Circulating angiogenic factor levels correlate with extent of disease and risk of recurrence in patients with soft tissue sarcoma

S. S. Yoon1,{dagger}, N. H. Segal1, A. B. Olshen2, M. F. Brennan1 and S. Singer1,*

1 Sarcoma Disease Management Team, Department of Surgery and 2 Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

* Correspondence to: Dr S. Singer, Sarcoma Disease Management Team, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Tel: +1-212-639-2940; Fax: +1-212-717-3053; Email: singers{at}mskcc.org


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background: Tumor angiogenesis, or new blood vessel formation, is regulated by a balance between pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and anti-angiogenic factors such as endostatin.

Patients and methods: To investigate this angiogenic balance in soft tissue sarcomas (STS), blood samples were collected from 76 STS patients and 15 healthy controls, and analyzed for VEGF, bFGF and endostatin using quantitative enzyme-linked immunosorbent assays (ELISA).

Results: Forty-one patients (54%) had primary tumors, 20 (26%) had local recurrences and 15 (20%) had metastatic disease with or without local disease. Levels of all three angiogenic factors were highly variable in STS patients. Mean levels of VEGF and bFGF were 12 and 14 times higher, respectively, in patients compared with controls (P<0.0001). VEGF levels correlated with size of tumor, with the highest levels found in tumors >10 cm in size. Patients with metastases had endostatin levels 45% lower than patients without metastases (P=0.047). In 54 patients who underwent resection of primary disease or local recurrence, low pre-operative bFGF level was associated with a higher risk of subsequent recurrence (P=0.044).

Conclusions: STS secrete widely variable levels of angiogenic factors, and levels of specific factors may correlate with extent of disease, predict risk of recurrence and possibly guide the use of anti-angiogenic agents.

Key words: angiogenesis, basic fibroblast growth factor, endostatin, soft tissue sarcoma, vascular endothelial growth factor


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Soft tissue sarcomas (STS) are a heterogeneous group of tumors derived from cells usually of mesenchymal origin. These tumors are relatively uncommon, occur throughout the body, and are classified into over 50 different histological subtypes [1Go, 2Go]. The biological behavior of these malignancies can range from low-grade indolent tumors with a propensity for local recurrence to high-grade aggressive tumors that metastasize early to distant sites such as the lung.

As with other types of tumors, STS require angiogenesis (the formation of new blood vessels from pre-existing blood vessels) in order to grow beyond a few millimeters in size [3Go]. The normal adult vasculature is relatively quiescent, with a turnover half-life of months to years [4Go]. Tumors must induce these inactive endothelial cells to proliferate and form a tumor neovasculature that supplies oxygen and nutrients and eliminates waste products.

A variety of factors secreted by tumors have been identified as promoting tumor angiogenesis, with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) being two of the most important factors [5Go]. VEGF naturally occurs in three isoforms containing 121, 165 and 189 amino acids, and binds to two tyrosine kinase receptors, VEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as KDR/Flk-1) [6Go]. VEGF expression is upregulated in the vast majority of tumors examined thus far, and circulating VEGF levels in cancer patients have correlated in several studies with advanced tumor stage or poor prognosis [7Go]. Inhibition of VEGF signaling by means of antibodies or small molecule inhibitors can inhibit tumor growth in animal models [8Go] and has had some success in recent clinical trials [9Go, 10Go].

Similarly, bFGF levels are elevated in patients with a variety of cancers [11Go]. bFGF (also known as FGF-2) was one of the first angiogenic factors to be characterized, and induces endothelial cell proliferation, migration and capillary tube formation [12Go]. The FGF family consists of at least 19 members that are all 18–30 kDa proteins and have high affinity for heparin [8Go]. Four different FGF receptors have been identified, and all are tyrosine kinase receptors [13Go]. Inhibition of bFGF can decrease tumor growth in mice, and agents that decrease bFGF production, such as interferon-{alpha} and thalidomide, have shown some efficacy in certain cancers [14Go].

The process of angiogenesis is regulated not only by pro-angiogenic factors, but rather by a balance between pro-angiogenic and anti-angiogenic factors [3Go]. Endostatin is a 20 kDa carboxy-terminal fragment of collagen XVIII, which is found in the basement membranes of blood vessels, smooth muscle and epithelium [15Go]. Tumors generate endostatin during invasive growth by releasing degradative enzymes such as matrix metalloproteinases and elastases, which break down collagen XVIII [16Go]. Endostatin strongly inhibits angiogenesis in animal tumor models [17Go], but efficacy in clinical trials has yet to be demonstrated.

