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Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma
Journal Pre-proof Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma Yuhuan Zhao, MD, Donglai Chen, MD, Wenjia Wang, MD, Ting Zhao, MD, Junmiao Wen, MD, Fuquan Zhang, MD, Shanzhou Duan, MD, Chang Chen, MD, PhD, Yonghua Sang, MD, PhD, Yongsheng Zhang, MD, Yongbing Chen, MD, PhD PII:
S0003-4975(20)30067-9
DOI:
https://doi.org/10.1016/j.athoracsur.2019.12.017
Reference:
ATS 33414
To appear in:
The Annals of Thoracic Surgery
Received Date: 28 May 2019 Revised Date:
15 November 2019
Accepted Date: 5 December 2019
Please cite this article as: Zhao Y, Chen D, Wang W, Zhao T, Wen J, Zhang F, Duan S, Chen C, Sang Y, Zhang Y, Chen Y, Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma, The Annals of Thoracic Surgery (2020), doi: https://doi.org/10.1016/ j.athoracsur.2019.12.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 by The Society of Thoracic Surgeons
Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma
Running Head: TIM-3 expression in esophageal cancer
Yuhuan Zhao, MD#1; Donglai Chen, MD#2; Wenjia Wang, MD#1; Ting Zhao, MD1; Junmiao Wen, MD3; Fuquan Zhang, MD1; Shanzhou Duan, MD1; Chang Chen, MD, PhD2; Yonghua Sang, MD, PhD*1; Yongsheng Zhang, MD*4; Yongbing Chen, MD, PhD*1
1. Department of Thoracic Surgery, the Second Affiliated Hospital of Soochow University, Suzhou, China
2. Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China
3. Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China
4. Department of Pathology, the Second Affiliated Hospital of Soochow University, Suzhou, China
# *Drs Yuhuan Zhao, Donglai Chen, and Wenjia Wang contributed equally to this work; Drs Yonghua Sang, Yongsheng Zhang, and Yongbing Chen are co-senior authors.
Correspondence to:
Yongbing Chen, Department of Thoracic Surgery, the Second Affiliated Hospital of Soochow University, Suzhou 215000, China, E-mail: [email protected]
Word Count: 4496 words
1
Abstract
Background: T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a promising checkpoint. However, its features and prognostic value remain undetermined in esophageal squamous cell carcinoma (ESCC). In this study, we evaluated the prognostic value of TIM-3 expression and its relationship with programmed cell death 1 (PD-1) and CD8+ tumor-infiltrating lymphocytes (TILs) in patients with surgically resected ESCC. Methods: Expression levels of TIM-3, PD-1 and CD8+ TILs in ESCC were determined by immunohistochemistry. The association between clinicopathological features or clinical outcomes and TIM-3 expression was analyzed.
Results: 183 patients with ESCC who had undergone esophagectomy without implementation of neoadjuvant therapy at the Second Affiliated Hospital of Soochow University from January 2009 to December 2014 were included. PD-1 positivity (p = 0.032) and high CD8+ TILs density (p = 0.035) significantly correlated with positive TIM-3 expression. TIM-3 positivity was an independent risk factor for recurrence-free survival (RFS) (p < 0.001) and overall survival (OS) (p < 0.001). Subgroup analysis revealed that TIM-3+PD-1+CD8 low group had the worst RFS and OS, whereas TIM-3−PD-1−CD8 high group had the best RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p < 0.001). Conclusions: Positive TIM-3 expression was associated with PD-1 positivity and high CD8+ TILs density, which was an independent risk factor for RFS and OS in ESCC. Furthermore, the combination of TIM-3 and/or PD-1 expression or CD8+ TILs density could further stratify patients into different groups with distinct prognosis. 2
Keywords: TIM-3; PD-1; Tumor infiltrating lymphocytes; Esophageal Squamous Cell Carcinoma; Prognosis
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Abbreviations
ESCC: esophageal squamous cell carcinoma
TIM-3: T-cell immunoglobulin and mucin-domain containing-3
PD-1, PDCD1: programmed cell death 1
TILs: tumor-Infiltrating lymphocytes
OR: odds ratio
CI: confidence interval
HR: hazard ratio
Galetin-9: Glycan-binding protein-9
Th1: T-helper cells 1
Tc1: T cytotoxic cells 1
PD-L1: programmed death ligand 1
EGFR: epidermal growth factor receptor
VEGF: vascular endothelial growth-factor receptor
Tregs: regulatory T cells
HAVCR2: hepatitis A virus cellular receptor 2
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Esophageal cancer is one of the most common malignant tumors and leading causes of cancer-related deaths worldwide [1]. Esophageal squamous cell carcinoma (ESCC) accounts for the overwhelming majority of esophageal cancer in China [2]. For early-stage ESCC, surgical resection is the first choice for radical treatment. However, postoperative recurrence and metastasis remain an intractable problem which compromises the curative effect of surgical resection. Despite the progress in chemotherapy, surgery and radiotherapy, the 5-year survival of ESCC is still unsatisfactory [3,4,5]. In recent years, immunotherapy has made a breakthrough in treating many types of cancer and is being increasingly utilized [6,7] which provides an alternative for adjuvant therapy. Immunotherapy can improve the ability of the immune system in recognition and elimination of cancer cells but can only benefit limited number of patients.
Immune checkpoints such as programmed cell death 1 (PD-1) have been shown as the most promising target, whose inhibitors have exhibited remarkable responses in a number of cancer types [8,9,10]. However, the efficacy of PD-1 inhibitors remains unproven as an alternative in adjuvant therapy for patients with operable ESCC. Moreover, a few studies on PD-1 inhibitors showed that adaptive resistance was inevitable in immunotherapy [11,12]. In order to promote and optimize the application of immunotherapy in operable ESCC in the future, we tried to further characterize other immune checkpoints, especially those associated with the adaptive resistance of PD-1 inhibitor therapy. T cell immunoglobulin mucin-3 (TIM-3) is a promising checkpoint which can exhaust T-helper cells 1 (Th1) and T cytotoxic cells 1 (Tc1) once combining with Galetin-9 expressed on tumor cells. So far, it has been undefined concerning TIM-3 expression and its relationships with clinicopathological characteristics and prognostic impact in ESCC patients. In addition, although the relationship between PD-1 expression and clinical outcomes of ESCC has been assessed, 5
conflict findings have been reported in different studies [13,14,15], which highlights the necessity of our further investigation.
