Prognostic value of peripheral blood T lymphocyte subsets in clear cell renal cell carcinoma.

BACKGROUND: To date, few studies have evaluated the role of peripheral blood T lymphocyte subsets in patients with clear cell renal cell carcinoma (ccRCC). Here we measured the levels of peripheral blood T lymphocyte subsets and evaluated its prognostic value in ccRCC.
METHODS: Data from 122 patients with RCC from January 2018 to January 2020 were collected. Preoperative peripheral blood T lymphocyte subsets and medical records were analyzed. Kaplan-Meier cures and log rank test were used for analyzing overall survival (OS). Univariate and multivariate survival analyses were underwent by performing the Cox proportional hazards models. Correlations were tested by Pearson's correlation analysis.
RESULTS: Of 122 patients, a total of 80 ccRCC patients was enrolled. Patients with low CD3+ T cells and low CD4+/CD8+ ratio displayed a worse OS than patients with high CD3+ T cells and high CD4+/CD8+ ratio (P=0.029 and 0.002, respectively). Multivariate analyses showed CD3+ T cells and CD4+/CD8+ ratio were independent predictive factors for the OS (HR: 0.295, 95% CI, 0.091-0.956; P=0.042 and HR: 0.244, 95% CI, 0.065-0.920; P=0.037, respectively). Moreover, NLR negatively correlated with both levels of CD3+ T cells and CD4+/CD8+ ratio (P<0.001, r=-0.398 and P=0.012, r=-0.280, respectively).
CONCLUSIONS: The findings of our study suggest that preoperative CD3+ T cells and CD4+/CD8+ ratio in peripheral blood are independent predictors for patients with ccRCC. 2021 Translational Andrology and Urology. All rights reserved.

Introduction

Renal cell carcinoma (RCC) accounts for approximately 90% of all renal tumors. Histologically, clear cell RCC (ccRCC) is the majority of RCC, accounting for 75–80% of total cases (1). Nowadays surgical treatment is still an effective measure option for early ccRCC, however the treatment of advanced and metastatic ccRCC remains challenging. What’s worse, more than 30% of ccRCC patients exhibit metastatic lesions at the time of initial diagnosis and the probability of metastases in the remaining patients is approximately 30% (2).

Poor host immune function is a main factor leading to ccRCC progression. Peripheral blood T lymphocyte subsets is an effective way to reflect systemic immune status (3,4). Several studies have shown the immunological effects of peripheral blood T lymphocyte subsets by different treatments in tumor patients. For example, anti-PD-1 treatment (nivolumab) declined CD4+ cell levels but increased the proportion of CD8+ T cell in oral cavity squamous cell carcinoma (5). For patients with esophageal cancer, CD3+ T cell as well as CD4+ T cells and CD4+/CD8+ ratio were significantly increased in peripheral blood after radiotherapy (6). Moreover, peripheral blood T lymphocyte subsets can be acted as prognostic indicators. For patients with small cell lung cancer, analysis of progression free survival showed that CD8+ T cells in circulating lymphocytes was an independent predictive factor (7).

Recent studies have demonstrated most RCC patients suffer from local or systemic immune deficiencies, leading to low immune functions (8,9). However, few studies have evaluated the role of peripheral blood T lymphocyte subsets in patients with ccRCC. In this study, we detected the level of peripheral blood T lymphocyte subsets in pathologically diagnosed ccRCC patients preoperatively and performed prognosis assessment. In addition, we also analyzed relevant factors that affect the level of peripheral blood T lymphocytes subsets. We present the following article in accordance with the REMARK reporting checklist (available at http://dx.doi.org/10.21037/tau-20-1066).

