Evaluation of Prognostic Parameters to Identify Aggressive Penile Carcinomas.

BACKGROUND: Advanced penile carcinoma is characterized by poor prognosis. Most data on prognostic factors are based on small study cohorts, and even meta-analyses are limited in patient numbers. Therefore, there is still a lack of evidence for clinical decisions. In addition, the most recent TNM classification is questionable; in line with previous studies, we found that it has not improved prognosis estimation. METHODS: We evaluated 297 patients from Germany, Russia, and Portugal. Tissue samples from 233 patients were re-analyzed by two experienced pathologists. HPV status, p16, and histopathological parameters were evaluated for all patients. RESULTS: Advanced lymph node metastases (N2, N3) were highly significantly associated with reductions in metastasis-free (MFS), cancer-specific (CS), and overall survival (OS) rates (p = <0.001), while lymphovascular invasion was a significant parameter for reduced CS and OS (p = 0.005; p = 0.007). Concerning the primary tumor stage, a significant difference in MFS was found only between pT1b and pT1a (p = 0.017), whereas CS and OS did not significantly differ between T categories. In patients without lymph node metastasis at the time of primary diagnosis, lymphovascular invasion was a significant prognostic parameter for lower MFS (p = 0.032). Histological subtypes differed in prognosis, with the worst outcome in basaloid carcinomas, but without statistical significance. HPV status was not associated with prognosis, either in the total cohort or in the usual type alone. CONCLUSION: Lymphatic involvement has the highest impact on prognosis in penile cancer, whereas HPV status alone is not suitable as a prognostic parameter. The pT1b stage, which includes grading, as well as lymphovascular and perineural invasion in the T stage, seems questionable; a revision of the TNM classification is therefore required.

[Importance of HPV status and p16 for the prognosis of penile carcinoma]. / Bedeutung von HPV-Status und p16 für die Prognose des Peniskarzinoms.

BACKGROUND: Penile cancer is a rare but often lethal tumour disease, especially in the metastatic stage. Most data on prognostic factors for penile cancer are based on small patient cohorts, and even meta-analyses are mostly limited in terms of patient numbers. There is a lack of sufficient parameters to predict the metastatic risk of these tumours. Furthermore, the role of the HPV status for the prognosis, and, in this regard, of p16INK4a is still unclear. MATERIAL AND METHODS: In this study, 236 patients from an international multicentre cohort were analysed with regard to histological subtypes, HPV and p16 status, and other clinical parameters. The HPV status was only graded as HPV-positive if HPV was detected by PCR and the p16 status defined by immunochemistry was positive. The statistical analysis was carried out using the Kaplan-Meier method as well as the log-rank test and a univariable and multivariable analysis using the Cox regression model. RESULTS: A positive HPV status was not a significant parameter for either metastasis-free (MFS), tumour-specific (CSS) or overall survival (OS). p16-positive tumours showed a significantly better MFS (p=0.026), which was also confirmed in the subgroup analysis of HPV-negative tumours (p=0.037) without differences in CSS or OS. In the usual type, there was also a trend towards an improved MFS, but without statistical significance (p=0.070). p16-positive tumours were associated with a highly significantly better MFS (hazard ratio 0.3; p=0.004) in the multivariable Cox regression, while patients with a pT1b tumour stage or advanced lymph node metastasis showed a significantly worse survival. In the multivariable analysis of HPV-negative tumours, p16 status was also confirmed as an independent predictor of MFS (Hazard ratio 0.2; p=0.007). CONCLUSION: HPV status alone seems to be lacking prognostic relevance. In contrast, p16 status was confirmed as an independent prognostic factor. Thus, the expression of p16INK4a is associated with a significantly better MFS. Especially in HPV-negative tumours, the p16 status should be evaluated with regard to the prognostic value and thus also with a view to the treatment decision.

The prominent role of the S100A8/S100A9-CD147 axis in the progression of penile cancer.

