Louis S. Liou

Identification of a MicroRNA Panel for Clear-cell Kidney Cancer

OBJECTIVES To identify a robust panel of microRNA signatures that can classify tumor from normal kidney using microRNA expression levels. Mounting evidence suggests that microRNAs are key players in essential cellular processes and that their expression pattern can serve as diagnostic biomarkers for cancerous tissues. METHODS We selected 28 clear-cell type human renal cell carcinoma (ccRCC), samples from patient-matched specimens to perform high-throughput, quantitative real-time polymerase chain reaction analysis of microRNA expression levels. The data were subjected to rigorous statistical analyses and hierarchical clustering to produce a discrete set of microRNAs that can robustly distinguish ccRCC from their patient-matched normal kidney tissue samples with high confidence. RESULTS Thirty-five microRNAs were found that can robustly distinguish ccRCC from their patient-matched normal kidney tissue samples with high confidence. Among this set of 35 signature microRNAs, 26 were found to be consistently downregulated and 9 consistently upregulated in ccRCC relative to normal kidney samples. Two microRNAs, namely, MiR-155 and miR-21, commonly found to be upregulated in other cancers, and miR-210, induced by hypoxia, were also identified as overexpressed in ccRCC in our study. MicroRNAs identified as downregulated in our study can be correlated to common chromosome deletions in ccRCC. CONCLUSIONS Our analysis is a comprehensive, statistically relevant study that identifies the microRNAs dysregulated in ccRCC, which can serve as the basis of molecular markers for diagnosis.

Previously unidentified changes in renal cell carcinoma gene expression identified by parametric analysis of microarray data

BackgroundRenal cell carcinoma is a common malignancy that often presents as a metastatic-disease for which there are no effective treatments. To gain insights into the mechanism of renal cell carcinogenesis, a number of genome-wide expression profiling studies have been performed. Surprisingly, there is very poor agreement among these studies as to which genes are differentially regulated. To better understand this lack of agreement we profiled renal cell tumor gene expression using genome-wide microarrays (45,000 probe sets) and compare our analysis to previous microarray studies.MethodsWe hybridized total RNA isolated from renal cell tumors and adjacent normal tissue to Affymetrix U133A and U133B arrays. We removed samples with technical defects and removed probesets that failed to exhibit sequence-specific hybridization in any of the samples. We detected differential gene expression in the resulting dataset with parametric methods and identified keywords that are overrepresented in the differentially expressed genes with the Fisher-exact test.ResultsWe identify 1,234 genes that are more than three-fold changed in renal tumors by t-test, 800 of which have not been previously reported to be altered in renal cell tumors. Of the only 37 genes that have been identified as being differentially expressed in three or more of five previous microarray studies of renal tumor gene expression, our analysis finds 33 of these genes (89%). A key to the sensitivity and power of our analysis is filtering out defective samples and genes that are not reliably detected.ConclusionsThe widespread use of sample-wise voting schemes for detecting differential expression that do not control for false positives likely account for the poor overlap among previous studies. Among the many genes we identified using parametric methods that were not previously reported as being differentially expressed in renal cell tumors are several oncogenes and tumor suppressor genes that likely play important roles in renal cell carcinogenesis. This highlights the need for rigorous statistical approaches in microarray studies.

Prognostic importance of resection margin width after nephron-sparing surgery for renal cell carcinoma

OBJECTIVES To examine the relationship between the width of the resection margin and disease progression in renal cell carcinoma (RCC) after nephron-sparing surgery (NSS). During NSS for RCC, it is standard practice to excise the tumor along with a surrounding margin of normal parenchyma (margin of resection) to ensure complete resection of the neoplasm. However, no agreement has been reached on how wide the margin of resection should be. METHODS We retrospectively reviewed the histopathologic sections and medical records of 69 patients with localized RCC who had undergone NSS between 1976 and 1988 to determine whether the resection margin, tumor size, TNM stage, and Fuhrman nuclear grade were associated with disease progression (defined as local tumor recurrence or metastasis). The mean postoperative follow-up interval was 8.5 years. RESULTS No association was found between the width of the resection margin and disease progression (P = 0.98, log-rank test). Both TNM stage and Fuhrman nuclear grade correlated with disease progression. Patients with T1-T2 tumors had lower progression (P <0.001, log-rank test), and increased Fuhrman nuclear grade correlated with more disease progression (P <0.001, log-rank test). CONCLUSIONS The width of the resection margin after NSS for RCC does not correlate with long-term disease progression. A histologic tumor-free margin of resection, irrespective of the width of the margin is sufficient to achieve complete local excision of RCC.

