3.1 Demographics of population in TCGA LUAD datasets
497 LUAD patients with both clinical and transcriptome profiling were downloaded from TCGA LUAD datasets. After excluding those with missing or insufficient data on age, gender or stage, we included 316 patients to further examine the prognostic role by Cox analysis and Logistic regression. We also included 325 LUAD patients from FDUSCC diagnosed between 2009 and 2015 to perform IHC microarrays. The baseline characteristics of these two cohorts were presented in Table 1.
Correlations between the clinical information and NRAS were analyzed by logistic analyses in TCGA cohort. The categorical dependent value is based on the median value NRAS expression. As shown in Table2, high expression of NRAS in LUAD was significantly associated with advanced stage (stage I vs stage III, OR=1.90(1.14-3.21), p=0.0059) and positive lymph nodes (positive vs negative, OR=2.00(1.26-3.20), p=0.0037).
3.2 NRAS expression is up-regulated in LUAD specimens
Wilcox T test was performed to estimate the relationship between NRAS expression and disease status of LUAD based on TCGA datasets. As shown in Fig. 1 A, the expression of NRAS was significantly upregulated in LUAD samples compared with normal samples (p=3.85e-04). Meanwhile, we paired the tumor tissue and adjacent tumor tissue of the same patient from TCGA LUAD datasets. The pair analysis shown in Fig. 1B indicated the same results as the former (p=5.19e-05). 11 LUAD tissue and 3 normal tissue IHC images were obtained from the HPA website. The staining intensity of LUAD tissue was medium-strong (2 of 11 were strong), while that of normal pneumocytes was weak. As shown in Fig.1C and Fig.1E (intensity: strong, staining: high, quantity>75%), the tumor cells were intensely stained. As shown in Fig. 1D and Fig. 1F (Macrophage: Intensity: medium, staining: moderate, quantity: >75%; Pneumocyte: Intensity: weak, staining: low, quantity: 75%-25%), the intensity of macrophage staining is moderate, and that of pneumocytes is weak in normal tissues. This indicates that NRAS acts as a pivotal role in LUAD progression.
3.3 NRAS expression predicted survival and could be used as an independent prognostic biomarker in LUAD patients
Patients were divided into high expression group and low expression group according to the median value of NRAS expression. As shown in Fig. 2A, Kaplan-Meier survival analysis shows that high NRAS expression group has a significantly poorer overall survival (OS) compared with low expression group. The univariable analysis shown in Table3a reveals that NRAS expression correlated significantly with a poor OS. Besides, clinical stage, tumor size and lymph nodes are significantly associated with OS. According to multivariable analysis (Table3b), the expression level of NRAS remains independently associated with OS (p=0.012). Besides, we verified the prognostic value of NRAS at the protein level. We evaluated the protein level of NRAS in completely resected tissues using TMA. A total of 325 LUAD patients, undergoing surgical resection in Fudan University Shanghai Cancer Center, were enrolled in this study. According to the IRS of IHC staining, we defined 0-6 as weak staining group, and 7-12 as high staining group. The representative IHC images of NRAS are shown in Fig. 2D. As shown in Fig. 2C, higher expression of NRAS is also associated with worse OS at the protein level (p=0.0072).
The prognostic value of NRAS in LUAD was also verified by three GEO datasets GSE50081 (including 181 LUAD samples), GSE42127 (176 LUAD samples), and GSE30219 (293 LUAD samples). Since each dataset used different genetic chips and algorithms, the cut-off value to divide patients into high or low expression group was based on the median value of NRAS expression in each dataset. As shown in Fig. S1 (GSE50081: p=0.028, GSE42127: p=0.0078, GSE30219: p=0.0026), we performed Kaplan-Meier survival analyses as a validation. Hence, NRAS expression level could be an independent prognostic factor for patients with LUAD.
3.4 GSEA identified NRAS-related signaling pathways
To further study the signaling pathways that NRAS might be involved in, we performed a GSEA between high and low NRAS expression phenotypes. The gene sets were enriched and analyzed by Hallmark (h.all.v6.2.symbols.gmt) and Go (c5.bp.v6.2.symbols.gmt) Collection.
11 Hallmark items (Fig. 4A) including G2M checkpoint, E2F targets, DNA repair, P13K AKT MTOR signaling, MTORC1 signaling pathways were significantly enriched in NRAS high expression phenotype. According to the enrichment of Go Collection (Fig. 4B and Fig. 4C), high NRAS expression gene sets were significantly correlated with 12 immune -related signaling pathways such as activation of innate immune response, negative regulation of B cell proliferation and T cell regulation signaling pathway. There is no signal pathways with low expression in NRAS enriched in neither GO nor Hallmark Collection based on NES, NOM P value, and FDR q value.
3.5 Correlation analysis between NRAS and TIM in LUAD
We estimated the relationship between the abundance of TIICs and NRAS expression via the TIMER algorithm. The results shown in Fig. 4A indicates that the expression of NRAS was positively correlated with CD8+ T cells (cor=0.27, p=1.06e-09), macrophages (cor=0.331, p=4.06e-14), and neutrophils (cor=0.373, p=8.81e-18), while it was negatively correlated with B cells (cor=-0.216, p=1.31e-0.6) and CD 4+ T cells (cor=-0.108, p=1.65e-02). In addition, we analyzed the correlation between immune-related biomarkers and NRAS expression. As shown in Fig. 4B, NRAS expression was positively correlated with CD274 (rho=0.333, p=8.59e-15), HAVCR2 (rho=0.239, p=4.01e-08), PDCD1 (rho=0.101, p=2.16e-02), CD47 (rho=0.189, p=1.64e-05) and CD24 (rho=0.227, p=1.82e−07). The results shown in Fig. 4C-E validated the correlation between NRAS and immune-related biomarkers at the protein level via IHC staining of TMAs. Analyzing the IRS score of these immune-related molecules between the NRAS strong staining group and weak staining group, we found that the IRS of PD-L1 (p=6.24e-3), PD-1 (p=1.37e04), and TIM-3 (p=2.47e-10) were significantly higher in NRAS strong staining group than weak group. The representative stainings were performed in Fig. S2.