ERBB2 Amplification as a Predictive and Prognostic Biomarker in Upper Tract Urothelial Carcinoma.

Upper tract urothelial carcinomas (UTUCs) occur in about 5-10% of all urothelial carcinomas and are frequently discovered in high-stage disease. We aimed to evaluate human epidermal growth factor receptor 2 (ERBB2) protein expression immunohistochemically and ERBB2 amplification in UTUCs by fluorescence in situ hybridization, applying a tissue microarray technique. ERBB2 overexpression and ERBB2 amplification were defined according to the recommendations of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) for breast cancer and gastric carcinoma (GC), revealing scores of 2+ and 3+ in 10.2% and 41.8% of UTUCs, respectively. The performance parameters showed obviously higher sensitivity of ERBB2 immunoscoring according to the ASCO/CAP criteria for GC. ERBB2 amplification was detected in 10.5% of UTUCs. ERBB2 overexpression was more likely to be found in high-grade tumors and was associated with tumor progression. Univariable Cox regression analysis revealed a significantly lower progression-free survival (PFS) in cases with ERBB2 immunoscores of 2+ or 3+ according to the ASCO/CAP guidelines for GC. UTUCs with ERBB2 amplification showed a significantly shorter PFS in the multivariable Cox regression analysis. Irrespective of their ERBB2 status, patients with UTUC treated with platin showed a significantly lower PFS than UTUC patients who had not received any platin-based therapy. In addition, UTUC patients with a normal ERBB2 gene status who had not received platin-based therapy showed significantly longer overall survival. The results suggest that ERBB2 is a biomarker for progression in UTUCs and may define a distinct subgroup of UTUCs. As previously shown, ERBB2 amplification is infrequent. However, the small number of patients diagnosed with ERBB2-amplified UTUC might benefit from ERBB2-targeted cancer therapy. In clinical-pathological routine diagnostics, the determination of ERBB2 amplification is an established method in some defined entities and also successful in small samples. Still, the simultaneous use of ERBB2 immunohistochemistry and ERBB2 in situ hybridization would be important in order to record the low rate of amplified UTUC cases as completely as possible.

Ceruloplasmin expression in renal cell carcinoma correlates with higher-grade and shortened survival.

Aim: We aimed to explore ceruloplasmin (CP) expression in clear cell renal cell carcinoma (ccRCC). Materials & methods: CP was analyzed in biofluid samples of 63 ccRCC patients, divided into three grading groups, and immunohistochemically, in 308 ccRCC. Results: Significant differences of mean plasma and urine CP levels in different grading groups were found. CP immunoreactivity was significantly linked to high-grade disease. Log rank tests showed a significant shorter overall survival rate in CP-positive cases (all p < 0.05). Conclusion: CP protein levels in biofluid samples confirmed differential CP expressions, depending on nuclear grade in ccRCC as previously seen in RNA expression analysis. CP expression was linked to high-grade disease and reduced survival rate in RCC.

Loss of Mismatch-repair Protein Expression and Microsatellite Instability in Upper Tract Urothelial Carcinoma and Clinicopathologic Implications.

BACKGROUND: Upper tract urothelial carcinoma (UTUC) may arise in the setting of hereditary non-polyposis colorectal cancer (Lynch syndrome [LS]) or sporadically. Variable frequencies of microsatellite instability (MSI) were found in UTUC. For advanced solid MSI tumors, targeted therapy with programmed death-ligand 1 inhibitors is available. Therefore, we aimed to determine the prevalence of mismatch repair (MMR) protein loss and MSI in UTUC using a tissue microarray approach and further molecular and correlation analysis. MATERIALS AND METHODS: We studied the immunohistochemical expression of MLH1, MSH2, MSH6, and PMS2 on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 128 patients with UTUC. MSI analysis was performed in 79 cases with deficient MMR protein expression, and/or in patients aged 60 years and below, and/or other tumors possibly related to LS. RESULTS: Loss of MMR protein expression was seen in 24 (18.8%) of 128 cases. MSI analysis revealed MSI-high in 29, MSI-low in 7 cases. The Fisher exact test demonstrated significant differences between MSI and loss of MMR protein expression, clinically possible LS, tumor growth pattern, inverted growth pattern, and death (P < .001, P < .001, P = .002, P = .003, and P = .033, respectively). MSI does not appear to influence survival (overall and progression-free), but there was a significant shorter progression-free survival in MSI-high versus MSS patients who had received chemotherapy. CONCLUSION: The frequency of MSI in UTUC was 36 (28.1%) of 128 patients with a good accuracy of immunohistochemistry. In daily practice, MSI screening especially is recommended in patients with advanced UTUC and inverted papillary tumor growth pattern with the aim of screening patients for possible targeted therapy.