Currently, no established biomarkers are recommended for the routine diagnosis of penile carcinoma (PeCa). The rising incidence of this human papillomavirus (HPV)-related cancer entity highlights the need for promising candidates. The Calprotectin subunits S100A8 and S100A9 mark myeloid-derived suppressor cells in other HPV-related entities while their receptor CD147 was discussed to identify patients with PeCa at a higher risk for poor prognoses and treatment failure. We thus examined their expression using immunohistochemistry staining of PeCa specimens from 74 patients on tissue microarrays of the tumor center, the invasion front, and lymph node metastases. Notably, whereas the tumor center was significantly more intensively stained than the invasion front, lymph node metastases were thoroughly positive for both S100 subunits. An HPV-positive status combined with an S100A8+S100A9+ profile was related with an elevated risk for metastases. We observed several PeCa specimens with S100A8+S100A9+-infiltrating immune cells overlapping with CD15 marking neutrophils. The S100A8+S100A9+CD15+ profile was associated with dedifferentiated and metastasizing PeCa, predominantly of HPV-associated subtype. These data suggest a contribution of neutrophil-derived suppressor cells to the progression of HPV-driven penile carcinogenesis. CD147 was elevated, expressed in PeCa specimens, prominently at the tumor center and in HPV-positive PeCa cell lines. CD147+HPV+ PeCa specimens were with the higher-frequency metastasizing cancers. Moreover, an elevated expression of CD147 of HPV-positive PeCa cell lines correlated negatively with the susceptibility to IgA-based neutrophil-mediated tumor cell killing. Finally, stratifying patients regarding their HPV/S100A8/S100A9/CD15/CD147 profile may help identify patients with progressing cancer and tailor immunotherapeutic treatment strategies.

Diagnostics and prognostic evaluation in renal cell tumors: the German S3 guidelines recommendations.

The German guidelines on renal cell carcinoma (RCC) have been developed at highest level of evidence based on systematic literature review. In this paper, we are presenting the current recommendations on diagnostics including preoperative imaging and imaging for stage evaluation as well as histopathological classification. The role of tumor biopsy is further discussed. In addition, different prognostic scores and the status of biomarkers in RCC are critically evaluated.

DKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells.

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear ß-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.

miRNA Expression Characterizes Histological Subtypes and Metastasis in Penile Squamous Cell Carcinoma.

Although microRNAs are described as promising biomarkers in many tumor types, little is known about their role in PSCC. Thus, we attempted to identify miRNAs involved in tumor development and metastasis in distinct histological subtypes considering the impact of HPV infection. In a first step, microarray analyses were performed on RNA from formalin-fixed, paraffin-embedded tumor (22), and normal (8) tissue samples. Microarray data were validated for selected miRNAs by qRT-PCR on an enlarged cohort, including 27 tumor and 18 normal tissues. We found 876 significantly differentially expressed miRNAs (p ≤ 0.01) between HPV-positive and HPV-negative tumor samples by microarray analysis. Although no significant differences were detected between normal and tumor tissue in the whole cohort, specific expression patterns occurred in distinct histological subtypes, such as HPV-negative usual PSCC (95 differentially expressed miRNAs, p ≤ 0.05) and HPV-positive basaloid/warty subtypes (247 differentially expressed miRNAs, p ≤ 0.05). Selected miRNAs were confirmed by qRT-PCR. Furthermore, microarray data revealed 118 miRNAs (p ≤ 0.01) that were significantly differentially expressed in metastatic versus non-metastatic usual PSCC. The lower expression levels for miR-137 and miR-328-3p in metastatic usual PSCC were validated by qRT-PCR. The results of this study confirmed that specific miRNAs could serve as potential diagnostic and prognostic markers in single PSCC subtypes and are associated with HPV-dependent pathways.

The HPV and p63 Status in Penile Cancer Are Linked with the Infiltration and Therapeutic Availability of Neutrophils.

