sLAG-3 in non-small-cell lung cancer patients' serum.

BACKGROUND: Anti-programmed cell death-1/programmed cell death ligand-1 monoclonal antibodies have been widely used in non-small-cell lung cancer (NSCLC), but not every patient can get benefits from them. Whether other molecular markers can predict the results of programmed cell death-1/programmed cell death ligand-1 inhibitors need to be explored. Lymphocyte-activation gene-3 (LAG-3) is another important immune checkpoint, which can inhibit tumor immunity. Soluble LAG-3 (sLAG-3) plays different functions from LAG-3. In this study, we detected the serum sLAG-3 level in NSCLC patients.
METHODS: sLAG-3 was detected in 247 hospitalized patients by enzyme-linked immunosorbent assay. Every sample was repeated three times.
RESULTS: Two-hundred forty-seven hospitalized patients were enrolled in this study. Of them, 71 had benign diseases and 176 were NSCLC patients. sLAG-3 in NSCLC serum was correlated with NSCLC stage. The sLAG-3 levels were significantly higher in stage I-II NSCLC than in stage III-IV (p<0.001).
CONCLUSION: The advanced NSCLC had the lower sLAG-3 expression. This might be related to the poor cancer immune response. Increasing sLAG-3 level might be a promising treatment in advanced NSCLC patients.

Introduction

The incidence and mortality of lung cancer are higher.1 Lung cancer patients are always diagnosed at an advanced stage.2 Chemotherapy and targeted therapy are the standard treatments for advanced non-small-cell lung cancer (NSCLC). But not all patients can get benefit from these therapies.3–5 Therefore, it is very necessary to search a new treatment for NSCLC patients.

Immunotherapy has a good result in lung cancer patients. Many clinical trials, such as Checkmate-017, Checkmate-057, Keynote-010, Keynote-024, and OAK, showed that anti-programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) monoclonal antibodies could improve the objective response rate (ORR) and prolong the overall survival (OS) in NSCLC patients. The anti-PD-1/PD-L1 monoclonal antibodies have been approved for the standard NSCLC treatment by the US Food and Drug Administration.6–10

Similar to the targeted therapy, not all lung cancer patients can benefit from anti-PD-1/PD-L1 monoclonal antibodies. Only ~20% of NSCLC patients can get benefit from the PD-1/PD-L1 inhibitors,7,8,10 and resistance is an inevitable problem.11 One study reported that when the patients faced the resistance of PD-1/PD-L1 inhibitor, T-cell immunoglobulin and mucin-domain containing-3 expression was significantly increased, which suggested that the resistance of PD-1 inhibitor might be related to other immunological checkpoints compensatory high expression.12

Lymphocyte-activation gene-3 (LAG-3; CD 223) is another important immune checkpoint in lung cancer,13 which can bind a non-holomorphic region of major histocompatibility complex class II (MHC class II) with greater affinity than CD 4.14,15 LAG-3 can inhibit the cancer immune response and play a complicated role in the immune system. Soluble LAG-3 (sLAG-3) can induce dendritic cells to mature and attack tumor cells.16 In our previous study, we reported that the LAG-3 expression was correlated with PD-1/PD-L1 expression in NSCLC patients and was related to poor prognosis. In this study, we detected the serum sLAG-3 level in NSCLC patients.

Methods

Patients

From January 2016 to June 2017, 247 hospitalized patients were continuously enrolled in this study from Shanghai Pulmonary Hospital. Most of the serum samples were collected within 2 days prior to test. Samples were stored at −80°C without repeated freeze and thaw. The feature of samples was blinded to the test before and after detecting sLAG-3. Blood samples were obtained before diagnosis and receiving any anticancer treatment. All participates signed the informed consent form. Pulmonary benign and NSCLC patients were diagnosed by histology. Lung cancer stages were categorized by seventh edition of International Association for the Study of Lung Cancer (IASLC) TNM staging system. The protocol was approved by the Shanghai Pulmonary Hospital, Tongji University.

ELISA for sLAG-3

sLAG-3 was detected by enzyme-linked immunosorbent assay (ELISA) kit (Uscn, Wuhan, China). Every sample was repeated three times.

Statistical analysis

We performed statistical analysis by SPSS 17.0. Chi-square tests were used to analyze the clinical data between benign diseases and NSCLC. The measurement material used the rank sum test. All statistics were two-sided, and statistical significance was defined as p<0.05.

Results

Patient characteristics

Two-hundred forty-seven hospitalized patients were continuously enrolled in this study from Shanghai Pulmonary Hospital. Of them, 71 had benign diseases and 176 were NSCLC patients. Table 1 shows the clinical data of all cases included in this study. In benign pulmonary diseases group, 53 (74.6%) were males and 18 (25.4%) were females. Eight (11.3%) patients were >70 years old. Thirty-nine (55.9%) patients had a smoking history. In the NSCLC group, there were 41 (23.3%) females. Nineteen (10.8%) were >70 years old. Ninety-nine (56.3%) patients had a smoking history.

