Prognostic Value of Combined Analysis of CTLA-4 and PLR in Esophageal Squamous Cell Carcinoma (ESCC) Patients

Objective The purpose of this study was to evaluate the prognostic role of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) expression level and the platelet lymphocyte ratio (PLR) level in esophageal squamous cell carcinoma (ESCC) patients. Methods 84 ESCC patients who received surgical treatment in our hospital were enrolled in the study. The correlation of each biomarker's level with ESCC patients' clinicopathological characteristics and overall survival (OS) was assessed. Results The elevated expression rate of T-CTLA-4 (tumor cell CTLA-4) and I-CTLA-4 (interstitial lymphocyte CTLA-4) was 48.8% and 44.0%, respectively. The number of enrolled patients with a higher PLR level (≥119) was 48. The prognostic value of T-CTLA-4, I-CTLA-4, and PLR in ESCC patients was not detected. However, patients with both a low T-CTLA-4 expression level and a low PLR level that had longer OS (p = 0.023) were found. The prognostic role of T-CTLA-4(-) +PLR (-) status in ESCC patients was also confirmed in multivariate analyses (p = 0.027). Conclusion These results demonstrated the potential prognostic value of combined analysis of CTLA-4 and PLR in ESCC patients.


Introduction
Esophageal squamous cell carcinoma (ESCC) is one of the most common types of cancer in northern China [1,2]. Although much progress has been made in surgical techniques and chemoradiotherapy, the survival rate of patients with ESCC is still low [3][4][5]. Therefore, developing novel prognostic factors and optimizing the treatment of ESCC patients are urgently needed [6].
Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a critical immune checkpoint that negatively regulates T cell activation of the immune system. It has been reported that CTLA-4 is overexpressed and correlated with poor prognosis in various types of cancer, including breast cancer [7], lung cancer [8], prostate cancer [9], and cervical cancer [10]. More recently, promising results were also shown by administration of CTLA-4 inhibitor in cancer treatment [11][12][13]. Hence, the expression level of CTLA-4 in cancer cells or microenvironments may constitute a potential predictive or prognostic biomarker of malignant disease patients. However, the potential role of CTLA-4 in ESCC patients has yet to be clearly defined [14].
Previous studies have shown that systemic inflammatory factors can be used as a predictive or prognostic marker of immunotherapy patients targeting the CTLA-4 pathway [15][16][17][18]. These results suggest that there may be an interaction between systemic inflammatory factors and immune checkpoints. Therefore, the combined analysis of systemic inflammatory factors and immune checkpoint molecules may provide a new perspective for the study of prognostic biomarkers in cancer patients. As a systemic inflammatory factor, the prognostic significance of platelet lymphocyte ratio (PLR) in patients with ESCC has been evaluated in a number of retrospective studies [19][20][21]. However, the potential relationship between CTLA-4 and PLR in ESCC patients and its prognostic significance have not been revealed.
In this study, the prognostic value of the CTLA-4 expression level and the PLR level was analyzed in ESCC patients. The results showed that neither CTLA-4 nor PLR could predict the treatment outcome of ESCC patients. However, patients with both a low T-CTLA-4 expression level and a low PLR level that had longer overall survival (OS) were found in this study.

Materials and Methods
2.1. Patient's Characteristics. We retrospectively analyzed 84 patients with ESCC who underwent radical esophagectomy at the General Hospital of the Ningxia Medical University from September 2008 to December 2010. All of the enrolled patients had complete medical records and sufficient paraffin-embedded tissue blocks. Clinical, laboratory, pathological, and treatment data were collected from the patients' medical records. There were 74 males and 10 females with a median age of 63 years (range, 38-80 years). Tumor staging was classified according to the seventh edition of the American Joint Committee on Cancer Staging Manual [22]. There were 23 cases of stage I, 31 cases of stage II, and 30 cases of stage III. Tumor differentiation was classified as well and moderately (n = 50) or poorly differentiated (n = 34). Ivor Lewis esophagectomy, including two-field lymphadenectomy with total mediastinal lymph node dissection, was performed. Patients with postoperative pathological diagnosis of stage T3 or lymph node metastasis received adjuvant radiotherapy or chemotherapy. The percentage of patients receiving adjuvant radiotherapy and chemotherapy was 29.8% and 27.4%, respectively. All the enrolled patients did not receive radiotherapy or chemotherapy before surgery. The study  protocol was approved by the Ethics Committee of the General Hospital of Ningxia Medical University.

PLR Level
Evaluation. PLR is defined as the ratio of absolute platelet count to lymphocyte count. In each patient, the whole blood cells were detected within one week before the surgery and the platelet and lymphocyte counts were obtained. The cut-off value of PLR was determined by receiver operating curve (ROC) analysis.

