Circulating lymphocyte subsets are prognostic factors in patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a geographically and racially variable disease that has a high incidence in Southeast China. According to previous studies on tumor immunity, we compared multiple clinical parameters and blood indexes with outcomes regarding to Epstein-Barr virus (EBV) status in NPC patients. According to the EBV load at diagnosis, 220 NPC patients who received concurrent chemoradiotherapy (CRT) were divided into two groups: EBV DNA ≥ 1500 copies/mL and EBV DNA < 1500 copies/mL, respectively. We compared clinical parameters with peripheral blood mononuclear cells, lymphocyte subsets and biochemical indexes. We also analyzed distant metastases and the overall survival rate regarding to these characteristics. In most cases, the two groups showed the same trends. Most blood indexes were decreased during CRT and the decrease of the absolute count was more significant than the percentage. Patients with younger age showed the higher CD3+ and CD3 + CD8+ percentages. Patients whose EBV DNA ≥ 1500 copies/mL showed a higher N classification than those with EBV DNA < 1500 copies/mL at first diagnosis. Within patients with EBV DNA ≥ 1500 copies/mL, a higher CD3 + CD8+ percentage or lower CD3-CD56+ percentage had better OS rates, and the CD3 + CD8+ percentage was an independent prognostic factor by multivariate survival analyses. CRT caused an overall decrease of blood cells in NPC patients. Among all the blood indexes, the CD3 + CD8+ percentage showed a correlation with age and was an independent prognostic factor in patients with EBV DNA ≥ 1500 copies/mL at first diagnosis, which is worthy for further large cohort study.


Background
Nasopharyngeal carcinoma (NPC) is one of malignant epithelial cell tumors at the top and lateral wall of the nasal cavity with different etiology and pathology [1]. According to the World Health Organization (WHO) classification, NPC is divided into keratinizing (K) carcinoma (squamous cell carcinoma; SCC), non-keratinizing (NK) carcinoma (including differentiated and undifferentiated variants) and basaloid squamous cell carcinoma. There are great differences in the geographical and ethnic distributions of NPC. The incidence of NPC is significantly higher in South China and Southeast Asia, mainly belonging to NK-NPC (90%). The population is sensitive to radiotherapy and the disease has almost 100% association with Epstein-Barr virus (EBV) [2]. EBV is a common linear double-stranded virus, about 122-180 nm, which contains a double helix DNA wrapped in the protein capsid. EBV infection has been found mainly in B lymphocytes and epithelial cells which can be cleaved and replicated [3]. The current research on other pathogenic factors of NPC is not thorough. Genetic susceptibility and consumption of pickled food are also important pathogenic factors [2]. Previous studies have demonstrated that high EBV DNA levels may indicate poor prognosis and reduced long-term survival in NPC patients [4,5]. However, tumor immunity at the circulating level in relation to NPC patient outcome remains to be explored.
Radiotherapy is the main treatment for NPC [6]. Radiotherapy alone is effective in the early stage, concurrent chemoradiotherapy (CRT) in the locally advanced stage, and systematic therapy in patients with metastases and recurrence [7]. In recent years, with the progress of molecular biology, targeted therapy and immunotherapy have become new trends, and have determined their positions as second-line treatments for recurrent/metastatic NPC. Moreover, the exploration of first-line combined chemotherapy also shows a good prospect [8,9].
Immune cell infiltration is a characteristic of almost all malignant tumors. Immune cells mainly include tumor-associated macrophages, lymphocytes, and mast cells. They play important roles in immune monitoring and contribute to the elimination of tumor cells [10]. Given the minimally invasive nature of blood sample collection compared to tumor infiltrating tissues, the regularity of various indexes in peripheral blood is more convenient for clinical application, such as T lymphocyte cells, B lymphocyte cells and NK cells. The inflammation index is the characteristic marker of tumor and plays an auxiliary role in determining the occurrence and progression of tumor [11]. Peripheral blood Neutrophil count/Lymphocyte count (NLR), Lymphocyte count/Monocyte count (LMR), Platelet count/Lymphocyte count (PLR), Platelet count×Neutrophil count/ Lymphocyte count (SII) are widely used indexes in the evaluation of inflammation [12]. In addition, albumin (ALB) and lactate dehydrogenase (LDH) are well-studied prognostic biomarkers of NPC [13].
This study was based on the status of EBV load in patients with NPC. We explored not only the relationship between EBV DNA load, lymphocyte subsets and inflammatory indexes in peripheral blood, but also the relationships between EBV DNA load, clinical parameters, distant metastases and OS. The aim was to investigate indicators with prognostic value in NPC patients, and these findings may hence significantly aid the clinical decision-making process.

