The VEGF- and PDGF-family of angiogenic markers have prognostic impact in soft tissue sarcomas arising in the extremities and trunk

  • Thomas K Kilvaer4Email author,

    Affiliated with

    • Eivind Smeland4,

      Affiliated with

      • Andrej Valkov4, 4,

        Affiliated with

        • Sveinung W Sorbye4, 4,

          Affiliated with

          • Roy M Bremnes4, 4,

            Affiliated with

            • Lill-Tove Busund4, 4 and

              Affiliated with

              • Tom Donnem4, 4

                Affiliated with

                BMC Clinical Pathology201414:5

                DOI: 10.1186/1472-6890-14-5

                Received: 2 November 2013

                Accepted: 16 January 2014

                Published: 20 January 2014

                Abstract

                Background

                Soft-tissue sarcomas are rare malignant tumors of mesenchymal lineage that can arise in any part of the body. Prognosis, and hence also treatment may vary according to histologic subtype and localization. Angiogenesis is the process of forming new blood vessels from pre-existing ones. The deregulation of this process is thought to be an important step in malignant transformation. This study investigates the prognostic impact of platelet derived growth factor- (PDGF), vascular endothelial growth factor- (VEGF) and fibroblast growth factor (FGF) families in soft-tissue sarcomas of the extremities & trunk (ET) and visceral & retroperitoneal (VR) locations.

                Methods

                Tumor samples from 181 patients (115 ET and 66 VR) with resected soft tissue sarcomas were collected and tissue microarrays were constructed. Immunohistochemistry was used to evaluate angiogenic marker expression. Recurrence-free survival (RFS), metastasis-free survival (MFS) and disease-specific survival (DSS) were used as endpoints in prognostic impact assessment.

                Results

                In univariate analyses, almost all investigated angiogenic markers had prognostic impact in the ET group. In contrast, only FGFR-1 showed any significant prognostic impact in the VR group. In the multivariate analyses, PDGF-D (HR = 1.863, 95% CI = 1.057-3.283, P = 0.031), VEGFR-1 (HR = 2.106, 95% CI = 1.038-4.272, P = 0.039) and VEGF-A (HR 2.095, 95% CI 1.028-4.271, P = 0.042) were independent negative prognosticators for DSS, MFS and RFS, respectively, in the ET group. FGFR-1 was an independent positive prognosticator for DSS (HR = 0.243, 95% CI = 0.095-0.618, P = 0.003) in the VR group.

                Conclusions

                Angiogenic molecules from the PDGF and VEGF families have prognostic impact in soft-tissue sarcomas arising in the ET, but not in VR locations. In the latter histological grade and resection margins are the most important prognostic factors.

                Keywords

                Angiogenesis Sarcoma Extremity Trunk FGF PDGF VEGF Visceral Retroperitoneal

                Background

                Soft tissue sarcomas (STS) constitute a highly heterogeneous collection of tumors comprising over 50 histological subtypes, arising from mesenchymal tissue and capable of forming tumors in all parts of the human body [1]. This group amounts to 0.5-1% of the annual tumor burden with a mortality of about 40-60%, resulting in an estimated 11 280 cases and 3 900 deaths in the US in 2012 [2]. It is good practice to distinguish between STSs arising in the extremity & trunk (ET), head & neck (HN) and visceral & retroperitoneal (VR) localizations as treatment and prognosis vary widely according to localization [3]. Further subdivision, according to histological type, malignancy grade, stage and vascular invasion among others, can be conducted [3]. Definitive treatment is radical surgery followed by radiotherapy in case of non-radical surgical margins [4]. Adjuvant chemotherapy for adult STS is still under investigation, and hence the routine use of such treatment is today limited to the palliative setting [5].

                Angiogenesis is the process of forming new blood vessels from pre-existing ones. Folkman and coworkers proved this to be a pivotal step in carcinogenesis by showing that tumors would not grow beyond > 2 mm in diameter without forming vasculature [6, 7]. In 2001, Hanahan and Weinberg, suggested angiogenesis as one of the hallmarks of cancer [8] and in the 2011 updated version angiogenesis was still considered one of the most important aspects of cancer progression [9].

