Comprehensive Analysis Identifies PI3K/Akt Pathway Alternations as an Immune-Related Prognostic Biomarker in Colon Adenocarcinoma Patients Receiving Immune Checkpoint Inhibitor Treatment

Abstract

Introduction: In recent years, immune checkpoint inhibitors (ICIs) have attracted widespread attention and made breakthroughs in progress towards the treatment of various cancers. However, ICI therapy is selective, and its effects on many patients are not ideal. It is therefore critical to identify prognostic biomarkers of response to ICI therapy. The PI3K/Akt pathway plays important roles in tumor formation and metastasis. However, there are no published reports clarifying the relationship between PI3K/Akt pathway mutations and prognosis for colon adenocarcinoma (COAD) patients receiving immunotherapy.

Methods: We collected data from a COAD cohort from The Cancer Genome Atlas (TCGA) database, including whole-exome sequencing (WES) data, RNA-seq data, and clinical data. We also collected data, including clinical prognosis and targeted sequencing data, from a cohort of COAD patients receiving immunotherapy. We collected 50 COAD patients (Local-COAD) from the Zhujiang Hospital of Southern Medical University and performed targeted sequencing. We analyzed the effects of PI3K/Akt pathway mutations on the patients' clinical prognosis, immunogenicity, and immune microenvironments. Gene set enrichment analysis (GSEA) was used to analyze the significantly upregulated and downregulated signaling pathways. We used these results to hypothesize potential mechanisms by which PI3K/Akt mutations could affect the prognosis of COAD patients.

Results: Univariate and multivariate Cox analyses and Kaplan-Meier (KM) survival curves showed that patients with PI3K-Akt mutations had better overall survival (OS) than those without PI3K-Akt mutations. Genes with significant mutation rates in the two cohorts were screened by panoramic view. CIBERSORT was used to analyze changes in 22 types of immune cells to identify immune activated cells. Similarly, patients in the PI3K/Akt-mutated type (PI3K/Akt-MT) group had significantly increased immunogenicity, including increases in tumor mutation burden (TMB), neoantigen load (NAL), and MANTIS score. Using GSEA, we identified upregulated pathways related to immune response.

Conclusion: PI3K/Akt pathway mutation status can be used as an independent predictor of response to ICI treatment in COAD patients. PI3K/Akt mutations are correlated with improved OS, higher immunogenicity, greater immune response scores, and increases in activated immune cells.

Figure 1

Results of the univariate and multivariate Cox regression analyses and KM survival curve of the immunotherapy cohort. (a) The results of the univariate and multivariate regression analyses are displayed with a forest map. The main part of the forest map is used to show the HR and 95% confidence intervals. The factors associated with good prognosis are HR < 1, and those associated with poor prognosis are HR > 1. (b) The KM curve of the immunotherapy cohort (Samstein et al.) predicted OS (P < 0.01, log-rank test).

Figure 2

Gene mutation panorama of colorectal cancer patients in the immunotherapy cohort (a) and TCGA-COAD cohort (b) and the relationship between PI3K/Akt pathway mutations and clinical characteristics (c). (a) The 20 genes with the highest mutation frequencies in COAD patients in the immunotherapy cohort and their corresponding clinical characteristics are displayed. The mutation frequencies of PIK3CA, ARID1A, and PTPRS were significantly increased in the PI3K/Akt-MT group. Yellow represents cleavage site mutations, blue represents missense mutations, orange represents frame shift mutations, green represents insertion/deletion mutations, and brown represents nonsense mutations. (b) The 20 genes with the highest mutation frequencies in patients in the TCGA-COAD cohort and their corresponding clinical characteristics are displayed. With the exception of KRAS, the mutation frequencies of all other genes changed significantly. Yellow represents cleavage site mutations, blue represents missense mutations, orange represents frame shift mutations, green represents insertion/deletion mutations, and brown represents nonsense mutations. (c) Relationship between PI3K/Akt pathway mutations and clinical characteristics such as age, sex, and sample type differences in the immunotherapy cohort (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; Fisher's exact test).

Figure 3

The relationship between PI3K/Akt pathway mutations and enhanced immunogenicity. (a) Comparison of DDR mutations in the PI3K/Akt-MT and PI3K/Akt-WT groups in the immunotherapy cohort. (b) Comparison of TMB in the PI3K/Akt-MT and PI3K/Akt-WT groups in the immunotherapy cohort. (c) Comparison of DDR mutations in the PI3K/Akt-MT and PI3K/Akt-WT groups in the TCGA cohort. (d) Comparison of TMB in the PI3K/Akt-MT and PI3K/Akt-WT groups in the Local-COAD cohort. (e) Comparison of TMB between PI3K/Akt-MT and PI3K/Akt-WT groups in the TCGA cohort. (f) Comparison of NAL between PI3K/Akt-MT and PI3K/Akt-WT groups in the TCGA cohort. (g) Comparison of MANTIS scores of the PI3K/Akt-MT and PI3K/Akt-WT groups in the TCGA cohort (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; Mann–Whitney U test).

Figure 4

The relationship between PI3K/Akt mutations and immune microenvironment. (a) CIBERSORT comparison of the content of 22 types of immune cells in the tumor microenvironments of PI3K/Akt-MT and PI3K/Akt-WT patients (algorithm running the lm22 signature and 1,000 permissions). (b) Difference in immune checkpoint gene expression between the PI3K/Akt-WT and PI3K/Akt-MT groups. (c) Difference in immune fractions between the PI3K/Akt-WT and PI3K/Akt-MT groups. (d) The functions of immune module genes involved in the PI3K/Akt pathway, including antigen presentation, CD8+ T cells, cytologic activity, inhibition, NK cells, stimulation, and type I IFN (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; Mann–Whitney U test).

Figure 5

(a) GSEA comparison of upregulated and downregulated genes in the PI3K/Akt-MT and PI3K/Akt-WT groups. The enrichment score greater than 0 indicates that the pathway is upregulated in the PI3K/Akt-MT group, and the enrichment score less than 0 indicates that the pathway is downregulated in the PI3K/Akt-MT group. The color of the pathway name is consistent with the color of the broken line in the figure; P value less than 0.05 is statistically significant. (b) The potential mechanism of PI3K/Akt-MT patients with colorectal cancer receiving ICI had a better prognosis.