Markers of angiogenesis and clinical features in patients with sarcoma
The growth and dissemination of sarcomas depends on angiogenesis. A number of measurable markers related to tumor angiogenesis have been studied in patients with sarcoma.
The available literature related to markers of angiogenesis and clinical features in patients with sarcoma was reviewed. Clinical features of interest included tumor size, tumor grade, tumor stage, presence of metastatic disease, and prognosis. In patients with soft-tissue sarcomas, tumor vascular endothelial growth factor (VEGF) expression correlates with stage, grade, and prognosis. Circulating VEGF levels also correlate with tumor grade. High circulating angiopoietin-2 levels are associated with increased tumor size in soft-tissue sarcoma. For patients with osteosarcoma, tumor VEGF expression correlates with outcome. Elevated tumor and circulating VEGF levels are associated with the development of lung metastases in osteosarcoma. Patients with Ewing sarcoma have increased circulating VEGF levels compared with controls. Angiogenesis markers correlate with important clinical features in patients with sarcomas ranging from soft-tissue sarcomas to bone sarcomas. Markers of angiogenesis may serve an important role in predicting a particular patient's clinical course and in identifying patients for possible antiangiogenic therapy. Cancer 2007 © 2006 American Cancer Society.
Sarcomas are mesenchymal-derived cancers. This heterogeneous group of tumors depends on new blood vessel development, also known as angiogenesis, for tumor growth and metastasis. Increasingly, antiangiogenic strategies are being utilized in patients with these tumors.1, 2 In addition, an increasing body of literature has demonstrated that measurable markers related to tumor angiogenesis correlate with specific clinical features in patients with sarcoma.3 These correlations are summarized in Table 1 and presented in full detail in this review.
Table 1. Reported Correlations Between Measures of Angiogenesis and Clinical Features in Patients With Sarcoma
|Microvessel density|| || || ||Chondrosarcoma|| || |
|Tumor VEGF expression|| || ||Soft-tissue sarcoma||Chondrosarcoma, Soft-tissue sarcoma||GIST, Osteosarcoma, Soft-tissue sarcoma||Ewing sarcoma, GIST, Osteosarcoma, Soft-tissue sarcoma|
|Circulating VEGF levels||Ewing sarcoma, GIST, Soft-tissue sarcoma||Bone sarcoma, Soft-tissue sarcoma|| ||Bone sarcoma, Soft-tissue sarcoma||Osteosarcoma|| |
|Tumor PDGF expression|| || || ||Soft-tissue sarcoma|| ||Osteosarcoma|
|Tumor PDGFR expression|| || || ||Chondrosarcoma|| ||Chondrosarcoma, Rhabdomyosarcoma|
|Tumor MMP expression|| || || || || ||Chondrosarcoma, Soft-tissue sarcoma|
|Circulating angiopoietin 2||Soft-tissue sarcoma||Soft-tissue sarcoma|| || || || |
|Circulating bFGF|| || || ||Soft-tissue sarcoma|| || |
Initial attempts at correlating angiogenesis with clinical features in soft-tissue sarcoma focused on microvessel density. In 1 large series of 119 patients with high-grade extremity soft-tissue sarcoma of multiple histologies, factor VIII immunostaining provided reproducible microvessel counts.4 Microvessel density did not correlate with risk of distant metastasis or overall survival. Another series of 115 patients with soft-tissue sarcoma confirmed that microvessel density does not correlate with prognosis.5 Similar findings have been reported in smaller series of patients with leiomyosarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumors, and synovial sarcoma.6–9 Recognizing that tumor microvessels may be less clustered in sarcomas, another group assessed microvessel density at several predetermined locations within soft-tissue sarcoma tumor specimens.10 Despite using methods to account for a more diffuse microvessel pattern, microvessel density again did not correlate with outcome.