                Vascular endothelial growth factors (VEGF) and receptors (VEGFR) are pivotal in endothelial cell proliferation and sprouting during angio- and lymphangiogenesis [10]. Platelet-derived growth factors (PDGF) and receptors (PDGFR) play an important part in the regulation of tumor stroma through the recruitment of pericytes and vascular smooth muscle cells helping to stabilize newly formed vessels and through stimulation of stromal cells to produce VEGF-A and thus drive angiogenesis [11, 12]. Fibroblast growth factors (FGF) and receptors (FGFR) drives endothelial cell proliferation and sprouting and activate several molecules involved in extracellular matrix remodelling including matrix metallo-proteinases and urokinase-like plasminogen activator [13].

                Our group has previously reported on the expression of VEGF, PDGF and FGF families of growth factors in STSs of all sites [1416]. This report investigates the differential impact of these growth factors in STSs arising in ET versus VR localizations.

                Methods

                Patients and clinical samples

                Primary tumor tissue from anonymized patients diagnosed with STS at the University Hospital of North-Norway and the Hospitals of Arkhangelsk County, Russia, from 1973 through 2006, were collected. In total 496 patients were registered from the hospital databases. Of these, 388 patients were excluded from the study because of: missing clinical data (n = 86), inadequate formalin-fixed paraffin-embedded (FFPE) tissue blocks (n = 161), no surgery performed and/or metastasis present at the time of diagnosis (n = 55) or head and neck sarcomas (n =13). Thus 115 patients with STSs of the extremities and trunk wall and 66 patients with STSs of visceral or retroperitoneal origin, with complete medical records and FFPE tissue blocks were eligible.

                This report includes follow-up data as of September 2009. The median follow-up was 53.9 (range 0.5-391.7) months for extremity and trunk patients and 59.4 (range 0.10-366.7) months for visceral and retroperitoneal patients. Complete demographic and clinical data were collected retrospectively. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the Departments of Pathology at the University Hospital of North-Norway and the Hospitals of Arkhangelsk County, Russia. The tumors were graded according to the French Fédération Nationale des centres de Lutte Contre le Cancer (FNCLCC) system and histologically subtyped according to the World Health Organization guidelines [1, 17]. Wide resection margins were defined as wide local resection with free microscopic margins or amputation of the affected limb or organ.

                Microarray construction

                All sarcomas were histologically reviewed by two trained pathologists (S. Sorbye and A. Valkov) and the most representative areas of tumor cells (neoplastic mesenchymal cells) were carefully selected and marked on the hematoxylin and eosin (H/E) slide and sampled for the tissue microarray (TMA) blocks. The TMAs were assembled using a tissue-arraying instrument (Beecher Instruments, Silver Springs, MD). The Detailed methodology has been previously reported [18]. Briefly, we used a 0.6 mm diameter stylet, and the study specimens were routinely sampled with four replicate core samples from different areas of neoplastic tissue. Normal tissue from the patients was used as staining control.

                To include all core samples, 12 TMA blocks were constructed. Multiple 5-μm sections were cut with a Micron microtome (HM355S) and stained by specific antibodies for immunohistochemistry (IHC) analysis.

                Immunohistochemistry

                The applied antibodies were subjected to in-house validation by the manufacturer for IHC analysis on paraffin-embedded material. The detailed methodology has previously been reported [1416].

                Scoring of immunohistochemistry

                The ARIOL imaging system (Genetix, San Jose, CA) was used to scan the slides of antibody staining of the TMAs and the dominant staining intensity was scored as: 0 = negative; 1 = weak; 2 = intermediate; 3 = strong semi-qantitively on computer screen. The detailed methodology has previously been reported and cut-off values chosen were the same as in our previous studies [1416]. High expression in tumor cells were defined as ≥ 1 (VEGF-C), ≥ 1.5 (PDGF-A, PDGF-C, PDGF-B, VEGF-A, VEGF-D, VEGFR-1-2 and -3) and ≥ 2 (PDGF-D, PDGFR-α, PDGFR-β, FGF2 and FGFR-1).

