Ann Surg Oncol DOI 10.1245/s10434-014-3601-1
ORIGINAL ARTICLE – UROLOGIC ONCOLOGY
Prognostic Value of Diametrically Polarized Tumor-Associated Macrophages in Renal Cell Carcinoma Le Xu, MD, PhD1, Yu Zhu, MD, PhD2, Lian Chen, MD3, Huimin An, MD1, Weijuan Zhang, PhD4, Guomin Wang, MD1, Zongming Lin, MD1, and Jiejie Xu, MD, PhD5,6,7 Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China; 2Department of Urology, Ninth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China; 3Department of Pathology, Children’s Hospital, Fudan University, Shanghai, China; 4Department of Immunology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; 5Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; 6Key Laboratory of Glycoconjugate Research, MOH, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; 7Key Laboratory of Medical Molecular Virology, MOE & MOH, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China 1
ABSTRACT Background. As the most abundant tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) are significant for fostering tumor progression. CD68? TAMs display diversely polarized programs comprising CD11c? proinflammatory macrophages (M1) and CD206? immunosuppressive macrophages (M2). The aim of this study was to determine the survival impact of diametrically polarized TAMs in clear-cell renal cell carcinoma (ccRCC) and their application to stratification of patients according to their prognostic values. Methods. The study included 185 consecutive patients with ccRCC who underwent nephrectomy between 1999 and 2001. CD68? total and diametrically polarized (CD11c? M1 and CD206? M2) TAM densities were assessed by immunohistochemistry, and the relationships
Drs. Le Xu, Yu Zhu, and Lian Chen contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-3601-1) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 28 December 2013 Z. Lin, MD e-mail: [email protected] J. Xu, MD, PhD e-mail: [email protected]
with clinicopathologic features and prognosis were evaluated. Results. Low CD11c? TAM density and high CD206? TAM density were associated with reduced cancer-specific survival (P = 0.043 and P = 0.017, respectively), whereas CD68? TAM density only had borderline prognostic significance (P = 0.062). Furthermore, combined analysis of CD11c? and CD206? TAMs (CD11c/CD206 signature) had a better power to predict patients’ outcome (P = 0.010). Together with TNM stage, tumor necrosis, and performance status, CD11c/CD206 signature was an independent prognostic factor (P = 0.010). When applied to the University of California Integrated Staging System intermediate-/high-risk group for localized ccRCC, CD11c/ CD206 signature could further distinguish patients with dismal prognosis (P = 0.004). Conclusions. Intratumoral balance of diametrically polarized TAMs is a novel independent predictor for survival in patients with ccRCC. Tipping the balance toward an antitumoral phenotype might be a promising target of postoperative adjuvant therapy. Renal cell carcinoma (RCC), which accounts for 2–3 % of all adult malignant neoplasms, is the most lethal urologic cancer. The estimated 5-year survival rate for patients with localized RCC is approximately 70–90 %. However, once metastatic disease develops, the prognosis is poor, with 5-year survival rate ranging between 0 and 20 %.1,2 The natural history of RCC is complex and is influenced by factors other than pathologic stage.3 Currently, several
L. Xu et al.
prognostic models have been proposed to identify patients at high risk of disease progression after nephrectomy. One widely used model is the University of California integrated staging system (UISS), which incorporates TNM stage, Fuhrman grade, and Eastern Cooperative Oncology Group performance status (ECOG-PS), resulting in 3 risk groups [low- (LR), intermediate- (IR), and high-risk (HR)] for localized and metastatic RCC.4,5 Macrophages are a major cellular component of human tumors, where they are commonly termed tumor-associated macrophages (TAMs).6 A wide array of biologically active molecules are present in the tumor microenvironment as products of malignant and stromal cells. Infiltrating macrophages respond to this milieu and adapt a range of activation states that can be classified within the M1/M2 polarization model.7,8 M1 macrophages, activated by toll-like receptor (TLR) ligands and interferon-c (IFN-c), have roles in antitumor immunity. In contrast, M2 macrophages, stimulated by interleukin-4 (IL-4) or IL-13, have the ability to promote tumor growth and progression.8 Therefore, it is plausible that incorporation of macrophage polarization into established predictive models would improve prognostic stratification. There has been growing interest in studying the prognostic importance of macrophage infiltrations in solid tumors. CD68, the most widely used macrophage marker, is expressed on all macrophages.9 However, CD68 does not allow for the discrimination between M1 and M2 subsets. Recently, cell surface scavenger receptors, such as mannose receptor (CD206) and hemoglobin/haptoglobin scavenger receptor (CD163) have been recognized as specific M2 macrophage markers.6 Thus, M2 macrophages can be phenotypically discriminated from their M1 counterparts as the former express high levels of CD206 and CD163, while the latter are characterized by high amounts of CD11c and TLR4 on their surface.10–12 Based on this knowledge, we hypothesized that intratumoral CD68? pan-macrophages comprise CD11c? M1 macrophages and CD206? M2 macrophages. We, therefore, performed immunohistochemical analyses of CD68, CD11c, and CD206 in RCC tissues. Our data showed that both low CD11? TAM density and high CD206? TAM density associated with poor survival, whereas CD68? TAM density only had borderline prognostic significance. Furthermore, combined analysis of CD11c and CD206 (designated as CD11c/CD206 signature) outperformed single macrophage markers in predicting prognosis of RCC patients after surgery. MATERIALS AND METHODS Patients We retrospectively recruited 185 consecutive patients undergoing nephrectomy for clear-cell RCC (ccRCC) at
Zhongshan Hospital, Shanghai, China between 1999 and 2001. This study was approved by the hospital’s ethics committee and informed consent was obtained from each patient. For each patient, the following clinicopathologic information was collected: age, gender, tumor size, TNM stage, Fuhrman grade, presence of histologic tumor necrosis, and ECOG-PS. Patients were staged using radiographic reports and postoperative pathological data and were reassigned according to 2010 AJCC TNM classification. Patients with N1 or M1 tumors were considered to have metastatic disease. The UISS predictive model was applied to the patients.4 There were 2 decision boxes based on the interaction of TNM stage, Fuhrman grade, and ECOG-PS used to classify patients with localized and metastatic RCC into LR, IR, or HR groups. Patients with localized RCC (n = 169) were treated with radical or partial nephrectomy. Patients with metastatic RCC (n = 16) were treated with cytoreductive nephrectomy followed by interferon-a-based immunotherapy. After surgery, patients were evaluated with physical examination, laboratory studies, chest imaging, and abdominal ultrasound or CT scan every 6 months for the first 2 years and annually thereafter for 5 years. Median follow-up was 70 months (range, 10–120 months). Cancerspecific survival (CSS) was the main endpoint of the study. We calculated CSS from the date of nephrectomy to the date of death from RCC or last follow-up. There were 4 patients who died from other causes and were censored for the CSS endpoint. Immunohistochemistry Immunohistochemical analysis is described in the supplemental material, in the section Supplemental Methods. Primary antibodies against human CD68 (Dako, Glostrup, Denmark), CD11c (Abcam, Cambridge, MA) and CD206 (Abcam, Cambridge, MA) were applied in the procedure. Statistical Analyses The ‘‘minimum P value’’ approach was applied to obtain the cutoff providing the best separation between the groups of patients related to their CSS (Fig. S1).13,14 The cutoff values for low and high CD68?, CD206?, and CD11c? TAM densities were 50 (30th percentile), 30 (30th percentile), and 55 (80th percentile) cells per field (9200), respectively. Correlations between immunohistochemical variables and clinicopathologic characteristics were analyzed with v2 and t tests. Kaplan–Meier method with log-rank test was applied to compare survival curves. All statistical tests were two sided and performed at a significance level of 0.05. Univariate and multivariate Cox regression models
Diametrically Polarized TAMs in RCC
FIG. 1 Representative photographs of CD68 (a, d, g, and j), CD11c (b, e, h, and k), and CD206 (c, f, i, and l) immunostaining in tissue microarrays (original magnification 9200). Case 13 (a–c) showed low densities of CD68?, CD11c?, and CD206? macrophages. Case 74 (d–f) showed high densities of CD68?, CD11c?, and CD206?
macrophages. Case 94 (g–i) showed high densities of CD68? and CD11c? macrophages, but low CD206? macrophage density. Case 180 (j–l) showed high densities of CD68? and CD206? macrophages, but low CD11c? macrophage density
were used to analyze the impact of prognostic factors on CSS, and P [ 0.10 was the removal criterion when performing backward stepwise variable deletions. The
predictive accuracy of various Cox regression models was quantified by Harrell’s concordance index (C-index). Data were analyzed using SPSS Statistics 17.0 and Stata 11.0.
