Int. J. Exp. Pathl. (2016), 97, 86–92
ORIGINAL ARTICLE
High-level expression of periostin is significantly correlated with tumour angiogenesis and poor prognosis in osteosarcoma Fei Hu*, Xi-Fu Shang*, Wei Wang†, Wei Jiang‡, Ce Fang*, Dong Tan* and Hang-Cheng Zhou§ *Department of Orthopedics, Anhui Provincial Hospital, Anhui Medical University, Hefei, China, †Department of Medical Oncology, Anhui Provincial Hospital, Anhui Medical University, Hefei, China, ‡Department of Orthopedics, the First Hospital Affiliated to Anhui Medicine University, Hefei, China and §Department of Pathology, Anhui Provincial Hospital, Anhui Medical University, Hefei, China
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY doi: 10.1111/iep.12171
Received for publication: 12 December 2014 Accepted for publication: 31 December 2015 Correspondence: Fei Hu Department of Orthopedic Anhui Provincial Hospital Anhui Medical University No.17 Lujiang Road Hefei 230001 China Tel.: +86 551 62283324 Fax: +86 551 62283324 E-mail: [email protected]
SUMMARY Periostin (PN), originally named as osteoblast-specific factor-2 (OSF-2), has been involved in regulating adhesion and differentiation of osteoblasts. Recently many studies have shown that high-level expression of PN is correlated significantly with tumour angiogenesis and prognosis in many kinds of human cancer. However, whether and how periostin expression influences prognosis in osteosarcoma remains unknown. This study aimed to examine the expression of PN in patients with osteosarcoma and explore the relationship of PN expression with clinicopathologic factors, tumour angiogenesis and prognosis. Immunohistochemistry was performed to determine the expression of PN in osteosarcoma and osteochondroma respectively. Vascular endothelial growth factor (VEGF) and CD34 were also examined in tissues from the osteosarcoma patients mentioned above. The results showed that PN expression was significantly (P < 0.05) higher in osteosarcoma (80.9%) than in osteochondroma (14.7%). Increased PN protein expression was associated with histological subtype (P = 0.000), Enneking stage (P = 0.027) and tumour size (P = 0.009). The result also showed that high expression of PN correlated with VEGF expression (r = 0.285; P = 0.019) and that tumours with PN-positive expression significantly had higher microvessal density (44.6 13.7 vs. 20.6 6.5; P = 0.000) compared to those in normal bone tissues. Additionally, the expression of PN was found to be an independent prognostic factor in osteosarcoma patients. In conclusion, our findings suggest that PN may have an important role in tumour progression and may be used as a prognostic biomarker for patients with osteosarcoma. Keywords angiogenesis, biomarker, osteosarcoma, periostin, prognosis
Osteosarcoma is the most common primary malignancy in orthopaedic surgery, which accounts for approximately 20% of primary malignancies of bone (Arndt & Crist 1999). Osteosarcomas tends to develop distant metastases frequently and ultimately this results in death, especially in cases where there is lung metastasis. About 20% of patients have visible metastases on imaging at diagnosis and a quarter of the patients have metastases during the course of treatment (Theoleyre et al. 2005). Despite recent advances in multimodality treatments consisting of aggressive neoadjuvant chemotherapy and wide tumour resection, metastatic
or recurrent disease still occurs in 30–40% of these patients and the majority of those succumb to the disease (Yang et al. 2014). Therefore, developing a novel biomarker to identify the invasive potential and prognosis of osteosarcoma is significantly crucial. Periostin (PN), also named osteoblast-specific factor-2 (OSF-2), was originally identified in 1993 as an 90 kDa protein acids, which was secreted from the mouse osteoblastic cell line MC3T3-E1 (Takeshita et al. 1993). This protein shows homologous with an insect cell adhesion protein named fasciclin I (FAS I) protein family, members of which © 2016 The Authors.
