Cancer Epidemiology 39 (2015) 539–544
Contents lists available at ScienceDirect
Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net
Clinical and prognostic significance of OPN and VEGF expression in patients with non-small-cell lung cancer Qunying Lin a,1, Lijing Guo a, Guosheng Lin a, Zhiwei Chen a, Tonghuan Chen a, Juan Lin a, Bo Zhang 1,b,c, Xiaobin Gu b,* a b c
Department of Respiratory Medicine, Affiliated Hospital of Putian University, 999 East zhendong Road, Licheng District, Putian, Fujian Province, China Cancer Center, Chinese PLA General Hospital and Chinese PLA Medical School, 28 Fuxing Road, Bejing 100853, China International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, New Building West 10-11th Floor, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 November 2014 Received in revised form 25 April 2015 Accepted 25 May 2015 Available online 27 June 2015
Background: Osteopontin (OPN) and vascular endothelial growth factor (VEGF) play important roles in cancer progression and angiogenesis. In the current study we aimed to investigate the clinical significance of OPN and VEGF expression in patients with non-small-cell lung cancer (NSCLC) and to investigate their prognostic value for NSCLC. Methods: Immunohistochemical staining was used to detect the expression of OPN and VEGF in 146 NSCLC patients undergoing surgical resection in our hospital between 2006 and 2008. The associations between OPN and VEGF expression and clinicopathological data were analyzed using chisquare test analysis. Survival analysis was performed using the Kaplan–Meier method. The prognostic values of OPN and VEGF were evaluated by univariate and multivariate Cox proportional hazard model analysis. Results: OPN and VEGF expression was positive in 94 and 86 out of 146 NSCLC specimens, respectively. OPN expression was significantly associated with gender (P = 0.002), TNM stage (P < 0.001) and tumor differentiation (P = 0.008). VEGF expression was significantly associated with TNM stage (P = 0.015), tumor differentiation (P = 0.032) and lymph-node status (P < 0.001). There was a significant correlation between OPN and VEGF expression (P = 0.035). Survival analysis indicated that OPN(+)/ VEGF(+) patients had the worst prognosis. Furthermore, univariate and multivariate analysis suggested that tumor stage, lymph-node metastases, OPN expression and VEGF expression were independent prognostic factors for NSCLC. Conclusion: The data suggest that OPN and VEGF expressions could serve as prognostic factors for NSCLC. ß 2015 Elsevier Ltd. All rights reserved.
Keywords: Non-small cell lung cancer Osteopontin Vascular endothelial growth factor Survival Prognosis
1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide. Its prognosis remains dismal due to early metastasis [1]. Non-small-cell lung cancer (NSCLC) accounts for approximately 80–85% of all lung cancers. Traditional prognostic factors – such as TNM stage, tumor size, serum lactate dehydrogenase (LDH) levels etc. – have been applied in the management of NSCLC patients; however, their prognostic efficiency is unsatisfactory. Therefore, there is an urgent need to identify new biomarkers that
* Corresponding author. Tel.: +86 10 66937592; fax: +86 10 66937592. E-mail address: [email protected] (X. Gu). 1 These authors contributed equally to this work as first co-authors. http://dx.doi.org/10.1016/j.canep.2015.05.010 1877-7821/ß 2015 Elsevier Ltd. All rights reserved.
