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.     

Identification of genes that correlate clear cell renal cell carcinoma and obesity and exhibit potential prognostic value

BackgroundRenal cell carcinoma (RCC) is a common urologic malignancy. Although the relationship between clear cell RCC (ccRCC) and obesity has been well-established by several large-scale retrospective studies, the molecular mechanisms and genetic characteristics behind this correlation remains unclear. In the current study, several bioinformatics tools were used to identify the key genes in ccRCC related to obesity.MethodsMicroarray data comparing ccRCC with normal renal tissues in patients with and without obesity were downloaded from the GEO database for screening of differentially expressed genes (DEGs). The DEGs were verified with expression level and survival analysis using several online bioinformatics tools.ResultsIn the current study, the differential expression of five genes correlated with both ccRCC and obesity; IGHA1 and IGKC as oncogenes, and MAOA, MUC20 and TRPM3 as tumor suppressor genes. These genes were verified by comparing the relationship between the expression levels and survival outcomes from open-source data in The Cancer Genome Atlas (TCGA) dataset.ConclusionsIn conclusion, the five genes differentially expressed in ccRCC and obesity are related to disease progression and prognosis, and therefore could provide prognostic value for patients with 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.     

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.     

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.     

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.     

5t4在肾透明细胞癌中的表达及意义

目的探讨滋养层糖蛋白5t4在肾透明细胞癌中的表达及其意义。方法采用免疫组织化学技术检测72例肾透明细胞癌组织标本、17例癌旁肾组织以及14例非癌因素的肾脏组织标本中5t4的表达,并对5t4在肾透明细胞癌的表达与组织学分级的关系进行分析。结果 5t4在肾细胞癌组织中的阳性率为70.8%,癌旁肾组织中的阳性率为41.2%,两者具有差异性(P0.05)。5t4在肾癌组织学分级中低分级与高分级的表达存在显著差异(P0.01)。结论 5t4可作为判断肾透明细胞癌分化程度的标志物。     

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.     

C3, C3AR1, HLA-DRA,and HLA-E as potential prognostic biomarkers for renal clear cell carcinoma

BackgroundPrognostic biomarkers play a vital role in the early detection of the cancer and assessment of prognosis. With advances in technology, a large number of biomarkers of kidney renal clear cell carcinoma (KIRC) have been discovered, but their prognostic value has not been fully investigated, and thus have not been widely used in clinical practice. We aimed to identify the reliable markers associated with the prognosis of KIRC patients.MethodsWe obtained 72 normal samples and 539 tumor samples from The Cancer Genome Atlas (TCGA), and 23 normal samples and 32 tumor samples from the Gene Expression Omnibus (GEO). Overlapping differentially expressed genes (ODEGs) were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, followed by construction of a protein-protein interaction (PPI) network to screen hub genes. Kaplan-Meier analysis, univariate Cox analysis, multivariate Cox analysis, Wilcoxon signed-rank test, Kruskal-Wallis test, and gene set enrichment analysis (GSEA) were performed to verify the prognostic value and function of the markers we selected. The relationships among gene expression level, tumor immune cell infiltration, and immune-checkpoints were also analyzed.ResultsA total of 910 genes were screened out, and C3, C3AR1, HLA-DRA, and HLA-E were identified as potential tumor markers. The expression of each gene was closely associated with tumor immune cell infiltration, survival rate, and the patients’ clinical characteristics (P<0.05). C3AR1, HLA-DRA, and HLA-E were also verified as independent prognostic factors of KIRC (P<0.05), and all these potential biomarkers had a close correlation with immune checkpoints.ConclusionsC3, C3AR1, HLA-DRA, and HLA-E could be reliable biomarkers of KIRC and may have a significant contribution to make in immunotherapy, thus playing an important role in the improvement of prognosis.     

小窝蛋白-1与血小板反应蛋白在肾透明细胞癌中的表达及其意义

目的研究小窝蛋白-1（CAV-1）和血小板反应蛋白（TSP）在肾透明细胞癌、癌旁组织、正常组织中的表达并探讨二者关系。方法应用免疫组织化学SP法对38例肾透明细胞癌、癌旁、正常组织中的CAV-1和TSP进行检测。结果CAV-1在肾透明细胞癌组织中表达明显高于其在癌旁、正常组织肾小管和肾小球中表达,三者呈递减关系,其差异有统计学意义（P〈0．001）;TSP在肾透明细胞癌中呈低表达,在正常组织肾小管中则呈高表达。CAV-1在肾透明细胞癌组织中的高表达,与肾透明细胞癌的组织学分级、临床分期密切相关,与TSP的低表达恰好相反,但两者之间的相关性无统计学意义（P〉0．05）,可能是两个独立的指标。结论CAV-1可能是促进肾透明细胞癌侵袭、生长及导致不良预后的一个潜在的因子。     

