mRNA and micro-RNA expression analysis in laser-capture microdissected prostate biopsies: Valuable tool for risk assessment and prevention trials
Introduction
Despite advances in the diagnosis and treatment, prostate cancer (PCa) remains the second leading cause of cancer-related deaths in men. The vast majority of PCa are detected by needle biopsies that provide a random and sparse sample of prostate tissue, which is archived in paraffin after formalin fixation. Although RNA is degraded in formalin-fixed paraffin-embedded (FFPE) tissue, recent studies have shown that meaningful gene and micro-RNA (miRNA) expression data can be obtained from these specimens (Ayala et al., 2003, Calin and Croce, 2006, Dakhova et al., 2009). The ability to analyze RNA expression in FFPE-bpx provides a useful tool for risk stratification for negative biopsies, identification of prognostic markers in positive biopsies and could allow gene expression profiling to become an important surrogate endpoint in PCa prevention trials.
Prostate biopsies contain a mixture of histological entities (stroma, benign epithelium, cancer, pre-malignancy, and atrophy). Therefore, it is imperative to separate well-characterized epithelial areas from the stroma by laser-capture microdissection (LCM) prior to RNA analysis. Shukla et al. (Shukla et al., 2008) used LCM to obtain reliable gene expression markers for epithelial and stromal compartments in radical prostatectomy (RP) and TURP samples. However, only one study to date combined LCM with analysis of the expression of gene transcripts in formalin-fixed prostate biopsies (Ayala et al., 2003). Although that study reported plausible results for expression of genes that are specifically associated with stroma, benign epithelium and PCa, the only direct comparison of FFPE to frozen tissue was made in tissue from a xenograft model and the number of genes was limited.
Here we report validation of RNA expression analysis in FFPE biopsies by comparison to the gold standard, frozen tissue obtained at radical prostatectomy (RP) from the same patient. We isolated pure populations of normal epithelium, normal stroma, and PCa epithelium and then compared matched FFPE and frozen tissue for gene expression of several compartment-specific and PCa-related mRNAs. We also examined the expression of several PCa-related miRNAs in normal and malignant epithelium in the matched sample types. Lastly, to determine whether our technique could quantify expression of mRNAs and miRNAs in multi-target panels, we compared fixed and frozen samples from the same patient using PCR arrays.
Section snippets
Prostate tissue
Archival formalin-fixed paraffin-embedded (FFPE) prostate needle biopsy (Bpx) specimens were used. Needle biopsy cores (18 G), eight per patient, were fixed in formalin immediately and paraffin embedded within 24 h. Biopsy specimens were collected from February to July 2007 and paired fresh-frozen tissue was acquired at radical prostatectomy (< 6 months from biopsy) by a pathologist during the gross examination and dissection of specimens. Tissue was snap frozen and stored at − 80 °C. Frozen
LCM-collected RNA
Three pairs of prostate specimens were selected from an existing set of radical prostatectomy patients. From each patient, we compared gene expression profiling from their pre-surgical FFPE prostate needle biopsies (FFPE-bpx) to their frozen surgically-removed radical prostatectomy tissue (frozen-RP). The patients were 53–56 years of age with similar Gleason scores (6–7), pre-operative PSA, identical clinical stage, and race. LCM was performed to separately collect the normal epithelium, normal
Discussion
In this study we directly compared mRNA and miRNA expression from LCM-collected FFPE and frozen tissues from the same patient. It was important to fully validate expression analysis from FFPE-RNA since the RNA is highly fragmented and not an ideal RT-PCR template. We found that small amplicon size (60–75 bp) TaqMan® assays worked well since non-specific primer–dimer products are not detected. Our previous attempts with small amplicon primer pairs and SYBR green resulted in an abundance of
Conclusions
We show that despite all of the challenges, with careful attention to technique and a full understanding of the limitations, expression profiling of mRNA gene sets by PCR arrays is readily achievable from archived biopsy material. MiRNA expression profiling from archived biopsies produced equivalent results to data obtained from frozen specimens, further supporting the utility of miRNAs as biomarkers in archived specimens.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank Vijayalakshmi Ananthanaryanan for providing her services as a pathologist in selecting the normal and tumor areas of the tissues, Erika Enk for her assistance in specimen selection, Dr. Debra Tonetti for the preamp primers and mRNA PCR array, and Dr. Charles J. Bieberich for the NKX3.1 antibody. This research was supported by NIH-NCI R03 CA131595 (Nonn) and NIH-NCI RO1 CA90759 (Gann).
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