00005 and < .00005), and IL-8 (P < .00005 and < .00005) were strongly correlated with a poor overall and progression-free survival, respectively. Multivariate analysis revealed stage of disease (P = .0238), tumor burden (P = .0347), VEGF (P = .0036), bFGF (P = .0252), and IL-8 (P = .0447) as independent predictive factors of overall survival. Tumor burden (P = .0081), VEGF (P = .0245), and IL-8 (P = .0089) were found as independent predictive factors of progression-free survival. CONCLUSION: Our data suggest that the angiogenic serum factors VEGF, bFGF, and IL-8 are useful predictive markers for overall and progression-free survival in melanoma patients.
THE INCIDENCE OF malignant melanoma has increased during the last decade at a rate faster than any other cancer.1 The main concern after removal of a primary melanoma is whether it has metastasized, and early markers that identify melanoma patients with high risk for disease progression may necessitate timely therapeutic intervention, which may further improve survival rates.2 Recent results indicate that tumor angiogenesis, the formation of new capillaries, is one of the most important biologic features that is closely related to tumor growth and tumor metastasis.3,4 A number of studies have reported an association between intratumoral microvessel density and tumor aggressiveness in different human cancers, including malignant melanoma.5-8 These data showed that the increased intratumoral microvessel density correlated closely with a higher incidence of metastasis and a poorer prognosis in various malignancies. Current results indicate that the switch to the angiogenic phenotype depends on a net balance between positive and negative angiogenic factors released by the tumor.4 To date, many angiogenic factors, such as fibroblast growth factor family members, vascular endothelial growth factor (VEGF), angiogenin, platelet-derived endothelial cell growth factor, and interleukin-8 (IL-8) have been identified and shown to be produced by a variety of different tumor cells, including malignant melanoma cells.9-14 Although elevated serum levels of angiogenic factors have been observed in sera taken from patients with different types of human cancer, little is known about its clinical significance, and only limited information exists on blood level and the prognostic value of these factors in patients with malignant melanoma. The potential clinical relevance prompted us to measure the level of a panel of angiogenic factors (angiogenin, VEGF, basic fibroblast growth factor [bFGF], and IL-8) in the serum of melanoma patients and to correlate these parameters with the clinical course of the disease. We found a stage-dependent increase of angiogenic factors in the serum of melanoma patients, and, moreover, that VEGF, bFGF, and IL-8 serum levels were highly correlated with the disease-free and overall survival.
Patients After informed consent was obtained, blood was drawn from 125 unselected patients presenting at the Department of Dermatology, The Saarland University Hospital, Homburg/Saar, Germany, with histologically confirmed malignant melanoma of different stages of disease and from 30 healthy controls matched in age and sex. Patients were enrolled onto this study between September 1997 and November 1998. Clinical staging of patients was performed according to modified criteria of the American Joint Committee on Cancer (AJCC) with the following modifications: stage I included patients with primary melanoma (stage I and stage II according to AJCC criteria); stage II included patients with regional lymph node and/or in-transit metastases (stage III according to AJCC criteria), and stage III included patients with distant metastases (stage IV according to AJCC criteria). Follow-up was performed at least in 3-month intervals and included physical examination, x-ray of the chest, ultrasound of the abdomen and lymph nodes, and blood chemistry. Patients in advanced stages of disease (III) additionally underwent computed tomography of the brain and scintography of the skeleton. Patients were treated according to therapy protocols of the Dermatologic Cooperative Oncology Group, including cytostatic (dacarbazine, cisplatin, temozolomide, vincristine) and immunomodulatory (interferon alfa [IFN-  ]) agents in different combinations and schedules. Detailed clinical characteristics of the patients enrolled onto this study are presented in Table 1. Sera of healthy controls were kindly provided by the Department of Hematology and Blood Transfusion of the Saarland University Hospital. All controls were blood donors undergoing regular physical and laboratory examinations.
