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Serum angiopoietin-1 as a prognostic marker in resected early stage lung cancer
Lung Cancer 66 (2009) 359–364
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Serum angiopoietin-1 as a prognostic marker in resected early stage lung cancer Joo Hun Park a,1 , Ho Choi b,1 , Young Bae Kim c , Young Sun Kim a , Seung Soo Sheen a , Jin-Hyuk Choi d , Hye Lim Lee a , Keu Sung Lee a , Woo Young Chung a , Sungsoo Lee b , Kyung Joo Park e , Sung Chul Hwang a , Kyi Beum Lee c , Kwang Joo Park a,∗ a
Department of Pulmonary and Critical Care Medicine, Ajou University School of Medicine, Youngtong-gu, Suwon, 443-721, South Korea Department of Thoracic and Cardiovascular Surgery, Ajou University School of Medicine, Suwon, South Korea c Department of Pathology, Ajou University School of Medicine, Suwon, South Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, South Korea e Department of Radiology, Ajou University School of Medicine, Suwon, South Korea b
a r t i c l e
i n f o
Article history: Received 5 December 2008 Received in revised form 12 February 2009 Accepted 1 March 2009 Keywords: Angiopoietin-1 Early stage Non-small cell lung cancer Survival Vascular endothelial growth factor
a b s t r a c t Purpose: We evaluated the clinical significance of angiopoietins and vascular endothelial growth factor (VEGF) in patients with resected early stage lung cancer. Patients and methods: The study enrolled 101 patients with completely resected non-small cell lung cancer (NSCLC) of stage I or II, along with 70 healthy volunteers. Serum concentrations of angiopoietin1, angiopoietin-2, and VEGF were measured with an ELISA. Immunohistochemical expression of angiopoietin-1 was compared with the microvessel density on the lung cancer tissues. Results: The patients had lower serum angiopoietin-1 (32.1 ± 9.9 ng/mL vs. 39.0 ± 10.8 ng/mL, p < 0.001), higher angiopoietin-2 (1949.2 ± 1099.4 pg/mL vs. 1498.6 ± 650.0 pg/mL, p < 0.01), and higher VEGF (565.1 ± 406.3 pg/ mL vs. 404.6 ± 254.8 pg/mL, p < 0.01) levels than the controls. The angiopoietin-2 level was higher in stage II than in stage I patients (p < 0.05). The levels of angiopoietin-1 (r = 0.28) and angiopoietin-2 (r = 0.36) each correlated with the VEGF level. Patients with a higher level of angiopoietin-1 (≥31.2 ng/mL) had better disease-specific and relapse-free survival than those with a lower angiopoietin-1 level (<31.2 ng/mL). Angiopoietin-1 expression negatively correlated with the microvessel density. Conclusion: Serum angiopoietin-1 is a potential marker for predicting postoperative survival and recurrence in patients with early stage NSCLC. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Surgical resection remains the most effective treatment for non-small cell lung cancer (NSCLC), especially in the early stages, but postoperative recurrence is a great concern [1,2]. Studies of various genetic, pathologic, and clinical markers to identify the group at high risk for postoperative recurrence have produced variable results [2–5]. Angiogenesis, which is an important process in tumorigenesis, has also been evaluated for this purpose, with promising results [6–8]. Angiogenesis is controlled by a complex interplay among positive and negative regulators [9]. Vascular endothelial growth factor (VEGF) and angiopoietins are among the most important angiogenic factors [10]. VEGF is upregulated in various malignancies
∗ Corresponding author. Tel.: +82 31 219 5121; fax: +82 31 219 5124. E-mail address: [email protected] (K.J. Park). 1 These authors contributed equally to this study. 0169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2009.03.002
and has prognostic implications [11,12]. Angiopoietins, cooperating with VEGF, are also involved in tumor angiogenesis [13,14], although their precise roles and interplay in tumor development and progression have not been clearly defined [10]. Angiopoeitin-1 (Ang-1) and angiopoietin-2 (Ang-2) are counteracting ligands for the endothelium-specific receptor Tie-2. Ang-1 promotes angiogenesis during development [15] and acts to stabilize the vasculature by promoting interactions between endothelial cells and the surrounding extracellular matrix [16,17], yet it inhibits vascular expansion in malignant tissues [14,18,19]. Ang-2 competitively binds to Tie-2 to destabilize the effects of Ang-1 and prime the tumor vasculature for the subsequent actions of pro-angiogenic factors such as VEGF [20,21]. Ang-2 is upregulated in various cancers, including lung cancer [22,23], and its expression is co-localized and correlated with that of VEGF [19]. The tissue expression level of Ang-2 is related to the stage progression and overall survival [23,24]. Serum Ang-2 has also been reported to be increased in breast cancer [25], prostate cancer [25], thyroid cancer [26], acute myeloid leukemia [27], and
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lung cancer [28], with prognostic implications in the latter two cancers. The role of Ang-1 in cancer is more controversial than that of Ang-2. Although Ang-1 has been shown to be upregulated in many cancer tissues [22,29–31], its expression was rather lower in some cancer types, including lung cancer, compared with normal tissues [19,32]. There are a few studies on serum Ang-1 levels, and these have reported conflicting results. Compared with the level in controls, the serum Ang-1 level was elevated in breast cancer [25] but lower in thyroid cancer [26]; the level was similar to the control level in prostate cancer [25] and acute myeloid leukemia [27]. As few studies have examined the clinical implications of serum angiopoietins in lung cancer, especially in early stage cases, we evaluated the clinical significance of serum angiopoietins along with VEGF in patients with resected early stage (stages I and II) lung cancer. 2. Patients and methods 2.1. Subjects The study comprised 101 patients diagnosed with NSCLC. All patients were in stage I or II and underwent complete resection between March 1999 and July 2007. Serum samples were taken from each lung cancer patient at the time of diagnosis, before any therapeutic measures were started. The patients had, at a minimum, a lobectomy with routine systematic nodal dissection of both hilar and mediastinal lymph nodes. There were no macroscopic or microscopic tumor residues as judged by tumor-free resection margins at the bronchus and pulmonary blood vessels. No patient received anti-cancer therapy before surgery or postoperative adjuvant therapy. The postoperative pathological stage was determined according to the TNM staging system [33]. Survival time was calculated from the date of surgery until the last date of contact or date of death. For disease-specific survival, only deaths from lung cancer were considered as events. However, we eliminated 1 case who expired from an obvious complication of the surgical procedure. Relapse time was calculated from the date of surgery to the date on which local recurrence or systemic metastasis was detected. Sera were also obtained from a healthy control group during the last 6 months of the study period. This group consisted of 70 healthy adults, as documented by a general health examination. Their basic laboratory tests and chest roentgenograms were normal, and no respiratory symptoms were indicated. Baseline demographic data are presented in Table 1. Table 1 Demographics of the patients with lung cancer and the controls.