In the current study, we evaluated the prognostic significance of TIM-3 expression and the relationship between TIM-3 expression and clinicopathological features in ESCC. We also evaluated the relationships between TIM-3 and PD-1 expression or CD8+ TILs density and further explored the distinct prognostic value of the different combinations of these three markers.
Patients and Methods
Patients
We retrospectively reviewed 261 patients with esophageal cancer who accepted surgical treatment in our department in the Second Affiliated Hospital of Soochow University from January 2009 to December 2014. The patients in our study had to meet the following inclusion criteria: (1) patients had an R0 resection (2) patients that had not undergone any systemic therapy before surgery and (3) patients who were pathologically confirmed with primary TxNxM0 squamous cell carcinoma according to the eighth edition of the TNM classification [16]. The exclusion criterion was (1) patients with autoimmune diseases and other kinds of esophageal cancer (e.g. adenocarcinoma), (2) patients lost to follow-up and (3) patients with concurrent multiple primary tumors or other malignancy. According to the criteria, 78 patients were excluded and the remaining 183 patients were included. This study was approved by the Institutional Review Board of the Second Affiliated Hospital of Soochow University.
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Surgical procedure and follow-up
In all cases, upper thoracic ESCC was treated by McKeown esophagectomy combined with three-field lymphadenectomy, while middle thoracic ESCC was treated by Ivor-Lewis esophagectomy combined with two-field lymphadenectomy, and lower thoracic ESCC was treated by Ivor-Lewis or Sweet esophagectomy combined with two-field lymphadenectomy. However, if cervical lymph nodes enlargement was found by preoperative examination, three-field lymphadenectomy was performed. Patients were followed up at outpatient department every 1 to 3 months after discharge in postoperative 2 years and every 6 months afterwards, while patients who were inconvenient to go to outpatient department were followed up by phone. The endpoints in our study are recurrence-free survival (RFS) and overall survival (OS). RFS was defined as the time between surgery and the date of diagnosed as recurrence. OS was defined as the time between surgery and death or the date of last follow-up.
Immunohistochemistry
Consecutive formalin-fixed and paraffin-embedded tissue sections were taken from the tumor specimen with 4-µm thickness. Firstly, these sections were deparaffinized and rehydrated. Afterwards, we performed antigen retrieval in antigen retrieval solution using a steamer autoclave at 121℃ for 15 minutes. Nonspecific proteins were blocked with 10% goat serum for 1 hour, and sections were then rinsed with 0.01 mol/L PBS for 3 minutes. The sections were then respectively incubated with Anti-TIM-3 (ab47997,Abcam; diluted 1: 200), Anti-PD-1 (ab52587, Abcam;diluted 1: 100), Anti-CD8 (ab4055, Abcam; diluted 1: 100) overnight at 4˚C. The sections were 7
counterstained with a DAB Horseradish Peroxidase Color Development Kit (Beyotime, China) after incubating with horseradish peroxidase conjugated secondary antibody at room temperature for 30 min. Finally, the sections were counterstained with hematoxylin and mounted.
All specimens were reviewed by two independent experienced pathologists (Li F. and Zhang Y.) who were blinded to patient outcomes. The staining intensity and the percentage of positively stained cells expressing TIM-3, PD-1 and CD8 were counted respectively in five microscopic fields under high magnification in three sections from each tumor tissue. If disagreement occurred, discussion was held until a consensus was reached. For TIM-3 and PD-1 expression, three grades were classed according to the percentage of the number of staining cells: 1, ≤33%; 2, >33% to ≤66%; 3, >66%. The staining intensity was also ranked into three grades: 1, absent or weak staining; 2, moderate staining; 3, strong staining. The immunostaining results were calculated by the sum of the scores for percentage of stained cells and staining intensity. Finally, score >3 was considered positive expression and score ≤3 was considered negative expression [17,18]. In addition, high CD8+ TILs density was defined as ≥10% staining in all non-epithelial cells as previously reported [19].
Statistical analysis
SPSS 25.0 software (IBM Corporation, Armonk, NY, USA) was used to analyze statistical data. The relationships between TIM-3 and clinicopathological features, PD-1 or CD8 were evaluated by Chi-square tests and Spearman´s rank correlation. Multiple comparisons were adjusted by Bonfferoni test. The independent predictors of TIM-3 expression were analyzed by the logistic 8
regression. The RFS rate and OS rate were examined by Kaplan-Meier method. The log-rank test was applied to compare survival differences. A time-dependent Cox proportional hazards regression model was used to evaluate the prognostic factors for RFS and OS. In our study, a two-sided p-value of less than 0.05 was considered statistically significant. We also made bioinformatic analyses using Tumor IMmune Estimation Resource (TIMER, a website based on the The Cancer Genome Atlas database, https://cistrome.shinyapps.io/timer/) for external validation.
Results
TIM-3 expression and clinicopathological features
TIM-3 is expressed not only on tumor cells but also on TILs. In our study, only TIM-3 expressed on TILs were counted (Figure 1B). Table 1 showed the relationships between TIM-3 expression and characteristics of the patients in ESCC. Among 183 patients, 87 (47.5%) were classified as TIM-3-positive. Significant association was found between TIM-3 expression and vascular invasion (p = 0.044). However, there were no significant association between TIM-3 expression and other clinicopathological characteristics.
Associations between TIM-3, PD-1 and CD8 expression
Representative stained fields on histopathological slides for PD-1 and CD8 expression are displayed in Figure 1C-D. PD-1 and CD8 are only expressed on TILs. Among all patients, 69 (37.7%) were classified as PD-1-positive and 88 (48.1%) were classified as high CD8+ TILs density.
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As shown in Table 1, positive TIM-3 expression was associated with PD-1 positivity (p = 0.012) and high CD8 density (p = 0.015) on TILs. Furthermore, in multivariate logistic analysis, PD-1 positivity (odds ratio [OR] = 2.006; 95% confidence interval [CI] = 1.063-3.787; p = 0.032) and high CD8+ TILs density (OR=1.970; 95% CI=1.048-3.705; p=0.035) were independent predictive factors for TIM-3 expression (Table 2).
Prognostic value of TIM-3 and PD-1 expression in ESCC
Kaplan-Meier analysis demonstrated that patients in TIM-3-positive group had significantly shorter RFS (log-rank test p < 0.001; Figure 2A) and OS (log-rank test p < 0.001; Figure 2B) than those in TIM-3-negative group. However, PD-1 expression was not significantly associated with RFS (log-rank test p =0.092; Supplementary Figure 1A) and OS (log-rank test p =0.060; Supplementary Figure 1B).