86
International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology
Expression of PN in osteosarcoma are involved in many biologic processes, such as cell motility, adhesion, metastatic growth and angiogenesis (Gillan et al. 2002; Bao et al. 2004; Shao et al. 2004). Initially, periostin has been shown to be expressed preferentially in the periosteum and periodontal ligaments, which act as a critical regulator for bone and tooth formation and maintenance (Litvin et al. 2004). Recently, accumulating evidence has revealed that high-level expression of PN is correlated significantly with various human cancers including liver, head and neck, neuroblastoma, breast, colon, oesophageal, ovary etc (Gillan et al. 2002; Bao et al. 2004; Shao et al. 2004; Kudo et al. 2006; Sasaki et al. 2002; Lv et al. 2013a; Wang et al. 2014). Furthermore, emerging evidence suggests that high expression of PN protein is closely correlated with tumour angiogenesis in some types of human cancer (Shao et al. 2004; Wang et al. 2014). Whether and how PN expression influences prognosis in osteosarcoma, however, remains unknown. In the present study, we used the immunohistochemistry to examine the expression of PN, vascular endothelial growth factor (VEGF) and microvessel density (MVD) in osteosarcoma tissues. Further, the survival analysis was observed by Kaplan–Meier method. A multivariate survival analysis was performed for all parameters that were significant in the univariate analysis using the Cox regression model. The objectives of this were to elucidate the expression of PN in osteosarcoma and to examine its correlation with clinicopathological characteristics and prognosis.
Materials and methods Patients and specimens Sixty-eight patients with osteosarcoma were selected at the Department of Orthopedics in Anhui Provincial Hospital and the First Hospital Affiliated to Anhui Medicine University between 2001 and 2011. The selection criteria were as follows: (i) a diagnosis of malignant osteosarcoma based on pathology; (ii) complete records of the cases and the followup information had been preserved; and (iii) no anti-angiogenesis drugs had been used. These patients with osteosarcoma were treated according to the standardized protocol consisting of neoadjuvant chemotherapy (four cycles of methotrexate and cisplatin were administered with a minimum of a 21-day interval between consecutive cycles), followed by appropriate surgical management and postoperative adjuvant chemotherapy. Follow-up was terminated on 6 May 2014. Sixty-eight osteochondroma tissue specimens were obtained from the same hospital as the controls. Two pathologists (Hang-Cheng Zhou, Jiang Zhu) who were blinded to the clinical information confirmed all of histological diagnoses and judged the degree of staining independently. This study was approved by the Institutional Review Board of the Anhui Provincial Hospital affiliated to Anhui Medical University. All patients involved in this study had signed the informed consent. The study protocol conformed to the ethical guide lines of the Declaration of Helsinki. International Journal of Experimental Pathology, 2016, 97, 86–92
87
Ethical approval Ethical approval for the use of human subjects was obtained from the research ethics committee of Anhui Medical University
Histopathological and immunohistochemical analyses The histopathological and immunohistochemical analyses were performed on biopsies prior to neoadjuvant chemotherapy. The expressions of PN, VEGF and CD34 were detected by immunohistochemistry using a two-step method according to the manufacturer’s instructions. Semiquantitative estimation was made to interpret the results of immunohistochemistry according to the percentage of staining cells per 100 cells in 10 microscopic fields with high-power (4009) microscope, as follows: 0–10%, negative ( ); 10–30%, weak positive (+); >30%, strong positive (++). MVD was quantified in five fields in which there was high expression using high-power lens (400 9) and values were expressed by average measurements.
Statistical analysis All statistical analyses were performed using SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA). The corrections between PN expression and clinicopathological parameters were assessed by v2 test or Fisher’s exact test. The Kaplan– Meier method was used for survival analysis, and differences in survival were estimated using the log-rank test. Cox proportional hazards regression model was used for multivariate survival analysis to assess the prognostic factors that were significant in the univariate analysis. P < 0.05 was considered statistically significant.