L. Xu et al.
Immunohistochemical Findings CD68, CD11c, and CD206 positive staining mainly appeared in the cytoplasm of macrophages (Fig. 1). Sporadic positive CD68 staining was observed in cancer cells; no CD11c or CD206 staining was detected in cancer cells. Statistics of immunohistochemical variables are shown in Table S2. Generally, CD68? cells were more abundant than CD11c? or CD206? cells, and CD11c? cells were outnumbered by CD206? cells in RCC tissues (all P \ 0.001). Survival Analyses with Single Macrophage Markers and Correlations Between These Markers and Clinicopathologic Features Kaplan–Meier analyses indicated that, as single variables, low CD11? TAM density and high CD206? TAM density associated with reduced survival (P = 0.043 and P = 0.017, respectively) (Fig. 2b, c), whereas CD68? TAM density only had borderline prognostic significance (P = 0.062) (Fig. 2a). As summarized in Table 1, patients with high CD68? TAM density were prone to having high-grade tumors and histologic necrosis (P = 0.022 and P = 0.035, respectively). Female patients tended to harbor more CD11c? TAMs (P = 0.017). CD11c? TAM density was positively correlated with Fuhrman grade (P = 0.021). CD206? TAM density was positively correlated with patient’s age and ECOG-PS (P = 0.014 and P = 0.015, respectively). Combined Analysis of CD11c and CD206 Has a Better Power to Predict Outcome Since M1 and M2 macrophage
Cancer-specific survival
As summarized in Table S1, median age was 60 years (range, 30–84 years), median tumor size was 5 cm (range, 1–14 cm), and histologic necrosis was presented in 42.7 % of cases. Also, 7 patients (3.8 %) had lymph node metastases and 10 patients (5.4 %) had distant metastases at the time of surgery. The distribution of TNM stages I, II, III, and IV was 64.9, 14.1, 15.7, and 5.4 %, respectively. Fuhrman grades were 1, 2, 3, and 4 in 7.0, 33.0, 43.8, and 16.2 % of cases, respectively. ECOG-PS was evaluated as C1 in 42.7 % of cases. Among 169 patients with localized RCC, UISS was classified as LR, IR, and HR in 19.5, 73.4, and 7.1 % of cases, respectively. Among 16 patients with metastatic RCC, UISS was classified as LR, IR, and HR in 50.0, 43.8, and 6.2 % of cases, respectively. At the time of last follow-up, 37.8 % of patients had died of RCC.
100 80 60 40
P = 0.062 Low CD68+ TAMs High CD68+ TAMs
20 0
24
0
48
72
96
120
Time after surgery (months) Number at risk Low CD68+ TAMs 55 High CD68+ TAMs 130
b Cancer-specific survival
Patient Characteristics
a
54
42
28
15
0
119
88
51
18
0
72
96
120
100 80 60 40
P = 0.043 Low CD11c+ TAMs High CD11c+ TAMs
20 0 0
24
48
Time after surgery (months) Number at risk Low CD11c+ TAMs 147 137 High CD11c+ TAMs 38 36
c Cancer-specific survival
RESULTS
105
60
28
0
25
19
5
0
72
96
120
100 80 60 40
P = 0.017 Low CD206+ TAMs High CD206+ TAMs
20 0 0
24
48
Time after surgery (months) Number at risk Low CD206+ TAMs 55 54 High CD206+ TAMs 130 119
44
35
14
0
86
44
19
0
FIG. 2 Kaplan-Meier curves showing CSS probabilities based on intratumoral CD68?, CD11c?, and CD206? macrophage densities (a, b, and c). TAMs tumor-associated macrophages
subtypes perform opposing functions, it is believed that M1/ M2 polarization in tumor tissue might have an impact on cancer progression. Thus, we proposed that combined analysis of CD11c and CD206 would allow for improved prognostic stratification of RCC patients by assessing both
Diametrically Polarized TAMs in RCC TABLE 1 Correlations between immunohistochemical variables and clinicopathologic characteristics CD68? TAMs
Variable
Mean age (years)a
CD11c? TAMs
Low (n = 55)
High (n = 130)
58.7
61.4
Gender
CD206? TAMs
P
Low (n = 147)
High (n = 38)
0.176
60.7
60.0
0.290
Male Female
23 (12.4 %) 31 (16.8 %)
46 (24.9 %) 84 (45.5 %)
5.0
5.4
I ? II
44 (23.8 %)
102 (55.1 %)
III ? IV
11 (5.9 %)
28 (15.2 %)
Mean tumor size (cm)a TNM stage
0.261
Low (n = 55)
High (n = 130)
P
0.736
57.2
62.0
0.014
23 (12.4 %) 32 (17.3 %)
47 (25.4 %) 83 (44.9 %)
5.4
5.2
46 (24.9 %)
100 (54.1 %)
9 (4.9 %)
30 (16.1 %)
0.017 62 (33.5 %) 85 (45.9 %)
8 (4.3 %) 30 (16.2 %)
5.2
5.8
117 (63.2 %)
29 (15.7 %)
30 (16.2 %)
9 (4.9 %)
0.815
Fuhrman grade
P
0.177
0.468
0.659
0.022
0.306
0.021
0.511
1?2
29 (15.7 %)
45 (24.3 %)
65 (35.2 %)
9 (4.7 %)
24 (13.0 %)
50 (27.0 %)
3?4
26 (14.1 %)
85 (45.9 %)
82 (44.4 %)
29 (15.7 %)
31 (16.8 %)
80 (43.2 %)
Absent
38 (20.5 %)
68 (36.8 %)
86 (46.5 %)
20 (10.8 %)
36 (19.5 %)
70 (37.8 %)
Present
17 (9.2 %)
62 (33.5 %)
61 (33.0 %)
18 (9.7 %)
19 (10.3 %)
60 (32.4 %)
42 (22.7 %)
61 (33.0 %)
13 (7.0 %)
69 (37.3 %)
12 (7.1 %)
21 (12.4 %)
Necrosis
0.035
ECOG-PS
0.514
0.145
0
39 (21.1 %)
64 (34.6 %)
C1
16 (8.6 %)
66 (35.7 %)
UISS (localized) LR
0.145
0.933 84 (45.3 %)
19 (10.3 %)
63 (34.1 %)
19 (10.3 %)
0.229
0.015
0.054
19 (11.2 %)
IR
34 (20.1 %)
90 (53.3 %)
93 (55.0 %)
31 (18.3 %)
38 (22.5 %)
86 (50.9 %)
HR
3 (1.8 %)
9 (5.3 %)
10 (5.9 %)
2 (1.2 %)
1 (0.6 %)
11 (6.5 %)
LR
2 (12.5 %)
6 (37.5 %)
6 (37.5 %)
2 (12.5 %)
1 (6.2 %)
7 (43.8 %)
IR ? HR
2 (12.5 %)
6 (37.5 %)
7 (43.8 %)
1 (6.2 %)
3 (18.8 %)
5 (31.2 %)
UISS (metastatic)
2 (1.2 %)
0.189
14 (8.3 %)
b
31 (18.3 %)
1.000
0.707
1.000
0.569
TAMs tumor-associated macrophages, ECOG-PS Eastern Cooperative Oncology Group performance status, UISS University of California Los Angeles Integrated Staging System, LR low risk, IR intermediate risk, HR high risk a b
Student’s t test Fisher exact test; Chi square test for all the other analyses
antitumoral M1 macrophages and protumoral M2 macrophages. Patients were classified into 4 groups according to their CD11c? and CD206? TAM densities: group I, high CD11c? but low CD206? TAM densities; group II, both low densities; group III, both high densities; group IV, low CD11c? but high CD206? TAM densities. Significant difference in CSS (P = 0.010) was found among the 4 groups (Fig. 3a). The 5-year CSS rates for groups I, II, III, and IV were 100, 84.5, 82.9, and 66.8 %, respectively. Thus, an immune profile characterized by CD11chigh/ CD206low may reflect an M1-skewed status in tumor tissue and is predictive of a favorable outcome. In contrast, an immune response characterized by CD11clow/CD206high may represent an M2-skewed status and a population of patients with poor prognosis. In groups II and III, the influence of CD11c? TAMs, low or high, on prognosis was probably counterbalanced by simultaneous low or high CD206? TAMs, and vice versa. Therefore, patients in
groups II and III had the intermediate survival data. When the analysis was restricted to localized RCC, a CD11chigh/ CD206low immune profile was correlated with a long-term CSS rate of 100 % (P = 0.013) (Fig. 3b). However, CD11c/ CD206 signature was not predictive in metastatic RCC in our study (P = 0.400) (Fig. S2). Table 2 lists the univariate and multivariate analyses of potential prognostic factors for CSS. After backward stepwise variable selection, TNM stage (P \ 0.001), tumor necrosis (P = 0.031), ECOG-PS (P = 0.003), and CD11c/ CD206 signature (P = 0.010) remained in the model as independent prognostic factors. These variables collectively yielded a predictive accuracy of 81.2 %. Extension of the UISS Prognostic Model with TAM Polarization For localized RCC, the 5-year CSS rates were 93.3, 80.0, and 38.9 % for UISS LR, IR, and HR, respectively. For metastatic RCC patients grouped according
L. Xu et al. b FIG. 3 Kaplan–Meier curves showing CSS probabilities based on
Cancer-specific survival
a
All RCC 100 80 60 I II III IV
40 20
P = 0.010
0 0
Number at risk Group I 10 Group II 39 Group III 28 Group IV 108
24
Cancer-specific survival
72
96
12 0
10
7
7
3
0
39
33
26
11
0
26
18
12
2
0
98
72
34
17
0
b
Localized RCC 100 80 60 I II III IV
40 20
