LAG-3 and PD-1/PD-L1 Inhibitors Might Become a Promising Treatment for Small Cell Lung Cancer

Background Immune therapy has achieved notable success in cancer treatment. A novel immunocheckpoint, Lymphocyte activing gene-3 (LAG-3), has shown promising therapeutic ecacy in non-small cell lung cancer (NSCLC). However, literature about LAG-3 in small cell lung cancer (SCLC) is scarce. We statistically analyzed the correlation of LAG-3 expression with programmed death 1 (PD-1) and programmed death ligand 1 (PD-1), its survival predictive signicance, and the possibility of becoming a treatment alternative of SCLC. Methods In this study, we included 102 patients diagnosed with SCLC. The expression of protein was evaluated by immunohistochemistry (IHC) staining. We performed correlation analysis and survival analysis with SPSS software (version SPSS, Inc.; Chicago, IL) Results In SCLC, none tumor cell expressed LAG-3. The expression of LAG-3 was remarkably associated to PD-1 and PD-L1 expression on TILs (p=0.006, p=0.001). LAG-3 was the only prediction for TILs PD-L1 expression (ORs=0.161, 95% CI: 0.063-0.412, p<0.01). Although LAG-3-positive patients had relatively longer RFS, LAG-3 expression had no statistically signicant differences in predicting prognosis (p=0.088). Conclusions We found a signicant correlation of LAG-3, PD-1, PD-L1 expression. LAG-3 is an important immune checkpoint closely linked to PD-1/PD-L1 and might be a promising novel alternative of immune therapy for SCLC.


Background
Among all cancer types, lung cancer ranks rst in both morbidity and mortality and poses an increasingly serious threat to human health [1,2] . Approximately 10% to 15% cases can be categorized as small-cell lung cancer (SCLC), a cancer type with high growth fraction, high recurrence rate, leading to poor prognosis [3][4][5] . Although chemotherapy is the standard rst-line treatment for SCLC [6] , resistance to chemotherapy hinders survival prolongation. Therefore, it is an urgency to explore some more effective therapeutic strategies for patients with SCLC.
Some tumor cells with less immunogenicity can escape from immune elimination and developed into cancers, which was reported can be reversed by suppressing certain immune checkpoints [10] . The notable success of some immune checkpoints inhibitors in treating cancers has been reported [11,12] . The PD-1/PD-L1 pathway inhibitor has been approved effective for treating NSCLC. It could also lead to signi cantly longer survival in SCLC patients with the rst-line chemotherapy combined [13][14][15][16] .
Likewise, LAG-3 shows stronger a nity to human leukocyte antigen II (HLA-II) expressed on antigen presenting cells (APCs) compared to CD4, thereby prohibits the binding of HLA-II with TILs, hindering antitumor response [25,26] . In HLA-II-positive melanoma tumors, this might pave the way for immune escape with bidirectional function [23] . LAG-3 serves as an essential marker of T cell exhaustion, promoting T-cell apoptosis, inhibiting proliferation, suppressing activation, declining cytokines secretion and increasing tolerance [27,28] . Elevated LAG-3-expression was observed on TILs of patients with various solid tumors, such as hepatocellular carcinoma, gastric carcinoma etc, as well as hematologic malignancies [29] .
Scholars regarded that LAG-3 has co-function with PD-L1 and PD-1 [11] . In vivo research suggested that T cells would get activated if one of the pathways was blocked. Strategy of blocking both pathways showed even more notable effect [30] . LAG-3 may become a new promise immune checkpoint in cancer treatment. Additionally, both LAG-3 and PD-1 pathways could make a promising therapeutic strategy better than CTLA-4 blockade for less toxicity [11] . Moreover, soluble LAG-3 can possibly be promising anticancer vaccine [31] .
Similar to other types of cancer, our recent study has found that some NSCLC patients had LAG-3-positive TILs. The expression of LAG-3 can be predicted by PD-1 expression and related to worse prognosis [23] . However, literature is scarce on the LAG-3 expression and how it effects survival in SCLC. In this study, we enrolled 102 patients with SCLC, investigated LAG-3 expression in TILs, conducted survival analysis and correlation analysis of clinic pathological traits and PD-L1, PD-1, LAG-3 expression.
Patients And Methods

Patients
We included 102 patients aged from 38 to 81 who were diagnosed as SCLC in Shanghai Pulmonary Hospital from March, 2017 to January, 2019. We reviewed the surgical histology reports and categorized lung cancer stages with 8th edition International Association for the Study of Lung Cancer (IASLC) TNM staging system. Fifty four of them received chemotherapy after diagnosis. All participators were competent to provide consent.

IHC Procedure
Dewaxing tissue slides with xylene, then alcohol, rinsing slides with distilled water. After recovering antigen, background staining reduction, we incubated the primary antibody in the slides at RT for 1 hour.
And then, rinsing with PBS, incubating with the HRP-conjugated goat anti-rabbit immunoglobulin G detective antibody at RT for 30 min. Rinsing with PBS, visualizing the antigen with DAB, cell nuclear counterstain with hematoxylin and slides mounting. IHC was performed by pathologist Liping Zhang.

Cutoff value determination of surface markers
We chose a value of at least 5% as LAG-3 cutoff for the best prediction of recurrence-free survival (RFS). The PD-1 on TILs staining was determined as positive when over 1%. Five percent staining was determined as lower limit of PD-L1 cutoff. The staining of FOXP3, CD3, CD4, CD8 was con rmed as positive when more than 10%, 40%, 30%, 30% individually.

