Identification of tumor antigens in renal cell carcinoma by serological proteome analysis

We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.     

Expression of CIDE proteins in clear cell renal cell carcinoma and their prognostic significance

Clear cell renal cell carcinoma (ccRCC) is the major and aggressive subtype of renal cell carcinoma. It is known to derive its histologic appearance from accumulation of abundant lipids and glycogens. The cell death-inducing DFF45-like effector (CIDE) family has been characterized as the lipid droplet proteins involved in the metabolism of lipid storage droplets. The purpose of this study was to evaluate the expression of CIDE proteins in ccRCC cells and to investigate their prognostic significance. We examined consecutive patients with sporadic ccRCC, who underwent nephrectomy, to measure their mRNA and protein expression of CIDE proteins. We found that Cidec and ADRP expression were significantly up-regulated in ccRCC, compared with normal kidney tissues. Cideb was down-regulated. We also found that Cideb was expressed more in low-grade ccRCC than in high-grade tumors. To further clarify the relationship between Cideb expression and patient prognosis, we evaluated 57 ccRCC patients followed up for 120 months. Reduced ccRCC Cideb expression was associated with a higher Fuhrman nuclear grade. Patients with high Cideb expression had better overall survival rate than those with low expression (p < 0.05). Cideb expression was an independent predictor of survival (p = 0.001). Although the biologic function of Cideb in ccRCC remains unknown, the expression level of Cideb might be a novel predictor of prognosis in ccRCC.     

Serological and proteomic evaluation of antibody responses in the identification of tumor antigens in renal cell carcinoma

Renal cell carcinoma (RCC) is relatively resistant to conventional chemotherapy and radiotherapy. However, reports of spontaneous regression along with promising results in clinical trials suggest that immunotherapuetic strategies may be of clinical benefit. Few RCC related antigens have been identified to date, and the technical difficulty and time constraints of current antigen identification techniques preclude the screening of large numbers of patients. A comparatively rapid strategy has been used to identify components of tumors that elicit an antibody response in the patient - the serological and proteomic evaluation of antibody responses (SPEAR) approach. This combines two-dimensional polyarylamide gel electrophoresis of tumor and normal kidney samples with immunoblotting using autologous patient sera and protein identification by mass spectrometry. Using the SPEAR approach to screen RCC patients for naturally occurring antitumor antibody responses, a number of candidate immunogens have been identified in patients with high-grade disease and their relative expression levels in tumor tissue compared to normal tissue have been studied. These proteins include annexins I and IV, thymidine phosphorylase (TP), carbonic anhydrase I, Mn-superoxide dismutase and major vault protein (MVP). Downstream analysis of the tissue expression of some of these proteins shows that MVP is up-regulated in 2/4 of RCC tumors but is also expressed in normal kidney whereas TP is up-regulated in 100% (11/11) of RCC cases examined with no or minimal expression in normal kidney, indicating a potential use as a therapeutic target.     

嫌色性肾细胞癌与嗜酸细胞瘤的临床病理分析与比较

目的探讨嫌色性肾细胞癌(chromophobe renal cell carcinoma,CRCC)和嗜酸细胞瘤(oncocytoma)的临床病理学特征,提高对二者的认识和诊疗水平。方法对手术切除的5例CRCC和2例Oncocytoma进行肉眼和光镜观察、免疫组织化学染色,分析其临床病理学差异。结果 CRCC以实体结构为主、胞质半透明或嗜酸性颗粒状、核皱缩且核周有空晕;Oncocytoma以巢状结构为主,胞质嗜酸颗粒状、核圆形、核仁小。免疫组化染色显示两者均呈E-cadherin、EMA、CK20、vimentin阳性;CRCC瘤细胞CD10和CK7强阳性而S-100蛋白阴性,5名患者随访8~32个月,其中1例手术12个月后由于转移死亡;Oncocytoma瘤细胞S-100蛋白强阳性、CD10和CK7阴性,2名患者随访41个月,均未出现复发和转移。结论 CRCC和oncocytoma二者形态学各有特征,免疫表型相似,二者鉴别诊断主要在于生长方式和细胞学特征。综上所述:CRCC为惰性肿瘤,Oncocytoma为良性肿瘤,预后较好,手术切除后极少发生复发和转移。     

Heterogeneity of tumor microenvironment is associated with clinical prognosis of non-clear cell renal cell carcinoma: a single-cell genomics study

