An automated, sensitive, high-throughput biomarker protocol for tissue microarrays containing archival prostate specimens: The prognostic potential of an ERG EMT panel.

Abstract

181 Background: Tissue microarrays (TMAs), linked to clinical databases containing clinical outcome data, offer a great potential for defining and validating new biomarkers in urological oncology. The long term follow up required for true clinical outcome prostate cancer results in the use of archival material for TMA construction. The use of such material has inherent problems which must be overcome to unlock the full potential of prostate TMAs. Methods: A prostate 2,450 core TMA containing normal, prostatic intraepithelial neoplasia and malignant prostate tissue from 524 archival patient specimens was constructed and analysed. Each core was independently verified histologically. An automated, high throughput fluorescent staining (Leica BOND-MAX), image capture (Carl Zeiss Microimaging Mirax scanner) and scoring (Definiens Tissue Studio) protocol was compared with standardised conventional staining and manual scoring techniques. Results: Image analysis showed excellent correlation (r=0.785) and absolute agreement between the automated and manual scoring protocols (ICC=0.769, 95% CI [0.629-0.877]). Replicate analysis demonstrated that the automated, high throughput protocol was more reliable and consistent than the standardised manual staining and scoring protocols. The increased sensitivity of fluorescent staining in the automated protocol highlighted significant (p>0.0001) antigen degradation (pan-cytokeratin), hence poorly persevered regions, in 50.7% of the archival TMA cores. Using well preserved cores the potential of combining Erg expression with EMT markers as a prognostic marker will be presented. Conclusions: This study shows that archival TMAs can be utilised for biomarker characterisation and validation. However there are caveats to their use, namely antigen preservation. The use of multicolour automated protocols, which utilise a house keeping antigen, circumvents these caveats and releases the full potential of TMAs in biomarker research.