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Quantitative determination of predictive cancer biomarkers in formalin-fixed, paraffin-embedded tissue using a new, fully automated method for RNA isolation.

11032 Background: Personalized cancer therapy depends on the evaluation of tissue-based biomarkers in routine tumor samples. As many new biomarkers are measured on the mRNA level, standardized procedures for mRNA analysis in formalin-fixed, paraffin-embedded (FFPE) tissue are needed. In this study, we present a novel, standardized, and fully automated method for fast isolation of total RNA from FFPE tissue sections.


METHODS
Tissue samples (n = 501) from 167 breast carcinomas which had been stored between 2 months and 21 years were investigated. Total RNA was extracted from tissue sections using a new method based on silica-coated iron oxide beads in combination with a specific liquid-handling robot. Novel and unique features of the method are an automated extraction-integrated deparaffinization step and a bead-based negative selection step to eliminate any undigested tissue. Yield of RNA was assessed using the Ribogreen assay. mRNA fragment lengths were estimated by reverse transcription PCR (RT-PCR) for G6PDH. Expression of the breast cancer biomarkers ESR1, PGR and HER2 was measured by kinetic RT-PCR (kPCR) and compared with immunohistochemistry (IHC).


RESULTS
RNA was successfully isolated from all samples, with a mean yield of 1.4 μg/sample (range: 0.1-7.8 μg) and fragment lengths of at least 150 bp in 99% of samples. PCR analyses could be performed in all samples. Investigating three sections of each tumor, we observed a low section-to-section variability of kPCR results (root of mean squared errors of relative ESR1, PGR or HER2 expression in three sections: 0.2-0.5 Ct values). Comparing kPCR results with IHC, we detected a good concordance between both methods, with agreements of 98.4% (standard error [SE]: 2.7%) for ESR1, 84.4% (SE: 5.3%) for PGR and 89.9% (SE: 8.9%) for HER2.


CONCLUSIONS
This novel RNA extraction method is a major technical improvement for implementation of reproducible, high-throughput and cost-efficient testing of cancer biomarkers in the clinical routine and in gene-expression research studies using archived FFPE material in molecular labs. [Table: see text].