Urologic Oncology: Seminars and Original Investigations 33 (2015) 112.e1–112.e8
Original article
Galectin-8 predicts postoperative recurrence of patients with localized T1 clear cell renal cell carcinoma Yidong Liu, Ph.D.a, Le Xu, M.D.b, Yu Zhu, M.D.c, Weijuan Zhang, Ph.D.d, Weisi Liu, Ph.D.a, Haiou Liu, Ph.D.a, Jiejie Xu, M.D., Ph.D.a,* a
Key Laboratory of Glycoconjugate Research, Ministery Of Health, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China b Department of Urology, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai, China c Department of Urology, Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China d Department of Immunology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China Received 16 August 2014; received in revised form 2 November 2014; accepted 3 November 2014
Abstract Background: Galectin-8 (Gal-8), belonging to a family of the “tandem repeat”–type galectins that contain 2 carbohydrate recognition domains, serves to retain cell surface residency and signaling of glycoproteins including cytokine and growth factor receptors, and thereby promoting development and progression of various malignancies. This study aims to evaluate the effect of Gal-8 expression on postoperative recurrence of patients with localized pathologic T1 (pT1) clear cell renal cell carcinoma (ccRCC). Patients and methods: In this retrospective study, we enrolled 244 patients (122 in group A and 122 in group B) with localized pT1 ccRCC undergoing nephrectomy at a single institution. Specimens from patients were collected from January 2003 to December 2008. Median follow-up was 71 months (range: 12–120 mo) in group A and 70 months (range: 12–119 mo) in group B. Overall, 14 patients experienced recurrence in group A (n ¼ 122) and 22 patients had recurrence in group B (n ¼ 122). Gal-8 expression was assessed by immunohistochemistry in clinical specimens. Kaplan-Meier method with log-rank test was performed to compare survival curves. Cox regression models were used to evaluate the prognostic values of variables on recurrence-free survival. Concordance index was calculated to assess prognostic accuracy. Results: In both groups, patients with high expression of Gal-8 were significantly inclined to have high rates of necrosis. High Gal-8 expression indicated early recurrence of patients with localized pT1 ccRCC. Gal-8 expression was determined to be an independent adverse prognostic indicator for recurrence. The accuracy of The Mayo Clinic Stage, Size, Grade, and Necrosis score and University of Los Angeles Integrated Staging System prognostic models was improved when Gal-8 expression was added. Conclusions: Gal-8 expression is a potential independent unfavorable prognostic indicator for postoperative recurrence of patients with localized pT1 ccRCC. r 2015 Elsevier Inc. All rights reserved.
Keywords: Clear cell renal cell carcinoma; Galectin-8; Recurrence-free survival; Prognostic biomarker
1. Introduction This work was supported by Grants from the National Basic Research Program of China (2012CB822104), the National Key Projects for Infectious Diseases of China (2012ZX10002-012), the Government National Natural Science Foundation of China (31100629, 31270863, 81471621, 81472227), the Program for New Century Excellent Talents in University, China (NCET-13-0146), and the 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. * Corresponding author. Tel.: þ86 21 54237332; fax: þ86 21 64437203. E-mail address: [email protected] (J. Xu). http://dx.doi.org/10.1016/j.urolonc.2014.11.001 1078-1439/r 2015 Elsevier Inc. All rights reserved.
Renal cell carcinoma (RCC), which accounts for approximately 3% of all human malignancies, is the most common malignant cancer in the adult kidney. According to the World Health Organization (WHO) classification, clear cell subtype (ccRCC) represents 80% to 90% of all RCCs [1]. At present, owing to the increased application of radiological diagnostic techniques and the interest in regular checkups, the clinical diagnosis of early-stage RCC has
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increased. RCC could be cured surgically if diagnosed at an early stage; however, approximately 30% of patients undergoing nephrectomy for localized RCC will have local recurrence or distant metastasis [2]. Patients who at a higher risk of recurrence or progression need to undergo a closer follow-up. Recent studies revealed that molecular predictors can not only assist in outcome prediction but also have the potential to serve as practical targets for development of therapeutic strategies. Thegalectins (Gals) are a structurally related family of proteins that function as animal lectins with at least one highly conserved carbohydrate recognition domain and an affinity for β-galactosides [3]. They are involved in various biological processes including cell growth, apoptosis, angiogenesis, cell migration, and inflammation [4,5]. Previous studies have indicated that elevated expression of Gal-1 might serve as a potential marker for RCC and is implicated in ccRCC progression via the hypoxia-inducible factors (HIF)/mammalian target of rapamycin (mTOR) signaling axis [6,7]. According to the previous studies, Gal-3 is also highly expressed in ccRCC and involved in tumor progression [8,9]. Similar to Gal-1 and Gal-3, Gal-8 is implicated in modulation of cell matrix and cell-cell interactions [10,11]. Studies have shown that Gal-8 expression is markedly related with certain neoplasms in tissues such as the pancreas, the colon, the skin, and the liver [12–15]. In the prostate and bladder carcinomas, there have been conflicting data documented so far. On one hand, high expression of Gal-8 might give neoplasms some growth or metastasis-related advantages [16,17]. On the other hand, Danguy et al. [12] showed that the expression level of Gal-8 is very low in normal prostatic tissues as well as in benign hyperplasias or adenocarcinomas. Another work reported that decreased Gal-8 is a marker for recurrence in bladder carcinoma [18]. Regarding the expression of Gal-8 in the kidney, no statistically significant differences of staining intensity were observed between RCC and normal tissues [12]. However, the low number of specimens and intertumoral and intratumoral heterogeneity prevent any final conclusion [12,19]. In this study, we analyzed the expression of Gal-8 by immunohistochemistry (IHC) in tumor tissues of patients with localized pathologic T1 (pT1) ccRCC and their associations with clinicopathologic variables and disease recurrence. We further evaluated the prognostic values of Gal-8 expression and attempted to assess the effect of Gal-8 expression on conventional prognostic models.
2. Patients and methods 2.1. Patients Specimens from 393 patients (260 patients in localized pT1 stage) with ccRCC treated with radical or partial
nephrectomy at Zhongshan Hospital, Fudan University (Shanghai, China), were collected in the previous work. Patients were selected based on the following criteria: (a) confirmed postoperative histopathology diagnosis, (b) no comorbidities, (c) complete available follow-up data, (d) no adjuvant anticancer therapy after surgery, and (e) tumor stage classification carried out according to the 2010 American Joint Committee on Cancer TNM classification [20]. Patients who were lost to follow-up, experienced bilateral disease and familial RCC, received preoperative neoadjuvant therapy or postoperative adjuvant therapy or both, or died within the first month after surgery were excluded. According to the aforementioned selection standard and our interest in low-risk group of patients with ccRCC, 244 patients with localized pT1 ccRCC were enrolled in the current retrospective study. Specimens from patients were collected from January 2003 to December 2008. The study was approved by the ethics committee of Fudan University, and each patient provided informed consent on the use of clinical specimens. Patients who received preoperative neoadjuvant were not recruited in the current study. Clinicopathologic information of each patient, including age, sex, tumor size, T stage, Fuhrman grade, necrosis, and Eastern Cooperative Oncology Group performance status (ECOG-PS), was obtained from patients' records. The Mayo Clinic Stage, Size, Grade, and Necrosis score (SSIGN) and University of Los Angeles Integrated Staging System (UISS) scores were used for the patients. Recurrence-free survival (RFS) was the main end point of this study. RFS was calculated from the date of surgery to the date of recurrence or to the date of the most recent follow-up. The 244 patients were randomly assigned into 2 groups: group A was the training group and group B was the validation group. Median follow-up was 71 months (range: 12–120 mo) in group A and 70 months (range: 12–119 mo) in group B. 2.2. Immunohistochemistry Tissue microarrays (TMAs) were performed as previously described [21]. Primary anti–Gal-8 antibody (diluted 1:100; Abcam, Cambridge, MA) was applied for IHC staining. The specificity of this antibody was determined by western blotting and IHC with peptide competition. The immunostaining intensity was evaluated by 2 independent pathologists without the knowledge of clinicopathologic data and clinical outcome of each patient. A semiquantitative IHC score (H-score) ranging from 0 to 300 for each specimen was calculated by multiplying the staining intensities (0—negative, 1—weak staining, 2—moderate staining, and 3—strong staining) by the distribution areas (0%–100%). Gal-8 expression was analyzed as continuous (with H-score) and categorical (by dichotomizing) variables. Patients in each group were dichotomized into lowand high-expression subgroups according to median intensity score of each group.
