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Syndecan-4 and fibronectin in osteosarcoma
Pathology (June 2012) 44(4), pp. 325–330
ANATOMICAL PATHOLOGY
Syndecan-4 and fibronectin in osteosarcoma KI YONG NA*, PATRIZIA BACCHINI{, FRANCO BERTONI§, YOUN WHA KIM*
AND
YONG-KOO PARK*{
*Department of Pathology, Graduate School of Medicine, Kyung Hee University, Seoul {Medical Science and Engineering Research Center for Bioreactions to Reactive Oxygen Species, Korea; zDepartment of Pathology, Villa Erbosa Hospital, Bologna, and §Department of Pathology, Rizzoli Institute, Bologna, Italy
Summary Aims: Syndecan-4 (SDC4) and fibronectin (FN), which belong to the cell adhesion molecules, have been reported to correlate with tumour growth and invasion in various carcinomas. We aimed to investigate the prognostic value of these molecules in osteosarcoma. Methods: Using immunohistochemistry, we compared the expression of these molecules in high grade osteosarcoma to low grade central osteosarcoma, osteoid osteoma and normal bone. Further, the expression of SDC4 and FN were analysed with prognostic factors of high grade osteosarcoma. Results: In high grade osteosarcoma, SDC4 was expressed in 50 of the 65 samples; of these, 32 of 65 showed strong expression profiles. FN was expressed in 46 of 65 samples, and 29 of 65 had evidence of strong expression of this molecule. SDC4 and FN expression were increased in high grade osteosarcoma as compared to other tissues. Strong SDC4 expression was associated with the occurrence of distant metastasis and a large tumour size, and strong FN expression was associated with the occurrence of distant metastasis. Strong expression of SDC4 or FN was associated with significantly shorter overall survival, respectively. Conslusions: Increased expression of SDC4 and FN may be underlying molecular alteration of osteosarcoma which accounts for more aggressive clinical behaviour. Key words: Fibronectin, immunohistochemistry, osteosarcoma, syndecan-4. Received 27 September, revised 1 November, accepted 2 November 2011
INTRODUCTION Osteosarcoma is the most common primary malignant bone tumour with the exception of myeloma and has a predominant peak incidence in childhood and adolescence. It is characterised by early metastasis, frequent vascular invasion and infiltrative growth to the adjacent soft tissue. Aggressive therapeutic modalities, including neoadjuvant chemotherapy followed by surgical excision and post-operative chemotherapy, have improved patient outcomes, with 5-year survival rates as high as 70%. However, the prognosis of patients with overt metastasis or recurrent disease remains unfavourable. Unfortunately, further improvements in survival have not occurred in the last decade, despite multiple attempts at intensifying chemotherapy and trials that utilise novel agents.1 Given the increase in data concerning the biomolecular mechanisms underlying the disease, a new search for alternative agents should be conducted. The extracellular matrix (ECM) regulates the growth, proliferation, movement and differentiation of the cells living
Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e328353447b
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within it. It is constantly remodelling itself, and its synthesis and degradation are associated with tumour invasion and metastasis. Furthermore, cell adhesion molecules support either an increase or a decrease in the ability of tumour cells to interact with the ECM.2 Fibronectin (FN) is a large ECM glycoprotein that binds to many molecules, including collagen, fibrin, proteoglycans and cell surface receptors. FN messenger RNA has two splice forms that give rise to cellular FN and plasma FN. Cellular FN is an adhesive protein that mediates the interactions between cells and their microenvironment. Integrin is recognised to be the primary receptor for FN. The interaction between FN and integrin regulates cell attachment and triggers a number of signalling pathways that regulate cell proliferation, survival and apoptosis, including mitogenactivated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase.3 The role of FN in carcinoma is well-documented. It has been shown that FN over-expression is associated with invasive growth in several types of carcinomas.4–6 Other key cell surface receptors of FN are the heparan sulfate proteoglycan (HSPG), referred to as syndecans, of which syndecan-4 (SDC4) is probably the most predominant. SDC4 has been recognised as a co-receptor for integrin. Mounting evidence suggests that SDC4 interacts with FN independent of intergrin and that their interaction regulates cell adhesion and migration. In addition, the role of SDC4 as a co-receptor for growth factors has been highlighted. SDC4 modulates cell adhesion, migration and proliferation in response to several growth factors, including fibroblast growth factor (FGF), transforming growth factor-b (TGF-b) and hepatocyte growth factor (HGF).7 Changes in SDC4 expression have been investigated in several types of malignant tumour, including liver,8 breast,9 ovary,10 and gestational trophoblastic tumours.11 Although much less is known about the role of FN and SDC4 in sarcoma, recent research supports the hypothesis that these biomarkers may be involved in the progression of sarcoma. In particular, the HSPG, which modulates many growth factor signals, has been implicated in the growth of rhabdomyosarcoma12 and fibrosarcoma13 cell lines. The interaction between FN and integrin has been proven to play a role in tumour growth and the migration of fibrosarcoma cell lines.14 In osteosarcoma cell lines, SDC4 and FN have been shown to be involved in tumour growth and migration.15–18 Another study in human tissue lines showed that osteosarcoma expressed high levels of FN as compared to normal bone.19 Because SDC4 is a cellular receptor for FN, we hypothesised
2012 Royal College of Pathologists of Australasia
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that SDC4 may be highly expressed in osteosarcoma. Furthermore, their expression may be associated with the aggressive behaviour of osteosarcoma, as in cell line experiments. However, to date, no study has investigated the prognostic significance of FN or SDC4 in osteosarcoma. Using immunohistochemistry, we analysed the expression of SDC4 and FN in addition to the clinicopathological features of osteosarcoma.