This study was approved by the Institutional Review Board of Ajou University Hospital. All patients provided consent before samples were taken. 2.2. Measurements of serum concentrations of Ang-1, Ang-2, and VEGF Enzyme-linked immunosorbent assays were used to measure the amounts of Ang-1, Ang-2, and VEGF (Quantikine; R&D Systems, Minneapolis, MN, USA). 2.3. Immunohistochemistry Immunohistochemical studies were conducted on 20 representative deparaffinized sections (among 101 cases), using the streptavidin–biotin-peroxidase method (UltraVision LP Large Volume Detection System, Thermo Fisher Scientific, Fremont, CA, USA). Briefly, 4-m-thick sections were cut from paraffin-embedded material, deparaffinized with xylene, and rehydrated through a graded series of ethanol. After the inhibition of endogenous peroxidase, the sections were exposed to primary antibody at 4 ◦ C overnight. Mouse monoclonal anti-human Ang-1 (diluted 1:20, R&D Systems, Minneapolis, MN, USA) and mouse monoclonal anti-human CD34 (diluted 1:100, NeoMarkers, Fremont, CA, USA) antibodies were used as primary antibodies. The immunoreactive proteins were visualized with 3,3 -diamino-benzidine, and the sections were counterstained with hematoxylin. For the negative controls, the primary antibody was omitted. 2.4. Evaluation of immunohistochemistry and microvessel density (MVD) The slides were examined independently by two pathologists who were blinded to both the clinical and pathologic data. The expression of Ang-1 was evaluated based on the extent of staining (percentage of positive tumor cells) using a visual grading scale of 0–3 (0 = none, 1 = 1–9%, 2 = 10–49%, 3 = ≥50%) and based on the intensity of staining using a scale of 0–3 (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining). A semi-quantitative immunohistochemical score was obtained by multiplying the staining extent and intensity grades. The median value of all the scores was chosen a priori as the cutoff value for dividing protein expression into high and low categories. MVD was determined as the number of microvessels in a defined area of the specimen. The number of CD34-positive vessels was counted in four selected hot spots in a 400× field (0.26 mm2 field area). The mean value of two independent counts for the same specimen was calculated, and the MVD was defined as the mean number of microvessels per 0.26 mm2 field area. 2.5. Statistical analysis
Lung cancer
Controls
p value
Number Age (years) Male, n (%)
101 63.0 ± 10.9 84 (83.2)
70 61.4 ± 6.9 51 (72.9)
NS NS
Stage Stage IA/IB Stage IIA/IIB
13/60 4/24
Cell type Squamous cell carcinoma, n (%) Adenocarcinoma, n (%) Large cell carcinoma, n (%) BAC, n (%) Othersa , n (%)
52 (51.5) 37 (36.6) 4 (4.0) 6 (5.9) 2 (2.0)
The data are presented as the means ± SD. BAC: bronchioloalveolar cell carcinoma; NS: not significant. a Others include two adenosquamous cell type.
SPSS for Windows (ver. 12; SPSS, Chicago, IL, USA) was used for the analysis. All values are given as means ± standard deviation except for the survival period, for which the mean ± standard error is reported. An unpaired Student’s t-test, Chi-square test, or analysis of variance with post hoc tests was performed as appropriate. Independent relationships between parameters were examined using Pearson’s or Spearman’s correlation analysis as appropriate. Univariate analysis of the survival data was performed using the Kaplan–Meier method, and the significance of differences between groups was analyzed using the log-rank test. To assess the independent effect of different pre-treatment variables on survival in the presence of other variables, multivariate analysis was carried out using the Cox proportional hazards model. Only significant variables from the univariate analysis were entered into the Cox
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Table 2 Comparison of serum angiopoietin and VEGF levels according to cell type. Cell type
n
Ang-1 (ng/mL)
Total Squamous cell carcinoma Adenocarcinoma Large cell carcinoma BAC Others
101 52 37 4 6 2
32.1 33.7 30.1 33.9 31.5 24.3
± ± ± ± ± ±
9.9 10.9 8.4 9.0 9.7 71.6
Ang-2 (pg/mL) 1949.2 2219.9 1673.5 1573.6 1592.8 1830.3
± ± ± ± ± ±
1099.4 1216.5 984.6 629.7 469.1 476.2
VEGF (pg/mL) 565.1 640.3 496.3 614.2 308.7 555.1
± ± ± ± ± ±
406.3 482.4 301.3 251.9 193.2 347.1
The data are presented as the means ± SD. Ang-1: angiopoietin-1; Ang-2: angiopoietin-2; VEGF: vascular endothelial growth factor.