Results Expression of PN in osteosarcoma and osteochondroma tissues Sixty-eight patients with osteosarcoma were comprised of 37 males and 31 females, with the mean age of 25 13 years old. Detailed histologic subtype included 63 cases of conventional osteosarcoma and five cases of special osteosarcoma (including four cases of low-grade central osteosarcoma and one case of parosteal osteosarcoma). As shown in Table 1, the positive rate of PN expression was 80.9% (55/68) in osteosarcoma and 14.7% (10/68) in osteochondroma samples. The protein expression level of PN was significantly
Table 1 Differential expression of periostin between osteosarcoma tissues and osteochondroma tissues (cases) Periostin Tissues
Case number
Positive
Negative
Positive rate (%)
Osteosarcoma Osteochondroma
68 68
55 10
13 58
80.9% 14.7%
88 (a)
F. Hu et al. (b)
Table 2 Periostin expression status in relation to selected clinicopathologic features in 68 osteosarcoma patients (cases) Periostin Clinicopathologic data
(c)
(e)
(d)
(f)
Figure 1 Representative immunohistochemical staining of periostin in osteosarcoma and osteochondroma tissues. Periostin (PN) mainly expressed in the cytoplasm of osteosarcoma tissues. (a) HE staining of osteosarcoma, 9100; (b) ++ for PN staining in osteosarcoma, 9100; (c) ++ for PN staining in osteosarcoma, 9400; (d) + for PN staining in osteosarcoma, 9100; (e) negative for PN staining in osteosarcoma, 940; (f) negative for PN staining in osteochondroma, 9100.
higher in osteosarcoma tissues than the level in osteochondroma tissues (P < 0.05). The distribution of positive expression area of PN was mainly localized in the cytoplasm, some tumour cells stained strongly, while others exhibited slightly or no staining at all (Figure 1). To elucidate its clinical significance, we also assessed the correlation between PN expression and clinicopathological parameters available for the patients (Table 2). Positive expression of PN in osteosarcoma was significantly correlated with histological subtype (P = 0.000), Enneking stage (P = 0.027) and tumour size (P = 0.009). However, there are no significant correlation with age, gender, location and alkaline phosphatase.
Expression of VEGF and MVD in osteosarcoma tissues and its correlation with PN The positive rate of VEGF expression was 66.18% (45/68), and the distribution of positive expression area was mainly
Case number
Sex Male 37 Female 31 Age at diagnosis (year)
ORIGINAL ARTICLE
High-level expression of periostin is significantly correlated with tumour angiogenesis and poor prognosis in osteosarcoma Fei Hu*, Xi-Fu Shang*, Wei Wang†, Wei Jiang‡, Ce Fang*, Dong Tan* and Hang-Cheng Zhou§ *Department of Orthopedics, Anhui Provincial Hospital, Anhui Medical University, Hefei, China, †Department of Medical Oncology, Anhui Provincial Hospital, Anhui Medical University, Hefei, China, ‡Department of Orthopedics, the First Hospital Affiliated to Anhui Medicine University, Hefei, China and §Department of Pathology, Anhui Provincial Hospital, Anhui Medical University, Hefei, China
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY doi: 10.1111/iep.12171
Received for publication: 12 December 2014 Accepted for publication: 31 December 2015 Correspondence: Fei Hu Department of Orthopedic Anhui Provincial Hospital Anhui Medical University No.17 Lujiang Road Hefei 230001 China Tel.: +86 551 62283324 Fax: +86 551 62283324 E-mail: [email protected]
SUMMARY Periostin (PN), originally named as osteoblast-specific factor-2 (OSF-2), has been involved in regulating adhesion and differentiation of osteoblasts. Recently many studies have shown that high-level expression of PN is correlated significantly with tumour angiogenesis and prognosis in many kinds of human cancer. However, whether and how periostin expression influences prognosis in osteosarcoma remains unknown. This study aimed to examine the expression of PN in patients with osteosarcoma and explore the relationship of PN expression with clinicopathologic factors, tumour angiogenesis and prognosis. Immunohistochemistry