Esophageal squamous cell carcinoma (ESCC) is one of the most common types of cancer in northern China [
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 [
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 [
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.
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 [
Immunohistochemistry (IHC) was performed as previously reported [
Tumor cells and interstitial lymphocytes were stained with CTLA-4. The expression levels of CTLA-4 were defined as follows: “−” (negative), “+” (weakly positive), “++” (moderately positive), and “+++” (strongly positive). Tissue sections with “++” and “+++” were defined as a high expression of CTLA-4 [
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.
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.
Pearson’s chi-squared test or Fisher’s exact test was used to analyze the relationship between CTLA-4 or PLR and clinicopathological features of ESCC patients. Spearman’s rank correlation analysis was used to analyze the correlation between T-CTLA-4 (tumor cell CTLA-4), I-CTLA-4 (interstitial lymphocyte CTLA-4), and PLR. The OS curve was drawn by the Kaplan-Meier method and compared between groups by the log-rank test. The median follow-up time was also analyzed by the Kaplan-Meier method. The Cox proportional hazard model was used for univariate and multivariate analyses. The minimum significant level was described as
T-CTLA-4 and I-CTLA-4 were mainly distributed in the cytoplasm. The cytoplasmic pattern of I-CTLA-4 was more homogeneous than that of T-CTLA-4 staining. Figure
Representative immunostaining for T-CTLA-4 and I-CTLA-4. Positive staining for CTLA-4 in tumor cells ((a) 200x magnification; (b) 400x magnification). Positive staining for CTLA-4 in interstitial lymphocytes ((c) 200x magnification; (d) 400x magnification).
The median PLR value of the whole group was 121.7. According to the results of ROC analysis (Figure
ROC curve. The optimal cut-off value of PLR was determined by ROC analysis.
The effects of the CTLA-4 expression level and the PLR level on the survival of ESCC patients were examined. Kaplan-Meier analysis did not find the effects of the T-CTLA-4 expression level (
The overall survival curve of ESCC patients with different levels of T-CTLA-4, I-CTLA-4, and PLR. The Kaplan-Meier survival curve for patients with ESCC whose tumors were classified as either higher or lower level for T-CTLA-4, I-CTLA-4, and PLR, respectively. T-CTLA-4 (
Considering the interaction between inflammatory diseases and CTLA-4 or PLR levels, 12 ESCC patients with concurrent inflammatory diseases were excluded in the subsequent analysis. The expression levels of T-CTLA-4 (
Patients with T-CTLA-4(-) +PLR (-) status had superior overall survival. Effects of different levels of T-CTLA-4 (a), I-CTLA-4 (b), PLR (c), and T-CTLA-4(-) +PLR (-) status (d) on the overall survival of ESCC patients without inflammatory diseases. Patients with T-CTLA-4(-) +PLR (-) status had significantly longer overall survival (
Univariate analysis showed that T stage (
Univariate and multivariate Cox regression analyses estimating the risk factors of OS in ESCC patients.
Variable | Univariate analyses | Multivariate analyses | ||||
---|---|---|---|---|---|---|
HR | 95% CI | HR | 95% CI | |||
Gender | 0.389 | 0.094-1.612 | 0.193 | |||
Age | 0.683 | 0.268-1.738 | 0.424 | |||
KPS | 1.237 | 0.674-2.271 | 0.493 | |||
Drinking | 0.943 | 0.505-1.760 | 0.854 | |||
Smoking | 1.449 | 0.762-2.756 | 0.258 | |||
Weight loss | 1.116 | 0.579-2.152 | 0.742 | |||
Histological grade | 0.832 | 0.454-1.527 | 0.553 | |||
Location | 2.706 | 0.652-11.237 | 0.171 | |||
Length | 1.991 | 0.949-4.177 | 0.069 | |||
T stage | 2.718 | 1.250-5.913 | 0.012 | 3.168 | 0.320-31.376 | 0.324 |
N stage | 3.464 | 1.841-6.517 | 0.000 | 5.194 | 0.547-49.265 | 0.151 |
TNM stage | 2.218 | 1.428-3.171 | 0.000 | 0.537 | 0.064-4.508 | 0.567 |
Resection margin | 0.935 | 0.226-3.875 | 0.926 | |||
Adjuvant radiotherapy | 2.107 | 1.116-3.978 | 0.022 | 2.104 | 1.109-3.993 | 0.023 |
Adjuvant chemotherapy | 1.829 | 0.941-3.556 | 0.075 | |||
T-CTLA-4(-) +PLR (-) | 2.031 | 1.083-3.807 | 0.027 | 2.025 | 1.071-3.826 | 0.030 |
Abbreviations: OS: overall survival; ESCC: esophageal squamous cell carcinoma; HR: hazard ratio; CI: confidence interval; KPS: Karnofsky; T-CTLA-4: tumor cell cytotoxic T-lymphocyte-associated antigen-4; PLR: platelet lymphocyte ratio.