For a given cancer, one means of determining which factors play pivotal roles in the angiogenic balance is to measure circulating levels of angiogenic factors. Although numerous studies have examined circulating levels of angiogenic factors in patients with cancer [11Go, 18Go], only a few small studies have specifically examined circulating angiogenic factors in STS patients [19Go–22Go]. In this study, we sought to measure the circulating levels of two pro-angiogenic factors, VEGF and bFGF, and one anti-angiogenic factor, endostatin, in STS patients with various stages of disease. We found that VEGF and bFGF levels were highly variable and in general elevated in STS patients compared with healthy controls. Interestingly, we also found that endostatin levels were significantly decreased in patients with metastatic disease compared with patients with localized disease. These findings may have significant implications in the application of anti-angiogenic therapies.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Blood sample collection
After obtaining informed consent, blood was collected from 76 patients with STS who were evaluated at a single institution (Memorial Sloan-Kettering Cancer Center), as well as from 15 healthy volunteer controls, under an Internal Review Board-approved protocol. Approximately 10 ml of blood was collected in EDTA- or heparin-containing tubes. Samples were centrifuged at 1000 g for 10 min, followed by collection of the plasma. Plasma samples were stored at –80°C until enzyme-linked immunosorbent assays (ELISA) were performed.

Enzyme-linked immunosorbent assays
Plasma samples were measured for VEGF, bFGF and endostatin using the following commercially available ELISA kits: Quantikine Human VEGF ELISA Kit (R&D Systems, Minneapolis, MN, USA), Quantikine bFGF HS ELISA Kit (R&D Systems) and CytElisa Human Endostatin (Cytimmune Sciences, Inc., College Park, MD, USA). Manufacturers' protocols were followed, and samples were measured in duplicate. ELISA plates were read using the Emax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Standard curves were generated using four-parameter curves for VEGF and bFGF and semi-log curves for endostatin. Samples with angiogenic factor levels below the sensitivity of the assay were assigned a value midway between 0 and the lower limit of detection (4.5 pg/ml for VEGF and 0.33 pg/ml for bFGF). The mean value of duplicate samples was used as the final concentration.

Clinical data
Clinical information regarding patients was obtained from the institution's prospective sarcoma database as well as from medical records. Patient demographic data at the time of blood collection included age and sex. Characteristics of the primary tumor were recorded, and included site, size and depth. Any tumor distal to the shoulder or buttock was considered an extremity lesion. Buttock, shoulder, abdominal wall and chest wall lesions were considered trunk tumors. Tumors invading or deep to the investing muscular fascia were considered deep tumors and all others were considered superficial tumors. All biopsy and surgical specimens were analyzed by experienced sarcoma pathologists, who determined histological subtype, grade (low or high) and margin of resection (positive or negative). At the time of blood collection, patients were divided by extent of disease into primary disease only, local recurrence only, primary disease with distant metastases, local recurrence with distant metastases and distant metastases only. Local recurrence was defined as recurrence of disease at the site of primary tumor resection, and distant metastasis was defined as disease at a site other than the site of the primary tumor.

Statistical analysis
Distributions of angiogenic factors were compared using the Wilcoxon rank sum test. Recurrence-free survival was evaluated for 54 patients who underwent resection of primary disease or local recurrence and had at least 6 months of follow-up. Survival curves were estimated using the Kaplan–Meier method [23Go]. Angiogenic factors and other clinical variables were associated with survival using the univariate Cox proportional hazards model [24Go]. Variables with associated P values <0.1 were entered into a multivariate Cox model.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
STS characteristics
The characteristics of the primary STS are listed in Table 1. About 80% of the primary STS were located in the extremity and abdomen (either retroperitoneal or intra-abdominal). The remaining STS were located in the trunk and head/neck. The median size of primary STS was 12 cm. Two-thirds of primary STS were high-grade, and the vast majority invaded or were deep to the investing muscular fascia. The most common histological subtypes were liposarcoma, malignant fibrous histiocytoma, fibrosarcoma and leiomyosarcoma.


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Table 1. Characteristics of 76 primary soft tissue sarcomas

 
Eighty per cent of patients had primary disease or local recurrence, while 20% of patients had distant metastases (Table 2). Of the patients with distant metastases, 10 patients had lung metastases and the remaining five patients had liver, intra-abdominal and/or lymph node metastases.


