High CD4⁺ T cell density is associated with poor prognosis in patients with non-muscle-invasive bladder cancer

Abstract

Purpose: The aim of this study was to investigate the clinical significance of CD4⁺ T cells in non-muscle-invasive bladder cancer (NMIBC) tissues in situ. Methods: Immunohistochemistry was used to examine the distribution of CD4⁺ T cells in 131 NMIBC tissues. Kaplan-Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS) and recurrence-free survival (RFS). Results: NMIBC patients were divided into two groups based on the median frequency of CD4⁺ T cells (median, 1/×400 high resolution). On univariate analysis, CD4⁺ T cell density was inversely associated with overall survival (P = 0.01). In those patients with high CD4⁺ T density, 5-year OS rates was only 77%, compared with 86% in those with low density, respectively. Although CD4⁺ T cell density showed no prognostic significance for RFS (P = 0.36), 5-year RFS rates of patients with high CD4⁺ T density (58%) was lower than those of patients with low CD4⁺ T density (65%, respectively). By multivariate analysis, tumor infiltrating CD4⁺ T cell density emerged as an independent prognostic factor for OS (HR, 2.75; P = 0.004). In addition, no association was found between CD4⁺ T cell density and any clinicopathological variables (P > 0.05). Conclusion: Our findings suggest that CD4⁺ T cells could potentially serve as a poor prognostic marker for patients with NMIBC.

Introduction

High-risk Non-muscle-invasive bladder cancer (NMIBC) is clinically characterized by high recurrence rates and represents one of the most costly cancers [1]. Bacillus Calmette-Guerin immune therapy has been used to prevent recurrence in patients with NMIBC for more than 40 years [2-4]. Although little is known about the exact mechanism for the effectiveness of Bacillus Calmette-Guerin immune therapy in NMIBC, induction of a local T cell-mediated immune response seems to be the most likely cause [5].

Tumor-infiltrating T cells may be an indicator of the host immune response to tumor and an attractive target for immunotherapy [6,7]. Most emphasis to date has been focused on CD8⁺ T cells, as they can be directly cytotoxic to tumor cells and their abundance inside tumors appears to indicate better prognosis [8]. Previous studies have shown that CD8⁺ T cells infiltration is associated with improved disease outcome in human bladder cancer [9] and other various tumors, including ovarian, renal, lung and colorectal carcinoma [10-13]. In contrast, the complex ways of CD4⁺ T cells influencing tumor immunity are not fully understood [8,14]. In mice models, tumor specific CD4⁺ T cells can reject myeloma by activating macrophages [15]. CD4⁺ T cells can also be more efficient than CD8⁺ T cells at rejecting solid tumors [16]. In fact, CD4⁺ T cells can either display anti- or pro-tumoral activity [17-19]. However, little is known about the clinical significance of CD4⁺ T cells in patients with NMIBC.

Herein, by using a new rabbit anti-human CD4 antibody, we investigated the prognostic significance of CD4⁺ T cells in 131 patients with NMIBC. The results showed that CD4⁺ T cells could predict a poor prognosis in human bladder cancer.

Materials and methods

Patients and tissue specimens

Tissue specimens were obtained from 131 patients who had pathologically confirmed NMIBC, at Tengzhou central people’s hospital of Shandong province during January 2003 and December 2009. No patient had a distant metastasis or received anticancer therapies prior to surgery. All tumors were graded according to the World Health Organization 2004 classification and staged according to the TNM classification (6th edition, 2002). Specimens were obtained immediately after surgical resection and fixed in 10% neutral formalin, then paraffin embedded and used for histological assays. This study was approved by the Ethics Committee of our institution, following the ethical guidelines of the Declaration of Helsinki, with informed consent in writing obtained from every patient.