difference in CSS (P = 0.004) was found among these 4 groups according to CD11c/CD206 signature (Fig. 3c). Thus, in UISS IR/HR group for localized RCC, the 5-year CSS rate was 100 % if tumors displayed a CD11chigh/CD206low immune profile in contrast to 65 % if a CD11clow/ CD206high profile was observed. The C-index was 0.718 when assessed with UISS outcome algorithm and was improved to 0.752 when CD11c/CD206 signature was added. However, in UISS LR group for localized RCC, CD11c/CD206 signature failed to further stratify patients with different prognosis (P = 0.786) (Fig. S3). DISCUSSION
P = 0.013
0 0
Number at risk Group I 9 Group II 36 Group III 26 Group IV 98
c Cancer-specific survival
48
Time after surgery (months)
the combination of CD11c? and CD206? TAM densities in all patients (a), patients with localized RCC (b), and patients in the UISS combined intermediate- and high-risk group for localized RCC (c). Patients were classified into 4 groups according to their CD11c? and CD206? TAM densities: group I, high CD11c? but low CD206? TAM densities; group II, both low densities; group III, both high densities; group IV, low CD11c? but high CD206? TAM densities. TAMs tumor-associated macrophages, RCC renal cell carcinoma, UISS University of California Los Angeles Integrated Staging System
24
48
72
96
12 0
Time after surgery (months) 9
6
6
3
0
36
33
26
11
0
25
17
11
2
0
92
70
34
17
0
UISS intermediate/high risk for localized RCC 100 80 60 I II III IV
40 20
P = 0.004
0 0
24
48
72
96
12 0
Time after surgery (months) Number at risk Group I 8 Group II 26 Group III 25 Group IV 77
8
5
5
3
0
26
23
19
6
0
24
16
11
2
0
71
51
26
10
0
to UISS LR and IR, the 5-year CSS rates were 22.5 and 12.5 %, respectively. Then we investigated whether incorporation of M1/M2 TAM polarization into the UISS outcome algorithm would improve prognostic stratification. In UISS IR/HR group for localized RCC, significant
In the present study, we showed that identification of intratumoral macrophage polarization using CD11c and CD206 immunostaining can predict outcomes for surgically treated RCC patients, especially for those with localized disease. Moreover, incorporation of CD11c/CD206 signature into the UISS predictive model could further stratify patients and provide more prognostic information. For example, patients with a CD11clow/CD206high immune profile may need to undergo a closer follow-up after surgery. However, the incorporation of TAM polarization into current prognostic models requires validation in an independent and larger population. In this study, we observed a relatively lower proportion of patients with metastatic disease compared with other studies. It is because we chose to investigate surgically treated RCC patients, while those patients with metastatic RCC who only received immunotherapy were not included in this study. Depending on external stimuli, macrophages can acquire different phenotypes with partly opposing properties.15 M1 macrophages mediate defense of the host from a variety of bacteria and viruses and play roles in anticancer immunity. M2 macrophages have anti-inflammatory function, favor angiogenesis, and promote tumor growth.8 It is important to note that ‘‘M1’’ and ‘‘M2’’ polarization states are extremes of a spectrum, macrophages often express a mixed M1/M2 phenotype in response to signals present within individual microenvironments.16,17 It is believed TAMs are predominantly polarized in the tumor microenvironment toward a protumoral M2 phenotype.18 Consistent with this notion, our data showed that
Diametrically Polarized TAMs in RCC TABLE 2 Univariate and multivariate Cox regression analyses of potential prognostic factors for CSS Variable
Cancer-specific survival Univariate analysis
Tumor size (cm)
HR (95 % CI)
Pc
C index
1.235 (1.135–1.345)
\0.001
0.653
\0.001
0.684
TNM stage a
Multivariate analysis
I vs. I III vs. Ia
1.547 (0.746–3.208) 2.131 (1.150–3.948)
0.241 0.016
IV vs. Ia
Pc
HR (95 % CI)
\0.001 1.746 (0.761–4.007) 1.739 (0.905–3.340)
0.188 0.097
15.015 (6.433–35.045)
\0.001
11.783 (5.720–24.272)
\0.001
Fuhrman grade (3 ? 4 vs. 1 ? 2a)
1.607 (1.196–2.161)
0.002
0.613
Tumor necrosis (yes vs. noa)
3.089 (1.903–5.015)
\0.001
0.645
1.846 (1.057–3.225)
0.031
ECOG-PS (C1 vs. 0 )
2.394 (1.479–3.873)
\0.001
0.600
2.121 (1.285–3.501)
0.003
CD68? TAMs (high vs. lowa)
1.688 (0.981–3.002)
0.067
0.562
a
CD11c? TAMs (high vs. lowa)
0.477 (0.228–0.996)
0.049
0.568
CD206? TAMs (high vs. lowa)
1.949 (1.112–3.418)
0.020
0.580
0.018
0.645
CD11c/CD206 signatureb II vs. Ia
3.068 (0.399–23.601)
0.282
a
3.288 (0.404–26.768)
IV vs. Ia
6.548 (0.903–47.488)
III vs. I C index
0.010 6.775 (0.853–53.829)
0.070
0.266
6.086 (0.698–53.048)
0.102
0.063
13.180 (1.722–100.87)
0.013
0.812
HR hazard ratio, 95 % CI 95 % confidence interval, ECOG-PS Eastern Cooperative Oncology Group performance status, TAMs tumorassociated macrophages, C index concordance index a
Reference group
b
Patients were classified into 4 groups according to their CD11c? and CD206? TAM densities: group I, high CD11c? but low CD206? TAM densities; group II, both low densities; group III, both high densities; group IV, low CD11c? but high CD206? TAM densities
c
An overall statistic (P) is provided for categorical variables. For these variables, hazard ratios and P values are provided for each variable level
intratumoral CD206? cells were more abundant than CD11c? cells in most cases, indicating an M2-polarized macrophage infiltration in RCC tissues. Conversely, CD11c? cells outnumbering CD206? cells was also observed in a few cases, suggesting the existence of TAMs with a relatively M1-skewed phenotype. However, the absolute cell numbers may not reflect actual macrophage polarization in the tumor microenvironment. Hence, the CD11c/CD206 signature was used in the study to delineate macrophage polarization and categorize patients. A CD11chigh/CD206low immune profile indicated an M1polarized macrophage infiltration and associated with favorable prognosis after nephrectomy. In contrast, a CD11clow/CD206high immune profile denoted an M2polarized macrophage infiltration and was predictive of inferior outcome. Additionally, CD11clow/CD206low and CD11chigh/CD206high profiles might suggested a mixed M1/M2 phenotype and associated with intermediate survival. The first studies attempting to correlate TAMs with prognosis generated heterogeneous results, most likely because macrophage infiltration was evaluated with antiCD68 antibodies, which do not distinguish between M1
and M2 subsets.19 Recently, clinical studies based on M2specific markers, such as CD163, have provided more consistent results. It has been reported that elevated amounts of intratumoral CD163? macrophages associated with poor prognosis in patients with melanoma, ccRCC, breast cancer, and lung cancer.20–23 Increasing studies have also shown that TAMs drive reparative mechanisms in tumors after treatment with vascular-targeting agents. Depletion of TAMs by clodronate-loaded liposomes (clodrolip) augmented the inhibitory effects of sorafenib on tumor angiogenesis and metastasis in hepatocellular carcinoma.24 Moreover, TAM depletion by clodrolip or a CSF1R inhibitor increased the antiangiogenic and antitumor effects of VEGF/VEGFR2 antibodies in subcutaneous tumor models.25,26 In the present study, we used CD11c and CD206 as the markers for M1 and M2 macrophages, respectively. CD11c was commonly used as an M1 marker in adipose tissue and was also regarded as a marker for myeloid dendritic cells (DCs), which may call into question whether these CD11c? cells are macrophages or DCs.27,28 A major problem in defining macrophages and DCs lies in the fact that they stem from common myeloid precursors and express common cell surface markers.8 It should be