                Statistical methods

                All statistical analyses were done using the statistical package SPSS (Chicago, IL), version 16. The IHC scores from each observer were compared for interobserver reliability by use of a two-way random effect model with absolute agreement definition. The intraclass correlation coefficient (reliability coefficient) was obtained from these results. The Chi-square test and Fishers Exact test were used to examine the association between molecular marker expression and various clinicopathological parameters. Univariate analyses were done using the Kaplan-Meier method, and statistical significance between survival curves was assessed by the log-rank test. Disease-specific survival (DSS) was determined from the date of diagnosis to the time of cancer related death. Metastasis-free survival (MFS) was defined from the date of diagnosis to the clinical appearance of the first metastasis. Recurrence-free survival (RFS), was defined from the date of diagnosis to the clinical appearance of the first recurrence. To assess the independent value of different pretreatment variables on survival, metastasis and local recurrence, in the presence of other variables, multivariate analyses were carried out using the Cox proportional hazards model. Only variables of significant value from the univariate analyses were entered into the Cox regression analysis. Probability for stepwise entry and removal was set at .05 and .10, respectively. The significance level used for all statistical tests was P < 0.05.

                Ethical clearance

                The Norwegian National Data Inspection Board and The Regional Committee for Research Ethics (Northern Norway) approved the study.

                Results

                Clinicopathological variables

                The clinicopathological variables are summarized in Table 1. In the ET group, comprising 115 patients, median age was 59 (range 0-89) years, 50% of the patients were male, 67 patients were Norwegian and 48 Russian and 68% of the tumors were located in the extremities. Of the histological subtypes represented, 48 were undifferentiated pleomorphic sarcomas, 18 liposarcomas, 12 fibrosarcomas, 10 synovial sarcomas, 9 leiomyosarcomas, 5 angiosarcomas, 5 rhabdomyosarcomas, 5 malignant peripheral nerve sheath tumors (MPNST) and 3 sarcoma not otherwise specified (NOS).
                Table 1

                Prognostic clinicopathological variables as predictors for disease-specific survival, metastasis and local recurrence in patients with resected Extremitiy & Trunk and Visceral & Retroperitoneal soft-tissue sarcomas (univariate analyses, log rank test, n = 115 and 66 respectively)

                 

                Extremity & trunk

                Visceral & retroperitoneal

                  

                Disease-specific survival

                Metastasis-free survival

                Recurrence-free survival

                 

                Disease-specific survival

                Metastasis-free survival

                Recurrence-free survival

                Characteristics

                Patients (n)

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                Patients (n)

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                Age

                              

                ≤ 20 years

                12

                42

                0.431

                42

                0.129

                73

                0.690

                1

                0

                <0.001

                100

                0.112

                100

                0.786

                21-60 years

                48

                61

                 

                63

                 

                69

                 

                36

                71

                 

                67

                 

                71

                 

                > 60 years

                55

                55

                 

                69

                 

                64

                 

                29

                43

                 

                44

                 

                62

                 

                Gender

                              

                Male

                57

                56

                0.298

                66

                0.368

                66

                0.759

                15

                79

                0.039

                86

                0.022

                66

                0.799

                Female

                58

                54

                 

                61

                 

                67

                 

                51

                51

                 

                48

                 

                68

                 

                Patient nationality

                              

                Norwegian

                67

                65

                0.004

                74

                0.008

                71

                0.249

                54

                62

                0.051

                59

                0.122

                66

                0.892

                Russian

                48

                42

                 

                48

                 

                57

                 

                12

                36

                 

                46

                 

                59

                 

                Histological entity

                              

                Pleomorphic sarcoma

                48

                42

                0.004

                61

                0.001

                56

                0.664

                6

                50

                0.917

                67

                0.264

                40

                0.274

                Leiomyosarcoma

                9

                100

                 

                78

                 

                78

                 

                39

                55

                 

                46

                 

                77

                 

                Liposarcoma

                18

                83

                 

                94

                 

                89

                 

                13

                62

                 

                81

                 

                54

                 

                Fibrosarcoma

                12

                57

                 

                67

                 

                55

                 

                0

                      

                Angiosarcoma

                5

                40

                 

                20

                 

                67

                 

                2

                50

                 

                50

                 

                50

                 

                Rhabdomyosarcoma

                5

                60

                 

                60

                 

                60

                 

                1

                  

                100

                 

                100

                 

                MPNST

                5

                53

                 

                60

                 

                60

                 

                4

                67

                 

                100

                 

                100

                 

                Synovial sarcoma

                10

                13

                 

                30

                 

                64

                 

                1

                100

                 