Cox regression analysis for RFS and OS
In univariate analysis，variables whose p value smaller than 0.20 were enrolled into multivariate Cox regression analysis. Univariate analysis revealed that age, pathologic differentiation, TNM stage, TIM-3, PD-1, and CD8 expression were potential independent factors for prognoses (Table 3). Further multivariate analysis confirmed that TIM-3 positivity was associated with poorer RFS (HR = 4.007; 95%CI = 2.450-6.554; p < 0.001) and OS (hazard ratio [HR] = 2.620; 95%CI = 1.569-4.375; p < 0.001) in addition to advanced TNM stage (RFS: HR = 2.020; 95%CI = 1.297,3.147; p = 0.002; OS: HR = 1.922; 95%CI = 1.183,3.124; p = 0.008). In 10
contrast, high CD8+ TILs density was associated with better RFS (HR = 0.311; 95%CI = 0.193-0.502; p < 0.001) and OS (HR = 0.353; 95%CI = 0.209-0.597; p < 0.001).
Relationships between a combination of TIM-3 and PD-1 expression and/or CD8+ TILs density and clinical outcomes As shown in Figure 3, patients with TIM-3+PD-1+ had the worst RFS and OS, patients with TIM-3-PD-1- had the best RFS and OS, and patients with TIM-3 or PD-1 single positive had moderate RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p = 0.003) (Figure 3A-B). Meanwhile, patients with TIM-3+CD8 low had the worst RFS and OS, patients with TIM-3-CD8 high had the best RFS and OS, and others had moderate RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p < 0.001) (Figure 3C-D). Subgroup analysis revealed that TIM-3+PD-1+CD8 low group had the worst RFS and OS, while TIM-3−PD-1−CD8 high group had the best RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p < 0.001) (Supplementary Figure 2).
Comment
Although progress has been made unceasingly in therapeutic strategies for ESCC, especially in molecular targeted therapies including anti-EGFR and anti-VEGF drugs [20,21], the clinical outcomes of patients with ESCC remain frustrating. The past decade witnessed the rapid rise of immunotherapy which brings incredible effects on various types of cancer via reactivation and proliferation of host immunocytes. Since the inhibition and exhaustion of T cells play important roles in the tumorigenesis of ESCC [22], it is urgent to assess the roles of different immune 11
checkpoints that compromise the anti-tumor effect of T cells in ESCC. To our best knowledge, this is the first study to synchronously characterize TIM-3 and PD-1 expression and their association with CD8+ TILs in surgically resected ESCC.
TIM-3 and PD-1 has been found to express in kinds of tumor types and function as important checkpoints. Previous studies revealed that positive TIM-3 expression was associated with poor prognosis in lung adenocarcinoma, acute myelogenous leukemia, and hepatocellular carcinomas [23,24,25] because of suppressed antitumor immunity by TIM-3. Differential gene analysis showed that different expression levels of TIM-3 (p < 0.001) and PD-1 (0.05 ≤ p < 0.1) in ESCC were respectively found between tumor tissues and adjacent normal tissues (Supplementary Figure 3). In this study, TIM-3 were stained in surgically resected ESCC tissues. 47.5% (n=87) of the included patients were identified as TIM-3-positive on TILs. TIM-3 positivity was independent risk factor for prognosis, whereas PD-1 was not. Conflict findings had been reported about the relationship between PD-1 expression and clinical outcomes of ESCC, a study was in agreement with our current result [13], but two other studies indicated that PD-1 positivity was related to poor prognosis [14,15]. These controversies may be due to the difference of patient cohort or the methods used to evaluate PD-1 expression. Further studies are needed in the future. As shown in Table 2, PD-1 positivity and high CD8+ TILs density were independent factors for TIM-3 expression. External validation analysis also supported our result (Supplementary Figure 4). In this study, we divided the patients into four groups according to the expression levels of TIM-3 and PD-1, in which TIM-3+PD-1+ patients had the worst RFS and OS. Since interactions between PD-1 and PD-L1 can inhibit the anti-tumor effect of CD8+ TILs and enhance the function of regulatory T cells (Tregs), immune escape and anti-apoptosis of tumor cells were inevitable with 12
the conjugation of PD-1/PD-L1 in ESCC. Moreover, there was an observed correlation between TIM-3 and PD-1 in the process of T cell exhausting [26]. Therefore the co-expression of TIM-3 and PD-1 on TILs indicated the severely inhibited functions of T cells which lost the capability of proliferation and releasing cytokines [27]. In subgroup analysis, patients with TIM-3+CD8 low had the worst RFS and OS. Studies had shown that high TIM-3 expression on CD8+ T cells indicated progression of tumors and poor prognosis [28], which partly support our results. Our analysis showed that patients with TIM-3+PD-1+CD8 low had the worst RFS and OS. Conversely, patients with TIM-3−PD-1−CD8 high had the best RFS and OS. To explain such a phenomenon, we therefore stained Ki67 to find out the cause. As shown in Supplementary Figure 5, the number of the patients with high Ki67 expression on tumor cells and high TILs density was almost half of the patients with high Ki67 expression on T cells and high TILs density. Notably, most of the patients with Ki67-high TILs have negative PD-1 and TIM-3 expression in their ESCC lesions, suggesting that these proliferative TILs have potentials to kill tumor cells which may account for their favorable survival. According to our data, immunotherapy via combined blockade of TIM-3 and PD-1 might exhibit more powerful effects on the ESCC patients with TIM-3+PD-1+ and high CD8+ TILs density compared with blocking TIM-3/Galetin-9 or PD-1/PD-L1 interaction alone.
There are several limitations in our study. First, due to the retrospective nature of our study, selection and performance biases were inevitable. Second, we only included patients from a single institution and did not include patients with esophageal adenocarcinoma. Third, no patients received neoadjuvant chemoradiation therapy in our current study which merits further investigation. Fourth, adjuvant therapy was not included in the analysis. Fifth, we did not investigate TIM-3 expression on tumor cells and the expression level of PD-L1. A multi-center study with large patient cohorts may 13
help to address these limitations in the future. In conclusion, positive TIM-3 expression was associated with PD-1 positivity and high CD8+ TILs density, which was an independent risk factor for RFS and OS in ESCC. Furthermore, the combination of TIM-3 and/or PD-1 expression or CD8+ TILs density could further stratify patients into different groups with distinct prognosis.
