Gene Discovery in Bladder Cancer Progression using cDNA Microarrays

Abstract

To identify gene expression changes along progression of bladder cancer, we compared the expression profiles of early-stage and advanced bladder tumors using cDNA microarrays containing 17,842 known genes and expressed sequence tags. The application of bootstrapping techniques to hierarchical clustering segregated early-stage and invasive transitional carcinomas into two main clusters. Multidimensional analysis confirmed these clusters and more importantly, it separated carcinoma in situ from papillary superficial lesions and subgroups within early-stage and invasive tumors displaying different overall survival. Additionally, it recognized early-stage tumors showing gene profiles similar to invasive disease. Different techniques including standard t-test, singlegene logistic regression, and support vector machine algorithms were applied to identify relevant genes involved in bladder cancer progression. Cytokeratin 20, neuropilin-2, p21, and p33ING1 were selected among the top ranked molecular targets differentially expressed and validated by immunohistochemistry using tissue microarrays (n ‫؍‬ 173). Their expression patterns were significantly associated with pathological stage, tumor grade, and altered retinoblastoma (RB) expression. Moreover, p33ING1 expression levels were significantly associated with overall survival. Analysis of the annotation of the most significant genes revealed the relevance of critical genes and pathways during bladder cancer progression, including the overexpression of oncogenic genes such as Transitional cell carcinomas (TCCs) of the bladder define a group of histologically and genetically diverse cancers that account for ϳ4% of all adult malignancies with an annual incidence of ϳ53,200 cases in the United States. Early-stage TCC has been classified into two groups with distinct clinical behavior and different molecular profiles. Superficial low-grade tumors (Ta) are always papillary and may recur but rarely progress, whereas high-grade tumors can be either papillary or flat lesions (Tis) and often progress to invasive disease.2 Clinically, patients diagnosed with localized stage have a 5-year relative survival rate of 93%. However, patients presenting with regional and distant stage have 5-year relative survival rates of 49% and 6%, respectively.1 Among the molecular events that characterize superficial papillary noninvasive bladder tumors are deletions affecting the long arm of chromosome 9, and activation of certain oncogenes, such as H-RAS, alterations identified only in a subset of invasive bladder neoplasms.2 Deletions of 13q at the RB locus and 17p at the TP53 locus, as well as 18q (DCC locus) and 5q (APC locus) losses have been reported in invasive bladder transitional carcinomas, but are absent in papillary noninvasive tumors.2,3 A remaining challenge in bladder cancer is to define targets characteristic of aggressive early-stage tumors before they recur or progress into invasive disease. The present study was designed to identify critical molecular targets altered along the...

Applications of array technology: identification of molecular targets in bladder cancer

“…Many data analysis tools have been described for target identification. They vary from t-test algorithms or standard hierarchical clustering (Thykjaer et al, 2001) to single logistic regression analysis, to take into account the dispersion of the data when comparing also two categories, or the application of bootstrapping techniques to evaluate the robustness of hierarchical clustering (Sanchez-Carbayo et al, 2003b). Bipartition of the data into training and validation sets and the use of supervised vector machine algorithms with or without factor analysis have been utilised for subtype classification (Sanchez-Carbayo et al, 2003b) and predictive purposes (Dyrskjot et al, 2003).…”

“…Bladder cancer studies using clinical material are also relatively limited by two studies using the oligonucleotide format (Thykjaer et al, 2001;Dyrskjot et al, 2003), and another study using cDNA microarrays (Sanchez-Carbayo et al, 2003b). These reports have focused mainly on tumour classification, finding satisfactory results to segregate superficial and invasive tumours using pools of samples representing different stages (Thykjaer et al, 2001), or individual tumours (Dyrskjot et al, 2003;Sanchez-Carbayo et al, 2003a, b).…”

“…These reports have focused mainly on tumour classification, finding satisfactory results to segregate superficial and invasive tumours using pools of samples representing different stages (Thykjaer et al, 2001), or individual tumours (Dyrskjot et al, 2003;Sanchez-Carbayo et al, 2003a, b). The first attempts to apply DNA microarrays for prediction of recurrence (Dyrskjot et al, 2003) and survival (Dyrskjot et al, 2003;Sanchez-Carbayo et al, 2003b) have recently been reported. Many clinical questions remain to be evaluated by the application of DNA microarrays in classification, prediction of overall survival, recurrence, progression, or chemosensitivity to the current intravesical treatment in superficial disease or to the chemotherapy utilised in invasive and metastatic disease.…”

“…For example, p33ING1 was found to be significantly associated with pathological stage, tumour grade, and overall survival, when validated by immunohistochemistry using tissue microarrays. Analysis of the annotation of the most significant genes revealed the relevance of critical genes and pathways during bladder cancer progression (Sanchez-Carbayo et al, 2003b).…”

Comparison of hypoxia transcriptome in vitro with in vivo gene expression in human bladder cancer

“…This contrasts with other expression arrays published for bladder cancer where a panel of four bladder cancer cell lines (Sanchez-Carbayo et al, 2003) or four normal bladder tissue samples (Dyrskjot et al, 2003) were used as reference. Using the in vitro induced genes, we were able to define for each tumour an in vivo hypoxia profile.…”