The present study was approved by the ethics committee of Shengjing Hospital affiliated to China Medical University (Shenyang, China; no. 2016PS256K), and the need for informed consent from patients was exempted due to the use of retrospective paraffin-embedded specimens. Pathological specimens were collected from 60 patients with SCLC who underwent surgery at Shengjing Hospital affiliated to China Medical University between 2008 and 2014, though five patients were lost to follow-up. The majority (43/60) of these patients with T2-3 or N1-2 (20) metastasis had not been diagnosed correctly due to the lack of effective pathological determination, and unnecessarily underwent surgery as a result of this. The nodules from certain patients with established SCLC were resected, and these patients were in the 1A stage (17/60 T1N0M0 in Tumor-Node-Metastasis staging) with no infiltration in the visceral pleura, main bronchus, surrounding lymph nodes or distant organs, and the tumor size was <3 cm. Tumor staging was based on serum tumor markers [carcinoembryonic antigen, Cyfra 21-1, neuron specific enolase (NSE) and squamous cell carcinoma antigen] and imaging [positron emission tomography/computed tomography (PET/CT) or chest CT scan, bone emission CT and brain contrast-enhanced magnetic resonance imaging] prior to surgery. Patients who had received neoadjuvant therapy or had an immune system-associated disease were excluded. Histological diagnoses were based on the guidelines of the World Health Organization (21).

NSE is one of the key markers used to evaluate the progression of patients with SCLC (22). Patients with SCLC may also present with hyponatremia, which is caused by inappropriate secretion of antidiuretic hormone or paraneoplastic syndrome. Hyponatremia predicts a poor outcome for patients with SCLC (23). Additionally, a number of patients with SCLC and hypercoagulability (high levels of D-dimer) also have a worse prognosis (24). Serum NSE levels, Na⁺ levels and D-dimer levels were measured 1 day following admission to hospital, at least 1 week prior to surgery. The serum levels of these components were measured using the NSE detection kit (Roche Diagnostics GmbH, Mannheim, Germany), the OLYMPUS K⁺ and AU640/5400/5800 assays (Beckman Coulter, Inc., Brea, CA, USA), and the HemosIL D-dimer HS 500 assay (Instrumentation Laboratory Co., Bedford, MA, USA), according to the manufacturers' protocols. Clinicopathological variables collected for analysis included sex, tumor location, age at diagnosis, tumor size, node involvement (N), NSE expression levels, Na⁺ levels and D-dimer expression levels. Disease recurrence and survival were observed in the follow-up clinic or obtained through telephone correspondence. Follow-up was until mortality or December 2015.

A total of 60 paraffin-embedded SCLC specimens were obtained from the Pathology Department of the Shengjing Hospital affiliated to China Medical University. The samples had been fixed in 10% formalin for 2 h at room temperature. IHC was performed on the resected SCLC tumor tissues (3 µm thickness) using primary antibodies: Anti-TIGIT antibody (1:50 dilution; cat. no. sc-103319), anti-CD155 antibody (1:100 dilution; cat. no. sc-514623; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-PD-1 antibody (1:100 dilution; cat. no. 66220-1-Ig) and anti-PD-L1 antibody (1:200 dilution; cat. no. 66248-1-Ig) (both ProteinTech Group, Inc., Chicago, IL, USA), and IHC kits containing horseradish peroxidase-conjugated affinipure rabbit anti-goat/goat anti-rabbit/goat anti-mouse secondary antibodies (cat. nos. ZB-2306, ZB-2301 and ZB-2305, respectively; dilutions as supplied; OriGene Technologies, Inc., Beijing, China) according to the manufacturer's protocols. The specimens were deparaffinized in 100% xylene for 15 min at room temperature, rehydrated in descending ethanol series for 5 min at room temperature, and incubated in 3% H₂O₂ for 1 h at room temperature. Antigen retrieval was achieved by heating the samples in citrate buffer (pH 6.0) for 10 min at 95°C. The specimens were incubated with the protein blocking solution provided by the IHC kits for 1–2 h at room temperature, then incubated with the primary antibodies in a humid chamber overnight at 4°C. The negative controls were treated with PBS instead of the primary antibodies. Following incubation with the secondary antibodies for 1 h at room temperature, the specimens were stained using a DAB kit (OriGene Technologies, Inc.). All sections were counterstained with 100% hematoxylin for 30 sec at room temperature. All images were recorded using a Nikon E800 fluorescent microscope and analyzed with NIS-Elements Br version 4.60.00 (Nikon Corporation, Tokyo, Japan). All IHC results were assessed by two pathologists independently in a blinded manner. Discordant opinions were settled by a third pathologist. The intensity of staining was defined as follows: No staining was considered a negative result (‘0’); positively stained sections were analyzed using the integrated optical density (IOD) and the areas of staining distribution with NIS-Elements Br version 4.60.00; the mean density was obtained by dividing the IOD value by the area, and an average from 5 representative fields was calculated (magnification, ×400).