can stratify patients according to risk and predict unfavorable clinical outcomes. Osteopontin (OPN) is a multifunctional protein secreted by various cells [2]. OPN plays an important role in the progression, development, and metastasis of cancers [3,4]. Circulating serum OPN levels have been shown to be associated with tumor stage and survival rate in NSCLC patients [5–7]. Angiogenesis is a necessary process in tumor invasion and metastasis. The accumulation of tumor mass must be accompanied by an increase in the microvasculature to provide nutrients and oxygen to meet the high energy demand of tumor metabolism. Vascular endothelial growth factor (VEGF) is the most important promoter of tumor angiogenesis [8]. VEGF can facilitate the growth of epithelial cells, promote vasculature formation, and stimulate blood vessel permeability [9]. Previous studies have shown that VEGF expression can predict poor outcome in NSCLC patients [10]. A recent study by Goutam
Q. Lin et al. / Cancer Epidemiology 39 (2015) 539–544
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et al. [11] indicated that OPN could induce VEGF expression and stimulate neovascularization in breast cancer. The prognostic significance of OPN and VEGF in NSCLC patients was investigated by Noriharu et al. [12]; however, they focused only on stage I lung adenocarcinoma, and the sample size was relatively small. Therefore, in the present study we aimed to investigate the prognostic role of OPN and VEGF expression in NSCLC patients of all stages. 2. Materials and methods 2.1. Patients and tissue samples The study was approved by the Research Ethics Committee of the Affiliated Hospital of Putian University, Fujian, China. Informed consent was obtained from all patients prior to surgery. A total of 146 primary NSCLC tissue samples were collected by the Affiliated Hospital of Putian University between 2006 and 2008. Patients did not receive any anti-cancer treatment (chemotherapy or radiotherapy) before surgery. Patient characteristics are shown in Table 1. NSCLC diagnosis was based on World Health Organization criteria. Tumor stage was determined according to the 2009 TNM classification of malignant tumors by the International Union Against Cancer and the American Joint Committee on Cancer. 2.2. Immunohistochemistry NSCLC tissue samples were fixed by immersion in 4% paraformaldehyde overnight at 4 8C, embedded in regular paraffin wax, and cut into 4-mm sections. Immunohistochemical analyses of OPN and VEGF expression were performed on formalin-fixed paraffin-embedded sections of surgical specimens. For immunohistochemistry, tissue sections were deparaffinized and rehydrated in phosphate-buffered saline (PBS). After antigen retrieval with target retrieval solution, endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide, and sections were then blocked in 10% fetal calf serum. Non-specific binding was blocked by pre-incubation with 10% fetal calf serum in PBS
with 0.01% sodium azide, and then the slides were incubated in a humidified chamber for 1 h with antibodies against OPN (titer 1:100, Santa Cruz, USA) and VEGF (titer 1:50, Abcam, UK). Finally, samples were incubated with peroxidase-conjugated streptavidin. Color was developed by incubating the slides for several minutes with diaminobenzidine, and nuclei were counterstained with hematoxylin. For substitute negative controls, the primary antibodies were replaced with PBS. Positive controls were provided by the kit supplier. The results of immunohistochemical staining were interpreted by two experienced pathologists, and the mean density of staining was calculated using the ImagePro Plus 6.0 software (ImagePro, Bethesda, MD). 2.3. Evaluation of immunohistochemical staining The percentage scoring of the immunoreactive NSCLC tissues was as follows: 0, no staining and less than 10% of tumor cells or stroma cells with membrane staining; 1+, more than 10% of tumor cells or stroma cells with faint partial membrane staining; 2+, more than 10% of tumor cells or stroma cells with weak to moderate partial membrane staining; 3+, more than 10% of tumor cells or stroma cells with strong partial membrane staining. Specimens with scores of 0 or 1+ were considered negative, and those with scores of 2+ or 3+ were considered positive for OPN expression. The VEGF staining was considered positive when at least 10% of the tumor cells were stained, as described elsewhere [13]. 2.4. Statistical analysis Statistical analysis of group differences was conducted using the x2 test. The Kaplan–Meier method was used for survival analysis, and differences in survival were estimated using the logrank test. The correlation analysis was performed using the Spearman test. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards regression model). The significant level was set at P < 0.05. All statistical analyses were performed using SPSS 19.0 software (SPSS Inc, Chicago, IL).
Table 1 Association between OPN, VEGF expression and clinicopathological variables of NSCLC patients. Clinicopathological variable Gender Male Female Age 60 years >60 years Smoking status Never smoked Smokers Histology type SCC AC TNM stage I II III IV Differentiation Well Moderate Poor Lymph node status Positive Negative
No. of patients
OPN positive (n = 94)
OPN negative (n = 52)
P-value
VEGF positive (n = 86)
VEGF negative (n = 60)
P-value
91 55
50 44
41 11
0.002
45 41
36 24
0.359
65 81
48 46
17 35
0.032
35 51
30 30
0.266
72 74
43 51
29 23
0.246
40 46
32 28
0.417
83 63
52 41
31 22
0.652
47 39
36 24
0.521
19 57 55 15
4 39 41 10
15 18 14 5
0.000
6 38 30 12
13 19 25 3
0.015
20 60 66
9 34 51
11 26 15
0.008
8 32 46
12 28 20
0.032
81 65
54 40
27 25
0.52
37 49
54 6
0.000
SCC, squamous cell carcinoma; AC, adenocarcinoma.
Q. Lin et al. / Cancer Epidemiology 39 (2015) 539–544
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Fig. 1. Immunohistochemical staining of osteopontin (OPN) and vascular endothelial growth factor (VEGF) in patients with non-small-cell lung cancer (NSCLC). (A) Negative OPN expression. (B) Positive OPN expression. (C) Negative VEGF expression. (D) Positive VEGF expression. Original magnification 200.