Squamous penile cancer displays a rare human papillomavirus (HPV)-associated tumor entity. Investigations on the molecular pathogenesis of HPV-driven penile cancer are impaired by the rareness of clinical specimens and, in particular, are missing relevant cell culture models. Here, we identified in HPV-positive penile cancer cell lines that HPV16 oncoproteins control TP63 expression by modulating critical regulators, while integration into the TP63 open reading frame facilitates oncogene expression. The resulting feed-forward loop leads to elevated p63 levels that in turn enhance the release of the neutrophil-recruiting chemokine CXCL8. Remarkably, elevated CXCL8 amounts lead to the increased surface exposition of the Fc receptor of human IgA antibodies, FcαRI, on neutrophils and correlated with a higher susceptibility to antibody-dependent neutrophil-mediated cytotoxicity (ADCC) using an EGFR-specific IgA2 antibody. IHC staining of tissue microarrays proved that elevated expression of p63 together with neutrophil infiltration were significantly more frequent in HPV-positive penile cancer displaying a higher tumor grade. In summary, we identified a promising marker profile of patients with penile cancer at higher risk for worse prognosis. However, these patients may benefit from immunotherapeutic approaches efficiently engaging neutrophils for tumor cell killing.

Challenging the prognostic impact of the new WHO and TNM classifications with special emphasis on HPV status in penile carcinoma.

The evidence concerning prognostic parameters for clinical decision-making in penile cancer is either weak or missing. We therefore analysed the prognostic value of the revised TNM and WHO classification systems on relapse and survival with special emphasis on HPV status. We collected clinical data and tissue samples of 121 patients from centres in Germany and Russia. HPV genotyping and p16INK4a immunostaining were performed. The histological subtype and TNM were reclassified by two experienced uropathologists. Survival analyses were performed by Kaplan-Meier estimator and log-rank test. Uni- and multivariable analyses were performed by Cox proportional hazard model and Fisher's exact test for contingency analysis. HPV status was not found to be an independent prognostic factor. Histological subtypes differ in prognosis with the best outcome found in warty and the worst in basaloid carcinomas. Patients with pT1b defined by poor differentiation or lymphovascular invasion (LVI) had the shortest metastasis-free survival compared with pT1a (log-rank, p = 0.02). Lymph node metastasis and LVI were significantly associated with poor metastasis-free, cancer-specific and overall survival and could be identified as the only independent prognostic parameters. Prognostic value of TNM could not be improved using the 8th versus the 7th edition. In contrast to HPV status, histological subtypes are of prognostic value and should be an essential part of pathologic reports. The impact of the HPV status needs to be analysed in a subtype-specific manner. Parameters describing lymphatic dissemination have the highest impact on prognosis. Inclusion of tumour grade and LVI into a single T-category (pT1b) seems questionable.

A three-gene methylation marker panel for the nodal metastatic risk assessment of muscle-invasive bladder cancer.

PURPOSE: In this study, we aimed to identify a DNA methylation pattern suitable for prognosis assessment of muscle-invasive bladder cancer and to investigate metastasis-associated processes regulated by DNA methylation. METHODS: Genome-wide methylation analysis was performed on 23 muscle-invasive bladder tumors by microarray analysis. Validation was performed by the qAMP technique in two different patient cohorts (n = 32 and n = 100). mRNA expression was analyzed in 12 samples. Protein expression was determined using tissue microarrays of 291 patients. Bladder cancer cell lines T24 and 253JB-V were used for functional analyses. RESULTS: Microarray analyses revealed KISS1R, SEPT9 and CSAD as putative biomarkers with hypermethylation in node-positive tumors. The combination of the three genes predicted the metastatic risk with sensitivity of 73% and specificity of 71% in cohort 1, and sensitivity of 82% and specificity of 54% in cohort 2. mRNA expression differences were detected for KISS1R (p = 0.04). Protein expression of KISS1R was significantly reduced (p < 0.001). Knockdown of SEPT9v3 resulted in increased cell migration by 28% (p = 0.04) and increased invasion by 22% (p = 0.004). KISS1R overexpression resulted in decreased cell migration (25%, p = 0.1). CONCLUSIONS: We identified a methylation marker panel suitable to differentiate between patients with positive and negative lymph nodes at time of cystectomy. This enables a risk assessment for patients who potentially benefit from extended lymph node resection as well as from neoadjuvant chemotherapy and could improve the survival rates. Furthermore, we examined the impact of putative markers on tumor behavior. Hence, KISS1R and SEPT9 could represent a starting point for the development of novel therapy approaches.

