Kenneth T. Pace

Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis.

OBJECTIVE We seek to identify the differentially expressed miRNAs in the clear cell subtype (ccRCC) of kidney cancer. DESIGN AND METHODS We performed a miRNA microarray analysis to compare the miRNA expression levels between ccRCC tissues and their normal counterpart. The top dysregulated miRNAs were validated by quantitative RT-PCR analysis. Bioinformatics analysis was also performed. RESULTS A total of 33 dysregulated miRNAs were identified in ccRCC, including 21 upregulated miRNAs and many of these miRNAs have been reported to be dysregulated in other malignancies and have a potential role in cancer pathogenesis. The miRNAs showed a significant correlation with reported chromosomal aberration sites. We also utilized target prediction algorithms to identify gene targets. Preliminary analyses showed these targets can be directly involved in RCC pathogenesis. CONCLUSION We identified miRNAs that are dysregulated in ccRCC and bioinformatics analysis suggests that these miRNAs may be involved in cancer pathogenesis and have the potential to be biomarkers.

miRNA profiling for clear cell renal cell carcinoma: biomarker discovery and identification of potential controls and consequences of miRNA dysregulation.

PURPOSE Renal cell carcinoma is the most common neoplasm of the adult kidney. Currently to our knowledge there are no biomarkers for diagnostic, prognostic or predictive applications for renal cell carcinoma. miRNAs are nonprotein coding RNAs that negatively regulate gene expression and are potential biomarkers for cancer. MATERIALS AND METHODS We analyzed 70 matched pairs of clear cell renal cell carcinoma and normal kidney tissues from the same patients by microarray analysis and validated our results by quantitative real-time polymerase chain reaction. We also performed extensive bioinformatic analysis to explore the role and regulation of miRNAs in clear cell renal cell carcinoma. RESULTS We identified 166 miRNAs that were significantly dysregulated in clear cell renal cell carcinoma, including miR-122, miR-155 and miR-210, which had the highest over expression, and miR-200c, miR-335 and miR-218, which were most down-regulated. Analysis of previously reported miRNAs dysregulated in RCC showed overall agreement in the direction of dysregulation. Extensive target prediction analysis revealed that many miRNAs were predicted to target genes involved in renal cell carcinoma pathogenesis. In renal cell carcinoma miRNA dysregulation can be attributed in part to chromosomal aberrations, co-regulation of miRNA clusters and co-expression with host genes. We also performed a preliminary analysis showing that miR-155 expression correlated with clear cell renal cell carcinoma size. This finding must be validated in a larger independent cohort. CONCLUSIONS Analysis showed that miRNAs are dysregulated in clear cell renal cell carcinoma and may contribute to kidney cancer pathogenesis by targeting more than 1 key molecule. We identified mechanisms that may contribute to miRNA dysregulation in clear cell renal cell carcinoma. Dysregulated miRNAs represent potential biomarkers for kidney cancer.

The miR-17-92 Cluster is Over Expressed in and Has an Oncogenic Effect on Renal Cell Carcinoma

PURPOSE miRNAs are small, nonprotein coding RNAs that are differentially expressed in many malignancies. We previously identified 80 miRNAs that are dysregulated in clear cell renal cell carcinoma. In this study we validated over expression of the miR-17-92 cluster in clear cell renal cell carcinoma and tested the effect of 2 members of this cluster (miR-17-5p and miR-20a) on tumor proliferation. We also elucidated the role of miRNA in clear cell renal cell carcinoma pathogenesis with bioinformatics. MATERIALS AND METHODS miRNA expression was validated by quantitative reverse transcriptase-polymerase chain reaction. The cell proliferation effect of miR-17-5p and miR-20a was tested in a renal adenocarcinoma cell line model. Multiple in silico analyses were done of dysregulated miRNAs. RESULTS We validated miR-71-92 cluster over expression in clear cell renal cell carcinoma by quantitative reverse transcriptase-polymerase chain reaction. Transfection of miR-20a inhibitor significantly decreased cell proliferation in a dose dependent manner. Transfection of miR-17-5p, which is not endogenously expressed in the ACHN cell line, led to increased cell proliferation compared to control values. This effect was suppressed by miR-17-5p inhibitor. Bioinformatics analysis identified 10 clusters of miRNAs dysregulated in clear cell renal cell carcinoma that followed the same expression patterns. We also identified matching patterns between reported chromosomal aberration in clear cell renal cell carcinoma and miRNA dysregulation for 37.5% of the miRNAs. Target prediction analysis was done using multiple algorithms. Many key molecules in clear cell renal cell carcinoma pathogenesis, including HIFs, mTOR, VEGF and VHL, were potential targets for dysregulated miRNAs. CONCLUSIONS A significant number of dysregulated proteins in clear cell renal cell carcinoma are potential miRNA targets. Also, many clear cell renal cell carcinoma dysregulated miRNAs are phylogenetically conserved.