MATERIALS AND METHODS Patients and tissue samples This study included 65 patients with primary high grade intramedullary osteosarcoma (Fig. 1), 20 with low grade central osteosarcoma, seven with osteoid osteoma, and seven normal bone tissues (Fig. 2). Seventy-eight patients with high grade intramedullary osteosarcoma who had been treated at the Kyung Hee University Hospital from 1987 to 2007 were retrospectively reviewed. To compare the occurrence of metastasis during follow-up and the expression profiles of SDC4 and FN, samples from patients with skip metastasis (n ¼ 2) and samples from patients with distant metastasis at the time of diagnosis (n ¼ 11) were excluded. Skip metastasis was revealed by pathological examination of the resected specimens. Lung nodules seen on the initial chest computed tomography (CT) scan were regarded as a haematogenous lung metastasis. After 13 patients were excluded, we selected the surgically resected specimens of 65 localised, primary high grade intramedullary osteosarcoma patients. Twenty samples of low grade osteosarcoma were archived from the Department of Pathology at the Rizzoli Institute, Italy. Seven samples of osteoid osteoma were obtained surgically. Seven samples of normal bone were obtained from the distal femur of patients undergoing surgical treatment for osteoarthritis. In the high grade osteosarcoma group, the age of patients ranged from 6 to 63 years of age (median age 26, mean age 24.6). The clinicopathological data, including age, gender, the primary tumour site, tumour size, the histological subtype, the follow-up period, the occurrence of distant metastasis and local recurrence are summarised in Table 1. Seventeen patients received neoadjuvant chemotherapy, and the remainder underwent operation without any preoperative therapy. After surgical excision, all patients received post-operative chemotherapy based on high-dose methotrexate, cisplatin and doxorubicin. Following chemotherapy, the patients were monitored with CT scans of the lung, which were performed every 3 months during the first 3 years after chemotherapy, every 4 months during the fourth and fifth years and every 6 months thereafter. The development of local recurrence and distant metastases were assessed using the CT scans. Our study protocol was reviewed and approved by the Kyung Hee University Institutional Review Board, and informed consent was obtained from each of the study participants. Two pathologists (KYN, YKP) reviewed all of the cases. Immunohistochemistry To conduct the immunohistochemical analysis, we used the Bond Polymer Intense Detection System (Vision BioSystems, Australia) on 4 mm thick tissue sections. The sections were deparaffinised with Bond Dewax Solution (Vision Biosystems), and an antigen retrieval procedure was performed using the Bond ER1 Solution (Vision Biosystems) for SDC4 antibody and the Bond ER2 Solution for the FN antibody for 30 min at 1008C. The endogenous peroxidase was quenched by incubation with hydrogen peroxide for 5 min. The sections
were incubated (1:500 dilution) with rabbit IgG polyclonal antibody for SDC4 (Santa Cruz Biotechnology, USA) and with rabbit IgG monoclonal antibody for FN (Santa Cruz Biotechnology) for 15 min at room temperature. The positive control samples consisted of invasive ductal carcinomas and oesophageal squamous cell carcinomas for the SDC4 and FN antibodies, respectively.5,9 Immunohistochemical evaluation SDC4 and FN immunoreactivity were analysed using a semi-quantitative scoring method, which has been described previously. Tumours were interpreted as positive for SDC4 when cytoplasmic staining of tumour cell was seen. The score was the sum of the percentage of positive tumour cells (0, none; 1, <1/100; 2, 1/100–1/10; 3, 1/10–1/3; 4, 1/3–2/3; and 5, >2/3) and the staining intensity (0, none; 1, weak; 2, moderate; 3, strong).9 Tumours were interpreted as positive for FN when cytoplasmic staining of tumour cell was detected. Cells with no positive staining were rated as ‘0’, ‘’ if there were only a few scattered positive cells, ‘þ’ if there was a cluster(s) of positively stained cells that accounted for less than 30% of the cells within a visual field and ‘þþ’ if there were cluster(s) of positively stained cells that accounted for more than 30% of the cells within a visual field.5 For the statistical analysis in the high grade osteosarcoma cells lines, the median score of each protein was used as the cut-off criterion. Using the median score, the patients were divided into a strong (> median score) expression group and a weak or negative ( median score) expression group. Statistical analysis The Chi-square test or Fisher’s exact test were performed to compare SDC4 and FN expression between high grade osteosarcoma, low grade central osteosarcoma, osteoid osteoma and normal bone tissue and to determine whether their expressions were correlated with the clinicopathological parameters of high grade osteosarcoma. The correlation between SDC4 and FN expression was determined using Spearman’s rank coefficient. Univariate and multivariate survival analyses were used to examine the prognostic significance of SDC4 and FN expressions using the Kaplan-Meier method, and differences were analysed using the log-rank test for univariate survival analysis. The parameters that were found to be significant on the univariate analysis were then input into the multivariate survival analysis using the Cox proportional hazards model (95% confidence interval) with a backward stepwise elimination method. Statistical analyses were performed using the SPSS Software (SPSS, USA). Statistical significance was defined as a p value less than 0.05.
RESULTS Of the 65 patients with high grade osteosarcoma, 19 (29.2%) experienced tumour recurrence; distant metastasis occurred in 28 patients (43.1%); and 34 (51.5%) patients died by the end of the follow-up period. The median follow-up time was 43 months (ranging from 3 to 87 months). For the entire cohort of 65 patients, the 1, 2 and 3 year overall survival rates were 84.6%, 60% and 55.3%, respectively. Regarding univariate analyses, the known clinical prognostic factors, including tumour size ( p < 0.001), tumour location ( p < 0.001), local recurrence ( p ¼ 0.011) and distant metastasis ( p < 0.001), showed a statistically significant association with overall survival. Seven of 13 patients (54.8%) with tumour necrosis <90% died; however, all four patients with tumour necrosis >90% were alive with no disease relapse during follow-up. The
Fig. 1 (A–D) Representative case of high grade osteosarcoma and immunoreactivity of SDC4 and FN. (A) Osteosarcoma of the distal femur metaphysis involving the epiphysis and forming an extraosseous mass with mineralisation. (B) H&E section shows a lace-like pattern of osteoid produced by anaplastic malignant tumour cells. (C) Cytoplasmic immunoreactivity of SDC4 and (D) FN.
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SYNDECAN-4 AND FIBRONECTIN IN OSTEOSARCOMA
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Fig. 2 A representative case of (A–C) low grade central osteosarcoma, (D–E) osteoid osteoma and (G–I) normal bone, showing H&E and immunoreactivity of SDC4 and FN. (A) H&E section, (B) cytoplasmic immunoreactivity of SDC4 and (C) FN in low grade central osteosarcoma. (D) H&E section, (E) negative immunoreactivity of SDC4 and (F) FN in osteoid osteoma. (G) H&E section, (H) negative immunoreactivity of SDC4 and (F) FN in normal bone.
Table 1 Clinicopathological characteristics of 65 patients with high grade osteosarcoma Characteristics Age at diagnosis (years) Range Median Mean Sex Male Female Tumour size <8 cm 8 cm Primary site Long bone of extremity Craniofacial Pelvic bone Histological subtype Osteoblastic Chondroblastic Fibroblastic Neoadjuvant CTx Yes No Tumour necrosis 90% <90% Follow-up (months) Range Median Distant metastasis Occurrence Absent Local recurrence Occurrence Absent CTx, chemotherapy.
No. cases (%)
6–63 26 24.6 45 (69.2) 20 (30.8) 31 (47.7) 34 (52.3) 52 (80.0) 4 (6.1) 9 (13.8) 53 (80.5) 6 (9.2) 6 (9.2) 17 (26.1) 48 (73.8) 4 (23.5) 13 (76.4) 3–87 43 28 (43.1) 37 (56.9) 19 (29.2) 46 (70.8)
lack of relationship between tumour necrosis and overall survival is considered to be due to the small number of patients. The other clinicopathological parameters had no significant influence on overall survival. The immunohistochemistry results are summarised in Table 2. In high grade osteosarcoma, SDC4 immunoreactivity was scored as 0–2 in 23% (n ¼ 15), 3–6 in 27.6% (n ¼ 18) and 7–8 in 49.2% (n ¼ 32) of samples (Fig. 1C). In the high grade osteosarcoma cells, FN immunoreactivity was scored as ‘–’ in 29.2% (n ¼ 19), ‘þ/–’ in 25.8% (n ¼ 17), ‘þ’ in 16.9% (n ¼ 11) and ‘þþ’ in 25.8% (n ¼ 17) of the samples (Fig. 1D). In low grade central osteosarcoma, SDC4 immunoreactivity was scored as 0–2 in 70% (n ¼ 14), 3–6 in 20% (n ¼ 4) and 7–8 in 10% (n ¼ 2) of the samples (Fig. 2B). In low grade central osteosarcoma, FN immunoreactivity was rated as ‘–’ in 85% (n ¼ 17), ‘þ/–’ in 10% (n ¼ 2) and ‘þ’ in 5% (n ¼ 1) of the samples (Fig. 2C). SDC4 and FN showed negative immunoreactivity in all 14 cases of osteoid osteoma (Fig. 2E and 2F, respectively) and normal bone (Fig. 2H and 2I, respectively). SDC4 and FN expression were increased in high grade osteosarcoma as compared to other tissues ( p < 0.001 and p < 0.001, respectively). Furthermore, a significant correlation was observed between the expression of SDC4 and FN in high grade osteosarcoma ( p ¼ 0.018). In high grade osteosarcoma, strong SDC4 expression was significantly associated with the occurrence of distant metastasis and a larger tumour size ( p ¼ 0.009 and p ¼ 0.001, respectively). Strong FN expression was significantly associated with the occurrence of distant metastasis ( p ¼ 0.001). The correlation between SDC4 or FN expression and clinicopathological factors is summarised in Table 3. Univariate analysis revealed that strong expression of SDC4 or FN was significantly associated with poor overall survival
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328 Table 2
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Immunoreactivity of SDC4 and FN in high grade osteosarcoma, low grade central osteosarcoma, osteoid osteoma and normal bone SDC4
Diagnosis (n) Normal bone (7) Osteoid osteoma (7) Low grade central OS (20) High grade OS (65)
FN
0–2
3–6
7–8
þ/
þ
þþ
7 7 14 15
0 0 4 18
0 0 2 32
7 7 17 19
0 0 2 17
0 0 1 11
0 0 0 18
SDC4, syndecan-4; FN, fibronectin; OS, osteosarcoma. High grade OS versus low grade central OS, osteoid osteoma and normal bone: SDC4, p < 0.001; FN, p < 0.001.
