Urologic Oncology: Seminars and Original Investigations 34 (2016) 5.e1–5.e9

Original article

Decreased expression of CTR2 predicts poor prognosis of patients with clear cell renal cell carcinoma Yu Xia, M.D.a,1, Li Liu, M.D., Ph.D.a,1, Qilai Long, M.D., Ph.D.a, Qi Bai, M.D.a, Jiajun Wang, M.D.a, Jiejie Xu, M.D., Ph.D.b,*, Jianming Guo, M.D., Ph.D.a,* a

b

Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China Received 26 May 2015; received in revised form 28 July 2015; accepted 24 August 2015

Abstract Purpose: Clear cell renal cell carcinoma (ccRCC) is well known for its hypervascularity due to the Von Hippel-Lindau/hypoxia-inducible factor dysregulation. Recent findings suggested that copper transporter 2 (CTR2) is also associated with angiogenesis through copper's modulation of the hypoxia-inducible factor pathway. Our group thus explored the prognostic role of CTR2 in patients with ccRCC. Materials and methods: A total of 331 patients with ccRCC who underwent nephrectomy were enrolled between February 2005 and June 2007 at a single institution. The median follow-up was 98.97 months (2.63–120.47 mo). Patients' samples were collected and stained for CTR2 by immunohistochemistry. The staining intensity was analyzed quantitatively and defined as high/low expression using X-tile software. Stage, Size, Grade, and Necrosis score and University of California Los Angeles Integrated Staging System score were applied to stratify patients' risks. Survival analyses were performed through the Kaplan-Meier method and Cox proportional hazard model. After integrating tumoral CTR2 expression with other clinical parameters, 2 nomograms were generated for overall survival (OS) and disease-free survival (DFS) prediction. Results: CTR2 expression in ccRCC was decreased compared with that in the peritumoral tissue (Po 0.001) and negatively correlated with many other clinical parameters. In survival analyses using the Kaplan-Meier method, low tumoral CTR2 expression displayed a dismal prognostic effect both in OS and DFS prediction (Po 0.001). Multivariate analyses also revealed the same result after adjusted with other clinical parameters (Po0.001). Stratifying patients into 3 risk levels using the Stage, Size, Grade, and Necrosis score and University of California Los Angeles Integrated Staging System score, decreased CTR2 expression associated with shorter OS and DFS in the low- and intermediate-risk groups. Moreover, the generated nomogram integrating tumoral CTR2 expression performed better in predicting patients' OS than using TNM stages alone (c-index ¼ 0.799; 95% CI: 0.752–0.846 vs. 0.691; 95% CI: 0.637–0.745). Conclusions: CTR2 is a novel prognostic marker for patients with ccRCC both in OS and DFS prediction, and could be incorporated with other clinical parameters for better patient risk stratification. r 2016 Elsevier Inc. All rights reserved.

Keywords: Clear cell renal cell carcinoma; Copper transporter 2; Overall survival; Disease-free survival; Prognostic factor

1. Introduction In the United States in 2015, there were estimated to be 61,560 new cases and 14,080 cancer-related deaths owing to kidney and renal pelvis cancer alone [1]. Renal cell carcinoma (RCC) is well known for its multiresistance to conventional cancer therapies. Moreover, patients with 1

These authors contributed equally to this work. Corresponding authors. Tel./fax: þ86-215-423-7332. E-mail addresses: [email protected] (J. Xu), [email protected] (J. Guo). *

http://dx.doi.org/10.1016/j.urolonc.2015.08.013 1078-1439/r 2016 Elsevier Inc. All rights reserved.

localized diseases often experience recurrences after curative surgeries [2]. Current clinical parameters such as Fuhrman grades and TNM stages could be used to evaluate the prognosis of patients with RCC [3,4], although these parameters are not entirely reliable [5]. Although it is well elucidated that clear cell RCC (ccRCC) formation is based on Von Hippel-Lindau mutation leading to an activation of hypoxia-inducible factor (HIF) signaling, this cannot perfectly explain why this tumor displays different biological behaviors [2]. Therefore, it is necessary to search for better biomarkers for more accurate RCC prognosis prediction.