The deparaffinization and antigen retrieval of the sections was carried out as described for the IHC method. Nonspecific immunoglobulin binding was blocked using 5% bovine serum albumin (cat. no. 8850; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 2 h at room temperature. Sections were incubated with primary anti-TIGIT antibody and anti-CD8 antibody (1:75 dilution; cat. no. 17335-1-AP; ProteinTech Group, Inc.), or with anti-PD-1 antibody and anti-CD8 antibody (1:75 dilution; cat. no. 17335-1-AP; ProteinTech Group, Inc.) at 4°C. Following overnight incubation, the slides were incubated for 4 h at room temperature with the following mixed fluorescent secondary antibodies: i) Tetramethylrhodamine (TRITC)-goat anti-rabbit secondary antibody (1:50 dilution; cat. no. ZF-0316); ii) fluorescein isothiocyanate (FITC)-goat anti-mouse second antibody (1:100 dilution; cat. no. ZF-0312; both OriGene Technologies, Inc.); iii) TRITC-donkey anti-goat secondary antibody (1:50 dilution; cat. no. sc-2094); and iv) FITC-donkey anti-rabbit secondary antibody (1:100 dilution; cat. no. sc-2090; both Santa Cruz Biotechnology, Inc.), followed by incubation with DAPI (Beijing Solarbio Science & Technology Co., Ltd.) for 5 min at room temperature. Finally, the images were observed and captured (×400 magnification) using a fluorescence microscope (Eclipse NI; Nikon Corporation).

The association between the marker expression levels and the clinicopathological features was analyzed using a Pearson's χ² test. The survival analysis for different groups was performed using a Kaplan-Meier survival (log-rank tests). The Cox regression model was used to perform multivariate analysis. The statistical results were performed using SPSS software, version 20.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

PD-1, PD-L1, TIGIT and CD155 expression levels were analyzed in 60 human SCLC tissues by IHC. PD-1/PD-L1 and TIGIT/CD155 were highly expressed, particularly in the cytoplasm and cell membrane of cancer cells and the matrix of tumor tissue (Fig. 2). The mean densities of PD-1, PD-L1, TIGIT and CD155 staining in the 60 SCLC samples from patients were 0.288, 0.316, 0.302 and 0.304, respectively (data not shown). Detailed clinicopathological characteristics are presented in Table I. It was reported that high expression levels PD-L1 and CD155 in tumors were associated with high levels of NSE expression (PD-L1, P=0.007; CD155, P=0.021), and larger tumor size was associated with high expression levels of PD-L1 (P=0.009). Therefore, it was hypothesized that NSE may be a useful factor when selecting patients with SCLC who may benefit from checkpoint (anti-PD-L1 or anti-CD155) targeting therapy. There was no significant association between high PD-L1 or CD155 expression levels and N, Na⁺ or D-dimer expression levels.

Patients were divided into two groups according to the median PD-L1 or CD155 expression levels; these groups were a PD-L1 high/low group and a CD155 high/low group, respectively. As presented in Fig. 3, patients with higher PD-L1 or CD155 expression levels tended to have shorter OS times (PD-L1, 16.26±2.91 months; CD155, 16.20±2.42 months) compared with patients in the low expression group (PD-L1, 36.43±6.46 months; CD155, 29.87±3.66 months) (PD-L1, P=0.001; CD155, P=0.002). However, in the PD-1 and TIGIT high and low expression groups, there were no significant associations with survival time.

Patients were subsequently divided into three groups: i) PD-L1 and CD155 low expression levels (n=14); ii) either PD-L1 or CD155 overexpression (n=22); or iii) PD-L1 and CD155 overexpression (n=24). Significant differences in OS were reported between groups (P<0.001 for OS, as presented in Fig. 3). The OS of patients with PD-L1 or CD155 overexpression alone (26.70±6.99 months) tended to be shorter compared with patients with low expression of the two (38.82±2.67 months), and the OS of patients who had high expression levels of PD-L1 and CD155 together was the poorest (13.13±2.53 months) (P<0.001).

To determine the prognostic value of the expression levels of these immune checkpoints, Kaplan-Meier survival calculations (log-rank tests) were used for sex, tumor location, age at diagnosis, tumor size, N status, PD-L1, CD155, PD-1, TIGIT, NSE, Na⁺, D-dimer and postoperative therapeutic methods. It was reported that PD-L1 (P=0.001), CD155 (P=0.002), N status (P=0.046), NSE (P=0.047) and postoperative therapeutic methods (P=0.004) were associated with the OS of patients with SCLC (Table II). Furthermore, a multivariate Cox regression model was used to analyze PD-L1, CD155, N status, NSE and postoperative therapeutic methods to determine their prognostic value. High expression levels of PD-L1 [hazard ratio (HR)=2.55, 95% confidence interval (CI)=1.18–5.51, P=0.017] and CD155 (HR=2.40, 95% CI=1.05–5.50, P=0.038) were independent predictors of poor OS in patients with SCLC (Table III).

Using immunofluorescence double staining, it was demonstrated that PD-1 and TIGIT were expressed on CD8⁺ TILs in certain specimens from SCLC patients (Fig. 4). These results confirmed that in SCLC, the receptors of CD155 and PD-L1, TIGIT and PD-1, were constitutively expressed on CD8⁺ TILs. It is possible that PD-1⁺ or TIGIT⁺ CD8⁺ TILs are involved in immune regulation by interacting with ligands expressed on tumor cells.