( p ¼ 0.041 and p ¼ 0.033, respectively). The Kaplan–Meier curves for patients with respect to overall survival are presented in Fig. 3. In the multivariate analysis, a larger tumour size ( p ¼ 0.014) and distant metastasis ( p ¼ 0.004) were independent predictors of a poor prognosis (Table 4).
DISCUSSION A previous study investigated mRNA expression and immunoreactivity of FN in seven mature bone tissues and seven osteosarcoma tissues. Whereas mRNA expression and immunoreactivity were observed for all cases of osteosarcoma, they were not detected in normal bone tissue.19 In line with these results, we observed increased FN expression in high grade osteosarcoma when compared to other tissues. FN plays a role in guiding cell adhesion, migration, cell cycle progression and cell differentiation.3 It is noteworthy that Table 3
strong FN expression was associated with invasive growth tumours in several cancers. FN over-expression was associated with extra-renal extension of renal cell carcinoma.6 Further, FN over-expression was correlated with deeper invasion in oesophageal carcinoma and bladder carcinoma.4,5 We demonstrated that strong FN expression was associated with the occurrence of distant metastasis in high grade osteosarcoma. FN–integrin interaction activates focal adhesion kinase (FAK), through the Rho-ROCK signalling pathway.20 This signalling cascade has been shown to be involved in the invasive growth of osteosarcoma.21 Increased FN level may determine an enhanced invasiveness potential of osteosarcoma. Functional studies have shown that a FN-rich matrix increased invasive growth in osteosarcoma cell lines.15,22 As with carcinomas, FN may be involved in the invasive growth of osteosarcoma. Little is known concerning the role of SDC4 in human malignancy. Several studies have demonstrated that SDC4
Associations of SDC4 and FN expression with clinicopathological characteristics of 65 patients with high grade osteosarcoma SDC4
Characteristics Age, years 14 <14 Sex Male Female Tumour size, cm <8 8 Histological subtype Osteoblastic Chondroblastic Fibroblastic Tumour location Axial Extremity Neoadjuvant CTx Yes No Tumour necrosis >90% <90% Distant metastasis Occurrence Absent Local recurrence Occurrence Absent
FN
Strong (%)
Weak/Neg (%)
p value
Strong (%)
Weak/Neg (%)
p value
17 (54.8) 15 (46.9)
19 (55.9) 14 (42.4)
0.456
17 (58.6) 12 (41.4)
19 (58.6) 17 (47.2)
0.413
24 (75.0) 8 (25.0)
21(63.6) 12 (36.4)
0.235
22 (75.9) 7 (24.1)
23 (63.9) 13 (36.1)
0.222
8 (25.0) 24 (75.0)
23 (69.7) 10 (30.3)
0.001
11 (37.9) 18 (62.1)
20 (55.6) 16 (44.4)
0.122
26 (81.3) 2 (6.2) 4 (12.4)
27 (81.8) 4 (12.4) 2 (6.2)
0.098
23 (75.9) 1 (3.1) 5 (15.5)
30 (86.1) 5 (15.5) 1 (3.1)
0.208
6 (18.8) 26 (81.3)
3 (9.1) 30 (90.9)
0.303
6 (20.7) 23 (79.3)
3 (8.3) 33 (91.7)
0.278
8 (25.0) 24 (75.0)
10 (30.3) 23 (69.7)
0.783
11 (37.9) 18 (62.1)
7 (19.4) 29 (80.6)
0.162
1 (12.5) 7 (87.5)
3 (33.3) 6 (66.7)
0.335
2 (57.1) 9 (81.8)
2 (33.3) 4 (66.7)
0.445
19 (59.4) 13 (40.6)
9 (27.3) 24 (72.7)
0.009
19 (65.5) 10 (34.5)
9 (25.0) 27 (75.0)
0.001
12 (37.5) 20 (62.5)
7 (21.2) 26 (78.8)
0.121
11 (37.9) 18 (62.1)
8 (22.2) 28 (77.8)
0.134
CTx; chemotherapy; FN, fibronectin; Neg, negative; SDC4, syndecan-4.
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SYNDECAN-4 AND FIBRONECTIN IN OSTEOSARCOMA
SDC4 expression
FN expression
1.0
1.0
Survival probability
0.6
Censored
Weak or negative
0.4 Strong
Weak or negative 0.4
0.2
0.0
0.0 20
40
60
80
100
Strong
0
B
Overall survival (month)
Censored
0.6
0.2
0
P = 0.033
0.8
Survival probability
P = 0.041
0.8
A
329
20
60
40
80
100
Overall survival (month)
Fig. 3 (A,B) The association between SDC4 or FN expression status and prognosis of patients with osteosarcoma. Kaplan–Meier curves for overall survival rate of patients with osteosarcoma based on (A) SDC4 or (B) FN expression.