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2.3. Statistical analysis Statistical analysis was conducted by MedCalc Software (version 11.4.2.0; MedCalc, Mariakerke, Belgium) and Stata 11.0 (StataCorp, College Station, TX). Student t test (continuous variable) and chi-square test (categorical variable) are used to evaluate the association of clinicopathologic variables with Gal-8 expression. Correlation analysis was conducted to evaluate the association between 2 continuous variables. Kruskal-Wallis test was performed when we assessed the association of Fuhrman grade with continuous Gal-8 expression. Survival curves were set by Kaplan-Meier method and compared by log-rank test. Hazard ratios (HRs) and 95% CIs of prognostic factors were evaluated by univariate and multivariate Cox proportional hazard models. The accuracy of the prognostic models was evaluated by Harrell's concordance index (c-index). Furthermore, the Akaike information criterion (AIC) value was calculated to evaluate the discriminatory ability of prognostic models, and smaller AIC values present a better predicting ability. P values of all statistical tests were 2 tailed, and P o 0.05 was considered significant.
3. Results 3.1. Association of Gal-8 expression with clinicopathologic variables in patients with ccRCC To determine the expression pattern and its potential clinical significance in tumor tissues of patients with ccRCC, we detected the expression of Gal-8 by immunohistochemistry analysis in 244 patients with localized pT1 ccRCC treated with radical or partial nephrectomy. As presented in Fig. 1, the positive staining of Gal-8 was mainly located in the cytoplasm of tumor cells, and the staining intensity was variable in different specimens from patients with ccRCC. According to the semiquantitative H-score, the median intensity score was 71 (range: 0–221)
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in group A, by which, the group was dichotomized into high (n ¼ 55) and low (n ¼ 67) Gal-8 expression subgroups. The median intensity score of group B, 76 (range: 0–233), was also applied to dichotomize the group B into highexpression (n ¼ 59) and low-expression (n ¼ 63) subgroups. As summarized in Table 1, there were no significant differences between the patients with high Gal-8 expression and those with low Gal-8 expression regarding age, sex, tumor size, T stage, Fuhrman grade, and ECOG-PS (P 4 0.05). However, patients with high Gal-8 expression were significantly inclined to have histologic necrosis (continuous Gal-8: P ¼ 0.032 in group A and P ¼ 0.003 in group B; dichotomous Gal-8: P ¼ 0.013 in group A and P ¼ 0.037 in group B). 3.2. High expression of Gal-8 is associated with recurrence in patients with localized pT1 ccRCC As recorded, among 14 patients who experienced recurrence in group A (n ¼ 122), 12 patients had local recurrence and 2 patients had distant metastasis (pulmonary metastasis [n ¼ 1] and brain metastasis [n ¼ 1]). However, in group B (n ¼ 122), among 22 patients who experienced recurrence, 18 patients had local recurrence and 4 patients had distant metastasis (pulmonary metastasis [n ¼ 3] and skin metastasis [n ¼ 1]). Kaplan-Meier survival analysis was performed to compare the clinical outcome between Gal-8 high-expression and lowexpression subgroups. As presented in Fig. 2A and B, high expression level of Gal-8 was significantly associated with increased risk of recurrence after surgery (P ¼ 0.008 in group A and P ¼ 0.003 in group B). To determine whether the association of Gal-8 expression with recurrence risk of patients was dependent on T stage, subgroups of T stage were analyzed. Patients with T1aN0M0 could not be stratified by Gal-8 expression (P ¼ 0.148 in group A, Fig. 2C; P ¼ 0.200 in group B, Fig. 2D), whereas patients with T1bN0M0 could be significantly
Fig. 1. Galectin-8 (Gal-8) expression in clear cell renal cell carcinoma (ccRCC) tissues. Representative Gal-8 immunohistochemistry (IHC) images of ccRCC tumor tissues with low expression level (A) and high expression level (B). Arrows indicate positive staining of Gal-8 in each image (original magnification, 200). Scale bar: 50 μm. (Color version of figure is available online.)
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Table 1 Association between Gal-8 expression and patient characteristics Characteristic
Group A (n ¼ 122)
Group B (n ¼ 122)
Continuous Gal-8 (H-score)
Number %
Mean ⫾ SD
Range
Age, y Mean ⫾ SD Range
54.76 ⫾ 11.29 22–80
87.41 ⫾ 50.11
0–221
Sex Female Male
32 90
81.94 ⫾ 47.81 89.36 ⫾ 51.02
0–190 0–221
87.41 ⫾ 50.11
0–221
87.30 ⫾ 46.05 87.55 ⫾ 55.08
9–208 0–221
Dichotomous Gal-8
0.728
Tumor size, cm Mean ⫾ SD 3.53 ⫾ 1.45 Range 0.50–7.00
55.85 ⫾ 12.27 31–80
11 44
21 46
67 55
54.92 45.08
Fuhrman grade 1 2 3 4
30 55 30 7
24.59 87.23 ⫾ 47.13 13–183 45.08 77.60 ⫾ 48.82 0–204 24.59 97.17 ⫾ 42.29 36–208 5.74 123.43 ⫾ 83.28 15–221
Necrosis Absent Present
103 19
84.43 83.23 ⫾ 49.44 0–208 15.57 110.05 ⫾ 48.87 39–221
ECOG-PS 0 Z1
110 12
90.16 9.84
3.38 ⫾ 1.29 1.00–6.00
3.65 ⫾ 1.56 0.50–7.00
Range
33 22
34 33
11 22 18 4
19 33 12 3
41 14
62 5
51 4
59 8
90.70 ⫾ 58.25
0–233
26 96
83.23 ⫾ 49.64 92.72 ⫾ 60.45
7–203 0–233
3.44 ⫾ 1.34 0.80–7.00
90.70 ⫾ 58.25
0–233
74 48
60.66 39.34
96.99 ⫾ 63.29 81.00 ⫾ 48.53
4–233 0–216
32 54 27 9
26.23 88.03 ⫾ 59.85 0–216 44.26 91.06 ⫾ 60.43 0–227 22.13 85.59 ⫾ 51.93 6–227 7.38 113.33 ⫾ 61.57 64–233
101 21
82.79 83.53 ⫾ 54.41 0–227 17.21 125.14 ⫾ 64.98 34–233
110 12
90.16 87.62 ⫾ 58.00 0–233 9.84 118.92 ⫾ 54.97 64–213
13 50
3.23 ⫾ 1.34 0.80–6.50
3.63 ⫾ 1.33 1.50–7.00
38 21
36 27
16 24 14 5
16 30 13 4
44 15
57 6
52 7
58 5
0.974
0.101
0.525
0.651
0.877
0.003
0.578
SD ¼ standard deviation. The bold characters indicate that these P values are considered statistically significant. * P o 0.05 is considered statistically significant.
