Identification of IRF-associated molecular subtypes in clear cell renal cell carcinoma to characterize immunological characteristics and guide therapy

Abstract

Background: Recently studies have identified a critical role for interferon regulatory factor (IRF) in modulating tumour immune microenvironment (TME) infiltration and tumorigenesis.

Methods: Based on IRF1-9 expression profiles, we classified all ccRCC samples into three molecular subtypes (clusters A-C) and characterized the prognosis and immune infiltration of these clusters. IRFscore constructed by principal component analysis was performed to quantify IRF-related subtypes in individual patients.

Results: We proved that IRFscore predicted multiple patient characteristics, with high IRFscore group having poorer prognosis, suppressed TME, increased T-cell exhaustion, increased TMB and greater sensitivity to anti- PD-1/CTLA-4 therapies. Furthermore, analysis of metastatic ccRCC (mccRCC) molecular subtypes and drug sensitivity proved that low IRFscore was more sensitive to targeted therapies. Moreover, IRFscore grouping can be well matched to the immunological and molecular typing of ccRCC. qRT-PCR showed differential expression of IRFs in different cell lines.

Conclusions: Evaluating IRF-related molecular subtypes in individual ccRCC patients not only facilitates our understanding of tumour immune infiltration, but also provides more effective clinical ideas for personalised treatment.

Keywords: IRF family; ccRCC; immunotherapy; t cell exhaustion; targeted therapy; tumour microenvironment.

Figure 1

Landscape of IRFs expression in ccRCC. (A-B) Boxplot of IRFs expression in ccRCC and normal tissues from TCGA database (A) and GSE36895 (B). (C) ROC curves demonstrate IRF family ability to differentiate between tumour and normal tissue. (D-E) Principal component analysis for the expression profiles of IRFs to distinguish tumours from normal samples in TCGA database (D) and GSE36895 (E). (F) The interaction between IRFs in ccRCC. (G) The PPI network of IRFs. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

RT-PCR analysis of IRF1-9 expression levels in 786-O, Caki-1 and HK-2 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

The IRF-related molecular subtypes in ccRCC and biological and immune characteristic of each pattern. (A) PCA for the transcriptome profiles of three IRF clusters. (B) Survival analyses of three IRF clusters. (C) The expression of IRF1-9 in three IRF clusters. (D-E) GSVA enrichment analysis showing the activation states of biological pathways in distinct clusters. (F) The abundance of each TME infiltrating cell in three clusters. (G) Box plot indicated the correlation between IRF clusters and immune scores, stromal scores and estimate scores. (H) The expression of most immune checkpoints among distinct IRF clusters. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

IRF gene clusters in ccRCC and biological characteristics of each gene cluster. (A) 547 IRF-associated DEGs shown in venn diagram. (B-C) GO (B) and KEGG (C) enrichment analysis on these DEGs. (D) Survival analyses of three IRF gene clusters. (E) The expression of IRFs in three gene clusters. (F) The abundance of each TME infiltrating cell in three gene clusters. **p < 0.01; ***p < 0.001.

Figure 5

Construction of IRF signatures. (A) Alluvial diagram showing the changes of IRF cluster, gene cluster, IRFscore and patient survival status. (B-C) Differences in IRFscore among three gene clusters (B) and IRF clusters (C). (D) The expression of IRF1-9 in two IRFscore groups. (E) Kaplan-Meier survival analysis for two IRFscore groups. (F) ROCs for 1-, 3-, 5-, and 7-year survival time based on IRFscore. (G) Nomograms incorporating IRFscore and clinical characteristics for predicting patient 1-, 3-, 5-year survival. ns, not significant; ***p < 0.001.

Figure 6

immune characteristics and somatic variants in IRFscore groups. (A) The abundance of each TME infiltrating cell in two IRFscore groups. (B) The immune scores, stromal scores and estimate score difference in high and low IRFscore groups. (C) The relative fraction of each TME-infiltrated cell in two IRFscore groups. (D) The differences in the receptors or ligands expressed by exhausted T cells between two IRFscore groups. (E) The differences in TLS-related markers between two IRFscore groups. (F) Kaplan-Meier survival analysis for two TMB score groups. (G) Kaplan-Meier survival analysis for patients stratified by IRFscore and TMB score. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

IRFscore in the role of ccRCC clinical therapies. (A-E) Box plot showing the sensitivity of patients with high and low IRFscore subgroups to chemotherapy drugs, including sunitinib (A), sorafenib (B), nilotinib (C), temsirolimus (D) and pazopanib (E). (F-I) The association between IPS and immune checkpoints in ccRCC patients with different IRFscore.