The clinicopathologic and follow-up data of patients were prospectively collected. All patients were evaluated every 3 months during the first year, every 6 months during the second year, and annually thereafter. Follow-up visits consisted of a history, physical examination, and routine biochemical analyses. Ultrasonography of the abdomen, urography, and chest X-rays were performed at 3, 6 and 12 months post operatively, and then annually unless otherwise clinically indicated. Abdominal/pelvic CT scans were performed 6 months postoperatively and annually thereafter. Overall survival (OS) was defined as the interval between surgery and death or between surgery and the last observation for surviving patients. Recurrence-free survival (RFS) was defined as the interval between surgery and recurrence or between surgery and the last observation for patients without recurrence.

Immunohistochemistry

Formalin-fixed and paraffin-embedded samples were cut into 4-μm sections, which then were processed for immunohistochemistry as previously described [20]. Briefly, paraffin sections were first deparaffinized and hydrated, then endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide containing double distilled water. Antigen retrieval was performed by microwave treatment in citrate buffer (pH 6.0). Sections were blocked with normal sera from the same species from which secondary antibodies were derived. After overnight incubation at 4°C with antibody against CD4 (1:500 dilution, Sino Biological Inc., Beijing, China), or control antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), sections were incubated with secondary antibody conjugated to horseradish peroxidase (Envision + Dual Link Kit, DAKO) for 30 min at 37°C. The enzymatic reactions were developed peroxidase and 3,3’-diaminobenzidine tetrahydrochloride. Sections were counterstained with hematoxylin (Zymed Laboratories, San Francisco, CA, USA) and mounted in nonaqueous mounting medium.

Evaluation of immunohistology

Tissue sections were analyzed by two independent observers who were blinded to the clinical outcome. To evaluate the density of tissue-infiltrating CD4⁺ T cells, tissue sections were screened at a low-power field (100×) and the five most representative fields were selected for analysis at 400× magnification (0.07 mm² per field) using a Nikon DS-Fi2 CCD camera (Nikon, Tokyo, Japan) that was installed on a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). The infiltrating cells per field were enumerated manually and counts were expressed as means ± SEM. Because macrophages occasionally stain with CD4 antibody, CD4⁺ T cells with apparent morphological appearance of macrophages were excluded from the count. A significant linear correlation existed between the counts of two independent observers and the average count of the two investigators was used in subsequent analyses to minimize inter-observer variability.

Statistical analyses

Statistical analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Cumulative survival time was calculated using the Kaplan-Meier method and was analyzed by the log-rank test. A multivariate Cox proportional hazards model was used to estimate the adjusted hazard ratios and 95% confidence intervals (CIs), and to identify independent prognostic factors. For categorical analyses, the median value was used as a cut-off to dichotomize continuous variables (for clinical applications). The relationships between categorical variables were analyzed using χ² tests. For such comparisons, two-tailed P-values < 0.05 were considered statistically significant.

Results

Study population

Clinicopathological characteristics of the 131 NMIBC patients are presented in Table 1. Median age of patients was 60 yr, 86.3% were male, 82.4% had tumors < 3 cm, 67.9% had multifocal disease, 23.7% had Ta stage, and 71.8% had low histological grade. The median follow-up for living patients was 76.9 months (range, 6.4-137.3 months). Of 131 patients, 37 (28.2%) patients died and 59 (45%) patients had evidence of disease recurrence during this follow-up period. The OS and RFS rates were 97% and 78% at 2 year, 79% and 62% at 5 years, respectively.

Table 1.

Clinicopathological characteristics of non-muscle-invasive bladder cancer patients

Variable	No.
No. of patients	131
Age, years (median, range)	62 (20-90)
Sex (male/female)	113/18 (86.3%/13.7%)
Tumor size (≤ 3 cm/> 3 cm)	108/23 (82.4%/17.6%)
Tumor number (unifocal/multifocal )	89/42 (67.9%/32.1%)
Tumor stage (Ta/T1)	31/100 (23.7%/76.3%)
Histological grade (low/high)	94/37 (71.8%/28.2%)