Copy Number Alterations with Prognostic Potential in Clear Cell Renal Cell Carcinoma.

OBJECTIVES: To detect chromosomal aberrations in a genome-wide manner with potential value for prognosis in groups of patients with different histopathological grading in clear cell renal carcinoma (ccRCC). MATERIAL AND METHODS: We performed a copy number alteration analysis using the Affymetrix platform and SNP 6.0 mapping arrays with samples from 48 ccRCC-patients. The data analysis was done using 3 different Software Platforms: Affymetrix Genotyping Console (version 4.1.3.840) and 2 open-source packages for validation: PennCNV and PICNIC. RESULTS: Consistent changes were found to divide the tumors into 4 groups: first group showed typical losses on 3p, second group losses on 3p plus gains on 5q, third group gains on chromosome 7 plus losses on chromosome 8; fourth group did not show any major changes. We selected the affected genes with the highest consistency and identified 13 different genes mapping in the SNP 6.0 results and Kyoto Encyclopedia of Genes and Genomes. Remarkable for further consideration were the phosphatidylinositol 3-kinase pathway, BRAF, MET, EGLN1; growth factors, for example, HGF, PGF and TGFB2. CONCLUSION: A multimodal approach with a well-defined workflow for detecting genomic aberrations by using array technologies and comparing the findings with different comprehensive databases may provide insights into functional tumor processes and help to identify potential new targets for more individualized future treatment.

Prognostic value of intraoperative measurements of renal tissue oxygenation and microcirculation on renal function in partial nephrectomy.

BACKGROUND: Partial nephrectomy (PNx) can be associated with macrocirculatory and microcirculatory alterations, ultimately leading to acute kidney injury (AKI). Measuring kidney tissue oxygenation (µHbO2) and microcirculation during open PNx might be feasible to early detect these alterations and prevent postoperative AKI. METHODS: µHbO2 and microcirculation were measured in 45 patients undergoing PNx by reflectance spectrophotometry and laser Doppler flowmetry (O2C™, Lea, Germany), related to ischemia time and tumour size. Pre- and postoperative creatinine levels were determined. RESULTS: µHbO2 was lower after reperfusion than before clamping (72 vs. 75%), while microcirculation and regional haemoglobin did not differ. Ischemia time was 15.7 min on average. µHbO2 was higher without ischemia (80 vs. 70%, p = 0.109) and in T1a- than T1b-tumours, independent of ischemia time and reperfusion. The renal collecting system (RCS) was opened in 19/45 patients with µHbO2 of 68% after reperfusion compared to 74% with intact RCS. Postoperative complications occurred in 6/45 patients (13%). µHbO2 was 68% before clamping vs. 75% without complications. Serum creatinine of patients with T1b was higher compared to T1a (103 vs. 87 µmol/L). Patients with larger tumours had higher postoperative creatinine levels (173 vs. 124 µmol/L; p = 0.052). CONCLUSION: We showed for the first time that the method is feasible to monitor renal tissue oxygenation at the level of microcirculation non-invasively and reproducibly during PNx. Tumour size seems to have a decisive influence on oxygenation and postoperative renal function. Our results imply that postoperative complications may be predicted by low intraoperative renal oxygenation and microcirculatory flow measurements.

Upper Urinary Tract Tumors: Which Diagnostic Methods Are Needed?