Table 1

Clinical data of all cases

Characteristic	Benign pulmonary diseases	NSCLC	p-value
Sex, n (%)
Female	18 (25.4)	41 (23.3)	0.743
Male	53 (74.6)	135 (76.7)
Age (years), n (%)
≤70	63 (88.7)	157 (89.2)	1.000
>70	8 (11.3)	19 (10.8)
Smoker, n (%)
Smoker	39 (55.9)	99 (56.3)	0.888
Non-smoker	32 (45.1)	77 (43.8)
Stage, n (%)
I–II	–	103 (58.5)
III–IV	–	73 (41.5)

Abbreviation: NSCLC, non-small-cell lung cancer.

sLAG-3 in different patients

We did a subanalysis in patients with benign pulmonary diseases; the sLAG-3 levels were non-significant between sex, age, and smoking status. In NSCLC patients, the sLAG-3 levels were non-significant between sex, age, smoking status, and pathology. The sLAG-3 levels were significantly higher in stage I–II NSCLC than in stage III–IV (p<0.001; Table 2).

Table 2

sLAG-3 in different groups

	Benign pulmonary diseases	p-value	NSCLC	p-value	Total	p-value
Sex, mean rank
Female	37.67	0.692	94.44	0.394	131.49	0.356
Male	35.43		86.70		121.65
Age (years), mean rank
≤70	35.94	0.942	88.63	0.924	124.10	0.948
>70	36.50		87.45		123.15
Smoker, mean rank
Smoker	34.11	0.484	87.25	0.774	120.33	0.473
Non-smoker	37.55		89.47		126.90
Stage, mean rank
I–II	–	–	111.77	<0.001	–	–
III–IV	–		55.66		–
Pathology, mean rank
Benign diseases	–	–	–	–	132.02	0.262
NSCLC	–		–		120.76
Subpathology, mean rank
Adenocarcinoma	–	–	94.79	0.158	–	–
Non-adenocarcinoma	–		83.83		–

Abbreviations: NSCLC, non-small-cell lung cancer; sLAG-3, soluble LAG-3.

Discussion

To our knowledge, this is the first study to analyze serum sLAG-3 in NSCLC patients. In our previous study, we detected LAG-3 expression in NSCLC by immunohistochemistry. We reported that LAG-3 expression was correlated with PD-1/PD-L1 expression and was related to poor prognosis in NSCLC patients.17 In this study, we found that the serum sLAG-3 levels were significantly higher in early-stage NSCLC.

Immunotherapy has been widely recognized in the treatment of lung cancer. Anti-PD-1/PD-L1 monoclonal antibodies have been widely used in NSCLC.6–10 Although anti-PD-1/PD-L1 monoclonal antibodies have a good result in NSCLC patients, not all patients can get benefits from them. Some biomarkers can partly predict the efficacy of anti PD-1/PD-L1 monoclonal antibodies, such as PD-1, PD-L1 expression, and tumor mutation burden.6–10 Whether other molecular markers can predict the results of PD-1/PD-L1 inhibitor needs to be explored. LAG-3 is another important immune checkpoint.13,17 LAG-3 can inhibit cancer immunity by negatively regulating T cell proliferation, activation, and homeostasis in cancer patients. In gastric cancer, LAG-3 and PD-1 expression on T cells impaired the postsurgery cell-mediated immunity.18 LAG-3 was highly expressed in hepatocellular carcinoma.19 Our group reported that LAG-3 was correlated with PD-1/PD-L1 expression, and higher expression of LAG-3 predicted poor prognosis in NSCLC.17 sLAG-3 plays different functions from LAG-3. sLAG-3 can induce dendritic cells attacking cancer cells.20,21 sLAG-3 can be detected in patient serum by ELISA.22 In this study, we found that sLAG-3 in NSCLC serum was correlated with the stages of NSCLC. The advanced lung cancer patients had the lower sLAG-3 expression. This might be related to the advanced disease patients having poor cancer immune response. Thus, increasing sLAG-3 level in advanced lung cancer might be a promising treatment in NSCLC patients.

There are some limitations in this study. First, we lacked the data of ORR and OS. Second, tumor tissue LAG-3, PD-1, and PD-L1 expressions should be analyzed in these patients. Furthermore, we will prospectively compare serum sLAG-3 level with tumor LAG-3, PD-1, and PD-L1 expressions and analyze the correlation of sLAG-3 with survival data in a large-scale study.

Conclusion

We detected sLAG-3 in NSCLC patients’ serum and found that sLAG-3 was significantly lowly expressed in advanced-stage NSCLC. LAG-3 is an important immune checkpoint and may have synergistic function with PD-1/PD-L1. sLAG-3 can improve the immune response of cancer. Whether sLAG-3 could become a new treatment and serve as a liquid biopsy biomarker for lung cancer immunotherapy need to be further explored.