was performed to determine the expression of PN in osteosarcoma and osteochondroma respectively. Vascular endothelial growth factor (VEGF) and CD34 were also examined in tissues from the osteosarcoma patients mentioned above. The results showed that PN expression was significantly (P < 0.05) higher in osteosarcoma (80.9%) than in osteochondroma (14.7%). Increased PN protein expression was associated with histological subtype (P = 0.000), Enneking stage (P = 0.027) and tumour size (P = 0.009). The result also showed that high expression of PN correlated with VEGF expression (r = 0.285; P = 0.019) and that tumours with PN-positive expression significantly had higher microvessal density (44.6 13.7 vs. 20.6 6.5; P = 0.000) compared to those in normal bone tissues. Additionally, the expression of PN was found to be an independent prognostic factor in osteosarcoma patients. In conclusion, our findings suggest that PN may have an important role in tumour progression and may be used as a prognostic biomarker for patients with osteosarcoma. Keywords angiogenesis, biomarker, osteosarcoma, periostin, prognosis
Osteosarcoma is the most common primary malignancy in orthopaedic surgery, which accounts for approximately 20% of primary malignancies of bone (Arndt & Crist 1999). Osteosarcomas tends to develop distant metastases frequently and ultimately this results in death, especially in cases where there is lung metastasis. About 20% of patients have visible metastases on imaging at diagnosis and a quarter of the patients have metastases during the course of treatment (Theoleyre et al. 2005). Despite recent advances in multimodality treatments consisting of aggressive neoadjuvant chemotherapy and wide tumour resection, metastatic
or recurrent disease still occurs in 30–40% of these patients and the majority of those succumb to the disease (Yang et al. 2014). Therefore, developing a novel biomarker to identify the invasive potential and prognosis of osteosarcoma is significantly crucial. Periostin (PN), also named osteoblast-specific factor-2 (OSF-2), was originally identified in 1993 as an 90 kDa protein acids, which was secreted from the mouse osteoblastic cell line MC3T3-E1 (Takeshita et al. 1993). This protein shows homologous with an insect cell adhesion protein named fasciclin I (FAS I) protein family, members of which © 2016 The Authors.
86
International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology
Expression of PN in osteosarcoma are involved in many biologic processes, such as cell motility, adhesion, metastatic growth and angiogenesis (Gillan et al. 2002; Bao et al. 2004; Shao et al. 2004). Initially, periostin has been shown to be expressed preferentially in the periosteum and periodontal ligaments, which act as a critical regulator for bone and tooth formation and maintenance (Litvin et al. 2004). Recently, accumulating evidence has revealed that high-level expression of PN is correlated significantly with various human cancers including liver, head and neck, neuroblastoma, breast, colon, oesophageal, ovary etc (Gillan et al. 2002; Bao et al. 2004; Shao et al. 2004; Kudo et al. 2006; Sasaki et al. 2002; Lv et al. 2013a; Wang et al. 2014). Furthermore, emerging evidence suggests that high expression of PN protein is closely correlated with tumour angiogenesis in some types of human cancer (Shao et al. 2004; Wang et al. 2014). Whether and how PN expression influences prognosis in osteosarcoma, however, remains unknown. In the present study, we used the immunohistochemistry to examine the expression of PN, vascular endothelial growth factor (VEGF) and microvessel density (MVD) in osteosarcoma tissues. Further, the survival analysis was observed by Kaplan–Meier method. A multivariate survival analysis was performed for all parameters that were significant in the univariate analysis using the Cox regression model. The objectives of this were to elucidate the expression of PN in osteosarcoma and to examine its correlation with clinicopathological characteristics and prognosis.
