Development and validation of the prognostic value of the immune-related genes in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is a highly heterogeneous tumor, resulting a challenge of developing target therapeutics. Not long ago, immune checkpoint blockade regimens combine with tyrosin kinase inhibitors have evolved frontline options in metastatic RCC, which implies arrival of the era of tumor immunotherapy. Studies have demonstrated immune-related genes (IRGs) could characterize tumor milieu and related to patient survival. Nevertheless, the clinical significance of classifier depending on IRGs in ccRCC has not been well established.MethodsThe R package limma, univariate and LASSO cox regression analysis were used to screen the prognostic related IRGs from TCGA database. Multivariate cox regression was utilized to establish a risk prediction model for candidate genes. Quantitative real-time PCR was used to confirm the expression of candidates in clinical samples from our institution. CIBERSORT algorithm and correlation analysis were applied to explore tumor-infiltrating immune cells signature between different risk groups. A clinical nomogram was also developed to predict OS by using the rms R package based on the risk prediction model and other independent risk factors. The ICGC data was used for external validation of either gene risk model or nomogram.ResultsWe identified 382 differentially expressed immune related genes. Four unique prognostic IRGs (CRABP2, LTB4R, PTGER1 and TEK) were finally affirmed to associate with tumor survival independently and utilized to establish the risk score model. All candidates’ expression was successfully laboratory confirmed by q-PCR. CIBERSORT analysis implied patients in unfavorable-risk group with high CD8 T cell, regulatory T cell and NK cell infiltration, as well as high expression of PD-1, CTLA4, TNFRSF9, TIGIT and LAG3. A nomogram combined IRGs risk score with age, gender, TNM stage, Fuhrman grade, necrosis was further generated to predict of 3- and 5-year OS, which exhibited superior discriminative power (AUCs were 0.811 and 0.795).ConclusionsOur study established and validated a survival prognostic model system based on 4 unique immune related genes in ccRCC, which expands knowledge in tumor immune status and provide a potent prediction tool in future.     

Increased phosphorylated CREB1 protein correlates with poor prognosis in clear cell renal cell carcinoma

BackgroundThis study aims to investigate the level of cAMP response element-binding protein 1 (phospho S133) (p-CREB1) protein in clear cell renal cell carcinoma (ccRCC) and evaluates its prognosis significance.MethodsImmunohistochemistry (IHC) method was performed to detect p-CREB1 staining in 233 ccRCC patients. Three or more high-power fields per tissue section were equally captured by a Leica DMRXA microphotographic system, and average staining intensity (optical density, OD) was analyzed by Leica Qwin Standard V2.6 system. Univariate and multivariate Cox proportional regression model was performed to assess the correlation of p-CREB1 staining and clinical outcomes.ResultsIHC proved that the level of p-CREB1 protein was significantly higher in tumor tissues than in adjacent normal tissues, and gradually increased from normal to tumor sections. On the basis of the receiver operating characteristic curve, patients were divided into low p-CREB1 staining (OD ≤0.28) and high p-CREB1 staining subgroup (OD >0.28) according to p-CREB1 protein staining intensity of tumor cells. Multivariate analyses showed that high p-CREB1staining was an independent risk factor for cancer-specific free survival, overall survival and progression-free survival.Conclusionsp-CREB1 protein is an independent prognostic biomarker for ccRCC patients.     

Clinicopathological and prognostic impact of somatic mutations in Chinese patients with clear cell renal cell carcinoma

BackgroundThe study of the genomic landscape of Chinese clear cell renal cell carcinoma (ccRCC) entered its nascence in recent years, and the clinical relevance of individual genes in Chinese ccRCC has not yet been researched. The study aimed to explore the relationships between somatic mutations and clinical behaviors in Chinese ccRCC.MethodsTumor tissue samples were obtained from 105 Chinese patients with ccRCC and deep sequencing targeting 556 cancer genes was performed. Correlation analysis, receiver operator characteristic (ROC) analysis and survival analysis were carried out using SPSS software.ResultsA total of 41 genes were used to investigate the relationship between genes and clinical behaviors. We found that different clinical indices were mutually correlated, and there were 12 genes associated with clinical indices. The Kaplan-Meier curves showed that high Fuhrman grade and metastatic disease at diagnosis were significantly associated with poor prognosis. Mutations in BAP1, PTEN, ERBB2, TP53, CDK8, TSC1, SETD2, or SPEN were significantly associated with poor prognosis, consistent with the results of The Cancer Genome Atlas (TCGA) cohort. Mutation of BTG1 occurred much more frequently in Chinese ccRCC (10.5%) than in the TCGA cohort (0.60%), and it was associated with a better prognosis.ConclusionsA total of 8 genes (BAP1, PTEN, ERBB2, TP53, CDK8, TSC1, SETD2, and SPEN) were found to be associated with poor prognosis of ccRCC, and a new gene (BTG1) was possibly associated with the good prognosis of Chinese ccRCC.     