Non-clear renal cell carcinomas (nccRCCs) are less frequent in kidney cancer with histopathological heterogeneity. A better understanding of the tumor biology of nccRCC can provide more effective treatment paradigms for different subtypes. To reveal the heterogeneity of tumor microenvironment (TME) in nccRCC, we performed 10x sing-cell genomics on tumor and normal tissues from patients with papillary renal cell carcinoma (pRCC), chromophobe RCC (chrRCC), collecting duct carcinoma (CDRCC) and sarcomatoid RCC (sarRCC). 15 tissue samples were finally included. 34561 cells were identified as 16 major cell clusters with 34 cell subtypes. Our study presented the sing-cell landscape for four types of nccRCC, and demonstrated that CD8+ T cells exhaustion, tumor-associated macrophages (TAMs) and sarcomatoid process were the pivotal factors in immunosuppression of nccRCC tissues and were closely correlated with poor prognosis. Abnormal metabolic patterns were present in both cancer cells and tumor-infiltrating stromal cells, such as fibroblasts and endothelial cells. Combined with CIBERSORTx tool, the expression data of bulk RNA-seq from TCGA were labeled with cell types of our sing-cell data. Calculation of the relative abundance of cell types revealed that greater proportion of exhausted CD8+ T cells, TAMs and sarRCC derived cells were correlated with poor prognosis in the cohort of 274 nccRCC patients. To the best of our knowledge, this is the first study that provides a more comprehensive sight about the heterogeneity and tumor biology of nccRCC, which may potentially facilitate the development of more effective therapies for nccRCC.Subject terms: Cancer genomics, Cancer microenvironment, Renal cell carcinoma     

The clinical significance of UBE2C gene in progression of renal cell carcinoma

Renal cell carcinoma (RCC), with high morbidity and mortality, is one of the top ten serious cancers. Due to limited therapies and little knowledge about the mechanism underlying RCC, overall survival of RCC patients is poor. UBE2C is a member of ubiquitin modification system and promotes carcinogenesis in cancer, but its role in RCC is unknown. Based on the TCGA (The Cancer Genome Atlas) data, UBE2C was over-expressed in a total of 525 RCC tissues and displayed higher expression in advanced tissues (stage IV vs stage I, p<0.05). RT-qPCR and IHC analysis confirmed over-expression of UBE2C in 90 of clinical RCC tissues. Further, UBE2C was associated with clinical factors including TNM stage, gender, and pathological stage. And higher UBE2C expression predicted shorter overall survival and progression-free survival. Both univariate and multivariate COX analysis suggested UBE2C as a critical gene in RCC. Then GO and KEGG analysis showed that cell cycle and DNA replication pathways were two top signaling pathways affected by UBE2C. In vitro assay showed that knockdown of UBE2C in 786-O cells inhibited proliferation and migration significantly. Therefore, this study proves that UBE2C is an important gene in RCC and is essential to proliferation and migration of RCC.Key words: UBE2C, GO analysis, KEGG analysis, renal cell carcinoma     

Prognostic significance of metallothionein expression in renal cell carcinoma

BACKGROUND: Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival. PATIENTS AND METHODS: The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data (histological cell type, tumor nuclear grade, pathologic stage and patients' survival). RESULTS: Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25-50% of tumor cells in 7 cases, and in a percentage of 50-75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival. CONCLUSIONS: The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter.     

Morphometric analysis of the tumor associated tissue eosinophilia in the oral squamous cell carcinoma using different staining techniques

In a previous study, we found tumor-associated tissue eosinophilia (TATE) to be a favourable prognostic indicator for oral squamous cell carcinomas. Special techniques such as autofluorescence or immunohistochemistry are reported to be sometimes necessary to detect the presence of intact and degranulating eosinophils within the tumors. The aim of this study was to compare the number of eosinophils identified routinely with hematoxylin and eosin stain and by immunohistochemistry in oral squamous cell carcinomas with TATE. Thirty specimens of oral squamous cell carcinoma of the tongue, floor of the mouth, retromolar area and inferior gingiva with TNM stages II and III were used for histopathological analysis. Three-micrometer sections were stained with hematoxylin and eosin and immunohistochemically with monoclonal anti-human granulocyte-associated antigen using a standard streptavidin-biotin-peroxidase complex technique. The number of eosinophils/mm2 in the invasive front of the tumors was automatically quantified in a x400 field using an image computer analyser. Univariate statistical analysis was carried out using Student's t test. The computer-assisted morphometric results showed that there was no statistically significant difference (p>0.05) in the number of eosinophils/mm2 identified by hematoxylin and eosin or immunostaining technique in oral squamous cell carcinomas with TATE. This result suggests that hematoxilyn and eosin routine stain is a useful technique for measuring eosinophils in squamous cell carcinoma with eosinophilic tumor infiltration.     