Later studies comparing tumor vascular endothelial growth factor (VEGF) expression with clinical features in patients with soft-tissue sarcoma have been more informative. Two studies reported a positive correlation between higher tumor VEGF expression by immunohistochemistry and higher tumor grade.11, 12 Only 1 of these studies evaluated the prognostic impact of high tumor VEGF expression and did not find an association.12 This finding is somewhat unexpected given the known association between tumor grade and prognosis in patients with soft-tissue sarcoma. Two other groups evaluated VEGF expression in gastrointestinal stromal tumor (GIST) using immunohistochemistry. Both groups reported that increased VEGF tumor expression correlated with inferior outcome, including an increased likelihood of developing liver metastases.13, 14
Another group used an enzyme-linked immunosorbent assay (ELISA)-based method to determine tumor VEGF levels more quantitatively in 115 patients with soft-tissue sarcoma.5 Higher tumor VEGF levels correlated with higher tumor stage, higher tumor grade, and increased risk of local and distant tumor recurrence. Not surprisingly, then, patients with tumor VEGF levels above the median level for the group had lower overall survival compared with patients with lower tumor VEGF levels. On multivariate analysis, tumor VEGF level was an independent predictor of overall survival and both local and distant tumor recurrence.5
These results indicate that microvessel density does not correlate with prognosis in soft-tissue sarcoma, whereas tumor VEGF level does correlate with prognosis. These results may reflect methodological difficulties in determining accurate microvessel counts. Alternatively, these results raise the possibility that VEGF impacts prognosis through a mechanism other than driving tumor vessel growth. For example, VEGF may facilitate tumor metastasis by increasing vascular permeability. Increased vascular permeability may also stimulate tumor growth by increasing nutrient availability. VEGF levels could serve as a surrogate marker of other cytokines involved in tumor growth or VEGF could play a role in directly stimulating tumor growth.
The possibility of direct VEGF stimulation of tumor growth is supported by findings in uterine leiomyosarcoma. VEGF, VEGF receptor-1 (VEGFR-1), VEGFR-2, and phosphorylated VEGFR-1 immunohistochemical staining was performed in 21 uterine leiomyosarcoma samples.15 VEGF, VEGFR-1, and VEGFR-2 tumor cell staining was observed at higher levels than in normal uterine smooth muscle cells, whereas phosphorylated VEGFR-1 staining was more prevalent in normal cells than in tumor cells. Patients with phosphorylated VEGFR-1 tumor cell staining had significantly decreased overall survival compared with patients without phosphorylated VEGFR-1 expression. VEGF, VEGFR-1, and VEGFR-2 staining did not correlate with overall survival.15 This report suggests an autocrine or paracrine mechanism in uterine leiomyosarcoma and also highlights the importance of evaluating the expression of activated VEGF receptors.
Angiogenesis mediators other than VEGF have also been evaluated for their clinical impact in soft-tissue sarcoma. Platelet-derived growth factor-B (PDGF-B) protein and mRNA expression appear to correlate with tumor grade and cell proliferation in soft-tissue sarcomas.16 One group evaluated platelet-derived growth factor receptor (PDGFR) gene expression in pediatric rhabdomyosarcoma and demonstrated decreased failure-free survival for patients with tumors that overexpressed either PDGFR-α or PDGFR-β mRNA.17 Several mediators of extracellular matrix breakdown have been shown to correlate with prognosis in patients with soft-tissue sarcoma. Increased levels of matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase plasminogen activator appear to confer a worse prognosis, whereas increased levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) are associated with improved outcomes.18–20
