Emmprin and survivin predict response and survival following cisplatin-containing chemotherapy in patients with advanced bladder cancer.

PURPOSE: Cisplatin-containing chemotherapy is the standard of care for patients with locally advanced and metastatic transitional cell carcinoma of the urothelium. The response rate is approximately 50% and tumor-derived molecular prognostic markers are desirable for improved estimation of response and survival. EXPERIMENTAL DESIGN: Affymetrix GeneChip expression profiling was carried out using tumor material from 30 patients. A set of genes with an expression highly correlated to survival time after chemotherapy was identified. Two genes were selected for validation by immunohistochemistry in an independent material of 124 patients receiving cisplatin-containing therapy. RESULTS: Fifty-five differentially expressed genes correlated significantly to survival time. Two of the protein products (emmprin and survivin) were validated using immunohistochemistry. Multivariate analysis identified emmprin expression (hazard ratio, 2.23; P < 0.0001) and survivin expression (hazard ratio, 2.46; P < 0.0001) as independent prognostic markers for poor outcome, together with the presence of visceral metastases (hazard ratio, 2.62; P < 0.0001). In the clinical good prognostic group of patients without visceral metastases, both markers showed significant discriminating power as supplemental risk factors (P < 0.0001). Within this group of patients, the subgroups of patients with no positive, one positive, or two positive immunohistochemistry scores (emmprin and survivin) had estimated 5-year survival rates of 44.0%, 21.1%, and 0%, respectively. Response to chemotherapy could also be predicted with an odds ratio of 4.41 (95% confidence interval, 1.91-10.1) and 2.48 (95% confidence interval, 1.1-5.5) for emmprin and survivin, respectively. CONCLUSIONS: Emmprin and survivin proteins were identified as strong independent prognostic factors for response and survival after cisplatin-containing chemotherapy in patients with advanced bladder cancer.

Gene expression signatures predict outcome in non-muscle-invasive bladder carcinoma: a multicenter validation study.

PURPOSE: Clinically useful molecular markers predicting the clinical course of patients diagnosed with non-muscle-invasive bladder cancer are needed to improve treatment outcome. Here, we validated four previously reported gene expression signatures for molecular diagnosis of disease stage and carcinoma in situ (CIS) and for predicting disease recurrence and progression. EXPERIMENTAL DESIGN: We analyzed tumors from 404 patients diagnosed with bladder cancer in hospitals in Denmark, Sweden, England, Spain, and France using custom microarrays. Molecular classifications were compared with pathologic diagnosis and clinical outcome. RESULTS: Classification of disease stage using a 52-gene classifier was found to be highly significantly correlated with pathologic stage (P < 0.001). Furthermore, the classifier added information regarding disease progression of T(a) or T(1) tumors (P < 0.001). The molecular 88-gene progression classifier was highly significantly correlated with progression-free survival (P < 0.001) and cancer-specific survival (P = 0.001). Multivariate Cox regression analysis showed the progression classifier to be an independently significant variable associated with disease progression after adjustment for age, sex, stage, grade, and treatment (hazard ratio, 2.3; P = 0.007). The diagnosis of CIS using a 68-gene classifier showed a highly significant correlation with histopathologic CIS diagnosis (odds ratio, 5.8; P < 0.001) in multivariate logistic regression analysis. CONCLUSION: This multicenter validation study confirms in an independent series the clinical utility of molecular classifiers to predict the outcome of patients initially diagnosed with non-muscle-invasive bladder cancer. This information may be useful to better guide patient treatment.

A molecular signature in superficial bladder carcinoma predicts clinical outcome.

PURPOSE: Cancer of the urinary bladder is a common malignant disease in the western countries. The majority of patients presents with superficial tumors with a high recurrence frequency, a minor fraction of these patients experience disease progression to a muscle invasive stage. No clinical useful molecular markers exist to identify patients showing later disease progression. The purpose of this study was to identify markers of disease progression using full-genome expression analysis. EXPERIMENTAL DESIGN: We did a full-genome expression analysis (59,619 genes and expressed sequence tags) of superficial bladder tumors from 29 bladder cancer patients (13 without later disease progression and 16 with later disease progression) using high-density oligonucleotide microarrays. We used supervised learning for identification of the optimal genes for predicting disease progression. The identified genes were validated on an independent test set (74 superficial tumor samples) using in house-fabricated 60-mer oligonucleotide microarrays. RESULTS: We identified a 45-gene signature of disease progression. By monitoring this progression signature in an independent test set, we found a significant correlation between our classifications and the clinical outcome (P < 0.03). The genes identified as differentially expressed were involved in regulating apoptosis, cell differentiation, and cell cycle and hence may represent potential therapeutic targets. CONCLUSIONS: Our results indicate that it may be possible to identify patients with a high risk of disease progression at an early stage using a molecular signature present already in the superficial tumors. In this way, better treatment and follow-up regimens could be assigned to patients suffering from superficial bladder cancer.