                0

                 

                0

                 

                Sarcoma NOS

                3

                100

                 

                67

                 

                67

                 

                0

                      

                Tumor size

                              

                < 5 cm

                38

                70

                0.048

                81

                0.053

                74

                0.085

                11

                82

                0.107

                73

                0.259

                89

                0.006

                5-10 cm

                45

                48

                 

                53

                 

                61

                 

                24

                62

                 

                62

                 

                70

                 

                > 10 cm

                30

                49

                 

                58

                 

                62

                 

                31

                45

                 

                45

                 

                49

                 

                Missing

                2

                             

                Malignancy grade

                              

                1

                29

                89

                <0.001

                89

                0.001

                85

                0.054

                23

                78

                0.005

                77

                0.051

                86

                0.046

                2

                41

                56

                 

                65

                 

                60

                 

                29

                46

                 

                42

                 

                58

                 

                3

                45

                35

                 

                43

                 

                57

                 

                14

                46

                 

                47

                 

                49

                 

                Vascular invasion

                              

                Absent

                64

                70

                <0.001

                85

                <0.001

                79

                <0.001

                43

                59

                0.656

                60

                0.847

                66

                0.675

                Present

                50

                35

                 

                33

                 

                43

                 

                20

                51

                 

                54

                 

                78

                 

                Missing

                1

                      

                3

                      

                Tumor depth

                              

                Superficial

                12

                91

                0.010

                100

                0.012

                91

                0.041

                32

                      

                Deep

                103

                51

                 

                59

                 

                63

                 

                34

                      

                Resection margins

                              

                Wide

                61

                66

                0.004

                72

                0.045

                82

                <0.001

                50

                65

                0.021

                59

                0.654

                90

                <0.001

                Non-wide

                54

                44

                 

                54

                 

                46

                 

                16

                50

                 

                53

                 

                44

                 

                Abbreviations: MPNST Malingnant peripheral nerve sheat tumor, NOS Not otherwise specified.

                In the VR group, median age was 58 (range 13-88) years, 23% of the patients were male and 54 patients were Norwegian and 12 Russian. Of the histological subtypes represented, 39 were leiomyosarcomas, 13 liposarcomas, 6 pleomorphic sarcomas, 4 neurofibrosarcomas/MPNSTs, 2 angiosarcomas, 1 rhabdomyosarcoma and 1 synovial sarcoma.

                Interobserver variability

                Interobserver scoring agreement was tested for PDGF-B, PDGFR-α, VEGF-C, VEGFR-3, FGF2 and FGFR1 and found to be good (0.77-0.90, P < 0.001) [1416].

                Univariate analyses

                The impact of the clinicopathological variables on DSS, MFS and RFS in the ET group are summarized in Table 1. Patient nationality (P = 0.004), histological entity (p = 0.004), tumor size (p = 0.048), malignancy grade (P < 0.001), vascular invasion (P <0.001), tumor depth (P = 0.010) and resection margins (P = 0.004) were all prognostic indicators of DSS. Patient nationality (P = 0.008), histological entity (P = 0.001), malignancy grade (P = 0.001), vascular invasion (P < 0.001), tumor depth (P = 0.012) and resection margins (P = 0.045) were prognostic indicators of MFS. Finally, vascular invasion (P < 0.001), tumor depth (P = 0.041) and resection margins (P < 0.001) were prognostic indicators of RFS.

                The impact of the angiogenic markers on DSS, MFS and RFS in the ET group are summarized in Table 2. PDGF-A (P = 0.035), PDGF-B (P = 0.006), PDGF-C (P = 0.032), PDGF-D (P = 0.003), PDGFR-α (P = 0.002), PDGFR-β (P = 0.029), VEGF-A (P = 0.001), VEGFR-1 (P = 0.001) and FGF2 (P = 0.033) were prognostic indicators of DSS. PDGF-A (P = 0.007), PDGF-B (P = 0.003), PDGFR-α (P = 0.002), PDGFR-β (P = 0.002), VEGF-A (P = 0.001), VEGFR-1 (P < 0.001) and VEGFR-3 (P = 0.008) were prognostic indicators of MFS. PDGF-A (P = 0.012), PDGF-B (P = 0.015), PDGFR-α (P = 0.011), VEGF-A (P = 0.002) and VEGFR-1 (P = 0.036) were prognostic indicators of RFS.
                Table 2