expression is increased in breast carcinoma,9 liver carcinoma,8 and malignant mesothelioma cells,23 as compared to their normal counterparts. In contrast, SDC4 expression was decreased in colon carcinoma cells.24 Thus, SDC4 expression in malignant tumours may be tissue-specific. In this study, increased SDC4 expression was detected in high grade osteosarcoma when compared to low grade osteosarcoma, osteoid osteoma and normal bone. SDC4 is a low affinity growth factor receptor that is able to impact the ability of several growth factors such as FGF, HGF and TGF-b to bind to its high affinity receptor.7 With regard to this role, SDC4 has been implicated in tumour growth in response to growth factors. The association between strong SDC4 expression and high proliferative activity of tumour cells has been suggested in breast carcinoma9 and liver carcinoma.8 As reported to be its effect on other cell systems, SDC4 modulated basic fibroblast growth factor (bFGF)-induced cell proliferation in both osteoblasts and osteosarcoma cells. In addition, increased SDC4 level enhanced the effects of bFGF on cell proliferation.17,18 Osteosarcoma is known for autocrine secretion of many growth factors such as TGF-b, bFGF and HGF, and these growth factors were highly Table 4
expressive in osteosarcoma tissues.25,26 Increase of SDC4 protein level may enhance the action of such growth factors. This study demonstrated the association of strong SDC4 expression and larger tumour size in the clinical setting of osteosarcoma and it may be related to the role of SDC4 as a co-receptor of growth factors. A relationship between SDC4 and metastatic potential has been shown in several cell line studies, including melanoma27 and hepatocellular carcinoma.28 In line with these findings, we observed an association between strong SDC4 expression and the occurrence of distant metastasis. Migration and invasion is considered to be the first step in metastasis.29 Thodeti et al. showed that SDC4 binds to a disintegrin and metalloprotease 12 (ADAM12), and then mediates cell spreading in osteosarcoma cell lines.30 Furthermore, the interaction of SDC4 and ADAM12 sequentially activated Rho and metalloproteinase 2 (MMP2), both of which have been shown to stimulate the invasion and motility of osteosarcoma cells.21 Further investigation in osteosarcoma cell lines is essential to elucidate the role of SDC4 in metastasis, and will provide the basis of future studies.
Univariate and multivariate survival analyses for 65 patients with high grade osteosarcoma Multivariate Univariate
Characteristics
p value
Sex, male vs. female Age, 14 vs. <14 years Histological type, OB vs. non-OB Tumour size, 8 vs. <8 cm Tumour location, axial vs. extremity Neoadjuvant CTx, yes vs. no Tumour necrosis, >90% vs. <90% Distant metastasis, occurrence vs. absent Local recurrence, occurrence vs. absent SDC4 expression, strong vs. weak or negative FN expression, strong vs. weak or negative
0.255 0.347 0.589 <0.001 <0.001 0.486 0.103 <0.001 0.011 0.041 0.033
HR (95%CI)
2.729 1.736
3.121 1.674 0.689 1.219
NA NA NA (1.181–6.306) (0.739–4.080) NA NA (1.390–7.008) (0.785–3.569) (0.296–1.606) (0.548–2.712)
p value
0.014 0.221
0.004 0.188 0.394 0.627
CI, confidence interval; CTx, chemotherapy; FN, fibronectin; HR, hazard ratio; NA, not available; OB, osteoblastic; SDC4, syndecan-4.
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In this study, FN or SDC4 expression was associated with worse prognosis. However, the expression of SDC4 or FN did not have significant prognostic value after taking other prognostic factors into account. Much less is known about the role of SDC4 and FN as a prognostic marker in osteosarcoma, however, the interpretation may be important based on the fact that their expression was closely related to distant metastasis. In our study, distant metastasis was the most strong prognostic factor. Therefore, the prognostic relevance of SDC4 or FN may be affected by distant metastasis. One interesting finding was that the expression of FN and SDC4 showed close relationship. SDC-4 can trigger signalling cascades required for cell spreading or homeostasis by exposing a cryptic binding site for FN.31 FN-SDC4 interaction activated PKCa signalling pathway and increased survival, proliferation and migration in osteoblasts.32 Several of the steps associated with metastatic progression have been linked to PKCa, including resistance to apoptosis, migration and invasion.33 At least in cases with strong expression for both SDC4 and FN, their strong expression may contribute to activation of PKCa signalling pathway and therefore enforce the metastatic progression. In conclusion, we compared the expression of FN and SDC4 in high grade osteosarcoma, low grade central osteosarcoma, osteoid osteoma and normal bone and analysed the prognostic value of these markers in high grade osteosarcoma. FN and SDC4 expression were increased in high grade osteosarcoma when compared to other tissues. In high grade osteosarcoma, strong SDC4 expression was significantly associated with a larger tumour size, distant metastasis and poor overall survival. In addition, strong FN expression was significantly associated with distant metastasis and poor overall survival. Our results support the role of SDC4 and FN in tumour growth and invasion; this finding has been suggested in other carcinomas as well. SDC4 and FN may be involved in tumour growth and invasion in osteosarcoma. Conflicts of interest and sources of funding: The authors declare no conflict of interest. This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No.20100028333).
Address for correspondence: Dr Y-K. Park, Department of Pathology, Kyung Hee University Hospital, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Korea. E-mail: [email protected]
References 1. Kong C, Hansen MF. Biomarkers in osteosarcoma. Expert Opin Med Diagn 2009; 3: 13–23. 2. Kim SH, Turnbull J, Guimond S. Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. J Endocrinol 2011; 209: 139–51. 3. Akiyama SK, Olden K, Yamada KM. Fibronectin and integrins in invasion and metastasis. Cancer Metastasis Rev 1995; 14: 173–89. 4. Ioachim E, Michael M, Stavropoulos NE, et al. A clinicopathological study of the expression of extracellular matrix components in urothelial carcinoma. BJU Int 2005; 95: 655–9. 5. Zhang J, Zhi H, Zhou C, et al. Up-regulation of fibronectin in oesophageal squamous cell carcinoma is associated with activation of the Erk pathway. J Pathol 2005; 207: 402–9. 6. Waalkes S, Atschekzei F, Kramer MW, et al. Fibronectin 1 mRNA expression correlates with advanced disease in renal cancer. BMC Cancer 2010; 10: 503.