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Figure Legends Figure 1. Immunochemistry for TIM-3, PD-1 and CD8+ TILs. Negative expression for TIM-3 (A) (magnification × 400); positive expression for TIM-3 (B), PD-1 (C) and CD8 (D) on TILs (magnification × 400). (TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; TILs, tumor-infiltrating lymphocytes)
Figure 2. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients based on TIM-3 expression. (TIM-3, T-cell immunoglobulin and mucin-domain containing-3)
Figure 3. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients according to TIM-3 and PD-1 expression. Kaplan–Meier curves for recurrence-free survival (C) and overall survival (D) in ESCC patients according to TIM-3 expression and the expression level of CD8+TILs. (TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; TILs, tumor-infiltrating lymphocytes)
Supplementary Figure 1. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients based on PD-1 expression. (PD-1, programmed cell death 1)
Supplementary Figure 2. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients according to the expression level of TIM-3, PD-1 and CD8+TILs. (TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; TILs, tumor-infiltrating lymphocytes)
Supplementary Figure 3. Differential gene analysis between tumor tissues and normal tissues for HAVCR2 (A) and PDCD1 (B). (p-value significant codes:0≤***<0.001≤**<0.01≤*<0.05≤·<0.1) 19
(HAVCR2, hepatitis A virus cellular receptor 2, a gene encodes for TIM-3; PDCD1, programmed cell death 1; ESCA, esophageal cancer)
Supplementary Figure 4. Gene correlation analysis between HAVCR2 and PDCD1 in esophageal cancer. (HAVCR2, hepatitis A virus cellular receptor 2, a gene encodes TIM-3; PDCD1, programmed cell death 1)
Supplementary Figure 5. Description of Ki67 and CD8 expression and their co-expression.
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Table 1 TIM-3 expression and clinicopathological features in ESCC
TIM-3 expression
Variables
Total
Negative
Positive
Number
183
96 (52.5%)
87 (47.5%)
Sex
p value
0.742
Male
147 (80.3%)
78 (81.2%)
69 (79.3%)
Female
36 (19.7%)
18 (18.8%)
18 (20.7%)
Age
0.473
≤65
106 (57.9%)
58 (60.4%)
48 (55.2%)
>65
77 (42.1%)
38 (39.6%)
39 (44.8%)
Smoking
0.606
Yes
110 (60.1%)
56 (58.3%)
54 (62.1%)
No
73 (39.9%)
40 (41.7%)
33 (37.9%)
Tumor location
0.565
Upper
8 (4.4%)
5 (5.2%)
3 (3.4%)
Middle
115 (62.8%)
57 (59.4%)
58 (66.7%)
Lower
60 (32.8%)
34 (35.4%)
26 (29.9%)
Surgical type
0.294
Sweet esophagectomy
66 (36.1%)
39 (40.6%)
27 (31.1%)
Ivor-Lewis esophagectomy
72 (39.3%)
33 (34.4%)
39 (44.8%)
Mckeown esophagectomy
45 (24.6%)
24 (25.0%)
21 (24.1%)
Pathologic differentiation
0.160
Poor
46 (25.1%)
19 (19.8%)
27 (31.0%)
Moderate
125 (68.3%)
69 (71.9%)
56 (64.4%)
Well
12 (6.6%)
8 (8.3%)
4 (4.6%)
pT stage
0.719
21
T1
11 (6.0%)
7 (7.3%)
4 (4.6%)
T2
74 (40.4%)
41 (42.7%)
33 (37.9%)
T3
85 (46.5%)
42 (43.8%)
43 (49.4%)
T4
13 (7.10%)
6 (6.2%)
7 (8.1%)
pN stage
0.060
N0
117 (63.9%)
69 (71.9%)
48 (55.2%)
N1
55 (30.1%)
22 (22.9%)
33 (37.9%)
N2
11 (6.0%)
5 (5.2%)
6 (6.9%)
Vascular invasion
0.044
Present
24 (13.1%)
8 (8.3%)
16 (18.4%)
Absent
159 (86.9%)
88 (91.7%)
71 (81.6%)
PD-1 expression
0.012
Negative
114 (62.3%)
68 (70.8%)
46 (52.9%)
Positive
69 (37.7%)
28 (39.2%)
41 (47.1%)
CD8 expression
0.015
Low
95 (51.9%)
58 (60.4%)
37 (42.5%)
High
88 (48.1%)
38 (39.6%)
50 (57.5%)
TIM-3, T-cell immunoglobulin and mucin-domain containing-3; ESCC, esophageal squamous cell carcinoma; PD-1, programmed cell death 1
22
Table 2 Logistic regression model for TIM-3 expression in ESCC
Multivariate
Variables
OR (95% CI)
p value
Pathologic differentiation (poor vs. high & moderate)
1.742 (0.848,3.579)
0.131
pT stage (T3-4 vs. T1-2)
0.963(0.500,1.857)
0.911
pN stage (N1-2 vs. N0)
1.718 (0.783,3.449)
0.128
Vascular invasion (present vs. absent)
1.677 (0.630,4.465)
0.301
PD-1 expression (positive vs. negative)
2.006 (1.063,3.787)
0.032
CD8 expression (high vs. low)
1.970 (1.048,3.705)
0.035
TIM-3, T-cell immunoglobulin and mucin-domain containing-3; ESCC, esophageal squamous cell carcinoma; PD-1, programmed cell death 1; OR, odds ratio; CI, confidence interval
23
Table 3 Cox regression analysis for recurrence-free survival and overall survival
Recurrence-free survival
Overall survival
Univariate
Multivariate
Univariate
Multivariate
Variables
p value
HR (95% CI)
p value
HR (95% CI)
p value
Sex (male vs. female)
0.724
Age (>65 vs. ≤65)
0.164
1.226 (0.760,1.976)
0.403
Smoking (yes vs. no)
0.243
0.240
Tumor location (middle vs. upper & lower)
0.379
0.477
Surgical type (Sweet & Ivor-lewis vs. McKeown)
0.485
0.387
Pathologic differentiation (poor vs. moderate & high)
0.152
1.370 (0.844,2.223)
0.202
0.416
TNM stage (III vs. I-II)
<0.001
2.020 (1.297,3.147)
0.002
<0.001
1.922 (1.183,3.124)
0.008
Vascular invasion (present vs. absent)
0.401
TIM-3 expression (positive vs. negative)
<0.001
4.007 (2.450,6.554)
<0.001
<0.001
2.620(1.569,4.375)
<0.001
PD-1 expression (positive vs. negative)
0.092
1.321 (0.828,2.107)
0.243
0.060
1.490 (0.908,2.444)
0.114
CD8 expression (high vs. low)
0.002
0.311 (0.193,0.502)
<0.001
0.001
0.353 (0.209,0.597)
<0.001
p value
0.844
1.183 (0.771,1.816)
0.441
0.153
0.282
TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; HR,hazard ratio; CI,confidence interval
24
S0003-4975(20)30067-9
DOI:
https://doi.org/10.1016/j.athoracsur.2019.12.017
Reference:
ATS 33414
To appear in:
The Annals of Thoracic Surgery
Received Date: 28 May 2019 Revised Date:
15 November 2019
Accepted Date: 5 December 2019
Please cite this article as: Zhao Y, Chen D, Wang W, Zhao T, Wen J, Zhang F, Duan S, Chen C, Sang Y, Zhang Y, Chen Y, Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma, The Annals of Thoracic Surgery (2020), doi: https://doi.org/10.1016/ j.athoracsur.2019.12.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 by The Society of Thoracic Surgeons
Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma
Running Head: TIM-3 expression in esophageal cancer
Yuhuan Zhao, MD#1; Donglai Chen, MD#2; Wenjia Wang, MD#1; Ting Zhao, MD1; Junmiao Wen, MD3; Fuquan Zhang, MD1; Shanzhou Duan, MD1; Chang Chen, MD, PhD2; Yonghua Sang, MD, PhD*1; Yongsheng Zhang, MD*4; Yongbing Chen, MD, PhD*1
1. Department of Thoracic Surgery, the Second Affiliated Hospital of Soochow University, Suzhou, China
2. Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China
3. Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China
4. Department of Pathology, the Second Affiliated Hospital of Soochow University, Suzhou, China
# *Drs Yuhuan Zhao, Donglai Chen, and Wenjia Wang contributed equally to this work; Drs Yonghua Sang, Yongsheng Zhang, and Yongbing Chen are co-senior authors.