The Overall Survival Benefit for Patients with T1 Renal Cell Carcinoma after Nephron-Sparing Surgery Depends on Gender and Age.

Due to the recommendations in the urological guidelines to perform nephron-sparing surgery in patients with organ-confined renal cell carcinoma (RCC), the customary therapy regimen changed, but it is not well studied yet whether partial nephrectomy (PN) especially in the elderly is beneficial. From 2000 to 2015, 3,592 patients from 7 clinics undergoing surgery in RCC were identified; 2,323 had T1 tumours. We retrospectively compared the overall survival benefit of patients with T1 RCC who underwent either PN or radical nephrectomy (RN) and studied effects of age and gender. RESULTS: In T1 RCC, PN was beneficial in male patients (p = 0.0006) independent of age, especially in those men ≤75 years of age (p = 0.0005); but PN was not beneficial for female patients (p = 0.0629) regardless of age and male patients older than 75 years (p = 0.736). The OS of female patients after RN and male patients after PN is the same, regardless of age. A life expectancy of more than 45 months at least is necessary to experience an overall survival benefit after PN. CONCLUSIONS: There should be harder proven indications for PN in female patients and especially in all patients older than 75 years, particularly with regard to perioperative risk factors.

Survival advantage of partial over radical nephrectomy in patients presenting with localized renal cell carcinoma.

BACKGROUND: Partial nephrectomy (PN) preserves renal function and has become the standard approach for T1a renal cell carcinoma (RCC). However, there is still an ongoing debate as to which patients will actually derive greater benefit from partial than from radical nephrectomy (RN). The aim of this study was to retrospectively evaluate the impact of the type of surgery on overall survival (OS) in patients with localized RCC. METHODS: Renal surgery was performed in 4326 patients with localized RCC (pT ≤ 3a N/M0) at six German tertiary care centers from 1980 to 2010: RN in 2955 cases (68.3%), elective (ePN) in 1108 (25.6%), and imperative partial nephrectomy (iPN) in 263 (6.1%) cases. The median follow-up for all patients was 63 months. Kaplan-Meier and Cox regression analyses were carried out to identify prognosticators for OS. RESULTS: PN was performed significantly more often than RN in patients presenting with lower tumor stages, higher RCC differentiation, and non-clear cell histology. Accordingly, the calculated 5 (10)-year OS rates were 90.0 (74.6)% for ePN, 83.9 (57.5)% for iPN, and 81.2 (64.7)% for RN (p < 0.001). However, multivariate analysis including age, sex, tumor diameter and differentiation, histological subtype, and the year of surgery showed that ePN compared to RN still qualified as an independent factor for improved OS (HR 0.79, 95% CI 0.66-0.94, p = 0.008). CONCLUSION: Even allowing for the weaknesses of this retrospective analysis, our multicenter study indicates that in patients with localized RCC, PN appears to be associated with better OS than RN irrespective of age or tumor size.

FISH analysis of washing urine from the upper urinary tract for the detection of urothelial cancers.

