Construction Of A Prognostic Model Related To Ferroptosis In Renal Chromophobe Cell Carcinoma And External Validation Of A Four-category Grading Syste

Part Ⅰ Identification and validation of a ferroptosis-related signature for prediction of the prognosis and tumor microenvironment in patients with chromophobe renal cell carcinoma Background:Ferroptosis is a novel form of regulated cell death that is different from other forms,which has an important role in tumor growth inhibition.The prognostic value of ferroptosisrelated genes(FRGs)in chromophobe renal cell carcinoma is unclear.The purpose of this study was to construct and validate a prognostic signature related to ferroptosis in chromophobe renal cell carcinoma(Ch RCC)and to explore its role in immune cell infiltration and systemic therapy.Methods:The gene expression profiles of Ch RCC patients obtained from The Cancer Genome Atlas(TCGA)database were used to identify prognostic FRGs by univariate Cox proportional hazards analyses.Ferroptosis molecular subtypes were obtained by consensus clustering analysis.Differentially expressed FRGs between Ch RCC and normal tissues were identified using the LIMMA R package.The FRG-based signature in the training set was established by least absolute shrinkage and selection operator analysis and verified in the testing set.The risk score was calculated by FRG expression and the corresponding coefficient.The patients were divided into high-and low-risk groups using the median of the risk scores as the cut-off value.Survival differences between high-and low-risk groups were shown by a Kaplan–Meier curve.Cox proportional hazard regression models were used in univariate and multivariate analyses of Ch RCC patients to assess the prognostic value of the FRG-based signature and clinical variables.Nomogram was used to predict overall survival.The association between molecular subtypes and the prognostic signature and immune microenvironment was explored to predict responses to immunotherapy by the single sample GSEA algorithm and ESTIMATE algorithm.The response of Ch RCC patients in high-and low-risk groups to drugs in GDSC database and Cell Miner database was evaluated.Sixty Ch RCC patients at the affiliated Hospital of Qingdao University were included in the study for external verification.Immunohistochemistry was used to verify expression of the FRG-based signature externally.Results:Ch RCC patients were divided into two FRG subtypes.Two FRGs(TFRC and SLC7A11)were identified to construct the prognostic signature.The high-risk group and cluster 2 had worse overall survival than the low-risk group and cluster 1,respectively.The low-risk group and cluster 1 had higher levels of immune cell infiltration and expression of MHC and immune checkpoint molecules than the high-risk group and cluster 2.The risk score was a predictor of overall survival and had a good predictive ability,which was verified in the testing set and evaluated by ROC and calibration curves.The different sensitivities of targeted drugs in patients with different risks were evaluated.External immunohistochemical analysis showed that TFRC and SLC7A11 were highly expressed in tumor tissues compared with para-cancer normal tissues,and the expression level was significantly associated with a more advanced stage and worse cancer-specific survival.Conclusion:An FRG signature was identified and validated to predict the prognosis of Ch RCC.A significant association between the signature and immune cell infiltration,immune checkpoint expression,and drug response is helpful to guide comprehensive treatment of Ch RCC.Part Ⅱ External validation of a four-tiered grading system for chromophobe renal cell carcinomaBackground: This study aimed to validate the prognostic value of a four-tiered grading system recently proposed by Avulova et al.and to explore the prognostic ability of another fourtiered classification grading system in which there is a separate Grade 3 for tumor necrosis.Grading of chromophobe renal cell carcinoma(Ch RCC)by the Fuhrman system is not feasible because of the inherent nuclear atypia in ChRCC.Methods: We collected relevant data of 263 patients with Ch RCC who had undergone surgery in our hospital from 2008 to 2020.Pathological information was based on reviews of the microscopic findings by two urologic pathologists who were blinded to patients’ survival outcomes.The Kaplan–Meier method was used to calculate the survival rate and Cox proportional hazard regression models to assess associations with cancer-specific survival(CSS)and distant metastasis-free survival(DMFS)by hazard ratios(HRs)and 95% confidence intervals(CIs).Results: Ten patients died from Ch RCC,and 12 developed metastases.The 5-year CSS rates were 95.9%.Grades 2(HR=11.6;95%Cl: 1.20-111.3;P = 0.03),3(HR = 24.9;95%Cl: 2.59-239.8;P = 0.005),and 4(HR = 302.9;95%Cl: 34.6-3250.8;P < 0.001)in a four-tiered grading system were significantly associated with CSS in a univariate setting.The associations of grade in the four-tiered system with CSS and DMFS were statistically significant after being adjusted for each characteristic.However,the difference in CSS between Grades 2 and 3 was not significant(HR = 2.14,95% CI 0.43–10.63;P = 0.35).The HRs of the associations between an exploratory grading system that includes a separate Grade 3 for tumor necrosis and CSS were as follows: Grade 2,10.2(Cl 1.06-97.9,P=0.045);Grade 3,11.4(Cl 1.18-109.6,P=0.04);Grade 4,267.9(Cl 27.6-2603.3,P< 0.001).Similarly,Grades 2 and 3 did not differ significantly.Conclusion: The four-tiered grading system studied is useful for predicting death from Ch RCC and metastasis.However,Grade 3 did not more accurately predict risk of death and metastasis than did Grade 2.This was also true for the other exploratory grading system that we investigated.