Correspondence to:
Yongbing Chen, Department of Thoracic Surgery, the Second Affiliated Hospital of Soochow University, Suzhou 215000, China, E-mail: [email protected]
Word Count: 4496 words
1
Abstract
Background: T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a promising checkpoint. However, its features and prognostic value remain undetermined in esophageal squamous cell carcinoma (ESCC). In this study, we evaluated the prognostic value of TIM-3 expression and its relationship with programmed cell death 1 (PD-1) and CD8+ tumor-infiltrating lymphocytes (TILs) in patients with surgically resected ESCC. Methods: Expression levels of TIM-3, PD-1 and CD8+ TILs in ESCC were determined by immunohistochemistry. The association between clinicopathological features or clinical outcomes and TIM-3 expression was analyzed.
Results: 183 patients with ESCC who had undergone esophagectomy without implementation of neoadjuvant therapy at the Second Affiliated Hospital of Soochow University from January 2009 to December 2014 were included. PD-1 positivity (p = 0.032) and high CD8+ TILs density (p = 0.035) significantly correlated with positive TIM-3 expression. TIM-3 positivity was an independent risk factor for recurrence-free survival (RFS) (p < 0.001) and overall survival (OS) (p < 0.001). Subgroup analysis revealed that TIM-3+PD-1+CD8 low group had the worst RFS and OS, whereas TIM-3−PD-1−CD8 high group had the best RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p < 0.001). Conclusions: Positive TIM-3 expression was associated with PD-1 positivity and high CD8+ TILs density, which was an independent risk factor for RFS and OS in ESCC. Furthermore, the combination of TIM-3 and/or PD-1 expression or CD8+ TILs density could further stratify patients into different groups with distinct prognosis. 2
Keywords: TIM-3; PD-1; Tumor infiltrating lymphocytes; Esophageal Squamous Cell Carcinoma; Prognosis
3
Abbreviations
ESCC: esophageal squamous cell carcinoma
TIM-3: T-cell immunoglobulin and mucin-domain containing-3
PD-1, PDCD1: programmed cell death 1
TILs: tumor-Infiltrating lymphocytes
OR: odds ratio
CI: confidence interval
HR: hazard ratio
Galetin-9: Glycan-binding protein-9
Th1: T-helper cells 1
Tc1: T cytotoxic cells 1
PD-L1: programmed death ligand 1
EGFR: epidermal growth factor receptor
VEGF: vascular endothelial growth-factor receptor
Tregs: regulatory T cells
HAVCR2: hepatitis A virus cellular receptor 2
4
Esophageal cancer is one of the most common malignant tumors and leading causes of cancer-related deaths worldwide [1]. Esophageal squamous cell carcinoma (ESCC) accounts for the overwhelming majority of esophageal cancer in China [2]. For early-stage ESCC, surgical resection is the first choice for radical treatment. However, postoperative recurrence and metastasis remain an intractable problem which compromises the curative effect of surgical resection. Despite the progress in chemotherapy, surgery and radiotherapy, the 5-year survival of ESCC is still unsatisfactory [3,4,5]. In recent years, immunotherapy has made a breakthrough in treating many types of cancer and is being increasingly utilized [6,7] which provides an alternative for adjuvant therapy. Immunotherapy can improve the ability of the immune system in recognition and elimination of cancer cells but can only benefit limited number of patients.
Immune checkpoints such as programmed cell death 1 (PD-1) have been shown as the most promising target, whose inhibitors have exhibited remarkable responses in a number of cancer types [8,9,10]. However, the efficacy of PD-1 inhibitors remains unproven as an alternative in adjuvant therapy for patients with operable ESCC. Moreover, a few studies on PD-1 inhibitors showed that adaptive resistance was inevitable in immunotherapy [11,12]. In order to promote and optimize the application of immunotherapy in operable ESCC in the future, we tried to further characterize other immune checkpoints, especially those associated with the adaptive resistance of PD-1 inhibitor therapy. T cell immunoglobulin mucin-3 (TIM-3) is a promising checkpoint which can exhaust T-helper cells 1 (Th1) and T cytotoxic cells 1 (Tc1) once combining with Galetin-9 expressed on tumor cells. So far, it has been undefined concerning TIM-3 expression and its relationships with clinicopathological characteristics and prognostic impact in ESCC patients. In addition, although the relationship between PD-1 expression and clinical outcomes of ESCC has been assessed, 5
conflict findings have been reported in different studies [13,14,15], which highlights the necessity of our further investigation.
In the current study, we evaluated the prognostic significance of TIM-3 expression and the relationship between TIM-3 expression and clinicopathological features in ESCC. We also evaluated the relationships between TIM-3 and PD-1 expression or CD8+ TILs density and further explored the distinct prognostic value of the different combinations of these three markers.