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7. Xian X, Gopal S, Couchman JR. Syndecans as receptors and organizers of the extracellular matrix. Cell Tissue Res 2010; 339: 31–46. 8. Roskams T, De Vos R, David G, et al. Heparan sulphate proteoglycan expression in human primary liver tumours. J Pathol 1998; 185: 290–7. 9. Baba F, Swartz K, van Buren R, et al. Syndecan-1 and syndecan-4 are overexpressed in an estrogen receptor-negative, highly proliferative breast carcinoma subtype. Breast Cancer Res Treat 2006; 98: 91–8. 10. Davies EJ, Blackhall FH, Shanks JH, et al. Distribution and clinical significance of heparan sulfate proteoglycans in ovarian cancer. Clin Cancer Res 2004; 10: 5178–86. 11. Crescimanno C, Marzioni D, Paradinas FJ, et al. Expression pattern alterations of syndecans and glypican-1 in normal and pathological trophoblast. J Pathol 1999; 189: 600–8. 12. Williamson D, Selfe J, Gordon T, et al. Role for amplification and expression of glypican-5 in rhabdomyosarcoma. Cancer Res 2007; 67: 57–65. 13. Peterfia B, Hollosi P, Szilak L, et al. Role of syndecan-1 proteoglycan in the invasiveness of HT-1080 fibrosarcoma. Magy Onkol 2006; 50: 115–20. 14. Reyes CD, Petrie TA, Garcia AJ. Mixed extracellular matrix ligands synergistically modulate integrin adhesion and signaling. J Cell Physiol 2008; 217: 450–8. 15. Harisi R, Dudas J, Timar F, et al. Invasive growth and topoisomeraseswitch induced by tumorous extracellular matrix in osteosarcoma cell culture. Cell Biol Int 2005; 29: 959–67. 16. Vial D, Monaghan-Benson E, McKeown-Longo PJ. Coordinate regulation of fibronectin matrix assembly by the plasminogen activator system and vitronectin in human osteosarcoma cells. Cancer Cell Int 2006; 6: 8. 17. Bodo M, Lilli C, Bellucci C, et al. Basic fibroblast growth factor autocrine loop controls human osteosarcoma phenotyping and differentiation. Mol Med 2002; 8: 393–404. 18. Song SJ, Cool SM, Nurcombe V. Regulated expression of syndecan-4 in rat calvaria osteoblasts induced by fibroblast growth factor-2. J Cell Biochem 2007; 100: 402–11. 19. Kilian O, Dahse R, Alt V, et al. Expression of EDAþ and EDBþ fibronectin splice variants in bone. Bone 2004; 35: 1334–45. 20. Pankov R, Yamada KM. Fibronectin at a glance. J Cell Sci 2002; 115: 3861–3. 21. Matsumoto Y, Tanaka K, Harimaya K, et al. Small GTP-binding protein, Rho, both increased and decreased cellular motility, activation of matrix metalloproteinase 2 and invasion of human osteosarcoma cells. Jpn J Cancer Res 2001; 92: 429–38. 22. Sotobori T, Ueda T, Myoui A, et al. Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin beta1 into lipid rafts. Exp Cell Res 2006; 312: 3927–38. 23. Gulyas M, Hjerpe A. Proteoglycans and WT1 as markers for distinguishing adenocarcinoma, epithelioid mesothelioma, and benign mesothelium. J Pathol 2003; 199: 479–87. 24. Park H, Kim Y, Lim Y, et al. Syndecan-2 mediates adhesion and proliferation of colon carcinoma cells. J Biol Chem 2002; 277: 29730–6. 25. Fieten H, Spee B, Ijzer J, et al. Expression of hepatocyte growth factor and the proto-oncogenic receptor c-Met in canine osteosarcoma. Vet Pathol 2009; 46: 869–77. 26. Kloen P, Gebhardt MC, Perez-Atayde A, et al. Expression of transforming growth factor-beta (TGF-beta) isoforms in osteosarcomas: TGF-beta3 is related to disease progression. Cancer 1997; 80: 2230–9. 27. O’Connell MP, Fiori JL, Kershner EK, et al. Heparan sulfate proteoglycan modulation of Wnt5A signal transduction in metastatic melanoma cells. J Biol Chem 2009; 284: 28704–12. 28. Charni F, Friand V, Haddad O, et al. Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells. Biochim Biophys Acta 2009; 1790: 1314–26. 29. PosthumaDeBoer J, Witlox MA, Kaspers GJ, et al. Molecular alterations as target for therapy in metastatic osteosarcoma: a review of literature. Clin Exp Metastasis 2011; 28: 493–503. 30. Thodeti CK, Albrechtsen R, Grauslund M, et al. ADAM12/syndecan-4 signaling promotes beta 1 integrin-dependent cell spreading through protein kinase Calpha and RhoA. J Biol Chem 2003; 278: 9576–84. 31. Woods A, Couchman JR. Signaling from the matrix to the cytoskeleton: role of cell surface proteoglycans in matrix assembly. Kidney Int Suppl 1996; 54: S64–7. 32. Wang Z, Telci D, Griffin M. Importance of syndecan-4 and syndecan -2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminasefibronectin complex. Exp Cell Res 2011; 317: 367–81. 33. Ren L, Hong SH, Cassavaugh J, et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene 2009; 28: 792–802.
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ANATOMICAL PATHOLOGY
Syndecan-4 and fibronectin in osteosarcoma KI YONG NA*, PATRIZIA BACCHINI{, FRANCO BERTONI§, YOUN WHA KIM*
AND
YONG-KOO PARK*{
*Department of Pathology, Graduate School of Medicine, Kyung Hee University, Seoul {Medical Science and Engineering Research Center for Bioreactions to Reactive Oxygen Species, Korea; zDepartment of Pathology, Villa Erbosa Hospital, Bologna, and §Department of Pathology, Rizzoli Institute, Bologna, Italy
Summary Aims: Syndecan-4 (SDC4) and fibronectin (FN), which belong to the cell adhesion molecules, have been reported to correlate with tumour growth and invasion in various carcinomas. We aimed to investigate the prognostic value of these molecules in osteosarcoma. Methods: Using immunohistochemistry, we compared the expression of these molecules in high grade osteosarcoma to low grade central osteosarcoma, osteoid osteoma and normal bone. Further, the expression of SDC4 and FN were analysed with prognostic factors of high grade osteosarcoma. Results: In high grade osteosarcoma, SDC4 was expressed in 50 of the 65 samples; of these, 32 of 65 showed strong expression profiles. FN was expressed in 46 of 65 samples, and 29 of 65 had evidence of strong expression of this molecule. SDC4 and FN expression were increased in high grade osteosarcoma as compared to other tissues. Strong SDC4 expression was associated with the occurrence of distant metastasis and a large tumour size, and strong FN expression was associated with the occurrence of distant metastasis. Strong expression of SDC4 or FN was associated with significantly shorter overall survival, respectively. Conslusions: Increased expression of SDC4 and FN may be underlying molecular alteration of osteosarcoma which accounts for more aggressive clinical behaviour. Key words: Fibronectin, immunohistochemistry, osteosarcoma, syndecan-4. Received 27 September, revised 1 November, accepted 2 November 2011
INTRODUCTION Osteosarcoma is the most common primary malignant bone tumour with the exception of myeloma and has a predominant peak incidence in childhood and adolescence. It is characterised by early metastasis, frequent vascular invasion and infiltrative growth to the adjacent soft tissue. Aggressive therapeutic modalities, including neoadjuvant chemotherapy followed by surgical excision and post-operative chemotherapy, have improved patient outcomes, with 5-year survival rates as high as 70%. However, the prognosis of patients with overt metastasis or recurrent disease remains unfavourable. Unfortunately, further improvements in survival have not occurred in the last decade, despite multiple attempts at intensifying chemotherapy and trials that utilise novel agents.1 Given the increase in data concerning the biomolecular mechanisms underlying the disease, a new search for alternative agents should be conducted. The extracellular matrix (ECM) regulates the growth, proliferation, movement and differentiation of the cells living
Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e328353447b
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within it. It is constantly remodelling itself, and its synthesis and degradation are associated with tumour invasion and metastasis. Furthermore, cell adhesion molecules support either an increase or a decrease in the ability of tumour cells to interact with the ECM.2 Fibronectin (FN) is a large ECM glycoprotein that binds to many molecules, including collagen, fibrin, proteoglycans and cell surface receptors. FN messenger RNA has two splice forms that give rise to cellular FN and plasma FN. Cellular FN is an adhesive protein that mediates the interactions between cells and their microenvironment. Integrin is recognised to be the primary receptor for FN. The interaction between FN and integrin regulates cell attachment and triggers a number of signalling pathways that regulate cell proliferation, survival and apoptosis, including mitogenactivated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase.3 The role of FN in carcinoma is well-documented. It has been shown that FN over-expression is associated with invasive growth in several types of carcinomas.4–6 Other key cell surface receptors of FN are the heparan sulfate proteoglycan (HSPG), referred to as syndecans, of which syndecan-4 (SDC4) is probably the most predominant. SDC4 has been recognised as a co-receptor for integrin. Mounting evidence suggests that SDC4 interacts with FN independent of intergrin and that their interaction regulates cell adhesion and migration. In addition, the role of SDC4 as a co-receptor for growth factors has been highlighted. SDC4 modulates cell adhesion, migration and proliferation in response to several growth factors, including fibroblast growth factor (FGF), transforming growth factor-b (TGF-b) and hepatocyte growth factor (HGF).7 Changes in SDC4 expression have been investigated in several types of malignant tumour, including liver,8 breast,9 ovary,10 and gestational trophoblastic tumours.11 Although much less is known about the role of FN and SDC4 in sarcoma, recent research supports the hypothesis that these biomarkers may be involved in the progression of sarcoma. In particular, the HSPG, which modulates many growth factor signals, has been implicated in the growth of rhabdomyosarcoma12 and fibrosarcoma13 cell lines. The interaction between FN and integrin has been proven to play a role in tumour growth and the migration of fibrosarcoma cell lines.14 In osteosarcoma cell lines, SDC4 and FN have been shown to be involved in tumour growth and migration.15–18 Another study in human tissue lines showed that osteosarcoma expressed high levels of FN as compared to normal bone.19 Because SDC4 is a cellular receptor for FN, we hypothesised
2012 Royal College of Pathologists of Australasia
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that SDC4 may be highly expressed in osteosarcoma. Furthermore, their expression may be associated with the aggressive behaviour of osteosarcoma, as in cell line experiments. However, to date, no study has investigated the prognostic significance of FN or SDC4 in osteosarcoma. Using immunohistochemistry, we analysed the expression of SDC4 and FN in addition to the clinicopathological features of osteosarcoma.