ADRENOCORTICAL CARCINOMA IN CHILDREN: Review and Recent Innovations

Adrenocortical carcinoma in childhood is a rare potentially fatal disease. Despite its often dramatic presentation, there typically has been a distressingly long delay between the onset of symptoms and the time of diagnosis. This delay undoubtedly has contributed to the historically poor prognosis in these children by permitting the disease to reach an advanced stage before treatment is started. It is imperative that the physician recognizes the endocrine manifestations of these tumors early and has a high index of suspicion. Although biochemical and histologic evaluations are helpful, they often cannot differentiate benign lesions from malignant neoplasms and should not unduly delay intervention. Aggressive complete surgical resection continues to be the mainstay of treatment and is the best prognosticator of overall survival. The role of adjuvant therapy and chemotherapy continues to evolve. Molecular studies have increased understanding of cancer biology and may provide possible novel therapeutic approaches in the future. It is hoped that increased familiarity with this unusual tumor will result in earlier detection, prompt intervention, and improved survival for children with adrenocortical carcinoma.

Differential protein profiling in renal-cell carcinoma

Characterizing the alterations of protein expression in cancer cells can be very useful in providing insight into the changes in the functional pathways and thus the fundamental mechanisms of cancer development at the molecular level. In this study, we profiled protein expressions in eleven pairs of primary cell cultures derived from renal‐cell carcinoma (RCC) tissues and patient‐matched normal kidney tissues utilizing two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Together with the immunoblot analysis of proteins from the RCC tissues, the study also demonstrated that the alterations of protein expression observed in RCC primary cell cultures reflected those observed in the original RCC tissues. We analyzed the expression profiles and identified proteins differentially expressed in RCC primary cell cultures by 2‐D PAGE and mass spectrometry (MS). We found sixteen proteins were overexpressed and seven proteins underexpressed in RCC. The deregulated expressions of proteins include those involved in metabolism, cellular morphology, heat shock response, cell growth, etc. Overexpression of three proteins, αβ‐crystallin, manganese superoxide dismutase (MnSOD), and annexin IV, most commonly observed in primary RCC cell cultures, were also observed by immunoblot analysis of proteins from the RCC tissues from which the primary cell cultures were derived. Semi‐quantitative reverse transcription (RT)‐polymerase chain reaction (PCR) analysis revealed the direct correlation between deregulated gene expression and the corresponding protein abundance in two of the three most commonly upregulated proteins we found in RCC.

Microarray gene expression profiling and analysis in renal cell carcinoma.

BackgroundRenal cell carcinoma (RCC) is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays.MethodsTotal RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC.ResultsSelected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR). Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved.ConclusionsThis is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most notably, genes involved in cell adhesion were dominantly up-regulated whereas genes involved in transport were dominantly down-regulated. This study reveals significant gene expression alterations in key biological pathways and provides potential insights into understanding the molecular mechanism of renal cell carcinogenesis.

LONG-TERM RENAL FUNCTIONAL EFFECTS OF SHOCK WAVE LITHOTRIPSY, PERCUTANEOUS NEPHROLITHOTOMY AND COMBINATION THERAPY: A COMPARATIVE STUDY OF PATIENTS WITH SOLITARY KIDNEY

PURPOSE We compared the long-term impact on renal function after shock wave lithotripsy, percutaneous nephrolithotomy or the 2 techniques combined in patients with a solitary kidney. MATERIALS AND METHODS A total of 45 women and 38 men 15 to 86 years old (mean age 56.1) with a solitary kidney were treated with shock wave lithotripsy (53), percutaneous nephrolithotomy (18) or the 2 techniques combined (12). Before and after treatment serum creatinine, blood pressure and the calculated glomerular filtration rate were determined, and raw and calculated data were compared by the Kruskal-Wallis, Fisher exact and Wilcoxon rank sum tests, and the Spearman correlation coefficient. Followup was 1 to 166.5 months (mean 53.0, median 46.9) overall and statistically equivalent in the 3 treatment arms. RESULTS Treatment groups were comparable in regard to patient age, sex distribution, weight, blood pressure and pretreatment serum creatinine. There was no significant difference in any evaluated pretreatment or posttreatment parameters and no difference in the change in any parameter after treatment. Stratifying patients to pretreatment serum creatinine less or greater than 2 mg./dl. likewise revealed no significant difference in the impact on long-term renal function. However, pretreatment serum creatinine positively and strongly correlated with a positive change in the glomerular filtration rate after therapy. CONCLUSIONS In this study there was no evidence that any of these 3 treatment modalities resulted in the deterioration of renal function even at long-term followup. This finding implies that shock wave lithotripsy, percutaneous nephrolithotomy and the 2 therapies combined are equally efficacious for preserving renal function when performed in patients with a solitary kidney.

