Due to the widespread use of PSA testing, diagnosis of PCa typically relies on needle biopsies, which provide sparse, random sampling of the prostate. About 70-80% of these biopsies are negative for cancer, and the non-malignant tissue samples, which may contain valuable risk information, are routinely archived in paraffin after formalin fixation. Although RNA is degraded in formalin-fixed paraffin-embedded (FFPE) tissue, recent studies have shown that, with adequate attention to methodological detail, meaningful gene and microRNA (miRNA) expression data can be obtained from these specimens. We have optimized and validated several methods to overcome the challenges of working with prostate biopsies, which requires the ability to work with extremely limited amounts of material as well as the ability to isolate relatively homogeneous cell populations. miRNA signatures are rapidly being identified for many cancers including prostate cancer. Compared to mRNAs, miRNAs are highly stable in FFPE tissue due their small size, and thus may be ideal biomarkers in prostate biopsies. Epithelial and stromal tissues were collected from FFPE prostate biopsies by laser capture microdissection (LCM) from three patients. Tissue was LCM-collected in a similar manner from paired frozen tissue from the same patients. Total RNA was isolated and the expression of mRNA and miRNAs analyzed by qRT-PCR (mention PreAmp). Expression of 7 epithelial or stromal-specific genes (K18, PSA, NKX3.1, AMACR, IGF1, TIMP3, Desmin) showed agreement between the FFPE-biopsies and the frozen tissue in all three patients. Normal and PCa tissue was available for two patients. Expression of three PCa-related genes (PCA3, AMACR and NKX3.1) also showed agreement between the FFPE-biopsies and frozen tissue. As predicted, PCA3 and AMACR were elevated and NKX3.1 decreased in PCa relative to normal areas. Analysis of three miRNAs reported to be down-regulated in PCa (miR-125b, miR-22, miR-16) showed that these miRNAs have higher expression in the stromal compartment. However, normal epithelium and PCa showed similar levels of miRNA expression. The difference between our methodology and the previously published reports for these miRNAs and PCa is that we collected the tissue by LCM. This suggests that previous findings showing decreased expression of these miRNAs in PCa may be an artifact resulting from decreased stromal tissue in the PCa lesions. This is the first study to directly compare RNA expression from FFPE and frozen tissues from the same patient and also the first study to examine miRNA expression in LCM-collected tissue from prostate biopsies. We show that with this methodology, RNA profiling of mRNA and miRNA from FFPE-biopsy material is quite feasible. More importantly, in the small set of samples, RNA expression in the FFPE-biopsy was reflective of the whole prostate and the relative expression was similar to frozen material. Our goal is to apply this methodology to archival prostate biopsy banks in order to identify RNA markers predictive of PCa. These methods will also facilitate measurement of RNA markers in prostate cancer prevention clinical trials in which pre and post-biopsy material has been collected.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):A69.

Seventh AACR International Conference on Frontiers in Cancer Prevention Research-- Nov 16-19, 2008; Washington, DC