A novel inflammation-associated prognostic signature for clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) are typically situated in a complex inflammatory and immune microenvironment, which has been reported to contribute to the unfavorable prognosis of patients with ccRCC. There would be beneficial clinical implications for elucidating the roles of its molecular characteristics in the inflammatory microenvironment. This is because it would facilitate the development of reliable biomarkers for pre-stratification prior to the designation of individualized treatment strategies. In the present study, RNA-sequencing data from 607 patients were retrospectively analyzed to elucidate the profile of inflammatory molecules. Based on this, an inflammatory prognostic signature (IPS) was developed and further validated using clinical ccRCC samples. Subsequently, the associated mechanisms in terms of the immune microenvironment and molecular pathways were then investigated. This proposed IPS was found to exhibit superior accuracy compared with the criterion of a good prognostic model for the prediction of patient prognosis from ccRCC [area under the receiver operating characteristic curve (AUC)=0.811] in addition to being an independent factor for prognostic risk stratification [hazard ratio: 11.73 (95% CI, 26.98-5.10); log-rank test, P<0.001]. Pathologically, ccRCC cells identified as high-risk according to their IPS presented with a more malignant tumor structure, including voluminous eosinophilic cytoplasm, acinar/lamellar/tubular growth patterns and atypic nuclei. High-risk ccRCC also exhibited higher infiltration levels by four types of immune cells, including T regulatory cells, but lower infiltration levels by mast cells. Pathways associated with immune-inflammation interaction, including the IL-17 pathway, were found to be upregulated in IPS-identified high-risk ccRCC. Furthermore, by combining the IPS with clinical factors, an integrated prognostic index was developed and validated for increasing the accuracy of patient risk-stratification for ccRCC (AUC=0.911). In conclusion, the complex regulatory mechanisms and molecular characteristics involved in ccRCC-inflammation interaction, coupled with their prognostic potential, were systematically elucidated in the present study. This may have important implications in furthering the understanding into the molecular mechanisms underlying this ccRCC-inflammation interaction, which can in turn be exploited for identifying high-risk patients with ccRCC prior to designing their clinical treatment strategy.

Keywords: clear cell renal cell carcinoma; immune cells; inflammation; precision medicine; prognostic signature.

Figure 1.

Results of feature selection using least absolute shrinkage and selection operator regression. (A) Coefficients and (B) partial likelihood deviance. The number of genes are presented at the top of each panel.

Figure 2.

Validation of IPS. Distribution plots for the IPS score, overall survival time and survival status of each patient with clear cell renal cell carcinoma in the (A) training cohort and (B) validation cohort. (C) Differential expression profiles of inflammation-associated genes constituting the IPS between IPS-identified high- and low-risk groups, with dots above indicating outliers. Box plots show the five-number summary of a set of data, including the minimum score, first (lower) quartile, median, third (upper) quartile and maximum score. (D) ROC curves of IPS in the training and validation cohort. Kaplan-Meier curves indicating the difference in survival probability between the IPS-identified high-risk and low-risk groups in (E) the training cohort and (F) validating cohort. Correlation analyses attested the association between the IPS score and survival time in (G) the training cohort and (H) validation cohort. IPS, inflammatory prognostic signature; AUC, area under the ROC curve; ROC, receiver operating characteristic. *P<0.05, **P<0.01 and ***P<0.001.

Figure 3.

Molecular mechanisms of inflammation-associated genes in the progression of clear cell renal cell carcinoma. (A) Enrichment results of the Gene Ontology annotation, including the top 10 terms in the categories BP, CC and MF. (B) Dysregulated pathways mainly regulated by the inflammation-associated genes. (C) Protein-protein interactive network of the inflammation-associated genes constituting the inflammatory prognostic signature. BP, Biological process; CC, Cellular component; MF, Molecular function.

Figure 4.

Comprehensive infiltration profiles of immune cells of clear cell renal cell carcinoma. Immune cell profiles in the (A) the training cohort and (B) validation cohort. NK, natural killer.

Figure 5.

Identified differential infiltrated immune cells in the IPS-identified high- and low-risk ccRCC groups. (A) Infiltration of six types of immune cells compared between the IPS-identified high- and low-risk ccRCC groups in the training cohort. (B) Correlations between those immune cell types and the IPS score in the training cohort. A violin plot is a hybrid of a box plot and a kernel density plot, which shows peaks in the data, and dots indicate each data point. Bars above and to the right of the plot represent frequency of the corresponding values. (C) Infiltration of six types of immune cells compared between the IPS-identified high- and low-risk ccRCC groups in the validation cohort. (D) Correlations between those immune cells and the IPS score in the validation cohort. ccRCC, clear cell renal cell carcinoma; IPS, inflammatory prognostic signature. *P<0.05, **P<0.01 and ***P<0.001.

Figure 6.

Construction and validation of an IPI. (A) Nomogram of the IPI. IPS score, overall survival time and survival status of each patient with ccRCC in (B) the training and (C) validation cohorts. Kaplan-Meier curves indicating the difference in survival probability between patients with ccRCC in the high- and low-risk groups from the (D) training and (E) validation cohorts. (F) ROC curve of the IPI. IPS, inflammatory prognostic signature; IPI, integrated prognostic index; ccRCC, clear cell renal cell carcinoma; AUC, area under the ROC curve; ROC, receiver operating characteristic.

Figure 7.

Clinical validation of the IPS. (A) H&E staining results of IPS-identified high- and low-risk ccRCC samples. Arrows indicate some macrophage examples. (B) IHC results of the top four most hazardous IGPs. All scale bars are 50 µm. (C) IGP expression and IPS score of each patient with ccRCC. (D) Quantification of IHC results of the top four most hazardous IGPs. ccRCC, clear cell renal cell carcinoma; IPS, inflammatory prognostic signature. CCL7, C-C motif chemokine ligand 7; LTB4R2, leukotriene B4 receptor 2. **P<0.01 and ***P<0.001.