Sodium Pentosan Polysulfate Reduces Urothelial Responses to Inflammatory Stimuli Via an Indirect Mechanism

PURPOSE Sodium pentosan polysulfate has been promoted as a urothelial cytoprotective agent for treating interstitial cystitis. The nuclear transcription factor nuclear factor kappaB is thought to have a role in mediating the urothelial inflammatory response of interstitial cystitis. We further defined a possible cytoprotective effect of sodium pentosan polysulfate by characterizing the effect of the drug on the expression of nuclear factor kappaB. MATERIALS AND METHODS For cell culture human urothelial cells were incubated in various concentrations of sodium pentosan polysulfate for 16 hours in keratinocyte serum-free medium. They were subsequently treated with the known nuclear factor kappaB stimulants tumor necrosis factor-alpha, lipopolysaccaride (LPS) and double-stranded RNA (dsRNA). Each stimulant was then incubated with sodium pentosan polysulfate separately and the mixture was used to treat cultured urothelial cells. For electrophoretic mobility shift assay total cell extracts were prepared and run in electrophoretic mobility shift assays using a radiolabeled nuclear factor kappaB consensus sequence as a probe. Western blot analysis was done to assess nuclear factor kappaB activation by measuring degradation of the inhibitory subunit of the nuclear factor kappaB complex. RESULTS Nuclear factor kappaB activation by tumor necrosis factor-alpha, LPS and dsRNA was unaltered when cultured cells were incubated in sodium pentosan polysulfate before treatment. In contrast, nuclear factor kappaB activation by LPS and dsRNA was suppressed when the stimulants were incubated with sodium pentosan polysulfate before cell treatment. This suppressive effect was confirmed by Western blot analysis. CONCLUSION Sodium pentosan polysulfate may have a nonspecific effect against the viral (dsRNA) and bacterial (LPS) activation of nuclear factor kappaB. The observed clinical effect of sodium pentosan polysulfate may be mediated by nonspecific binding of sodium pentosan polysulfate molecules and the inflammatory stimulants of urothelial activation. These findings suggest a mechanism of action for sodium pentosan polysulfate that occurs in the urine rather than at the mucosal membrane by direct interaction of the drug with potential interstitial cystitis inducing inflammatory agents.

Identification of Novel Epigenetic Markers for Clear Cell Renal Cell Carcinoma

PURPOSE We identified significantly hypermethylated genes in clear cell renal cell carcinoma. MATERIALS AND METHODS We previously identified a set of under expressed genes in renal cell carcinoma tissue through transcriptional profiling and a robust computational screen. We selected 19 of these genes for hypermethylation analysis using a rigorous search for the best candidate regions, considering CpG islands and transcription factor binding sites. The genes were analyzed for hypermethylation in the DNA of 38 matched clear cell renal cell carcinoma and normal samples using matrix assisted laser desorption ionization time-of-flight mass spectrometry. The significance of hypermethylation was assessed using 3 statistical tests. We validated the down-regulation of significantly hypermethylated genes at the RNA and protein levels in a separate set of patients using reverse transcriptase-polymerase chain reaction, immunohistochemistry and Western blots. RESULTS We found 7 significantly hypermethylated regions from 6 down-regulated genes, including SFRP1, which was previously shown to be hypermethylated in renal cell carcinoma and other cancer types. CONCLUSIONS To our knowledge we report for the first time that another 5 genes (SCNN1B, SYT6, DACH1, and the tumor suppressors TFAP2A and MT1G) are hypermethylated in renal cell carcinoma. Robust computational screens and the high throughput methylation assay resulted in an enriched set of novel genes that are epigenetically altered in clear cell renal cell carcinoma. Overall the detection of hypermethylation in these highly down-regulated genes suggests that assaying for their methylation using cells from urine or blood could provide the basis for a viable diagnostic test.

Somatic cell mapping of conglutinin (CGN1) to cattle syntenic group U29 and fluorescence in situ localization to Chromosome 28

A 260-bp genomic PstI fragment, which encodes a portion of the carbohydrate recognition domain, was used along with hybrid somatic cells to map the conglutinin gene (CGN1) to domestic cow (Bos taurus) syntenic group U29. In turn, a cosmid containing the entire bovine CGN1 was used with fluorescence in situ hybridization to sublocalize this gene to cattle chromosome (Chr) (BTA) 28 band 18. Since BTA 28 and several of the other small acrocentric autosomes of cattle are difficult to discriminate, we have also chromosomally sublocalized CGN1 to the p arm of the lone biarmed autosome of the gaur (Bos gaurus). The use of the gaur 2/28 Robertsonian as a marker chromosome and our assignment of CGN1 to BTA 28 should help resolve some of the nomenclatural questions involving this cattle chromosome.