3. Results 3.1. Patient characteristics There were 146 NSCLC patients in our study, including 91 men and 55 women, with a mean age of 60.5 years. In respect of clinical features, 83 cases were squamous-cell carcinoma (SCC) and 63 were adenocarcinoma (AC). There were 19 patients in stage I, 57 patients in stage II, 55 patients in stage III and 15 patients in stage IV. Regarding tumor differentiation, 20 patients were staged as well differentiated, 60 as moderately differentiated, and 66 as poorly differentiated. The detailed characteristics of the NSCLC patients are shown in Table 1. 3.2. OPN and VEGF expression in NSCLC tissues and its correlation with clinicopathological features OPN expression was positive in 94 out of 146 NSCLC specimens (64.4%) and negative in the remaining 52 specimens (35.6%). VEGF expression was positive in 86 out of 146 NSCLC specimens (58.9%) and negative in the remaining 60 samples (41.1%). The representative immunohistochemical staining of OPN and VEGF is shown in Fig. 1. In addition, as summarized in Table 1, OPN expression was significantly associated with gender (P = 0.002), TNM stage (P < 0.001) and tumor differentiation (P = 0.008), but was not correlated with age, smoking status, histology type or lymph-node status (all P > 0.05). Furthermore, VEGF expression was significantly associated with TNM stage (P = 0.015), tumor differentiation (P = 0.032) and lymph-node status (P < 0.001), while there was no significant relationship between VEGF expression and variables such as age, gender, tumor histology and smoking status (all P > 0.05), as shown in Fig. 1. Moreover, 49 patients were positive for both OPN and VEGF expression, 45 patients were positive for OPN but negative for VEGF expression, 37 patients were positive
for VEGF but negative for OPN expression, and the remaining 15 patients were negative for both OPN and VEGF. There was a significant correlation between OPN and VEGF expressions (P = 0.035, Table 2). 3.3. Influence of OPN and VEGF expression on survival According to the Kaplan–Meier survival analysis, the 5-year overall survival rate was significantly lower in patients with positive OPN expression than in those with negative OPN expression (Fig. 2, P < 0.001). Similarly, NSCLC patients with high VEGF expression had worse survival rates than patients with low VEGF expression (Fig. 2, P < 0.01). In addition, when patients were stratified as OPN(+)/VEGF(+), OPN(+)/VEGF( ), OPN( )/VEGF(+), and OPN( )/VEGF( ), the survival analysis indicated that OPN(+)/ VEGF(+)patients had the worst prognosis among these groups (Fig. 2, P < 0.001), while OPN( )/VEGF( ) patients had the highest survival rate. To test the impact of OPN and VEGF expression and other clinicopathological parameters in NSCLC patients, univariate and multivariate analyses were performed using the Cox proportional hazards model. Univariate analysis identified that tumor stage (P = 0.004), lymph-node status (P = 0.012), OPN expression (P = 0.023) and VEGF expression (P = 0.003) were significantly associated with overall survival (Table 3). Multivariate Cox regression analyses showed that tumor stage (P = 0.006), OPN expression (P = 0.029) and VEGF expression (P = 0.014) were Table 2 Correlation between OPN and VEGF expression. OPN expression
Positive Negative
VEGF expression
P-value
Positive
Negative
49 37
45 15
0.035
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Fig. 2. Kaplan–Meier analysis shows overall survival for osteopontin (OPN) (A), vascular endothelial growth factor (VEGF) (B), and combined OPN and VEGF (C) in patients with non-small-cell lung cancer (NSCLC).