regression analysis, in a stepwise manner. The p values of less than 0.05 were considered to be statistically significant. 3. Results 3.1. Comparison of serum Ang-1, Ang-2, and VEGF levels between lung cancer patients and controls Patients with lung cancer had a lower serum Ang-1 level (32.1 ± 9.9 ng/mL vs. 39.0 ± 10.8 ng/mL, p < 0.0001) and higher serum Ang-2 level (1949.2 ± 1099.4 pg/mL vs. 1498.6 ± 650.0 pg/ mL, p < 0.01) compared with the control group. The serum VEGF level was higher in patients with lung cancer than in the control group (565.1 ± 406.3 pg/mL vs. 404.6 ± 254.8 pg/mL, p < 0.01). 3.2. Evaluation of serum Ang-1, Ang-2, and VEGF levels in lung cancer patients No notable difference was found in the serum Ang-1, Ang-2, or VEGF level depending on the cell type of NSCLC. The serum Ang2 level tended to be higher in squamous cell carcinoma than in adenocarcinoma, without statistical significance (Table 2). The serum Ang-2 level was higher in stage II than in stage I (stage I: 1746.8 ± 923.6 pg/mL vs. stage II: 2330.6 ± 1288.7 pg/mL, p < 0.05), whereas no difference between stages I and II was found in the serum Ang-1 level (stage I: 31.9 ± 10.5 ng/mL vs. stage II: 31.9 ± 7.9 ng/mL) or the VEGF level (stage I: 518.3 ± 375.1 pg/mL vs. stage II: 651.2 ± 472.3 pg/mL). In the correlation analysis, the serum Ang-1 (r = 0.28, p < 0.01) and Ang-2 levels (r = 0.36, p < 0.01) were each correlated with the VEGF level (Fig. 1). The correlation between the Ang-1 and Ang-2 levels was not significant. 3.3. Analysis of disease-specific survival in lung cancer patients according to Ang-1, Ang-2, and VEGF levels For the survival analysis, the serum Ang-1, Ang-2, and VEGF levels were dichotomized using median values. The disease-specific
survival period was obtained in 100 patients with NSCLC. Twentynine patients died during the follow-up period. The patients in the group with higher serum Ang-1 (≥31.2 ng/ mL; n = 50; mean survival time = 85.4 ± 7.0 months) survived longer than those in the lower serum Ang-1 group (<31.2 ng/mL; n = 50; mean survival time = 58.9 ± 7.5 months, p < 0.05; Fig. 2A). In the subgroup analysis, stage I patients in the higher serum Ang-1 group (n = 36, mean survival time = 85.5 ± 4.1 months) survived longer than stage I patients in the lower serum Ang-1 group (n = 36, mean survival time = 65.5 ± 9.3 months, p < 0.05; Fig. 2B). However, for the subgroup of patients with stage II, the survival period did not differ between the serum Ang-1 groups, perhaps because of the small number of patients (p = 0.157; Fig. 2C). In the analysis of serum Ang-2 and VEGF levels, no significant difference in survival was found by analyzing the total patients or those in each stage. Patients with stage I survived longer than those with stage II (76.7 ± 7.5 months vs. 54.1 ± 9.1 months, p < 0.05). Following a univariate analysis to evaluate the prognostic value of clinical and experimental markers on cancer-related survival, we found that performance status and clinical stage were significantly associated with a worse prognosis, as was a higher serum level of Ang-1. In a Cox multivariate analysis, only serum Ang-1 maintained its significance (Table 3). 3.4. Analysis of postoperative recurrence in lung cancer patients according to the Ang-1, Ang-2, and VEGF levels Ninety-eight patients were included in the analysis for recurrence; 2 of the 100 cases in the disease-specific survival analysis were excluded because the exact information about relapse could not be determined. Twenty-six patients suffered recurrence after the operation. Among the 26 cases with postoperative recurrence, 18 patients were in the lower Ang-1 group, and 8 were in the higher Ang-1 group. In the analysis of the relapse-free survival, patients with a higher serum Ang-1 level had significantly longer relapse-free periods than those with a lower serum Ang-1 level (82.9 ± 6.4 months vs. 56.8 ± 8.0 months, p < 0.05; Fig. 2D).
Fig. 1. Correlations of the (A) serum angiopoietin-1 (Ang-1) and (B) angiopoietin-2 (Ang-2) levels with the serum vascular endothelial growth factor (VEGF) level in all lung cancer patients.
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Fig. 2. Kaplan–Meier curves for disease-specific survival in (A) all patients and in (B) stage I patients, in (C) stage II patients, and (D) relapse-free survival in all patients with lung cancer according to serum angiopoietin-1 (Ang-1) levels. The patients were divided into higher and lower Ang-1 groups using the median value of 31.2 ng/mL.