L. Xu et al.
noted that this problem is not unique to this field, as the distinction between macrophages and DCs is controversial among immunologists even in well-defined lymphoid tissues.29,30 It is interesting that CD11c? TAM density was positively correlated with Fuhrman grade in this study. One possible explanation was that high-grade tumors are poorly differentiated and probably express more molecular patterns and antigens that the immune system could recognize. In this regard, high-grade tumors are more immunogenic than low-grade tumors and thus harbor more proinflammatory CD11c? macrophages. Since our data showed that most RCC patients (94.6 %) displayed an M2-skewed phenotype or a mixed M1/M2 phenotype in this study, tipping the macrophage balance toward a tumoricidal M1 phenotype by immunological intervention represents a promising adjuvant therapy for RCC. Therefore, IFN-c could be administered locally at the tumor site to reverse the immunosuppressive and protumoral properties of M2 macrophages and thus enhance the efficacy of active and adoptive antitumor immunotherapies.31 In addition, suppressing nuclear factor-jB (NF-jB) pathway can switch TAMs to an M1-polarized phenotype that is cytotoxic to malignant cells.32 Recently, monoclonal antibodies targeting the immune inhibitory co-receptor programmed death1 (PD-1) protein and 1 of its ligands, PD-L1, have demonstrated increased antitumor responses and evident therapeutic benefits in patients with advanced cancer.33,34 Macrophages and DCs also express PD-L1 that could suppress T cell activation. Thus, blocking PD-L1 on TAMs might enhance the efficacy of anticancer therapies. In conclusion, our results indicate that the concurrence of low CD11c? macrophages and high CD206? macrophages in RCC tissues suggests an M2-polarized status and associates with reduced survival. This finding provides a novel independent predictor for prognosis and may improve current predictive models with regard to counseling patients, selecting patients for adjuvant therapies, and customizing follow-up after surgery. ACKNOWLEDGMENT This work was supported by grants from National Key Projects for Infectious Diseases of China (2012ZX10002-012 to J.X.), National Natural Science Foundation of China (31100629 to W.Z., 31270863 to J.X., 81372755 to Z.L.), Program for New Century Excellent Talents in University (NCET-130146 to J.X.), and Shanghai Rising-Star Program (13QA1400300 to J.X.). We thank Drs. Yumei Wen and Jianxin Gu (Shanghai Medical College, Fudan University) for helpful discussions, and Dr. Lingli Chen (Zhongshan Hospital, Fudan University) for technical assistance.
REFERENCES 1. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma. N Engl J Med. 1996;335:865–75. 2. Figlin RA. Renal cell carcinoma: management of advanced disease. J Urol. 1999;161:381–6; discussion 386–7.
3. Sun M, Shariat SF, Cheng C, Ficarra V, Murai M, Oudard S, et al. Prognostic factors and predictive models in renal cell carcinoma: a contemporary review. Eur Urol. 2011;60:644–61. 4. Zisman A, Pantuck AJ, Wieder J, Chao DH, Dorey F, Said JW, et al. Risk group assessment and clinical outcome algorithm to predict the natural history of patients with surgically resected renal cell carcinoma. J Clin Oncol. 2002;20:4559–66. 5. Patard JJ, Kim HL, Lam JS, Dorey FJ, Pantuck AJ, Zisman A, et al. Use of the University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: an international multicenter study. J Clin Oncol. 2004;22:3316–22. 6. De Palma M, Lewis CE. Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell. 2013;23:277–86. 7. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306. 8. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 2011;11:723–37. 9. Falini B, Flenghi L, Pileri S, Gambacorta M, Bigerna B, Durkop H, et al. PG-M1: a new monoclonal antibody directed against a fixative-resistant epitope on the macrophage-restricted form of the CD68 molecule. Am J Pathol. 1993;142:1359–72. 10. Morris DL, Singer K, Lumeng CN. Adipose tissue macrophages: phenotypic plasticity and diversity in lean and obese states. Curr Opin Clin Nutr Metab Care. 2011;14:341–6. 11. Ito A, Suganami T, Yamauchi A, Degawa-Yamauchi M, Tanaka M, Kouyama R, et al. Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue. J Biol Chem. 2008;283:35715–23. 12. Nishimura S, Manabe I, Nagasaki M, Eto K, Yamashita H, Ohsugi M, et al. CD8? effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. Nat Med. 2009;15:914–20. 13. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313:1960–4. 14. Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, 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. 15. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122:787–95. 16. Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010;11:889–96. 17. Lawrence T, Natoli G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol. 2011;11:750–61. 18. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002;23:549–55. 19. Senovilla L, Vacchelli E, Galon J, Adjemian S, Eggermont A, Fridman WH, et al. Trial watch: Prognostic and predictive value of the immune infiltrate in cancer. Oncoimmunology. 2012;1:1323–43. 20. Jensen TO, Schmidt H, Moller HJ, Hoyer M, Maniecki MB, Sjoegren P, et al. Macrophage markers in serum and tumor have prognostic impact in American Joint Committee on Cancer stage I/II melanoma. J Clin Oncol. 2009;27:3330–7. 21. Komohara Y, Hasita H, Ohnishi K, Fujiwara Y, Suzu S, Eto M, et al. Macrophage infiltration and its prognostic relevance in clear cell renal cell carcinoma. Cancer Sci. 2011;102:1424–31. 22. Medrek C, Ponten F, Jirstrom K, Leandersson K. The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients. BMC Cancer. 2012;12:306.
Diametrically Polarized TAMs in RCC 23. Zhang BC, Gao J, Wang J, Rao ZG, Wang BC, Gao JF. Tumorassociated macrophages infiltration is associated with peritumoral lymphangiogenesis and poor prognosis in lung adenocarcinoma. Med Oncol. 2011;28:1447–52. 24. Zhang W, Zhu XD, Sun HC, Xiong YQ, Zhuang PY, Xu HX, et al. Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects. Clin Cancer Res. 2010;16:3420–30. 25. Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA. Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach. Br J Cancer. 2006;95:272–81. 26. Priceman SJ, Sung JL, Shaposhnik Z, Burton JB, Torres-Collado AX, Moughon DL, et al. Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy. Blood. 2010;115:1461–71. 27. Nguyen MT, Favelyukis S, Nguyen AK, Reichart D, Scott PA, Jenn A, et al. A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. J Biol Chem. 2007;282:35279–92. 28. Wu H, Perrard XD, Wang Q, Perrard JL, Polsani VR, Jones PH, et al. CD11c expression in adipose tissue and blood and its role in
29. 30.
31.
32.
33.
34.
diet-induced obesity. Arterioscler Thromb Vasc Biol. 2010;30: 186–92. Hume DA. Macrophages as APC and the dendritic cell myth. J Immunol. 2008;181:5829–35. Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol. 2010;10:453–60. Duluc D, Corvaisier M, Blanchard S, et al. Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages. Int J Cancer. 2009;125:367–73. Fong CH, Bebien M, Didierlaurent A, Nebauer R, Hussell T, Broide D, et al. An antiinflammatory role for IKKbeta through the inhibition of ‘‘classical’’ macrophage activation. J Exp Med. 2008;205:1269–76. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443–54. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455–65.