Table 3 Univariate and multivariate analysis of prognostic factors in NSCLC patients. Variables
HR
Univariate analysis
P-value
HR
95% CI Gender Age Smoking status Histology type TNM stage Differentiation Lymph node status OPN VEGF
1.305 1.472 1.396 1.582 1.945 1.295 1.578 1.481 1.643
0.735–2.614 0.679–2.781 0.484–2.933 1.175–2.349 1.276–4.139 0.734–2.283 1.231–3.357 1.075–2.796 1.297–3.215
Multivariate analysis
P-value
95% CI 0.557 0.693 0.328 0.147 0.004 0.245 0.012 0.023 0.003
1.578
1.045–3.756
0.006
1.437 1.304 1.575
0.958–2.729 0.995–2.534 1.014–2.467
0.127 0.029 0.014
Q. Lin et al. / Cancer Epidemiology 39 (2015) 539–544
independent prognostic factors (Table 3). Thus, OPN and VEGF expression may be useful for predicting the overall survival of NSCLC patients. 4. Discussion In the present study we examined the expression of OPN and VEGF in tissues using immunohistochemical staining, and we investigated the correlations between OPN and VEGF expression and clinicopathological characteristics in NSCLC patients. Our data showed that OPN and VEGF were overexpressed in tumor tissues, and that OPN and VEGF expression was significantly associated with clinical features indicating tumor progression and metastasis, such as tumor grade and lymph-node status. Furthermore, survival analysis showed that patients with high OPN or VEGF expression had a poor clinical outcome, and the OPN(+)/VEGF(+) patients had the least favorable prognosis. Multivariate analysis indicated that OPN and VEGF expressions had independent prognostic value for NSCLC patients and may serve as potential biomarkers in clinical practice. The present study reconfirmed the prognostic significance of OPN and VEGF expression in NSCLC patients, which was previously reported by Noriharu et al. [12]. More importantly, the NSCLC patients in our study contained squamous-cell carcinoma (SCC) and adenocarcinoma (AC) of all TNM stages from stage I to stage IV. Thus, the patient population in our study is more representative and our results are therefore more convincing. OPN is an arginine–glycine–aspartate-containing adhesive glycoprotein with the characteristics of both a matrix protein and a cytokine. OPN has multifaceted functions and is involved in tissue reconstruction, malignant transformation, and immunerelated reaction. Previous reports have shown that OPN expression is enhanced in a variety of solid tumors such as breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, and lung cancer [14–18]. The frequency of positivity seems to increase with the clinical stage of the disease and is associated with a worse prognosis [19]. Recently, Monalisa et al. [20] suggested that OPN could be a therapeutic target for cancer treatment, and our results strengthen this concept by confirming the role of OPN in cancer promotion. What’s more, we determined the prognostic value of OPN expression in NSCLC. VEGF acts as the predominant regulator of angiogenesis; it promotes the accumulation and proliferation of endothelial cells. Therefore, OPN plays a critical role in angiogenesis during tumor progression [21]. Elevated VEGF expression is reported in various malignancies [22]. Previous studies have also reported the prognostic role of VEGF expression in breast cancer and gastric cancer [23,24]. Furthermore, several studies have shown the interaction between OPN and VEGF in tumor development [25]. An elegant study conducted by Goutam et al. [11] showed that OPN can induce the accumulation of VEGF via autocrine and paracrine mechanisms, and that VEGF subsequently facilitates angiogenesis and promotes tumor growth. Similarly, we detected the significant association between OPN and VEGF expressions in our study. In conclusion, our study reveals that OPN and VEGF expression are highly correlated with the prognosis of NSCLC patients. In univariate and multivariate analyses, the high levels of expression of OPN and VEGF are identified as independent prognostic factors of worse overall survival. Our study suggests that OPN and VEGF might be considered as targets in NSCLC treatment. Conflict of interests The authors declare that they have no conflict of interests.
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Authorship contribution XBG designed the study, developed IHC, collected data, helped in statistical analysis, wrote the manuscript. QYL and BZ conceived the study, evaluated IHC sections, participated in data analysis and manuscript drafting. LG and GSL performed statistical analysis and wrote the manuscript. ZC, JHC and JL reviewed the data and contributed to the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81201605) and the Natural Science Foundation of Fujian Province of China (Grant No. 2013J01390). References [1] A. Jemal, R. Siegel, J.Q. Xu, E. Ward, Cancer Statistics, 2010, CA-Cancer J. Clin. 60 (5) (2010) 277–300, http://dx.doi.org/10.1002/caac.20073. [2] J. Wu, P. Pungaliya, E. Kraynov, B. 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Ellis, Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis, J. Clin. Oncol. 