Introduction
Lung adenocarcinoma (LUAD) is the most common variant of non–small cell lung cancer (NSCLC) across the world. LUAD has become significantly more prevalent over the past two decades, with a similar decrease in squamous cell carcinoma during the same period [
1]. There have been advances in the current strategy of combining various therapies with tyrosine kinase inhibitors (TKIs) to treat patients with LAUD. However, the 5-year overall survival (OS) rate for patients with LUAD remains poor [
2,
3]. At present, there is an urgent need to identify specific prognostic biomarkers in patients with LAUD, to enable the development of optimized, appropriate treatment protocols and clinical management for patients with different elements of risk. Several studies have attempted to predict and evaluate prognosis in patients with LAUD, through diverse gene expression profiles and bioinformatics approaches [
4‐
6]
. However, these studies usually incorporate genes from the entire genome or transcriptome and do not consider the intrinsic biological functions. This may give rise to the fact that many of these signatures remain merely mathematical but unable to truly reflect the real characters inherent in the tumor.
In recent years, the treatment of LUAD has developed rapidly. In addition to traditional surgery, radiotherapy, and chemotherapy, molecular targeted therapy, novel strategy like immunotherapy has been becoming of increasingly great concern [
7,
8]. The introduction of immune checkpoint-blocking antibodies that target programmed death 1 (PD-1) receptor as well as against its ligand, programmed death-ligand 1 (PD-L1) of the B7-CD28 family has significantly improved survival rates in patients with advanced lung cancer. These antibodies are available for clinical use [
9‐
11]. However, beneficial immunotherapeutic effects were only observed in a subgroup of patients with response rates of 17–21% [
12], suggesting that there may be other immune checkpoints in the LAUD tumor microenvironment (TME).
In addition to these star targets of the B7-CD28 family, several emerging and promising immune checkpoints have been identified [
13]. Most of these novel immune checkpoints are from the TNF superfamily, including the TNF ligand superfamily (TNFSF) as well as the TNF receptor superfamily (TNFRSF), which are responsible for regulating pathways related to cell survival, differentiation, and non-immunogenic death. Most notably, these novel immune checkpoints belonging to the TNF superfamily also play a critical function in regulating the immune system, providing co-stimulatory or co-inhibitory signals vital for natural and adaptive immunity with an emphasis on T-cell responsiveness [
14,
15].
These novel immune checkpoints molecules can trigger inflammatory activities in several cells in the TME, including cells like T and B lymphocytes as well as tissue-resident cells such as epithelial and fibroblasts cells [
16]. Therefore, modulating and controlling the interaction of these novel immune checkpoints would be a novel therapeutic target with great potential for tumor treatment. Meanwhile, many cancer therapies targeting these novel immune checkpoints are in full swing, including preclinical and clinical trials. For example, therapies targeting OX40, CD40, and CD27 have achieved favorable progress in various tumor treatments, including for patients with lung cancer [
14,
17,
18]. This observation inspired us to explore and establish a novel immune checkpoints-based prognosis system for LUAD, revealing immune features for patients with LUAD.
Herein, we firstly enrolled 1883 LUAD samples from nine independent cohorts, including eight public cohorts and an independent cohort. Then, we systematically explored these novel immune checkpoints in LAUD and filtered out genes with the most prognostic value. From this, we constructed a signature based on the combination of LTA, CD160, and CD40LG, which was also well-validated in different cohorts. Finally, we further examined the clinical characteristics, immunotherapy responses, and immune cells infiltration of the prognostic signature. The novel combination of LTA, CD160, and CD40LG, may help us understand the immune status of patients with LUAD, but also optimize available tumor immunotherapies.
Discussion
High-throughput sequencing and genomics research has ushered in the identification of emerging biomarkers and therapeutic targets. These advancements have also improved our understanding of tumors. However, there is little information on which biomarkers may predict immune therapy responses and prognoses, revealing tumor immune phenotypes in LAUD. To improve our understanding of the co-stimulatory signals important in LAUD, we combined three crucial novel immune checkpoints (LTA, CD160, and CD40LG) to create a novel signature. We used the TCGA database as a training cohort and systematically explored the association between the gene expressions of some novel immune checkpoint members and prognostic outcomes in patients with LUAD. In the process, we identified a novel immune checkpoints-based risk signature, which is closely related to OS and RFS of patients with LAUD. This novel risk signature was validated in seven different publicly available cohorts as well as 102 samples of frozen tumor tissues using qPCR data. The validity of our signature was further confirmed using a meta-analysis. Our results indicated that the risk signature was well-validated and significantly related to OS in various important clinical and mutation subgroups. Our novel signature independently predicted prognosis in patients with LAUD. We also explored and analyzed relevant mechanisms, immune predictors for immunotherapy, and immune cells infiltration of this risk signature. Thus, this signature may contribute to a deep and comprehensive understanding of precision immunotherapy for LAUD.
