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1 60 Available online at Serum Angiopoietin-2 as a Clinical Marker for Lung Cancer in Patients with Solitary Pulmonary Nodules Chunhua Xu 1,2, Wei Wang 1,2, Yuchao Wang 1,2, Xiuwei Zhang 3, Jun Yan 4, and Like Yu 1,2 1 Endoscopic Center of Nanjing Chest Hospital, Nanjing, Jiangsu, 2 Clinical Center of Nanjing Respiratory Diseases and Imaging, Nanjing, Jiangsu, 3 Department of Respiratory Medicine, Nanjing Jiangning Hospital, Nanjing, Jiangsu, and 4 MOE Key Laboratory, Model Animal Research Center, Nanjing University, Nanjing, Jiangsu, China Abstract. To evaluate the clinical significance of angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) in lung cancer patients with solitary pulmonary nodule (SPN). The study enrolled 128 patients with malignant SPN, 60 patients with benign SPN, along with 40 healthy volunteers. Serum concentrations of Ang-2 and VEGF were measured by ELISA. Serum Ang-2 and VEGF levels in patients with malignant SPN were significantly higher than those in patients with benign SPN (p<0.01), and those in healthy volunteers (p<0.05). With a cutoff of pg/ml, the sensitivity and specificity of Ang-2 in differentiating between patients with malignant SPN and patients with benign SPN, and between patients with malignant SPN and healthy volunteers was 69.5 and 92.5%, and 80.5 and 97.5%, respectively. Additionally, higher levels of Ang-2 were associated with higher tumor stage (p<0.05). The levels of Ang-2 correlated with the VEGF level (p<0.01). In conclusion, measurement of serum Ang-2 might be a useful diagnostic marker for SPN patients. Keywords: Angiopoietin-2, solitary pulmonary nodule, diagnosis, biomarker. Introduction Solitary pulmonary nodule (SPN) is a round or oval opacity smaller than 3 cm in diameter that is completely surrounded by pulmonary parenchyma and is not associated with lymphadenopathy, atelectasis, or pneumonia [1,2]. Most SPN are detected incidentally on chests X-rays or computerized tomography (CT) scans that are usually performed for some other purpose [3]. SPN can be divided into benign and malignant. The early differentiation of malignant from benign SPN is extremely critical because it represents a different approach regarding treatment and prediction of prognosis [4,5]. Some SPN are indicated pathologically in the early stages of lung cancers [6,7]. Identification of malignant nodules is crucial because they represent a localized and potentially curable form of lung cancer. A few plasma biomarkers have been used to screen and diagnose cancers [8-10]. Currently, there is no single convenient and reliable biomarker proven effective for the diagnosis of lung cancer [11]. Address correspondence to Like Yu, Endoscopic Center of Nanjing Chest Hospital, 215 Guangzhou Road, Nanjing, , China; phone/fax: ; e mail: lkyu54@126.com. Angiopoietins (Ang), novel endothelial factors, were found to be ligands for the endothelium-specific tyrosin kinase receptor Tie-2 [12]. Ang included Ang-1, Ang-2, Ang-3 and Ang-4, the best characterized were Ang-1 and Ang-2. Ang-2 was soluble 70-ku factors, which consist of an amino-terminal coiled-coil domain and a carboxy-terminal fibrinogen-like domain. Ang-2 is upregulated in various cancers, including lung cancer [13,14], and its expression is co-localized and correlated with that of VEGF [15]. Serum Ang-2 has also been reported to be increased in breast cancer [16], prostate cancer [16], thyroid cancer [17] and acute myeloid leukemia [18]. However, the serum levels of Ang-2 have not been evaluated in SPN. We measured the serum levels of Ang-2 and VEGF in SPN patients and assessed their interrelationship and clinical significance. Materials and Methods Patients. The study included 188 patients with SPN who were recruited from the Nanjing Chest Hospital from January 2009 to December The patients consisted of 128 individuals who had malignant SPN and 60 with benign SPN. Forty healthy volunteers without SPN were also recruited as controls. Tumors were /16/ by the Association of Clinical Scientists, Inc.