Statistical analysis
We conducted correlation analysis and survival analysis with SPSS software (version 17.0; SPSS, Inc.; Chicago, IL). Spearman's rank correlation was applied to calculate relativity of PD-L1, PD-1, LAG-3 expression. We used Chi-square tests to evaluate the relativity of clinicopathological traits and PD-L1, PD-1, LAG-3 expression. Logistic regression model was applied to gure out whether LAG-3 made meaningful expression prediction for PD-L1 and PD-1, gender, age, smoking status, lung cancer stage, history of chemotherapy were all included as well. Kaplan-Meier method was implemented to estimate survival curves, and Cox regression model to the correlation analysis on RFS and clinical features, including age, gender, history of chemotherapy, smoking status, staging of lung cancer, PD-L1 on tumor cells, PD-L1 on TILs, PD-1, LAG-3 on TILs and CD3, CD4, CD8, FOXP3. Statistical signi cance was de ned as P < 0.05. All statistics were 2-sided.

Survival analysis
With Kaplan-Meier analysis, patients who were LAG-3-positive had longer RFS, but there was no signi cantly statistic difference in comparison with LAG-3-negative patients (p=0.088) (Figure 1).

Discussion
As a novel immune checkpoint, literature is scarce on LAG-3 expression in SCLC and the correlation with survival. To our knowledge, this is the rst indication of the possible immunotherapeutic effect for SCLC focused on LAG-3.
As is stated above, LAG-3 serves as an essential marker of T cell exhaustion [27,28] . TILs are regarded as crucial components in anti-tumor immune response and directly related to the development of cancer [32] .
The function of CD4+, CD8+ T cells, DCs, Tregs etc. is regulated by inhibitory and active receptors, remarkably impact cancer immune escape [33] .
From a mechanistic standpoint, LAG-3 blockade is equal to the blockade of the binding between LAG-3 and HLA-II molecules, which increases the binding of HLA-II to TILs, thus enhances anti-tumor responses [34] . High LAG-3 expression can be observed on TILs in hematologic malignancies and various solid tumors, including hepatocellular carcinoma, gastric cancer, renal cell carcinomas and ovarian cancer [29] . In our study, LAG-3-positive TILs existed in 39.2% of patients; with none tumor cells expressed LAG-3. Additionally, research indicated that if PD-1 or LAG-3 pathway alone was blocked, TILs would get more activated and lead to prolonged survival, either by antibodies or knocking down [35] . Built on existing studies, we used to consider LAG-3 a novel alternative of immune-based treatment for cancers. In our recent study, we found that NSCLC patients with LAG-3-negative TILs had longer survival [23] . But in SCLC LAG-3 could not predict survival.
Immune escaping pathways are closely associated with one another [36] . LAG-3 is co-expressed on TILs with PD-1 and have remarkable synergy with it, disrupting immune responses to cancer cells [37] . Based on research before, compared with blocking either LAG-3 or PD-1 alone, blocking both pathways showed much more remarkable therapeutic e cacy for cancers [30,35] . In the meanwhile, due to the upregulated expression of LAG-3 in patients insensitive to PD-1 blocking treatment, application of combined strategy will improve the prognosis [29,36] . This has already been proved in treating melanoma patients with this condition [38,39] .
Given the different impact the above-mentioned checkpoints on NSCLC and SCLC survival, we take the immune mechanism into consideration. Different from over-expressed on variety of tumors including NSCLC, PD-L1 expression is suppressed in SCLC [40] . In our study, there were only 4 patients (3.9%) with PD-L1-positive tumor cells, consistent with previously reported results in an immunohistochemical study of SCLC which found PD-L1-positive tumor cells was lower than 20% [41] . This might be one of the reasons why PD-1/PD-L1 pathway inhibition therapy alone cannot lead to notably improved prognosis in SCLC [42] . On the other hand, worse outcomes of SCLC was also considered to linked with higher FOXP3+T cells in ltrates [43] . Furthermore, the immune microenvironment of SCLC seems to be distinct from that of other solid tumors, for instance, NSCLC [40] . In this context, we can infer that combined immunotherapy is especially important in SCLS.
There were some limitations should be taken into consideration. Firstly, this study was a retrospective one. Secondly, the sample size was not large enough and more data are needed to support our indication.
Researchers are moving forward in the investigation of immunotherapy in recent years, aiming to more favorable prognosis for patients with SCLC. It is noteworthy that the characteristics of the SCLC immune microenvironment remain unclear. Researchers are moving forward in LAG-3 function and its interaction with other immunomarkers, yet there are still questions remain to work out. What role does LAG-3 play in the development of SCLC? Which immune checkpoint serves as the key regulator in anti-tumor responses in SCLC? How will immune responses change during the progress of SCLC? Is it possible that different major effector cells are involved in NSCLC and SCLC? Further studies are needed.

Conclusions
Immunocheckpoints play signi cant role in tumor immune escape with pathways closely interrelated to one another. Some SCLC patients have LAG-3-positive TILs. LAG-3 is prominently co-functioned with PD-1/PD-L1, and one of the meaningful predictions for their expression. Combination of LAG-3 and PD-1/PD-L1 inhibitors might become a promising immunotherapy for SCLC.

List Of Abbreviations
Lymphocyte activing gene-3 (LAG-3), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), tumor-in ltrating lymphocytes (TILs) Declarations Ethics approval and consent to participate The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This research was approved by the ethics committee of the Shanghai Pulmonary Hospital, Tongji University (No. K20-022). Written consent was given by all patients, and the experiment con rmed with the tenets of the Declaration of Helsinki.

Not applicable
Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests

Authors' contributions
Liping Zhang preformed all IHC staining and checked the results. Yi Xu was responsible for all the statistic analysis, and was a major contributor in writing the manuscript. All authors read and approved the nal manuscript.    Figure 1 Lymphocyte -activating gene 3 (LAG-3) and relapse free survival (RFS) in SCLC