Stone Attenuation and Skin-to-Stone Distance on Computed Tomography Predicts for Stone Fragmentation by Shock Wave Lithotripsy

OBJECTIVES To determine whether stone attenuation and the skin-to-stone distance (SSD) can predict for stone fragmentation by SWL independently. Identifying the factors predictive of shock wave lithotripsy (SWL) outcome would help streamline the care of patients with stones. METHODS A retrospective review was performed of 111 patients undergoing initial SWL for a solitary, 5-20 mm, renal calculus. Stone size, location, attenuation value, and SSD were determined on pretreatment noncontrast computed tomography. The outcome was categorized as stone free, complete fragmentation <5 mm, and incomplete fragmentation >or=5 mm or unchanged at 2 weeks on kidney/ureter/bladder radiography. RESULTS After SWL, 44 (40%) were stone free, 27 (24%) had complete fragmentation, and 40 (36%) of 111 patients had incomplete fragmentation. The stone attenuation of the successfully treated patients (stone free and complete fragmentation groups) was 837 +/- 277 Hounsfield units (HU) vs 1092 +/- 254 HU for those with treatment failure (incomplete fragmentation; P < .01). The mean SSD also differed: 9.6 cm +/- 2.0 vs 11.1 cm +/- 2.5 for the successful treatment group vs the treatment failure group, respectively (P = .01). On multivariate analysis, the factors that independently predicted the outcome were stone attenuation, SSD, and stone composition. When patients were stratified into 4 risk groups (stone <900 HU and SSD <9.0 cm, stone <900 HU and SSD >or=9.0 cm, stone >or=900 HU and SSD <9.0 cm, and stone >or=900 HU and SSD >or=9.0 cm), the SWL success rate was 91%, 79%, 58%, and 41%, respectively (odds ratio 7.1, 95% confidence interval 1.6-32 for <900 HU and SSD <9.0 cm group vs other 3 risk groups; P = .01). CONCLUSIONS The results of our study have shown that a stone attenuation of <900 HU, SSD of <9 cm, and stone composition predict for SWL success, independent of stone size, location, and body mass index. These factors will be considered important in the prospective design of a SWL treatment nomogram at our center.

Limitations to Ultrasound in the Detection and Measurement of Urinary Tract Calculi

OBJECTIVES To evaluate differences in stone measurement using computed tomography (CT) and ultrasound (US). Axial unenhanced helical CT is the reference-standard imaging modality for the assessment of urinary tract calculi; however, US is also commonly used. Differences in stone measurement using these techniques are poorly described and contributors to measurement error remain unknown. METHODS All patients at our institution undergoing both abdominal CT and renal US less than 1 month apart since June 2004 were reviewed. Solitary renal calculi were identified on both CT and US in all cases. RESULTS We identified 71 calculi in 60 patients. Compared with CT, US overestimated stone size, an effect that was more pronounced with smaller calculi. The mean stone measurement on CT was 7.4 +/- 4.4 mm and on US it was 9.2 +/- 4.5 mm (P = .018). For stones </=5 mm, US measurements were a mean of 1.9 +/- 1.2 mm greater than CT (P <.001). US and CT measurements were discordant for 60% of stones </=5 mm. Discordance was associated with US measurement of skin-to-stone distance (P = .018), but not body mass index (P = .189) or location within the urinary tract (P = .161). Review of the literature revealed that US has a pooled sensitivity and specificity of 45% and 94%, respectively, for the detection of ureteric calculi and 45% and 88%, respectively, for renal calculi. CONCLUSIONS US overestimates stone size in urolithiasis, a finding that may have implications for stone management. Discordance in stone measurement varies with size and is greatest in stones </=5 mm. US measurement of skin-stone-distance is an important determinant of error in US measurement of renal calculi.

MECHANICAL PERCUSSION, INVERSION AND DIURESIS FOR RESIDUAL LOWER POLE FRAGMENTS AFTER SHOCK WAVE LITHOTRIPSY: A PROSPECTIVE, SINGLE BLIND, RANDOMIZED CONTROLLED TRIAL

PURPOSE We compare the effectiveness of mechanical percussion and inversion with observation for eliminating lower caliceal fragments 3 months after shock wave lithotripsy. MATERIALS AND METHODS At 3 months after shock wave lithotripsy 69 patients with residual lower caliceal fragments 4 mm. or less were randomized to receive either mechanical percussion and inversion or observation for 1 month. The observation group then received crossover mechanical percussion and inversion if fragments persisted. All patients were followed with plain film of the kidneys, ureters and bladder to assess the stone area and stone-free status, and renal tomography or noncontrast spiral computerized tomography to confirm stone-free status. A blinded radiologist reviewed all films. Patients were treated with a mechanical chest percussor applied to the flank while inverted to greater than 60 degrees after receiving 20 mg. furosemide. RESULTS A total of 35 patients were randomized to receive immediate mechanical percussion and inversion therapy and 34 observation. Of the patients in the observation group 28 subsequently received mechanical percussion and inversion after completing the observation period. The groups were not different in gender, body mass index, side affected, stone location or renal anatomical features. The mechanical percussion and inversion group had a substantially higher stone-free rate than the observation group (40% versus 3%, respectively, p <0.001). The mechanical percussion and inversion group also had a greater improvement in total stone area than controls (-63.3% versus +2.7%, respectively, p <0.001). No significant adverse effects were noted in the mechanical percussion and inversion group. CONCLUSIONS Mechanical percussion and inversion is a safe and effective treatment option for residual lower caliceal fragments 3 months after shock wave lithotripsy. Nearly 50% of patients become stone-free, and stone burden is decreased by 50% in the remainder.