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Y. Xia et al. / Urologic Oncology: Seminars and Original Investigations 34 (2016) 5.e1–5.e9

Copper transporter 2 (CTR2) is the second member of the mammalian SLC31 copper transporter family, sharing similar structures with its homologous companion CTR1, which has been well defined as a high-affinity Cuþ importer [6]. However, little is known about CTR2 functionalities. Some experiments revealed that CTR2 serves as a lowaffinity Cuþ importer, a lysosomal Cuþ exporter, or as a regulator of cellular macropinocytosis [7–9]. This molecule was recently identified to be an important assistant for the degradation of CTR1, and thereby could indirectly lower the copper concentration in target cells [10]. Copper has been known for its pro-angiogenesis abilities for more than 2 decades [11]. Clinical trials with Cu chelation therapy have generated promising data for various cancer types [12]. Accumulating evidence supports the hypothesis that copper stimulates angiogenesis through regulation of the HIF-1α/hypoxia response element binding process and thus could activate downstream signaling molecules, especially the vascular endothelial growth factor [13,14]. Copper and its transporters might also interact with the HIF-2α [15,16], which was recently considered as a more important stimulator for RCC proliferation [17]. As copper and its transporters might interact with the HIF pathway, which is crucial for ccRCC formation [2], we wondered whether these transporters would associate with the outcome of patients with ccRCC. Moreover, a recent study highlighted CTR2, in company with CD133, as a putative stem cell marker in ccRCC [18]. Thus, by immunohistochemistry, we evaluated the relationship between CTR2 expression and the survival of patients with ccRCC. To predict paitents' overall survival (OS) and disease-free survival (DFS), 2 nomograms integrating tumoral CTR2 expression with other clinical parameters were formed.

2. Materials and methods 2.1. Patients Between February 2005 and June 2007, 331 patients with ccRCC were enrolled from the Department of Urology, Zhongshan Hospital, Fudan University. The hospital's ethics committee approved this study. Informed consent was obtained from each patient. Patients were followed up every 3 months and the last follow-up was on January 30, 2015, for all available patients. Based on imaging tests or histopathology information, metastasis or recurrences were defined. The inclusion criteria were as follows: no history of other malignant tumors, no history of anticancer therapy, pathologically proven ccRCC, and patients after radical or partial nephrectomy. Patients with pathologically confirmed mixed type of primary renal cancer, tumors necrosis area 480%, or patients who died within the first month after surgery were excluded. Data collected included age, gender, Eastern Cooperative Oncology Group Performance Status

(ECOG PS), tumor size, TNM stage, Fuhrman grade, and presence of tumor necrosis. Tumor histologic type, differentiation, and stage were reassessed by 2 independent urology pathologists and 1 urologist according to the 2004 WHO criteria and the American Joint Committee on Cancer 2010 TNM classification [3,4]. DFS is defined as the time between curative surgery and recurrence or metastasis. OS is defined as the interval from date of curative surgery until death from any cause. Patients' risk stratifications were performed using the Stage, Size, Grade, and Necrosis (SSIGN) score, SSIGN (localized) score, and the University of California Los Angeles Integrated Staging System (UISS) score [19–21]. 2.2. Immunohistochemistry and evaluation Immunohistochemical staining was performed on tissue microarray as previously described [22]. In tissue microarray construction, representative areas were chosen away from necrosis and hemorrhage. Basically, peritumoral tissue was normal renal cortex collected 5 cm away from tumor capsule. For those kidneys with larger tumor size, peritumoral specimens were taken furthest away from visible lesions. As for samples limitations, the number of peritumoral samples was less than the tumoral one (168 vs. 303). In all, 147 specimens got paired in available tissue blocks. For immunohistochemistry, Rabbit polyclonal anti-CTR2 primary antibody (1:100 dilution, ab58777, Abcam, Cambridge, MA) and EnVision Detection System (Dako) were applied in this procedure. An Olympus CDD (charge coupled device) camera and a Nikon eclipse Ti-s microscope were then used to record the staining results. Samples were scanned at high-power magnification (200) and recorded by NIS-Elements F3.2 software. We took 3 independent shots with the strongest CTR2 staining in each specimen for analysis. For peritumoral assessment, only renal cortex tissue was taken into account. All data were recorded under the same circumstance. The staining intensity was analyzed by Image-Pro Plus version 6.0 software (Media Cybernetics Inc., Bethesda, MD) and presented in the form of integrated optical density (IOD). For every specimen, the mean IOD of those 3 strongest staining photos was regarded as the CTR2 expression intensity. These slides were evaluated by 2 observers unaware of the patients' clinical features and outcomes. 2.3. Statistical analyses Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA) and SPSS 19.0 (SPSS Inc., Chicago, IL). Po 0.05 was regarded as statistically significant. As the IOD scores of tumor tissue did not accord with normal distribution, we used the Wilcoxon matched-pair signed-rank test to evaluate the CTR2 expression differences among these 147 paired