13 46
0.139
0.013
0.962
54.17 ⫾ 10.14 34–81
0.093
0.209
0.032
0.913 53.95 ⫾ 12.54 24–77
0.464 21.31 78.69
0.401
0.091
Dichotomous Gal-8
P value* High (n ¼ 59) Low (n ¼ 63) P value* 0.454
54.07 ⫾ 11.32 24–81
0.305
0.979
87.48 ⫾ 50.27 0–221 86.75 ⫾ 50.82 20–201
Mean ⫾ SD
0.226
0.633
T stage T1a T1b
Continuous Gal-8 (H-score)
0.241 53.44 ⫾ 9.92 22–73
0.474 26.23 73.77
Patients
P value* High (n ¼ 55) Low (n ¼ 67) P value* Number %
0.037
0.077
0.672
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Patients
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Fig. 2. Recurrence-free survival (RFS) analysis of patients with localized pT1 renal cell carcinoma (ccRCC) based on Gal-8 expression. Kaplan-Meier analysis of RFS in group A (n ¼ 122) (A) and in group B (n ¼ 122) (B); in pT1a subgroup of group A (n ¼ 67) (C) and group B (n ¼ 74) (D); and in pT1b subgroup of group A (n ¼ 55) (E) and group B (n ¼ 48) (F). P value was calculated by log-rank test. (Color version of figure is available online.)
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Gal-8: HR ¼ 1.022, CI: 1.007–1.038, P ¼ 0.005 in group A; HR ¼ 1.020, CI: 1.010–1.030, P o 0.001 in group B; dichotomous Gal-8: HR ¼ 11.761, CI: 2.334–59.268, P ¼ 0.003 in group A; HR ¼ 7.481, CI: 2.447–22.870, P o 0.001 in group B).
stratified by Gal-8 expression (P ¼ 0.004 in group A, Fig. 2E; P o 0.001 in group B, Fig. 2F) in RFS analysis. 3.3. High expression of Gal-8 is an independent indicator of recurrence in patients with localized pT1 ccRCC Univariate Cox analysis was performed to evaluate the prognostic significance of clinicopathologic variables for RFS. As shown in Table 2, tumor size (HR ¼ 1.820, CI: 1.268–2.612, P ¼ 0.001 in group A; HR ¼ 1.564, CI: 1.169–2.093, P ¼ 0.003 in group B), T stage (HR ¼ 7.723, CI: 1.741–34.253, P ¼ 0.008 in group A; HR ¼ 3.138, CI: 1.320–7.463, P ¼ 0.010 in group B), Fuhrman grade (HR ¼ 4.209, CI: 1.418–12.492, P ¼ 0.010 in group A; HR ¼ 2.489, CI: 1.083–5.721, P ¼ 0.033 in group B), necrosis (HR ¼ 3.215, CI: 1.083–9.541, P ¼ 0.036 in group A; HR ¼ 2.488, CI: 1.018–6.081, P ¼ 0.047 in group B), ECOG-PS (HR ¼ 4.664, CI: 1.465– 14.852, P ¼ 0.010 in group A; HR ¼ 4.416, CI: 1.731– 11.268, P ¼ 0.002 in group B), high expression of continuous Gal-8 (HR ¼ 1.015, CI: 1.006–1.024, P ¼ 0.002 in group A; HR ¼ 1.010, CI: 1.004–1.016, P ¼ 0.002 in group B), and high expression of dichotomous Gal-8 (HR ¼ 4.798, CI: 1.347–17.089, P ¼ 0.016 in group A; HR ¼ 4.069, CI: 1.508–10.977, P ¼ 0.006 in group B) were identified as risk factors, which might indicate recurrence in patients with localized pT1 ccRCC. Then multivariate Cox regression analysis involving potential risk factors identified by univariate Cox analysis was performed. Besides tumor size, Fuhrman grade, and ECOG-PS, Gal-8 expression was identified as an independent prognostic factor for recurrence (Table 2, continuous
3.4. Extension of prognostic models with Gal-8 expression for patients with localized pT1 ccRCC To further assess the prognostic power of Gal-8 expression, we constructed prognostic models combining Gal-8 expression with SSIGN score and UISS score and compared the prognostic accuracy of these models by c-index and AIC analysis. As presented in Table 3, in group A, the c-indices were 0.865 and 0.707 when assessed with SSIGN and UISS outcome algorithms and were improved to 0.891 and 0.798 when continuous Gal-8 signature was added and to 0.873 and 0.778 when dichotomous Gal-8 signature was added. Likewise, in group B, the c-indices were improved from 0.743 and 0.671 to 0.833 and 0.787 when continuous Gal-8 signature was added and to 0.791 and 0.754 when dichotomous Gal-8 signature was added. Furthermore, each combined model showed a lower AIC value than corresponding conventional model alone.
4. Discussion To our knowledge, the study is the first to put forward an association between high expression of Gal-8 and an incremental risk of recurrence in patients with localized pT1 ccRCC after surgery. Meanwhile, patients with
Table 2 Univariate and multivariate Cox regression analysis of recurrence-free survival Characteristic
Univariate
Multivariate Continuous Gal-8 (H-score)
Dichotomous Gal-8 (high vs. low) Hazard ratio (95% CI)
Hazard ratio (95% CI)
P*
Hazard ratio (95% CI)
Group A Tumor size (cm) T stage (T1b vs. T1a) Fuhrman grade (3 þ 4 vs. 1 þ 2) Necrosis (present vs. absent) ECOG PS (Z1 vs. 0) Continuous Gal-8 (H-score) Dichotomous Gal-8 (high vs. low)
1.820 7.723 4.209 3.215 4.664 1.015 4.798
(1.268–2.612) (1.741–34.253) (1.418–12.492) (1.083–9.541) (1.465–14.852) (1.006–1.024) (1.347–17.089)
0.001 0.008 0.010 0.036 0.010 0.002 0.016
2.661 1.144 5.734 1.067 4.303 1.022 –
(1.301–5.443) (0.155–8.426) (1.786–18.406) (0.311–3.661) (1.207–15.337) (1.007–1.038)
0.008 0.896 0.004 0.919 0.025 0.005 –
Group B Tumor size, cm T stage (T1b vs. T1a) Fuhrman grade (3 þ 4 vs. 1 þ 2) Necrosis (present vs. absent) ECOG PS (Z1 vs. 0) Continuous Gal-8 (H-score) Dichotomous Gal-8 (high vs. low)
1.564 3.138 2.489 2.488 4.416 1.010 4.069
(1.169–2.093) (1.320–7.463) (1.083–5.721) (1.018–6.081) (1.731–11.268) (1.004 –1.016) (1.508–10.977)
0.003 0.010 0.033 0.047 0.002 0.002 0.006
1.898 1.687 3.734 1.222 3.490 1.020 –
(1.027–3.509) (0.379–7.504) (1.425–9.785) (0.472–3.167) (1.183–10.297) (1.010–1.030)
0.042 0.494 0.008 0.681 0.024 o0.001 –
The bold characters indicate that these P values are considered statistically significant. o 0.05 is considered statistically significant.