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。     

E2F1在肾透明细胞癌中的表达及意义

目的：探讨E2F1在肾透明细胞癌中的表达及意义。方法：通过实时定量PCR和Western blot方法检测E2F1在肾透明细胞癌及对应瘤旁组织中mRNA和蛋白的表达情况,并分析E2F1的mRNA水平与临床病理资料的关系及表达相关性。结果：与对应瘤旁组织相比,E2FJ的mRNA水平在肾透明细胞癌手术标本中表达明显升高（P=0．0002）,相应的蛋白水平对比与mRNA变化一致;E2F1的mRNA水平在不同年龄、性别组间差异无统计学意义（P〉0．05）,而在组织学分级、肿瘤直径大小、T分期、临床分期和大血管浸润与否差异有统计学意义（P〈0．05）。结论：E2F1表达上调可能在肾透明细胞癌的肿瘤形成中发挥作用,且E2F1表达升高可能促进肾透明细胞癌的恶性进展。     

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。     

MMP-2和CXCR4mRNA在人肾透明细胞癌中的表达及意义

目的研究人肾透明细胞癌(ccRCC)组织中基质金属蛋白酶-2(MMP-2)和CXC趋化因子受体4(CXCR4)的表达情况及相关性,探讨二者与临床病理因素的关系。方法应用反转录-聚合酶链式反应(RT-PCR)方法检测68例ccRCC组织和36例癌旁正常肾组织中MMP-2与CXCR4mRNA的表达水平。结果在ccRCC组织中MMP-2和CXCR4mRNA的半定量值分别为0.848±0.367和0.939±0.336,高于在正常肾组织中的半定量值0.379±0.138和0.418±0.146,差异有统计学意义(t=7.375、8.860,P0.05);在ccRCC中MMP-2和CXCR4mRNA的表达与肿瘤的临床分期、分化程度和淋巴结转移情况有关(t=2.169~3.852,P0.05),而与患者的年龄、性别及肿瘤大小无关(t=0.201~0.630,P0.05);在ccRCC组织中MMP-2和CXCR4mRNA的表达呈正相关(r=0.832,P0.05)。结论 MMP-2和CXCR4可能共同参与了人ccRCC的发生、发展、侵袭和转移,并且在发挥作用时具有协同作用,联合检测MMP-2和CXCR4可能有助于协助ccRCC的早期诊断和病情评估。     

Comprehensive analysis of glutathione peroxidase-1 (GPX1) expression and prognostic value in three different types of renal cell carcinoma

BackgroundGlutathione peroxidase-1 (GPX1) is generally expressed in tissues with high oxygen tension such as the kidneys and lungs, and its main function is to degrade reactive oxygen species (ROS) and protect cells from oxidative stress. Studies have shown that GPX1 is upregulated in many tumor tissues and is closely related to tumor progression and metastasis. This study aimed to explore the possibility of GPX1 as a biomarker for kidney chromophobe cell carcinoma (KICH), kidney renal papillary cell carcinoma (KIRP), and kidney renal clear cell carcinoma (KIRC).MethodsThe Oncomine and GEPIA databases were used to analyze the GPX1 expression differences between tumor and normal tissues, and the UALCAN, GEPIA and DriverDBv3 databases were used to perform the survival analyses. The GeneMANIA interactive tool was then used to find the GPX1-related protein-protein interaction (PPI). Following this, the LinkedOmics database was used for the enrichment analysis of GPX1, and the Timer database was used to estimate the abundance of immune infiltration. Finally, quantitative polymerase chain reaction (qPCR) was performed on patient specimens collected in the clinic to confirm the database findings.ResultsIn our study, we found that the expression of GPX1 in three types of renal cell carcinoma (RCC) were upregulated, and the high expression of GXP1 was related to the poor prognosis of patients with KICH and KIRC. On the contrary, KIRP patients with a high expression of GPX1 had a better prognosis. In addition, GPX1 was related to the abundance of immune cell infiltration. The results of the qPCR analysis confirmed that the expression of GPX1 in RCC was increased compared with the control group (P<0.05).ConclusionsOur results indicate that the expression of GPX1 is related to the prognosis of three types of RCC. As such, GPX1 expression could be a reliable diagnostic and prognostic biomarker for RCC and, more importantly, may provide a new direction for therapeutic strategies.