Serum Analyses Sera harvested by centrifugation at 1,500 x g were stored at -70°C until tested. Commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) were used for quantitative determination of VEGF, IL-8, angiogenin, and bFGF serum concentrations according to the manufacturer’s instructions. Standard curves were constructed using serial dilutions of recombinant angiogenin, bFGF, VEGF or IL-8. The minimum detectable concentrations were determined as 3 pg/mL for bFGF, 6 pg/mL for angiogenin, 5 pg/mL for VEGF, and 10 pg/mL for IL-8. The intra- and interassay variations of these assays were less than 10%. Each sample was tested in duplicate. Optical densities were quantified using a microtiter plate reader (Multiskan MCC/340; Labsystems, Helsinki, Finland).
Statistical Methods
Serum Angiogenic Factors Are Elevated in Melanoma Patients Sera were obtained from 125 patients diagnosed with malignant melanoma. They included 14 male patients with a mean age of 52.8 ± 2.8 years (mean ± SEM) and 17 female patients with a mean age of 51.3 ± 3.3 years in stage I (primary melanoma), 19 male patients with a mean age of 55.7 ± 2.3 years and 27 female patients with a mean age of 56.1 ± 3.0 years in stage II (regional lymph node and/or in-transit metastases), and 30 male patients with a mean age of 60.2 ± 1.7 years and 18 female patients with a mean age of 56.3 ± 2.5 years in stage III (distant metastases). The healthy controls consisted of 15 male patients with a mean age of 54 ± 5.3 years and 15 female patients with a mean age of 52 ± 7.5 years. As listed in Table 1, the mean serum levels of angiogenin (P < .0005), bFGF (P = .004), VEGF (P = .037), and IL-8 (P = .004) were significantly increased in melanoma patients compared with healthy donors. No relationship was observed between serum angiogenic factors and sex (Table 1) or age (data not shown) of the patients tested, except angiogenin, which was significantly (P = .001) elevated in males compared with females (Table 1).
Stage of Disease and Tumor Burden Are Correlated With Serum Concentrations of bFGF, VEGF and IL-8
Differential Effects of Therapy Modalities on Serum Levels of Angiogenic Factors
Predictive Value of Angiogenic Factors for Overall Survival
Predictive Value of Angiogenic Factors for Progression-Free Survival Because the probability of progression, besides overall survival, is one of the most important parameters in melanoma patients regarding the frequency of follow-up examinations and the choice of a suitable mode of therapy, we analyzed the predictive value of angiogenic serum factor concentrations for the probability of progression-free survival. As shown in Fig 3, increased serum concentrations of bFGF (P < .00005), VEGF (P = .0036), and IL-8 (P < .00005) were strongly correlated with a higher probability of progression of the disease in melanoma patients, as revealed by Kaplan-Meier analysis with the date of serum sampling used as the starting point of calculation. The same analysis considering the time point of diagnosis as the starting point revealed similar results (data not shown). Serum concentrations of angiogenin had no impact on progression-free survival. Testing the prognostic factors stage of disease and tumor burden together with the angiogenic serum factors by multivariate analysis using the proportional hazards model of Cox, we found tumor burden (P = .0081), VEGF (P = .0245), and IL-8 (P = .0089) as independent predictive factors of progression-free survival of melanoma patients. Stage of disease (P = .3555), serum angiogenin (P = .3831), and bFGF (P = .0729) showed no independent predictive value for patients’ progression-free survival.