Methods

Patients

A cohort of 122 patients with RCC was admitted to the fifth affiliated hospital of Sun Yat-sen University from January 2018 to January 2020. Eligible patients were pathologically diagnosed as ccRCC, peripheral blood T lymphocyte subsets and computed tomography were underwent preoperatively. Finally, a total of 80 patients was enrolled into this study. All of the 80 ccRCC patients received neither neoadjuvant chemotherapy nor immunity therapy preoperatively, and no patients had any coexisting immune system disorders or known active infections before treatment. All procedures performed in this study were in accordance with the Declaration of Helsinki (as revised in 2013) and approved by the Ethics Committee of the Ethics Committee of the fifth affiliated hospital of Sun Yat-sen University (No.: 2020#L101-1). Because of the retrospective nature of the research, the requirement for informed consent was waived.

Data collection

Patient information and laboratory data were collected from the patient records. Preoperative peripheral blood T lymphocyte subsets and complete blood counts within a week were analyzed. The levels of peripheral blood T lymphocyte subsets were determined using the Beckman DxFLEX flow cytometer (California, USA). The subsets of T lymphocyte cells include CD3+, CD4+, CD8+ T cells and CD4+/CD8+ ratio. Complete blood counts were performed using an automated hematology analyzer (Sysmex XN-10, Kobe, Japan).

Follow-up

All patients were followed up every 3–6 months, with a range of 3–28 months and a mean follow-up of 13.3 months. Physical examination, routine blood test and chemistry analysis, chest radiography and abdominal ultrasonography or computed tomography were performed at each follow-up. The study endpoint was patient death. Overall survival (OS) was calculated as the time from the date of enrollment to death or the date of the last follow-up.

Statistical analysis

All statistical analyses were performed using the SPSS, version 19.0 (Chicago, IL, USA). Chi-square test was adopted for the comparisons between groups. Independent predictors of OS were analyzed using the univariate and multivariate Cox proportional hazards regression analyses. Survival analyses of T lymphocyte cell subsets were performed using Kaplan–Meier curve and log-rank test. The Spearman correlation coefficient were used to detect the linear correlations. P<0.05 suggested that the difference was statistically significant.

Results

Distribution of T lymphocyte subsets in patients with ccRCC

As reported, cancer patients are often accompanied by decreased CD3+, CD4+, CD8+ T cells and CD4+/CD8+ ratio (10). In our study, 22 patients were presented with low CD3+ T cells, and low CD4+ T cells, low CD8+ T cells and low CD4+/CD8+ ratio were found in 33, 15 and 15 patients, respectively. The distribution of T lymphocyte subsets of 80 patients with ccRCC was shown in Table S1.

Clinicopathological characteristics and T lymphocyte subsets of ccRCC patients

Among the 80 patients with ccRCC, 51 (63.8%) patients were male and 29 (36.2%) patients were female, with a mean age of 54.9 years (range, 26–84 years). The distributions of clinicopathological characteristics in T lymphocyte subset group were shown in .

Table 1

Clinical and pathological characteristics stratified by CD3+ T cell count, CD4+ T cell count and CD4/CD8 ratio in patients with ccRCC