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specimens. The IOD scores cut-points for determining high/ low expression in tumoral and peritumoral staining were evaluated separately by X-tile software through the minimum P-value method [23]. The connections between CTR2 high/low expression and patients' clinical features were analyzed using the χ2 test, Fisher's exact method, or Cochran-Mantel-Haenszel χ2 test. Kaplan-Meier analyses and log-rank tests were applied for DFS and OS evaluation. Cox univariate analyses were performed and those parameters with statistical significance were brought into a multivariate Cox proportional hazards model. R software version 3.0.2 with the “rms” package (R Foundation for Statistical Computing, Vienna, Austria) was applied for nomogram formation and calibration. The Harrell's concordance index (c-index) was formed to measure the nomogram prognostic accuracy.

3. Results 3.1. Patient characteristics Table S1 presents the major characteristics of the cohort. The median follow-up was 98.97 months (2.63–120.47 mo) and the median age was 55 years (23–86 y). Regional lymph node metastasis was identified in only 2 people in this cohort and 16 patients developed distant metastatic disease before surgery. For patients' risk stratifications, the SSIGN (localized) and UISS scores were applied for DFS evaluations. In OS analyses, owing to limited number of patients and the complexity of SSIGN score, we finally simplified this system into 3 risk groups (low: 0–2, intermediate: 3–4,

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and high: 5–10). For the same reasons, UISS scores 3 to 6 were also combined as the high-risk group in OS assessment. Related Kaplan-Meier analyses for OS and DFS are shown in Fig. S1. 3.2. CTR2 expression in ccRCC The expressions of CTR2 in tumoral and peritumoral tissue were recorded by immunohistochemistry as low expression or high expression (Fig. 1A–D). Positive CTR2 staining was detected on the membrane or in the cytoplasm of both the tumoral and peritumoral tissue cells. CTR2 selectively presented in the nephric tubule in peritumoral tissue (Fig. 1D). By analyzing the IOD of 147 paired specimens, we found that the CTR2 expression intensities between ccRCC and peritumoral tissue were predominantly different (Po 0.001). The IOD medians were 16,523.6 (Q1 ¼ 12,316.5; Q3 ¼ 24,774.3; range: 1,259.165,505.8) in tumor tissue and 24,065.3 (Q1 ¼ 17,186.8; Q3 ¼ 31,006.5; range: 670.871,252.7) in nontumor tissue (Fig. 1E). The mean and SD were 18,864.5 ⫾ 10,165.2 for tumor tissue and 24,568.6 ⫾ 11,136.9 for nontumor tissue, respectively. 3.3. Associations between clinicopathological features and CTR2 expression The associations between patient's clinicopathological features and CTR2 expression are described in Table 1. In 303 ccRCC specimens, CTR2 expression strongly correlated with age (P ¼ 0.033), ECOG PS (P ¼ 0.011), T classification (P ¼ 0.008), distant metastasis (P ¼ 0.025), TNM stage (P ¼ 0.001), and tumor size (P ¼ 0.004) and

Fig. 1. Immunohistochemical analyses of CTR2 expression in human samples. Representative CTR2 immunohistochemical staining in clear cell renal cell carcinoma (200 magnification). (A) Low CTR2 expression in tumor tissue; (B) high CTR2 expression in tumor tissue; (C) low CTR2 expression in nontumor tissue; (D) high CTR2 expression in nontumor tissue; and (E) IOD score of CTR2 expression in paired tumor and nontumor tissue. The horizontal line: Q1, Q3, and median. P-value, calculated by Wilcoxon matched-pair signed-rank test, o0.05 was regarded as statistically significant.