Patients and Methods
Patients
We retrospectively reviewed 261 patients with esophageal cancer who accepted surgical treatment in our department in the Second Affiliated Hospital of Soochow University from January 2009 to December 2014. The patients in our study had to meet the following inclusion criteria: (1) patients had an R0 resection (2) patients that had not undergone any systemic therapy before surgery and (3) patients who were pathologically confirmed with primary TxNxM0 squamous cell carcinoma according to the eighth edition of the TNM classification [16]. The exclusion criterion was (1) patients with autoimmune diseases and other kinds of esophageal cancer (e.g. adenocarcinoma), (2) patients lost to follow-up and (3) patients with concurrent multiple primary tumors or other malignancy. According to the criteria, 78 patients were excluded and the remaining 183 patients were included. This study was approved by the Institutional Review Board of the Second Affiliated Hospital of Soochow University.
6
Surgical procedure and follow-up
In all cases, upper thoracic ESCC was treated by McKeown esophagectomy combined with three-field lymphadenectomy, while middle thoracic ESCC was treated by Ivor-Lewis esophagectomy combined with two-field lymphadenectomy, and lower thoracic ESCC was treated by Ivor-Lewis or Sweet esophagectomy combined with two-field lymphadenectomy. However, if cervical lymph nodes enlargement was found by preoperative examination, three-field lymphadenectomy was performed. Patients were followed up at outpatient department every 1 to 3 months after discharge in postoperative 2 years and every 6 months afterwards, while patients who were inconvenient to go to outpatient department were followed up by phone. The endpoints in our study are recurrence-free survival (RFS) and overall survival (OS). RFS was defined as the time between surgery and the date of diagnosed as recurrence. OS was defined as the time between surgery and death or the date of last follow-up.
Immunohistochemistry
Consecutive formalin-fixed and paraffin-embedded tissue sections were taken from the tumor specimen with 4-µm thickness. Firstly, these sections were deparaffinized and rehydrated. Afterwards, we performed antigen retrieval in antigen retrieval solution using a steamer autoclave at 121℃ for 15 minutes. Nonspecific proteins were blocked with 10% goat serum for 1 hour, and sections were then rinsed with 0.01 mol/L PBS for 3 minutes. The sections were then respectively incubated with Anti-TIM-3 (ab47997,Abcam; diluted 1: 200), Anti-PD-1 (ab52587, Abcam;diluted 1: 100), Anti-CD8 (ab4055, Abcam; diluted 1: 100) overnight at 4˚C. The sections were 7
counterstained with a DAB Horseradish Peroxidase Color Development Kit (Beyotime, China) after incubating with horseradish peroxidase conjugated secondary antibody at room temperature for 30 min. Finally, the sections were counterstained with hematoxylin and mounted.
All specimens were reviewed by two independent experienced pathologists (Li F. and Zhang Y.) who were blinded to patient outcomes. The staining intensity and the percentage of positively stained cells expressing TIM-3, PD-1 and CD8 were counted respectively in five microscopic fields under high magnification in three sections from each tumor tissue. If disagreement occurred, discussion was held until a consensus was reached. For TIM-3 and PD-1 expression, three grades were classed according to the percentage of the number of staining cells: 1, ≤33%; 2, >33% to ≤66%; 3, >66%. The staining intensity was also ranked into three grades: 1, absent or weak staining; 2, moderate staining; 3, strong staining. The immunostaining results were calculated by the sum of the scores for percentage of stained cells and staining intensity. Finally, score >3 was considered positive expression and score ≤3 was considered negative expression [17,18]. In addition, high CD8+ TILs density was defined as ≥10% staining in all non-epithelial cells as previously reported [19].
Statistical analysis
SPSS 25.0 software (IBM Corporation, Armonk, NY, USA) was used to analyze statistical data. The relationships between TIM-3 and clinicopathological features, PD-1 or CD8 were evaluated by Chi-square tests and Spearman´s rank correlation. Multiple comparisons were adjusted by Bonfferoni test. The independent predictors of TIM-3 expression were analyzed by the logistic 8
regression. The RFS rate and OS rate were examined by Kaplan-Meier method. The log-rank test was applied to compare survival differences. A time-dependent Cox proportional hazards regression model was used to evaluate the prognostic factors for RFS and OS. In our study, a two-sided p-value of less than 0.05 was considered statistically significant. We also made bioinformatic analyses using Tumor IMmune Estimation Resource (TIMER, a website based on the The Cancer Genome Atlas database, https://cistrome.shinyapps.io/timer/) for external validation.
Results
TIM-3 expression and clinicopathological features
TIM-3 is expressed not only on tumor cells but also on TILs. In our study, only TIM-3 expressed on TILs were counted (Figure 1B). Table 1 showed the relationships between TIM-3 expression and characteristics of the patients in ESCC. Among 183 patients, 87 (47.5%) were classified as TIM-3-positive. Significant association was found between TIM-3 expression and vascular invasion (p = 0.044). However, there were no significant association between TIM-3 expression and other clinicopathological characteristics.
Associations between TIM-3, PD-1 and CD8 expression
Representative stained fields on histopathological slides for PD-1 and CD8 expression are displayed in Figure 1C-D. PD-1 and CD8 are only expressed on TILs. Among all patients, 69 (37.7%) were classified as PD-1-positive and 88 (48.1%) were classified as high CD8+ TILs density.
9
As shown in Table 1, positive TIM-3 expression was associated with PD-1 positivity (p = 0.012) and high CD8 density (p = 0.015) on TILs. Furthermore, in multivariate logistic analysis, PD-1 positivity (odds ratio [OR] = 2.006; 95% confidence interval [CI] = 1.063-3.787; p = 0.032) and high CD8+ TILs density (OR=1.970; 95% CI=1.048-3.705; p=0.035) were independent predictive factors for TIM-3 expression (Table 2).
Prognostic value of TIM-3 and PD-1 expression in ESCC
Kaplan-Meier analysis demonstrated that patients in TIM-3-positive group had significantly shorter RFS (log-rank test p < 0.001; Figure 2A) and OS (log-rank test p < 0.001; Figure 2B) than those in TIM-3-negative group. However, PD-1 expression was not significantly associated with RFS (log-rank test p =0.092; Supplementary Figure 1A) and OS (log-rank test p =0.060; Supplementary Figure 1B).
