Van-Duc Luu

Mining Tissue Microarray Data to Uncover Combinations of Biomarker Expression Patterns that Improve Intermediate Staging and Grading of Clear Cell Renal Cell Cancer

Purpose: Tumor stage and nuclear grade are the most important prognostic parameters of clear cell renal cell carcinoma (ccRCC). The progression risk of ccRCC remains difficult to predict particularly for tumors with organ-confined stage and intermediate differentiation grade. Elucidating molecular pathways deregulated in ccRCC may point to novel prognostic parameters that facilitate planning of therapeutic approaches. Experimental Design: Using tissue microarrays, expression patterns of 15 different proteins were evaluated in over 800 ccRCC patients to analyze pathways reported to be physiologically controlled by the tumor suppressors von Hippel-Lindau protein and phosphatase and tensin homologue (PTEN). Tumor staging and grading were improved by performing variable selection using Cox regression and a recursive bootstrap elimination scheme. Results: Patients with pT2 and pT3 tumors that were p27 and CAIX positive had a better outcome than those with all remaining marker combinations. A prolonged survival among patients with intermediate grade (grade 2) correlated with both nuclear p27 and cytoplasmic PTEN expression, as well as with inactive, nonphosphorylated ribosomal protein S6. By applying graphical log-linear modeling for over 700 ccRCC for which the molecular parameters were available, only a weak conditional dependence existed between the expression of p27, PTEN, CAIX, and p-S6, suggesting that the dysregulation of several independent pathways are crucial for tumor progression. Conclusions: The use of recursive bootstrap elimination, as well as graphical log-linear modeling for comprehensive tissue microarray (TMA) data analysis allows the unraveling of complex molecular contexts and may improve predictive evaluations for patients with advanced renal cancer. Clin Cancer Res; 16(1); 88–98

Loss of VHL and hypoxia provokes PAX2 up-regulation in clear cell renal cell carcinoma.

Purpose: The paired box gene 2, PAX2, encodes for a transcription factor that is up-regulated during nephrogenesis and becomes silenced in mature epithelium of the glomeruli, the proximal, and distal tubules. Reactivation of PAX2 has been frequently observed in clear cell renal cell carcinoma (ccRCC), a tumor type characterized by loss of von Hippel-Lindau (VHL) tumor suppressor function. The regulation of PAX2 expression in ccRCC is unknown. Experimental Design: We applied reporter gene assays to investigate PAX2 promoter regulation. Furthermore, PAX2 expression was determined in ccRCC cell lines under normoxic and hypoxic condition in a VHL wild-type and mutated background. PAX2 expression was also assessed in 831 human ccRCC and correlated with hypoxia-inducible factor α (HIFα) and clinical parameters. Results: Here, we show that both loss of VHL protein (pVHL) function and hypoxia leads to strong PAX2 reexpression. Using luciferase reporter gene assays, no induction was obtained in spite of six hypoxia response element motifs identified in the promoter of PAX2. Comprehensive immunohistochemical analyses showed significant correlations between PAX2, HIF1α, and HIF2α—target CCND1 expression patterns in ccRCC patients. Notably, PAX2 expression was highly associated with early-stage, well-differentiated ccRCC and, consequently, better clinical outcome (P < 0.0001 each). Additional analyses indicated that PAX2 repressor WT1 and cancer-linked hypomethylation are not important for transcriptional regulation of PAX2 in ccRCC. Conclusion: We conclude that in ccRCC, PAX2 reactivation is driven by HIF-dependent mechanisms following pVHL loss.

Integrative genome-wide expression profiling identifies three distinct molecular subgroups of renal cell carcinoma with different patient outcome.

BackgroundRenal cell carcinoma (RCC) is characterized by a number of diverse molecular aberrations that differ among individuals. Recent approaches to molecularly classify RCC were based on clinical, pathological as well as on single molecular parameters. As a consequence, gene expression patterns reflecting the sum of genetic aberrations in individual tumors may not have been recognized. In an attempt to uncover such molecular features in RCC, we used a novel, unbiased and integrative approach.MethodsWe integrated gene expression data from 97 primary RCC of different pathologic parameters, 15 RCC metastases as well as 34 cancer cell lines for two-way nonsupervised hierarchical clustering using gene groups suggested by the PANTHER Classification System. We depicted the genomic landscape of the resulted tumor groups by means of Single Nuclear Polymorphism (SNP) technology. Finally, the achieved results were immunohistochemically analyzed using a tissue microarray (TMA) composed of 254 RCC.ResultsWe found robust, genome wide expression signatures, which split RCC into three distinct molecular subgroups. These groups remained stable even if randomly selected gene sets were clustered. Notably, the pattern obtained from RCC cell lines was clearly distinguishable from that of primary tumors. SNP array analysis demonstrated differing frequencies of chromosomal copy number alterations among RCC subgroups. TMA analysis with group-specific markers showed a prognostic significance of the different groups.ConclusionWe propose the existence of characteristic and histologically independent genome-wide expression outputs in RCC with potential biological and clinical relevance.