Ethics statement and the criteria for patient inclusion
A retrospective single-institution cohort design was used, and a total of 220 patients with NPC who signed an informed consent form and have completed treatment were enrolled in this study. Patients with a history of other malignant tumors were excluded. This study was approved by the local Ethics Committee of the Zhejiang Cancer Hospital (IRB-2021-326). Grading and staging were undertaken according to the 8th edition of the International Union for Cancer Control/American Joint Committee on Cancer (UICC/AJCC) cancer staging system for NPC. Patients who survived were censored at their last date of follow-up. The OS rate was calculated from the date of diagnosis to the date of death.

Therapy regimen
None of the patients underwent surgery. The tissue sent for pathological diagnosis was obtained by nasopharyngeal biopsy. All patients underwent concurrent CRT. Chemotherapy regimen: Nedaplatin plus Docetaxel; Chemotherapy interval: once every 21 days. Radiotherapy dose: 2.0 Gy. Radiotherapy interval: once every 5 days, suspended for 2 days. Circulating blood samples were collected before CRT. The first time was before therapy, named T1; the second time was during therapy, named T2; the third time was before the last therapy, named T3.

Blood samples
Circulating peripheral blood mononuclear cells (PBMCs) were obtained during the period between January 2016 and December 2020. Two milliliters of venous blood was collected in a vacuum tube prefilled with EDTA-K 2 (Zhejiang Gongdong Medical Technology Co., Ltd. China) for EBV DNA detection, white blood cell (WBC) count, neutrophil count, total lymphocyte count and platelet count. Two milliliters of venous blood was collected in a vacuum tube prefilled with heparin sodium (Kangshi Medical Inc., China) for lymphocyte subset detection. Two milliliters of venous blood was collected in a vacuum tube prefilled with coagulant (Zhejiang Gongdong Medical Technology Co., Ltd. China) for ALB and LDH. After centrifugation in Biocoll Separating Solution (Merck, Germany), PBMCs were separated and recovered, washed twice in PBS and stored at 4 °C. Lymphocyte subset detection was performed within 24 h. The patient characteristics are summarized in Table 1.

EBV DNA and different blood indexes detection
EBV DNA was analyzed by Roche Lightcycler 480 fluorescence quantitative PCR (Roche, USA) with Daan EBV DNA detection reagent (Daan Gene Co., Ltd. China). WBC count, neutrophil count, total lymphocyte count and platelet count were analyzed by a Mindray CAL 8000 automatic blood cell analysis pipeline (Mindray, China) immediately after blood collection. The vacuum tube prefilled with coagulant was centrifuged at 3500 r/min for 5 min, and serum was detected by a Hitachi 7600 automatic biochemical analyzer (HITACHI, Japan). For flow cytometry analysis, PBMCs in 50 μL PBS supplemented with 0.5% BSA (Thermo Fisher, USA) (1 × 10 6 cells/mL) were incubated with anti-human monoclonal antibodies (mAbs) for 15 min. The following mAbs were used for flow cytometry: anti-CD45/CD4/CD8/CD3, anti-CD45/CD56/CD19/ CD3, anti-CD4, anti-CD45RA, anti-CD45RO, anti-CD8 and anti-CD38 (Beckman Coulter, USA) (Supplementary Table 1). OptiLyse C lysing solution (Beckman Coulter, USA) was used for hemolysis for 15 min, and PBMCs were resuspended in PBS (Beckman Coulter, USA). The results were acquired by a Beckman Coulter FC500 flow cytometer and analyzed by CXP analysis software with the recommended reference ranges [12,14,15]. The reference ranges were adjusted in our laboratory (Supplementary Table 2).
We divided the patients into two groups: a low-risk group (EBV DNA < 1500 copies/mL at T1) and a highrisk group (EBV DNA ≥ 1500 copies/mL at T1) by a previously defined EBV-DNA cutoff value [16,17]. We mainly compared the differences between the two groups in the study.