Survival analysis

On univariate analysis (Table 2), sex, tumor size, tumor number and tumor stage showed no prognostic significance for OS and RFS. In contrast, age was predictor for OS (P < 0.001) and histological grade was predictor for RFS (P = 0.04). For further analysis, the counting data of CD4⁺ T cells were divided into 2 groups: high density and low density. Kaplan-Meier survival curves were then plotted to investigate further the association with survival (Figure 1). The log-rank statistic was used to compare survival rates. There was an inverse correlation between CD4⁺ T cell density and OS (P = 0.01, Figure 1A). Patients with higher CD4⁺ T cell density had significantly shorter OS (median 75.9 months) than those with lower CD4⁺ T cell density (median 82.4 months). In those patients with a CD4⁺ T density above the median, 5-year and 10-year OS rates were only 77% and 54%, compared with 86% and 71% in those below the median, respectively. Although CD4⁺ T cell density showed no prognostic significance for RFS (P = 0.36, Figure 1B), 5-year and 10-year RFS rates of patients with a CD4⁺ T density above the median (58% and 32%, respectively) were lower than those of patients with a CD4⁺ T density below the median (65% and 54%, respectively). However, no association was found between CD4⁺ T cell density and clinicopathological variables including age, sex, tumor size, tumor number, tumor stage and histological grade (Table 3).

Table 2.

Univariate analysis of the factors associated with survival and recurrence

	Overall survival			Recurrence-free survival
Variable	HR	95% CI	P	HR	95% CI	P
Age, years (> 62/≤ 62)	4.843	2.283-10.277	< 0.001	0.888	0.527-1.495	0.654
Sex (female/male)	0.762	0.27-2.153	0.608	1.127	0.554-2.292	0.742
Tumor size (> 3 cm/≤ 3 cm)	1.8	0.849-3.819	0.125	0.912	0.448-1.856	0.8
Tumor number (multifocal/unifocal)	0.856	0.414-1.771	0.675	1.556	0.919-2.633	0.1
Tumor stage (T1/Ta)	0.604	0.285-1.281	0.189	0.812	1.449-1.47	0.492
Histological grade (high/low)	1.615	0.831-3.14	0.158	1.75	1.025-2.988	0.04
CD4+ T cells (high/low)	2.487	1.248-4.955	0.01	1.43	0.856-2.389	0.172

Abbreviations: HR, hazard ratio; CI, confidence interval; NA, not applicable. Note: Univariate and multivariate analysis. Cox proportional hazards regression model. HR > 1, risk for death increased; HR < 1, risk for death reduced.

Figure 1.

Prognostic significance of CD4⁺ T cells in NMIBC (n = 131). A, B: Representative pictures of CD4⁺ T cells. Patients were divided into two groups according to the median number of CD4⁺ T cells per ×400 field (median = 1). Bars, 50 μm. C, D: Cumulative overall survival and recurrence-free survival curves were calculated by the Kaplan-Meier method and analyzed by the log-rank test. Dash lines, low group; Solid lines, high group.

Table 3.

Associations between CD4⁺ T cell density and the clinicopathological characteristics

	CD4+ T cell density
Variable	Low	High	R	P
No. of patients	67 (51%)	64 (49%)
Age, years			-0.01	0.907
≤ 62	37 (55%)	36 (56%)
> 62	30 (45%)	28 (44%)
Sex			0.009	0.917
Male	58 (87%)	55 (86%)
Female	9 (13%)	9 (14%)
Tumor size			-0.009	0.914
≤ 3 cm	55 (82%)	53 (83%)
> 3 cm	12 (18%)	11 (17%)
Tumor number			0.016	0.858
Unifocal	46 (69%)	43 (67%)
Multifocal	21 (31%)	21 (33%)
Tumor stage			-0.031	0.728
Ta	15 (22%)	16 (25%)
T1	52 (78%)	48 (75%)
Histological grade			-0.003	0.977
Low	48 (72%)	46 (72%)
High	19 (28%)	18 (28%)

We next assessed whether CD4⁺ T cell density could serve as independent predictor of OS. A multivariate Cox proportional hazards analysis was performed, and those variables that were associated with survival by univariate analysis were adopted as covariates (Table 4). The density of CD4⁺ T was an independent prognostic factor for OS (HR, 2.75; P = 0.004). Moreover, age remained associated with OS (HR, 5.169; P < 0.001).

