Comparative gene expression profiling analysis of urothelial carcinoma of the renal pelvis and bladder

Abstract

Background: Urothelial carcinoma (UC) can arise at any location along the urothelial tract, including the urethra, bladder, ureter, or renal pelvis. Although tumors arising in these various locations have similar morphology, it is unclear whether the gene expression profiles are similar between the upper-tract (ureter and renal pelvis) and lower-tract (bladder and urethra) carcinomas. Because differences may facilitate different screening and treatment modalities, we sought to examine the relationship between urothelial carcinoma of the renal pelvis (rUC) and urothelial carcinoma of the bladder (bUC).

Methods: Fresh tumor tissue was collected from patients with bUC (n = 10) and benign mucosa from the bladder of individuals undergoing resection for non-UC conditions (n = 7). Gene expression profiles from these samples were determined using high-throughput Affymetrix gene expression microarray chips. Bioinformatic approaches were used to compare the gene expression profiles of these samples with those of rUC samples and normal kidney samples that had been described previously.

Results: Using unsupervised analytic approaches, rUC and bUC were indistinguishable. Yet when a supervised analytic approach was used, a small number of differentially expressed genes were identified; these differences were most likely limited to a single pathway--the chloride ion binding activity pathway--which was more frequently activated in rUC than in bUC.

Conclusions: We found that the gene expression profiles of UCs from the upper and lower tract were extremely similar, suggesting that similar pathogenic mechanisms likely function in the development of these tumors. The differential expression of genes in the identified pathway may represent a new avenue for detection of upper-tract tumors.

Figure 1

The summarization of gene expressions grouped on physical locations of genomic cytobands, relative to normal samples from bladder. bNO: normal samples from bladder; rNO: normal samples from renal pelvis; bUC: bladder UC; rUC: renal pelvis UC. A) Unsupervised clustering result of the samples using raw summarization data. The majority of tumor and normal samples are separated, while the rUCs and bUCs are mixed with each other, showing indistinguishable summarization profiles. B) The summarization plot reflects underlying genomic imbalances in our study population and correctly identifies the most frequently observed cytogenetic alterations previously documented in UC (marked on right margin of the plot).

Figure 2

Unsupervised clustering analysis. A) Phylogenetic plot of the samples using all genes in the database (17,589 in total). B) Similar plot based on 3518 genes filtered by interquartile range (IQR > 0.9) and coefficient of variation (CV > 0.1) to remove genes with small intensity variations among the studied samples. C) Consensus plot based on the filtered genes; 500 clustering trees were generated by bootstrapped genes. The numbers on the branching lines are the percentage of times that the specific cluster occurred among the 500 trees. Normal samples from either renal pelvis or bladder always clustered with their respective groups (100%), whereas upper- and lower-tract UCs demonstrated frequent overlap. D) Heat map of SAM-selected genes that are the most differentiated between UC samples in the C3 and C4 groups identified in the unsupervised study (Figure 2A and B).

Figure 3

Supervised analysis studies to identify genes differentially expressed. A) Twenty-one genes that best distinguish UC of the renal pelvis and UC of bladder. q-values were obtained by the FDR controlling procedure on the p-values with Wilcoxon rank sum and signed rank tests. B-C) Gene expression profiles of the genes CLCA2 and GABRA, showing significantly overexpression in bUC relative to rUC (potential biomarkers for distinguishing between bUC and rUC). Bars show the median values for each class. D) Normal samples from bladder and renal pelvis, 778 genes identified, see Additional File 4: Supplementary Table S2, and E) normal bladder and combined UC samples (bUC+rUC as a single class), 558 genes. See Additional File 5: Supplementary Table S3. F) Venn diagram of gene lists in A, D, E).