OBJECTIVES: We reviewed the data of patients with upper urinary tract (UUT) tumors to evaluate the effectiveness of diagnostic procedures. METHODS: This retrospective study evaluated tumor characteristics, imaging procedures, epidemiological and follow-up data of 113 patients. We analyzed the importance of non-invasive and endoscopic diagnosis in addition to imaging as well as the influence of stage and grade on recurrence rate. RESULTS: Most tumors were urothelial carcinomas (92.9%). The cardinal symptoms were hematuria (40.7%), flank pain (2.7%), and urinary obstruction (14.2%). Forty-seven patients received intravenous urograms (IVUs), 57 retrograde ureteropyelography (RUP), 89 CTs, 6 an MRI. The correct positive tumor identification was reached by IVU in 27/47 patients, by RUP in 50/57, by CT in 74/89, and by MRI in 3/6 patients representing sensitivities of 57.4% (IVU), 87.7% (RUP), 83.1% (CT), and 50% (MRI). Sixty-four patients had urine cytology, which was correctly positive in 60.9% and 56 had a diagnostic ureterorenoscopy, which was correctly positive in 83.9%. During follow-up more than 20% of patients developed a recurrence. CONCLUSION: In patients with hematuria and flank pain, UUT must be considered a differential diagnosis. UUT to the extent of 76.6% showed more invasive growth (>Ta). Thus, rapid and efficient diagnosis based primarily on imaging is required. Contrast CT scan seems to be the imaging modality with the best performance. However, often only a combination of diagnostic procedures gives a certain diagnosis. Due to the high recurrence rate, close follow-up is needed.

TOP2A, HELLS, ATAD2, and TET3 Are Novel Prognostic Markers in Renal Cell Carcinoma.

OBJECTIVE: To identify and validate novel prognostic marker genes in clear cell renal cell carcinoma (RCC) that are increasingly expressed during tumor progression. METHODS: Total RNA was isolated from normal renal tissue, primary G1 and G3 tumors, 14 samples each, and 32 metastases from RCC patients. Expression profiles were created using oligonucleotide microarrays. Significant gene expression differences (P < .05) were identified among normal kidney, primary tumor, and metastases. For all filtered genes, univariate survival analysis was carried out. Genes for which lower expression was significantly associated with longer survival were further analyzed using multivariate analysis. Expression of the best candidate markers was further validated in an independent cohort of 55 primary tumors using quantitative real-time polymerase chain reaction. RESULTS: Fifty-nine genes exhibited increased expression in primary RCC compared with normal kidney, and in metastases compared with primary tumors. In univariate or multivariate survival analysis, upregulation of 15 genes was significant. Expression of 8 genes was validated by quantitative real-time polymerase chain reaction. Survival analysis in an independent cohort of 55 RCC patients based on expression in primary RCC showed that TOP2A (hazard ratio [HR] = 4.3, P = .005), HELLS (HR = 3.7, P = .007), ATAD2 (HR = 3.7, P = .019), and TET3 (HR = 2.8, P = .035) represent independent predictors for cancer-specific survival. The proteins encoded by these genes function as topoisomerase, helicase, chromatin modifier, and methyl cytosine dioxygenase, respectively. They are involved in proliferation, transcription, and epigenetic modification. CONCLUSION: High mRNA levels of TOP2A, HELLS, ATAD2, and TET3 are independent predictors of poor outcome in RCC patients and may be used for individual risk-adapted therapy in the future.

Expression and clinicopathological correlations of retinoid acid receptor responder protein 1 in renal cell carcinomas.

AIM: To evaluate the expression and prognostic value of RARRES1 at protein level in renal cell carcinoma (RCC). MATERIALS & METHODS: Expression profile of RARRES1 was analyzed in 903 documented RCC followed by clinicopathological correlations and survival analysis. RESULTS: RARRES1 expression was seen in 72.5% of RCC. A stronger RARRES1 expression was seen in high grade compared with low grade RCC (p < 0.001). Logrank tests revealed shorter overall survival in RARRES1 positive RCC (p = 0.006) and in pT1/2 tumors with RARRES1 expression (p = 0.002). CONCLUSION: The variable expression profile in low and high grade RCC may reflect and confirm the differences of previous gene expression analysis. There was a significant prognostic value of RARRES1 expression in patients with RCC, especially in pT1/2 tumors.

Pneumocystis pneumonia (PCP) and Pneumocystis jirovecii carriage in renal transplantation patients: a single-centre experience.