Several groups have evaluated circulating proangiogenic and antiangiogenic factors in patients with soft-tissue sarcoma. In 1 small series of 46 patients, serum VEGF levels measured by ELISA did not differ between patients with soft-tissue sarcoma and healthy controls.21 Five other studies have demonstrated that circulating VEGF levels are significantly elevated in patients with soft-tissue sarcoma.22–26 Patients with GIST have higher serum VEGF levels than healthy controls.27 Most studies have reported that elevated circulating VEGF levels correlate with higher tumor grade and increased local disease burden, but not with the presence of metastatic disease.22, 24–26 Preoperative circulating VEGF levels did not have prognostic significance in the 2 studies evaluating this issue.23, 25 Another study followed serum VEGF levels after surgical or medical therapy for soft-tissue sarcoma and found that rising VEGF levels heralded recurrent or refractory disease.24
Circulating angiopoietin-2 and basic fibroblast growth factor (bFGF) levels are also elevated in patients with soft-tissue sarcoma compared with healthy controls.22, 23, 25, 26 In 1 series, higher circulating levels of angiopoietin-2 were seen in large tumors and in tumors arising in the trunk.26 Circulating bFGF levels were higher in patients with fibrosarcoma and leiomyosarcoma compared with the other histologies represented in 1 series of 108 patients with soft-tissue sarcoma.26 Elevated bFGF levels were generally associated with higher tumor grade, but not with tumor size or the presence of metastases.22, 23, 25, 26 The impact of circulating bFGF on outcome is currently unclear. One study measured plasma bFGF levels in 76 patients and found that patients with high bFGF had a higher recurrence-free survival compared with patients with lower plasma bFGF levels.25 In contrast, serum bFGF levels did not correlate with outcome in another study of 25 patients with soft-tissue sarcoma.23 These conflicting results may reflect differences in patient populations or differences between plasma and serum bFGF levels.
Preoperative circulating endostatin levels have been evaluated in patients with soft-tissue sarcoma, also with somewhat mixed results. In 1 study, plasma endostatin levels were not significantly different between patients and healthy controls.25 Patients with metastatic disease at the time of surgery had lower plasma endostatin levels compared with healthy controls and patients without metastases. Plasma endostatin levels did not correlate with outcome in this study.25 In another study, serum endostatin levels were significantly higher in patients than in controls.23 In this study patients with serum endostatin levels greater than 2 standard deviations above the mean for the group had worse disease-free survival compared with patients with lower serum endostatin levels.23 The use of endostatin as a marker of metastasis or prognosis will require prospective evaluation.
Several groups have evaluated tumor microvessel density and outcome in osteosarcoma. The largest series evaluating this issue included 60 adult and pediatric patients with both localized and metastatic osteosarcoma treated at a single institution.28 Microvessel counts based on CD31 staining were obtained retrospectively on pretreatment biopsy specimens. Patients with high microvessel density had significantly greater overall survival, recurrence-free survival, and tumor necrosis after neoadjuvant chemotherapy compared with patients with low microvessel density. The effect of microvessel density on overall survival was also found in a favorable subgroup of patients with localized disease who had achieved a surgical remission. High microvessel density was found to be an independent favorable prognostic feature in this series.28
Conflicting results were obtained from a series of 39 pediatric patients with nonmetastatic osteosarcoma.29 Microvessel density was determined from von Willebrand factor staining in pretreatment biopsy specimens. High microvessel density correlated with lower overall and disease-free survival as well as larger tumor size and higher incidence of developing distant metastases. A smaller series of 29 pediatric patients with nonmetastatic osteosarcoma treated at a single institution evaluated microvessel density using CD34 staining.30 Microvessel density did not differ between patients who survived and patients who died from osteosarcoma. These 3 conflicting reports highlight the difficulty in drawing conclusions from subjective measures of angiogenesis, particularly in the setting of different microvessel stains between studies.
