Establishment of a Lymph Node Metastasis-Associated Prognostic Signature for Lung Adenocarcinoma

Figures

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  Figure 1. Identification of differentially expressed genes (DEGs) between metastasis (M) and nonmetastasis (NM) groups. (a) Heatmap of DEGs between M and NM groups. Blue box indicates the M group and red box indicates NM group. (b) Volcano plot showing the DEGs between M and NM groups in TCGA cohort. Blue node represents a lower expression of gene in the M group, and red node represents a higher expression of gene in the M group.

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  Figure 2. Functional enrichment analysis of DEGs. Lilac stands for gene ontology (GO)_biological processes (BP) category, orange stands for GO_cellular component (CC) category, modena stands for GO_molecular function (MF) category, and green stands for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The number in the box represents the count of genes enriched in the term.

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  Figure 3. Results of weighted gene coexpression network analysis. (a) Left panel shows adjacency matrix weight parameter power selection plot. X axis represents the power value and Y axis represents the square of the correlation coefficient between log (k) and log (p (k)) in the corresponding network. Horizontal red line: 0.85. Right panel shows the mean connectivity (Y-axis) as a function of the soft-thresholding power (Y-axis). (b) Cluster dendrogram of the coexpression network modules. Each color represents a different module. (c) Association between the gene modules and metastasis state. The left column is lymph node metastasis and right column is no lymph node metastasis. (d) Venn diagram of the intersection of DEGs and yellow module genes.

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  Figure 4. Identification of prognostic signature in the TCGA cohort. (a) Calculation of LASSO coefficient for each lambda. Each line represents a gene confidence value. (b) Partial likelihood deviance of LASSO coefficient. The two vertical dashed lines represent lambda.min (left line) and lambda.1se (right line). Horizontal axis represents the log (λ) value, while vertical axis represents the partial likelihood deviance of the log (λ) value.

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  Figure 5. Construction and validation of metastasis-associated signature model in the training and three validation cohorts. (a) The distribution and survival state of samples in the TCGA cohort. (b) Kaplan–Meier (KM) survival curves for samples in the high-risk (HR) and low-risk (LR) groups of TCGA cohort. (c) Receiver operating characteristic (ROC) analysis of overall survival at 1, 3, and 5 years in TCGA cohort. (d) The distribution and survival state of samples in the GSE68465. (e) KM survival curves showing survival outcomes of GSE68465. (f) ROC analysis of GSE68465. (g) Distribution of patients in GSE42127 based on the median RS and survival status for each case. (h) Overall survival curves for patients in LR and HR groups in GSE42127 dataset. (i) ROC curve showed the predictive efficiency of the RS in GSE42127. (j) RS distribution and survival status distribution of patients in GSE50081. (k) KM curves of overall survival in GSE50081. (l) ROC curves for 1-, 3-, and 5-year survival in GSE50081.

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  Figure 6. Functional enrichment analysis based on the RS model by GSEA. Six significantly enriched KEGG pathways in the HR group.

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  Figure 7. KM survival analysis of patients divided into the low- and high-expression groups of eight signature genes in the TCGA cohort. (a) ANGPTL4; (b) KRT6A; (c) TCN1; (d) TNS4; (e) PTPRH; (f) RGS20; (g) GPR98; (h) BARX2. Blue line represents low-expression group and red line represents high-expression group.

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  Figure 8. Correlation analysis of RS and clinical characteristics. (a) RS in subgroups of the T stages (T1 + T2 vs. T3 + T4), N stages (N0 vs. N1–N3), or pathological stages (I + II vs. III + IV). (b) Heatmaps showing the mRNA expression of eight selected genes in TCGA cohort. (c) Correlation of each gene and clinical characteristics (T stages, N stages, and pathological stages). Ns, no significance; ∗p value < 0.05; ∗∗p value < 0.01; ∗∗∗p value < 0.001; ∗∗∗∗p value < 0.0001.

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  Figure 9. Validation of the expression level of genes in the RS model. (a) Protein expression level of five genes in LUAD and normal tissues based on immunohistochemistry results from the human protein atlas (HPA) database. (b) mRNA expression level of RS model genes in M and NM groups in GSE68465. Blue indicates NM sample and yellow indicates M sample.