3.5. Immunohistochemical assessment of Ang-1 expression and MVD The overall expression of Ang-1 tended to be low and heterogeneous. However, one consistent finding was that the expression of Ang-1 was inversely correlated with the MVD (rs = 0.61, p < 0.05). Areas with high microvascularity in tumor sections showed low expression of Ang-1, and vice versa (Fig. 3). We were not able to present full survival analysis data because of the small number of immunohistochemistry cases. When MVD and Ang-1 expression scores were divided using median values, patients with higher MVD showed longer disease-specific survival than those with lower MVD in a univariate analysis (p < 0.05), while Ang-1 expression levels failed to show a significant survival difference (p = 0.24). Serum Ang-1 levels tended to be elevated in patients with higher Ang-1 expression scores than those with lower scores (34.1 ± 5.7 ng/mL vs. 30.2 ± 4.0 ng/mL, p = 0.099). 4. Discussion The main finding of our study is that the serum Ang-1 level is a useful parameter for predicting overall survival and recurrence in
patients with resected early stage lung cancer, whereas the Ang-2 and VEGF levels lacked prognostic implications. Ang-1 has dual contrasting functions. In normal or developing tissues, Ang-1 promotes angiogenesis [16,21,34,35], whereas Ang-1 inhibits angiogenesis and tumor growth and metastasis in cancerous tissues [18,36,37]. However, instead of contrasting actions, these dual functions can be considered to have a consistent action with different manifestations, depending on the given conditions, for the maintenance of the normal vasculature. From this perspective, an association in lung cancer patients between better survival and higher Ang-1 levels appears plausible. The depletion of Ang-1 might disrupt the homeostatic control over tumor neoangiogenesis and represent a loss of anti-angiogenic activity, which might be associated with biological tumor aggressiveness. Serum Ang-1 levels have not been evaluated often in cancers. Our results concur to an extent with a previous report that measured serum angiopoietin levels in thyroid cancer patients [26]. In that report, thyroid cancer patients had lower serum Ang-1 and higher serum Ang-2 levels compared with controls, although the prognostic significance was not evaluated. However, an earlier study found variable serum Ang-1 levels according to different types of cancers [25].
Table 3 Univariate and multivariate analyses for disease-specific survival in all lung cancer patients. Variable
Univariate analysis (p value)
Age (years) Gender (male/female) Smoking (smoker/never smoked) Histology (cell type) ECOG Performance status (0/≥1) Stage (I/II) Serum Ang-1 levels (<31.2/≥31.2 ng/mL)
0.123 0.280 0.144 0.280 0.020 0.020 0.013
Multivariate analysis Hazard ratio
CI: confidence interval; ECOG: Eastern Cooperative Oncology Group.
2.53 2.05 0.30
95% CI
1.129–5.686 0.897–4.672 0.153–1.008
p value
0.062 0.078 0.039
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Fig. 3. Immunohistochemical staining of angiopoietin-1 (Ang-1) and CD34 in non-small cell lung cancer, showing their inverse relationship (400×). (A) High microvascularity (high CD34 expression) and (B) low Ang-1 expression are concomitantly observed in the same portion of a squamous cell carcinoma. Similarly, (C) low microvascularity (low CD34 expression) is accompanied by (D) high Ang-1 expression in the same portion of an adenocarcinoma.
In tissue studies, Ang-1 is upregulated in various malignancies, with a few exceptions [22,29,38]. In lung cancer tissues, the expression pattern of Ang-1 is variable and its clinical significance is controversial [19,22,24,39]. The overall contradictory findings regarding Ang-1 expression may not be explained adequately by the differences in laboratory methodologies and study populations. One more possible explanation is provided by reports that have consistently shown extremely variable and heterogeneous expression of Ang-1, even in the same tissue [24,39]. The heterogeneity of Ang1 expression may underlie the variable results of tissue expression studies, because small sample pieces from a tissue may not represent the entire tissue. Although we evaluated tissue expression of Ang-1 in only a small number of cases, its expression was also found to be low and heterogeneous. Therefore, our tissue results should be limited to only the inverse relationship between tumor neovascularization and the Ang-1 expression level, which may correspond to the serum Ang-1 results as well as to the pathophysiological background. In a previous study assessing the clinical implications of the serum Ang-2 level in lung cancer at all stages, we found that Ang2 levels were elevated in the more advanced stages; the results implied that Ang-2 was a potential prognosticator with a significant, but not independent, impact on survival [28]. In the present study, although Ang-2 maintained its stage-discriminating power in the early stages, the prognostic significance of Ang-2 did not extend to the early stages of lung cancer. Nevertheless, the prognostic implications of Ang-2 should not be nullified solely by this result. Given that Ang-2 reflected the stage progression, studies involving a greater number of lung cancer cases in each stage may show Ang-2 to useful, along with other markers, in predicting prognosis. Many studies have evaluated the prognostic significance of serum VEGF in lung cancer, and most have shown positive results, but not consistently [8]. Serum VEGF has rarely been evaluated
specifically in resected early stage lung cancer. We found that serum VEGF was not a significant prognosticator in that group. The results of the correlation analysis, although statistically weak, correspond to the pathophysiological concept that Ang-1 and Ang-2 interact closely with VEGF [10,14]. We acknowledge several limitations of our study. First, the patient population was not distributed evenly, especially with regard to cancer stage. This shortcoming and the overall small numbers of patients prevented a detailed subgroup analysis, which requires further investigation of more cases in the future. Also, some of the eligible patients (mostly stage II patients) were excluded during the study period for various reasons such as missed sampling, postoperative adjuvant therapy, and incomplete resection. Second, correlation analysis of the serum data with tissue expression is important to validate our results. However, our immunohistochemical data are very limited, and further tissue evaluation with more cases should be undertaken in the future. In conclusion, our analysis of markers of tumor angiogenesis indicated that serum Ang-1 may be a useful parameter for predicting survival and recurrence after the resection of early stage NSCLC. Further studies are necessary to clarify its clinical validity and prognostic implications.
Conflict of interest The authors declare that there is no conflict of interests.
