Abstract 2886: Simultaneous profiling of multiple endogenous signaling regulators in microdissected grade specific tumour populations derived from archival diagnostic prostate cancer needle biopsies
Authors: JOSEPH AJAY , Kachroo Naveen , Murphy Tania , Warren Ann Y. , Gnanapragasam Vincent J. 
Published: June 2012

Abstract
Background: There is emerging evidence that endogenous signaling regulators are altered in the transition from benign to malignant cells. These regulators have been implicated in cancer progression across diverse tumour types. Signalling regulators however have primarily been previously studied in isolation. Here we report simultaneous expression analysis of a panel of signalling regulators in tumours derived from archival formal fixed paraffin embedded (FFPE) diagnostic prostate tissue biopsies.
Methods: Archival FFPE diagnostic prostate needle biopsies were subjected to laser microdissection to isolate grade specific tumour populations. An optimised protocol was established to extract RNA from 24 individual biopsies (n=6 each; Gleason Grade 3, 4, 5 and benign epithelium). Total RNA was converted to cDNA and a pre-amplification step performed using protocols validated for FFPE derived degraded RNA. Expression of a multi-gene panel was tested by real time PCR. Spred protein expression was studied by optimised immunohistochemistry.
Results: Expression of housekeeping genes (RPL13, GAPDH and β-actin) and 18S ribosomal RNA was detected in all samples within acceptable Ct ranges. In epithelial samples there was no detectable expression of the stromal marker COL6A3. All cancers expressed significantly higher levels of the tumour markers PCA3, KI67 EZH2, and E2F3. We next tested expression of a panel of endogenous signalling regulators (Sef, Sprouty 2, RKIP, DUSP1, Spred 1 & 2). Expression of Sef, DUSP1 and RKIP showed a progressive reduction in expression with increasing cancer grade. Sprouty 2 expression was retained in grade 3 and 4 tumours but reduced in grade 5 tumours. Spred 1 & 2 expression has not previously been reported in prostate cancer. In this analysis we observed comparable levels of Spred 2 in benign and grade 3 tumours but significantly lower levels in grade 4 & 5 tumours. Unexpectedly, we observed increased levels of Spred 1 in tumours compared to benign samples. This converse trend was also observed in interrogation of Spred 1 & 2 mRNA expression using the MSKCC c-Bio Cancer Genomics portal. We further validated this observation at the protein level in a panel of benign (n=93) and malignant (n=143) cancer biopsies.
Conclusion: In this study we demonstrate the study of multiple genes in microdissected archival diagnostic FFPE prostate needle biopsies. Our findings report the first parallel profiling of multiple signalling regulators in prostate cancer and first data on specific alterations in Spred 1 & 2 expression in different tumour grades.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2011-2886