ORIGINAL ARTICLE – UROLOGIC ONCOLOGY
Prognostic Value of Diametrically Polarized Tumor-Associated Macrophages in Renal Cell Carcinoma Le Xu, MD, PhD1, Yu Zhu, MD, PhD2, Lian Chen, MD3, Huimin An, MD1, Weijuan Zhang, PhD4, Guomin Wang, MD1, Zongming Lin, MD1, and Jiejie Xu, MD, PhD5,6,7 Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China; 2Department of Urology, Ninth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China; 3Department of Pathology, Children’s Hospital, Fudan University, Shanghai, China; 4Department of Immunology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; 5Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; 6Key Laboratory of Glycoconjugate Research, MOH, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; 7Key Laboratory of Medical Molecular Virology, MOE & MOH, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China 1
ABSTRACT Background. As the most abundant tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) are significant for fostering tumor progression. CD68? TAMs display diversely polarized programs comprising CD11c? proinflammatory macrophages (M1) and CD206? immunosuppressive macrophages (M2). The aim of this study was to determine the survival impact of diametrically polarized TAMs in clear-cell renal cell carcinoma (ccRCC) and their application to stratification of patients according to their prognostic values. Methods. The study included 185 consecutive patients with ccRCC who underwent nephrectomy between 1999 and 2001. CD68? total and diametrically polarized (CD11c? M1 and CD206? M2) TAM densities were assessed by immunohistochemistry, and the relationships
Drs. Le Xu, Yu Zhu, and Lian Chen contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-3601-1) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 28 December 2013 Z. Lin, MD e-mail: [email protected] J. Xu, MD, PhD e-mail: [email protected]
with clinicopathologic features and prognosis were evaluated. Results. Low CD11c? TAM density and high CD206? TAM density were associated with reduced cancer-specific survival (P = 0.043 and P = 0.017, respectively), whereas CD68? TAM density only had borderline prognostic significance (P = 0.062). Furthermore, combined analysis of CD11c? and CD206? TAMs (CD11c/CD206 signature) had a better power to predict patients’ outcome (P = 0.010). Together with TNM stage, tumor necrosis, and performance status, CD11c/CD206 signature was an independent prognostic factor (P = 0.010). When applied to the University of California Integrated Staging System intermediate-/high-risk group for localized ccRCC, CD11c/ CD206 signature could further distinguish patients with dismal prognosis (P = 0.004). Conclusions. Intratumoral balance of diametrically polarized TAMs is a novel independent predictor for survival in patients with ccRCC. Tipping the balance toward an antitumoral phenotype might be a promising target of postoperative adjuvant therapy. Renal cell carcinoma (RCC), which accounts for 2–3 % of all adult malignant neoplasms, is the most lethal urologic cancer. The estimated 5-year survival rate for patients with localized RCC is approximately 70–90 %. However, once metastatic disease develops, the prognosis is poor, with 5-year survival rate ranging between 0 and 20 %.1,2 The natural history of RCC is complex and is influenced by factors other than pathologic stage.3 Currently, several
L. Xu et al.
prognostic models have been proposed to identify patients at high risk of disease progression after nephrectomy. One widely used model is the University of California integrated staging system (UISS), which incorporates TNM stage, Fuhrman grade, and Eastern Cooperative Oncology Group performance status (ECOG-PS), resulting in 3 risk groups [low- (LR), intermediate- (IR), and high-risk (HR)] for localized and metastatic RCC.4,5 Macrophages are a major cellular component of human tumors, where they are commonly termed tumor-associated macrophages (TAMs).6 A wide array of biologically active molecules are present in the tumor microenvironment as products of malignant and stromal cells. Infiltrating macrophages respond to this milieu and adapt a range of activation states that can be classified within the M1/M2 polarization model.7,8 M1 macrophages, activated by toll-like receptor (TLR) ligands and interferon-c (IFN-c), have roles in antitumor immunity. In contrast, M2 macrophages, stimulated by interleukin-4 (IL-4) or IL-13, have the ability to promote tumor growth and progression.8 Therefore, it is plausible that incorporation of macrophage polarization into established predictive models would improve prognostic stratification. There has been growing interest in studying the prognostic importance of macrophage infiltrations in solid tumors. CD68, the most widely used macrophage marker, is expressed on all macrophages.9 However, CD68 does not allow for the discrimination between M1 and M2 subsets. Recently, cell surface scavenger receptors, such as mannose receptor (CD206) and hemoglobin/haptoglobin scavenger receptor (CD163) have been recognized as specific M2 macrophage markers.6 Thus, M2 macrophages can be phenotypically discriminated from their M1 counterparts as the former express high levels of CD206 and CD163, while the latter are characterized by high amounts of CD11c and TLR4 on their surface.10–12 Based on this knowledge, we hypothesized that intratumoral CD68? pan-macrophages comprise CD11c? M1 macrophages and CD206? M2 macrophages. We, therefore, performed immunohistochemical analyses of CD68, CD11c, and CD206 in RCC tissues. Our data showed that both low CD11? TAM density and high CD206? TAM density associated with poor survival, whereas CD68? TAM density only had borderline prognostic significance. Furthermore, combined analysis of CD11c and CD206 (designated as CD11c/CD206 signature) outperformed single macrophage markers in predicting prognosis of RCC patients after surgery. MATERIALS AND METHODS Patients We retrospectively recruited 185 consecutive patients undergoing nephrectomy for clear-cell RCC (ccRCC) at
Zhongshan Hospital, Shanghai, China between 1999 and 2001. This study was approved by the hospital’s ethics committee and informed consent was obtained from each patient. For each patient, the following clinicopathologic information was collected: age, gender, tumor size, TNM stage, Fuhrman grade, presence of histologic tumor necrosis, and ECOG-PS. Patients were staged using radiographic reports and postoperative pathological data and were reassigned according to 2010 AJCC TNM classification. Patients with N1 or M1 tumors were considered to have metastatic disease. The UISS predictive model was applied to the patients.4 There were 2 decision boxes based on the interaction of TNM stage, Fuhrman grade, and ECOG-PS used to classify patients with localized and metastatic RCC into LR, IR, or HR groups. Patients with localized RCC (n = 169) were treated with radical or partial nephrectomy. Patients with metastatic RCC (n = 16) were treated with cytoreductive nephrectomy followed by interferon-a-based immunotherapy. After surgery, patients were evaluated with physical examination, laboratory studies, chest imaging, and abdominal ultrasound or CT scan every 6 months for the first 2 years and annually thereafter for 5 years. Median follow-up was 70 months (range, 10–120 months). Cancerspecific survival (CSS) was the main endpoint of the study. We calculated CSS from the date of nephrectomy to the date of death from RCC or last follow-up. There were 4 patients who died from other causes and were censored for the CSS endpoint. Immunohistochemistry Immunohistochemical analysis is described in the supplemental material, in the section Supplemental Methods. Primary antibodies against human CD68 (Dako, Glostrup, Denmark), CD11c (Abcam, Cambridge, MA) and CD206 (Abcam, Cambridge, MA) were applied in the procedure. Statistical Analyses The ‘‘minimum P value’’ approach was applied to obtain the cutoff providing the best separation between the groups of patients related to their CSS (Fig. S1).13,14 The cutoff values for low and high CD68?, CD206?, and CD11c? TAM densities were 50 (30th percentile), 30 (30th percentile), and 55 (80th percentile) cells per field (9200), respectively. Correlations between immunohistochemical variables and clinicopathologic characteristics were analyzed with v2 and t tests. Kaplan–Meier method with log-rank test was applied to compare survival curves. All statistical tests were two sided and performed at a significance level of 0.05. Univariate and multivariate Cox regression models
Diametrically Polarized TAMs in RCC
FIG. 1 Representative photographs of CD68 (a, d, g, and j), CD11c (b, e, h, and k), and CD206 (c, f, i, and l) immunostaining in tissue microarrays (original magnification 9200). Case 13 (a–c) showed low densities of CD68?, CD11c?, and CD206? macrophages. Case 74 (d–f) showed high densities of CD68?, CD11c?, and CD206?
macrophages. Case 94 (g–i) showed high densities of CD68? and CD11c? macrophages, but low CD206? macrophage density. Case 180 (j–l) showed high densities of CD68? and CD206? macrophages, but low CD11c? macrophage density
were used to analyze the impact of prognostic factors on CSS, and P [ 0.10 was the removal criterion when performing backward stepwise variable deletions. The
predictive accuracy of various Cox regression models was quantified by Harrell’s concordance index (C-index). Data were analyzed using SPSS Statistics 17.0 and Stata 11.0.