Patient Follow-Up.
Follow-up began from the date of completed surgery with or without chemoradiotherapy and continued until the last follow-up date or death of the patient. Follow-ups were conducted every three months in the first year, every six months in the next two years, and annually thereafter. OS is defined as the interval between the diagnosis of ESCC and the date of death or censored. The median follow-up time was 79 months.   The median PLR value of the whole group was 121.7. According to the results of ROC analysis (Figure 2), the cut-off value of PLR was determined to be 119. The number of patients with higher PLR levels was 48.The correlation between PLR and the expression of T-CTLA-4 or I-CTLA-4 was analyzed by Spearman's rank correlation. There was a   (Figure 4(c)) still had no effect on the OS in patients with ESCC without the concurrent inflammatory diseases. However, patients with both low T-CTLA-4 levels and low PLR levels (T-CTLA-4 (-) +PLR (-)) had superior OS (p < 0 023) than other patients (Figure 4(d)). Univariate analysis showed that T stage (p = 0 012), N stage (p < 0 001), TNM stage (p < 0 001), adjuvant radiotherapy (p = 0 022), and T-CTLA-4(-) +PLR (-) status (p = 0 027 ) were significantly associated with OS. The multivariate analysis results showed that adjuvant radiotherapy (p = 0 023) and T-CTLA-4(-) +PLR (-) status (p = 0 030) were two independent prognostic factors (Table 1). However, no correlation between T-CTLA-4(-) +PLR (-) status and clinical characteristics were found in Pearson's chi-squared test or Fisher's exact test (Table 2).

Discussion
The CTLA-4 blockade has become a progressive treatment strategy and has opened up an exciting way for cancer management, including esophageal cancer [25]. The combination of immunotherapy and traditional treatment strategy can improve the therapeutic effect and prolong the life of patients with esophageal cancer. The analysis of the expression level of CTLA-4 in tumor cells and interstitial lymphocytes can provide a basis for the selection of patients with the most effective immunotherapy [26]. In addition, the prognostic value of the CTLA-4 expression in patients with ESCC should also be confirmed.
In this study, we first evaluated the prognostic value of CTLA-4 in patients with ESCC. The elevated expression rate of T-CTLA-4 and I-CTLA-4 was 48.8% and 44.0%, respectively. The prognostic value of T-CTLA-4 and I-CTLA-4 in ESCC was not detected. In considering the interaction between inflammation and the CTLA-4 expression, we excluded 12 ESCC patients with concurrent inflammatory diseases. However, T-CTLA-4 and I-CTLA-4 still had no effect on the outcome of the rest of the patients. In addition, no correlation was found between the expression of CTLA-4 and the clinical characteristics of patients with ESCC. So far, there is only one report about the expression of CTLA-4 in esophageal cancer, in which the expression level of CTLA-4 protein in primary esophageal cancer lesions has a potential prognostic value [14]. However, the relationship between the high expression of CTLA-4 and clinicopathological factors has not been reported. The difference between the published study and our study may be due to heterogeneous study samples, technical problems, and different cut-off values of the CTLA-4 expression. Therefore, the prognostic value of CTLA-4 in patients with ESCC remains to be evaluated in further studies. Inflammation plays a key role in the development of cancer [27]. Chronic inflammation induced by environmental exposure can activate the proinflammatory signaling pathway, thereby promoting tumor growth, progression, angiogenesis, and metastasis [28,29]. As an indicator of systemic inflammation, PLR has been widely studied in patients with ESCC, and its prognostic effect has been documented [19][20][21]. However, the results of different studies are controversial. In the current study, we have not revealed the prognostic value of PLR in patients with ESCC. In a recent meta-analysis, the authors suggest that a combined analysis of PLR and other biomarkers may play a role in distinguishing ESCC patients with different treatment outcomes [30].
Immune status has a great impact on the prognosis of cancer patients [31,32]. The immune effect of the host and the cancer itself is two important aspects of the immune status that affect the cancer prognosis, and there is an interaction between them [33]. Understanding the interaction between the cancer and the host immune system holds a great promise to develop the biomarkers for the diagnosis, treatment, and prognosis evaluation of cancer patients [34]. In fact, studies have shown that there is an interaction between PLR, which reflects the host immune and inflammatory levels, and CTLA-4, which reflects the immune status of cancer [35][36][37][38]. Combined analysis of PLR and CTLA-4 can improve the effectiveness of prognostic evaluation in patients with ESCC. Therefore, we analyzed the CTLA-4 expression levels and the PLR levels in patients with ESCC in this study. From this analysis, we identified a subgroup of patients with a lower T-CTLA-4 level and a lower PLR level as having the best OS compared with other patients. Multivariate survival analysis also showed the effect of T-CTLA-4(-) +PLR (-) status on prognosis.

Conclusion
To the best of our knowledge, this is the first study to analyze the effect of CTLA-4 and PLR on the prognosis of patients with ESCC. However, further prospective studies are needed to confirm the results of this study and to explore whether it can guide personalized immunotherapy in patients with ESCC.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
The authors declare that they have no conflict of interest.

Authors' Contributions
Conception and design were done YY Wang and R Zhao. Collection and assembly of data were done by CY Zhang and J Zhang. Data analysis and interpretation were done by H Zhe, YF Ma, and YY Wang. Manuscript writing was done by CY Zhang and YY Wang. All authors did the final approval of the manuscript. Cui-Ying Zhang and Juan Zhang contributed equally to this work.