INTRODUCTION: To evaluate FISH analysis of washing urine from the upper urinary tract (UUT) in comparison with cytology (Cyt) for the detection of urothelial cancers. PATIENTS AND METHODS: In 82 patients with symptoms or abnormalities of the UUT sampling of washing urine for FISH and Cyt and a stepwise diagnostic work-up (e.g. retrograde ureteropyelography, ureterorenoscopy and endoscopic biopsy) were performed. In case of endoscopically and/or histologically proven malignancy patients either underwent nephroureterectomy, partial ureterectomy or local treatment. Sensitivity and specificity for FISH and Cyt as well as its combination were determined. RESULTS: Urothelial cancer of the UUT was detected in 20 patients. Eleven patients underwent nephroureterectomy, six partial ureterectomy and three endoscopic tumour treatment. This revealed nine pTa, three pT1 and seven muscle-invasive tumours. Twelve tumours were classified as low and seven as high-grade tumours. In one patient with a macroscopic unequivocal finding of tumour, endoscopic laser ablation without histologic confirmation was performed. FISH was evaluable in 76 patients and detected 16 tumours with a sensitivity and specificity of 84.2 and 91.1 %, respectively. Cyt was performed in 79 and was evaluable in 78 patients. It detected ten tumours with a sensitivity and specificity of 52.6 and 91.4 %, respectively. Cyt and FISH together detected 19 tumours with (sensitivity 100 % and specificity 83.6 %). CONCLUSION: FISH was more sensitive than and equally specific to Cyt in the detection of urothelial cancers of the UUT. Both markers in combination revealed the best sensitivity, making it a possible approach in future settings.

Urine protein profiling identified alpha-1-microglobulin and haptoglobin as biomarkers for early diagnosis of acute allograft rejection following kidney transplantation.

PURPOSE: Early diagnosis of acute rejection and effective immunosuppressive therapy lead to improvement in graft survival following kidney transplantation. In this study, we aimed to establish a urinary protein profile suitable to distinguish between patients with rejection and stable graft function and to predict acute rejection based on postoperatively collected urine samples. A further objective was to identify candidate proteins for the use as biomarkers in clinical practice. METHODS: Urine samples of 116 kidney recipients were included. Rejection was proven by biopsy (n = 58), and stable transplant function was monitored for at least 2 years (n = 58). Postoperative urine samples were collected between 3rd and 10th day following transplantation. Urinary protein profiles were obtained by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein identification and validation were performed using multiplex fluorescence 2DE, peptide mass fingerprinting and enzyme-linked immunosorbent assay. RESULTS: A protein profile including four mass peaks differentiated acute rejection from stable transplants at the time point of rejection and at the postoperative state with 73 % sensitivity and 88 % specificity. Alpha-1-microglobulin (A1MG) and Haptoglobin (Hp) were identified as putative rejection biomarkers. Protein levels were significantly higher in postoperative urine from patients with rejection (A1MG 29.13 vs. 22.06 µg/ml, p = 0.001; Hp 628.34 vs. 248.57 ng/ml, p = 0.003). The combination of both proteins enabled the diagnosis of early rejection with 85 % sensitivity and 80 % specificity. CONCLUSION: Protein profiling using mass spectrometry is suitable for noninvasive detection of rejection-specific changes following kidney transplantation. A specific protein profile enables the prediction of early acute allograft rejection in the immediate postoperative period. A1MG and Hp appear to be reliable rejection biomarkers.

MicroRNAs with prognostic potential for metastasis in clear cell renal cell carcinoma: a comparison of primary tumors and distant metastases.

BACKGROUND: MicroRNAs (miRNAs) are regulators of gene expression in tumor development and progression. However, their influence on metastasis of clear cell renal cell carcinoma (ccRCC) is less understood. To determine the role of miRNAs in metastatic progression, miRNA expression in primary ccRCC was compared to distant metastases. METHODS: Total RNA of 53 primary ccRCCs, 35 distant metastases from lung, bone, brain, and abdomen, as well as 17 normal kidney tissues was isolated from fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) samples. The miRNA microarrays were performed based on fresh frozen tissue. Results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on fresh frozen tissue and FFPE samples. Real-time cell analyses and transwell invasion assays were carried out after transient transfection of microRNA-30c (miR-30c) in cell line 786-O. RESULTS: There were 14 miRNAs differently expressed in metastatic primary ccRCC and distant metastases compared to non-metastatic primary tumors. A strong correlation of miRNAs to progression-free- and cancer-specific 5-year-survival was determined. Specific miRNAs were differently expressed in distant metastases compared to primary ccRCC. A miRNA signature distinguished lung metastases from other metastatic sites. Overexpression of miR-30c increased adherence and decreased migration and invasion in the ccRCC cell line. CONCLUSIONS: MiRNAs are deregulated in metastatic primary ccRCC and could be promising prognostic markers for an early prediction of metastasis. Alterations in miRNA expression characterize distant metastases of different metastatic sites. Furthermore, our study suggests a functional role of miR-30c in metastasis. The miRNAs could be a helpful tool for individual follow-up prediction and personalized therapy selection.

Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells.

Epithelial-mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRß) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRß in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRß. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRß to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFß1 induced myofibroblasts were the strongest attractants, while aFGF or TGFß1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects.

Differential vascular expression and regulation of oncofetal tenascin-C and fibronectin variants in renal cell carcinoma (RCC): implications for an individualized angiogenesis-related targeted drug delivery.

The study was aimed at determining the vascular expression of oncofetal fibronectin (oncfFn) and tenascin-C (oncfTn-C) isoforms in renal cell carcinoma (RCC) and its metastases which are well-known targets for antibody-based pharmacodelivery. Furthermore, the influence of tumour cells on endothelial mRNA expression of these molecules was investigated. Evaluation of vascular ED-A(+) and ED-B(+) Fn as well as A1(+) and C(+) Tn-C was performed after immunofluorescence double and triple staining using human recombinant antibodies on clear cell, papillary and chromophobe primary RCC and metastases. The influence of hypoxic RCC-conditioned medium on oncfFn and oncfTn-C mRNA expression was examined in human umbilical vein endothelial cells (HUVEC) by real time RT-PCR. There are RCC subtype specific expression profiles of vascular oncfFn and oncfTn-C and corresponding patterns when comparing primary tumours and metastases. Within one tumour, there are different vessel populations with regard to the incorporation of oncfTn-C and oncfFn into the vessel wall. In vitro tumour-derived soluble mediators induce an up regulation of oncfTn-C and oncfFn mRNA in HUVEC which can be blocked by Avastin(®). Vascular expression of oncFn and oncTn-C variants depends on RCC subtype and may reflect an individual tumour stroma interaction or different stages of vessel development. Therefore, oncFn or oncTn-C variants can be suggested as molecular targets for individualized antibody based therapy strategies in RCC. Tumour-derived VEGF could be shown to regulate target expression.

Identification of genomic alterations associated with metastasis and cancer specific survival in clear cell renal cell carcinoma.

PURPOSE: We identified regions of DNA copy number changes that are significantly associated with metastasis and clinical outcome in patients with clear cell renal cell carcinoma. MATERIALS AND METHODS: We analyzed 53 primary clear cell renal cell carcinomas, including 31 metastasized and 22 nonmetastasized tumors, by array comparative genomic hybridization with a median resolution of 1 to 1.5 Mbp. To validate copy number aberrations with potential prognostic value we performed fluorescence in situ hybridization analysis using commercially available fluorescent probes. RESULTS: We identified 5 recurrent chromosomal aberrations that were significantly associated with metastasis, including gains of 1q21.3, 12q13.12, 12q13.3q14.1 and 20q11.21q13.2, and loss of 9p21.3p24.1. The most prominent of them with the highest OR for metastatic risk were loss of 9p21.3p24.1, and gains of 1q21.3 and 20q11.21q13.32. Eight alterations involving chromosomes 7, 9, 12, 16 and 20 significantly correlated with shortened cancer specific survival. The lowest p values on Kaplan-Meier analysis showed losses of 9p21.3p24.1 and 9q32q33.1, and gains of 7q36.3 and 20q11.21q13.32. Fluorescence in situ hybridization done in the same cohort for the 4 select regions 1q21.3, 7q36.3, 9p21.3p24.1 and 20q11.21q13.32 clearly confirmed the results of array comparative genomic hybridization. CONCLUSIONS: Data suggest that specific chromosomal alterations in clear cell renal cell carcinoma can be used to predict metastasis and cancer specific survival in patients with clear cell renal cell carcinoma. It seems possible to design a combined fluorescence in situ hybridization assay based on these genetic targets for outcome prediction, which can be used for routine diagnostics.