Variables	CD3+ T cell count				CD4+ T cell count				CD8+ T cell count				CD4+/CD8+ ratio
	Low (<770)	High (>770)	P		Low (<500)	High (>500)	P		Low (<238)	High (>238)	P		Low (<1.0)	High (>1.0)	P
Age (y)			0.614				0.994				0.630				0.020
<65	16	47			26	37			13	50			8	55
>65	6	11			7	10			2	15			7	10
Sex			0.612				0.624				0.392				0.034
Male	15	36			20	31			11	40			6	45
Female	7	22			13	16			4	25			9	20
Hypertension			0.787				0.921				0.025				0.912
Yes	8	23			13	18			2	29			6	25
No	14	35			20	29			13	36			9	40
Diabetes mellitus			0.984				1.000				0.865				0.865
Yes	3	6			4	5			1	8			1	8
No	19	52			29	42			14	57			14	57
Location			0.291				0.698				0.014				0.031
Left	12	24			14	22			11	25			6	34
Right	10	34			19	25			4	40			9	31
Surgical approach			0.075				0.004				0.026				0.519
NSS	14	24			22	16			11	27			6	32
RN	8	34			11	31			4	38			9	33
BMI (kg/m2)			0.358				0.578				0.005				1.000
<25	15	33			21	27			12	36			9	39
>25	7	25			12	20			3	29			6	26
NLR			<0.001				<0.001				0.166				0.166
<3	8	49			16	41			8	49			8	49
>3	14	9			17	6			7	16			7	16
PLR			0.002				0.001				0.007				0.694
<132	5	36			9	32			3	38			7	34
>132	17	22			24	15			12	27			8	31
CD3+ T cell (cells/μL)			-				<0.001				<0.001				0.128
<770	22	0			22	0			14	8			7	15
>770	0	58			11	47			1	57			8	50
CD4+ T cell (cells/μL)			<0.001				–				<0.001				0.102
<500	22	11			33	0			14	19			9	24
>500	0	47			0	47			1	46			6	41
CD8+ T cell (cells/μL)			<0.001				<0.001				–				0.035
<238	14	1			14	1			15	0			1	14
>238	8	57			19	46			0	65			14	51
CD4+/CD8+			0.128				0.102				0.335				–
<1.0	7	8			9	6			1	14			15	0
>1.0	15	50			24	41			14	51			0	65
MAP score			0.201				0.882				0.519				0.099
0–2	9	33			17	25			9	33			5	37
3–5	13	25			16	22			6	32			10	28
RENAL score			0.006				0.403				0.039				0.702
4–6	2	24			9	17			1	25			6	20
7–12	20	34			24	30			14	40			9	45
Tumor size (cm)			0.001				0.063				0.002				0.452
<5	4	35			12	27			2	37			6	33
>5	18	23			21	20			13	28			9	32
T stage			0.081				0.464				0.509				0.925
T1-2	15	51			26	40			11	55			13	53
T3-4	7	7			7	7			4	10			2	12
Fuhrman grade			0.007				0.103				0.047				0.801
1–2	7	38			15	30			5	40			8	37
3–4	15	20			18	17			10	25			7	28

BMI, body mass index; MAP, Mayo adhesive probability; NLR, neutrophil to lymphocyte ratio; NSS, nephron sparing surgery; PLR, platelet to lymphocyte ratio; RN, radical nephrectomy.

PLR was significantly higher among the CD3+ low group, the CD4+ low group and the CD8+ low group than those among the CD3+ high group, the CD4+ high group and the CD8+ high group (P=0.002, 0.001 and 0.007, respectively), and NLR was significantly higher in the CD3+ low group and the CD4+ low group than those in the CD3+ high group and the CD4+ high group (P<0.001, for both). Patients in the CD3+ low group and the CD8+ low group were significantly more likely to have higher RENAL score, bigger tumor size and higher Fuhrman grade. Age and sex are significantly associated with the level of CD4+/CD8+ ratio (P=0.020 and 0.034, respectively). Additionally, larger BMI and a positive history of hypertension were found in patients with high level of CD8+ T cells than those with low level of CD8+ T Cells (P=0.005 and 0.025, respectively), however with no difference among the other three T lymphocyte subgroups.

Survival analysis with T lymphocyte cell subsets

As shown in , patients with low CD3+ T cells displayed a worse OS than patients with high CD3+ T cells (P=0.029). Similarly, the survival outcomes for patients in CD4+/CD8+ low group were also more negative (P=0.002) (). However, patients with low CD4+ T cells and low CD8+ T cells showed no significantly difference in OS compared with patients with high CD4+ T cells and high CD8+ T cells (P=0.549 and 0.199, respectively) ().

Figure 1

Kaplan-Meier analysis for overall survival (OS) in patients with ccRCC. (A) OS for patients with CD3+ T cells; (B) OS for patients with CD4+ T cells; (C) OS for patients with CD8+ T cells; (D) OS for patients with CD4+/CD8+ ratio.