Currently, apart from B7-CD28 family members, there are several emerging and potential immune checkpoints for immunotherapy, such as some TNF superfamily members [
16]. After analyzing the association between these novel immune checkpoints and prognoses in LUAD, we determined the most significant prognostic genes in these novel immune checkpoints, including protective (
LTA) and risky (
CD160 and
CD40LG) genes. Regarding the protective gene,
LTA, a proinflammatory cytokine, belongs to the TNF superfamily, involved in the inflammatory and immune responses [
34]. This molecule also can assist lymphocytes and stromal cells to induce cytotoxic effects on tumor cells [
34,
35]. It was reported that the polymorphisms of
the LTA gene are closely related to cancer risk, including LAUD and other adenocarcinoma malignancies [
36]. Meanwhile, researchers indicated that depleting LTA-expressing lymphocytes with LTA-specific monoclonal antibodies may be useful in treating autoimmune diseases [
37]. Also, in terms of risky genes,
CD160—also known as
BY55, is an immunoglobulin-like, glycosylphosphatidylinositol-anchored protein and expressed on natural killer cells, γδ T-cells, and a subset of CD4 + and CD8 + T-cells [
38,
39]. CD160 was proven to bind to herpesvirus entry mediator (HEVM) with high affinity, which induces robust natural cells effector activity and suppressed T-cell responses in vitro [
40,
41]. B and T lymphocyte attenuator (BTLA) is a novel checkpoint receptor for immunotherapy, while CD160 shares the same ligands with it as BTLA repaired receptor, suggesting that CD160 inhibitory also may be a promising target for immunotherapy [
27]. CD40LG is the ligand of CD40, and after they get combined, it will stimulate proinflammatory gene expression, including interleukins (IL)-1, IL-6, IL-8, IL-12, TNF-α, IFN-γ, and monocytic chemoattractant protein (MCP)-1. The activation of the CD40/CD40LG system is a major contributor to carcinogenesis. When CD40LG antibodies are used to disrupt the CD40/CD4OLG system’s function, it leads to the suppression of tumor cells [
42]. However, some researchers demonstrated that the membrane-stable CD40LG mutant gene transfer into the CD40-positive LUAD cell line. This gene transfer reduces cell proliferation and enhances apoptosis [
43]. Thus, the role of CD40LG in LUAD is still uncertain and requires further in-depth study and exploration. Similarly, the functions of LTA and CD160 in LUAD are unclear, and more relevant research are urgently needed.
We also investigated the potential genetic mechanisms of this signature. Risk-score-related genes were mainly enriched in immune response and leukocyte activation processes and pathways after correlation analysis. We analyzed seven immune-related metagenes to better understand the relationship between risk signature and immune responses. We found that risk score was negatively associated with LCK and MHC_II clusters, suggesting that high-risk patients have decreased B-cell function and impaired antigen-presentation ability. We also found higher infiltration of resting type CD4 + T-cells in high-risk patients, suggestive of immunosuppression. Next, we found that the patient risk score was positively associated with the expression of CD276. CD276 is a crucial immune checkpoint member of the B7-28 family that plays a pivotal role in inhibiting T-cell function, and immunotherapies that target CD276 have achieved positive results in various tumors [
33]. This showed that patients at elevated risk may benefit from immunotherapies that target CD276. We also found that patients at elevated risk exhibited higher average TMB levels, more T-cell dysfunction, greater exclusion scores, lower TIDE scores. Of interest, despite this trend, PD-L1 protein expression differed across risk groups. PD-L1 and TMB are currently the most reliable biomarkers for predicting responses to PD-1/PD-L1 immunotherapy [
44,
45]. These findings suggest that high-risk patients are mostly immunosuppressed and are therefore better candidates for immunotherapy.
Although this signature was successfully validated across multiple cohorts and appeared to act as an independent prognostic factor for LUAD, this study had several limitations. Firstly, it was a retrospective study that should be validated in prospective, large-scale cohorts. Secondly, we only examined some novel immune checkpoint genes, which may limit our signature's predictive capacity. However, this new classifier also provides more information on the state of the TME. Finally, none of the patients in this study underwent immunotherapy, thus our prediction of immunotherapy responsiveness is merely theoretical. Future studies should address these limitations.
In conclusion, we found that analyzing tumor expression of LTA, CD160, and CD40LG represents a novel immune signature that may be useful for predicting prognosis and response to immunotherapy in patients with LUAD. Further validation of these findings may improve the ability to shed some light on screening appropriate patients for are most likely to benefit from immunotherapy, enabling increasingly personalized, evidence-based care.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.