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Table 2. Extent of disease

 
Plasma levels of VEGF, bFGF and endostatin
Plasma levels of VEGF were found to occur over a much wider range in STS patients compared with controls (Figure 1A). In addition, mean levels of VEGF were over 12 times higher in patients compared with controls (Table 3), and this difference was highly significant. A similar pattern was found for plasma bFGF levels. Plasma bFGF levels in STS patients were highly variable (Figure 1B), and the mean level was over 14 times higher in patients compared with controls (Table 3).



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Figure 1. (A) Scatterplot of plasma vascular endothelial growth factor levels in patients and controls. (B) Scatterplot of plasma basic fibroblast growth factor levels in patients and controls. Bars represent the mean.

 

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Table 3. Plasma levels of angiogenic factors

 
Plasma endostatin levels also varied greatly in STS patients, while healthy controls had a much narrower range (Figure 2A). Mean plasma levels of endostatin were 22% higher in sarcoma patients compared with controls (Table 3). However, this difference did not attain statistical significance. Interestingly, the 61 patients without metastatic disease had a mean endostatin level 34% higher than controls, while the 15 patients with metastatic disease had mean endostatin levels 26% lower than controls and 45% lower than patients without metastatic disease (Figure 2B). The group of patients with metastases included 10 patients with primary disease or local recurrence as well as distant metastases, and five patients with distant metastases only. Four patients in the former group had higher than average endostatin levels, while all five patients in the latter group had very low levels of endostatin.



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Figure 2. (A) Scatterplot of plasma endostatin levels in patients and controls. (B) Scatterplot of plasma endostatin levels in patients without metastases, patients with metastases and controls. Bars represent the mean. *Circled patients all had local disease as well as distant metastases.

 
Subgroup analyses were performed for VEGF, bFGF and endostatin levels based on site, size and grade (data not shown). The only significant difference found was in VEGF levels according to size of tumor. Patients with tumors ≤5 cm in size had significantly lower levels of VEGF than patients with tumors >10 cm in size (mean ± SD: 127.2 ± 96.3 versus 205.3 ± 186.7 pg/ml; P=0.043), while patients with tumors 5–10 cm in size had intermediate VEGF levels (179.6 ± 152.1 pg/ml).

Recurrence-free survival
Recurrence-free survival was analyzed for 54 patients who had blood samples drawn and subsequently underwent resection of primary disease or local recurrence. The median follow-up after surgical resection in these patients was 22 months. Overall recurrence-free survival is shown in Figure 3A. Univariate analysis was performed for factors that might influence subsequent recurrence (Table 4). As seen in prior studies [25Go], intra-abdominal or retroperitoneal (versus extremity or trunk) site, local recurrence (versus primary disease), and positive resection margin were predictive of recurrence (P<0.001, 0.035 and 0.006, respectively). VEGF and endostatin levels were not significantly associated with subsequent recurrence. However, bFGF level was inversely correlated with recurrence-free survival, with patients with low levels much more likely to recur than patients with high levels (Figure 3B). All variables with P values <0.10 from univariate analysis were entered into a multivariate Cox model. The final model chosen using a stepwise procedure included only the site variable as significant.



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Figure 3. (A) Recurrence-free survival in 54 patients with primary or local recurrence resected and with at least 6 months of follow-up. Dotted lines represent 95% confidence intervals. (B) Recurrence-free survival stratified by plasma basic fibroblast growth factor (bFGF) level. Low bFGF, moderate bFGF and high bFGF represents patients with bFGF levels <20, 20–30 and >30 pg/ml, respectively.

 

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Table 4. Univariate analysis for recurrence-free survival

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Tumor angiogenesis represents a highly complex process involving precise communication between tumors cells and their host organ or tissue. This interplay is regulated by a wide variety of factors. Further study into this regulation may provide insight into how angiogenesis inhibitors can best be utilized as anticancer agents. We sought to examine the angiogenic process in STS by measuring circulating levels of three different angiogenic factors.

VEGF and bFGF levels are widely variable in patients with STS, and the mean levels in this patient cohort were 12–14 times higher than controls. These findings are similar to other studies examining levels of these factors in patients with STS [19Go, 20Go] and other cancers [11Go, 18Go]. The wide differences in observed levels of angiogenic factors in this study may be due in part to the heterogeneity of this STS population, as we included a wide range of patients with various primary sites, sizes, grades, histologies and extent of disease. Nonetheless, this series represents the largest series of STS patients in which circulating angiogenic factor levels have been measured.