Identification of a novel immune-related microRNA prognostic model in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is a type of kidney cancer, and one of the most common malignant tumors. Many studies have shown that certain microRNAs (miRNAs) play an important role in the occurrence and development of ccRCC. Nevertheless, the prognosis of ccRCC patients is very rarely based on these “immuno-miRs”. Our aim was thus to determine the relationship between immune-related miRNA signatures and ccRCC.MethodsWe downloaded the miRNA expression data from 521 KIRC and 71 normal tissues in The Cancer Genome Atlas (TCGA). We used “limma” package and univariate Cox regression analysis to identify differentially expressed miRNAs (DEMs) that related to overall survival (OS). We applied lasso and multivariate Cox regression analyses to construct a prognostic model based on immuno-miRs. We evaluated the performance of model by using the Kaplan-Meier method. Furthermore, Cox regression analysis was used to determine independent prognostic signatures in ccRCC.ResultsA total of 59 significant immuno-miRs were identified. We use univariate Cox regression analysis to acquire 18 immune-related miRNAs which were markedly related to OS of ccRCC patients in the training set. We then constructed the 9-immune-related-miRNA prognostic model (miR-21, miR-342, miR-149, miR-130b, miR-223, miR-365a, miR-9-1, and miR-146b) by using lasso and multivariate Cox regression. Further analysis suggested that the immune-related prognostic model could be an independent prognostic indicator for patients with ccRCC. The prognostic performance of the 9-immune-related-miRNA prognostic model was further validated successfully in the testing set.ConclusionsWe established a novel immune-based prognostic model of ccRCC based on potential prognostic immune-related miRNAs. Our results indicated that the 9-miRNA signature could be a practical and reliable prognostic tool for ccRCC.     

Assessment of the prognostic value of SPOCK1 in clear cell renal cell carcinoma: a bioinformatics analysis

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most common urological malignancies, and once metastasis occurs, it often has a poor prognosis and lacks effective treatment. Therefore, there is an urgent need to screen some new biomarkers and explore their molecular mechanisms to improve the early clinical diagnosis and targeted therapy of ccRCC. SPOCK1 (SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1) is a conserved multi-domain proteoglycan that plays an important role in the development of multiple cancer types; however, its prognostic value in ccRCC has not been investigated. The study of the prognostic value of SPOCK1 in ccRCC is a good complement to the study of ccRCC biomarkers.MethodsDatabases of this study included Oncomine, Kaplan-Meier Plotter, GEPIA, GeneMANIA, cBioPortal, and TIMER. Student’s t-test was used to analyze the differences in SPOCK1 expression in ccRCC tissues compared with tumor-adjacent normal tissues. Kaplan-Meier curves for survival analysis were used to assess the correlation between the expression of SPOCK1 and the prognostic outcomes. Correlation module drew the expression scatterplots between SPOCK1 and immune cell infiltration in ccRCC, together with the Spearman’s rho value and estimated statistical significance.ResultsThe SPOCK1 mRNA expression was significantly higher in ccRCC tissues (mean expression ± SD: 920.2±195.2) than in normal tissues (mean expression ± SD: 358.4±29.1, P=0.008), and high SPOCK1 expression significantly and positively correlated with the pathological stage of ccRCC patients (F value =10.2, P<0.001). Higher expression of SPOCK1 was also associated with significantly shorter overall survival (OS) and disease-free survival (DFS) in ccRCC patients (GEPIA: P=0.046, P<0.001, respectively; Kaplan-Meier Plotter: P=0.002, P=0.0022, respectively). The function of SPOCK1 is mainly related to tumor development and extracellular matrix remodeling, and it may participate in the epithelial-mesenchymal transition process. SPOCK1 expression significantly and positively correlated with infiltration of several immune cells in ccRCC, including cancer-associated fibroblasts (CAFs) (Rho =0.333, P=2.16×10⁻¹³), tumor-associated macrophages (TAMs) (Rho =0.18, P=1.02×10⁻⁴), and tumor-associated neutrophils (TANs) (Rho =0.165, P=3.83×10⁻⁴). Conversely, there was a significant and negative correlation between SPOCK1 expression and infiltration of CD4⁺ T cells (Rho =−0.113, P=0.015).ConclusionsSPOCK1 may be a potential prognostic biomarker in ccRCC.     