Statistical analysis
Statistical analyses were performed using STATA 14.2 (Stata Corp LLC, Texas, USA). Categorical variables are described as percentages, and numerical variables are represented as the mean ± SD. Qualitative data were compared using the Chi-square test. Univariate and multivariate analyses were performed by the cox proportional hazards model. Survival was determined using the Kaplan-Meier (KM) method. A p value of < 0.05 was regarded as statistically significant.

Patient characteristics
A total of 220 patients were included in this study, with a mean age of 52.31 ± 11.10 (ranging from 17 to 79). There were 166 males and 54 females in the study. It takes an average of 3.6 months from the onset of physical discomfort to diagnosis. The clinicopathological parameters of the patients are summarized in Table 1 We divided the patients into two groups by EBV DNA load, 149 cases belonged to low-risk group and 71 cases belonged to high-risk group. The two groups showed different in N classification (p < 0.001), and the high-risk group showed a higher N classification. The data are shown in Table 1.
Distant metastases were found in 44 patients (20.00%). Among all metastases, 17 were bone, 9 were lung, 6 were liver, 4 were brain, 2 were stomach, 2 were mouth, 1 was uterus and 3 were multiple metastases. All patients with metastatic disease had TNM stage IV disease.

The relationships between clinical parameters and blood indexes
We measured EBV DNA load, peripheral blood indexes such as WBC count, neutrophil count, total lymphocyte count, platelet count; lymphocyte subsets such as CD3+ percentage/count, CD3 + CD4+ percentage/ count, CD3 + CD8+ percentage/count; biochemical indexes ALB and LDH. The flow cytometry results are shown in Fig. 1A-J.
As shown in Tables 2-3, we studied the relationships between clinical parameters such as age, sex, smoking history, drinking history, family history of cancer, pathological type, clinical stage and the changes of blood indexes in each group. The calculations were carried out at T1. Among the clinical parameters, age was more correlated with lymphocyte subsets. The significant differences throughout CRT were CD3+ percentage (the younger age, the higher CD3+ percentage), and CD3 + CD8+ percentage (the younger age, the higher CD3 + CD8+ percentage). The other clinical parameters and blood indexes didn't always show the same rule throughout the therapy. For example, in the high-risk group, patients with higher N classification showed higher WBC count and neutrophil count. In low-risk group, patients with higher N classification showed higher CD4 + CD45RAcount and CD4 + CD45RO+ count.

The relationships between EBV DNA and blood indexes
The relationships between EBV DNA load and lymphocyte subsets at T1 were as follows: a higher EBV DNA load was correlated with higher CD4 + CD45RA-percentage (p = 0.015), CD4 + CD45RO+ percentage (p = 0.019) and lower CD4 + CD45RA+/CD4 + CD45RA-ratio (p = 0.034) (Supplementary Table 3). Additionally, patients with a higher EBV load (high-risk group) had higher NLR (p = 0.009), PLR (p = 0.021) and SII (p = 0.004) compared to the low-risk group. Furthermore, the high-risk group demonstrated higher plasma LDH levels compared to the low-risk group (p < 0.001) (Supplementary Table 3). At T2 and T3, these correlations were not observed. Data were presented as the mean value and were shown in Supplementary Tables 3, 4, 5 and 6.

Expression of blood indexes over time
When we compared the high-risk group and the lowrisk group, we found that the two groups were consistent during CRT. The decrease in absolute count was more significant than that in percentage, and all lymphocyte subset counts were decreased at the end of therapy. Additionally, the CD4/CD8 ratio and the CD4 + CD45RA+/ CD4 + CD45RA-ratios were significantly decreased during therapy. But NLR (p < 0.001), PLR (p < 0.001) and SII (p < 0.001) showed different patterns with that ratios increased over time. These changes were more significant in the low-risk group. Data were shown in Supplementary Tables 3, 4, 5 and 6.

