The development of optimized
therapy strategies against malignant
tumors is critically dependent on the assessment of tissue-based
biomarkers in routine diagnostic tissue samples. We investigated a novel, fully automated, and
xylene-free method for
RNA isolation and
biomarker determination using
formalin-fixed
paraffin-embedded (FFPE) tissue. The aim was to show that this approach is feasible and gives results that are comparable to the current
gold standards. Expression of the
breast cancer biomarkers ESR1, PGR, and HER2 was measured in a total of 501 FFPE tissue samples from 167
breast carcinomas, which had been stored for up to 21 years. Total
RNA was extracted from tissue sections and
biomarker expression was measured by kinetic RT-PCR (RT-kPCR). The results of the new method were compared with immunohistochemistry as the current gold standard.
RNA was successfully isolated from all samples, with a mean yield of 1.4 μg/sample and fragment lengths of at least 150 bp in 99% of samples. RT-kPCR analysis of ESR1, PGR, and HER2 was possible in all samples. Comparing RT-kPCR results with standard IHC, we found a good concordance for ESR1 (agreement: 98.4%), PGR (84.4%), and HER2 (89.8%). We observed a low section-to-section variability of kPCR results for all 3
biomarkers (root of mean squared errors: 0.2 to 0.5 Ct values). The new approach is a reliable high-throughput instrument for standardized testing of
biomarkers in clinical routine and for research studies on archived FFPE material up to 21 years old. For the assessment of ESR1, PGR, and HER2 the results are comparable to the current gold-standard.