2 Table 1. The characteristics of patients with malignant SPN, patients with benign SPN, and healthy volunteers. Variables Malignant SPN Benign SPN Healthy volunteers p value Subject, NO Age, year 54.3± ± ±11.7 >0.05 Male/Female 80/48 35/25 18/12 >0.05 Malignant SPN ADC 72 SCC 48 SCLC 8 Benign SPN Tuberculosis 30 Granuloma 10 Inflammation 20 ADC, adenocarcinoma; SCC, squamous cell carcinoma; SCLC, small cell lung carcinoma. Table 2. Serum Ang-2 and VEGF levels in the three groups. Group Malignant SPN Benign SPN Healthy volunteers p value (n=124) (n=60) (n=40) Ang-2(pg/mL) ± ± ± * VEGF (pg/ml) 512.9± ± ± * *Significant difference. Angiopoietin-2 and Solitary Pulmonary Nodules 61 Table 3. Relationship between serum Ang-2 and VEGF levels and clinicopathological characteristics in malignant SPN. Characteristics All cases Ang-2 (pg/ml) p VEGF (pg/ml) p Age (years) ± ±124.4 > ± ±171.6 Gender Male ± ±169.2 Female ± ±116.7 Smoking condition Non-smoker ± ±171.4 Smoker ± ±117.9 Histology ADC ± ±172.6 SCC ± ±120.8 SCLC ± ±156.7 Differentiation Well-moderate ± ±166.0 Poor ± ±104.8 TNM stage 0.033* I ± ±164.2 II ± ±132.1 ADC, adenocarcinoma; SCC, squamous cell carcinoma; SCLC, small cell lung carcinoma. *Significant difference.

3 62 Figure 1. Levels of Ang-2 and VEGF in SPN. Among 128 malignant SPN patients, (A) the serum levels of Ang-2 were ( ±599.1) pg/ml, which were significantly higher than those of benign SPN group (1491.7±288.6) pg/ml and healthy volunteers group (1441.2±325.7) pg/ml (p<0.01), (B) the serum levels of VEGF were (512.9± 133.2) pg/ml, which were significantly higher than those of benign SPN group (421.9±121.7) pg/ml and healthy volunteers group (406.1±124.2) pg/ml (p<0.05). radiation). Samples were centrifuged at 1500 g for 10 min at -4 C. The supernatant was stored at -80 C. Enzyme-linked immunosorbent assays were used to measure the amounts of Ang-2 and VEGF (Quantikine; R&D Systems, Minneapolis, MN, USA). All assays were run in duplicate, with dilutions as appropriate, and the technicians were blinded to clinical data. Figure 2. Correlation between serum Ang-2 and VEGF levels in patients with SPN. There is a significant correlation between Ang-2 and VEGF levels in SPN patients (r=0.441, p<0.001). staged pathologically according to the Union for International Cancer Control (UICC-7) staging system for lung cancer [19]. Histological type was determined according to the classification by the World Health Organization. The demographic and clinical variables of the cases and controls are shown in Table 1. All patients were selected based on presence of SPN on chest CT scan. Final diagnoses were confirmed with histopathologic examinations of specimens obtained by CT-guided transthoracic needle biopsy, transbronchial biopsy, videotape-assisted thoracoscopic surgery, or surgical resection. The study protocol was approved by the ethics committee of Nanjing Chest Hospital. All patients provided written informed consent before enrollment. Sample collection and determination of Ang-2 and VEGF levels. Serum samples from each individual were obtained at the time of diagnosis, before any therapeutic measures were started (surgery, chemotherapy, or Statistical analysis. Statistical software (SPSS for Windows, version 18; SPSS; Chicago, IL) was used for the analysis. All values are given as means± standard deviation except for the survival period in which the mean± standard error was used. The values did not fit a standard distribution, so nonparametric analysis was performed. The Mann-Whitney U test was used to compare the patients and control groups, and the Kruskal-Wallis test was used to compare all groups. Spearman correlation of rank coefficient was used to analyze correlations between parameters. The cutoff value for the serum concentrations of the tested parameters was calculated using a receiver operating characteristic (ROC) curve. The p values of less than 0.05 were considered to be statistically significant. Results Serum levels of Ang-2 and VEGF were elevated in the SPN group. Patients with malignant SPN had higher serum Ang-2 levels than the benign SPN group ( ±599.1 pg/ml vs ±288.6pg/ ml, p<0.01), and the control group ( ±599.1 pg/ml vs 441.2±325.7pg/mL, p<0.01 Figure 1A). Serum VEGF levels were also higher in patients with malignant SPN than in the benign SPN group and the control group (p<0.05, Figure 1B and Table 2). Correlation between serum Ang-2 and VEGF levels. In all patients with SPN, serum Ang-2 significantly correlates with VEGF (r=0.367, p<0.01; Figure 2). Further analysis revealed a significant correlation between serum Ang-2 and VEGF in the malignant SPN group (r=0.486, p<0.01), but not in the benign SPN group.