BACKGROUND: The Pneumocystis pneumonia is an increasing problem in transplanted patients: up to 25% suffer from Pneumocystis pneumonia, occurring during the first 6 months after transplantation. METHODS: From 2001 to 2009, we investigated 21 patients with pneumonia after renal transplantation for the presence of Pneumocystis jirovecii. The laboratory diagnosis was established by Grocott and Giemsa staining methods and Pneumocystis-specific mitochondrial transcribed large subunit nested polymerase chain reaction (PCR). The PCR was also used for the differentiation of Pneumocystis pneumonia from Pneumocystis carriage. RESULTS: Of 21 patients, 7 had a Pneumocystis pneumonia, 6 were Pneumocystis carriers and 8 patients were negative. Four out of seven Pneumocystis pneumonia patients and two out of six patients with Pneumocystis carriage had a delayed graft function. An acute cytomegalovirus infection after transplantation was not detectable in the patients with Pneumocystis pneumonia, but in three patients with Pneumocystis carriage. CONCLUSIONS: Pneumocystis pneumonia was present in 33.3% of transplanted patients with suspected pneumonia. An association between acute rejection or co-infections and Pneumocystis pneumonia or carriage in patients after renal transplantation cannot be excluded. In three out of seven Pneumocystis pneumonia patients, an overlapping of hospitalisation times and an onset of Pneumocystis pneumonia 6 months after transplantation was found. Thus, person-to-person transmission seems probable in these cases.

C-kit overexpression is not associated with KIT gene mutations in chromophobe renal cell carcinoma or renal oncocytoma.

INTRODUCTION: C-kit overexpression has previously been described in chromophobe renal cell carcinoma (cpRCC) and renal oncocytoma (RO). However, so far no KIT mutations have been found. The objective of our study was to analyse c-kit in a large cohort of renal tumors and to perform KIT mutation analysis in a subset cpRCC and RO cases with overexpression of c-kit. MATERIALS AND METHODS: We studied the immunohistochemical expression of c-kit on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 948 patients with renal tumors. CpRCC and RO cases with c-kit overexpression (n=23) were analyzed for KIT mutations in exons 9, 11, 13, 14, 15, and 17. RESULTS: Expression of c-kit was found in 6/642 (0.9%) clear cell RCC, 3/154 (1.9%) papillary RCC, 54/69 (78.3%) cpRCC, 37/45 (82.2%) RO and 2/30 (6.7%) of other unclassified tumor types. In none of the RO and cpRCC cases analyzed, a KIT gene mutation was found. CONCLUSION: C-kit expression is found in the majority of cpRCC and RO, but these tumors do not harbor the usual c-kit activating mutations. This may have implications for the use of tyrosine kinase inhibitors in patients with advanced cpRCC and c-kit expression.

Expression profiling of metastatic renal cell carcinoma using gene set enrichment analysis.

OBJECTIVE: To identify complex changes in cell biology occurring during metastatic progression of renal cell carcinoma using a novel gene expression analysis algorithm. METHODS: Whole genome expression profiling was carried out on 32 snap-frozen samples of clear-cell renal cell carcinoma metastases, 29 primary tumors (14 low grade, 15 high grade) and 14 samples of normal kidney tissue using oligonucleotide microarrays. These data were analyzed with the gene set enrichment analysis method, which is able to detect even small, but significant, expression changes in functionally connected genes that cannot be shown by gene-by-gene comparisons. RESULTS: There were 95 gene sets (pathways) with significant upregulation in metastases compared with normal kidney tissue (P < 0.01), and 77 gene sets with significant downregulation, respectively. Low-grade and high-grade tumors showed deregulation of various pathways that have previously not been described in renal cell carcinoma. There were significant changes of genes involved in cell cycle control, apoptosis, cell motility, metabolism, cell adhesion and cytoskeleton. Some promising new potential therapy targets were identified in renal cell carcinoma metastases; for example, aurora-kinase A and flap structure-specific endonuclease 1. CONCLUSION: Expression profiling of metastatic renal cell carcinoma using the gene set enrichment analysis pathway analysis method provides new and detailed insights in alterations occurring in renal cell carcinoma during malignant transformation and progression. These data can help to develop new and specifically targeted renal cell carcinoma therapies.

Prognostic and diagnostic implications of epithelial cell adhesion/activating molecule (EpCAM) expression in renal tumours: a retrospective clinicopathological study of 948 cases using tissue microarrays.