Specific protein patterns characterize metastatic potential of advanced bladder cancer.

PURPOSE: The prognosis in patients with metastasized bladder cancer is still poor. Clinical and histopathological parameters have limited ability to predict the risk of tumor progression. Thus, we identified specific protein patterns associated with tumor progression to differentiate specimens with and without metastasis. MATERIALS AND METHODS: We analyzed 46 metastasized and 42 nonmetastasized muscle invasive bladder cancers by ProteinChip® technology surface enhanced laser desorption/ionization time of flight mass spectrometry. Cell lysis was done after laser capture microdissection from cryostat sections to achieve high tumor cell purity. Surface enhanced laser desorption/ionization time of flight mass spectrometry was completed with 2 matrices (Q10 and CM10). Bioinformatic analysis was performed by XLMiner® clustering using the Fuzzy c-means method. Differentially expressed proteins were identified and verified by 2-dimensional gel electrophoresis, tryptic in gel digest, peptide mapping, immunodepletion assay and Western blot analysis. RESULTS: By combining data on 2 chip surfaces (Q10 and CM10) results showed 86% sensitivity and 89% specificity in the training set, and 63% sensitivity and 88% specificity in the validation set. The relevant protein peaks 10.83, 14.68, 16.15 and 27.85 Da were identified as S100A8, MAP-1LC3, MUC-1S1 and GST-M1, respectively. CONCLUSIONS: We defined specific protein patterns with ProteinChip technology using bioinformatic evaluation software, which allowed differentiation between nonmetastasized and metastasized bladder tumor samples with high sensitivity and specificity. We identified 4 differentially expressed proteins. Thus, it seems possible to identify patients at high metastasized risk even at a clinically localized stage, leading to individual therapy decisions.

A specific gene expression signature characterizes metastatic potential in clear cell renal cell carcinoma.

PURPOSE: The discovery of metastasis markers in clear cell renal cell carcinoma is of critical importance to define individual metastatic risk and select patients for new targeted therapies. We identified potential biomarkers for metastatic clear cell renal cell carcinoma by gene expression analysis. MATERIALS AND METHODS: We performed transcriptional profiling of 16 primary metastatic and 18 nonmetastatic clear cell renal cell carcinomas with PIQOR™ microarrays. Differentially expressed genes were validated by quantitative real-time polymerase chain reaction. RESULTS: Genes discriminating between metastatic and nonmetastatic tumors were identified at q <0.001 by significance analysis of microarrays. The metastatic signature contained 127 transcripts. In metastatic samples a greater than 4-fold decrease in expression was detected for the genes CD151 and IKBA (t/F statistic p <0.0001) while the genes MMP16, B7-H1, BCL2L2 and FRA2 showed greater than 4-fold increase of expression in metastatic primary tumors (p <0.0001). Quantitative real-time polymerase chain reaction revealed significant differences in expression among all metastatic tumors, including synchronously and metachronously metastasized tumors, and nonmetastatic tumors for FRA2 (p = 0.032) and CD151 (p = 0.005). In addition, the genes B7-H1 (p = 0.040), FRA2 (p = 0.035), CD151 (p = 0.004) and BCL2L2 (p = 0.035) showed significantly higher expression in early metastasized than in nonmetastatic tumor samples. Different B7-H1 (p = 0.002) and BCL2L2 (p = 0.007) expression levels were found in samples with late metastasis compared to those in synchronously metastasized tumors. CONCLUSIONS: We determined a metastatic signature of clear cell renal cell carcinoma by microarray analysis. Our data provide the possibility of defining the metastatic potential of primary clear cell renal cell carcinoma based on a select number of genes even in a localized situation.