Prostate cancer-associated gene expression alterations determined from needle biopsies

Abstract

Purpose: To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia.

Experimental design: Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR.

Results: Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy.

Conclusions: Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.

Figure 1

The heatmap of selective genes that either have been reported to exhibit differential expression in prostate cancer (AMACR, Hepsin and GSPP1) or represent a new observation (ID1 JMJD2A). A, upregulation. B, downregulation. Red: upregulated in tumor; Green: downregulated in tumor. Row: genes; Column: patients.

Figure 2

The downregulation of tumor ID1 and ID3 mRNA was confirmed by Real-time qRT-PCR in paired tumor and benign epithelial cDNA from 31 patients. ΔCT was calculated as the difference of cycle threshold value (CT) of ID1 and ID3 relative to the CT value of GAPDH. For both genes, p < 0.001, paired-t test.

Figure 3

The mRNA level of androgen receptor in paired tumor and benign epithelium from 31 patients was measured by qRT-PCR. P = 0.103, paired-t test.

Figure 4

The mRNA levels of three AR associated histone demethylase genes in 31 patients' cancer and adjacent benign epithelium were compared by qRT-PCR. ΔCT was calculated as the difference of cycle threshold value (CT) of JMJD1A, JMJD2C and LSD1 relative to the CT value of GAPDH. The p values (indicated in the graph) were calculated by paired-t test.