                Angiogenic markers as predictors for disease-specific survival, metastasis and local recurrence in patients with resected soft-tissue sarcomas of the extremities or trunk (univariate analyses, log rank test, n = 115)

                  

                Disase-specific survival

                Metastasis-free survival

                Recurrence-free survival

                Marker expression

                Patients (n)

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                PDGF-A

                       

                Low

                54

                60

                0.035

                74

                0.007

                79

                0.012

                High

                58

                52

                 

                51

                 

                55

                 

                Missing

                3

                      

                PDGF-B

                       

                Low

                44

                68

                0.006

                78

                0.003

                82

                0.015

                High

                68

                48

                 

                52

                 

                56

                 

                Missing

                3

                      

                PDGF-C

                       

                Low

                31

                71

                0.032

                68

                0.214

                68

                0.564

                High

                80

                50

                 

                61

                 

                65

                 

                Missing

                4

                      

                PDGF-D

                       

                Low

                73

                67

                0.003

                70

                0.051

                77

                0.002

                High

                40

                34

                 

                49

                 

                42

                 

                Missing

                2

                      

                PDGFR-α

                       

                Low

                69

                67

                0.002

                74

                0.002

                77

                0.011

                High

                43

                38

                 

                42

                 

                45

                 

                Missing

                3

                      

                PDGFR-β

                       

                Low

                85

                64

                0.029

                72

                0.002

                69

                0.825

                High

                24

                32

                 

                35

                 

                58

                 

                Missing

                6

                      

                VEGF-A

                       

                Low

                60

                65

                0.001

                75

                0.001

                77

                0.002

                High

                51

                43

                 

                48

                 

                51

                 

                Missing

                4

                      

                VEGF-C

                       

                Low

                69

                55

                0.476

                68

                0.083

                68

                0.232

                High

                38

                60

                 

                56

                 

                63

                 

                Missing

                8

                      

                VEGF-D

                       

                Low

                84

                57

                0.131

                67

                0.081

                70

                0.177

                High

                29

                50

                 

                51

                 

                55

                 

                Missing

                2

                      

                VEGFR-1

                       

                Low

                67

                63

                0.002

                77

                <0.001

                72

                0.036

                High

                44

                46

                 

                43

                 

                58

                 

                Missing

                4

                      

                VEGFR-2

                       

                Low

                78

                58

                0.332

                67

                0.189

                71

                0.240

                High

                28

                52

                 

                53

                 

                57

                 

                Missing

                9

                      

                VEGFR-3

                       

                Low

                75

                60

                0.053

                70

                0.008

                70

                0.159

                High

                34

                45

                 

                46

                 

                57

                 

                Missing

                6

                      

                FGF2

                       

                Low

                75

                61

                0.033

                66

                0.214

                70

                0.648

                High

                35

                49

                 

                56

                 

                67

                 

                Missing

                6

                      

                FGFR-1

                       

                Low

                83

                58

                0.460

                64

                0.411

                66

                0.768

                High

                26

                47

                 

                55

                 

                71

                 

                Missing

                6

                      

                Abbreviations: PDGF Platelet-derived growth factor, PDGFR Platelet-derived growth factor receptor, VEGF Vascular endothelial growth factor, VEGFR Vascular endothelial growth factor receptor, FGF Fibroblast growth factor, FGFR Fibroblast growth factor receptor.

                The impact of the clinicopathological variables on DSS, MFS and RFS in the VR group are summarized in Table 1. Age (P < 0.001), gender (P = 0.039), malignancy grade (P = 0.005) and resection margins (P = 0.021) were prognostic indicators of DSS. Gender (P = 0.022) was a prognostic indicator of MFS and tumor size (P = 0.006), malignancy grade (P = 0.046) and resection margins (P < 0.001) were prognostic indicators of RFS.