OBJECTIVE: To evaluate the expression and prognostic value of epithelial cell adhesion/activating molecule (EpCAM) in a large set of renal cell carcinomas (RCCs) using a tissue microarray (TMA) approach. MATERIAL AND METHODS: We studied the immunohistochemical expression and overexpression of EpCAM on TMAs containing formalin-fixed, paraffin-embedded samples of 948 patients with documented renal tumours. EpCAM expression was defined as the presence of a specific membranous staining in >5% of the tumour cells. EpCAM overexpression was specified by calculating a total staining score (score range 0-12) as the product of a proportion score and an intensity score, and defined as a score >4. RESULTS: Of 948 cases, 927 (97.8%) were evaluable morphologically (haematoxylin and eosin stain). EpCAM expression was found in 233/642 (36.3%), 126/155 (81.3%), 54/68 (78.3%), 17/45 (37.8%), 13/30 (43.3%) of clear-cell RCC, papillary RCC (pRCC), chromophobe RCC (cpRCC), oncocytomas and other unclassified tumour types, respectively. Log-rank tests showed a significantly longer overall survival (OS [P = 0.047]) and a trend of EpCAM expression to be associated with a longer progression-free survival (PFS) in all RCC entities (P = 0.065). EpCAM overexpression was significantly correlated with a better PFS in all RCC subtypes, cpRCC and pRCC (P = 0.011, 0.043 and 0.025, respectively). In multivariate analysis EpCAM overexpression was an independent marker for longer PFS in all RCC entities as well as in high grade RCC (P = 0.009 and P = 0.010, respectively). CONCLUSIONS: The histological subtypes associated with a high rate of EpCAM expression were cpRCC and pRCC. This retrospective analysis demonstrated a trend towards longer OS and PFS for all major RCC subtypes. EpCAM expression had significant prognostic value in patients with cpRCC and pRCC. Furthermore, EpCAM overexpression in high grade RCC may be a helpful marker for prognostication.

Expression and prognostic significance of a comprehensive epithelial-mesenchymal transition gene set in renal cell carcinoma.

PURPOSE: Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance. MATERIALS AND METHODS: We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis. RESULTS: The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma. CONCLUSIONS: Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.

Development of a rat model for investigation of experimental splinted uretero-ureterostomy, ureteral stenting and stenosis.

BACKGROUND AND AIM: The objective of this study was to establish a rat model to develop hypertrophic fibrosis for subsequent safe application of ureteral stents in order to investigate new treatment options for ureteral strictures. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats were used. Group 1: Sham surgery; group 2: surgery with uretero-ureteral anastomosis and stenting. Histopathological evaluation was carried out using 5-bromo-2-deoxyuridine administration before the animals were sacrificed. RESULTS: A total of thirty-one animals reached the final end-point. The most common surgical complications were urine extravasation and stent dislocations. Histological examination showed full regeneration of urothelium after 28 days and development of a scarring process. With stent insertion, moderate hypertrophia was seen. In contrast, the sham group had no evidence of significant scarring or stricture formations. CONCLUSION: Our rat model allows for investigation of the wound healing processes of urothelium of the ureteral wall and the study of the application of new miniature stents as drainage and drug carriers.

Prognostic value of intraoperative renal tissue oxygenation measurement on early renal transplant function.

Ischemia time is a prognostic factor in renal transplantation for postoperative graft function and survival. Kidney transplants from living donors have a higher survival rate than deceased donor kidneys probably because of shorter ischemia time. We hypothesized that measurement of intraoperative kidney oxygenation (µHbO(2) ) and microvascular perfusion predicts postoperative graft function. We measured microvascular hemoglobin oxygen saturation by reflectance spectrophotometry and microcirculatory kidney perfusion by laser Doppler flowmetry 5 and 30min after kidney reperfusion on the organ surface in 53 renal transplant patients including 19 grafts from living donors. These values were related to systemic hemodynamics, cold ischemia time (cit), early postoperative graft function and length of hospital stay. µHbO(2) improved 30 min after reperfusion compared to 5 min (from 67% to 71%, P < 0.05). µHbO(2) correlated with mean arterial blood pressure and central venous pH (P < 0.01). Most importantly, µHbO(2) was significantly higher in kidneys from living compared with deceased donors (74% vs. 63%) and in kidneys without vs. with biopsy-proven postoperative rejection (71% vs. 45%, P < 0.001). Finally, µHbO(2) correlated positively with cit and postoperative creatinine clearance and negatively with postoperative plasma creatinine, need for hemodialysis and length of hospital stay. Our results suggest higher oxygen extraction and thus oxygen demand of the grafts shortly after reperfusion. The intraoperative measurement of tissue oxygenation in kidney transplants is predictive of early postoperative graft function. Future studies should evaluate the potential effect of intraoperative therapeutic maneuvers to improve organ tissue oxygenation in renal transplantation.

