Matthias Maruschke

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.

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 (μHbO2) 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 30 min 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. μHbO2 improved 30 min after reperfusion compared to 5 min (from 67% to 71%, P < 0.05). μHbO2 correlated with mean arterial blood pressure and central venous pH (P < 0.01). Most importantly, μHbO2 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, μHbO2 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

What’s known on the subject? and What does the study add?

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

To identify complex changes in cell biology occurring during metastatic progression of renal cell carcinoma using a novel gene expression analysis algorithm.

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.

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.

Putative biomarker genes for grading clear cell renal cell carcinoma.

Objective: The initial objective of this renal cancer study was to identify gene sets in clear cell renal cell carcinoma (ccRCC) to support grading of ccRCC histopathology. Materials and Methods: Preselected ccRCC tumor tissues of grade 1 (G1, n = 14) and grade 3 (G3, n = 15) as well es 14 normal kidney tissues thereof were subjected to microarray expression analysis using Human Genome U133 Plus 2.0 Array. Event ratio scoring, hierarchical clustering and principal component analysis were used to determine gene sets that distinguish expression profiles from normal kidney tissue, G1 and G3 tumor tissues. Results: An initial set of 73 genes provided seven gene subclusters (SC01 to SC07) that distinguish RNA expression profiles from G1, G3 tumor and normal kidney tissues. A ranked list of 24 genes was determined that separated G1 from G3 tumors in high concordance with histopathological grading confirmed by immunohistochemical analysis of ceruloplasmin protein expression. Conclusion: A final set of 24 genes has been determined awaiting further validation on the RNA as well as on the protein level by studying an additional cohort of ccRCC patients. A reliable separation of G1 and G3 tumor grades will be instrumental to foster and direct the administration of upcoming targeted therapeutics of ccRCC tumors in a more predictive and reliable manner.

Traumatic Penile Injuries – Mechanisms and Treatment

Injuries of the penis are uncommon but represent urological emergencies. We report 3 cases of penile trauma illustrating the diversity of penile injury presentation. Radiological investigations are only mandatory if there is any suspicion of a urethral injury. Primary surgical approach is recommended in cases of penile fracture or local wound infection. An immediate operative exploration with removal of hematoma and primary defect closure should be done in every case. Surgical management should not be delayed to avoid late complications.

Immunohistochemical expression of retinoblastoma protein and p16 in renal cell carcinoma.

Introduction: The regulation proteins retinoblastoma protein (pRb) and p16 play an important role in the cell cycle as tumor suppressors. pRb is the main substrate for the function of cyclin-dependent kinases during the cell cycle in the transition from G1 to S phase. In this study, the immunohistochemical expression of pRb and p16 in renal cell carcinoma (RCC) were examined. Methods: Paraffin-embedded specimens from 94 patients with RCC were examined immunohistologically, using primary antibodies for p16 and pRb (Novocastra) and a biotin-conjugated anti-mouse IgG secondary antibody. Microscopically, the expression of p16 and pRb was evaluated by examination of the staining intensity of 100 cells of each specimen, and compared with epidemiological parameters (tumor size, TNM, nuclear grade and follow-up). Statistical analyses were conducted by SPSS, version 15.0 (SPSS® Inc., Chicago, Ill., USA), the χ2 test (Fisher’s exact test), the Kaplan-Meier method and Mantel’s log rank test. Results: All 94 tumors showed a positive reaction for pRb (weakly positive in 67.0%; strongly positive in 33.0%). p16 was expressed in only 52.1% (weakly positive in 48.9%; intermediately positive in 3.2%; no strongly positive expression). The expression of p16-positive tumors was significantly associated with the expression of pRb (p = 0.040). Tumor size, grading, lymph node and distant metastases did not correlate with p16/pRb expression. Conclusion: pRb and p16 control the cell cycle as tumor suppressors. Therefore, in many tumors they are dysregulated. There are distinct differences in expression in various individual RCC. However, in a limited number of cases there was no significant correlation with clinical parameters.

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.

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.