                The impact of angiogenic markers on DSS, MFS and RFS in the VR group is summarized in Table 3. FGRF-1 (P = 0.023) was the only prognostic indicator for DSS and PDGF-C (P = 0.045) for RFS.
                Table 3

                Angiogenic markers as predictors for disease-specific survival, metastasis and local recurrence in patients with resected visceral & retroperitoneal soft-tissue sarcomas (univariate analyses, log rank test, n = 66)

                  

                Disase-specific survival

                Metastasis-free survival

                Recurrence-free survival

                Marker expression

                Patients (n)

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                5-Year survival (%)

                P

                PDGF-A

                       

                Low

                23

                49

                0.473

                63

                0.593

                65

                0.315

                High

                39

                63

                 

                54

                 

                73

                 

                Missing

                4

                      

                PDGF-B

                       

                Low

                14

                54

                0.604

                82

                0.088

                61

                0.291

                High

                48

                59

                 

                51

                 

                73

                 

                Missing

                4

                      

                PDGF-C

                       

                Low

                20

                39

                0.297

                59

                0.986

                53

                0.045

                High

                41

                65

                 

                56

                 

                76

                 

                Missing

                5

                      

                PDGF-D

                       

                Low

                48

                52

                0.078

                52

                0.197

                70

                0.343

                High

                15

                80

                 

                73

                 

                59

                 

                Missing

                3

                      

                PDGFR-α

                       

                Low

                41

                56

                0.672

                61

                0.527

                71

                0.761

                High

                21

                61

                 

                51

                 

                66

                 

                Missing

                4

                      

                PDGFR-β

                       

                Low

                58

                57

                0.360

                54

                0.532

                70

                0.766

                High

                4

                75

                 

                75

                 

                75

                 

                Missing

                4

                      

                VEGF-A

                       

                Low

                34

                51

                0.326

                64

                0.719

                64

                0.054

                High

                29

                68

                 

                53

                 

                79

                 

                Missing

                3

                      

                VEGF-C

                       

                Low

                34

                66

                0.402

                69

                0.071

                62

                0.051

                High

                29

                50

                 

                45

                 

                84

                 

                Missing

                3

                      

                VEGF-D

                       

                Low

                34

                60

                0.856

                62

                0.388

                63

                0.116

                High

                30

                55

                 

                51

                 

                78

                 

                Missing

                2

                      

                VEGFR-1

                       

                Low

                37

                55

                0.724

                63

                0.358

                70

                0.510

                High

                25

                63

                 

                49

                 

                71

                 

                Missing

                3

                      

                VEGFR-2

                       

                Low

                44

                55

                0.858

                63

                0.446

                69

                0.821

                High

                19

                67

                 

                48

                 

                75

                 

                Missing

                3

                      

                VEGFR-3

                       

                Low

                36

                54

                0.552

                59

                0.821

                65

                0.220

                High

                25

                61

                 

                52

                 

                76

                 

                Missing

                5

                      

                FGF2

                       

                Low

                39

                56

                0.805

                51

                0.214

                74

                0.748

                High

                20

                65

                 

                67

                 

                66

                 

                Missing

                7

                      

                FGFR-1

                       

                Low

                43

                45

                0.023

                56

                0.385

                68

                0.448

                High

                20

                89

                 

                63

                 

                78

                 

                Missing

                3

                      

                Abbreviations: PDGF Platelet-derived growth factor, PDGFR Platelet-derived growth factor receptor, VEGF Vascular endothelial growth factor, VEGFR Vascular endothelial growth factor receptor, FGF Fibroblast growth factor, FGFR Fibroblast growth factor receptor.

                Multivariate cox proportional hazards analysis

                Table 4 presents multivariate analyses of clinicopathological and angiogenic marker variables with respect to DSS, MFS and RFS in the ET and VR groups, respectively.
                Table 4

                Multivariate analyses of clinopathological variables and angiogenic markers as prognostic values for disease-specific survival, metastasis and local recurrence in patients with resected soft-tissue sarcomas of the trunk or extremities (cox proportional hazards test)

                 

                Disase-specific survival

                 

                Metastasis-free survival

                 

                Recurrence-free survival

                 

                Variable

                HR

                95% CI

                P

                HR

                95% CI

                P

                HR

                95% CI

                P

                Extremity & trunk

                Malignancy grade

                         

                1

                1.000

                 

                <0.001*

                      

                2

                4.066

                1.389-11.901

                0.010

                      

                3

                6.025

                2.058-17.634

                0.001

                      

                Vascular invasion

                         

                Absent

                1.000

                  

                1.000

                  

                1.000

                  