Expression of VEGF has been used as a more objective means of evaluating the prognostic importance of angiogenesis in osteosarcoma. One group found that 63% of osteosarcoma samples demonstrated VEGF immunohistochemical staining in tumor cells.31 Patients with VEGF-positive tumors had a higher incidence of developing pulmonary metastases compared with patients with VEGF-negative tumors (82% vs 10%, respectively). Not surprisingly, patients with VEGF-positive tumors had significantly worse overall and disease-free survival.31
Another study in 30 patients with nonmetastatic osteosarcoma assessed VEGF expression using reverse-transcriptase polymerase chain reaction (RT-PCR) on pretreatment tumor samples.32 VEGF mRNA was expressed in all tumor samples, with 80% of tumors expressing the VEGF165 isoform. The remaining 20% of tumors expressed only the VEGF121 isoform. Of the patients with VEGF165-positive tumors, 83% developed pulmonary metastases compared with 16% of patients with VEGF165-negative tumors. Overall survival was significantly worse for patients with VEGF165-positive tumors. VEGFR-1 and VEGFR-2 were expressed in 57% and 67% of tumors, respectively. VEGFR expression did not correlate with survival.32
Unlike VEGFR-1 and VEGFR-2, which can bind any of the VEGF isoforms, the neuropilin family of receptors demonstrate VEGF isoform-specific binding.33 Neuropilin-2 binds VEGF145 and VEGF165, but not VEGF121. Neuropilin receptors are thought to modulate VEGF signaling through VEGFR-1 and VEGFR-2.33 In 1 series, 80% of osteosarcoma samples expressed neuropilin-2 mRNA. Patients with neuropilin-2 tumor expression had decreased overall survival compared with patients without neuropilin-2 tumor expression.34 These findings suggest a possible autocrine or paracrine mechanism by which angiogenesis and tumor growth in osteosarcoma are driven by neuropilin-2 augmentation of VEGF165 signaling. These findings further implicate VEGF165 isoform expression in defining a subset of patients with osteosarcoma and a poor prognosis.
Angiogenesis-related factors other than VEGF have also been evaluated as possible markers of prognosis in osteosarcoma. One group evaluated MMP expression by immunohistochemistry in 47 pretreatment osteosarcoma specimens.35 Increased membrane type-1 MMP (MT1-MMP) expression correlated with decreased overall survival. MT1-MMP plays a role in MMP-2 activation, but interestingly, MMP-2 expression did not correlate with survival in this study. Expression of MMP-9 and TIMP-2 also did not correlate with survival.35 Another group evaluated MMP expression in 55 osteosarcoma specimens after neoadjuvant chemotherapy.36 MMP-9 tumor cell expression by immunohistochemistry correlated with decreased overall and disease-free survival on both univariate and multivariate analyses. MT1-MMP expression was not assessed in this study.36 Given that MMP-9 tumor cell expression after neoadjuvant chemotherapy may be confounded by tumor necrosis, further investigation of the prognostic impact of pretreatment MMP-9 expression is necessary.
PDGF expression may also influence prognosis in osteosarcoma. In 1 series of 57 patients with osteosarcoma, PDGF-AA homodimer expression was assessed by immunohistochemistry.37 Patients with PDGF-AA expression above the mean level for the group had decreased disease-survival in both univariate and multivariate analyses, with a relative risk of recurrence of 2.7 for patients with high PDGF-AA tumor expression. PDGFR-α expression did not correlate with outcome, although patients older than 20 years of age had higher levels of PDGFR-α expression.37
Additional research has focused on circulating markers of angiogenesis in bone sarcoma. In 1 study of 27 patients, serum VEGF levels were measured in patients with osteosarcoma, Ewing sarcoma, and chondrosarcoma and compared with healthy controls.38 Only patients with Ewing sarcoma had a mean VEGF level significantly greater than the mean level for healthy controls. A larger study evaluated serum VEGF and bFGF levels in 72 patients with nonmetastatic bone sarcoma compared with healthy controls and patients with benign bone tumors.39 Patients with bone sarcomas had higher levels of serum VEGF and bFGF than healthy controls but not greater than patients with benign bone tumors. Higher serum VEGF levels were observed in bone sarcoma patients with larger tumors, higher grade tumors, and more locally invasive tumors. Serum VEGF and bFGF levels did not correlate with prognosis and did not differ between patients with osteosarcoma, Ewing sarcoma, or chondrosarcoma.39
The prognostic impact of serum VEGF, bFGF, and placental growth factor levels was evaluated in a series of 16 patients with osteosarcoma.40 Patients with metastatic disease at diagnosis or who developed metastatic disease within 1 year of diagnosis had significantly higher serum levels of VEGF than patients without early metastatic disease. The sensitivity and specificity of a serum VEGF level above the mean for the group in identifying early metastatic disease were 100% and 88.9%, respectively. Serum bFGF and placental growth factor levels did not differ between patients with and without early metastatic disease. Serum VEGF levels did not correlate with primary tumor size.40 The utility of tissue and serum VEGF in identifying patients with osteosarcoma at high risk of recurrence despite conventional chemotherapy will need to be evaluated in larger prospective studies.