Acknowledgement This work was supported by 2002 grant from Department of Medical Sciences, The Graduate School, Ajou University.
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Serum angiopoietin-1 as a prognostic marker in resected early stage lung cancer Joo Hun Park a,1 , Ho Choi b,1 , Young Bae Kim c , Young Sun Kim a , Seung Soo Sheen a , Jin-Hyuk Choi d , Hye Lim Lee a , Keu Sung Lee a , Woo Young Chung a , Sungsoo Lee b , Kyung Joo Park e , Sung Chul Hwang a , Kyi Beum Lee c , Kwang Joo Park a,∗ a
Department of Pulmonary and Critical Care Medicine, Ajou University School of Medicine, Youngtong-gu, Suwon, 443-721, South Korea Department of Thoracic and Cardiovascular Surgery, Ajou University School of Medicine, Suwon, South Korea c Department of Pathology, Ajou University School of Medicine, Suwon, South Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, South Korea e Department of Radiology, Ajou University School of Medicine, Suwon, South Korea b
a r t i c l e
i n f o
Article history: Received 5 December 2008 Received in revised form 12 February 2009 Accepted 1 March 2009 Keywords: Angiopoietin-1 Early stage Non-small cell lung cancer Survival Vascular endothelial growth factor
a b s t r a c t Purpose: We evaluated the clinical significance of angiopoietins and vascular endothelial growth factor (VEGF) in patients with resected early stage lung cancer. Patients and methods: The study enrolled 101 patients with completely resected non-small cell lung cancer (NSCLC) of stage I or II, along with 70 healthy volunteers. Serum concentrations of angiopoietin1, angiopoietin-2, and VEGF were measured with an ELISA. Immunohistochemical expression of angiopoietin-1 was compared with the microvessel density on the lung cancer tissues. Results: The patients had lower serum angiopoietin-1 (32.1 ± 9.9 ng/mL vs. 39.0 ± 10.8 ng/mL, p < 0.001), higher angiopoietin-2 (1949.2 ± 1099.4 pg/mL vs. 1498.6 ± 650.0 pg/mL, p < 0.01), and higher VEGF (565.1 ± 406.3 pg/ mL vs. 404.6 ± 254.8 pg/mL, p < 0.01) levels than the controls. The angiopoietin-2 level was higher in stage II than in stage I patients (p < 0.05). The levels of angiopoietin-1 (r = 0.28) and angiopoietin-2 (r = 0.36) each correlated with the VEGF level. Patients with a higher level of angiopoietin-1 (≥31.2 ng/mL) had better disease-specific and relapse-free survival than those with a lower angiopoietin-1 level (<31.2 ng/mL). Angiopoietin-1 expression negatively correlated with the microvessel density. Conclusion: Serum angiopoietin-1 is a potential marker for predicting postoperative survival and recurrence in patients with early stage NSCLC. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Surgical resection remains the most effective treatment for non-small cell lung cancer (NSCLC), especially in the early stages, but postoperative recurrence is a great concern [1,2]. Studies of various genetic, pathologic, and clinical markers to identify the group at high risk for postoperative recurrence have produced variable results [2–5]. Angiogenesis, which is an important process in tumorigenesis, has also been evaluated for this purpose, with promising results [6–8]. Angiogenesis is controlled by a complex interplay among positive and negative regulators [9]. Vascular endothelial growth factor (VEGF) and angiopoietins are among the most important angiogenic factors [10]. VEGF is upregulated in various malignancies
∗ Corresponding author. Tel.: +82 31 219 5121; fax: +82 31 219 5124. E-mail address: [email protected] (K.J. Park). 1 These authors contributed equally to this study. 0169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2009.03.002
and has prognostic implications [11,12]. Angiopoietins, cooperating with VEGF, are also involved in tumor angiogenesis [13,14], although their precise roles and interplay in tumor development and progression have not been clearly defined [10]. Angiopoeitin-1 (Ang-1) and angiopoietin-2 (Ang-2) are counteracting ligands for the endothelium-specific receptor Tie-2. Ang-1 promotes angiogenesis during development [15] and acts to stabilize the vasculature by promoting interactions between endothelial cells and the surrounding extracellular matrix [16,17], yet it inhibits vascular expansion in malignant tissues [14,18,19]. Ang-2 competitively binds to Tie-2 to destabilize the effects of Ang-1 and prime the tumor vasculature for the subsequent actions of pro-angiogenic factors such as VEGF [20,21]. Ang-2 is upregulated in various cancers, including lung cancer [22,23], and its expression is co-localized and correlated with that of VEGF [19]. The tissue expression level of Ang-2 is related to the stage progression and overall survival [23,24]. Serum Ang-2 has also been reported to be increased in breast cancer [25], prostate cancer [25], thyroid cancer [26], acute myeloid leukemia [27], and
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lung cancer [28], with prognostic implications in the latter two cancers. The role of Ang-1 in cancer is more controversial than that of Ang-2. Although Ang-1 has been shown to be upregulated in many cancer tissues [22,29–31], its expression was rather lower in some cancer types, including lung cancer, compared with normal tissues [19,32]. There are a few studies on serum Ang-1 levels, and these have reported conflicting results. Compared with the level in controls, the serum Ang-1 level was elevated in breast cancer [25] but lower in thyroid cancer [26]; the level was similar to the control level in prostate cancer [25] and acute myeloid leukemia [27]. As few studies have examined the clinical implications of serum angiopoietins in lung cancer, especially in early stage cases, we evaluated the clinical significance of serum angiopoietins along with VEGF in patients with resected early stage (stages I and II) lung cancer. 2. Patients and methods 2.1. Subjects The study comprised 101 patients diagnosed with NSCLC. All patients were in stage I or II and underwent complete resection between March 1999 and July 2007. Serum samples were taken from each lung cancer patient at the time of diagnosis, before any therapeutic measures were started. The patients had, at a minimum, a lobectomy with routine systematic nodal dissection of both hilar and mediastinal lymph nodes. There were no macroscopic or microscopic tumor residues as judged by tumor-free resection margins at the bronchus and pulmonary blood vessels. No patient received anti-cancer therapy before surgery or postoperative adjuvant therapy. The postoperative pathological stage was determined according to the TNM staging system [33]. Survival time was calculated from the date of surgery until the last date of contact or date of death. For disease-specific survival, only deaths from lung cancer were considered as events. However, we eliminated 1 case who expired from an obvious complication of the surgical procedure. Relapse time was calculated from the date of surgery to the date on which local recurrence or systemic metastasis was detected. Sera were also obtained from a healthy control group during the last 6 months of the study period. This group consisted of 70 healthy adults, as documented by a general health examination. Their basic laboratory tests and chest roentgenograms were normal, and no respiratory symptoms were indicated. Baseline demographic data are presented in Table 1. Table 1 Demographics of the patients with lung cancer and the controls.