Materials and methods Patients and specimens Sixty-eight patients with osteosarcoma were selected at the Department of Orthopedics in Anhui Provincial Hospital and the First Hospital Affiliated to Anhui Medicine University between 2001 and 2011. The selection criteria were as follows: (i) a diagnosis of malignant osteosarcoma based on pathology; (ii) complete records of the cases and the followup information had been preserved; and (iii) no anti-angiogenesis drugs had been used. These patients with osteosarcoma were treated according to the standardized protocol consisting of neoadjuvant chemotherapy (four cycles of methotrexate and cisplatin were administered with a minimum of a 21-day interval between consecutive cycles), followed by appropriate surgical management and postoperative adjuvant chemotherapy. Follow-up was terminated on 6 May 2014. Sixty-eight osteochondroma tissue specimens were obtained from the same hospital as the controls. Two pathologists (Hang-Cheng Zhou, Jiang Zhu) who were blinded to the clinical information confirmed all of histological diagnoses and judged the degree of staining independently. This study was approved by the Institutional Review Board of the Anhui Provincial Hospital affiliated to Anhui Medical University. All patients involved in this study had signed the informed consent. The study protocol conformed to the ethical guide lines of the Declaration of Helsinki. International Journal of Experimental Pathology, 2016, 97, 86–92
87
Ethical approval Ethical approval for the use of human subjects was obtained from the research ethics committee of Anhui Medical University
Histopathological and immunohistochemical analyses The histopathological and immunohistochemical analyses were performed on biopsies prior to neoadjuvant chemotherapy. The expressions of PN, VEGF and CD34 were detected by immunohistochemistry using a two-step method according to the manufacturer’s instructions. Semiquantitative estimation was made to interpret the results of immunohistochemistry according to the percentage of staining cells per 100 cells in 10 microscopic fields with high-power (4009) microscope, as follows: 0–10%, negative ( ); 10–30%, weak positive (+); >30%, strong positive (++). MVD was quantified in five fields in which there was high expression using high-power lens (400 9) and values were expressed by average measurements.
Statistical analysis All statistical analyses were performed using SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA). The corrections between PN expression and clinicopathological parameters were assessed by v2 test or Fisher’s exact test. The Kaplan– Meier method was used for survival analysis, and differences in survival were estimated using the log-rank test. Cox proportional hazards regression model was used for multivariate survival analysis to assess the prognostic factors that were significant in the univariate analysis. P < 0.05 was considered statistically significant.
Results Expression of PN in osteosarcoma and osteochondroma tissues Sixty-eight patients with osteosarcoma were comprised of 37 males and 31 females, with the mean age of 25 13 years old. Detailed histologic subtype included 63 cases of conventional osteosarcoma and five cases of special osteosarcoma (including four cases of low-grade central osteosarcoma and one case of parosteal osteosarcoma). As shown in Table 1, the positive rate of PN expression was 80.9% (55/68) in osteosarcoma and 14.7% (10/68) in osteochondroma samples. The protein expression level of PN was significantly
Table 1 Differential expression of periostin between osteosarcoma tissues and osteochondroma tissues (cases) Periostin Tissues
Case number
Positive
Negative
Positive rate (%)
Osteosarcoma Osteochondroma
68 68
55 10
13 58
80.9% 14.7%
88 (a)
F. Hu et al. (b)
Table 2 Periostin expression status in relation to selected clinicopathologic features in 68 osteosarcoma patients (cases) Periostin Clinicopathologic data
(c)
(e)
(d)
(f)
Figure 1 Representative immunohistochemical staining of periostin in osteosarcoma and osteochondroma tissues. Periostin (PN) mainly expressed in the cytoplasm of osteosarcoma tissues. (a) HE staining of osteosarcoma, 9100; (b) ++ for PN staining in osteosarcoma, 9100; (c) ++ for PN staining in osteosarcoma, 9400; (d) + for PN staining in osteosarcoma, 9100; (e) negative for PN staining in osteosarcoma, 940; (f) negative for PN staining in osteochondroma, 9100.
higher in osteosarcoma tissues than the level in osteochondroma tissues (P < 0.05). The distribution of positive expression area of PN was mainly localized in the cytoplasm, some tumour cells stained strongly, while others exhibited slightly or no staining at all (Figure 1). To elucidate its clinical significance, we also assessed the correlation between PN expression and clinicopathological parameters available for the patients (Table 2). Positive expression of PN in osteosarcoma was significantly correlated with histological subtype (P = 0.000), Enneking stage (P = 0.027) and tumour size (P = 0.009). However, there are no significant correlation with age, gender, location and alkaline phosphatase.
Expression of VEGF and MVD in osteosarcoma tissues and its correlation with PN The positive rate of VEGF expression was 66.18% (45/68), and the distribution of positive expression area was mainly
Case number
Sex Male 37 Female 31 Age at diagnosis (year)