MATERIALS AND METHODS Patients and tissue samples This study included 65 patients with primary high grade intramedullary osteosarcoma (Fig. 1), 20 with low grade central osteosarcoma, seven with osteoid osteoma, and seven normal bone tissues (Fig. 2). Seventy-eight patients with high grade intramedullary osteosarcoma who had been treated at the Kyung Hee University Hospital from 1987 to 2007 were retrospectively reviewed. To compare the occurrence of metastasis during follow-up and the expression profiles of SDC4 and FN, samples from patients with skip metastasis (n ¼ 2) and samples from patients with distant metastasis at the time of diagnosis (n ¼ 11) were excluded. Skip metastasis was revealed by pathological examination of the resected specimens. Lung nodules seen on the initial chest computed tomography (CT) scan were regarded as a haematogenous lung metastasis. After 13 patients were excluded, we selected the surgically resected specimens of 65 localised, primary high grade intramedullary osteosarcoma patients. Twenty samples of low grade osteosarcoma were archived from the Department of Pathology at the Rizzoli Institute, Italy. Seven samples of osteoid osteoma were obtained surgically. Seven samples of normal bone were obtained from the distal femur of patients undergoing surgical treatment for osteoarthritis. In the high grade osteosarcoma group, the age of patients ranged from 6 to 63 years of age (median age 26, mean age 24.6). The clinicopathological data, including age, gender, the primary tumour site, tumour size, the histological subtype, the follow-up period, the occurrence of distant metastasis and local recurrence are summarised in Table 1. Seventeen patients received neoadjuvant chemotherapy, and the remainder underwent operation without any preoperative therapy. After surgical excision, all patients received post-operative chemotherapy based on high-dose methotrexate, cisplatin and doxorubicin. Following chemotherapy, the patients were monitored with CT scans of the lung, which were performed every 3 months during the first 3 years after chemotherapy, every 4 months during the fourth and fifth years and every 6 months thereafter. The development of local recurrence and distant metastases were assessed using the CT scans. Our study protocol was reviewed and approved by the Kyung Hee University Institutional Review Board, and informed consent was obtained from each of the study participants. Two pathologists (KYN, YKP) reviewed all of the cases. Immunohistochemistry To conduct the immunohistochemical analysis, we used the Bond Polymer Intense Detection System (Vision BioSystems, Australia) on 4 mm thick tissue sections. The sections were deparaffinised with Bond Dewax Solution (Vision Biosystems), and an antigen retrieval procedure was performed using the Bond ER1 Solution (Vision Biosystems) for SDC4 antibody and the Bond ER2 Solution for the FN antibody for 30 min at 1008C. The endogenous peroxidase was quenched by incubation with hydrogen peroxide for 5 min. The sections
were incubated (1:500 dilution) with rabbit IgG polyclonal antibody for SDC4 (Santa Cruz Biotechnology, USA) and with rabbit IgG monoclonal antibody for FN (Santa Cruz Biotechnology) for 15 min at room temperature. The positive control samples consisted of invasive ductal carcinomas and oesophageal squamous cell carcinomas for the SDC4 and FN antibodies, respectively.5,9 Immunohistochemical evaluation SDC4 and FN immunoreactivity were analysed using a semi-quantitative scoring method, which has been described previously. Tumours were interpreted as positive for SDC4 when cytoplasmic staining of tumour cell was seen. The score was the sum of the percentage of positive tumour cells (0, none; 1, <1/100; 2, 1/100–1/10; 3, 1/10–1/3; 4, 1/3–2/3; and 5, >2/3) and the staining intensity (0, none; 1, weak; 2, moderate; 3, strong).9 Tumours were interpreted as positive for FN when cytoplasmic staining of tumour cell was detected. Cells with no positive staining were rated as ‘0’, ‘’ if there were only a few scattered positive cells, ‘þ’ if there was a cluster(s) of positively stained cells that accounted for less than 30% of the cells within a visual field and ‘þþ’ if there were cluster(s) of positively stained cells that accounted for more than 30% of the cells within a visual field.5 For the statistical analysis in the high grade osteosarcoma cells lines, the median score of each protein was used as the cut-off criterion. Using the median score, the patients were divided into a strong (> median score) expression group and a weak or negative ( median score) expression group. Statistical analysis The Chi-square test or Fisher’s exact test were performed to compare SDC4 and FN expression between high grade osteosarcoma, low grade central osteosarcoma, osteoid osteoma and normal bone tissue and to determine whether their expressions were correlated with the clinicopathological parameters of high grade osteosarcoma. The correlation between SDC4 and FN expression was determined using Spearman’s rank coefficient. Univariate and multivariate survival analyses were used to examine the prognostic significance of SDC4 and FN expressions using the Kaplan-Meier method, and differences were analysed using the log-rank test for univariate survival analysis. The parameters that were found to be significant on the univariate analysis were then input into the multivariate survival analysis using the Cox proportional hazards model (95% confidence interval) with a backward stepwise elimination method. Statistical analyses were performed using the SPSS Software (SPSS, USA). Statistical significance was defined as a p value less than 0.05.
RESULTS Of the 65 patients with high grade osteosarcoma, 19 (29.2%) experienced tumour recurrence; distant metastasis occurred in 28 patients (43.1%); and 34 (51.5%) patients died by the end of the follow-up period. The median follow-up time was 43 months (ranging from 3 to 87 months). For the entire cohort of 65 patients, the 1, 2 and 3 year overall survival rates were 84.6%, 60% and 55.3%, respectively. Regarding univariate analyses, the known clinical prognostic factors, including tumour size ( p < 0.001), tumour location ( p < 0.001), local recurrence ( p ¼ 0.011) and distant metastasis ( p < 0.001), showed a statistically significant association with overall survival. Seven of 13 patients (54.8%) with tumour necrosis <90% died; however, all four patients with tumour necrosis >90% were alive with no disease relapse during follow-up. The
Fig. 1 (A–D) Representative case of high grade osteosarcoma and immunoreactivity of SDC4 and FN. (A) Osteosarcoma of the distal femur metaphysis involving the epiphysis and forming an extraosseous mass with mineralisation. (B) H&E section shows a lace-like pattern of osteoid produced by anaplastic malignant tumour cells. (C) Cytoplasmic immunoreactivity of SDC4 and (D) FN.