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Table 1 Clinical characteristics of patients according to CTR2 expression in tumor tissue and nontumor tissue Characteristics

All patients Age, ya r55 455 Gender Female Male ECOG PS 0 1 2 3 4 Fuhrman nuclear grade 1 2 3 4 Necrosis Absent Present T classification T1 T2 T3 T4 N classification N0 N1 Distant metastasis No Yes TNM stage I II III IV Tumor size, cma r4 44

CTR2 expression in tumor tissue (n ¼ 303)

CTR2 expression in nontumor tissue (n ¼ 168)

High

Low

High

Low

193

110

70

98

P value 0.033b

107 86

47 63

57 136

30 80

0.322b 46 24

57 41

21 49

29 69

0.676b

0.955b

0.011c 151 36 3 2 1

72 27 9 1 1

0.688c 57 9 2 2 0

78 13 5 0 2

0.068c 24 145 23 1

8 82 19 1

168 25

95 15

0.047c 11 55 4 0

10 74 12 2

64 6

86 12

0.866b

0.448b

0.008c 137 11 42 3

58 14 37 1

23 1

15 1

0.032c 56 0 14 0

62 4 31 1

– –

– –

1.000b

0.025b 187 6

100 10

135 9 41 8

54 11 34 11

69 1

94 4

56 0 13 1

60 3 31 4

0.014c

0.004b 53 57

– 0.403b

0.001c

126 67

P value

0.037b 59 11

69 29

P o 0.05 was regarded as statistically significant. a Split at median. b 2 χ Test or Fisher's exact test. c Cochran-Mantel-Haenszel χ2 test.

marginally with Fuhrman nuclear grade (P ¼ 0.068). In 168 nontumor tissue samples, CTR2 expression also correlated with Fuhrman nuclear grade (P ¼ 0.047), T classification (P ¼ 0.032), TNM stage (P ¼ 0.014), and tumor size (P ¼ 0.037). No patient has developed regional lymph node metastasis in the peritumoral set. 3.4. Kaplan-Meier analyses Kaplan-Meier analyses were applied to assess patients' OS and DFS. The results showed that low expression of CTR2 in tumor tissue was associated with dismal outcomes,

both in the OS (Fig. 2A, Po 0.001) and DFS (Fig. 2B, Po 0.001) analyses. In all, 20 patients were excluded owing to regional lymph node metastasis, preoperative distant metastasis, or death from other causes in the DFS analysis, as such patients were not suited for DFS evaluation (Table S5). Additionally, we applied the SSIGN, SSIGN (localized), and UISS score to set patients apart into low-, intermediate-, and high-risk groups and analyzed them separately. The results revealed low tumoral CTR2 expression as a poor prognostic factor in both the low- (OS, Po 0.001; DFS, Po 0.001) and intermediate-risk (OS, P ¼ 0.008; DFS,

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Fig. 2. Kaplan-Meier analyses of overall survival and disease-free survival in ccRCC according to tumoral CTR2 expression. (A) Overall survival according to tumoral CTR2 expression and (B) disease-free survival according to tumoral CTR2 expression. P-value, calculated by log-rank test, o0.05 was regarded as statistically significant.

P ¼ 0.007) groups for the SSIGN and SSIGN (localized) score. As for UISS stratification, low tumoral CTR2 expression also correlated with a shorter survival time in both the low- (OS, P ¼ 0.002; DFS, P ¼ 0.004) and intermediate-risk (OS, Po 0.001; DFS, Po 0.001) groups (Fig. 3). However, in the high-risk groups, no significant prognostic value of tumoral CTR2 expression was found. Corresponding univariate analyses in different risk groups revealed the same result (Tables S3 and S4). In the high-risk group for SSIGN (localized) score, there was no peritumoral specimen for DFS evaluation. 3.5. Univariate and multivariate analyses The univariate analyses highlighted the dismal prognostic value of low tumoral CTR2 expression in both OS (Po 0.001) and DFS (Po 0.001) predictions. Peritumoral CTR2 expression also revealed a similar result (OS, P = 0.007; DFS, P = 0.002) (Table S2). Owing to little prognostic difference between Fuhrman grades 1 and 2, we combined them into a single category as a reference. In multivariate analysis, low expression of CTR2 in ccRCC is associated with a higher risk of death (hazard ratio ¼ 3.362, 95% CI: 2.0495.515, Po 0.001), and correlated with a shorter DFS (hazard ratio ¼ 3.838, 95% CI: 2.2826.455, Po 0.001) (Table 2). These results suggested low tumoral CTR2 expression as an independent poor prognostic marker in ccRCC adjusted with age, ECOG PS, Fuhrman grade, necrosis, T stage, and distant metastasis. 3.6. Nomograms for predicting OS and DFS We set up 2 nomograms for both OS and DFS predictions (Figs. 4A and S2A). Characteristics included were independent factors determined in the univariate analyses. We did not bring tumor size and TNM stage in

