Abstract
The use of archival formalin-fixed paraffin-embedded (FFPE) material to analyse gene expression is limited by the low quality of extracted RNA. In this paper, we utilised an RNA based assay to quantify expression of luminal and basal markers, together with ERBB2 probes, in FFPE archival tissue from 2009 to 2010, all of which had clinical and therapeutic information of more than 5 years. Receptor status of the patients was characterised using the QuantiGene® Plex assay with 100% concordance to immunohistochemical (IHC) and fluorescence in situ hybridisation (FISH) results. A panel of molecular markers known to classify luminal and basal tumours were used and correlated with receptor status of the tumours. As expected, the triple negative breast cancer (TNBC) samples were classified as basal and oestrogen receptor (ER) positive cases as luminal. In summary, the QuantiGene® Plex technology provides a platform to quantitate novel panels of biomarkers on archival material. Moreover, multiplex analysis allows the use of minimal amounts of material providing an opportunity to utilise laser micro-dissected material. FFPE tissue samples are an invaluable resource for retrospective studies to interrogate current novel biomarkers, particularly to generate disease free survival and overall survival graphs to measure predictive value using well annotated retrospective samples with full clinical and pharmacological outcomes.
Highlights
Investigations using RNA from archival formalin-fixed paraffinembedded (FFPE) material is challenging due to the extent of degradation in these samples [1] and due to the processing variables during and following cut-up of the surgical samples
We evaluated the possibility to use haematoxylin and eosin (H&E) staining to microdissect tumour sites with higher precision
Criteria to select FFPE-derived RNA with sufficient quality for gene expression studies show that less than 25% of the extracted RNA could be used for microarray analysis [12]
Summary
Investigations using RNA from archival formalin-fixed paraffinembedded (FFPE) material is challenging due to the extent of degradation in these samples [1] and due to the processing variables during and following cut-up of the surgical samples. Despite improvements in quantitative PCR (qPCR) technology [3,4], the poor quality of RNA extracted from FFPE material hinders the potential of gene expression studies in this invaluable resource available in pathology archives. The branched-chain DNA (bDNA) assay QuantiGene® technology provides a platform to perform expression studies from minimal amount of archival FFPE material without amplification of target sequences [5], overcoming the limiting factor of expression studies using degraded RNA. This assay replaces enzymatic amplification of target RNA with hybridization of specific probes followed by amplification of the reporter signal, through DNA molecule scaffold formation on bound probe-target sequences. Derive the best combination of genes to normalize the target expression values and enhance the power of prediction
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