Role of activating fibroblast growth factor receptor 3 mutations in the development of bladder tumors.

PURPOSE: Bladder tumors develop through different molecular pathways. Recent reports suggest activating mutations of the fibroblast growth factor receptor 3 (FGFR3) gene as marker for the "papillary" pathway with good prognosis, in contrast to the more malignant "carcinoma in situ" (CIS) pathway. The aim of this clinical follow-up study was to investigate the role of FGFR3 mutations in bladder cancer development in a longitudinal study. EXPERIMENTAL DESIGN: We selected 85 patients with superficial bladder tumors, stratified into early (stage T(a)/grade 1-2, n = 35) and more advanced (either stage T(1) or grade 3, n = 50) developmental stages. The patients were followed prospectively, and metachronous tumors were included. We did screening for FGFR3 and TP53 mutations by direct bidirectional sequencing and for genome-wide molecular changes with microarray technology. RESULTS: A total of 43 of 85 cases (51%) showed activating mutations of FGFR3. The mutations were associated with papillary tumors of early developmental stage. However, after stratifying for developmental stage, FGFR3-mutated tumors showed the same malignant potential as wild-type tumors. Tumors with concomitant CIS were generally FGFR3 wild type. They were characterized by different patterns of chromosomal changes and gene expression signatures compared with FGFR3-mutated tumors, indicating different molecular pathways. CONCLUSIONS: FGFR3 mutations seem to have a central role in the early development of papillary bladder tumors. These tumors follow a common molecular pathway, which is different from tumors with concomitant CIS. FGFR3 mutations do not seem to play a role in bladder cancer progression.

Gene expression in the urinary bladder: a common carcinoma in situ gene expression signature exists disregarding histopathological classification.

The presence of carcinoma in situ (CIS) lesions in the urinary bladder is associated with a high risk of disease progression to a muscle invasive stage. In this study, we used microarray expression profiling to examine the gene expression patterns in superficial transitional cell carcinoma (sTCC) with surrounding CIS (13 patients), without surrounding CIS lesions (15 patients), and in muscle invasive carcinomas (mTCC; 13 patients). Hierarchical cluster analysis separated the sTCC samples according to the presence or absence of CIS in the surrounding urothelium. We identified a few gene clusters that contained genes with similar expression levels in transitional cell carcinoma (TCC) with surrounding CIS and invasive TCC. However, no close relationship between TCC with adjacent CIS and invasive TCC was observed using hierarchical cluster analysis. Expression profiling of a series of biopsies from normal urothelium and urothelium with CIS lesions from the same urinary bladder revealed that the gene expression found in sTCC with surrounding CIS is found also in CIS biopsies as well as in histologically normal samples adjacent to the CIS lesions. Furthermore, we also identified similar gene expression changes in mTCC samples. We used a supervised learning approach to build a 16-gene molecular CIS classifier. The classifier was able to classify sTCC samples according to the presence or absence of surrounding CIS with a high accuracy. This study demonstrates that a CIS gene expression signature is present not only in CIS biopsies but also in sTCC, mTCC, and, remarkably, in histologically normal urothelium from bladders with CIS. Identification of this expression signature could provide guidance for the selection of therapy and follow-up regimen in patients with early stage bladder cancer.

Construction and validation of the APOCHIP, a spotted oligo-microarray for the study of beta-cell apoptosis.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a autoimmune disease caused by a long-term negative balance between immune-mediated beta-cell damage and beta-cell repair/regeneration. Following immune-mediated damage the beta-cell fate depends on several genes up- or down-regulated in parallel and/or sequentially. Based on the information obtained by the analysis of several microarray experiments of beta-cells exposed to pro-apoptotic conditions (e.g. double stranded RNA (dsRNA) and cytokines), we have developed a spotted rat oligonucleotide microarray, the APOCHIP, containing 60-mer probes for 574 genes selected for the study of beta-cell apoptosis. RESULTS: The APOCHIP was validated by a combination of approaches. First we performed an internal validation of the spotted probes based on a weighted linear regression model using dilution series experiments. Second we profiled expression measurements in ten dissimilar rat RNA samples for 515 genes that were represented on both the spotted oligonucleotide collection and on the in situ-synthesized 25-mer arrays (Affymetrix GeneChips). Internal validation showed that most of the spotted probes displayed a pattern of reaction close to that predicted by the model. By using simple rules for comparison of data between platforms we found strong correlations (rmedian= 0.84) between relative gene expression measurements made with spotted probes and in situ-synthesized 25-mer probe sets. CONCLUSION: In conclusion our data suggest that there is a high reproducibility of the APOCHIP in terms of technical replication and that relative gene expression measurements obtained with the APOCHIP compare well to the Affymetrix GeneChip. The APOCHIP is available to the scientific community and is a useful tool to study the molecular mechanisms regulating beta-cell apoptosis.