L. Xu et al.
Immunohistochemical Findings CD68, CD11c, and CD206 positive staining mainly appeared in the cytoplasm of macrophages (Fig. 1). Sporadic positive CD68 staining was observed in cancer cells; no CD11c or CD206 staining was detected in cancer cells. Statistics of immunohistochemical variables are shown in Table S2. Generally, CD68? cells were more abundant than CD11c? or CD206? cells, and CD11c? cells were outnumbered by CD206? cells in RCC tissues (all P \ 0.001). Survival Analyses with Single Macrophage Markers and Correlations Between These Markers and Clinicopathologic Features Kaplan–Meier analyses indicated that, as single variables, low CD11? TAM density and high CD206? TAM density associated with reduced survival (P = 0.043 and P = 0.017, respectively) (Fig. 2b, c), whereas CD68? TAM density only had borderline prognostic significance (P = 0.062) (Fig. 2a). As summarized in Table 1, patients with high CD68? TAM density were prone to having high-grade tumors and histologic necrosis (P = 0.022 and P = 0.035, respectively). Female patients tended to harbor more CD11c? TAMs (P = 0.017). CD11c? TAM density was positively correlated with Fuhrman grade (P = 0.021). CD206? TAM density was positively correlated with patient’s age and ECOG-PS (P = 0.014 and P = 0.015, respectively). Combined Analysis of CD11c and CD206 Has a Better Power to Predict Outcome Since M1 and M2 macrophage
Cancer-specific survival
As summarized in Table S1, median age was 60 years (range, 30–84 years), median tumor size was 5 cm (range, 1–14 cm), and histologic necrosis was presented in 42.7 % of cases. Also, 7 patients (3.8 %) had lymph node metastases and 10 patients (5.4 %) had distant metastases at the time of surgery. The distribution of TNM stages I, II, III, and IV was 64.9, 14.1, 15.7, and 5.4 %, respectively. Fuhrman grades were 1, 2, 3, and 4 in 7.0, 33.0, 43.8, and 16.2 % of cases, respectively. ECOG-PS was evaluated as C1 in 42.7 % of cases. Among 169 patients with localized RCC, UISS was classified as LR, IR, and HR in 19.5, 73.4, and 7.1 % of cases, respectively. Among 16 patients with metastatic RCC, UISS was classified as LR, IR, and HR in 50.0, 43.8, and 6.2 % of cases, respectively. At the time of last follow-up, 37.8 % of patients had died of RCC.
100 80 60 40
P = 0.062 Low CD68+ TAMs High CD68+ TAMs
20 0
24
0
48
72
96
120
Time after surgery (months) Number at risk Low CD68+ TAMs 55 High CD68+ TAMs 130
b Cancer-specific survival
Patient Characteristics
a
54
42
28
15
0
119
88
51
18
0
72
96
120
100 80 60 40
P = 0.043 Low CD11c+ TAMs High CD11c+ TAMs
20 0 0
24
48
Time after surgery (months) Number at risk Low CD11c+ TAMs 147 137 High CD11c+ TAMs 38 36
c Cancer-specific survival
RESULTS
105
60
28
0
25
19
5
0
72
96
120
100 80 60 40
P = 0.017 Low CD206+ TAMs High CD206+ TAMs
20 0 0
24
48
Time after surgery (months) Number at risk Low CD206+ TAMs 55 54 High CD206+ TAMs 130 119
44
35
14
0
86
44
19
0
FIG. 2 Kaplan-Meier curves showing CSS probabilities based on intratumoral CD68?, CD11c?, and CD206? macrophage densities (a, b, and c). TAMs tumor-associated macrophages
subtypes perform opposing functions, it is believed that M1/ M2 polarization in tumor tissue might have an impact on cancer progression. Thus, we proposed that combined analysis of CD11c and CD206 would allow for improved prognostic stratification of RCC patients by assessing both
Diametrically Polarized TAMs in RCC TABLE 1 Correlations between immunohistochemical variables and clinicopathologic characteristics CD68? TAMs
Variable
Mean age (years)a
CD11c? TAMs
Low (n = 55)
High (n = 130)
58.7
61.4
Gender
CD206? TAMs
P
Low (n = 147)
High (n = 38)
0.176
60.7
60.0
0.290
Male Female
23 (12.4 %) 31 (16.8 %)
46 (24.9 %) 84 (45.5 %)
5.0
5.4
I ? II
44 (23.8 %)
102 (55.1 %)
III ? IV
11 (5.9 %)
28 (15.2 %)
Mean tumor size (cm)a TNM stage
0.261
Low (n = 55)
High (n = 130)
P
0.736
57.2
62.0
0.014
23 (12.4 %) 32 (17.3 %)
47 (25.4 %) 83 (44.9 %)
5.4
5.2
46 (24.9 %)
100 (54.1 %)
9 (4.9 %)
30 (16.1 %)
0.017 62 (33.5 %) 85 (45.9 %)
8 (4.3 %) 30 (16.2 %)
5.2
5.8
117 (63.2 %)
29 (15.7 %)
30 (16.2 %)
9 (4.9 %)
0.815
Fuhrman grade
P
0.177
0.468
0.659
0.022
0.306
0.021
0.511
1?2
29 (15.7 %)
45 (24.3 %)
65 (35.2 %)
9 (4.7 %)
24 (13.0 %)
50 (27.0 %)
3?4
26 (14.1 %)
85 (45.9 %)
82 (44.4 %)
29 (15.7 %)
31 (16.8 %)
80 (43.2 %)
Absent
38 (20.5 %)
68 (36.8 %)
86 (46.5 %)
20 (10.8 %)
36 (19.5 %)
70 (37.8 %)
Present
17 (9.2 %)
62 (33.5 %)
61 (33.0 %)
18 (9.7 %)
19 (10.3 %)
60 (32.4 %)
42 (22.7 %)
61 (33.0 %)
13 (7.0 %)
69 (37.3 %)
12 (7.1 %)
21 (12.4 %)
Necrosis
0.035
ECOG-PS
0.514
0.145
0
39 (21.1 %)
64 (34.6 %)
C1
16 (8.6 %)
66 (35.7 %)
UISS (localized) LR
0.145
0.933 84 (45.3 %)
19 (10.3 %)
63 (34.1 %)
19 (10.3 %)
0.229
0.015
0.054
19 (11.2 %)
IR
34 (20.1 %)
90 (53.3 %)
93 (55.0 %)
31 (18.3 %)
38 (22.5 %)
86 (50.9 %)
HR
3 (1.8 %)
9 (5.3 %)
10 (5.9 %)
2 (1.2 %)
1 (0.6 %)
11 (6.5 %)
LR
2 (12.5 %)
6 (37.5 %)
6 (37.5 %)
2 (12.5 %)
1 (6.2 %)
7 (43.8 %)
IR ? HR
2 (12.5 %)
6 (37.5 %)
7 (43.8 %)
1 (6.2 %)
3 (18.8 %)
5 (31.2 %)
UISS (metastatic)
2 (1.2 %)
0.189
14 (8.3 %)
b
31 (18.3 %)
1.000
0.707
1.000
0.569
TAMs tumor-associated macrophages, ECOG-PS Eastern Cooperative Oncology Group performance status, UISS University of California Los Angeles Integrated Staging System, LR low risk, IR intermediate risk, HR high risk a b
Student’s t test Fisher exact test; Chi square test for all the other analyses
antitumoral M1 macrophages and protumoral M2 macrophages. Patients were classified into 4 groups according to their CD11c? and CD206? TAM densities: group I, high CD11c? but low CD206? TAM densities; group II, both low densities; group III, both high densities; group IV, low CD11c? but high CD206? TAM densities. Significant difference in CSS (P = 0.010) was found among the 4 groups (Fig. 3a). The 5-year CSS rates for groups I, II, III, and IV were 100, 84.5, 82.9, and 66.8 %, respectively. Thus, an immune profile characterized by CD11chigh/ CD206low may reflect an M1-skewed status in tumor tissue and is predictive of a favorable outcome. In contrast, an immune response characterized by CD11clow/CD206high may represent an M2-skewed status and a population of patients with poor prognosis. In groups II and III, the influence of CD11c? TAMs, low or high, on prognosis was probably counterbalanced by simultaneous low or high CD206? TAMs, and vice versa. Therefore, patients in
groups II and III had the intermediate survival data. When the analysis was restricted to localized RCC, a CD11chigh/ CD206low immune profile was correlated with a long-term CSS rate of 100 % (P = 0.013) (Fig. 3b). However, CD11c/ CD206 signature was not predictive in metastatic RCC in our study (P = 0.400) (Fig. S2). Table 2 lists the univariate and multivariate analyses of potential prognostic factors for CSS. After backward stepwise variable selection, TNM stage (P \ 0.001), tumor necrosis (P = 0.031), ECOG-PS (P = 0.003), and CD11c/ CD206 signature (P = 0.010) remained in the model as independent prognostic factors. These variables collectively yielded a predictive accuracy of 81.2 %. Extension of the UISS Prognostic Model with TAM Polarization For localized RCC, the 5-year CSS rates were 93.3, 80.0, and 38.9 % for UISS LR, IR, and HR, respectively. For metastatic RCC patients grouped according
L. Xu et al. b FIG. 3 Kaplan–Meier curves showing CSS probabilities based on
Cancer-specific survival
a
All RCC 100 80 60 I II III IV
40 20
P = 0.010
0 0
Number at risk Group I 10 Group II 39 Group III 28 Group IV 108
24
Cancer-specific survival
72
96
12 0
10
7
7
3
0
39
33
26
11
0
26
18
12
2
0
98
72
34
17
0
b
Localized RCC 100 80 60 I II III IV
40 20