23 (5) (2005) 1011–1027, http:// dx.doi.org/10.1200/jco.2005.06.081. [10] H. Imoto, T. Osaki, S. Taga, A. Ohgami, Y. Ichiyoshi, K. Yasumoto, Vascular endothelial growth factor expression in non-small-cell lung cancer: prognostic significance in squamous cell carcinoma, J. Thorac. Cardiovasc. Surg. 115 (5) (1998) 1007–1014. [11] G. Chakraborty, S. Jain, G.C. Kundu, Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth and angiogenesis via autocrine and paracrine mechanisms, Cancer Res. 68 (1) (2008) 152–161, http://dx.doi.org/ 10.1158/0008-5472.can-07-2126. [12] N. Shijubo, T. Uede, S. Kon, M. Maeda, T. Segawa, A. Imada, et al., Vascular endothelial growth factor and osteopontin in stage I lung adenocarcinoma, Am. J. Respir. Crit. Care Med. 160 (4) (1999) 1269–1273, http://dx.doi.org/ 10.1164/ajrccm.160.4.9807094. [13] H. Saito, S. Tsujitani, M. Ikeguchi, M. Maeta, N. Kaibara, Relationship between the expression of vascular endothelial growth factor and the density of dendritic cells in gastric adenocarcinoma tissue, Br. J. Cancer 78 (12) (1998) 1573–1577. [14] T.J. Yeatman, A.F. Chambers, Osteopontin and colon cancer progression, Clin. Exp. Metastasis 20 (1) (2003) 85–90, http://dx.doi.org/10.1023/a:1022502805474. [15] V. Donati, L. Boldrini, M. Dell’Omodarme, M.C. Prati, P. Faviana, T. Camacci, et al., Osteopontin expression and prognostic significance in non-small cell lung cancer, Clin. Cancer Res. 11 (18) (2005) 6459–6465, http://dx.doi.org/10.1158/10780432.ccr-05-0541. [16] S.F. Shang, A. Plymoth, S.K. Ge, Z.D. Feng, H.R. Rosen, S. Sangrajrang, et al., Identification of osteopontin as a novel marker for early hepatocellular carcinoma, Hepatology 55 (2) (2012) 483–490, http://dx.doi.org/10.1002/hep.24703. [17] M. Mirza, E. Shaughnessy, J.K. Hurley, K.A. Vanpatten, G.A. Pestano, B. 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Contents lists available at ScienceDirect
Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net
Clinical and prognostic significance of OPN and VEGF expression in patients with non-small-cell lung cancer Qunying Lin a,1, Lijing Guo a, Guosheng Lin a, Zhiwei Chen a, Tonghuan Chen a, Juan Lin a, Bo Zhang 1,b,c, Xiaobin Gu b,* a b c
Department of Respiratory Medicine, Affiliated Hospital of Putian University, 999 East zhendong Road, Licheng District, Putian, Fujian Province, China Cancer Center, Chinese PLA General Hospital and Chinese PLA Medical School, 28 Fuxing Road, Bejing 100853, China International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, New Building West 10-11th Floor, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 November 2014 Received in revised form 25 April 2015 Accepted 25 May 2015 Available online 27 June 2015
Background: Osteopontin (OPN) and vascular endothelial growth factor (VEGF) play important roles in cancer progression and angiogenesis. In the current study we aimed to investigate the clinical significance of OPN and VEGF expression in patients with non-small-cell lung cancer (NSCLC) and to investigate their prognostic value for NSCLC. Methods: Immunohistochemical staining was used to detect the expression of OPN and VEGF in 146 NSCLC patients undergoing surgical resection in our hospital between 2006 and 2008. The associations between OPN and VEGF expression and clinicopathological data were analyzed using chisquare test analysis. Survival analysis was performed using the Kaplan–Meier method. The prognostic values of OPN and VEGF were evaluated by univariate and multivariate Cox proportional hazard model analysis. Results: OPN and VEGF expression was positive in 94 and 86 out of 146 NSCLC specimens, respectively. OPN expression was significantly associated with gender (P = 0.002), TNM stage (P < 0.001) and tumor differentiation (P = 0.008). VEGF expression was significantly associated with TNM stage (P = 0.015), tumor differentiation (P = 0.032) and lymph-node status (P < 0.001). There was a significant correlation between OPN and VEGF expression (P = 0.035). Survival analysis indicated that OPN(+)/ VEGF(+) patients had the worst prognosis. Furthermore, univariate and multivariate analysis suggested that tumor stage, lymph-node metastases, OPN expression and VEGF expression were independent prognostic factors for NSCLC. Conclusion: The data suggest that OPN and VEGF expressions could serve as prognostic factors for NSCLC. ß 2015 Elsevier Ltd. All rights reserved.
Keywords: Non-small cell lung cancer Osteopontin Vascular endothelial growth factor Survival Prognosis
1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide. Its prognosis remains dismal due to early metastasis [1]. Non-small-cell lung cancer (NSCLC) accounts for approximately 80–85% of all lung cancers. Traditional prognostic factors – such as TNM stage, tumor size, serum lactate dehydrogenase (LDH) levels etc. – have been applied in the management of NSCLC patients; however, their prognostic efficiency is unsatisfactory. Therefore, there is an urgent need to identify new biomarkers that
* Corresponding author. Tel.: +86 10 66937592; fax: +86 10 66937592. E-mail address: [email protected] (X. Gu). 1 These authors contributed equally to this work as first co-authors. http://dx.doi.org/10.1016/j.canep.2015.05.010 1877-7821/ß 2015 Elsevier Ltd. All rights reserved.