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.     

Bacterial phospholipide antigens and their taxonomic significance

The investigation of interrelationships between the phospholipides of various microorganisms (33 strains of corynebacteria, mycobacteria and staphylococci) using crossed antibody neutralization reactions with phospholipide antigenic erythrocyte diagnostic was used for the assessment of the degree of antigenic propinquity and antigenic differences between the phospholipides of bacteria of the same species, genus, and of different genera. The role of the determinants of the corresponding (their own) and "foreign" genera in the antigenic differences between the phospholipides of the microorganisms investigated was established. On the basis of the results obtained the conclusion has been drawn that the method of assessment of antigenic interrelationships between phospholipides can be used for the study of some taxonomic problems.     

Fifteen hub genes associated with progression and prognosis of clear cell renal cell carcinoma identified by coexpression analysis

Renal cell carcinoma (RCC) is the most common type of renal tumor, and the clear cell renal cell carcinoma (ccRCC) is the most frequent subtype. In this study, our aim is to identify potential biomarkers that could effectively predict the prognosis and progression of ccRCC. First, we used The Cancer Genome Atlas (TCGA) RNA-sequencing (RNA-seq) data of ccRCC to identify 2370 differentially expressed genes (DEGs). Second, the DEGs were used to construct a coexpression network by weighted gene coexpression network analysis (WGCNA). Moreover, we identified the yellow module, which was strongly related to the histologic grade and pathological stage of ccRCC. Then, the functional annotation of the yellow module and single-samples gene-set enrichment analysis of DEGs were performed and mainly enriched in cell cycle. Subsequently, 18 candidate hub genes were screened through WGCNA and protein–protein interaction (PPI) network analysis. After verification of TCGA’s ccRCC data set, Gene Expression Omnibus (GEO) data set (GSE73731) and tissue validation, we finally identified 15 hub genes that can actually predict the progression of ccRCC. In addition, by using survival analysis, we found that patients of ccRCC with high expression of each hub gene were more likely to have poor prognosis than those with low expression. The receiver operating characteristic curve showed that each hub gene could effectively distinguish between localized and advanced ccRCC. In summary, our study indicates that 15 hub genes have great predictive value for the prognosis and progression of ccRCC, and may contribute to the exploration of the pathogenesis of ccRCC.     

肾透明细胞癌预后相关miRNAs的生物信息学分析

目的寻找可作为肾透明细胞癌（ccRCC）生物标志物的miRNA,以及ccRCC与正常组织间miRNA差异表达情况。 方法利用TCGA数据库下载ccRCC中miRNA表达数据,分析肿瘤与正常组织间差异表达miRNA。使用Kaplan-Meier曲线对患者进行生存分析,筛选出表达情况与临床预后相关的miRNA。通过生物信息学对miRNA的靶基因进行预测,然后运用FunRich软件和ClueGO对靶基因进行GO和KEGG富集分析。 结果通过TCGA数据库分析发现,ccRCC较正常组织差异表达miRNA共54个,其中上调33个,下调21个。通过生存分析发现hsa-miR-21和hsa-miR-155与患者预后相关,P≤0.05。进一步通过Perl软件在Targetscan、miRDB、miRTarBase、miRPath这四个数据库中预测miRNA靶基因并将结果取交集,共发现129个靶基因。GO和KEGG分析结果表明,这些靶基因主要与转录因子活性、信号转导以及FoxO、TNF等信号通路密切相关。 结论通过生物信息学分析发现了ccRCC与正常组织的差异表达miRNA;其中hsa-miR-21和hsa-miR-155与患者总体生存率相关,并通过调控靶基因参与相关的信号通路进而影响ccRCC的发生发展进程,提示hsa-miR-21和hsa-miR-155可能是ccRCC潜在的生物标志物。