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SYNDECAN-4 AND FIBRONECTIN IN OSTEOSARCOMA
327
Fig. 2 A representative case of (A–C) low grade central osteosarcoma, (D–E) osteoid osteoma and (G–I) normal bone, showing H&E and immunoreactivity of SDC4 and FN. (A) H&E section, (B) cytoplasmic immunoreactivity of SDC4 and (C) FN in low grade central osteosarcoma. (D) H&E section, (E) negative immunoreactivity of SDC4 and (F) FN in osteoid osteoma. (G) H&E section, (H) negative immunoreactivity of SDC4 and (F) FN in normal bone.
Table 1 Clinicopathological characteristics of 65 patients with high grade osteosarcoma Characteristics Age at diagnosis (years) Range Median Mean Sex Male Female Tumour size <8 cm 8 cm Primary site Long bone of extremity Craniofacial Pelvic bone Histological subtype Osteoblastic Chondroblastic Fibroblastic Neoadjuvant CTx Yes No Tumour necrosis 90% <90% Follow-up (months) Range Median Distant metastasis Occurrence Absent Local recurrence Occurrence Absent CTx, chemotherapy.
No. cases (%)
6–63 26 24.6 45 (69.2) 20 (30.8) 31 (47.7) 34 (52.3) 52 (80.0) 4 (6.1) 9 (13.8) 53 (80.5) 6 (9.2) 6 (9.2) 17 (26.1) 48 (73.8) 4 (23.5) 13 (76.4) 3–87 43 28 (43.1) 37 (56.9) 19 (29.2) 46 (70.8)
lack of relationship between tumour necrosis and overall survival is considered to be due to the small number of patients. The other clinicopathological parameters had no significant influence on overall survival. The immunohistochemistry results are summarised in Table 2. In high grade osteosarcoma, SDC4 immunoreactivity was scored as 0–2 in 23% (n ¼ 15), 3–6 in 27.6% (n ¼ 18) and 7–8 in 49.2% (n ¼ 32) of samples (Fig. 1C). In the high grade osteosarcoma cells, FN immunoreactivity was scored as ‘–’ in 29.2% (n ¼ 19), ‘þ/–’ in 25.8% (n ¼ 17), ‘þ’ in 16.9% (n ¼ 11) and ‘þþ’ in 25.8% (n ¼ 17) of the samples (Fig. 1D). In low grade central osteosarcoma, SDC4 immunoreactivity was scored as 0–2 in 70% (n ¼ 14), 3–6 in 20% (n ¼ 4) and 7–8 in 10% (n ¼ 2) of the samples (Fig. 2B). In low grade central osteosarcoma, FN immunoreactivity was rated as ‘–’ in 85% (n ¼ 17), ‘þ/–’ in 10% (n ¼ 2) and ‘þ’ in 5% (n ¼ 1) of the samples (Fig. 2C). SDC4 and FN showed negative immunoreactivity in all 14 cases of osteoid osteoma (Fig. 2E and 2F, respectively) and normal bone (Fig. 2H and 2I, respectively). SDC4 and FN expression were increased in high grade osteosarcoma as compared to other tissues ( p < 0.001 and p < 0.001, respectively). Furthermore, a significant correlation was observed between the expression of SDC4 and FN in high grade osteosarcoma ( p ¼ 0.018). In high grade osteosarcoma, strong SDC4 expression was significantly associated with the occurrence of distant metastasis and a larger tumour size ( p ¼ 0.009 and p ¼ 0.001, respectively). Strong FN expression was significantly associated with the occurrence of distant metastasis ( p ¼ 0.001). The correlation between SDC4 or FN expression and clinicopathological factors is summarised in Table 3. Univariate analysis revealed that strong expression of SDC4 or FN was significantly associated with poor overall survival
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328 Table 2
Pathology (2012), 44(4), June
NA et al.
Immunoreactivity of SDC4 and FN in high grade osteosarcoma, low grade central osteosarcoma, osteoid osteoma and normal bone SDC4
Diagnosis (n) Normal bone (7) Osteoid osteoma (7) Low grade central OS (20) High grade OS (65)
FN
0–2
3–6
7–8
þ/
þ
þþ
7 7 14 15
0 0 4 18
0 0 2 32
7 7 17 19
0 0 2 17
0 0 1 11
0 0 0 18
SDC4, syndecan-4; FN, fibronectin; OS, osteosarcoma. High grade OS versus low grade central OS, osteoid osteoma and normal bone: SDC4, p < 0.001; FN, p < 0.001.
( p ¼ 0.041 and p ¼ 0.033, respectively). The Kaplan–Meier curves for patients with respect to overall survival are presented in Fig. 3. In the multivariate analysis, a larger tumour size ( p ¼ 0.014) and distant metastasis ( p ¼ 0.004) were independent predictors of a poor prognosis (Table 4).
DISCUSSION A previous study investigated mRNA expression and immunoreactivity of FN in seven mature bone tissues and seven osteosarcoma tissues. Whereas mRNA expression and immunoreactivity were observed for all cases of osteosarcoma, they were not detected in normal bone tissue.19 In line with these results, we observed increased FN expression in high grade osteosarcoma when compared to other tissues. FN plays a role in guiding cell adhesion, migration, cell cycle progression and cell differentiation.3 It is noteworthy that Table 3
strong FN expression was associated with invasive growth tumours in several cancers. FN over-expression was associated with extra-renal extension of renal cell carcinoma.6 Further, FN over-expression was correlated with deeper invasion in oesophageal carcinoma and bladder carcinoma.4,5 We demonstrated that strong FN expression was associated with the occurrence of distant metastasis in high grade osteosarcoma. FN–integrin interaction activates focal adhesion kinase (FAK), through the Rho-ROCK signalling pathway.20 This signalling cascade has been shown to be involved in the invasive growth of osteosarcoma.21 Increased FN level may determine an enhanced invasiveness potential of osteosarcoma. Functional studies have shown that a FN-rich matrix increased invasive growth in osteosarcoma cell lines.15,22 As with carcinomas, FN may be involved in the invasive growth of osteosarcoma. Little is known concerning the role of SDC4 in human malignancy. Several studies have demonstrated that SDC4
Associations of SDC4 and FN expression with clinicopathological characteristics of 65 patients with high grade osteosarcoma SDC4
Characteristics Age, years 14 <14 Sex Male Female Tumour size, cm <8 8 Histological subtype Osteoblastic Chondroblastic Fibroblastic Tumour location Axial Extremity Neoadjuvant CTx Yes No Tumour necrosis >90% <90% Distant metastasis Occurrence Absent Local recurrence Occurrence Absent
FN
Strong (%)
Weak/Neg (%)
p value
Strong (%)
Weak/Neg (%)
p value
17 (54.8) 15 (46.9)
19 (55.9) 14 (42.4)
0.456
17 (58.6) 12 (41.4)
19 (58.6) 17 (47.2)
0.413
24 (75.0) 8 (25.0)
21(63.6) 12 (36.4)
0.235
22 (75.9) 7 (24.1)
23 (63.9) 13 (36.1)
0.222
8 (25.0) 24 (75.0)
23 (69.7) 10 (30.3)
0.001
11 (37.9) 18 (62.1)
20 (55.6) 16 (44.4)
0.122
26 (81.3) 2 (6.2) 4 (12.4)
27 (81.8) 4 (12.4) 2 (6.2)
0.098
23 (75.9) 1 (3.1) 5 (15.5)
30 (86.1) 5 (15.5) 1 (3.1)
0.208
6 (18.8) 26 (81.3)
3 (9.1) 30 (90.9)
0.303
6 (20.7) 23 (79.3)
3 (8.3) 33 (91.7)
0.278
8 (25.0) 24 (75.0)
10 (30.3) 23 (69.7)
0.783
11 (37.9) 18 (62.1)
7 (19.4) 29 (80.6)
0.162
1 (12.5) 7 (87.5)
3 (33.3) 6 (66.7)
0.335
2 (57.1) 9 (81.8)
2 (33.3) 4 (66.7)
0.445
19 (59.4) 13 (40.6)
9 (27.3) 24 (72.7)
0.009
19 (65.5) 10 (34.5)
9 (25.0) 27 (75.0)
0.001
12 (37.5) 20 (62.5)
7 (21.2) 26 (78.8)
0.121
11 (37.9) 18 (62.1)
8 (22.2) 28 (77.8)
0.134
CTx; chemotherapy; FN, fibronectin; Neg, negative; SDC4, syndecan-4.