4 Relationship between serum Ang-2 and VEGF levels and clinicopathological characteristics in malignant SPN. The relationships between Ang-2 and VEGF levels and clinicopathological characteristics of malignant SPN patients are shown in Table 3. The levels of Ang-2 significantly correlate with tumor stage (p=0.033). Conversely, serum VEGF levels did not increase with the stage, although serum VEGF levels in malignant SPN patients with stage II tended to be higher than in those with I stage (p=0.056). The serum Ang-2 and VEGF levels did not differ significantly with age (p=0.981 and 0.206), gender (p=0.932 and 0.766), smoking history (p=0.556 and 0.406), tumor grade (p=0.688 and 0.983) or histology (p=0.943 and 0.133). Diagnostic performance of Ang-2 in malignant SPN. To assess the performance of Ang-2 as a serum marker, ROC curves were used to calculate the sensitivity of this marker in separating malignant SPN patients from healthy volunteers or patients with benign SPN. As shown in Figure 3, an area under the curve (AUC) value for Ang-2 reached and 0.692, respectively. With a cutoff of pg/ml, the sensitivity and specificity of Ang-2 in differentiating between patients with malignant SPN and patients with healthy volunteers, and between patients with malignant SPN and benign SPN was, 80.5 and 97.5%, and 69.5 and 92.5%, respectively. These results indicated that serum Ang-2 was a valuable biomarker for SPN diagnosis (Table 4). Angiopoietin-2 and Solitary Pulmonary Nodules 63 Discussion Figure 3. ROC analysis of Ang-2 for differentiation of patients with malignant SPN from healthy volunteers (A) and from patients with benign SPN (B). The analysis resulted in an AUC of (patients with malignant SPN vs. healthy volunteers) and (patients with malignant SPN vs. patients with benign SPN), respectively. Differentiating malignant SPN from benign SPN has always been a challenge, as most patients are usually asymptomatic. Early detection and diagnosis of SPN are crucial for effective treatment. So far, chest X-ray is usually performed to identify pulmonary nodules as a routine screening test. CT scans and positron emission tomography (PET) scans are both more sensitive and specific than chest X-ray, and are thus employed for further imaging when pulmonary nodules are seen on chest X-rays [20]. However, imaging characteristics cannot distinguish malignant SPN from benign SPN in all patients. So far, a tissue biopsy is the gold standard diagnostic test that can confirm whether the nodules are cancerous or not [5]. However, due to its high cost and invasive nature, most patients are reluctant to undergo tissue biopsy. Therefore, searching for a minimally invasive test to differentiate malignant from benign SPN is very important. Ang plays an important role in cancer growth, maintenance, and metastasis. Ang-1 and 2 act as ligands for Tie2, which is a tyrosine kinase receptor specifically expressed on endothelial cells [21]. Ang-2 antagonizes the stabilizing action of Ang-1 by binding to Tie2 competitively, which destabilizes vessels. It also promotes tumor angiogenesis by priming the vasculature and potentiating the effects of cytokines at the front of active neovascularization [22]. Recent studies have shown that the expression pattern of Ang-2 is strongly associated with that of VEGF in the process of tumor angiogenesis, VEGF and Ang-2 seemed to play complementary and coordinated roles in the development of new blood vessels [23]. Our study aimed to evaluate the diagnostic and prognostic utility of serum Ang-2 levels in SPN patients. We found that serum Ang-2 levels were

5 64 Table 4. ROC curves for serumang-2 to distinguish patients with malignant SPN and healthy volunteers, and patients with malignant SPN and benign SPN. Group Cutoff value Sensitivity Specificity AUC p value 95% CI vs.healthy volunteers pg/mL 80.5% 97.5% vs. benign SPN pg/mL 69.5% 92.5% AUC, Area under the curve; CI, Confidence interval. significantly increased in malignant SPN patients compared with benign SPN and healthy volunteers, making them potential adjunctive tools for diagnosis of SPN. Furthermore, with a cutoff value of pg/ml, serum Ang-2 showed excellent diagnostic performance. Importantly, the serum Ang-2 levels did not differ significantly with age, gender and histology but significantly correlated with stage. This data further reinforced the diagnostic utility of Ang-2 levels in SPN patients. Our study showed a statistical correlation between Ang-2 and VEGF levels in malignant SPN patients. The levels of Ang-2 were related to tumor stage and vascular involvement. Ang-2 overexpression by newly formed tumor blood vessels may lead to vessel destabilization, which could drive the release of VEGF. In conclusion, we have demonstrated the presence of high serum Ang-2 levels in patients with malignant SPN. Ang-2 level can be used as a new diagnostic tool for SPN. Further studies are necessary to clarify the exact role of Ang-2 in malignant SPN growth. References 1. Ost D, Fein AM, Feinsilver SH. Clinical practice. The solitary pulmonary nodule. N Engl J Med 2003; 348: Sim YT, Poon FW. Imaging of solitary pulmonary nodule-a clinical review. Quant Imaging Med Surg 2013; 3: Brandman S, Ko JP. Pulmonary nodule detection, characterization, and management with multidetector computed tomography. J Thorac Imaging 2011; 26: Gould MK, Fletcher J, Iannettoni MD, et al. Evaluation of patients with pulmonary nodules: when is it lung cancer?: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 2007; 132: 108S-130S. 5. 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