Clinicopathological characteristics of ESCC patients.
Clinicopathological characteristics | T-CTLA-4(-) +PLR (-) | Other | ||
---|---|---|---|---|
Gender | ||||
Male | 66 | 30 | 36 | |
Female | 6 | 2 | 4 | 0.686 |
Age (years) | ||||
<70 | 62 | 27 | 35 | |
≥70 | 10 | 5 | 5 | 0.703 |
KPS | ||||
>80% | 39 | 17 | 22 | |
≤80% | 33 | 15 | 18 | 0.874 |
Weight loss (kg) | ||||
<5 | 50 | 22 | 28 | |
≥5 | 22 | 10 | 12 | 0.909 |
Drinking | ||||
Yes | 32 | 13 | 19 | |
No | 40 | 19 | 21 | 0.560 |
Smoking | ||||
Yes | 46 | 21 | 25 | |
No | 26 | 11 | 15 | 0.784 |
Histological differentiation | ||||
Well and moderate | 39 | 15 | 24 | |
Poor | 33 | 17 | 16 | 0.267 |
Location | ||||
Proximal third | 7 | 3 | 4 | |
Middle third | 43 | 17 | 26 | |
Distal third | 22 | 12 | 10 | 0.513 |
Tumor length (cm) | ||||
<3 | 18 | 10 | 8 | |
≥3 | 54 | 22 | 32 | 0.273 |
Infiltration depth | ||||
T1 and T2 | 21 | 11 | 10 | |
T3 and T4 | 51 | 21 | 30 | 0.384 |
Lymph node status | ||||
N0 | 47 | 23 | 24 | |
N1 | 25 | 9 | 16 | 0.293 |
pTNM staging | ||||
I and II | 48 | 23 | 25 | |
III | 24 | 9 | 15 | 0.402 |
Resection margin | ||||
Positive | 3 | 1 | 2 | |
Negative | 69 | 31 | 38 | 1.000 |
Adjuvant radiotherapy | ||||
Yes | 19 | 7 | 12 | |
No | 53 | 25 | 28 | 0.437 |
Adjuvant chemotherapy | ||||
Yes | 16 | 5 | 11 | |
No | 56 | 27 | 29 | 0.228 |
Abbreviations: T-CTLA-4: tumor cell cytotoxic T-lymphocyte-associated antigen-4; PLR: platelet lymphocyte ratio; ESCC: esophageal squamous cell carcinoma; KPS: Karnofsky.
The CTLA-4 blockade has become a progressive treatment strategy and has opened up an exciting way for cancer management, including esophageal cancer [
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 [
Inflammation plays a key role in the development of cancer [
Immune status has a great impact on the prognosis of cancer patients [
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.
The data used to support the findings of this study are available from the corresponding authors upon request.
The authors declare that they have no conflict of interest.
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.
We thank Timothy King and Dennis Wilson for the language editing of this manuscript. This work was supported by the National Natural Science Foundation of China (81502094) and the First-Class Discipline Construction Founded Project of Ningxia Medical University and the School of Clinical Medicine (NXYLXK2017A05).
Table S1: comparison of clinicopathologic characteristics between T-CTLA-4(-) +PLR (-) and other group of ESCC patients without concurrent inflammatory disease.