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SYNDECAN-4 AND FIBRONECTIN IN OSTEOSARCOMA
SDC4 expression
FN expression
1.0
1.0
Survival probability
0.6
Censored
Weak or negative
0.4 Strong
Weak or negative 0.4
0.2
0.0
0.0 20
40
60
80
100
Strong
0
B
Overall survival (month)
Censored
0.6
0.2
0
P = 0.033
0.8
Survival probability
P = 0.041
0.8
A
329
20
60
40
80
100
Overall survival (month)
Fig. 3 (A,B) The association between SDC4 or FN expression status and prognosis of patients with osteosarcoma. Kaplan–Meier curves for overall survival rate of patients with osteosarcoma based on (A) SDC4 or (B) FN expression.
expression is increased in breast carcinoma,9 liver carcinoma,8 and malignant mesothelioma cells,23 as compared to their normal counterparts. In contrast, SDC4 expression was decreased in colon carcinoma cells.24 Thus, SDC4 expression in malignant tumours may be tissue-specific. In this study, increased SDC4 expression was detected in high grade osteosarcoma when compared to low grade osteosarcoma, osteoid osteoma and normal bone. SDC4 is a low affinity growth factor receptor that is able to impact the ability of several growth factors such as FGF, HGF and TGF-b to bind to its high affinity receptor.7 With regard to this role, SDC4 has been implicated in tumour growth in response to growth factors. The association between strong SDC4 expression and high proliferative activity of tumour cells has been suggested in breast carcinoma9 and liver carcinoma.8 As reported to be its effect on other cell systems, SDC4 modulated basic fibroblast growth factor (bFGF)-induced cell proliferation in both osteoblasts and osteosarcoma cells. In addition, increased SDC4 level enhanced the effects of bFGF on cell proliferation.17,18 Osteosarcoma is known for autocrine secretion of many growth factors such as TGF-b, bFGF and HGF, and these growth factors were highly Table 4
expressive in osteosarcoma tissues.25,26 Increase of SDC4 protein level may enhance the action of such growth factors. This study demonstrated the association of strong SDC4 expression and larger tumour size in the clinical setting of osteosarcoma and it may be related to the role of SDC4 as a co-receptor of growth factors. A relationship between SDC4 and metastatic potential has been shown in several cell line studies, including melanoma27 and hepatocellular carcinoma.28 In line with these findings, we observed an association between strong SDC4 expression and the occurrence of distant metastasis. Migration and invasion is considered to be the first step in metastasis.29 Thodeti et al. showed that SDC4 binds to a disintegrin and metalloprotease 12 (ADAM12), and then mediates cell spreading in osteosarcoma cell lines.30 Furthermore, the interaction of SDC4 and ADAM12 sequentially activated Rho and metalloproteinase 2 (MMP2), both of which have been shown to stimulate the invasion and motility of osteosarcoma cells.21 Further investigation in osteosarcoma cell lines is essential to elucidate the role of SDC4 in metastasis, and will provide the basis of future studies.
Univariate and multivariate survival analyses for 65 patients with high grade osteosarcoma Multivariate Univariate
Characteristics
p value
Sex, male vs. female Age, 14 vs. <14 years Histological type, OB vs. non-OB Tumour size, 8 vs. <8 cm Tumour location, axial vs. extremity Neoadjuvant CTx, yes vs. no Tumour necrosis, >90% vs. <90% Distant metastasis, occurrence vs. absent Local recurrence, occurrence vs. absent SDC4 expression, strong vs. weak or negative FN expression, strong vs. weak or negative
0.255 0.347 0.589 <0.001 <0.001 0.486 0.103 <0.001 0.011 0.041 0.033
HR (95%CI)
2.729 1.736
3.121 1.674 0.689 1.219
NA NA NA (1.181–6.306) (0.739–4.080) NA NA (1.390–7.008) (0.785–3.569) (0.296–1.606) (0.548–2.712)
p value
0.014 0.221
0.004 0.188 0.394 0.627
CI, confidence interval; CTx, chemotherapy; FN, fibronectin; HR, hazard ratio; NA, not available; OB, osteoblastic; SDC4, syndecan-4.
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In this study, FN or SDC4 expression was associated with worse prognosis. However, the expression of SDC4 or FN did not have significant prognostic value after taking other prognostic factors into account. Much less is known about the role of SDC4 and FN as a prognostic marker in osteosarcoma, however, the interpretation may be important based on the fact that their expression was closely related to distant metastasis. In our study, distant metastasis was the most strong prognostic factor. Therefore, the prognostic relevance of SDC4 or FN may be affected by distant metastasis. One interesting finding was that the expression of FN and SDC4 showed close relationship. SDC-4 can trigger signalling cascades required for cell spreading or homeostasis by exposing a cryptic binding site for FN.31 FN-SDC4 interaction activated PKCa signalling pathway and increased survival, proliferation and migration in osteoblasts.32 Several of the steps associated with metastatic progression have been linked to PKCa, including resistance to apoptosis, migration and invasion.33 At least in cases with strong expression for both SDC4 and FN, their strong expression may contribute to activation of PKCa signalling pathway and therefore enforce the metastatic progression. In conclusion, we compared the expression of FN and SDC4 in high grade osteosarcoma, low grade central osteosarcoma, osteoid osteoma and normal bone and analysed the prognostic value of these markers in high grade osteosarcoma. FN and SDC4 expression were increased in high grade osteosarcoma when compared to other tissues. In high grade osteosarcoma, strong SDC4 expression was significantly associated with a larger tumour size, distant metastasis and poor overall survival. In addition, strong FN expression was significantly associated with distant metastasis and poor overall survival. Our results support the role of SDC4 and FN in tumour growth and invasion; this finding has been suggested in other carcinomas as well. SDC4 and FN may be involved in tumour growth and invasion in osteosarcoma. Conflicts of interest and sources of funding: The authors declare no conflict of interest. This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No.20100028333).
Address for correspondence: Dr Y-K. Park, Department of Pathology, Kyung Hee University Hospital, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Korea. E-mail: [email protected]
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