                Present

                2.141

                1.188-3.859

                0.011

                5.284

                2.418-11.544

                <0.001

                2.135

                1.019-4.475

                0.045

                Resection margins

                         

                Wide

                1.000

                     

                1.000

                  

                Non-wide

                1.818

                1.032-3.203

                0.039

                   

                2.687

                1.289-5.602

                0.008

                PDGF-B

                         

                Low

                      

                1.000

                  

                High

                      

                2.099

                0.937-4.706

                0.072

                PDGF-D

                         

                Low

                1.000

                     

                1.000

                  

                High

                1.863

                1.057-3.283

                0.031

                   

                1.844

                0.931-3.653

                0.079

                VEGF-A

                         

                Low

                      

                1.000

                  

                High

                      

                2.095

                1.028-4.271

                0.042

                VEGFR-1

                         

                Low

                   

                1.000

                     

                High

                   

                2.106

                1.038-4.272

                0.039

                   

                Visceral & retroperitoneal

                Gender

                         

                Male

                   

                1.000

                     

                Female

                   

                4.612

                1.089-19.536

                0.038

                   

                Malignancy grade

                         

                1

                1.000

                 

                0.003*

                   

                1.000

                 

                0.061

                2

                4.812

                1.823-12.705

                0.002

                   

                2.069

                0.630-6.794

                0.231

                3

                5.646

                1.790-17.804

                0.003

                   

                5.665

                1.330-24.123

                0.019

                Resection margins

                         

                Wide

                1.000

                     

                1.000

                  

                Non-wide

                2.712

                1.222-6.018

                0.014

                   

                11.996

                3.128-46.005

                <0.001

                PDGF-C

                         

                Low

                      

                1.000

                  

                High

                      

                0.413

                0.157-1.089

                0.074

                FGFR-1

                         

                Low

                1.000

                        

                High

                0.243

                0.095-0.618

                0.003

                      

                Abbreviations: HR Hazard ratio, CI Confidence interval, VEGF Vascular endothelial growth factor, VEGFR Vascular endothelial growth factor receptor, PDGFR Platelet-derived growth factor receptor, FGFR Fibroblast growth factor receptor, *overall significance as prognostic factor.

                In the ET group, high malignancy grade (P < 0.001), the presence of vascular invasion (P = 0.011), non-wide resection margins (P = 0.039) and high expression of PDGF-D (HR = 1.863, 95% CI = 1.057-3.283, P = 0.031) were significant independent prognostic indicators of DSS. Further, the presence of vascular invasion (P < 0.001) and high expression of VEGFR-1 (HR = 2.106, 95% CI = 1.038-4.272, P = 0.039) were significant independent prognostic factors of MFS, while the presence of vascular invasion (P = 0.045), non-wide resection margins (P = 0.008) and high expression of VEGF-A (HR 2.095, 95% CI 1.028-4.271, P = 0.042) were significant independent prognostic factors of RFS.

                In the VR group, high malignancy grade (P = 0.003) and non-wide resection margins (P = 0.014) were significant independent adverse prognostic indicators of DSS whereas high FGFR-1 expression (HR = 0.243, 95% CI = 0.095-0.618, P = 0.003) was an independent positive prognostic indicator of DSS. Female gender (P = 0.038) was an independent negative prognostic indicator of MFS while non-wide resection margins (P < 0.001) was an independent negative prognostic indicator of RFS.

                Discussion and conclusions

                In our univariate analyses high expression of most examined angiogenic markers were prognosticators of DSS and/or MFS and/or RFS in the ET group. Further, PDGF-D was an independent negative prognostic indicator of DSS, VEGFR-1 an independent negative prognostic indicator of MFS and VEGF-A an independent negative prognostic indicator of RFS. In contrast, only FGFR-1 was a prognosticator of DSS in both the univariate and multivariate analyses of the VR group. To our knowledge, this is the first comparison of the expression of angiogenic molecules in ET versus VR STSs.