Expression of blood indexes in patients with or without distant metastases
In 44 patients with distant metastases, 14 belonged to the high-risk group and 30 belonged to the low-risk group. In the high-risk group, monocyte count showed lower in patients with distant metastases at T1. In lowrisk group, the expression of CD3+ percentage and PLR were higher while CD3-CD56+ count and CD8 + CD38+ count were lower in patients with distant metastases at T1. The data were shown in Supplementary Tables 7 and 8.

Overall survival
In the high-risk group, after a median follow-up of 39.24 months, the OS rate was 83.10%. In the low-risk group, after a median follow-up of 42.44 months, the OS rate was 87.92%. EBV DNA load was not an independent prognostic factor for NPC in our analysis (data not shown).

Discussion
With the improved understanding of the pathogenesis of EBV associated NPC and plasma EBV DNA test for population screening as well as personalized therapy strategies, the mortality rate of NPC has been greatly reduced. However, the early diagnosis of NPC remains a challenge. Therefore, exploring therapeutic and prognostic biomarkers for NPC is necessary. In this study, NPC patients were divided into a high-risk group and a low-risk group according to the EBV DNA load before the first CRT. By comparing clinicopathological data and peripheral blood indexes between the high-risk group and the low-risk group, we hope to find potential biomarkers that can help to predict prognosis and guide NPC therapy.
In our study, the initial EBV DNA load was related to N classification that is patients with a higher initial EBV load had a higher N classification. This finding was consistent to previous studies [4,18]. In addition, we found that EBV DNA load was associated with a variety of lymphocyte subsets, especially CD3+ percentage and CD3 + CD8+ percentage. Similar to our results, Mo et al [19] reported the relationship between EBV DNA load and CD4 + CD25+ T cells and CD8+ cells in patients with NPC. A higher CD8+ percentage tends to have a better prognosis, which may due to the fact that CD8+ cells play an important role in establishing effective immune surveillance.
Previous studies have shown that EBV DNA and LDH are independent prognostic factors for locoregionally advanced NPC [16,17]. The circulating levels of EBV DNA and LDH in combination with other risk factors can predict recurrence and overall survival for NPC patients in established statistic nomogram models [13,20]. In our analysis, patients with higher initial EBV load referring to high-risk group had a higher LDH level compared to the low-risk group. However, multivariate survival analyses of overall survival combining circulating lymphocyte subsets did not show the significant prognostic value of LDH in high-risk patients. Thus, additional studies are warranted to explore the role of circulating level of LDH when NPC patients are stratified by plasma EBV DNA load, ideally from large prospective studies.
During CRT, we also found that lymphocyte subset counts were decreased before last therapy compared to initial time point before therapy in both high-risk group and low-risk group. This phenomenon has been observed during CRT in other tumors. For example, Lee et al reported pancytopenia after CRT in rectal cancer [21]. It may relate to myelosuppression [22]. Schuler et al [23] reported that the number of CD4+ T cells declined after CRT in patients with head and neck cancer, because CD4+ T cells were sensitive to CRT.
Inflammation plays a crucial role in cancer development [24]. Peripheral blood neutrophils and lymphocytes not only constitute the predominant proportion, but also play an important role in the immune response. The blood neutrophil-to-lymphocyte ratio has been found to be associated with disease progression in a number of malignancies [25]. During CRT, we found NLR, PLR and SII were increased, mainly because lymphocytes were more sensitive to CRT and the decrease of lymphocytes was greater than that in other white blood cells [24].
Cancer metastases are the main cause of cancer mortality. One important question regarding which patients are likely to develop distant metastases remains to be answered. We found differences of lymphocyte subsets before therapy between the highrisk group and the low-risk group when comparing patients who developed metastases or not. For example, patients who developed metastases in the highrisk group had lower monocyte counts while CD3+ percentage, CD3-CD56+ count and CD8 + CD38+ count were decreased in the low-risk group. These differences changed after therapy. EBV may attack immune cells and interfere with the immune system cell function [26]. EBV induces the apoptosis of monocyte precursors and therefore inhibit the development of dendritic cells [27]. Our findings warrant further investigation.