Cox regression analysis for RFS and OS
In univariate analysis，variables whose p value smaller than 0.20 were enrolled into multivariate Cox regression analysis. Univariate analysis revealed that age, pathologic differentiation, TNM stage, TIM-3, PD-1, and CD8 expression were potential independent factors for prognoses (Table 3). Further multivariate analysis confirmed that TIM-3 positivity was associated with poorer RFS (HR = 4.007; 95%CI = 2.450-6.554; p < 0.001) and OS (hazard ratio [HR] = 2.620; 95%CI = 1.569-4.375; p < 0.001) in addition to advanced TNM stage (RFS: HR = 2.020; 95%CI = 1.297,3.147; p = 0.002; OS: HR = 1.922; 95%CI = 1.183,3.124; p = 0.008). In 10
contrast, high CD8+ TILs density was associated with better RFS (HR = 0.311; 95%CI = 0.193-0.502; p < 0.001) and OS (HR = 0.353; 95%CI = 0.209-0.597; p < 0.001).
Relationships between a combination of TIM-3 and PD-1 expression and/or CD8+ TILs density and clinical outcomes As shown in Figure 3, patients with TIM-3+PD-1+ had the worst RFS and OS, patients with TIM-3-PD-1- had the best RFS and OS, and patients with TIM-3 or PD-1 single positive had moderate RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p = 0.003) (Figure 3A-B). Meanwhile, patients with TIM-3+CD8 low had the worst RFS and OS, patients with TIM-3-CD8 high had the best RFS and OS, and others had moderate RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p < 0.001) (Figure 3C-D). Subgroup analysis revealed that TIM-3+PD-1+CD8 low group had the worst RFS and OS, while TIM-3−PD-1−CD8 high group had the best RFS and OS (RFS: log-rank test p < 0.001; OS: log-rank test p < 0.001) (Supplementary Figure 2).
Comment
Although progress has been made unceasingly in therapeutic strategies for ESCC, especially in molecular targeted therapies including anti-EGFR and anti-VEGF drugs [20,21], the clinical outcomes of patients with ESCC remain frustrating. The past decade witnessed the rapid rise of immunotherapy which brings incredible effects on various types of cancer via reactivation and proliferation of host immunocytes. Since the inhibition and exhaustion of T cells play important roles in the tumorigenesis of ESCC [22], it is urgent to assess the roles of different immune 11
checkpoints that compromise the anti-tumor effect of T cells in ESCC. To our best knowledge, this is the first study to synchronously characterize TIM-3 and PD-1 expression and their association with CD8+ TILs in surgically resected ESCC.
TIM-3 and PD-1 has been found to express in kinds of tumor types and function as important checkpoints. Previous studies revealed that positive TIM-3 expression was associated with poor prognosis in lung adenocarcinoma, acute myelogenous leukemia, and hepatocellular carcinomas [23,24,25] because of suppressed antitumor immunity by TIM-3. Differential gene analysis showed that different expression levels of TIM-3 (p < 0.001) and PD-1 (0.05 ≤ p < 0.1) in ESCC were respectively found between tumor tissues and adjacent normal tissues (Supplementary Figure 3). In this study, TIM-3 were stained in surgically resected ESCC tissues. 47.5% (n=87) of the included patients were identified as TIM-3-positive on TILs. TIM-3 positivity was independent risk factor for prognosis, whereas PD-1 was not. Conflict findings had been reported about the relationship between PD-1 expression and clinical outcomes of ESCC, a study was in agreement with our current result [13], but two other studies indicated that PD-1 positivity was related to poor prognosis [14,15]. These controversies may be due to the difference of patient cohort or the methods used to evaluate PD-1 expression. Further studies are needed in the future. As shown in Table 2, PD-1 positivity and high CD8+ TILs density were independent factors for TIM-3 expression. External validation analysis also supported our result (Supplementary Figure 4). In this study, we divided the patients into four groups according to the expression levels of TIM-3 and PD-1, in which TIM-3+PD-1+ patients had the worst RFS and OS. Since interactions between PD-1 and PD-L1 can inhibit the anti-tumor effect of CD8+ TILs and enhance the function of regulatory T cells (Tregs), immune escape and anti-apoptosis of tumor cells were inevitable with 12
the conjugation of PD-1/PD-L1 in ESCC. Moreover, there was an observed correlation between TIM-3 and PD-1 in the process of T cell exhausting [26]. Therefore the co-expression of TIM-3 and PD-1 on TILs indicated the severely inhibited functions of T cells which lost the capability of proliferation and releasing cytokines [27]. In subgroup analysis, patients with TIM-3+CD8 low had the worst RFS and OS. Studies had shown that high TIM-3 expression on CD8+ T cells indicated progression of tumors and poor prognosis [28], which partly support our results. Our analysis showed that patients with TIM-3+PD-1+CD8 low had the worst RFS and OS. Conversely, patients with TIM-3−PD-1−CD8 high had the best RFS and OS. To explain such a phenomenon, we therefore stained Ki67 to find out the cause. As shown in Supplementary Figure 5, the number of the patients with high Ki67 expression on tumor cells and high TILs density was almost half of the patients with high Ki67 expression on T cells and high TILs density. Notably, most of the patients with Ki67-high TILs have negative PD-1 and TIM-3 expression in their ESCC lesions, suggesting that these proliferative TILs have potentials to kill tumor cells which may account for their favorable survival. According to our data, immunotherapy via combined blockade of TIM-3 and PD-1 might exhibit more powerful effects on the ESCC patients with TIM-3+PD-1+ and high CD8+ TILs density compared with blocking TIM-3/Galetin-9 or PD-1/PD-L1 interaction alone.
There are several limitations in our study. First, due to the retrospective nature of our study, selection and performance biases were inevitable. Second, we only included patients from a single institution and did not include patients with esophageal adenocarcinoma. Third, no patients received neoadjuvant chemoradiation therapy in our current study which merits further investigation. Fourth, adjuvant therapy was not included in the analysis. Fifth, we did not investigate TIM-3 expression on tumor cells and the expression level of PD-L1. A multi-center study with large patient cohorts may 13
help to address these limitations in the future. In conclusion, positive TIM-3 expression was associated with PD-1 positivity and high CD8+ TILs density, which was an independent risk factor for RFS and OS in ESCC. Furthermore, the combination of TIM-3 and/or PD-1 expression or CD8+ TILs density could further stratify patients into different groups with distinct prognosis.