because they might be confounding factors for T stage and M stage. In the nomogram, a higher score predicts a worse outcome. Bootstrap validations were performed for calibration (Figs. 4B and C, and S2B and C). For nomogram predicting OS, the Harrell c-index was 0.799 (95% CI: 0.7520.846) compared with that of TNM stage (0.691; 95% CI:, 0.6370.745), indicating a better performance for OS prediction. 4. Discussion RCC is well characterized by its angiogenic behaviors and hypervascular microenvironments owing to the Von Hippel-Lindau/HIF dysregulation [2]. Meanwhile, copper has been validated for many years as an angiogenic stimulator through the up-regulation of HIF pathway [13,14]. Recent research works indicate that CTR2 plays an important role in regulating cellular copper uptake and mobilization [10]. Thus we explored the prognostic role of CTR2 expression in patients with ccRCC. In our study the CTR2 expression was found to be positive both on plasma membrane and in intracellular area, consistent with previous findings [7,9]. More importantly, our findings demonstrated that low expression of CTR2 was an independent poor prognostic factor for patients with ccRCC. The tumoral CTR2 expression negatively correlated with various clinical characteristics and displayed independent prognostic effects on both the OS and DFS predictions in multivariate analyses. After classifying patients into several risk groups using the SSIGN, SSIGN (localized), and UISS score, tumoral CTR2 expression could distinguish the outcome differences in both the lowrisk and intermediate-risk groups. Data revealed no prognostic value of tumoral CTR2 expression in high-risk groups, probably owing to the limited number of patients with advanced ccRCC in our database. Moreover, we generated 2 nomograms by integrating tumoral CTR2 expression with other clinical parameters to predict OS

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Fig. 3. Kaplan-Meier analyses of overall survival and disease-free survival in ccRCC according to tumoral CTR2 expression stratified by the SSIGN/SSIGN (localized) and UISS score. (A)–(C) Patients in the SSIGN low/intermediate/high-risk groups for overall survival; (D)–(F) patients in the UISS low/ intermediate/high-risk groups for overall survival; (G)–(I) patients in the SSIGN (localized) low/intermediate/high-risk groups for disease-free survival; (J)–(L) patients in the UISS low/intermediate/high-risk groups for disease-free survival. SSIGN ¼ Stage, Size, Grade, and Necrosis, UISS ¼ University of California Los Angeles Integrated Staging System, P-value, calculated by log-rank test, o0.05 was regarded as statistically significant.

and DFS for patients with ccRCC, and these nomograms performed better than using TNM stages alone did. It was worth noting that the T stage parameter displayed a reversal between T2 and T3, which might be due to the relatively larger tumor size and burden in T2 compared with T3 in patients with ccRCC. Owing to the limited number of peritumoral specimens compared with tumoral samples (168 vs. 303), we did not further investigate the prognostic value of peritumoral CTR2 expression, though it might have some effects on outcomes of patients with ccRCC (Table S2).

CTR2 is encoded by gene hCTR2 (SLC31A2), located on chromosome 9q31/32 together with its homolog hCTR1 (SLC31A1) [6]. CTR1 has been well identified as a highaffinity Cuþ importer and possesses platinum uptake ability [24,25], whereas CTR2 functionalities have not been confirmed yet. Initially, researchers found that high CTR2 and low CTR1 expressions are significantly associated with resistance to platinum-based chemotherapy and shorter survival in ovarian cancer, indicating a crosstalk between these 2 homologous molecules [26]. A recent work has partially revealed the mechanism by which CTR2 could help the

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Table 2 Multivariate analyses of characteristics associated with overall survival and disease-free survival Characteristics