In the present study, we evaluated the serum concentration and the prognostic significance of a panel of angiogenic factors in melanoma patients of different clinical stages. We found significantly elevated serum levels of angiogenin, VEGF, bFGF, and IL-8 in melanoma patients compared with healthy controls. Blood values of VEGF, bFGF, and IL-8 were positively correlated with the stage of disease and the tumor burden, whereas angiogenin serum levels were not significantly different in patients with or without metastases. Furthermore, elevated serum levels of VEGF, bFGF, and IL-8 were strongly correlated with poor overall survival and a high probability of progression. Increased VEGF and IL-8 serum levels have been reported previously in smaller series of melanoma patients.15,16 However, this is the first study that demonstrates that angiogenic factors have a strong impact on patient prognosis. A more frequent and enhanced expression of VEGF has been detected in metastatic melanoma cells compared with primary melanoma, indicating a relationship between tumor tissue VEGF levels and melanoma progression.11 Increased VEGF concentrations may play a role in the angiogenic response necessary for melanoma growth and metastases but seems to be also involved in the inhibition of antitumor response by inhibiting the maturation of antigen-presenting cells.17,18 Prolonged exposure of immunoreactive cells to high blood levels of VEGF may therefore lead to the suppression of tumor-specific immunoresponse in advanced-stage melanoma patients. In correlation, VEGF-rich tumors were recently shown to be associated with early relapse in primary breast cancer and with a poor outcome in patients with small-cell lung carcinoma, non-Hodgkin’s lymphoma, gastric cancer, and esophageal squamous cell carcinoma.19-26 IL-8 and bFGF both were identified as essential autocrine growth factors for melanoma cells, and a direct correlation between the level of expression in different cell lines and their metastatic potential was observed.27 Transfection of nonmetastatic and IL-8–negative melanoma cells with the IL-8 gene rendered them highly tumorigenic and increased their metastatic potential in nude mice.14 On the other hand, antisense targeting of bFGF in melanoma cells completely blocked tumor cell growth and inhibited intratumoral angiogenesis in vivo.28 A recent study demonstrated expression of IL-8 in 50% of the vertical growth phase melanoma lesions and in 100% of metastatic lesions, whereas none of the radial growth phase tumors expressed IL-8.10 Also, invasive primary melanoma and metastatic melanoma were found to express bFGF mRNA, whereas melanomas in situ and benign melanocytic nevi were negative.29 In addition, the metastatic potential and invasiveness of human melanoma cells were found to be markedly increased on transduction with the bFGF gene.30 In agreement with these studies, we observed that high serum levels of bFGF and IL-8 were strongly associated with a poor outcome in melanoma patients. It is noteworthy that no bFGF and IL-8 were detectable in the serum of healthy controls, further indicating that enhanced serum concentrations of these factors in melanoma patients are derived from tumor cells in vivo. In this context, recent evidence indicates that the production of the angiogenic factors IL-8 and VEGF by melanoma cells may be upregulated through specific cytokines secreted by tumor-infiltrating monocytes/macrophages.31 Angiogenin is another potent blood vessel–inducing protein that was found to be expressed in human tumor cells as well as in various types of nonmalignant cells.32 A recent study showed that angiogenin is induced by hypoxia in human melanoma cells and that enhanced expression is correlated with tumor aggressiveness in vitro and in vivo.33 Increased angiogenin expression and serum concentration was observed in patients with colorectal carcinoma, pancreatic carcinoma, and urothelial carcinoma and was found to be correlated with tumor progression.12,34,35 Moreover, the disease-free survival of urothelial carcinoma patients with elevated angiogenin serum levels was significantly reduced compared with that of patients with normal serum concentrations.12 However, to the best of our knowledge, no studies have been performed until now concerning the angiogenin serum level and its prognostic relevance in melanoma patients. The present analysis demonstrates that angiogenin is elevated in the serum of melanoma patients but is not correlated with tumor load and prognosis. Our results also indicate that the serum concentration of certain angiogenic factors may be influenced by various therapeutic regimens, because the serum level of angiogenin, bFGF, and IL-8 was found to be significantly higher in melanoma patients under treatment with chemotherapy and/or immunotherapy compared with patients without any treatment. The increase of blood concentrations of angiogenic factors might in part result from therapy-induced cell damage and increased release of angiogenic factors to the circulation. The reason why VEGF was exceptional in that the level of VEGF serum concentration was not significantly influenced by any treatment regimen remains unclear. In conclusion, our study demonstrates increased concentrations of angiogenin, VEGF, bFGF, and IL-8 in serum of melanoma patients. We show that elevated serum levels of VEGF, bFGF, and IL-8 are strongly correlated with poor clinical outcome and therefore could be potent new predictive factors of disease progression and prognosis in individual melanoma patients. Our results suggest that bFGF and IL-8 may appear as better prognostic serum markers compared with VEGF. Additional investigations of a larger panel of melanoma patients are needed to confirm these findings. Also, several types of molecules with an antiangiogenic function have been recently developed, and clinical trials are in progress.36 The evaluation of serum levels of angiogenic factors may therefore play an important role in selecting melanoma patients for antiangiogenic therapy.
We thank Karin Hilgert and Alexandra Stark for excellent technical assistance and Thomas Georg (Institute of Medical Biometrics, Epidemiology and Medical Informatics, University of the Saarland) for helpful support on statistical analyses.
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