can stratify patients according to risk and predict unfavorable clinical outcomes. Osteopontin (OPN) is a multifunctional protein secreted by various cells [2]. OPN plays an important role in the progression, development, and metastasis of cancers [3,4]. Circulating serum OPN levels have been shown to be associated with tumor stage and survival rate in NSCLC patients [5–7]. Angiogenesis is a necessary process in tumor invasion and metastasis. The accumulation of tumor mass must be accompanied by an increase in the microvasculature to provide nutrients and oxygen to meet the high energy demand of tumor metabolism. Vascular endothelial growth factor (VEGF) is the most important promoter of tumor angiogenesis [8]. VEGF can facilitate the growth of epithelial cells, promote vasculature formation, and stimulate blood vessel permeability [9]. Previous studies have shown that VEGF expression can predict poor outcome in NSCLC patients [10]. A recent study by Goutam
Q. Lin et al. / Cancer Epidemiology 39 (2015) 539–544
540
et al. [11] indicated that OPN could induce VEGF expression and stimulate neovascularization in breast cancer. The prognostic significance of OPN and VEGF in NSCLC patients was investigated by Noriharu et al. [12]; however, they focused only on stage I lung adenocarcinoma, and the sample size was relatively small. Therefore, in the present study we aimed to investigate the prognostic role of OPN and VEGF expression in NSCLC patients of all stages. 2. Materials and methods 2.1. Patients and tissue samples The study was approved by the Research Ethics Committee of the Affiliated Hospital of Putian University, Fujian, China. Informed consent was obtained from all patients prior to surgery. A total of 146 primary NSCLC tissue samples were collected by the Affiliated Hospital of Putian University between 2006 and 2008. Patients did not receive any anti-cancer treatment (chemotherapy or radiotherapy) before surgery. Patient characteristics are shown in Table 1. NSCLC diagnosis was based on World Health Organization criteria. Tumor stage was determined according to the 2009 TNM classification of malignant tumors by the International Union Against Cancer and the American Joint Committee on Cancer. 2.2. Immunohistochemistry NSCLC tissue samples were fixed by immersion in 4% paraformaldehyde overnight at 4 8C, embedded in regular paraffin wax, and cut into 4-mm sections. Immunohistochemical analyses of OPN and VEGF expression were performed on formalin-fixed paraffin-embedded sections of surgical specimens. For immunohistochemistry, tissue sections were deparaffinized and rehydrated in phosphate-buffered saline (PBS). After antigen retrieval with target retrieval solution, endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide, and sections were then blocked in 10% fetal calf serum. Non-specific binding was blocked by pre-incubation with 10% fetal calf serum in PBS
with 0.01% sodium azide, and then the slides were incubated in a humidified chamber for 1 h with antibodies against OPN (titer 1:100, Santa Cruz, USA) and VEGF (titer 1:50, Abcam, UK). Finally, samples were incubated with peroxidase-conjugated streptavidin. Color was developed by incubating the slides for several minutes with diaminobenzidine, and nuclei were counterstained with hematoxylin. For substitute negative controls, the primary antibodies were replaced with PBS. Positive controls were provided by the kit supplier. The results of immunohistochemical staining were interpreted by two experienced pathologists, and the mean density of staining was calculated using the ImagePro Plus 6.0 software (ImagePro, Bethesda, MD). 2.3. Evaluation of immunohistochemical staining The percentage scoring of the immunoreactive NSCLC tissues was as follows: 0, no staining and less than 10% of tumor cells or stroma cells with membrane staining; 1+, more than 10% of tumor cells or stroma cells with faint partial membrane staining; 2+, more than 10% of tumor cells or stroma cells with weak to moderate partial membrane staining; 3+, more than 10% of tumor cells or stroma cells with strong partial membrane staining. Specimens with scores of 0 or 1+ were considered negative, and those with scores of 2+ or 3+ were considered positive for OPN expression. The VEGF staining was considered positive when at least 10% of the tumor cells were stained, as described elsewhere [13]. 2.4. Statistical analysis Statistical analysis of group differences was conducted using the x2 test. The Kaplan–Meier method was used for survival analysis, and differences in survival were estimated using the logrank test. The correlation analysis was performed using the Spearman test. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards regression model). The significant level was set at P < 0.05. All statistical analyses were performed using SPSS 19.0 software (SPSS Inc, Chicago, IL).