Gene expression analysis in clear cell renal cell carcinoma using gene set enrichment analysis for biostatistical management.

OBJECTIVE: To improve the workflow for standardizing the statistical interpretation provides an opportunity for the analysis of gene expression in clear cell renal cell carcinoma (ccRCC). RCC as a solid tumour entity represents a very suitable tumour model for such investigations. Although it is possible to investigate expression profiles by microarray technologies, the main problem is how to adequately interpret the accumulated mass of data derived from microarray technologies. There is a clear lack of a defined, consistent and comparable biostatistical analysis system, with no specific biostatistical standard methodology being available to compare the results of microarray analyses. We used the gene set enrichment analysis (GSEA) method to analyze microarray data from RCC tissue. The present study aimed to analyze differential expression profiles and establish biomarkers suitable for prognostication at the time of renal surgery by comparing RCC patients with long-term survival data against RCC samples of patients with poorly differentiated (grade 3) RCC, concomitant metastatic disease and short survival. PATIENTS AND METHODS: In the present study, a total of 29 ccRCC fresh-frozen tissue samples were used; 14 samples from grade 1 (G1) RCC patients without metastatic disease and 15 from grade 3 (G3) RCC patients with synchronous metastatic disease. Expression profiling was performed with the Human Genome U133 Plus 2.0 Array (Affymetrix Corp., Santa Clara, CA, USA). Clinical data and long-term follow-up were obtained for all patients. The primary probe level analysis was performed using the Affymetrix MAS 5 algorithm. Further statistical processing was carried out by GSEA, using the Molecular Signatures Database, MSigDB (http://www.broad.mit.edu/gsea/msigdb/index.jsp). After selecting gene sets with the highest leading edge subsets, a cluster and a further analyses based on MSigDB data bank analysis was performed. RESULTS: In total, 15 poorly G3 ccRCC, 14 well differentiated G1 ccRCC and 14 normal renal tissue samples were analyzed for comparative gene expression profiling. There were 12 of 15 G3 ccRCC patients who had synchronous metastatic disease at the time of surgery (pN+ and/or distant metastases: pN+ only = 4, M+ only = 11 and pN+M+ = 3). The GSEA identified 700 gene sets. Out of these, 120 sets with the highest leading edge subset were selected monitored by hierarchical clustering G1 vs G3. Comparative analysis using the the MSigDB data bank for pathway network identified 16 gene sets that were differentially strongly over- or underexpressed in G3 vs G1 tumours and are involved in various aspects of tumour physiology, such as metastases and cell motility, signalling and cell proliferation, as well as gene products that are involved in the building of the extracellular matrix and as cell surface markers. CONCLUSIONS: We analyzed microarray data of gene expression in ccRCC comparing poorly differentiated and well differentiated tumour tissue samples. Using GSEA, we found a number of genes set candidates relevant to biological network processes with high complexity; conspicuously, these comprised members of the interleukin- and chemokine-family, cyclin-dependent kinases, angiogenic growth factors and transcriptional factors. This suggests that, in poorly differentiated aggressive ccRCC, there may be a limited number of gene sets that are responsible for the very aggressive biological behaviour. This comparison performed at a gene set level enables the identification of such congruency between different gene sets and whole data sets with respect to a specific biological question. GSEA embedded in the statistical workflow procedure for the suitable preparation of expression data may improve the analysis and avoid missing changes at the molecular level. A systematic approach such as GSEA is clearly needed to analyze raw data from microarray analyses, although these data can only be descriptive and the mass of raw data is derived from a relatively small number of tissue samples. However, consistent alterations of gene expression found in specific tumour entities may allow a better understanding of certain aspects of specific tumour biology. Therefore, the molecular characterization of individual tumours may potentially be useful for the better individual assessment of prognosis and, finally, the identification of biomarkers and targets of specific treatments may eventually help to improve treatment.