NDUFA4L2 expression predicts poor prognosis in clear cell renal cell carcinoma patients

Background: NDUFA4L2 is overexpressed in VHL-deficient cell lines and neuroblastoma. The clinical significance of NDUFA4L2 in clear cell renal cell carcinoma (ccRCC) has not been well studied. Therefore, we evaluated the prognostic value of NDUFA4L2 in ccRCC patients.

Methods: In our study, NDUFA4L2 expression in 86 cases of ccRCC and adjacent normal tissues was monitored by immunohistochemistry, semi-quantitative RT-PCR, and Western blot analyses. The relationship between NDUFA4L2 expression and the clinical features of ccRCC was assessed.

Results: The results showed that NDUFA4L2 protein expression was found to be higher in ccRCC tissues 81.4% (70/86) than in normal tissues 26.7% (23/86) (p?=?0.021). The average level of NDUFA4L2 mRNA expression was found to be 122.23?±?6.018 and 21.34?±?1.036 in ccRCC tissue and adjacent normal tissue (p?Conclusions: Our study has provided the significant clinical relevance of NDUFA4L2 in ccRCC and suggested that ccRCC patients with NDUFA4L2 overexpression may be suitable as a potential therapeutic target for ccRCC patients.     

血管生成相关基因多态性与肾透明细胞癌的相关性研究

目的 探讨血管生成相关基因多态性与肾透明细胞癌发病风险的关系. 方法 选取2005年6月至2012年9月收治的859例经病理检查证实的肾透明细胞癌患者及1 004例健康对照人群,采集研究对象外周血,在7个与血管生成有关的基因中(HIF1A,EPAS1,VEGFA,VEGFR1,VEGFR2,VEGFR3和PDGFRB)筛选出24个候选单核苷酸多态性位点,采用单碱基微测序技术进行基因分型.应用Logistic回归模型调整混杂因素后,分析各基因型与肾透明细胞癌发生的关系.结果 24个SNP位点中19个对照组基因型均符合遗传平衡定律.VEGFA基因rs2010963位点,VEGFR1基因rs3812867位点,VEGFR3基因rs1382302、rs448012、rs 1049095位点等5个位点的多态性与肾透明细胞癌发生显著相关(P＜0.05).多重检验校正后,仅有VEGFA基因rs2010963位点和VEGFR3基因的rs448012位点与肾透明细胞癌发生存在相关性(P＜0.05).VEGFA基因rs2010963位点携带CC/GC基因型的患者较携带GG基因型患者的肾透明细胞癌发病率显著升高(FDR=0.048,OR=1.38,95％ CI:1.13 ～ 1.69),VEGFR3基因rs448012位点携带CC/GC基因型的患者肾透明细胞癌的发病率显著高于携带GG基因型的患者(FDR=0.045,OR=1.36,95％CI:1.12～1.66). 结论 VEGFA基因的rs2010963位点与VEGFR3基因的rs448012位点多态性与肾透明细胞癌发病相关.     

Validation of the World Health Organization/International Society of Urological Pathology grading for Chinese patients with clear cell renal cell carcinoma

BackgroundThis study aimed to compare the World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading system and the Fuhrman grading system and to verify the WHO/ISUP grade as a prognostic parameter of clear cell renal cell carcinoma (ccRCC) in a Chinese population.MethodsThe study consisted of 753 ccRCC patients treated with curative surgery between 2010 and 2018 at Xiangya Hospital Central South University (Changsha, China). All pathologic data were retrospectively reviewed by two pathologists. Cancer-specific survival (CSS) and recurrence-free survival (RFS) were examined as clinical outcomes.ResultsAccording to the WHO/ISUP grading system (ISUP group), nephrectomy type, pT stage and WHO/ISUP grade were independent risk factors for CSS (P<0.0001, P=0.0127 and P<0.0001, respectively) and RFS (P<0.0001, P=0.0077, and P<0.0001, respectively). In the Fuhrman group, nephrectomy type, pT stage and Fuhrman grade were independent risk factors for CSS (P<0.0001, P=0.0004, and P<0.0001, respectively) and RFS (P<0.0001, P=0.0001, and P<0.0001, respectively). The C-index for CSS and RFS using the Fuhrman grading system was 0.6323 and 0.6342, respectively, and that using the WHO/ISUP grading system was 0.6983 and 0.7005, respectively, both higher than the former (P=0.0185, and P=0.0172, respectively). In addition, upgrading from Fuhrman grade 2 to ISUP grade 3 resulted in worse CSS and RFS for ccRCC patients (P=0.0033 and P =0.0003, respectively).ConclusionsWe first verified correlations between the postoperative prognosis and WHO/ISUP grade of ccRCC in a Chinese population and confirmed that the ability to predict clinical outcomes with the WHO/ISUP grading system was superior to that with the Fuhrman grading system.     