14
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Figure Legends Figure 1. Immunochemistry for TIM-3, PD-1 and CD8+ TILs. Negative expression for TIM-3 (A) (magnification × 400); positive expression for TIM-3 (B), PD-1 (C) and CD8 (D) on TILs (magnification × 400). (TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; TILs, tumor-infiltrating lymphocytes)
Figure 2. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients based on TIM-3 expression. (TIM-3, T-cell immunoglobulin and mucin-domain containing-3)
Figure 3. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients according to TIM-3 and PD-1 expression. Kaplan–Meier curves for recurrence-free survival (C) and overall survival (D) in ESCC patients according to TIM-3 expression and the expression level of CD8+TILs. (TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; TILs, tumor-infiltrating lymphocytes)
Supplementary Figure 1. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients based on PD-1 expression. (PD-1, programmed cell death 1)
Supplementary Figure 2. Kaplan-Meier curves for recurrence-free survival (A) and overall survival (B) in ESCC patients according to the expression level of TIM-3, PD-1 and CD8+TILs. (TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; TILs, tumor-infiltrating lymphocytes)
Supplementary Figure 3. Differential gene analysis between tumor tissues and normal tissues for HAVCR2 (A) and PDCD1 (B). (p-value significant codes:0≤***<0.001≤**<0.01≤*<0.05≤·<0.1) 19
(HAVCR2, hepatitis A virus cellular receptor 2, a gene encodes for TIM-3; PDCD1, programmed cell death 1; ESCA, esophageal cancer)
Supplementary Figure 4. Gene correlation analysis between HAVCR2 and PDCD1 in esophageal cancer. (HAVCR2, hepatitis A virus cellular receptor 2, a gene encodes TIM-3; PDCD1, programmed cell death 1)
Supplementary Figure 5. Description of Ki67 and CD8 expression and their co-expression.
20
Table 1 TIM-3 expression and clinicopathological features in ESCC
TIM-3 expression
Variables
Total
Negative
Positive
Number
183
96 (52.5%)
87 (47.5%)
Sex
p value
0.742
Male
147 (80.3%)
78 (81.2%)
69 (79.3%)
Female
36 (19.7%)
18 (18.8%)
18 (20.7%)
Age
0.473
≤65
106 (57.9%)
58 (60.4%)
48 (55.2%)
>65
77 (42.1%)
38 (39.6%)
39 (44.8%)
Smoking
0.606
Yes
110 (60.1%)
56 (58.3%)
54 (62.1%)
No
73 (39.9%)
40 (41.7%)
33 (37.9%)
Tumor location
0.565
Upper
8 (4.4%)
5 (5.2%)
3 (3.4%)
Middle
115 (62.8%)
57 (59.4%)
58 (66.7%)
Lower
60 (32.8%)
34 (35.4%)
26 (29.9%)
Surgical type
0.294
Sweet esophagectomy
66 (36.1%)
39 (40.6%)
27 (31.1%)
Ivor-Lewis esophagectomy
72 (39.3%)
33 (34.4%)
39 (44.8%)
Mckeown esophagectomy
45 (24.6%)
24 (25.0%)
21 (24.1%)
Pathologic differentiation
0.160
Poor
46 (25.1%)
19 (19.8%)
27 (31.0%)
Moderate
125 (68.3%)
69 (71.9%)
56 (64.4%)
Well
12 (6.6%)
8 (8.3%)
4 (4.6%)
pT stage
0.719
21
T1
11 (6.0%)
7 (7.3%)
4 (4.6%)
T2
74 (40.4%)
41 (42.7%)
33 (37.9%)
T3
85 (46.5%)
42 (43.8%)
43 (49.4%)
T4
13 (7.10%)
6 (6.2%)
7 (8.1%)
pN stage
0.060
N0
117 (63.9%)
69 (71.9%)
48 (55.2%)
N1
55 (30.1%)
22 (22.9%)
33 (37.9%)
N2
11 (6.0%)
5 (5.2%)
6 (6.9%)
Vascular invasion
0.044
Present
24 (13.1%)
8 (8.3%)
16 (18.4%)
Absent
159 (86.9%)
88 (91.7%)
71 (81.6%)
PD-1 expression
0.012
Negative
114 (62.3%)
68 (70.8%)
46 (52.9%)
Positive
69 (37.7%)
28 (39.2%)
41 (47.1%)
CD8 expression
0.015
Low
95 (51.9%)
58 (60.4%)
37 (42.5%)
High
88 (48.1%)
38 (39.6%)
50 (57.5%)
TIM-3, T-cell immunoglobulin and mucin-domain containing-3; ESCC, esophageal squamous cell carcinoma; PD-1, programmed cell death 1
22
Table 2 Logistic regression model for TIM-3 expression in ESCC
Multivariate
Variables
OR (95% CI)
p value
Pathologic differentiation (poor vs. high & moderate)
1.742 (0.848,3.579)
0.131
pT stage (T3-4 vs. T1-2)
0.963(0.500,1.857)
0.911
pN stage (N1-2 vs. N0)
1.718 (0.783,3.449)
0.128
Vascular invasion (present vs. absent)
1.677 (0.630,4.465)
0.301
PD-1 expression (positive vs. negative)
2.006 (1.063,3.787)
0.032
CD8 expression (high vs. low)
1.970 (1.048,3.705)
0.035
TIM-3, T-cell immunoglobulin and mucin-domain containing-3; ESCC, esophageal squamous cell carcinoma; PD-1, programmed cell death 1; OR, odds ratio; CI, confidence interval
23
Table 3 Cox regression analysis for recurrence-free survival and overall survival
Recurrence-free survival
Overall survival
Univariate
Multivariate
Univariate
Multivariate
Variables
p value
HR (95% CI)
p value
HR (95% CI)
p value
Sex (male vs. female)
0.724
Age (>65 vs. ≤65)
0.164
1.226 (0.760,1.976)
0.403
Smoking (yes vs. no)
0.243
0.240
Tumor location (middle vs. upper & lower)
0.379
0.477
Surgical type (Sweet & Ivor-lewis vs. McKeown)
0.485
0.387
Pathologic differentiation (poor vs. moderate & high)
0.152
1.370 (0.844,2.223)
0.202
0.416
TNM stage (III vs. I-II)
<0.001
2.020 (1.297,3.147)
0.002
<0.001
1.922 (1.183,3.124)
0.008
Vascular invasion (present vs. absent)
0.401
TIM-3 expression (positive vs. negative)
<0.001
4.007 (2.450,6.554)
<0.001
<0.001
2.620(1.569,4.375)
<0.001
PD-1 expression (positive vs. negative)
0.092
1.321 (0.828,2.107)
0.243
0.060
1.490 (0.908,2.444)
0.114
CD8 expression (high vs. low)
0.002
0.311 (0.193,0.502)
<0.001
0.001
0.353 (0.209,0.597)
<0.001
p value
0.844
1.183 (0.771,1.816)
0.441
0.153
0.282
TIM-3, T-cell immunoglobulin and mucin-domain containing-3; PD-1, programmed cell death 1; HR,hazard ratio; CI,confidence interval
24