Background
Immune checkpoint inhibitors (ICIs), including monoclonal antibodies that target the programmed cell death protein (ligand) 1 [PD-(L)1] and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have revolutionized treatments across multiple cancer types [
1‐
3]. However, despite the impressive success of ICIs, durable clinical responses vary among patients [
4]. Thus, predictive biomarkers of ICI response are needed to deliver precise medical treatment [
5].
As of today, PD-L1 expression, high microsatellite instability (MSI-H), tumor mutation burden (TMB), copy number alteration (CNA), neoantigen load (NAL), tumor immune microenvironment (TIME), gene expression profiles (GEPs), and some specific gene mutations were found associated with ICI response [
6‐
11]. Among them, only a few biomarkers have been clinically validated and even those validated ones still had their limitations [
3,
10,
12]. For example, in the CheckMate 568 study, 44–50% of patients with high TMB or high PD-L1 expression did not respond to ICIs while nearly 12–15% of patients with low TMB or low PD-L1 expression achieved a partial or complete response [
10]. Therefore, exploration of novel precise biomarkers is required to maximize the clinical benefits.
As the largest family of receptor tyrosine kinases (RTKs), the erythropoietin-producing hepatocellular carcinoma (Eph) receptors are involved in a wide range of physiological activities, especially tumorigenesis, tumor immunity, and tumor angiogenesis [
13‐
15]. Tumor angiogenesis is associated with immunosuppression [
16]. Recent clinical trials showed that combination therapy of anti-angiogenesis and ICIs achieved more favorable outcomes than monotherapy in different cancers [
17‐
19]. With the ability to promote tumor angiogenesis, Eph receptors have potential impacts on the efficacy of immunotherapy. Moreover, Eph receptors play important roles in anti-tumor immunity. For example, Eph receptors are source of tumor-associated antigen (TAA), which could elicit selective anti-tumor immunity [
20]. Also, Yang et al. demonstrated Eph receptor-mediated cell contact-dependent juxtacrine signaling could reduce T cell-mediated anti-tumor immunity by upregulating PD-L1 expression [
21]. Eph receptors are closely associated with immune response, and EPH genes are frequently mutated in various cancers [
14]. Accordingly, genetic status of Eph receptors has potential predictive values in immunotherapy. However, the association between the genomic alterations of Eph receptor-related genes and ICI response has not been revealed.
In this study, we performed a comprehensive analysis of the predictive function of mutations in Eph receptor-related genes. And we uncovered that mutated EPHA7 was predictive of better clinical outcomes in patients receiving ICI therapy and strongly associated with enhanced anti-tumor immunity across multiple cancer types.
Discussion
In our study, we systematically collected and consolidated both clinical and genomic data to evaluate the association between EPH gene status and clinical responses in ICI-treated cancer patients. Then, we carefully validated our findings in another independent cohort and thoroughly explored the corresponding TIME. We found EPHA7-MUT was significantly associated with better clinical outcomes in ICI-treated patients and enhanced anti-tumor immunity. Remarkably, this predicting value of EPHA7-MUT was independent of TMB and CNA. This is the first study performing a comprehensive analysis of the relationship between EPH gene mutation status and clinical outcomes in ICI-treated patients across multiple cancer types.
We found some meaningful changes in biological functions of EPHA7-MUT tumors, including intercellular communication, angiogenesis, and metabolism. First of all, Eph receptors and their ligands (ephrin) have been proven essential in the cell communication system [
14]. Hence, it is reasonable to observe a downregulation of intercellular communication in EPHA7-MUT tumors in our analysis. Previous study showed that inhibiting intercellular communication by targeting EPHA10 could boost anti-tumor immunity by reducing PD-L1 expression [
21]. However, we found an upregulation of PD-L1 expression in EPHA7-MUT tumors with decreased cell-cell contact. This finding suggested that decreased intercellular communication in EPHA7-MUT tumors may have other underlying mechanisms that enhance anti-tumor immunity rather than depressing PD-L1 expression. Secondly, Eph-ephrin signaling promotes tumor angiogenesis [
14]. As expected, we found the angiogenesis pathway was significantly downregulated in EPHA7-MUT tumors. Angiogenesis and immunosuppression are closely related. Tortuous tumor vasculature causes a hypoxic tumor environment and hinders the infiltration of lymphocytes [
39]. Decreased angiogenesis in EPHA7-MUT tumors could promote lymphocyte infiltration. Finally, metabolic changes in EPHA7-MUT tumors could also strengthen anti-tumor immunity. Fatty acids and glucose promote the survival of immune cells in the tumor microenvironment, while cholesterol and lactate function reversely [
40]. Accumulation of fatty acids and glucose, and depletion of cholesterol and lactate within EPHA7-MUT tumor were detected in our analysis, which could create a better TIME and enable cytotoxic lymphocytes to work more effectively. Overall, EPHA7-MUT tumors are more likely to provide a friendly living environment for those effective immune cells and thus enhance the anti-tumor immunity.