difference in CSS (P = 0.004) was found among these 4 groups according to CD11c/CD206 signature (Fig. 3c). Thus, in UISS IR/HR group for localized RCC, the 5-year CSS rate was 100 % if tumors displayed a CD11chigh/CD206low immune profile in contrast to 65 % if a CD11clow/ CD206high profile was observed. The C-index was 0.718 when assessed with UISS outcome algorithm and was improved to 0.752 when CD11c/CD206 signature was added. However, in UISS LR group for localized RCC, CD11c/CD206 signature failed to further stratify patients with different prognosis (P = 0.786) (Fig. S3). DISCUSSION
P = 0.013
0 0
Number at risk Group I 9 Group II 36 Group III 26 Group IV 98
c Cancer-specific survival
48
Time after surgery (months)
the combination of CD11c? and CD206? TAM densities in all patients (a), patients with localized RCC (b), and patients in the UISS combined intermediate- and high-risk group for localized RCC (c). Patients were classified into 4 groups according to their CD11c? and CD206? TAM densities: group I, high CD11c? but low CD206? TAM densities; group II, both low densities; group III, both high densities; group IV, low CD11c? but high CD206? TAM densities. TAMs tumor-associated macrophages, RCC renal cell carcinoma, UISS University of California Los Angeles Integrated Staging System
24
48
72
96
12 0
Time after surgery (months) 9
6
6
3
0
36
33
26
11
0
25
17
11
2
0
92
70
34
17
0
UISS intermediate/high risk for localized RCC 100 80 60 I II III IV
40 20
P = 0.004
0 0
24
48
72
96
12 0
Time after surgery (months) Number at risk Group I 8 Group II 26 Group III 25 Group IV 77
8
5
5
3
0
26
23
19
6
0
24
16
11
2
0
71
51
26
10
0
to UISS LR and IR, the 5-year CSS rates were 22.5 and 12.5 %, respectively. Then we investigated whether incorporation of M1/M2 TAM polarization into the UISS outcome algorithm would improve prognostic stratification. In UISS IR/HR group for localized RCC, significant
In the present study, we showed that identification of intratumoral macrophage polarization using CD11c and CD206 immunostaining can predict outcomes for surgically treated RCC patients, especially for those with localized disease. Moreover, incorporation of CD11c/CD206 signature into the UISS predictive model could further stratify patients and provide more prognostic information. For example, patients with a CD11clow/CD206high immune profile may need to undergo a closer follow-up after surgery. However, the incorporation of TAM polarization into current prognostic models requires validation in an independent and larger population. In this study, we observed a relatively lower proportion of patients with metastatic disease compared with other studies. It is because we chose to investigate surgically treated RCC patients, while those patients with metastatic RCC who only received immunotherapy were not included in this study. Depending on external stimuli, macrophages can acquire different phenotypes with partly opposing properties.15 M1 macrophages mediate defense of the host from a variety of bacteria and viruses and play roles in anticancer immunity. M2 macrophages have anti-inflammatory function, favor angiogenesis, and promote tumor growth.8 It is important to note that ‘‘M1’’ and ‘‘M2’’ polarization states are extremes of a spectrum, macrophages often express a mixed M1/M2 phenotype in response to signals present within individual microenvironments.16,17 It is believed TAMs are predominantly polarized in the tumor microenvironment toward a protumoral M2 phenotype.18 Consistent with this notion, our data showed that
Diametrically Polarized TAMs in RCC TABLE 2 Univariate and multivariate Cox regression analyses of potential prognostic factors for CSS Variable
Cancer-specific survival Univariate analysis
Tumor size (cm)
HR (95 % CI)
Pc
C index
1.235 (1.135–1.345)
\0.001
0.653
\0.001
0.684
TNM stage a
Multivariate analysis
I vs. I III vs. Ia
1.547 (0.746–3.208) 2.131 (1.150–3.948)
0.241 0.016
IV vs. Ia
Pc
HR (95 % CI)
\0.001 1.746 (0.761–4.007) 1.739 (0.905–3.340)
0.188 0.097
15.015 (6.433–35.045)
\0.001
11.783 (5.720–24.272)
\0.001
Fuhrman grade (3 ? 4 vs. 1 ? 2a)
1.607 (1.196–2.161)
0.002
0.613
Tumor necrosis (yes vs. noa)
3.089 (1.903–5.015)
\0.001
0.645
1.846 (1.057–3.225)
0.031
ECOG-PS (C1 vs. 0 )
2.394 (1.479–3.873)
\0.001
0.600
2.121 (1.285–3.501)
0.003
CD68? TAMs (high vs. lowa)
1.688 (0.981–3.002)
0.067
0.562
a
CD11c? TAMs (high vs. lowa)
0.477 (0.228–0.996)
0.049
0.568
CD206? TAMs (high vs. lowa)
1.949 (1.112–3.418)
0.020
0.580
0.018
0.645
CD11c/CD206 signatureb II vs. Ia
3.068 (0.399–23.601)
0.282
a
3.288 (0.404–26.768)
IV vs. Ia
6.548 (0.903–47.488)
III vs. I C index
0.010 6.775 (0.853–53.829)
0.070
0.266
6.086 (0.698–53.048)
0.102
0.063
13.180 (1.722–100.87)
0.013
0.812
HR hazard ratio, 95 % CI 95 % confidence interval, ECOG-PS Eastern Cooperative Oncology Group performance status, TAMs tumorassociated macrophages, C index concordance index a
Reference group
b
Patients were classified into 4 groups according to their CD11c? and CD206? TAM densities: group I, high CD11c? but low CD206? TAM densities; group II, both low densities; group III, both high densities; group IV, low CD11c? but high CD206? TAM densities
c
An overall statistic (P) is provided for categorical variables. For these variables, hazard ratios and P values are provided for each variable level
intratumoral CD206? cells were more abundant than CD11c? cells in most cases, indicating an M2-polarized macrophage infiltration in RCC tissues. Conversely, CD11c? cells outnumbering CD206? cells was also observed in a few cases, suggesting the existence of TAMs with a relatively M1-skewed phenotype. However, the absolute cell numbers may not reflect actual macrophage polarization in the tumor microenvironment. Hence, the CD11c/CD206 signature was used in the study to delineate macrophage polarization and categorize patients. A CD11chigh/CD206low immune profile indicated an M1polarized macrophage infiltration and associated with favorable prognosis after nephrectomy. In contrast, a CD11clow/CD206high immune profile denoted an M2polarized macrophage infiltration and was predictive of inferior outcome. Additionally, CD11clow/CD206low and CD11chigh/CD206high profiles might suggested a mixed M1/M2 phenotype and associated with intermediate survival. The first studies attempting to correlate TAMs with prognosis generated heterogeneous results, most likely because macrophage infiltration was evaluated with antiCD68 antibodies, which do not distinguish between M1
and M2 subsets.19 Recently, clinical studies based on M2specific markers, such as CD163, have provided more consistent results. It has been reported that elevated amounts of intratumoral CD163? macrophages associated with poor prognosis in patients with melanoma, ccRCC, breast cancer, and lung cancer.20–23 Increasing studies have also shown that TAMs drive reparative mechanisms in tumors after treatment with vascular-targeting agents. Depletion of TAMs by clodronate-loaded liposomes (clodrolip) augmented the inhibitory effects of sorafenib on tumor angiogenesis and metastasis in hepatocellular carcinoma.24 Moreover, TAM depletion by clodrolip or a CSF1R inhibitor increased the antiangiogenic and antitumor effects of VEGF/VEGFR2 antibodies in subcutaneous tumor models.25,26 In the present study, we used CD11c and CD206 as the markers for M1 and M2 macrophages, respectively. CD11c was commonly used as an M1 marker in adipose tissue and was also regarded as a marker for myeloid dendritic cells (DCs), which may call into question whether these CD11c? cells are macrophages or DCs.27,28 A major problem in defining macrophages and DCs lies in the fact that they stem from common myeloid precursors and express common cell surface markers.8 It should be