*P
P*
1.927 (1.096–3.388) 2.643 (0.383–18.258) 6.130 (1.803–20.838) 1.656 (0.529–5.187) 10.226 (2.270–46.074) – 11.761 (2.334–59.268) 1.863 1.134 2.703 1.354 3.303 – 7.481
(1.011–3.431) (0.247–5.205) (1.126–6.491) (0.522–3.507) (1.186–9.201) (2.447–22.870)
P* 0.024 0.327 0.004 0.389 0.003 – 0.003 0.047 0.872 0.027 0.535 0.023 – o0.001
Y. Liu et al. / Urologic Oncology: Seminars and Original Investigations 33 (2015) 112.e1–112.e8 Table 3 Comparison of the accuracy of the prognostic models and Gal-8 expression for recurrence-free survival Model
Continuous Gal-8 (H-score) Dichotomous Gal-8 (high vs. low) SSIGN SSIGN þ continuous Gal-8 (H-score) SSIGN þ dichotomous Gal-8 (high vs. low) UISS UISS þ continuous Gal-8 (H-score) UISS þ dichotomous Gal-8 (high vs. low)
Group A
Group B
c-Index AIC
c-Index AIC
0.731 0.678 0.865 0.891 0.873
123.963 126.007 114.772 114.316 112.008
0.696 0.677 0.743 0.833 0.791
192.439 192.071 187.004 179.023 175.701
0.707 0.798 0.778
122.690 0.671 118.165 0.787 119.830 0.754
191.903 186.547 185.886
localized pT1 ccRCC could be stratified with Gal-8 expression in T1b stage in RFS analysis. High Gal-8 expression was positively associated with histologic necrosis. Furthermore, the accuracy of SSIGN and UISS prognostic models was improved when Gal-8 expression was added. It has been reported that the expression of Gal-1 and Gal-3 could be regulated by HIF1 [22,23]. In addition, von Hippel– Lindau (VHL) inactivation is a common event in ccRCC. Because Gal-8 exerts functions similar to Gal-1 and Gal-3 in certain tumors and has been validated to play a critical role in ccRCC in this study, we consider that Gal-8 expression might also be modulated by HIF1 and involved in VHL/HIF/HRG (hypoxia responsive gene) signaling axis. As presented in this study, Gal-8 expression could be applied as an independent prognostic factor in patients with early-stage ccRCC, especially in T1b stage. The profound molecular roles of Gal-8 in ccRCC tumorigenesis await further investigation. Besides the well-documented ability to mediate cell adhesion [10], Gal-8 could modulate endothelial cell migration and promote angiogenesis [24]. The proangiogenesis properties of Gal-8 further indicated its potential role in VHL/HIF/vascular endothelial growth factor (VEGF) signaling pathway. Another study reported that Gal-8 could promote cytoskeletal rearrangement via activation of Rho signaling [25]. A recent study concluded that Gal-8 exerted immunosuppressive function not only by inducing apoptosis of activated T cells but also via negatively mediating the critical function of lymphocyte function-associated antigen-1 in the immune system [26]. Numerous researches have determined that cytotoxic T-lymphocyte antigen-4 and programmed death-1 expressed on T cells, functioning as immune checkpoints, also exert immunosuppressive actions. Nowadays, anticytotoxic T-lymphocyte antigen-4 and anti-programmed death-1 monoclonal antibodies have presented striking antitumor activity in large phase I studies [27,28]. So Gal8 expression might also be identified as a potential predictive biomarker on treatment response of these
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immune checkpoints inhibitors, which needs further explorations. Gal-1 might crosslink glycans within glycoproteins on endothelial cell surface to compensate for the absence of cognate ligand and maintain angiogenesis in response to VEGF blockade, which leads to anti-VEGF refractory tumors [29]. Considering that Gal-8 might be involved in VHL/HIF/VEGF signaling pathway and promote angiogenesis, we supposed that Gal-8 could also be involved in tumor resistance to anti-VEGF therapy. In summary, Gal-8 could be exploited as a new therapeutic target in combination with anti-VEGF treatment in ccRCC. Although the clinical significance of Gal-8 expression in patients with localized T1 ccRCC has been concluded and revealed in this study, some limitations remain to be resolved. Considering that all specimens from patients were collected from a single institution, the results need to be further validated or revised in larger data sets and external heterogeneous cohorts. Although duplicate 1.0-mm tissue cores from 2 different areas were used to construct the TMA, the deviation of TMA analysis could not be avoided owing to the heterogeneous natural history of ccRCC. Moreover, the molecular role of Gal-8 in ccRCC remains to be fully elucidated in our future study. 5. Conclusions In summary, our present study identified Gal-8 expression as a potential independent unfavorable prognostic indicator for postoperative recurrence of patients with localized pT1 ccRCC. Combining Gal-8 expression with conventional prognostic models could improve their prognostic accuracy. Further researches might attempt to verify whether Gal-8 could be developed as a new therapeutic target. Acknowledgments The authors thank Ms. Haiying Zeng (Department of Pathology, Zhongshan Hospital, Shanghai Medical College of Fudan University) for technical assistance. References [1] Ljungberg B, Hanbury DC, Kuczyk MA, et al. Renal cell carcinoma guideline. Eur Urol 2007;51:1502–10. [2] Rini BI, Campbell SC, Escudier B. Renal cell carcinoma. Lancet 2009;373:1119–32. [3] Barondes SH, Castronovo V, Cooper DN, et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994;76:597–8. [4] Liu FT, Patterson RJ, Wang JL. Intracellular functions of galectins. Biochim Biophys Acta 2002;1572:263–73. [5] Yang RY, Rabinovich GA, Liu FT. Galectins: structure, function and therapeutic potential. Expert Rev Mol Med 2008;10:e17. [6] Huang CS, Tang SJ, Chung LY, et al. Galectin-1 upregulates CXCR4 to promote tumor progression and poor outcome in kidney cancer. J Am Soc Nephrol 2014;25:1486–95.