CD3+ T cells and CD4+/CD8+ ratio are independent predictive factors for the OS

On univariate Cox regression analysis, longer OS was noted in patient with younger age (HR =3.438, 95% CI: 1.085–10.894, P=0.036), higher CD3+ T cells (HR =0.302, 95% CI: 0.096–0.951, P=0.041) and higher CD4+/CD8+ ratio (HR =0.185, 95% CI: 0.055–0.626, P=0.007) (). Then multivariate survival analysis was performed to find out the independent factors affecting OS. The results indicated that CD3+ T cells (HR =0.295, 95% CI: 0.091–0.956, P=0.042) and CD4+/CD8+ ratio (HR =0.244, 95% CI: 0.065–0.920, P=0.037) were independent prognostic factors for OS ().

Table 2

Univariate and multivariate analysis regarding overall survival in patients with ccRCC

Variables	Univariate				Multivariate
	OR	95% CI	P		OR	95% CI	P
Age (y)
<65 vs. >65	3.438	1.085–10.894	0.036		2.871	0.817–10.082	0.100
Sex
Male vs. female	1.352	0.427–4.277	0.608
Hypertension
Yes vs. no	0.806	0.259–2.522	0.709
Diabetes mellitus
Yes vs. no	25.613	0.020–32,358.570	0.373
Location
Left vs. right	3.086	0.829–11.487	0.093
Surgical approach
NSS vs. RN	3.172	0.854–11.782	0.085
BMI (kg/m2)
<25 vs. >25	0.315	0.069–1.445	0.137
NLR
<3 vs. >3	1.936	0.621–6.032	0.255
PLR
<132 vs. >132	1.018	0.321–3.231	0.975
CD3+ T cell (cells/μL)
<770 vs. >770	0.302	0.096–0.951	0.041		0.295	0.091–0.956	0.042
CD4+ T cell (cells/μL)
<500 vs. >500	0.704	0.221–2.241	0.553
CD8+ T cell (cells/μL)
<238 vs. >238	0.475	0.148–1.523	0.211
CD4+/CD8+ ratio
<1.0 vs. >1.0	0.185	0.055–0.626	0.007		0.244	0.065–0.920	0.037
MAP score
0–2 vs. 3–5	1.622	0.511–5.147	0.412
RENAL score
4–6 vs. 7–12	4.475	0.576–34.770	0.152
Tumor size (cm)
<5 vs. >5	4.431	0.965–20.353	0.056
T stage
T1–2 vs. T3–4	1.779	0.475–6.665	0.392
Fuhrman grade
1–2 vs. 3–4	1.259	0.403–3.928	0.692

BMI, body mass index; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; MAP, Mayo adhesive probability.

Correlation between clinicopathological characteristics and T lymphocyte cell subsets

We further analyzed the correlation between clinicopathological characteristics and T lymphocyte cell subsets. Our results displayed that NLR was both negatively correlated with CD3+ T cells (r=−0.398, P<0.001) and CD4+/CD8+ ratio (r=−0.280, P =0.012) (). A positive correlation was observed between BMI and CD3+ T cells (r=0.301, P=0.007) and a negative correlation was observed between PLR and CD3+ T cells (r=−0.483, P<0.0001) (), however no significantly correlations were found neither between BMI and CD4+ T cells nor between PLR and CD4+ T cells ().

Figure 2

Correlation between clinicopathological characteristics and T lymphocyte cell subsets. (A) Correlation between clinicopathological characteristics and levels of CD3+ T cells. (B) Correlation between clinicopathological characteristics and levels of CD4+/CD8+ ratio.

Discussion

Prior studies have indicated that the prognostic and predictive value of T cells infiltrated in formalin-fixed and parrffin-embedded specimens in RCC (11,12). However, to the best of our knowledge, the present study is the first to identify the relationship between peripheral blood T lymphocyte subsets and the prognosis of patients with ccRCC. The current study shows that low CD3+ T cells and low CD4+/CD8+ ratio in peripheral blood were negatively associated with the OS in ccRCC patients.