Natural history and growth kinetics of clear cell renal cell carcinoma in sporadic and von Hippel-Lindau disease

BackgroundTo evaluate and compare the natural history and growth kinetics of sporadic clear cell renal cell carcinoma (ccRCC) with those of ccRCC in von Hippel-Lindau disease (VHL).MethodsSixty patients in the sporadic group with 61 tumors and 15 patients in the VHL group with 30 tumors whom all underwent delayed surgery after at least 12 months of active surveillance (AS) were enrolled to conduct a retrospective cohort study. The growth rate was calculated, and the growth kinetics between the sporadic and VHL groups were compared. The patient and tumor characteristics were reviewed, and their correlation with growth rate was analyzed.ResultsThe mean growth rate of sporadic ccRCC was 0.91 cm/year (ranging from 0–4.74 cm/year) and that of VHL ccRCC was 0.47 cm/year (ranging from 0.04–1.89 cm/year). The growth rate of sporadic ccRCC showed a tendency of being faster than that of VHL ccRCC but did not reach statistical significance (P=0.07). The factors affecting the growth rate were different between the two groups. For VHL ccRCC, the only factor that correlated with growth rate was initial tumor diameter (P<0.001), but for sporadic ccRCC, the only factor was pathological nuclear grade (P<0.001).ConclusionsThe growth rate of VHL-associated ccRCC might be slower than that of sporadic ccRCC. Furthermore, we identified a disparity in growth kinetics between sporadic and VHL-associated ccRCC.     

Prognostic value of peripheral blood T lymphocyte subsets in clear cell renal cell carcinoma

BackgroundTo date, few studies have evaluated the role of peripheral blood T lymphocyte subsets in patients with clear cell renal cell carcinoma (ccRCC). Here we measured the levels of peripheral blood T lymphocyte subsets and evaluated its prognostic value in ccRCC.MethodsData from 122 patients with RCC from January 2018 to January 2020 were collected. Preoperative peripheral blood T lymphocyte subsets and medical records were analyzed. Kaplan-Meier cures and log rank test were used for analyzing overall survival (OS). Univariate and multivariate survival analyses were underwent by performing the Cox proportional hazards models. Correlations were tested by Pearson’s correlation analysis.ResultsOf 122 patients, a total of 80 ccRCC patients was enrolled. Patients with low CD3⁺ T cells and low CD4⁺/CD8⁺ ratio displayed a worse OS than patients with high CD3⁺ T cells and high CD4⁺/CD8⁺ ratio (P=0.029 and 0.002, respectively). Multivariate analyses showed CD3⁺ T cells and CD4⁺/CD8⁺ ratio were independent predictive factors for the OS (HR: 0.295, 95% CI, 0.091–0.956; P=0.042 and HR: 0.244, 95% CI, 0.065–0.920; P=0.037, respectively). Moreover, NLR negatively correlated with both levels of CD3⁺ T cells and CD4⁺/CD8⁺ ratio (P<0.001, r=−0.398 and P=0.012, r=−0.280, respectively).ConclusionsThe findings of our study suggest that preoperative CD3⁺ T cells and CD4⁺/CD8⁺ ratio in peripheral blood are independent predictors for patients with ccRCC.     

Galectin-8 predicts postoperative recurrence of patients with localized T1 clear cell renal cell carcinoma