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[18] Kramer MW, Waalkes S, Serth J, et al. Decreased galectin-8 is a strong marker for recurrence in urothelial carcinoma of the bladder. Urol Int 2011;87:143–50. [19] Oka N, Takenaka Y, Raz A. Galectins and urological cancer. J Cell Biochem 2004;91:118–24. [20] Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010;17:1471–4. [21] Zhu XD, Zhang JB, Zhuang PY, et al. High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma. J Clin Oncol 2008;26:2707–16. [22] Zhao XY, Chen TT, Xia L, et al. Hypoxia inducible factor-1 mediates expression of galectin-1: the potential role in migration/invasion of colorectal cancer cells. Carcinogenesis 2010;31:1367–75. [23] Zeng Y, Danielson KG, Albert TJ, et al. HIF-1 alpha is a regulator of galectin-3 expression in the intervertebral disc. J Bone Miner Res 2007;22:1851–61. [24] Delgado VM, Nugnes LG, Colombo LL, et al. Modulation of endothelial cell migration and angiogenesis: a novel function for the “tandem-repeat” lectin galectin-8. FASEB J 2011;25:242–54. [25] Diskin S, Chen WS, Cao Z, et al. Galectin-8 promotes cytoskeletal rearrangement in trabecular meshwork cells through activation of Rho signaling. PLoS One 2012;7:e44400. [26] Vicuna L, Pardo E, Curkovic C, et al. Galectin-8 binds to LFA-1, blocks its interaction with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated from lupus patients. Biol Res 2014;46:275–80. [27] Hodi FS, O'Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711–23. [28] Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of antiPD-L1 antibody in patients with advanced cancer. N Engl J Med 2012;366:2455–65. [29] Croci DO, Cerliani JP, Dalotto-Moreno T, et al. Glycosylationdependent lectin-receptor interactions preserve angiogenesis in antiVEGF refractory tumors. Cell 2014;156:744–58.
Original article
Galectin-8 predicts postoperative recurrence of patients with localized T1 clear cell renal cell carcinoma Yidong Liu, Ph.D.a, Le Xu, M.D.b, Yu Zhu, M.D.c, Weijuan Zhang, Ph.D.d, Weisi Liu, Ph.D.a, Haiou Liu, Ph.D.a, Jiejie Xu, M.D., Ph.D.a,* a
Key Laboratory of Glycoconjugate Research, Ministery Of Health, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China b Department of Urology, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai, China c Department of Urology, Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China d Department of Immunology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China Received 16 August 2014; received in revised form 2 November 2014; accepted 3 November 2014
Abstract Background: Galectin-8 (Gal-8), belonging to a family of the “tandem repeat”–type galectins that contain 2 carbohydrate recognition domains, serves to retain cell surface residency and signaling of glycoproteins including cytokine and growth factor receptors, and thereby promoting development and progression of various malignancies. This study aims to evaluate the effect of Gal-8 expression on postoperative recurrence of patients with localized pathologic T1 (pT1) clear cell renal cell carcinoma (ccRCC). Patients and methods: In this retrospective study, we enrolled 244 patients (122 in group A and 122 in group B) with localized pT1 ccRCC undergoing nephrectomy at a single institution. Specimens from patients were collected from January 2003 to December 2008. Median follow-up was 71 months (range: 12–120 mo) in group A and 70 months (range: 12–119 mo) in group B. Overall, 14 patients experienced recurrence in group A (n ¼ 122) and 22 patients had recurrence in group B (n ¼ 122). Gal-8 expression was assessed by immunohistochemistry in clinical specimens. Kaplan-Meier method with log-rank test was performed to compare survival curves. Cox regression models were used to evaluate the prognostic values of variables on recurrence-free survival. Concordance index was calculated to assess prognostic accuracy. Results: In both groups, patients with high expression of Gal-8 were significantly inclined to have high rates of necrosis. High Gal-8 expression indicated early recurrence of patients with localized pT1 ccRCC. Gal-8 expression was determined to be an independent adverse prognostic indicator for recurrence. The accuracy of The Mayo Clinic Stage, Size, Grade, and Necrosis score and University of Los Angeles Integrated Staging System prognostic models was improved when Gal-8 expression was added. Conclusions: Gal-8 expression is a potential independent unfavorable prognostic indicator for postoperative recurrence of patients with localized pT1 ccRCC. r 2015 Elsevier Inc. All rights reserved.
Keywords: Clear cell renal cell carcinoma; Galectin-8; Recurrence-free survival; Prognostic biomarker
1. Introduction This work was supported by Grants from the National Basic Research Program of China (2012CB822104), the National Key Projects for Infectious Diseases of China (2012ZX10002-012), the Government National Natural Science Foundation of China (31100629, 31270863, 81471621, 81472227), the Program for New Century Excellent Talents in University, China (NCET-13-0146), and the 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. * Corresponding author. Tel.: þ86 21 54237332; fax: þ86 21 64437203. E-mail address: [email protected] (J. Xu). http://dx.doi.org/10.1016/j.urolonc.2014.11.001 1078-1439/r 2015 Elsevier Inc. All rights reserved.
Renal cell carcinoma (RCC), which accounts for approximately 3% of all human malignancies, is the most common malignant cancer in the adult kidney. According to the World Health Organization (WHO) classification, clear cell subtype (ccRCC) represents 80% to 90% of all RCCs [1]. At present, owing to the increased application of radiological diagnostic techniques and the interest in regular checkups, the clinical diagnosis of early-stage RCC has
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increased. RCC could be cured surgically if diagnosed at an early stage; however, approximately 30% of patients undergoing nephrectomy for localized RCC will have local recurrence or distant metastasis [2]. Patients who at a higher risk of recurrence or progression need to undergo a closer follow-up. Recent studies revealed that molecular predictors can not only assist in outcome prediction but also have the potential to serve as practical targets for development of therapeutic strategies. Thegalectins (Gals) are a structurally related family of proteins that function as animal lectins with at least one highly conserved carbohydrate recognition domain and an affinity for β-galactosides [3]. They are involved in various biological processes including cell growth, apoptosis, angiogenesis, cell migration, and inflammation [4,5]. Previous studies have indicated that elevated expression of Gal-1 might serve as a potential marker for RCC and is implicated in ccRCC progression via the hypoxia-inducible factors (HIF)/mammalian target of rapamycin (mTOR) signaling axis [6,7]. According to the previous studies, Gal-3 is also highly expressed in ccRCC and involved in tumor progression [8,9]. Similar to Gal-1 and Gal-3, Gal-8 is implicated in modulation of cell matrix and cell-cell interactions [10,11]. Studies have shown that Gal-8 expression is markedly related with certain neoplasms in tissues such as the pancreas, the colon, the skin, and the liver [12–15]. In the prostate and bladder carcinomas, there have been conflicting data documented so far. On one hand, high expression of Gal-8 might give neoplasms some growth or metastasis-related advantages [16,17]. On the other hand, Danguy et al. [12] showed that the expression level of Gal-8 is very low in normal prostatic tissues as well as in benign hyperplasias or adenocarcinomas. Another work reported that decreased Gal-8 is a marker for recurrence in bladder carcinoma [18]. Regarding the expression of Gal-8 in the kidney, no statistically significant differences of staining intensity were observed between RCC and normal tissues [12]. However, the low number of specimens and intertumoral and intratumoral heterogeneity prevent any final conclusion [12,19]. In this study, we analyzed the expression of Gal-8 by immunohistochemistry (IHC) in tumor tissues of patients with localized pathologic T1 (pT1) ccRCC and their associations with clinicopathologic variables and disease recurrence. We further evaluated the prognostic values of Gal-8 expression and attempted to assess the effect of Gal-8 expression on conventional prognostic models.