Table 1 Association between OPN, VEGF expression and clinicopathological variables of NSCLC patients. Clinicopathological variable Gender Male Female Age 60 years >60 years Smoking status Never smoked Smokers Histology type SCC AC TNM stage I II III IV Differentiation Well Moderate Poor Lymph node status Positive Negative
No. of patients
OPN positive (n = 94)
OPN negative (n = 52)
P-value
VEGF positive (n = 86)
VEGF negative (n = 60)
P-value
91 55
50 44
41 11
0.002
45 41
36 24
0.359
65 81
48 46
17 35
0.032
35 51
30 30
0.266
72 74
43 51
29 23
0.246
40 46
32 28
0.417
83 63
52 41
31 22
0.652
47 39
36 24
0.521
19 57 55 15
4 39 41 10
15 18 14 5
0.000
6 38 30 12
13 19 25 3
0.015
20 60 66
9 34 51
11 26 15
0.008
8 32 46
12 28 20
0.032
81 65
54 40
27 25
0.52
37 49
54 6
0.000
SCC, squamous cell carcinoma; AC, adenocarcinoma.
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Fig. 1. Immunohistochemical staining of osteopontin (OPN) and vascular endothelial growth factor (VEGF) in patients with non-small-cell lung cancer (NSCLC). (A) Negative OPN expression. (B) Positive OPN expression. (C) Negative VEGF expression. (D) Positive VEGF expression. Original magnification 200.
3. Results 3.1. Patient characteristics There were 146 NSCLC patients in our study, including 91 men and 55 women, with a mean age of 60.5 years. In respect of clinical features, 83 cases were squamous-cell carcinoma (SCC) and 63 were adenocarcinoma (AC). There were 19 patients in stage I, 57 patients in stage II, 55 patients in stage III and 15 patients in stage IV. Regarding tumor differentiation, 20 patients were staged as well differentiated, 60 as moderately differentiated, and 66 as poorly differentiated. The detailed characteristics of the NSCLC patients are shown in Table 1. 3.2. OPN and VEGF expression in NSCLC tissues and its correlation with clinicopathological features OPN expression was positive in 94 out of 146 NSCLC specimens (64.4%) and negative in the remaining 52 specimens (35.6%). VEGF expression was positive in 86 out of 146 NSCLC specimens (58.9%) and negative in the remaining 60 samples (41.1%). The representative immunohistochemical staining of OPN and VEGF is shown in Fig. 1. In addition, as summarized in Table 1, OPN expression was significantly associated with gender (P = 0.002), TNM stage (P < 0.001) and tumor differentiation (P = 0.008), but was not correlated with age, smoking status, histology type or lymph-node status (all P > 0.05). Furthermore, VEGF expression was significantly associated with TNM stage (P = 0.015), tumor differentiation (P = 0.032) and lymph-node status (P < 0.001), while there was no significant relationship between VEGF expression and variables such as age, gender, tumor histology and smoking status (all P > 0.05), as shown in Fig. 1. Moreover, 49 patients were positive for both OPN and VEGF expression, 45 patients were positive for OPN but negative for VEGF expression, 37 patients were positive
for VEGF but negative for OPN expression, and the remaining 15 patients were negative for both OPN and VEGF. There was a significant correlation between OPN and VEGF expressions (P = 0.035, Table 2). 3.3. Influence of OPN and VEGF expression on survival According to the Kaplan–Meier survival analysis, the 5-year overall survival rate was significantly lower in patients with positive OPN expression than in those with negative OPN expression (Fig. 2, P < 0.001). Similarly, NSCLC patients with high VEGF expression had worse survival rates than patients with low VEGF expression (Fig. 2, P < 0.01). In addition, when patients were stratified as OPN(+)/VEGF(+), OPN(+)/VEGF( ), OPN( )/VEGF(+), and OPN( )/VEGF( ), the survival analysis indicated that OPN(+)/ VEGF(+)patients had the worst prognosis among these groups (Fig. 2, P < 0.001), while OPN( )/VEGF( ) patients had the highest survival rate. To test the impact of OPN and VEGF expression and other clinicopathological parameters in NSCLC patients, univariate and multivariate analyses were performed using the Cox proportional hazards model. Univariate analysis identified that tumor stage (P = 0.004), lymph-node status (P = 0.012), OPN expression (P = 0.023) and VEGF expression (P = 0.003) were significantly associated with overall survival (Table 3). Multivariate Cox regression analyses showed that tumor stage (P = 0.006), OPN expression (P = 0.029) and VEGF expression (P = 0.014) were Table 2 Correlation between OPN and VEGF expression. OPN expression
Positive Negative
VEGF expression
P-value
Positive
Negative
49 37
45 15
0.035
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Fig. 2. Kaplan–Meier analysis shows overall survival for osteopontin (OPN) (A), vascular endothelial growth factor (VEGF) (B), and combined OPN and VEGF (C) in patients with non-small-cell lung cancer (NSCLC).