Patients with Ewing sarcoma have increased serum VEGF levels compared with healthy controls.38, 41 The impact of serum VEGF levels on the clinical course of patients with Ewing sarcoma has not been evaluated. In contrast, 1 study reported that VEGF tumor expression in Ewing sarcoma correlates with outcome.42 Using immunohistochemistry staining of pretreatment tumor specimens, 31 patients with Ewing sarcoma were divided into a VEGF-positive and a VEGF-negative group. VEGF-positive patients had significantly worse overall survival compared with VEGF-negative patients.42
Several markers of angiogenesis correlate with tumor grade in chondrosarcoma. In 1 series of 26 cartilage-derived tumors, pericartilage microvessels were observed in all of the tumors, including enchondroma and all grades of chondrosarcoma.43 Intracartilage microvessels were not seen in any of the enchondroma samples, whereas intracartilage microvessel counts were much higher in grade 2 and 3 chondrosarcoma than in grade 1 chondrosarcoma. Similarly, chondrocyte VEGF expression measured by immunohistochemistry increased with increasing tumor grade.43
Based on preclinical data indicating a role for PDGF in chondrocyte proliferation, another group evaluated 67 chondrosarcoma tumor samples for PDGF-AA homodimer and PDGFR-α expression by immunohistochemistry.44 PDGFR-α expression increased with increasing tumor grade. On univariate analysis, patients with high PDGFR-α expression had significantly worse overall survival compared with patients with low PDGFR-α expression. On multivariate analysis, though, the effect of PDGFR-α expression on survival was not independent of tumor grade. PDGF-AA expression did not correlate with tumor grade or survival.44
MMP-1 expression also correlates with outcome in chondrosarcoma. Patients with chondrosarcoma and a high MMP-1 to TIMP-1 ratio had a significantly worse disease-free survival compared with patients with a low MMP-1 to TMP-1 ratio.45 These results suggest that proangiogenic factors, including VEGF, PDGF, and MMPs, correlate with important clinical features in chondrosarcoma.
Surprisingly little data correlate clinical features in vascular sarcomas with markers of angiogenesis. In angiosarcoma, serum VEGF levels were not elevated and did not correlate with extent of disease in most of the patients included in 1 small series.46 In 1 patient with an angiosarcoma harboring a p53 gene mutation, serum VEGF levels were greater than in healthy controls and increased in concert with the development of metastatic disease.46, 47 In another small series of patients with cutaneous angiosarcoma, higher serum bFGF levels were reported in patients with nodular lesions than in patients with macular lesions or healthy controls.48 In 2 children with hemangioendothelioma, plasma VEGF levels were elevated compared with unaffected children.49
Angiogenesis markers correlate with important clinical features in patients with sarcomas ranging from soft-tissue sarcomas to bone sarcomas. These markers may play an increasing role in the management of patients with sarcoma. Markers of angiogenesis may serve an important role in predicting a particular patient's clinical course. In addition, angiogenesis markers may help to guide which patients with sarcoma may benefit most from antiangiogenic therapies and to monitor patients receiving these therapies. Future research will need to confirm existing findings, but also evaluate other measures of angiogenesis such as circulating endothelial cells.