Heba W.Z. Khella

The Clinical Utility of miR-21 as a Diagnostic and Prognostic Marker for Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. Increasing evidence suggests that microRNAs are dysregulated in RCC and are important factors in RCC pathogenesis. miR-21 is a known oncogene with tumor-promoting effects in many types of cancer. In this study, we analyzed miR-21 in 121 cases of healthy kidney and different RCC subtypes, including clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC), and oncocytoma. Total RNA was extracted, and the expression of miR-21 was measured with real-time quantitative RT-PCR using miR-21-specific probes. The expression of miR-21 was significantly up-regulated in RCC compared with healthy kidney. There was a significant difference in the expression levels between RCC subtypes, with the highest levels of expression in ccRCC and pRCC subtypes. miR-21 expression distinguished ccRCC and pRCC from chRCC and oncocytoma with 90% specificity (95% CI, 63.9% to 98.1%) and 83% sensitivity (95% CI, 53.5% to 97.6%). Significantly higher miR-21 levels were associated with higher stage and grade. Patients who were miR-21 positive had statistically significant shorter disease-free and overall survival rates. Thus, miR-21 is up-regulated in RCC, and its expression levels can be used as a diagnostic marker to distinguish ccRCC and pRCC from chRCC and oncocytoma. Moreover, it has potential as a prognostic marker in RCC, although it is not independent of tumor stage and grade.

Multilevel Whole-Genome Analysis Reveals Candidate Biomarkers in Clear Cell Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. We conducted an integrated analysis of copy number, gene expression (mRNA and miRNA), protein expression, and methylation changes in clear cell renal cell carcinoma (ccRCC). We used a stepwise approach to identify the most significant copy number aberrations (CNA) and identified regions of peak and broad copy number gain and loss, including peak gains (3q21, 5q32, 5q34-q35, 7p11, 7q21, 8q24, 11q13, and 12q14) and deletions (1p36, 2q34-q37, 3p25, 4q33-q35, 6q23-q27, and 9p21). These regions harbor novel tumor-related genes and miRNAs not previously reported in renal carcinoma. Integration of genome-wide expression data and gene set enrichment analysis revealed 75 gene sets significantly altered in tumors with CNAs compared with tumors without aberration. We also identified genes located in peak CNAs with concordant methylation changes (hypomethylated in copy number gains such as STC2 and CCND1 and hypermethylated in deletions such as CLCNKB, VHL, and CDKN2A/2B). For other genes, such as CA9, expression represents the net outcome of opposing forces (deletion and hypomethylation) that also significantly influences patient survival. We also validated the prognostic value of miRNA let-7i in RCCs. miR-138, located in chromosome 3p deletion, was also found to have suppressive effects on tumor proliferation and migration abilities. Our findings provide a significant advance in the delineation of the ccRCC genome by better defining the impact of CNAs in conjunction with methylation changes on the expression of cancer-related genes, miRNAs, and proteins and their influence on patient survival.

Exploring the role of miRNAs in renal cell carcinoma progression and metastasis through bioinformatic and experimental analyses

Metastasis results in most of the cancer deaths in clear cell renal cell carcinoma (ccRCC). MicroRNAs (miRNAs) regulate many important cell functions and play important roles in tumor development, metastasis and progression. In our previous study, we identified a miRNA signature for metastatic RCC. In this study, we validated the top differentially expressed miRNAs on matched primary and metastatic ccRCC pairs by quantitative polymerase chain reaction. We performed bioinformatics analyses including target prediction and combinatorial analysis of previously reported miRNAs involved in tumour progression and metastasis. We also examined the co-expression of the miRNAs clusters and compared expression of intronic miRNAs and their host genes. We observed significant dysregulation between primary and metastatic tumours from the same patient. This indicates that, at least in part, the metastatic signature develops gradually during tumour progression. We identified metastasis-dysregulated miRNAs that can target a number of genes previously found to be involved in metastasis of kidney cancer as well as other malignancies. In addition, we found a negative correlation of expression of miR-126 and its target vascular endothelial growth factor (VEGF)-A. Cluster analysis showed that members of the same miRNA cluster follow the same expression pattern, suggesting the presence of a locus control regulation. We also observed a positive correlation of expression between intronic miRNAs and their host genes, thus revealing another potential control mechanism for miRNAs. Many of the significantly dysregulated miRNAs in metastatic ccRCC are highly conserved among species. Our analysis suggests that miRNAs are involved in ccRCC metastasis and may represent potential biomarkers.

MicroRNAs in Kidney Disease: An Emerging Understanding

MicroRNAs (miRNAs) are short noncoding RNA molecules that function by negatively regulating the expression of their target genes in a tightly controlled manner. Accumulating evidence, based in part on effects seen after miRNA overexpression and/or knockdown, points to the critical involvement of miRNAs in kidney function in health and disease. In this review, we provide a quick overview of the biogenesis of miRNAs and their potential involvement in kidney development and normal function. We also discuss the current literature that has begun to uncover the role of miRNAs in the pathogenesis of kidney diseases, including diabetic nephropathy, hypertension, glomerulonephritis, and cancer. As such, miRNAs have potential utility in the clinical realm as disease biomarkers. Moreover, miRNAs represent an attractive therapeutic target for a number of kidney diseases. We close by discussing a number of potential challenges that face the field of miRNA research and clinical use.

miR-210 is a prognostic marker in clear cell renal cell carcinoma.

Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary ccRCC and compared its expression in 40 pairs of adjacent normal and cancerous tissues. We assessed its expression in primary and metastatic tumors, in the common RCC subtypes, and the benign oncocytoma. The results were validated with an independent data set from The Cancer Genome Atlas. miR-210 was significantly overexpressed in ccRCC compared with normal kidney. miR-210(+) patients had a statistically higher chance of disease recurrence [hazard ratio (HR), 1.82; P = 0.018] and shorter overall survival (HR, 2.46; P = 0.014). In multivariate analysis, miR-210 lost its statistically significant association with shorter disease-free survival and overall survival after adjusting for tumor size and tumor, node, metastasis stage. Papillary RCC showed comparable miR-210 overexpression, whereas decreased up-regulation was seen in chromophobe RCC and oncocytoma. A number of predicted targets that might be involved in carcinogenesis and aggressive tumor behavior were identified. miR-210 is a potential therapeutic target and independent marker of poor prognosis of ccRCC.

miR-221/222 Are Involved in Response to Sunitinib Treatment in Metastatic Renal Cell Carcinoma

Sunitinib is a multitargeting tyrosine kinase inhibitor used for metastatic renal cancer. There are no biomarkers that can predict sunitinib response. Such markers are needed to avoid administration of costly medication with side effects to patients who would not benefit from it. We compared global miRNA expression between patients with a short (≤12 months) versus prolonged (>12 months) progression-free survival (PFS) under sunitinib as first-line therapy for metastatic renal cell carcinoma. We identified a number of differentially expressed miRNAs and developed miRNA statistical models that can accurately distinguish between the two groups. We validated our models in the discovery set and an independent set of 57 patients. Target prediction and pathway analysis showed that these miRNAs are involved in vascular endothelial growth factor (VEGF), TGFβ, and mammalian target of rapamycin (mTOR)-mediated signaling and cell-cell communication. We tested the effect of these miRNAs on cellular proliferation and angiogenesis. We validated the negative correlation between miR-221 and its target, VEGFR2.miR-221 overexpression was associated with a poor PFS while its target, VEGFR2 was associated with longer survival. Gain of function experiments showed that miR-221 and miR-222 decreased angiogenesis and cellular proliferation in human umbilical vein endothelial cells (HUVEC) while increasing cellular proliferation in ACHN cells. miRNAs represent potential predictive markers for sunitinib response.

MicroRNA-194 is a Marker for Good Prognosis in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most prevalent adult kidney cancer. Prognostic markers are needed to guide patient management toward aggressive versus more conservative approaches, especially for small tumors ≤4 cm. miR‐194 was reported to be downregulated in several cancers and is involved in epithelial to mesenchymal transition. We evaluated miR‐194 as a prognostic marker in ccRCC. In a cohort of 234 patients with primary ccRCC, we correlated miR‐194 expression level with multiple clinicopathological features including disease‐free and overall survival, tumor size, clinical stage, and histological grade. Our results shows a stepwise decrease in miR‐194 expression from normal kidney to primary ccRCC (P = 0.0032) and a subsequent decrease from primary to metastatic lesions. Additionally, patients with higher miR‐194 expression has significantly longer disease‐free survival (P = 0.041) and overall survival (P = 0.031) compared to those with lower expression. In multivariate analysis, miR‐194‐positive tumors retain significance in disease‐free survival and overall survival, suggesting miR‐194 is an independent marker for good prognosis in ccRCC. Moreover, miR‐194 is a marker for good prognosis for patients with small renal masses (P = 0.014). These findings were validated on an independent data set from The Cancer Genome Atlas. We also compared miR‐194 expression between RCC subtypes. ccRCC had the highest levels, whereas chromophobe RCC and oncocytoma had comparable lower levels. Target prediction coupled with pathway analysis show that miR‐194 is predicted to target key molecules and pathways involved in RCC progression. miR‐194 represents a prognostic biomarker in ccRCC.

Exosomal MicroRNAs Are Diagnostic Biomarkers and Can Mediate Cell–Cell Communication in Renal Cell Carcinoma