                Current knowledge of the importance of tumor localization (ET versusVR tumors) when it comes to the prognostic impact of angiogenic markers in STSs is limited. Yudoh et. al. investigated the level of VEGF-A in tissue from ET patients and found high levels to predict survival, local recurrence and metastasis [18]. We have previously reported on the expression of PDGFs, VEGFs and FGFs in a larger cohort of STS of mixed sites and histology and found high expression of VEGFR-3, PDGF-B and FGF2 to have independent negative prognostic impact on DSS [1416]. When comparing the expression of angiogenic markers based on tumor location, it becomes apparent that these variables almost exclusively have prognostic impact in STS arising in the ET group (Tables 2, 3 and 4). This difference could to some extent be due to a smaller number of patients in the VR group, with a resulting increased risk of false negative results. However, near all angiogenic markers showed significant prognostic impact in the univariate analyses of the ET group, whereas only FGFR-1 showed prognostic impact in the VR group. Table 1 summarizes the clinopathological values in the ET and VR groups and it is apparent that the VR group contains a higher percentage of leiomysarcomas and liposarcomas. The different distribution of histologies between the ET and VR groups might suggest that angiogenic markers have higher impact in STSs arising in ET locations. Another explanation may be that ET tumors, even the slow growing ones, will produce symptoms when they reach a certain size due to limits created by connective and muscle tissue and blood and lymph vessels. VR tumors could in contrast grow to significant size before producing symptoms. This may explain our results as VR tumors in many cases only are found after the angiogenic switch have occurred, thus the impact of angiogenic markers have been negated in these tumors.

                In the PDGF-axis, all markers were prognosticators of DSS, all but PDGF-C were prognosticators of MFS and all but PDGF-C and PDGFR-β were prognosticators of RFS in the ET group (Table 2), while none of the PDGFs were prognosticators in the VR group. Further, PDGF-D was found to be an independent negative prognostic factor for DSS in the ET group. In our previous study, PDGF-B was an independent prognosticator of DSS [15], and in this study PDGF-D is an independent prognosticator of DSS. PDGF-B binds all PDGFRs while PDGF-D binds PDGFR-αβ and-ββ [11]. Both PDGF-B and PDGF-D has been shown to exhibit similar and extensive angiogenic and transforming abilities [19, 20]. Although our results cannot distinguish whether PDGF signalling drives tumor development through angiogenesis or other pathways, they strongly suggest PDGF signalling to be an important part of STS growth and progression.

                In the VEGF-axis, VEGF-A, and VEGFR-1 were prognosticators of DSS, MFS and RFS in the ET group, while none of the VEGFs were prognosticators in the VR group (Table 2). Further, VEGFR-1 was an independent prognostic indicator of MFS and VEGF-A was an independent prognostic indicator of RFS in the ET group. VEGF-A signalling is the major angiogenic pathway, and high tumor expression and availability in serum has previously been associated with malignancy grade, metastasis, local recurrence and worse overall survival in STS patients [18, 2126]. VEGFR-1 is thought to modulate VEGF-A signalling through VEGFR-2, has anti-angiogenic properties in its soluble form, and has been linked to metastasis in experimental studies suggesting a feasible biological link for our finding in these STS patients [27, 28]. This latter finding is quite interesting as antibodies and small-molecules targeting VEGFR-1 are being developed [29, 30].

                In the FGF-axis, FGF-2 was an unfavorable prognostic indicator of DSS in ET group. FGF2 is thought to drive cell-cycling, activate extracellular matrix remodelling and to rescue PDGF-B and VEGF-A driven angiogenesis in the presence of their respective inhibitors [13, 31, 32]. Surprisingly, FGFR-1 was an independent positive indicator of DSS in the VR group. To our knowledge these are new data, but these results have to be validated before a firm conclusion may be drawn due to the low number of patients.

                This study enhances our current knowledge on angiogenic prognosticators in STSs, strongly indicates the involvement of the PDGF and VEGF pathways in ET STS development and adds to the growing body of evidence suggesting that STSs of different sites and histology should be analyzed independently in future studies. Further emphasis should also be put on validating VEGFR-1 as a predictor of MFS in ET STS patients, as these patients may benefit from adjuvant therapy targeting VEGFR-1.

                Declarations

                Authors’ Affiliations

                (1)
                Department of Oncology, University Hospital of North Norway
                (2)
                Institute of Medical Biology, University of Tromso
                (3)
                Department of Clinical Pathology, University Hospital of North Norway
                (4)
                Institute of Clinical Medicine, University of Tromso

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                33. Pre-publication history

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                © Kilvaer et al.; licensee BioMed Central Ltd. 2014

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