This study was approved by the Institutional Review Board of Ajou University Hospital. All patients provided consent before samples were taken. 2.2. Measurements of serum concentrations of Ang-1, Ang-2, and VEGF Enzyme-linked immunosorbent assays were used to measure the amounts of Ang-1, Ang-2, and VEGF (Quantikine; R&D Systems, Minneapolis, MN, USA). 2.3. Immunohistochemistry Immunohistochemical studies were conducted on 20 representative deparaffinized sections (among 101 cases), using the streptavidin–biotin-peroxidase method (UltraVision LP Large Volume Detection System, Thermo Fisher Scientific, Fremont, CA, USA). Briefly, 4-m-thick sections were cut from paraffin-embedded material, deparaffinized with xylene, and rehydrated through a graded series of ethanol. After the inhibition of endogenous peroxidase, the sections were exposed to primary antibody at 4 ◦ C overnight. Mouse monoclonal anti-human Ang-1 (diluted 1:20, R&D Systems, Minneapolis, MN, USA) and mouse monoclonal anti-human CD34 (diluted 1:100, NeoMarkers, Fremont, CA, USA) antibodies were used as primary antibodies. The immunoreactive proteins were visualized with 3,3 -diamino-benzidine, and the sections were counterstained with hematoxylin. For the negative controls, the primary antibody was omitted. 2.4. Evaluation of immunohistochemistry and microvessel density (MVD) The slides were examined independently by two pathologists who were blinded to both the clinical and pathologic data. The expression of Ang-1 was evaluated based on the extent of staining (percentage of positive tumor cells) using a visual grading scale of 0–3 (0 = none, 1 = 1–9%, 2 = 10–49%, 3 = ≥50%) and based on the intensity of staining using a scale of 0–3 (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining). A semi-quantitative immunohistochemical score was obtained by multiplying the staining extent and intensity grades. The median value of all the scores was chosen a priori as the cutoff value for dividing protein expression into high and low categories. MVD was determined as the number of microvessels in a defined area of the specimen. The number of CD34-positive vessels was counted in four selected hot spots in a 400× field (0.26 mm2 field area). The mean value of two independent counts for the same specimen was calculated, and the MVD was defined as the mean number of microvessels per 0.26 mm2 field area. 2.5. Statistical analysis
Lung cancer
Controls
p value
Number Age (years) Male, n (%)
101 63.0 ± 10.9 84 (83.2)
70 61.4 ± 6.9 51 (72.9)
NS NS
Stage Stage IA/IB Stage IIA/IIB
13/60 4/24
Cell type Squamous cell carcinoma, n (%) Adenocarcinoma, n (%) Large cell carcinoma, n (%) BAC, n (%) Othersa , n (%)
52 (51.5) 37 (36.6) 4 (4.0) 6 (5.9) 2 (2.0)
The data are presented as the means ± SD. BAC: bronchioloalveolar cell carcinoma; NS: not significant. a Others include two adenosquamous cell type.
SPSS for Windows (ver. 12; SPSS, Chicago, IL, USA) was used for the analysis. All values are given as means ± standard deviation except for the survival period, for which the mean ± standard error is reported. An unpaired Student’s t-test, Chi-square test, or analysis of variance with post hoc tests was performed as appropriate. Independent relationships between parameters were examined using Pearson’s or Spearman’s correlation analysis as appropriate. Univariate analysis of the survival data was performed using the Kaplan–Meier method, and the significance of differences between groups was analyzed using the log-rank test. To assess the independent effect of different pre-treatment variables on survival in the presence of other variables, multivariate analysis was carried out using the Cox proportional hazards model. Only significant variables from the univariate analysis were entered into the Cox
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Table 2 Comparison of serum angiopoietin and VEGF levels according to cell type. Cell type
n
Ang-1 (ng/mL)
Total Squamous cell carcinoma Adenocarcinoma Large cell carcinoma BAC Others
101 52 37 4 6 2
32.1 33.7 30.1 33.9 31.5 24.3
± ± ± ± ± ±
9.9 10.9 8.4 9.0 9.7 71.6
Ang-2 (pg/mL) 1949.2 2219.9 1673.5 1573.6 1592.8 1830.3
± ± ± ± ± ±
1099.4 1216.5 984.6 629.7 469.1 476.2
VEGF (pg/mL) 565.1 640.3 496.3 614.2 308.7 555.1
± ± ± ± ± ±
406.3 482.4 301.3 251.9 193.2 347.1
The data are presented as the means ± SD. Ang-1: angiopoietin-1; Ang-2: angiopoietin-2; VEGF: vascular endothelial growth factor.