Differential protein expressions in renal cell carcinoma: new biomarker discovery by mass spectrometry.

Renal cell carcinoma (RCC) is the most common neoplasm in the adult kidney. Unfortunately, there are currently no biomarkers for the diagnosis of RCC. In addition to early detection, biomarkers have a potential use for prognosis, for monitoring recurrence after treatment, and as predictive markers for treatment efficiency. In this study, we identified proteins that are dysregulated in RCC, utilizing a quantitative mass spectrometry analysis. We compared the protein expression of kidney cancer tissues to their normal counterparts from the same patient using LC-MS/MS. iTRAQ labeling permitted simultaneous quantitative analysis of four samples (cancer, normal, and two controls) by separately tagging the peptides in these samples with four cleavable mass-tags (114, 115, 116, and 117 Da). The samples were then pooled, and the tagged peptides resolved first by strong cation exchange chromatography and then by nanobore reverse phase chromatography coupled online to nanoelectrospray MS/MS. We identified a total of 937 proteins in two runs. There was a statistically significant positive correlation of the proteins identified in both runs (r(p) = 0.695, p < 0.001). Using a cutoff value of 0.67 fold for underexpression and 1.5 fold for overexpression, we identified 168 underexpressed proteins and 156 proteins that were overexpressed in RCC compared to normal tissues. These dysregulated proteins in RCC were statistically significantly different from those of transitional cell carcinoma and end-stage glomerulonephritis. We performed an in silico validation of our results using different tools and databases including Serial Analysis of Gene Expression (SAGE), UniGene EST ProfileViewer, Cancer Genome Anatomy Project, and Gene Ontology consortium analysis.

A Clinical Nomogram to Predict the Successful Shock Wave Lithotripsy of Renal and Ureteral Calculi

PURPOSE Although shock wave lithotripsy is dependent on patient and stone related factors, there are few reliable algorithms predictive of its success. In this study we develop a comprehensive nomogram to predict renal and ureteral stone shock wave lithotripsy outcomes. MATERIALS AND METHODS During a 5-year period data from patients treated at our lithotripsy unit were reviewed. Analysis was restricted to patients with a solitary renal or ureteral calculus 20 mm or less. Demographic, stone, patient, treatment and 3-month followup data were collected from a prospective database. All patients were treated using the Philips Lithotron® lithotripter. RESULTS A total of 422 patients (69.7% male) were analyzed. Mean stone size was 52.3±39.3 mm2 for ureteral stones and 78.9±77.3 mm2 for renal stones, with 95 (43.6%) of the renal stones located in the lower pole. The single treatment success rates for ureteral and renal stones were 60.3% and 70.2%, respectively. On univariate analysis predictors of shock wave lithotripsy success, regardless of stone location, were age (p=0.01), body mass index (p=0.01), stone size (p<0.01), mean stone density (p<0.01) and skin to stone distance (p<0.01). By multivariate logistic regression for renal calculi, age, stone area and skin to stone distance were significant predictors with an AUC of 0.75. For ureteral calculi predictive factors included body mass index and stone size (AUC 0.70). CONCLUSIONS Patient and stone parameters have been identified to create a nomogram that predicts shock wave lithotripsy outcomes using the Lithotron lithotripter, which can facilitate optimal treatment based decisions and provide patients with more accurate single treatment success rates for shock wave lithotripsy tailored to patient specific situations.

A Comparison of Patient-controlled Sedation Using Either Remifentanil or Remifentanil-propofol for Shock Wave Lithotripsy

Patient-controlled sedation (PCS) has been used for extracorporeal shock wave lithotripsy (SWL) because it allows for rapid individualized titration of anesthetics. Because of its sedating effects, the addition of propofol to remifentanil may improve patient tolerance of SWL with PCS. One hundred twenty patients were randomly assigned to receive remifentanil 10 &mgr;g or remifentanil 10 &mgr;g plus propofol 5 mg for PCS with zero-lockout interval. Nine patients in the Remifentanil group and three patients in the Remifentanil-Propofol group required additional sedatives to complete their SWL (P = 0.128). Compared with the Remifentanil group, the Remifentanil-Propofol group required less remifentanil, had a decreased incidence of postoperative nausea and vomiting, and had a better overall satisfaction level. However, they had an increased incidence of transient apnea and oxygen desaturation. The incidence of apnea was 15% in the Remifentanil group and 52% in the Remifentanil-Propofol group (P < 0.001). All patients were able to move themselves to the stretcher at the end of SWL, and median time to home discharge was <70 min in both groups. Both remifentanil and remifentanil-propofol were useful for PCS during SWL.

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.