Molecular Classification of Breast Cancer Patients Using Formalin-fixed Paraffin-embedded Derived RNA Samples

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.

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

“…The described method has been applied for the simultaneous measurement of 40 transcripts in H&E stained ( Figure 1 ), microdissected ( Figure 2 ) highly degraded FFPE material. Using this method, we show the accurate characterization of receptor status ( Figure 3A ), classification of tumors into luminal and basal molecular subtypes 17 , and differential expression of the mesenchymal marker, FN1, when comparing tumor and matched control tissue ( Figure 3B ), in the various receptor positive and negative subtypes.…”

“…[Pearson Correlation p -value = 5.34E-26]. Reproduced with permission 17 . Please click here to view a larger version of this figure.…”