2. Patients and methods 2.1. Patients Specimens from 393 patients (260 patients in localized pT1 stage) with ccRCC treated with radical or partial
nephrectomy at Zhongshan Hospital, Fudan University (Shanghai, China), were collected in the previous work. Patients were selected based on the following criteria: (a) confirmed postoperative histopathology diagnosis, (b) no comorbidities, (c) complete available follow-up data, (d) no adjuvant anticancer therapy after surgery, and (e) tumor stage classification carried out according to the 2010 American Joint Committee on Cancer TNM classification [20]. Patients who were lost to follow-up, experienced bilateral disease and familial RCC, received preoperative neoadjuvant therapy or postoperative adjuvant therapy or both, or died within the first month after surgery were excluded. According to the aforementioned selection standard and our interest in low-risk group of patients with ccRCC, 244 patients with localized pT1 ccRCC were enrolled in the current retrospective study. Specimens from patients were collected from January 2003 to December 2008. The study was approved by the ethics committee of Fudan University, and each patient provided informed consent on the use of clinical specimens. Patients who received preoperative neoadjuvant were not recruited in the current study. Clinicopathologic information of each patient, including age, sex, tumor size, T stage, Fuhrman grade, necrosis, and Eastern Cooperative Oncology Group performance status (ECOG-PS), was obtained from patients' records. The Mayo Clinic Stage, Size, Grade, and Necrosis score (SSIGN) and University of Los Angeles Integrated Staging System (UISS) scores were used for the patients. Recurrence-free survival (RFS) was the main end point of this study. RFS was calculated from the date of surgery to the date of recurrence or to the date of the most recent follow-up. The 244 patients were randomly assigned into 2 groups: group A was the training group and group B was the validation group. Median follow-up was 71 months (range: 12–120 mo) in group A and 70 months (range: 12–119 mo) in group B. 2.2. Immunohistochemistry Tissue microarrays (TMAs) were performed as previously described [21]. Primary anti–Gal-8 antibody (diluted 1:100; Abcam, Cambridge, MA) was applied for IHC staining. The specificity of this antibody was determined by western blotting and IHC with peptide competition. The immunostaining intensity was evaluated by 2 independent pathologists without the knowledge of clinicopathologic data and clinical outcome of each patient. A semiquantitative IHC score (H-score) ranging from 0 to 300 for each specimen was calculated by multiplying the staining intensities (0—negative, 1—weak staining, 2—moderate staining, and 3—strong staining) by the distribution areas (0%–100%). Gal-8 expression was analyzed as continuous (with H-score) and categorical (by dichotomizing) variables. Patients in each group were dichotomized into lowand high-expression subgroups according to median intensity score of each group.
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2.3. Statistical analysis Statistical analysis was conducted by MedCalc Software (version 11.4.2.0; MedCalc, Mariakerke, Belgium) and Stata 11.0 (StataCorp, College Station, TX). Student t test (continuous variable) and chi-square test (categorical variable) are used to evaluate the association of clinicopathologic variables with Gal-8 expression. Correlation analysis was conducted to evaluate the association between 2 continuous variables. Kruskal-Wallis test was performed when we assessed the association of Fuhrman grade with continuous Gal-8 expression. Survival curves were set by Kaplan-Meier method and compared by log-rank test. Hazard ratios (HRs) and 95% CIs of prognostic factors were evaluated by univariate and multivariate Cox proportional hazard models. The accuracy of the prognostic models was evaluated by Harrell's concordance index (c-index). Furthermore, the Akaike information criterion (AIC) value was calculated to evaluate the discriminatory ability of prognostic models, and smaller AIC values present a better predicting ability. P values of all statistical tests were 2 tailed, and P o 0.05 was considered significant.
3. Results 3.1. Association of Gal-8 expression with clinicopathologic variables in patients with ccRCC To determine the expression pattern and its potential clinical significance in tumor tissues of patients with ccRCC, we detected the expression of Gal-8 by immunohistochemistry analysis in 244 patients with localized pT1 ccRCC treated with radical or partial nephrectomy. As presented in Fig. 1, the positive staining of Gal-8 was mainly located in the cytoplasm of tumor cells, and the staining intensity was variable in different specimens from patients with ccRCC. According to the semiquantitative H-score, the median intensity score was 71 (range: 0–221)
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in group A, by which, the group was dichotomized into high (n ¼ 55) and low (n ¼ 67) Gal-8 expression subgroups. The median intensity score of group B, 76 (range: 0–233), was also applied to dichotomize the group B into highexpression (n ¼ 59) and low-expression (n ¼ 63) subgroups. As summarized in Table 1, there were no significant differences between the patients with high Gal-8 expression and those with low Gal-8 expression regarding age, sex, tumor size, T stage, Fuhrman grade, and ECOG-PS (P 4 0.05). However, patients with high Gal-8 expression were significantly inclined to have histologic necrosis (continuous Gal-8: P ¼ 0.032 in group A and P ¼ 0.003 in group B; dichotomous Gal-8: P ¼ 0.013 in group A and P ¼ 0.037 in group B). 3.2. High expression of Gal-8 is associated with recurrence in patients with localized pT1 ccRCC As recorded, among 14 patients who experienced recurrence in group A (n ¼ 122), 12 patients had local recurrence and 2 patients had distant metastasis (pulmonary metastasis [n ¼ 1] and brain metastasis [n ¼ 1]). However, in group B (n ¼ 122), among 22 patients who experienced recurrence, 18 patients had local recurrence and 4 patients had distant metastasis (pulmonary metastasis [n ¼ 3] and skin metastasis [n ¼ 1]). Kaplan-Meier survival analysis was performed to compare the clinical outcome between Gal-8 high-expression and lowexpression subgroups. As presented in Fig. 2A and B, high expression level of Gal-8 was significantly associated with increased risk of recurrence after surgery (P ¼ 0.008 in group A and P ¼ 0.003 in group B). To determine whether the association of Gal-8 expression with recurrence risk of patients was dependent on T stage, subgroups of T stage were analyzed. Patients with T1aN0M0 could not be stratified by Gal-8 expression (P ¼ 0.148 in group A, Fig. 2C; P ¼ 0.200 in group B, Fig. 2D), whereas patients with T1bN0M0 could be significantly
Fig. 1. Galectin-8 (Gal-8) expression in clear cell renal cell carcinoma (ccRCC) tissues. Representative Gal-8 immunohistochemistry (IHC) images of ccRCC tumor tissues with low expression level (A) and high expression level (B). Arrows indicate positive staining of Gal-8 in each image (original magnification, 200). Scale bar: 50 μm. (Color version of figure is available online.)