One characteristic of most patients with malignant tumor is immune dysfunction, and inhibition of host immune system facilitates tumor progression. Increasing studies have demonstrated a prognostic and predictive role of systemic and local immunological markers in cancer patients (10,13). Clear cell renal cell carcinoma (ccRCC), representing the majority of RCC, is characterized by strong immunogenicity. As a result of it, immunotherapies targeting and blocking immune checkpoint, such as CTLA-4 and PD-1 are newly applied to patients with ccRCC during the last decade. CD3+ tissue infiltrating lymphocytes (TILs) forming memory T-cell and CD8+ TILs forming cytotoxic T-cell play a key role in immune defense (14). Local immune response in ccRCC, such as CD3+ and CD8+ TILs, has been analyzed in recent studies. It was reported that higher density of CD8+ TILs was associated with prolonged progression-free survival in ccRCC (15). In a cohort of 756 patients with ccRCC, the percentage of tumor-infiltrating CD3+ T cells in patients with better response to nivolumab was significantly increased (16). Moreover, lower T cell infiltrate significantly increased the recurrence of localized ccRCC following surgery (12).

Compared to local immunological markers by detecting tumor tissues, peripheral blood T lymphocyte subsets are indicators of systemic immune response and can be conveniently acquired. Changes of the peripheral blood T lymphocyte subsets response to various treatment and clinical outcomes are observed. For patients with various types of cancers after hyperthermia treatment, such as ovarian, gastric or endometrial cancer, the proportion of CD8+/CD28+ T cells was increased, while that of CD4+/CD25+/CD127+ cells was decreased (17). Excitingly, the prognostic role of the peripheral blood T lymphocyte subsets was also noted. The correlation between increased peripheral blood CD3+ and CD8+ T cells and improved survival was significantly positive in patients with pancreatic cancer after chemotherapy with 5FU (18). Similarly, for patient with small cell lung cancer, higher CD8+ T cells demonstrated better progression free survival (7).

The available evidence regarding the possible role of CD4+/CD8+ ratio in different cancer patients is conflicting. On one hand, the declined in CD4+/CD8+ ratio was associated with the progression of lung cancer patients, as the percentage of CD4+/CD8+ in high stage was lower than that in low stage (19). On the other hand, ovarian cancer patients with a decreased CD4+/CD8+ ratio were found to have a better OS than those with an increased ratio (20). In the present study, our results showed that declined CD3+ T cells and CD4+/CD8+ ratio were correlated with shorter OS in patients with ccRCC. To further explore the reduction of CD3+ T cells and CD4+/CD8+ ratio in patients with worse prognosis, their correlation with clinicopathological characteristics were detected. Our results showed that patients who were immunosuppressive were more likely to have an activated inflammatory response, such as a high level of NLR and PLR.

NLR and PLR are widely used as inflammation-associated indexes. Recent studies have demonstrated their prognostic role in various types of cancers, including RCC (21,22). Moreover, higher NLR and PLR are associated with worse prognosis in immunotherapy for patients with RCC (23,24). High activity of inflammatory response is noted with elevated NLR and PLR, while chronic inflammation usually leads to tumor development by suppressing the immune system. Our data suggested that CD3+ T cells and CD4+/CD8+ ratio were correlated with NLR and PLR levels, which is consistent with the above studies (23,24).

The present study also has certain limitations. Firstly, our study was conducted in a single institution with a small sample size of 80 patients and a short-term follow-up of 3–28 months. To provide a better evidence, our results require larger samples and longer follow-up in a multi-center cohorts. Secondly, our study only focused on the T lymphocyte subsets in peripheral blood to reflect the immune status. Our results should combine the B lymphocyte cell and natural killer cell to monitor systemic immune function. Besides, TILs were not detected in our study. A better understanding of the correlation between peripheral blood T lymphocyte subsets and TILs will help to explain the prognostic role of T lymphocyte subsets in ccRCC patients.

Conclusions

In conclusion, our study indicates that CD3+ T cells and CD4+/CD8+ ratio in peripheral blood are independent predictors for patients with ccRCC. With a low cost and conveniently acquire, CD3+ T cells and CD4+/CD8+ ratio are promising biomarkers that facilitate the clinical application in ccRCC. Therefore, the value of CD3+ T cells and CD4+/CD8+ ratio as prognostic indicators is worthy of further exploration in future clinical studies.

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