BackgroundGalectin-8 (Gal-8), belonging to a family of the “tandem repeat”–type galectins that contain 2 carbohydrate recognition domains, serves to retain cell surface residency and signaling of glycoproteins including cytokine and growth factor receptors, and thereby promoting development and progression of various malignancies. This study aims to evaluate the effect of Gal-8 expression on postoperative recurrence of patients with localized pathologic T1 (pT1) clear cell renal cell carcinoma (ccRCC).Patients and methodsIn this retrospective study, we enrolled 244 patients (122 in group A and 122 in group B) with localized pT1 ccRCC undergoing nephrectomy at a single institution. Specimens from patients were collected from January 2003 to December 2008. Median follow-up was 71 months (range: 12–120 mo) in group A and 70 months (range: 12–119 mo) in group B. Overall, 14 patients experienced recurrence in group A (n = 122) and 22 patients had recurrence in group B (n = 122). Gal-8 expression was assessed by immunohistochemistry in clinical specimens. Kaplan-Meier method with log-rank test was performed to compare survival curves. Cox regression models were used to evaluate the prognostic values of variables on recurrence-free survival. Concordance index was calculated to assess prognostic accuracy.ResultsIn both groups, patients with high expression of Gal-8 were significantly inclined to have high rates of necrosis. High Gal-8 expression indicated early recurrence of patients with localized pT1 ccRCC. Gal-8 expression was determined to be an independent adverse prognostic indicator for recurrence. The accuracy of The Mayo Clinic Stage, Size, Grade, and Necrosis score and University of Los Angeles Integrated Staging System prognostic models was improved when Gal-8 expression was added.ConclusionsGal-8 expression is a potential independent unfavorable prognostic indicator for postoperative recurrence of patients with localized pT1 ccRCC.     

长链非编码 RNA MIAT 在肾透明细胞癌中的表达情况和预后意义

目的 探讨长链非编码RNAMIAT在肾透明细胞癌中的表达情况及与患者临床指标的相关性,分析其作为肾透明细胞癌分子标记物的可能性。方法 通过荧光定量PCR方法 检测MIAT在40例肾透明细胞癌组织和40例癌旁正常组织中的表达情况,同时结合TCGA数据库分析MIAT表达水平与肾透明细胞癌患者临床指标和预后的关系。结果 MIAT在肾透明细胞癌组织中的表达明显高于癌旁正常组织,在肾癌细胞系中的表达明显高于正常肾小管上皮细胞,差异均具有统计学意义（P<0.05）。TCGA数据库资料分析表明,MIAT表达水平与肾癌患者T分期（P<0.001）、M分期呈正相关（P<0.05）。Kaplan-Meier生存分析表明,高表达MIAT的肾癌患者总体生存时间明显低于低表达MIAT的肾癌患者（Log-rankP<0.05）。结论 MIAT在肾透明细胞癌组织和肾癌细胞系中高表达,有可能成为肾透明细胞癌的分子标记物。     

基于TCGA数据库分析BIRC5在肾透明细胞癌中的表达及预后

目的通过分析杆状病毒凋亡抑制蛋白5(BIRC5)在肾透明细胞癌(ccRCC)组织中的表达,阐明BIRC5对其早期诊断及作为预后预测因子的作用。方法利用GEO、TCGA数据库和HPA分析BIRC5在ccRCC组织中mRNA和蛋白质水平的变化。运用UALCAN和LinkedOmics数据库阐述BIRC5表达与ccRCC临床病理学参数的相关性及对预后的影响。采用GEPIA、Kaplan-Meier Plotter、SurvExpress分析BIRC5表达与ccRCC预后的关系。结果BIRC5 mRNA在ccRCC中高表达,并且与ccRCC患者TNM分期相关(P<0.05),与ccRCC进展高度相关,高表达BIRC5mRNA是ccRCC患者不良预后指标。免疫组织化学结果证实,与正常肾组织相比,ccRCC中BIRC5蛋白表达量显著升高。结论在ccRCC中,BIRC5高表达是一种重要的早期诊断及不良预后指标,有望成为ccRCC早期诊断和预后预测标志物。     

同侧肾脏同时发生透明细胞型与乳头状肾细胞癌

目的探讨同侧肾脏发病并且相对独立的透明细胞型和肾乳头状细胞癌的临床病理特点及免疫表型,提高对该肿瘤的认识和诊断水平。方法本研究回顾了2例病理诊断为透明细胞型合并肾乳头状细胞癌的临床资料,通过光镜和免疫组织化学染色,针对肾细胞癌相关蛋白标志物[包括 Vimentin、CD10、CK（AE1/AE3）、CK7、CK8/18、PAX2、PAX8、CAⅨ、AMACR]进行了观察和分析。结果2例患者为男性,年龄分别为70、63岁。2例患者的两处独立肿瘤均位于左侧肾脏,镜下观察均可见两处独立肿瘤,肿瘤间隔有正常肾脏组织,分别为乳头状肾细胞癌及透明细胞型肾细胞癌,且免疫组化显示2例患者肿瘤的表型一致。结论单侧肾脏肾透明细胞癌合并肾乳头状细胞癌是一种少见的临床现象,这种现象的存在以及类似的免疫组化表型提示透明细胞型肾细胞癌和乳头状肾细胞癌在发生过程中可能存在着内部的联系。