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Table 1 Association between Gal-8 expression and patient characteristics Characteristic
Group A (n ¼ 122)
Group B (n ¼ 122)
Continuous Gal-8 (H-score)
Number %
Mean ⫾ SD
Range
Age, y Mean ⫾ SD Range
54.76 ⫾ 11.29 22–80
87.41 ⫾ 50.11
0–221
Sex Female Male
32 90
81.94 ⫾ 47.81 89.36 ⫾ 51.02
0–190 0–221
87.41 ⫾ 50.11
0–221
87.30 ⫾ 46.05 87.55 ⫾ 55.08
9–208 0–221
Dichotomous Gal-8
0.728
Tumor size, cm Mean ⫾ SD 3.53 ⫾ 1.45 Range 0.50–7.00
55.85 ⫾ 12.27 31–80
11 44
21 46
67 55
54.92 45.08
Fuhrman grade 1 2 3 4
30 55 30 7
24.59 87.23 ⫾ 47.13 13–183 45.08 77.60 ⫾ 48.82 0–204 24.59 97.17 ⫾ 42.29 36–208 5.74 123.43 ⫾ 83.28 15–221
Necrosis Absent Present
103 19
84.43 83.23 ⫾ 49.44 0–208 15.57 110.05 ⫾ 48.87 39–221
ECOG-PS 0 Z1
110 12
90.16 9.84
3.38 ⫾ 1.29 1.00–6.00
3.65 ⫾ 1.56 0.50–7.00
Range
33 22
34 33
11 22 18 4
19 33 12 3
41 14
62 5
51 4
59 8
90.70 ⫾ 58.25
0–233
26 96
83.23 ⫾ 49.64 92.72 ⫾ 60.45
7–203 0–233
3.44 ⫾ 1.34 0.80–7.00
90.70 ⫾ 58.25
0–233
74 48
60.66 39.34
96.99 ⫾ 63.29 81.00 ⫾ 48.53
4–233 0–216
32 54 27 9
26.23 88.03 ⫾ 59.85 0–216 44.26 91.06 ⫾ 60.43 0–227 22.13 85.59 ⫾ 51.93 6–227 7.38 113.33 ⫾ 61.57 64–233
101 21
82.79 83.53 ⫾ 54.41 0–227 17.21 125.14 ⫾ 64.98 34–233
110 12
90.16 87.62 ⫾ 58.00 0–233 9.84 118.92 ⫾ 54.97 64–213
13 50
3.23 ⫾ 1.34 0.80–6.50
3.63 ⫾ 1.33 1.50–7.00
38 21
36 27
16 24 14 5
16 30 13 4
44 15
57 6
52 7
58 5
0.974
0.101
0.525
0.651
0.877
0.003
0.578
SD ¼ standard deviation. The bold characters indicate that these P values are considered statistically significant. * P o 0.05 is considered statistically significant.
13 46
0.139
0.013
0.962
54.17 ⫾ 10.14 34–81
0.093
0.209
0.032
0.913 53.95 ⫾ 12.54 24–77
0.464 21.31 78.69
0.401
0.091
Dichotomous Gal-8
P value* High (n ¼ 59) Low (n ¼ 63) P value* 0.454
54.07 ⫾ 11.32 24–81
0.305
0.979
87.48 ⫾ 50.27 0–221 86.75 ⫾ 50.82 20–201
Mean ⫾ SD
0.226
0.633
T stage T1a T1b
Continuous Gal-8 (H-score)
0.241 53.44 ⫾ 9.92 22–73
0.474 26.23 73.77
Patients
P value* High (n ¼ 55) Low (n ¼ 67) P value* Number %
0.037
0.077
0.672
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Patients
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Fig. 2. Recurrence-free survival (RFS) analysis of patients with localized pT1 renal cell carcinoma (ccRCC) based on Gal-8 expression. Kaplan-Meier analysis of RFS in group A (n ¼ 122) (A) and in group B (n ¼ 122) (B); in pT1a subgroup of group A (n ¼ 67) (C) and group B (n ¼ 74) (D); and in pT1b subgroup of group A (n ¼ 55) (E) and group B (n ¼ 48) (F). P value was calculated by log-rank test. (Color version of figure is available online.)
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Gal-8: HR ¼ 1.022, CI: 1.007–1.038, P ¼ 0.005 in group A; HR ¼ 1.020, CI: 1.010–1.030, P o 0.001 in group B; dichotomous Gal-8: HR ¼ 11.761, CI: 2.334–59.268, P ¼ 0.003 in group A; HR ¼ 7.481, CI: 2.447–22.870, P o 0.001 in group B).
stratified by Gal-8 expression (P ¼ 0.004 in group A, Fig. 2E; P o 0.001 in group B, Fig. 2F) in RFS analysis. 3.3. High expression of Gal-8 is an independent indicator of recurrence in patients with localized pT1 ccRCC Univariate Cox analysis was performed to evaluate the prognostic significance of clinicopathologic variables for RFS. As shown in Table 2, tumor size (HR ¼ 1.820, CI: 1.268–2.612, P ¼ 0.001 in group A; HR ¼ 1.564, CI: 1.169–2.093, P ¼ 0.003 in group B), T stage (HR ¼ 7.723, CI: 1.741–34.253, P ¼ 0.008 in group A; HR ¼ 3.138, CI: 1.320–7.463, P ¼ 0.010 in group B), Fuhrman grade (HR ¼ 4.209, CI: 1.418–12.492, P ¼ 0.010 in group A; HR ¼ 2.489, CI: 1.083–5.721, P ¼ 0.033 in group B), necrosis (HR ¼ 3.215, CI: 1.083–9.541, P ¼ 0.036 in group A; HR ¼ 2.488, CI: 1.018–6.081, P ¼ 0.047 in group B), ECOG-PS (HR ¼ 4.664, CI: 1.465– 14.852, P ¼ 0.010 in group A; HR ¼ 4.416, CI: 1.731– 11.268, P ¼ 0.002 in group B), high expression of continuous Gal-8 (HR ¼ 1.015, CI: 1.006–1.024, P ¼ 0.002 in group A; HR ¼ 1.010, CI: 1.004–1.016, P ¼ 0.002 in group B), and high expression of dichotomous Gal-8 (HR ¼ 4.798, CI: 1.347–17.089, P ¼ 0.016 in group A; HR ¼ 4.069, CI: 1.508–10.977, P ¼ 0.006 in group B) were identified as risk factors, which might indicate recurrence in patients with localized pT1 ccRCC. Then multivariate Cox regression analysis involving potential risk factors identified by univariate Cox analysis was performed. Besides tumor size, Fuhrman grade, and ECOG-PS, Gal-8 expression was identified as an independent prognostic factor for recurrence (Table 2, continuous
3.4. Extension of prognostic models with Gal-8 expression for patients with localized pT1 ccRCC To further assess the prognostic power of Gal-8 expression, we constructed prognostic models combining Gal-8 expression with SSIGN score and UISS score and compared the prognostic accuracy of these models by c-index and AIC analysis. As presented in Table 3, in group A, the c-indices were 0.865 and 0.707 when assessed with SSIGN and UISS outcome algorithms and were improved to 0.891 and 0.798 when continuous Gal-8 signature was added and to 0.873 and 0.778 when dichotomous Gal-8 signature was added. Likewise, in group B, the c-indices were improved from 0.743 and 0.671 to 0.833 and 0.787 when continuous Gal-8 signature was added and to 0.791 and 0.754 when dichotomous Gal-8 signature was added. Furthermore, each combined model showed a lower AIC value than corresponding conventional model alone.