L. Xu et al.
noted that this problem is not unique to this field, as the distinction between macrophages and DCs is controversial among immunologists even in well-defined lymphoid tissues.29,30 It is interesting that CD11c? TAM density was positively correlated with Fuhrman grade in this study. One possible explanation was that high-grade tumors are poorly differentiated and probably express more molecular patterns and antigens that the immune system could recognize. In this regard, high-grade tumors are more immunogenic than low-grade tumors and thus harbor more proinflammatory CD11c? macrophages. Since our data showed that most RCC patients (94.6 %) displayed an M2-skewed phenotype or a mixed M1/M2 phenotype in this study, tipping the macrophage balance toward a tumoricidal M1 phenotype by immunological intervention represents a promising adjuvant therapy for RCC. Therefore, IFN-c could be administered locally at the tumor site to reverse the immunosuppressive and protumoral properties of M2 macrophages and thus enhance the efficacy of active and adoptive antitumor immunotherapies.31 In addition, suppressing nuclear factor-jB (NF-jB) pathway can switch TAMs to an M1-polarized phenotype that is cytotoxic to malignant cells.32 Recently, monoclonal antibodies targeting the immune inhibitory co-receptor programmed death1 (PD-1) protein and 1 of its ligands, PD-L1, have demonstrated increased antitumor responses and evident therapeutic benefits in patients with advanced cancer.33,34 Macrophages and DCs also express PD-L1 that could suppress T cell activation. Thus, blocking PD-L1 on TAMs might enhance the efficacy of anticancer therapies. In conclusion, our results indicate that the concurrence of low CD11c? macrophages and high CD206? macrophages in RCC tissues suggests an M2-polarized status and associates with reduced survival. This finding provides a novel independent predictor for prognosis and may improve current predictive models with regard to counseling patients, selecting patients for adjuvant therapies, and customizing follow-up after surgery. ACKNOWLEDGMENT This work was supported by grants from National Key Projects for Infectious Diseases of China (2012ZX10002-012 to J.X.), National Natural Science Foundation of China (31100629 to W.Z., 31270863 to J.X., 81372755 to Z.L.), Program for New Century Excellent Talents in University (NCET-130146 to J.X.), and Shanghai Rising-Star Program (13QA1400300 to J.X.). We thank Drs. Yumei Wen and Jianxin Gu (Shanghai Medical College, Fudan University) for helpful discussions, and Dr. Lingli Chen (Zhongshan Hospital, Fudan University) for technical assistance.
REFERENCES 1. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma. N Engl J Med. 1996;335:865–75. 2. Figlin RA. Renal cell carcinoma: management of advanced disease. J Urol. 1999;161:381–6; discussion 386–7.
3. Sun M, Shariat SF, Cheng C, Ficarra V, Murai M, Oudard S, et al. Prognostic factors and predictive models in renal cell carcinoma: a contemporary review. Eur Urol. 2011;60:644–61. 4. Zisman A, Pantuck AJ, Wieder J, Chao DH, Dorey F, Said JW, et al. Risk group assessment and clinical outcome algorithm to predict the natural history of patients with surgically resected renal cell carcinoma. J Clin Oncol. 2002;20:4559–66. 5. Patard JJ, Kim HL, Lam JS, Dorey FJ, Pantuck AJ, Zisman A, et al. Use of the University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: an international multicenter study. J Clin Oncol. 2004;22:3316–22. 6. De Palma M, Lewis CE. Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell. 2013;23:277–86. 7. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306. 8. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 2011;11:723–37. 9. Falini B, Flenghi L, Pileri S, Gambacorta M, Bigerna B, Durkop H, et al. PG-M1: a new monoclonal antibody directed against a fixative-resistant epitope on the macrophage-restricted form of the CD68 molecule. Am J Pathol. 1993;142:1359–72. 10. Morris DL, Singer K, Lumeng CN. Adipose tissue macrophages: phenotypic plasticity and diversity in lean and obese states. Curr Opin Clin Nutr Metab Care. 2011;14:341–6. 11. Ito A, Suganami T, Yamauchi A, Degawa-Yamauchi M, Tanaka M, Kouyama R, et al. Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue. J Biol Chem. 2008;283:35715–23. 12. Nishimura S, Manabe I, Nagasaki M, Eto K, Yamashita H, Ohsugi M, et al. CD8? effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. Nat Med. 2009;15:914–20. 13. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313:1960–4. 14. Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, 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. 15. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122:787–95. 16. Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010;11:889–96. 17. Lawrence T, Natoli G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol. 2011;11:750–61. 18. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002;23:549–55. 19. Senovilla L, Vacchelli E, Galon J, Adjemian S, Eggermont A, Fridman WH, et al. Trial watch: Prognostic and predictive value of the immune infiltrate in cancer. Oncoimmunology. 2012;1:1323–43. 20. Jensen TO, Schmidt H, Moller HJ, Hoyer M, Maniecki MB, Sjoegren P, et al. Macrophage markers in serum and tumor have prognostic impact in American Joint Committee on Cancer stage I/II melanoma. J Clin Oncol. 2009;27:3330–7. 21. Komohara Y, Hasita H, Ohnishi K, Fujiwara Y, Suzu S, Eto M, et al. Macrophage infiltration and its prognostic relevance in clear cell renal cell carcinoma. Cancer Sci. 2011;102:1424–31. 22. Medrek C, Ponten F, Jirstrom K, Leandersson K. The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients. BMC Cancer. 2012;12:306.
Diametrically Polarized TAMs in RCC 23. Zhang BC, Gao J, Wang J, Rao ZG, Wang BC, Gao JF. Tumorassociated macrophages infiltration is associated with peritumoral lymphangiogenesis and poor prognosis in lung adenocarcinoma. Med Oncol. 2011;28:1447–52. 24. Zhang W, Zhu XD, Sun HC, Xiong YQ, Zhuang PY, Xu HX, et al. Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects. Clin Cancer Res. 2010;16:3420–30. 25. Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA. Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach. Br J Cancer. 2006;95:272–81. 26. Priceman SJ, Sung JL, Shaposhnik Z, Burton JB, Torres-Collado AX, Moughon DL, et al. Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy. Blood. 2010;115:1461–71. 27. Nguyen MT, Favelyukis S, Nguyen AK, Reichart D, Scott PA, Jenn A, et al. A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. J Biol Chem. 2007;282:35279–92. 28. Wu H, Perrard XD, Wang Q, Perrard JL, Polsani VR, Jones PH, et al. CD11c expression in adipose tissue and blood and its role in
29. 30.
31.
32.
33.
34.
diet-induced obesity. Arterioscler Thromb Vasc Biol. 2010;30: 186–92. Hume DA. Macrophages as APC and the dendritic cell myth. J Immunol. 2008;181:5829–35. Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol. 2010;10:453–60. Duluc D, Corvaisier M, Blanchard S, et al. Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages. Int J Cancer. 2009;125:367–73. Fong CH, Bebien M, Didierlaurent A, Nebauer R, Hussell T, Broide D, et al. An antiinflammatory role for IKKbeta through the inhibition of ‘‘classical’’ macrophage activation. J Exp Med. 2008;205:1269–76. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443–54. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455–65.