Pseudo-likelihood analysis of codon substitution models with neighbor-dependent rates.

Recently, Markov processes for the evolution of coding DNA with neighbor dependence in the instantaneous substitution rates have been considered. The neighbor dependency makes the models analytically intractable, and previously Markov chain Monte Carlo methods have been used for statistical inference. Using a pseudo-likelihood idea, we introduce in this paper an approximative estimation method which is fast to compute. The pseudo-likelihood estimates are shown to be very accurate, and from analyzing 348 human-mouse coding sequences we conclude that the incorporation of a CpG effect improves the fit of the model considerably.

Hydrogen bond rotations as a uniform structural tool for analyzing protein architecture.

Proteins fold into three-dimensional structures, which determine their diverse functions. The conformation of the backbone of each structure is locally at each C(α) effectively described by conformational angles resulting in Ramachandran plots. These, however, do not describe the conformations around hydrogen bonds, which can be non-local along the backbone and are of major importance for protein structure. Here, we introduce the spatial rotation between hydrogen bonded peptide planes as a new descriptor for protein structure locally around a hydrogen bond. Strikingly, this rotational descriptor sampled over high-quality structures from the protein data base (PDB) concentrates into 30 localized clusters, some of which correlate to the common secondary structures and others to more special motifs, yet generally providing a unifying systematic classification of local structure around protein hydrogen bonds. It further provides a uniform vocabulary for comparison of protein structure near hydrogen bonds even between bonds in different proteins without alignment.

Pressure load: the main factor for altered gene expression in right ventricular hypertrophy in chronic hypoxic rats.

BACKGROUND: The present study investigated whether changes in gene expression in the right ventricle following pulmonary hypertension can be attributed to hypoxia or pressure loading. METHODOLOGY/PRINCIPAL FINDINGS: To distinguish hypoxia from pressure-induced alterations, a group of rats underwent banding of the pulmonary trunk (PTB), sham operation, or the rats were exposed to normoxia or chronic, hypobaric hypoxia. Pressure measurements were performed and the right ventricle was analyzed by Affymetrix GeneChip, and selected genes were confirmed by quantitative PCR and immunoblotting. Right ventricular systolic blood pressure and right ventricle to body weight ratio were elevated in the PTB and the hypoxic rats. Expression of the same 172 genes was altered in the chronic hypoxic and PTB rats. Thus, gene expression of enzymes participating in fatty acid oxidation and the glycerol channel were downregulated. mRNA expression of aquaporin 7 was downregulated, but this was not the case for the protein expression. In contrast, monoamine oxidase A and tissue transglutaminase were upregulated both at gene and protein levels. 11 genes (e.g. insulin-like growth factor binding protein) were upregulated in the PTB experiment and downregulated in the hypoxic experiment, and 3 genes (e.g. c-kit tyrosine kinase) were downregulated in the PTB and upregulated in the hypoxic experiment. CONCLUSION/SIGNIFICANCE: Pressure load of the right ventricle induces a marked shift in the gene expression, which in case of the metabolic genes appears compensated at the protein level, while both expression of genes and proteins of importance for myocardial function and remodelling are altered by the increased pressure load of the right ventricle. These findings imply that treatment of pulmonary hypertension should also aim at reducing right ventricular pressure.

Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro.

microRNAs (miRNA) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Here, we profiled the expression of 290 unique human miRNAs in 11 normal and 106 bladder tumor samples using spotted locked nucleic acid-based oligonucleotide microarrays. We identified several differentially expressed miRNAs between normal urothelium and cancer and between the different disease stages. miR-145 was found to be the most down-regulated in cancer compared with normal, and miR-21 was the most up-regulated in cancer. Furthermore, we identified miRNAs that significantly correlated to the presence of concomitant carcinoma in situ. We identified several miRNAs with prognostic potential for predicting disease progression (e.g., miR-129, miR-133b, and miR-518c*). We localized the expression of miR-145, miR-21, and miR-129 to urothelium by in situ hybridization. We then focused on miR-129 that exerted significant growth inhibition and induced cell death upon transfection with a miR-129 precursor in bladder carcinoma cell lines T24 and SW780 cells. Microarray analysis of T24 cells after transfection showed significant miR-129 target down-regulation (P = 0.0002) and pathway analysis indicated that targets were involved in cell death processes. By analyzing gene expression data from clinical tumor samples, we identified significant expression changes of target mRNA molecules related to the miRNA expression. Using luciferase assays, we documented a direct link between miR-129 and the two putative targets GALNT1 and SOX4. The findings reported here indicate that several miRNAs are differentially regulated in bladder cancer and may form a basis for clinical development of new biomarkers for bladder cancer.