Table 4.

Multivariate analysis of the factors associated with survival

Variable	HR	95% CI	P
Age, years (> 62/≤ 62)	5.169	2.43-10.992	< 0.001
CD4+ T cells (High/Low)	2.75	1.375-5.497	0.004

Note: Multivariate analysis. Cox proportional hazards regression model. Variables associated with survival by univariate analyses were adopted as covariates in multivariate analyses. HR > 1, risk for death increased; HR < 1, risk for death reduced.

Discussion

The available prognostic tool that was based on clinicopathological variables, such as the European Organisation for Research and Treatment of Cancer (EORTC) risk tables, had insufficient discriminative ability to accurately predict the risk of disease recurrence and progression at the level of the individual patient [21]. New biomarkers are needed to predict the outcome of bladder cancer, in addition to commonly used clinicopathological parameters [22].

Biomarkers may help to identify patients with NMIBC at higher risk for disease progression [23]. However, to date, such biomarkers have remained substantially limited. In this study, we found high CD4⁺ T cell density was an independent predictor of poor OS for patients with NMIBC. These findings identify CD4⁺ T cells as a potential prognostic marker that may help guide individual therapeutic management of patients with NMIBC.

The adaptive immune system may play an important role in the tumor progression [14,24,25]. Improved clinical outcome has been associated with the presence of intratumoral T cells in ovarian, renal, colorectal, and hepatocelluar carcinoma [8-13]. By contrast, other reports have demonstrated an inverse clinical correlation between regulatory T cell density and the local immune control of tumors from patients with ovarian, breast, and hepatocellular carcinomas [27-29]. Herein, we observed that the density of CD4⁺ T cells was higher in NMIBC tissues, a high number of CD4⁺ T cells was associated with poor OS of NMIBC patients. Although CD4⁺ T cell density had no association with RFS, patients with higher CD4⁺ T cell density had a 10-year median RFS rate 1. 7 times shorter than patients with lower CD4⁺ T cell density. These results suggested that the recurrence rate sustained increasing duing the long time follow-up period and CD4⁺ T cells may play important role in promoting bladder cancer recurrence and progression. Notably, the anti-CD4 antibody shows robust immunoreactivity in bladder formalin-fixed, paraffin-embedded tissues that can be treated with diverse antigen retrieval methods with good inter-observer reproducibility (data not shown). Therefore, immunohistochemical detection of CD4⁺ T cells could represent a novel prognostic indicator for NMIBC.

CD4⁺ T cells play crucial roles for host defense and immune-mediated disease by their ability to differentiate into specialized subpopulations, such as Th1 cells, Th2 cells, Th17 cells and regulatory T cells [27]. Among them, Th1 cells are strongly associated with good clinical outcome for almost all cancer types [8]. Regulatory T cells, characterized by the CD4⁺ CD25⁺ FOXP3⁺ phenotype, are believed to dampen T-cell immunity and to be the main obstacle tempering immunotherapy [7]. In the present study, we found that all CD4⁺ T cells could be an independent adverse predictor for patients with NMIBC. A possible explanation for the detrimental effect of CD4⁺ T cells on patient prognoses is that protumoral T cell subpopulations (regulatory T cells) might be more prevalent than those with antitumoral phenotypes (e.g. Th1 cells) in NMIBC tissues. Testing this hypothesis may be the subject of further investigations.

There are some limitations in this study. Firstly, the samples selected from a single institution, and the number of samples enrolled may not enough for subgroup analysis. Subsequently, this is a retrospective study. Last, the information on chemotherapy or radiotherapy is inadequate to draw a conclusion about the potential role of CD4⁺ T cells expression to therapeutic sensitivity.

In conclusion, our results demonstrate that CD4⁺ T cells are associated with a poor prognosis in NMIBC. These results may provide a novel independent predictor for prognosis, and suggest that a more detailed characterization of distinct CD4⁺ T cell subpopulations might provide an opportunity to eliminate protumoral CD4⁺ T cells or harness antitumoral CD4⁺ T cells in human cancers.

Disclosure of conflict of interest

None.