Feldman et al. [19Go] measured circulating levels of endostatin in patients with STS and found them to be significantly elevated. We similarly measured endostatin levels in STS and found a non-significant elevation in endostatin levels. However, we included in our study 15 patients with metastatic disease, and further analysis revealed that these 15 patients had a mean endostatin level that was actually lower than that of controls. The lowest levels were found in patients with distant metastases only, and no primary or recurrent tumor. There are several possible explanations for this finding. First, primary tumors may generate more endostatin than metastatic tumors. The only patients with metastases to have elevated endostatin levels were those with local tumors as well as distant metastases. Secondly, endostatin levels may be directly correlated with gross volume of disease, and the patients in this study with metastases generally had a lower tumor burden. Lastly, primary sarcomas that generate high levels of endostatin may inhibit the growth of distant micrometastases, as described in animal models [17Go, 26Go]. While, this last point is of scientific interest, clinical trials of endostatin have not yet demonstrated significant efficacy against distant metastases [27Go, 28Go].

Levels of circulating VEGF and bFGF correlate with tumor progression or survival in a variety of cancers including breast, lung, colorectal and prostate [11Go]. One study of STS patients found high endostatin levels to be associated with an increased risk of tumor recurrence after resection [19Go]. We did not find any correlation between VEGF or endostatin levels and recurrence-free survival in this group of STS patients. bFGF levels were inversely correlated with recurrence-free survival. Similar findings have been described in patients with breast, lung and ovarian carcinomas [29Go–31Go]. It is possible that tumors which utilize bFGF-driven angiogenesis are less aggressive and thus less likely to recur. Alternatively, tumors that secrete low levels of bFGF may utilize alternative angiogenic pathways that make them more aggressive.

Recent clinical trials in patients with metastatic renal cell cancer and colorectal cancer have demonstrated that inhibition of VEGF can inhibit tumor progression [9Go, 10Go]. There is currently an ongoing clinical trial combining an anti-VEGF antibody (bevacizumab) and doxorubicin for patients with metastatic STS (G. Demetri, personal communication). Given that tumor angiogenesis is highly variable depending on the specific tumor and the site in which the tumor is growing, measuring levels of circulating VEGF and other factors may not only help determine which factors are most important for tumor angiogenesis, but also help predict which tumors will be most responsive to specific anti-angiogenic therapies.

In summary, this study has taken an initial step toward examining the factors that regulate angiogenesis in STS. Circulating levels of VEGF, bFGF and endostatin were observed to be widely variable in STS patients compared with controls, with mean levels of both VEGF and bFGF dramatically higher in STS patients. Endostatin levels in patients with metastases were significantly lower than in patients without metastases. Further research will help elucidate the complex biology of tumor angiogenesis and may help define rational approaches to anti-angiogenic cancer therapies.


    Acknowledgements
 
We thank Rachel Jacobi, Beata Korytowski and Kara Detwiller for help with handling blood samples, and Monica Cha for expert database management. This study was supported by NIH grant K12CA87723-02 (to S.S.Y.) and the Kristen Ann Carr Fund (to S.S.Y., M.F.B. and S.S.).


    Notes
 
{dagger} Present address: Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital, 100 Blossom Street, Boston, MA 02114, USA Back

Received for publication January 26, 2004. Revision received March 23, 2004. Accepted for publication March 29, 2004.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1. Brennan MF, Alektiar KM, Maki RG. Soft tissue sarcoma. In DeVita VT Jr, Hellman S, Rosenberg SA (eds): Cancer: Principles and Practice of Oncology, 6th edition. Philadelphia, PA: Lippincott Williams & Wilkins 2001; 1841–1980.

2. Singer S, Demetri GD, Baldini EH, Fletcher CD. Management of soft-tissue sarcomas: an overview and update. Lancet Oncol 2000; 1: 75–85.[CrossRef][Medline]

3. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86: 353–364.[ISI][Medline]

4. Hobson B, Denekamp J. Endothelial proliferation in tumours and normal tissues: continuous labelling studies. Br J Cancer 1984; 49: 405–413.[ISI][Medline]

5. Ribatti D, Vacca A, Presta M. The discovery of angiogenic factors: a historical review. Gen Pharmacol 2000; 35: 227–231.[CrossRef][Medline]

6. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 2002; 20: 4368–4380.[Abstract/Free Full Text]

7. Ferrara N. The role of vascular endothelial growth factor in angiogenesis. In Voest EE, D'Amore PA (eds): Tumor Angiogenesis and Microcirculation. New York, NY: Marcel Dekkar, Inc. 2001; 361–374.

8. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol 2001; 61: 253–270.[CrossRef][ISI][Medline]

9. Kabbinavar F, Hurwitz HI, Fehrenbacher L et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003; 21: 60–65.[Abstract/Free Full Text]

10. Yang JC, Haworth L, Sherry RM et al. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003; 349: 427–434.[Abstract/Free Full Text]

11. Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol 2001; 19: 1207–1225.[Abstract/Free Full Text]

12. Shing Y, Folkman J, Sullivan R et al. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science 1984; 223: 1296–1299.[ISI][Medline]

13. Johnson DE, Williams LT. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res 1993; 60: 1–41.[ISI][Medline]

14. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci 2001; 22: 201–207.[CrossRef][ISI][Medline]

15. Tomono Y, Naito I, Ando K et al. Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes. Cell Struct Funct 2002; 27: 9–20.[CrossRef][ISI][Medline]

16. Wen W, Moses MA, Wiederschain D et al. The generation of endostatin is mediated by elastase. Cancer Res 1999; 59: 6052–6056.[Abstract/Free Full Text]

17. O'Reilly MS, Boehm T, Shing Y et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277–285.[ISI][Medline]

18. Kuroi K, Toi M. Circulating angiogenesis regulators in cancer patients. Int J Biol Markers 2001; 16: 5–26.[ISI][Medline]

19. Feldman AL, Pak H, Yang JC et al. Serum endostatin levels are elevated in patients with soft tissue sarcoma. Cancer 2001; 91: 1525–1529.[CrossRef][ISI][Medline]

20. Graeven U, Andre N, Achilles E et al. Serum levels of vascular endothelial growth factor and basic fibroblast growth factor in patients with soft-tissue sarcoma. J Cancer Res Clin Oncol 1999; 125: 577–581.[CrossRef][ISI][Medline]

21. Kuhnen C, Lehnhardt M, Tolnay E et al. Patterns of expression and secretion of vascular endothelial growth factor in malignant soft-tissue tumours. J Cancer Res Clin Oncol 2000; 126: 219–225.[CrossRef][ISI][Medline]

22. Linder C, Linder S, Munck-Wikland E, Strander H et al. Independent expression of serum vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in patients with carcinoma and sarcoma. Anticancer Res 1998; 18: 2063–2068.[ISI][Medline]

23. Kaplan E, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958; 457–481.

24. Cox D. Regression models and life-tables. J R Stat Soc 1972; 34: 187–220.[ISI]

25. Kattan MW, Leung DH, Brennan MF. Postoperative nomogram for 12-year sarcoma-specific death. J Clin Oncol 2002; 20: 791–796.[Abstract/Free Full Text]

26. Yoon SS, Eto H, Lin CM et al. Mouse endostatin inhibits the formation of lung and liver metastases. Cancer Res 1999; 59: 6251–6256.[Abstract/Free Full Text]

27. Eder JP Jr, Supko JG, Clark JW et al. Phase I clinical trial of recombinant human endostatin administered as a short intravenous infusion repeated daily. J Clin Oncol 2002; 20: 3772–3784.[Abstract/Free Full Text]

28. Herbst RS, Hess KR, Tran HT et al. Phase I study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol 2002; 20: 3792–3803.[Abstract/Free Full Text]

29. Brattstrom D, Bergqvist M, Larsson A et al. Basic fibroblast growth factor and vascular endothelial growth factor in sera from non-small cell lung cancer patients. Anticancer Res 1998; 18: 1123–1127.[ISI][Medline]

30. Colomer R, Aparicio J, Montero S et al. Low levels of basic fibroblast growth factor (bFGF) are associated with a poor prognosis in human breast carcinoma. Br J Cancer 1997; 76: 1215–1220.[ISI][Medline]

31. Obermair A, Speiser P, Reisenberger K et al. Influence of intratumoral basic fibroblast growth factor concentration on survival in ovarian cancer patients. Cancer Lett 1998; 130: 69–76.[CrossRef][ISI][Medline]