Ephrin receptors form a large family of receptor tyrosine kinase and regulate various biological functions. Both oncogenic and tumor suppressive roles have been reported for specific ephrin receptors [
14]. Particularly, EPHA7 has been previously identified as a tumor suppressive gene that inhibits tumor growth and progression in various cancers [
41,
42]. Survival analysis in both non-ICI-treated cohort and TCGA cohort revealed longer median OS in EPHAT-WT instead of EPHA7-MUT patients, indicating that EPHA7-MUT might potentially have a worse prognostic impact on cancer patients. Accordingly, the clinical benefits of EPHA7-MUT patients with ICIs should be the result that the ICI treatment benefits of EPHA7-MUT outweighed its harmful prognostic impact. We then further analyzed cancer subgroups in TCGA cohort, which showed a generally and numerically worse prognosis of EPHA7-MUT patients within each cancer type (Additional file
7: Figure S3). Colorectal cancer with EPHA7-MUT had the worst prognosis in TCGA cohort (HR = 5.21 [95% CI, 2.22–12.21], adjusted
P < 0.0001). This could partially explain why EPHA7-MUT colorectal cancer was the only one that presented with worse OS in the validation cohort, because the harmful prognostic impact of EPHA7-MUT outweighed its ICI treatment benefits in colorectal cancer. Also, besides the aforementioned sample size of the discovery cohort, this assumption could be another reason why only numerical survival advantage of EPHA7-MUT patients was observed in the discovery cohort, since the harmful prognostic impact of EPHA7-MUT could partly mask the ICI therapy benefits. Notably, these findings supported our previous assumption and further stressed the importance of ICI therapy in EPHA7-MUT patients, which could potentially turn the harmful prognostic impact of EPHA7-MUT patients into an overall survival benefit.
In the initial screening process, EPHA3 was ruled out since its FDR adjusted
P value of DCB was 0.053. However, given this borderline
P value, EPHA3 was worth following up. Hence, we have also done the survival analysis of EPHA3 in both discovery cohort and validation cohort. Results could be found in the supplementary material (Additional file
8: Figure S4). However, only numerical survival benefits were observed in both discovery and validation cohort for EPHA3-MUT patients. Therefore, EPHA3 might not be as effective as EPHA7 in predicting the efficacy of immunotherapy in current analysis.
In our primary analysis, it is individual EPH gene that was evaluated rather than cumulative effects of all 14 EPH genes. To investigate the combined effects of all 14 EPH genes, we performed further analysis combining all 14 genes (Additional file
9: Figure S5). EPH-MUT was defined as at least one EPH gene has mutation among 14 genes, while EPH-WT was defined as none of EPH genes has mutation. Although EPH-MUT patients presented with a higher ORR than EPH-WT patients (40.8% vs 24.8%), there were no significant differences between EPH-MUT and EPH-WT patients regarding DCB, PFS, and OS. Activation of different Eph receptors can have highly varied impacts on cellular processes, but exact function of each Eph receptor has not been fully understood. Therefore, it would be more reasonable to test the combined predictive value of EPH genes that have synergic effect in the future rather than all EPH genes.
This retrospective analysis also has several limitations. Firstly, only four out of fourteen EPH genes are included in the MSK-IMPACT panel. To analyze all EPH genes, we only included cohorts with WES data in the discovery cohort. Considering the limited sample size of the discovery cohort (n = 386), we should not completely exclude the predictive function of other EPH genes. Secondly, mutation rate of EPHA7 in melanoma was nearly 2.5 times higher than in other cancer types. The majority of EPHA-MUT samples were melanoma (33/38) in the discovery cohort, which is a major confounding factor causing bias. However, the survival advantages across multiple cancers in the validation cohort as well as the general upregulation of anti-tumor immunity in various cancers could compensate the bias to some degree. Still, the predictive value of EPHA7 mutation with regard to cancer types needs to be verified in future prospective trials. Additionally, the possible TIME and molecular mechanisms of EPHA7-MUT were demonstrated based on GSEA, which requires further molecular researches to validate. Finally, gene expression data has not been included in both the discovery and validation cohorts. Therefore, combination analysis of EPHA7 and other predictive biomarkers (e.g., expression of PD-L1) has not been performed. Clinical trials with expression data are needed to expand our findings and test the added value of tumor-infiltrating lymphocytes in the survival analysis of EPHA7-MUT.
Importantly, these limitations do not preclude the favorable clinical outcomes derived from immunotherapy in EPHA7-MUT patients. Unlike continuous variables such as TMB, CNA, or PD-L1 expression, EPHA7-MUT are easily detected by NGS and clearly classify patients into two groups that are associated with immunotherapy response. The scope of EPHA7-MUT falls in compensating the existing biomarkers to detect those patients who are most likely to benefit from immunotherapy. Our study not only paved the way for precise treatments tailored to molecular subtypes, but also indicated the association between Eph receptor-related TIME and immunotherapy response. Biological functions mediated by Eph receptors, especially tumor angiogenesis, intercellular contact, and tumor metabolism, should be better characterized in future studies. Although there are some researches or ongoing trials co-targeting these pathways and tumor immunity [
18,
40], our study introduces a novel angle that Eph receptors might influence the immunotherapy response through corresponding biological functions. Further elucidation of the molecular mechanism between Eph receptors and anti-tumor immunity is warranted to validate the predictive value as well as help to identify potential therapeutic targets.
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