OS

DFS b

Hazard ratio

95% CI

P value

Hazard ratio

95% CI

P valueb

1.510

0.899–2.537

1.000 1.682 1.489 2.941 3.477

Reference 0.958–2.953 0.634–3.494 0.391–22.099 0.452–26.772

0.119 0.252 – 0.070 0.361 0.295 0.231

1.000 5.682 9.055

Reference 3.109–10.386 1.063–77.140

o0.001 – o0.001 0.044

1.039

0.539–2.004

1.000 3.384 1.877 22.622

Reference 1.660–6.898 1.100–3.204 2.668–191.804

0.909 o0.001 – 0.001 0.021 0.004

Age, ya 455 vs. r55 ECOG PS 0 1 2 3 4

1.464

0.8942.398

1.000 1.820 2.246 3.392 3.621

Reference 1.077–3.075 1.010–4.995 0.457–25.191 0.481–27.268

0.130 0.059 – 0.025 0.047 0.232 0.212

Fuhrman grade 12 3 4

1.000 2.375 8.373

Reference 1.331–4.235 1.815–38.620

0.002 – 0.003 0.006

1.403

0.749–2.628

1.000 2.177 1.602 5.934

Reference 1.130–4.193 0.954–2.690 1.761–19.997

0.291 0.005 – 0.020 0.074 0.004

1.210–4.776

0.012







2.049–5.515

o0.001

3.838

2.282–6.455

o0.001

Necrosis Present vs. absent T stage T1 T2 T3 T4

Distant metastasis Yes vs. no 2.404 Intratumoral CTR2 (n ¼ 303) Low vs. high 3.362

P o 0.05 was regarded as statistically significant. Split at median. b Data obtained from the Cox proportional hazards model. a

degradation of CTR1 into a cleaved form, which would thereby import copper less efficiently and stimulate Cu mobilization, and finally lower the copper concentration in target cells [10]. As copper has an important role in facilitating angiogenesis partially based on the activation of HIF pathway [13,14], which is a dominant driving force for ccRCC initiation, and many evidence suggest that copper and caeruloplasmin levels are up-regulated in ccRCC [27], it is tempting to speculate that in our research, the dismal prognostic effect of low CTR2 expression might have something to do with the CTR1 transporter, copper level, and HIF pathway in ccRCC cells. However, this hypothesis remains to be validated rigorously through further experiments. In the last few years, antiangiogenic therapies targeting the HIF/vascular endothelial growth factor pathway have benefited many patients with metastatic RCC. As copper also uses this pathway for vascularization, a depletion of copper might have some effect on the remission of ccRCC. Indeed, there was a phase II clinical trial using tetrathiomolybdate, an oral copper chelator, in treating patients with metastatic RCC [28]. However, the clinical responses were limited to stable disease for a median of 34 weeks in one-third of the patients. As CTR2 displayed a prognostic role in patients with ccRCC, treatments targeting the CTR1/CTR2 regulation complex might bring a new angle for ccRCC management.

Owing to the complexity of copper homeostasis regulation and its numerous transporter families, studies of their relationships with ccRCC are far from being sufficient. As the number of patients enrolled in this study was relatively small, a larger, multi-centered, prospective study is required for validating these results. Besides, the prognostic significances of other copper transporters such as CTR1 need further investigation. 5. Conclusions Our group has identified that decreased tumoral CTR2 expression was an independent poor prognostic factor in patients with ccRCC. This novel biomarker could be incorporated with other clinical parameters to generate nomograms for better patient risk stratification. 6. Author contributions Y. Xia—acquisition of data, analysis and interpretation of data, statistical analysis, and drafting of the manuscript; L. Liu, Q. Long, Q. Bai, and J. Wang—technical and material support; J. Xu and J. Guo—study concept and design, analysis and interpretation of data, drafting of the

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Fig. 4. Nomogram for predicting 5- and 8-year overall survival in patients with clear cell renal cell carcinoma. (A) Nomogram for predicting clinical outcomes integrating intratumoral CTR2 expression, age, ECOG PS, T stage, distant metastasis, Fuhrman nuclear grade, and necrosis; (B) calibration plot for predicted and observed 5-year overall survival rate; and (C) calibration plot for predicted and observed 8-year overall survival rate. The gray line: ideal model, vertical bars: 95% CI, and ECOG PS ¼ Eastern Cooperative Oncology Group performance status. (Color version of figure is available online.)

manuscript, obtaining funds, and study supervision. All authors read and approved the final manuscript. Acknowledgments This work was supported by Grants from National Basic Research Program of China (2012CB822104), National Natural Science Foundation of China (31100629, 31270863, 31300671, 81372755, 31470794, 81401988, 81402082, 81402085, 81471621, 81472227, 81472376, 31570803 and 81572352), Program for New Century Excellent Talents in University, China (NCET-13-0146), Shanghai Health and Family Planning Commission (14ZR1406300), and Shanghai Rising-Star Program (13QA1400300). All these study sponsors have no roles in the study design and in the collection, analysis, and interpretation of data.

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. urolonc.2015.08.013.

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Decreased expression of CTR2 predicts poor prognosis of patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is well known for its hypervascularity due to the Von Hippel-Lindau/hypoxia-inducible factor dysregulation. Re...
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