Table 3 Univariate and multivariate analysis of prognostic factors in NSCLC patients. Variables
HR
Univariate analysis
P-value
HR
95% CI Gender Age Smoking status Histology type TNM stage Differentiation Lymph node status OPN VEGF
1.305 1.472 1.396 1.582 1.945 1.295 1.578 1.481 1.643
0.735–2.614 0.679–2.781 0.484–2.933 1.175–2.349 1.276–4.139 0.734–2.283 1.231–3.357 1.075–2.796 1.297–3.215
Multivariate analysis
P-value
95% CI 0.557 0.693 0.328 0.147 0.004 0.245 0.012 0.023 0.003
1.578
1.045–3.756
0.006
1.437 1.304 1.575
0.958–2.729 0.995–2.534 1.014–2.467
0.127 0.029 0.014
Q. Lin et al. / Cancer Epidemiology 39 (2015) 539–544
independent prognostic factors (Table 3). Thus, OPN and VEGF expression may be useful for predicting the overall survival of NSCLC patients. 4. Discussion In the present study we examined the expression of OPN and VEGF in tissues using immunohistochemical staining, and we investigated the correlations between OPN and VEGF expression and clinicopathological characteristics in NSCLC patients. Our data showed that OPN and VEGF were overexpressed in tumor tissues, and that OPN and VEGF expression was significantly associated with clinical features indicating tumor progression and metastasis, such as tumor grade and lymph-node status. Furthermore, survival analysis showed that patients with high OPN or VEGF expression had a poor clinical outcome, and the OPN(+)/VEGF(+) patients had the least favorable prognosis. Multivariate analysis indicated that OPN and VEGF expressions had independent prognostic value for NSCLC patients and may serve as potential biomarkers in clinical practice. The present study reconfirmed the prognostic significance of OPN and VEGF expression in NSCLC patients, which was previously reported by Noriharu et al. [12]. More importantly, the NSCLC patients in our study contained squamous-cell carcinoma (SCC) and adenocarcinoma (AC) of all TNM stages from stage I to stage IV. Thus, the patient population in our study is more representative and our results are therefore more convincing. OPN is an arginine–glycine–aspartate-containing adhesive glycoprotein with the characteristics of both a matrix protein and a cytokine. OPN has multifaceted functions and is involved in tissue reconstruction, malignant transformation, and immunerelated reaction. Previous reports have shown that OPN expression is enhanced in a variety of solid tumors such as breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, and lung cancer [14–18]. The frequency of positivity seems to increase with the clinical stage of the disease and is associated with a worse prognosis [19]. Recently, Monalisa et al. [20] suggested that OPN could be a therapeutic target for cancer treatment, and our results strengthen this concept by confirming the role of OPN in cancer promotion. What’s more, we determined the prognostic value of OPN expression in NSCLC. VEGF acts as the predominant regulator of angiogenesis; it promotes the accumulation and proliferation of endothelial cells. Therefore, OPN plays a critical role in angiogenesis during tumor progression [21]. Elevated VEGF expression is reported in various malignancies [22]. Previous studies have also reported the prognostic role of VEGF expression in breast cancer and gastric cancer [23,24]. Furthermore, several studies have shown the interaction between OPN and VEGF in tumor development [25]. An elegant study conducted by Goutam et al. [11] showed that OPN can induce the accumulation of VEGF via autocrine and paracrine mechanisms, and that VEGF subsequently facilitates angiogenesis and promotes tumor growth. Similarly, we detected the significant association between OPN and VEGF expressions in our study. In conclusion, our study reveals that OPN and VEGF expression are highly correlated with the prognosis of NSCLC patients. In univariate and multivariate analyses, the high levels of expression of OPN and VEGF are identified as independent prognostic factors of worse overall survival. Our study suggests that OPN and VEGF might be considered as targets in NSCLC treatment. Conflict of interests The authors declare that they have no conflict of interests.
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Authorship contribution XBG designed the study, developed IHC, collected data, helped in statistical analysis, wrote the manuscript. QYL and BZ conceived the study, evaluated IHC sections, participated in data analysis and manuscript drafting. LG and GSL performed statistical analysis and wrote the manuscript. ZC, JHC and JL reviewed the data and contributed to the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81201605) and the Natural Science Foundation of Fujian Province of China (Grant No. 2013J01390). References [1] A. Jemal, R. Siegel, J.Q. Xu, E. Ward, Cancer Statistics, 2010, CA-Cancer J. Clin. 60 (5) (2010) 277–300, http://dx.doi.org/10.1002/caac.20073. [2] J. Wu, P. Pungaliya, E. Kraynov, B. 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