Ferroptosis biomarkers predict tumor mutation burden's impact on prognosis in HER2-positive breast cancer

Figure 1 Flowchart for collecting and analyzing data from TCGA and METABRIC databases. scRNA-seq: Single-cell RNA sequencing; TMB: Tumor mutation burden.

Figure 2 Identification of candidate genes associated with ferroptosis in the TCGA cohort. A: Heatmap of candidate gene expression differences between tumor and normal tissues; B: Volcano map showing up-regulated and down-regulated genes; C: A Venn diagram to identify differentially expressed genes associated with ferroptosis between tumor and normal tissues; D: Forest plot showing univariate Cox regression analysis results of correlation between candidate gene expression and overall survival.

Figure 3 Prognostic analysis of four ferroptosis-related gene signature models in the TCGA cohort. A: The optimal lambda resulted in nine nonzero coefficients; B: The partial likelihood deviation curve was plotted vs lambda; C: Risk curves were plotted in the TCGA cohort; D: Kaplan–Meier curves for the overall survival of patients in the high-risk and low-risk groups in the TCGA cohort; E: The area under the curves of time-dependent receiver operating characteristic curves verified the prognostic performance of the risk score in the TCGA cohort; F: T-SNE analysis of the TCGA cohort. OS: Overall survival.

Figure 4 Results of univariate and multivariate Cox regression analyses. A: Results of the univariate and multivariate Cox analyses of overall survival (OS) in the TCGA derivation cohort; B: Results of univariate and multivariate Cox analyses of OS in the METABRIC derivation cohort. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. HR: Hazard ratio.

Figure 5 The four ferroptosis-related prognostic gene signature models for predicting 3-, 5-, and 8-year overall survival in HER2+ breast cancer patients. A: Independent risk factors were used to build a risk estimation nomogram to predict the probability of overall survival in HER2+ breast cancer patients; B: Calibration plots for 3-, 5-, and 8-year survival probabilities in the TCGA cohort; C: Restricted mean survival time Curve in the TCGA cohort. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. OS: Overall survival; RMS time: Restricted mean survival time.

Figure 6 The immune-related analysis of the four ferroptosis-related prognostic gene signature models in HER2+ breast cancer. A: The Immune cells infiltration of differential risk groups in TCGA; B: Heatmaps represent the correlation between immune cells and prognostic genes; C-E: The immune, stromal, and estimate scores were significantly distinct statistically between low- and high-risk subgroups; F: Expression of immune cell pathways in low- and high-risk subgroups; G:The seven well-known immune checkpoint genes were differentially expressed between low- and high-risk subgroups; H: Correlation chord diagram of 7 immune checkpoints. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 7 The mutation profile of different subgroups in the TCGA cohort. A: Relationship between tumor mutation burden (TMB) and risk scores; B: Relationship between TMB groups and risk groups; C: TMB in low- and high-risk subgroups; D: Kaplan–Meier survival analysis of a new subset in a cohort of HER2+ breast cancer patients combined with the risk group and TMB. TMB: Tumor mutation burden; NS: Not significant; BC: Breast cancer.

Figure 8 Overview of single cells from tumor samples and standard samples. A: Umap of 42 cell clusters; B: Umap of two different types of samples; C: Marker genes identified cell types; D: Proportion of cell types in patients' and normal people's samples; E-H: Expression of essential marker genes.