regression analysis, in a stepwise manner. The p values of less than 0.05 were considered to be statistically significant. 3. Results 3.1. Comparison of serum Ang-1, Ang-2, and VEGF levels between lung cancer patients and controls Patients with lung cancer had a lower serum Ang-1 level (32.1 ± 9.9 ng/mL vs. 39.0 ± 10.8 ng/mL, p < 0.0001) and higher serum Ang-2 level (1949.2 ± 1099.4 pg/mL vs. 1498.6 ± 650.0 pg/ mL, p < 0.01) compared with the control group. The serum VEGF level was higher in patients with lung cancer than in the control group (565.1 ± 406.3 pg/mL vs. 404.6 ± 254.8 pg/mL, p < 0.01). 3.2. Evaluation of serum Ang-1, Ang-2, and VEGF levels in lung cancer patients No notable difference was found in the serum Ang-1, Ang-2, or VEGF level depending on the cell type of NSCLC. The serum Ang2 level tended to be higher in squamous cell carcinoma than in adenocarcinoma, without statistical significance (Table 2). The serum Ang-2 level was higher in stage II than in stage I (stage I: 1746.8 ± 923.6 pg/mL vs. stage II: 2330.6 ± 1288.7 pg/mL, p < 0.05), whereas no difference between stages I and II was found in the serum Ang-1 level (stage I: 31.9 ± 10.5 ng/mL vs. stage II: 31.9 ± 7.9 ng/mL) or the VEGF level (stage I: 518.3 ± 375.1 pg/mL vs. stage II: 651.2 ± 472.3 pg/mL). In the correlation analysis, the serum Ang-1 (r = 0.28, p < 0.01) and Ang-2 levels (r = 0.36, p < 0.01) were each correlated with the VEGF level (Fig. 1). The correlation between the Ang-1 and Ang-2 levels was not significant. 3.3. Analysis of disease-specific survival in lung cancer patients according to Ang-1, Ang-2, and VEGF levels For the survival analysis, the serum Ang-1, Ang-2, and VEGF levels were dichotomized using median values. The disease-specific
survival period was obtained in 100 patients with NSCLC. Twentynine patients died during the follow-up period. The patients in the group with higher serum Ang-1 (≥31.2 ng/ mL; n = 50; mean survival time = 85.4 ± 7.0 months) survived longer than those in the lower serum Ang-1 group (<31.2 ng/mL; n = 50; mean survival time = 58.9 ± 7.5 months, p < 0.05; Fig. 2A). In the subgroup analysis, stage I patients in the higher serum Ang-1 group (n = 36, mean survival time = 85.5 ± 4.1 months) survived longer than stage I patients in the lower serum Ang-1 group (n = 36, mean survival time = 65.5 ± 9.3 months, p < 0.05; Fig. 2B). However, for the subgroup of patients with stage II, the survival period did not differ between the serum Ang-1 groups, perhaps because of the small number of patients (p = 0.157; Fig. 2C). In the analysis of serum Ang-2 and VEGF levels, no significant difference in survival was found by analyzing the total patients or those in each stage. Patients with stage I survived longer than those with stage II (76.7 ± 7.5 months vs. 54.1 ± 9.1 months, p < 0.05). Following a univariate analysis to evaluate the prognostic value of clinical and experimental markers on cancer-related survival, we found that performance status and clinical stage were significantly associated with a worse prognosis, as was a higher serum level of Ang-1. In a Cox multivariate analysis, only serum Ang-1 maintained its significance (Table 3). 3.4. Analysis of postoperative recurrence in lung cancer patients according to the Ang-1, Ang-2, and VEGF levels Ninety-eight patients were included in the analysis for recurrence; 2 of the 100 cases in the disease-specific survival analysis were excluded because the exact information about relapse could not be determined. Twenty-six patients suffered recurrence after the operation. Among the 26 cases with postoperative recurrence, 18 patients were in the lower Ang-1 group, and 8 were in the higher Ang-1 group. In the analysis of the relapse-free survival, patients with a higher serum Ang-1 level had significantly longer relapse-free periods than those with a lower serum Ang-1 level (82.9 ± 6.4 months vs. 56.8 ± 8.0 months, p < 0.05; Fig. 2D).
Fig. 1. Correlations of the (A) serum angiopoietin-1 (Ang-1) and (B) angiopoietin-2 (Ang-2) levels with the serum vascular endothelial growth factor (VEGF) level in all lung cancer patients.
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Fig. 2. Kaplan–Meier curves for disease-specific survival in (A) all patients and in (B) stage I patients, in (C) stage II patients, and (D) relapse-free survival in all patients with lung cancer according to serum angiopoietin-1 (Ang-1) levels. The patients were divided into higher and lower Ang-1 groups using the median value of 31.2 ng/mL.