BACKGROUND Apart from an invasive biopsy, currently no tools are available to confirm the diagnosis of clear cell renal cell carcinoma (ccRCC); this resulted in approximately 30% of patients being diagnosed with metastatic disease. OBJECTIVE To determine whether urinary microRNAs (miRNAs) can serve as biomarkers to confirm the diagnosis of ccRCC. DESIGN, SETTING, AND PARTICIPANTS Global miRNA expression was assessed in 28 preoperative urine samples from patients with ccRCC and 18 healthy participants. The independent validation set consisted of 81 ccRCC patients, 24 patients with benign lesions, and 33 healthy participants. We extracted both cell-free and exosomal RNA for miRNA expression analysis using miRNA-specific polymerase chain reaction assays. We also investigated exosomal miRNA secretion in cell line models and performed exosome transfer between RCC and endothelial cell types. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Receiver operating characteristic analysis was applied to identify the discrimination power of miRNAs. RESULTS AND LIMITATIONS Overall, miR-126-3p combined with miR-449a or with miR-34b-5p could significantly distinguish ccRCC patients from healthy participants (miR-126-3p-miR-449a: area under the curve [AUC]: 0.84; 95% confidence interval [CI], 0.7620-0.9151; p<0.001; miR-126-3p-miR-34b-5p: AUC: 0.79; 95% CI, 0.7013-0.8815; p<0.001). The combination of miR-126-3p and miR-34b-5p was also able to distinguish small renal masses (pT1a, ≤4cm) from healthy controls (AUC: 0.79; 95% CI, 0.6848-0.8980; p<0.001). Using miR-126-3p and miR-486-5p in combination, we were able to differentiate between benign lesions and ccRCC (AUC: 0.85; 95% CI, 0.7295-0.9615; p<0.01). The expression of a number of miRNAs returned to a level comparable with health after surgery. Kidney cancer cell lines were found to secrete exosomal miR-126-3p, miR-17-5p, miR-21-3p, and miR-25-3p, and these miRNAs were found to be internalized by other cell types. CONCLUSIONS We identified exosomal miRNAs as potential noninvasive diagnostic urinary biomarkers for ccRCC and provided evidence that miRNAs are secreted by the tumor and can function as a tool for intercellular communication. PATIENT SUMMARY We identified urinary microRNAs that can serve as diagnostic biomarkers for clear cell renal cell carcinoma.

miR-10b is a prognostic marker in clear cell renal cell carcinoma

Aims Clear cell renal cell carcinoma (ccRCC) is the most common adult kidney cancer. It is an aggressive tumour with unpredictable outcome. The currently used clinical parameters are not always accurate for predicting disease behaviour. miR-10b is dysregulated in different malignancies including RCC. Methods We assessed the clinical utility of miR-10b as a prognostic marker in 250 patients with primary ccRCC. We examined the correlation between miR-10b and clinicopathological parameters. We compared miR-10b expression among different RCC subtypes and normal kidney tissue. Results We observed a stepwise decrease of miR-10b expression from normal kidney to primary ccRCC and a further decrease from primary to metastatic RCC. miR-10b expression was significantly lower in stages III/IV compared with stages I/II (p=0.038). Using a binary cut-off, miR-10b-positive patients had significantly longer disease-free survival (HR=0.47, CI 0.28 to 0.79, p=0.004). In the subgroup of patients with tumour size >4 cm, higher miR-10b expression was associated with significant longer disease-free and overall survival (p=0.001 and p=0.036, respectively). miR-10b was significantly downregulated in ccRCC compared with normal kidney (p<0.0001), and oncocytoma (p=0.031). It was also downregulated in chromophobe RCC. In addition, we identified a number of miR-10b-predicted targets and pathways that are involved in tumourigenesis. Conclusions Our data point to miR-10b as a promising prognostic marker in ccRCC with potential therapeutic applications.

A miRNA-based classification of renal cell carcinoma subtypes by PCR and in situ hybridization

Renal cell carcinoma (RCC) constitutes an array of morphologically and genetically distinct tumors the most prevalent of which are clear cell, papillary, and chromophobe RCC. Accurate distinction between the typically benign-behaving renal oncocytoma and RCC subtypes is a frequent challenge for pathologists. This is critical for clinical decision making. Subtypes also have different survival outcomes and responses to therapy. We extracted RNA from ninety formalin-fixed paraffin-embedded (FFPE) tissues (27 clear cell, 29 papillary, 19 chromophobe, 4 unclassified RCC and 11 oncocytomas). We quantified the expression of six miRNAs (miR-221, miR-222, miR-126, miR-182, miR-200b and miR-200c) by qRT-PCR, and by in situ hybridization in an independent set of tumors. We developed a two-step classifier. In the first step, it uses expression of either miR-221 or miR-222 to distinguish the clear cell and papillary subtypes from chromophobe RCC and oncocytoma (miR-221 AUC: 0.96, 95% CI: 0.9132–1.014, p < 0.0001 and miR-222 AUC: 0.91, 95% CI: 0.8478–0.9772, p < 0.0001). In the second step, it uses miR-126 to discriminate clear cell from papillary RCC (AUC: 1, p < 0.0001) and miR-200b to discriminate chromophobe RCC from oncocytoma (AUC: 0.95, 95% CI: 0.8933–1.021, p < 0.0001). In situ hybridization showed a nuclear staining pattern. miR-126, miR-222 and miR-200b were significantly differentially expressed between the subtypes by in situ hybridization. miRNA expression could distinguish RCC subtypes and oncocytoma. miRNA expression assessed by either PCR or in situ hybridization can be a clinically useful diagnostic tool to complement morphologic renal tumor classification, improving diagnosis and patient management.