Development of a Predictive Immune-Related Gene Signature Associated With Hepatocellular Carcinoma Patient Prognosis

Abstract

Background: Hepatocellular carcinoma (HCC) remains the third leader cancer-associated cause of death globally, but the etiological basis for this complex disease remains poorly clarified. The present study was thus conceptualized to define a prognostic immune-related gene (IRG) signature capable of predicting immunotherapy responsiveness and overall survival (OS) in patients with HCC.

Methods: Five differentially expressed IRG associated with HCC were established the immune-related risk model through univariate Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses. Patients were separated at random into training and testing cohorts, after which the association between the identified IRG signature and OS was evaluated using the "survival" R package. In addition, maftools was leveraged to assess mutational data, with tumor mutation burden (TMB) scores being calculated as follows: (total mutations/total bases) × 10⁶. Immune-related risk term abundance was quantified via "ssGSEA" algorithm using the "gsva" R package.

Results: HCC patients were successfully stratified into low-risk and high-risk groups based upon a signature composed of 5 differentially expressed IRGs, with overall survival being significantly different between these 2 groups in training cohort, testing cohort and overall patient cohort (P = 1.745e-06, P = 1.888e-02, P = 4.281e-07). No association was observed between TMB and this IRG risk score in the overall patient cohort (P = 0.461). Notably, 19 out of 29 immune-related risk terms differed substantially in the overall patient dataset. These risk terms mainly included checkpoints, human leukocyte antigens, natural killer cells, dendritic cells, and major histocompatibility complex class I.

Conclusion: In summary, an immune-related prognostic gene signature was successfully developed and used to predict survival outcomes and immune system status in patients with HCC. This signature has the potential to help guide immunotherapeutic treatment planning for patients affected by this deadly cancer.

Keywords: hepatocellular carcinoma; immune landscape; immune-related genes; prognostic signature; tumor mutation burden.

Figure 1.

Differentially expressed immune-related gene identification. (A) DE IRGs associated with liver cancer were identified using a Venn diagram to analyze the intersection between the DE gene and IRG datasets. (B) GO analysis results. (C) The top 6 most significantly enriched KEGG pathways.

Figure 2.

Development of a prognostic immune-related risk signature associated with HCC patient outcomes. (A-B) Using LASSO regression analyses, 5 genes associated with HCC patient OS were identified, and 10-round cross-validation was conducted to avoid overfitting. (C) High- and low-risk HCC patient OS was evaluated using Kaplan–Meier curves. (D) Time-dependent ROC analyses of the identified immune-related risk signature in the training cohort.

Figure 3.

Immune-related risk signature validation. (A) High- and low-risk liver cancer patient OS was evaluated in the testing cohort. (B) A time-dependent ROC analysis of the immune-related risk signature in the testing cohort. (C) The OS of high- and low-risk liver cancer patients was assessed via Kaplan–Meier curve analyses in the overall cohort. (D) A time-dependent ROC analysis of the immune-related risk signature in the overall cohort.

Figure 4.

Correlations between immune-related risk signatures and liver cancer patient PFS and DFS. (A) The PFS of high- and low-risk liver cancer patients in the overall patient cohort was assessed using Kaplan-Meier curves. (B) The DFS of high- and low-risk liver cancer patients in the overall patient cohort was assessed using Kaplan-Meier curves.

Figure 5.

The association between immune-related risk signatures and HCC patient clinical characteristics. (A, B) Differences in risk scores as a function of patient age, sex, clinical stage, T stage, N stage, and M stage were assessed.

Figure 6.

High- and low-risk HCC patient mutation profiles and TMB values. (A) Summarized mutational data from 327 patients. (B, C) Mutational frequencies in the top 20 genes in the training and testing cohorts. (D) The relationship between immune-related risk scores and TMB. (E) The OS of high- and low-risk TMB liver cancer patient groups was assessed using Kaplan-Meier curves.

Figure 7.

The relationship between immune-related risk signatures, immune cell infiltration, and immune functionality in liver cancer patients. (A) The relative enrichment of 29 immune-related risk terms in high- and low-risk liver cancer patients. (B) HLA family gene expression in high- and low-risk liver cancer patients. (C-D) CD80 and CD86 expression levels in low- and high-risk liver cancer patients.

Figure 8.

The association between immune-related risk scores and 19 differentially expressed immune-related terms.