3.5. Immunohistochemical assessment of Ang-1 expression and MVD The overall expression of Ang-1 tended to be low and heterogeneous. However, one consistent finding was that the expression of Ang-1 was inversely correlated with the MVD (rs = 0.61, p < 0.05). Areas with high microvascularity in tumor sections showed low expression of Ang-1, and vice versa (Fig. 3). We were not able to present full survival analysis data because of the small number of immunohistochemistry cases. When MVD and Ang-1 expression scores were divided using median values, patients with higher MVD showed longer disease-specific survival than those with lower MVD in a univariate analysis (p < 0.05), while Ang-1 expression levels failed to show a significant survival difference (p = 0.24). Serum Ang-1 levels tended to be elevated in patients with higher Ang-1 expression scores than those with lower scores (34.1 ± 5.7 ng/mL vs. 30.2 ± 4.0 ng/mL, p = 0.099). 4. Discussion The main finding of our study is that the serum Ang-1 level is a useful parameter for predicting overall survival and recurrence in
patients with resected early stage lung cancer, whereas the Ang-2 and VEGF levels lacked prognostic implications. Ang-1 has dual contrasting functions. In normal or developing tissues, Ang-1 promotes angiogenesis [16,21,34,35], whereas Ang-1 inhibits angiogenesis and tumor growth and metastasis in cancerous tissues [18,36,37]. However, instead of contrasting actions, these dual functions can be considered to have a consistent action with different manifestations, depending on the given conditions, for the maintenance of the normal vasculature. From this perspective, an association in lung cancer patients between better survival and higher Ang-1 levels appears plausible. The depletion of Ang-1 might disrupt the homeostatic control over tumor neoangiogenesis and represent a loss of anti-angiogenic activity, which might be associated with biological tumor aggressiveness. Serum Ang-1 levels have not been evaluated often in cancers. Our results concur to an extent with a previous report that measured serum angiopoietin levels in thyroid cancer patients [26]. In that report, thyroid cancer patients had lower serum Ang-1 and higher serum Ang-2 levels compared with controls, although the prognostic significance was not evaluated. However, an earlier study found variable serum Ang-1 levels according to different types of cancers [25].
Table 3 Univariate and multivariate analyses for disease-specific survival in all lung cancer patients. Variable
Univariate analysis (p value)
Age (years) Gender (male/female) Smoking (smoker/never smoked) Histology (cell type) ECOG Performance status (0/≥1) Stage (I/II) Serum Ang-1 levels (<31.2/≥31.2 ng/mL)
0.123 0.280 0.144 0.280 0.020 0.020 0.013
Multivariate analysis Hazard ratio
CI: confidence interval; ECOG: Eastern Cooperative Oncology Group.
2.53 2.05 0.30
95% CI
1.129–5.686 0.897–4.672 0.153–1.008
p value
0.062 0.078 0.039
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Fig. 3. Immunohistochemical staining of angiopoietin-1 (Ang-1) and CD34 in non-small cell lung cancer, showing their inverse relationship (400×). (A) High microvascularity (high CD34 expression) and (B) low Ang-1 expression are concomitantly observed in the same portion of a squamous cell carcinoma. Similarly, (C) low microvascularity (low CD34 expression) is accompanied by (D) high Ang-1 expression in the same portion of an adenocarcinoma.
In tissue studies, Ang-1 is upregulated in various malignancies, with a few exceptions [22,29,38]. In lung cancer tissues, the expression pattern of Ang-1 is variable and its clinical significance is controversial [19,22,24,39]. The overall contradictory findings regarding Ang-1 expression may not be explained adequately by the differences in laboratory methodologies and study populations. One more possible explanation is provided by reports that have consistently shown extremely variable and heterogeneous expression of Ang-1, even in the same tissue [24,39]. The heterogeneity of Ang1 expression may underlie the variable results of tissue expression studies, because small sample pieces from a tissue may not represent the entire tissue. Although we evaluated tissue expression of Ang-1 in only a small number of cases, its expression was also found to be low and heterogeneous. Therefore, our tissue results should be limited to only the inverse relationship between tumor neovascularization and the Ang-1 expression level, which may correspond to the serum Ang-1 results as well as to the pathophysiological background. In a previous study assessing the clinical implications of the serum Ang-2 level in lung cancer at all stages, we found that Ang2 levels were elevated in the more advanced stages; the results implied that Ang-2 was a potential prognosticator with a significant, but not independent, impact on survival [28]. In the present study, although Ang-2 maintained its stage-discriminating power in the early stages, the prognostic significance of Ang-2 did not extend to the early stages of lung cancer. Nevertheless, the prognostic implications of Ang-2 should not be nullified solely by this result. Given that Ang-2 reflected the stage progression, studies involving a greater number of lung cancer cases in each stage may show Ang-2 to useful, along with other markers, in predicting prognosis. Many studies have evaluated the prognostic significance of serum VEGF in lung cancer, and most have shown positive results, but not consistently [8]. Serum VEGF has rarely been evaluated
specifically in resected early stage lung cancer. We found that serum VEGF was not a significant prognosticator in that group. The results of the correlation analysis, although statistically weak, correspond to the pathophysiological concept that Ang-1 and Ang-2 interact closely with VEGF [10,14]. We acknowledge several limitations of our study. First, the patient population was not distributed evenly, especially with regard to cancer stage. This shortcoming and the overall small numbers of patients prevented a detailed subgroup analysis, which requires further investigation of more cases in the future. Also, some of the eligible patients (mostly stage II patients) were excluded during the study period for various reasons such as missed sampling, postoperative adjuvant therapy, and incomplete resection. Second, correlation analysis of the serum data with tissue expression is important to validate our results. However, our immunohistochemical data are very limited, and further tissue evaluation with more cases should be undertaken in the future. In conclusion, our analysis of markers of tumor angiogenesis indicated that serum Ang-1 may be a useful parameter for predicting survival and recurrence after the resection of early stage NSCLC. Further studies are necessary to clarify its clinical validity and prognostic implications.
Conflict of interest The authors declare that there is no conflict of interests.
Acknowledgement This work was supported by 2002 grant from Department of Medical Sciences, The Graduate School, Ajou University.
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