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response

The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1α. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

[Von-Hippel-Lindau gene mutation types. Association of gene expression signatures in clear cell renal cell carcinoma]

AIMS The von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC. METHODS Total RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis. RESULTS By applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations. CONCLUSIONS The results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.ZusammenfassungFragestellungDer Von-Hippel-Lindau- (VHL-)Tumorsuppressor ist ein multifunktionelles Protein. VHL-Mutationen treten häufig auf im klarzelligen Nierenzellkarzinom (kNZK). Verschiedene Mutationstypen führen vermutlich zu spezifischen pVHL-Funktionsveränderungen, die wiederum einen signifikanten Einfluss auf die Genexpression und schließlich auf den Krankheitsverlauf haben dürften. Ziel der vorliegenden Studie ist die Korrelation von Genexpressionssignaturen mit spezifischen VHL-Mutationstypen im kNZK.MethodikTranskriptomanalyse wurde für 94 kNZK und 21 papilläre NZK (pNZK) mittels Affymetrix HG U133A Genchips durchgeführt. Alle 94 kNZK wurden auf VHL-Mutationen analysiert.ErgebnisseEin „hierarchical clustering“ anhand der zwischen kNZK und pNZK differenziell regulierten Gene zeigt eine deutliche Stratifizierung der beiden histologischen Subtypen. 186 Gene wurden zwischen VHL-Wildtyp kNZK und kNZK mit mutiertem VHL-Gen differenziell exprimiert.SchlussfolgerungUnsere Resultate weisen auf eine signifikante Auswirkung von VHL-Mutationen auf die Genexpression im NZK hin.AbstractAimsThe von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC.MethodsTotal RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis.ResultsBy applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations.ConclusionsThe results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.

Von-Hippel-Lindau-Gen-Mutationstypen

AIMS The von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC. METHODS Total RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis. RESULTS By applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations. CONCLUSIONS The results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.ZusammenfassungFragestellungDer Von-Hippel-Lindau- (VHL-)Tumorsuppressor ist ein multifunktionelles Protein. VHL-Mutationen treten häufig auf im klarzelligen Nierenzellkarzinom (kNZK). Verschiedene Mutationstypen führen vermutlich zu spezifischen pVHL-Funktionsveränderungen, die wiederum einen signifikanten Einfluss auf die Genexpression und schließlich auf den Krankheitsverlauf haben dürften. Ziel der vorliegenden Studie ist die Korrelation von Genexpressionssignaturen mit spezifischen VHL-Mutationstypen im kNZK.MethodikTranskriptomanalyse wurde für 94 kNZK und 21 papilläre NZK (pNZK) mittels Affymetrix HG U133A Genchips durchgeführt. Alle 94 kNZK wurden auf VHL-Mutationen analysiert.ErgebnisseEin „hierarchical clustering“ anhand der zwischen kNZK und pNZK differenziell regulierten Gene zeigt eine deutliche Stratifizierung der beiden histologischen Subtypen. 186 Gene wurden zwischen VHL-Wildtyp kNZK und kNZK mit mutiertem VHL-Gen differenziell exprimiert.SchlussfolgerungUnsere Resultate weisen auf eine signifikante Auswirkung von VHL-Mutationen auf die Genexpression im NZK hin.AbstractAimsThe von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC.MethodsTotal RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis.ResultsBy applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations.ConclusionsThe results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.

Von-Hippel-Lindau-Gen-Mutationstypen@@@Von-Hippel-Lindau gene mutation types: Assoziation mit Genexpressionssignaturen in klarzelligen Nierenzellkarzinomen@@@Association of gene expression signatures in clear cell renal cell carcinoma

AIMS The von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC. METHODS Total RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis. RESULTS By applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations. CONCLUSIONS The results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.ZusammenfassungFragestellungDer Von-Hippel-Lindau- (VHL-)Tumorsuppressor ist ein multifunktionelles Protein. VHL-Mutationen treten häufig auf im klarzelligen Nierenzellkarzinom (kNZK). Verschiedene Mutationstypen führen vermutlich zu spezifischen pVHL-Funktionsveränderungen, die wiederum einen signifikanten Einfluss auf die Genexpression und schließlich auf den Krankheitsverlauf haben dürften. Ziel der vorliegenden Studie ist die Korrelation von Genexpressionssignaturen mit spezifischen VHL-Mutationstypen im kNZK.MethodikTranskriptomanalyse wurde für 94 kNZK und 21 papilläre NZK (pNZK) mittels Affymetrix HG U133A Genchips durchgeführt. Alle 94 kNZK wurden auf VHL-Mutationen analysiert.ErgebnisseEin „hierarchical clustering“ anhand der zwischen kNZK und pNZK differenziell regulierten Gene zeigt eine deutliche Stratifizierung der beiden histologischen Subtypen. 186 Gene wurden zwischen VHL-Wildtyp kNZK und kNZK mit mutiertem VHL-Gen differenziell exprimiert.SchlussfolgerungUnsere Resultate weisen auf eine signifikante Auswirkung von VHL-Mutationen auf die Genexpression im NZK hin.AbstractAimsThe von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC.MethodsTotal RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis.ResultsBy applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations.ConclusionsThe results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.