4. Discussion To our knowledge, the study is the first to put forward an association between high expression of Gal-8 and an incremental risk of recurrence in patients with localized pT1 ccRCC after surgery. Meanwhile, patients with
Table 2 Univariate and multivariate Cox regression analysis of recurrence-free survival Characteristic
Univariate
Multivariate Continuous Gal-8 (H-score)
Dichotomous Gal-8 (high vs. low) Hazard ratio (95% CI)
Hazard ratio (95% CI)
P*
Hazard ratio (95% CI)
Group A Tumor size (cm) T stage (T1b vs. T1a) Fuhrman grade (3 þ 4 vs. 1 þ 2) Necrosis (present vs. absent) ECOG PS (Z1 vs. 0) Continuous Gal-8 (H-score) Dichotomous Gal-8 (high vs. low)
1.820 7.723 4.209 3.215 4.664 1.015 4.798
(1.268–2.612) (1.741–34.253) (1.418–12.492) (1.083–9.541) (1.465–14.852) (1.006–1.024) (1.347–17.089)
0.001 0.008 0.010 0.036 0.010 0.002 0.016
2.661 1.144 5.734 1.067 4.303 1.022 –
(1.301–5.443) (0.155–8.426) (1.786–18.406) (0.311–3.661) (1.207–15.337) (1.007–1.038)
0.008 0.896 0.004 0.919 0.025 0.005 –
Group B Tumor size, cm T stage (T1b vs. T1a) Fuhrman grade (3 þ 4 vs. 1 þ 2) Necrosis (present vs. absent) ECOG PS (Z1 vs. 0) Continuous Gal-8 (H-score) Dichotomous Gal-8 (high vs. low)
1.564 3.138 2.489 2.488 4.416 1.010 4.069
(1.169–2.093) (1.320–7.463) (1.083–5.721) (1.018–6.081) (1.731–11.268) (1.004 –1.016) (1.508–10.977)
0.003 0.010 0.033 0.047 0.002 0.002 0.006
1.898 1.687 3.734 1.222 3.490 1.020 –
(1.027–3.509) (0.379–7.504) (1.425–9.785) (0.472–3.167) (1.183–10.297) (1.010–1.030)
0.042 0.494 0.008 0.681 0.024 o0.001 –
The bold characters indicate that these P values are considered statistically significant. o 0.05 is considered statistically significant.
*P
P*
1.927 (1.096–3.388) 2.643 (0.383–18.258) 6.130 (1.803–20.838) 1.656 (0.529–5.187) 10.226 (2.270–46.074) – 11.761 (2.334–59.268) 1.863 1.134 2.703 1.354 3.303 – 7.481
(1.011–3.431) (0.247–5.205) (1.126–6.491) (0.522–3.507) (1.186–9.201) (2.447–22.870)
P* 0.024 0.327 0.004 0.389 0.003 – 0.003 0.047 0.872 0.027 0.535 0.023 – o0.001
Y. Liu et al. / Urologic Oncology: Seminars and Original Investigations 33 (2015) 112.e1–112.e8 Table 3 Comparison of the accuracy of the prognostic models and Gal-8 expression for recurrence-free survival Model
Continuous Gal-8 (H-score) Dichotomous Gal-8 (high vs. low) SSIGN SSIGN þ continuous Gal-8 (H-score) SSIGN þ dichotomous Gal-8 (high vs. low) UISS UISS þ continuous Gal-8 (H-score) UISS þ dichotomous Gal-8 (high vs. low)
Group A
Group B
c-Index AIC
c-Index AIC
0.731 0.678 0.865 0.891 0.873
123.963 126.007 114.772 114.316 112.008
0.696 0.677 0.743 0.833 0.791
192.439 192.071 187.004 179.023 175.701
0.707 0.798 0.778
122.690 0.671 118.165 0.787 119.830 0.754
191.903 186.547 185.886
localized pT1 ccRCC could be stratified with Gal-8 expression in T1b stage in RFS analysis. High Gal-8 expression was positively associated with histologic necrosis. Furthermore, the accuracy of SSIGN and UISS prognostic models was improved when Gal-8 expression was added. It has been reported that the expression of Gal-1 and Gal-3 could be regulated by HIF1 [22,23]. In addition, von Hippel– Lindau (VHL) inactivation is a common event in ccRCC. Because Gal-8 exerts functions similar to Gal-1 and Gal-3 in certain tumors and has been validated to play a critical role in ccRCC in this study, we consider that Gal-8 expression might also be modulated by HIF1 and involved in VHL/HIF/HRG (hypoxia responsive gene) signaling axis. As presented in this study, Gal-8 expression could be applied as an independent prognostic factor in patients with early-stage ccRCC, especially in T1b stage. The profound molecular roles of Gal-8 in ccRCC tumorigenesis await further investigation. Besides the well-documented ability to mediate cell adhesion [10], Gal-8 could modulate endothelial cell migration and promote angiogenesis [24]. The proangiogenesis properties of Gal-8 further indicated its potential role in VHL/HIF/vascular endothelial growth factor (VEGF) signaling pathway. Another study reported that Gal-8 could promote cytoskeletal rearrangement via activation of Rho signaling [25]. A recent study concluded that Gal-8 exerted immunosuppressive function not only by inducing apoptosis of activated T cells but also via negatively mediating the critical function of lymphocyte function-associated antigen-1 in the immune system [26]. Numerous researches have determined that cytotoxic T-lymphocyte antigen-4 and programmed death-1 expressed on T cells, functioning as immune checkpoints, also exert immunosuppressive actions. Nowadays, anticytotoxic T-lymphocyte antigen-4 and anti-programmed death-1 monoclonal antibodies have presented striking antitumor activity in large phase I studies [27,28]. So Gal8 expression might also be identified as a potential predictive biomarker on treatment response of these
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immune checkpoints inhibitors, which needs further explorations. Gal-1 might crosslink glycans within glycoproteins on endothelial cell surface to compensate for the absence of cognate ligand and maintain angiogenesis in response to VEGF blockade, which leads to anti-VEGF refractory tumors [29]. Considering that Gal-8 might be involved in VHL/HIF/VEGF signaling pathway and promote angiogenesis, we supposed that Gal-8 could also be involved in tumor resistance to anti-VEGF therapy. In summary, Gal-8 could be exploited as a new therapeutic target in combination with anti-VEGF treatment in ccRCC. Although the clinical significance of Gal-8 expression in patients with localized T1 ccRCC has been concluded and revealed in this study, some limitations remain to be resolved. Considering that all specimens from patients were collected from a single institution, the results need to be further validated or revised in larger data sets and external heterogeneous cohorts. Although duplicate 1.0-mm tissue cores from 2 different areas were used to construct the TMA, the deviation of TMA analysis could not be avoided owing to the heterogeneous natural history of ccRCC. Moreover, the molecular role of Gal-8 in ccRCC remains to be fully elucidated in our future study. 5. Conclusions In summary, our present study identified Gal-8 expression as a potential independent unfavorable prognostic indicator for postoperative recurrence of patients with localized pT1 ccRCC. Combining Gal-8 expression with conventional prognostic models could improve their prognostic accuracy. Further researches might attempt to verify whether Gal-8 could be developed as a new therapeutic target. Acknowledgments The authors thank Ms. Haiying Zeng (Department of Pathology, Zhongshan Hospital, Shanghai Medical College of Fudan University) for technical assistance. References [1] Ljungberg B, Hanbury DC, Kuczyk MA, et al. Renal cell carcinoma guideline. Eur Urol 2007;51:1502–10. [2] Rini BI, Campbell SC, Escudier B. Renal cell carcinoma. Lancet 2009;373:1119–32. [3] Barondes SH, Castronovo V, Cooper DN, et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994;76:597–8. [4] Liu FT, Patterson RJ, Wang JL. Intracellular functions of galectins. Biochim Biophys Acta 2002;1572:263–73. [5] Yang RY, Rabinovich GA, Liu FT. Galectins: structure, function and therapeutic potential. Expert Rev Mol Med 2008;10:e17. [6] Huang CS, Tang SJ, Chung LY, et al. Galectin-1 upregulates CXCR4 to promote tumor progression and poor outcome in kidney cancer. J Am Soc Nephrol 2014;25:1486–95.
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