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Quantitative Determination of Estrogen Receptor, Progesterone Receptor, and HER2 mRNA in Formalin-fixed Paraffin-embedded Tissue—A New Option for Predictive Biomarker Assessment in Breast Cancer

Müller, Berit Maria MD; Kronenwett, Ralf MD; Hennig, Guido PhD; Euting, Heike PhD; Weber, Karsten PhD; Bohmann, Kerstin PhD; Weichert, Wilko PhD; Altmann, Gabriela; Roth, Claudia; Winzer, Klaus-Jürgen MD; Kristiansen, Glen MD; Petry, Christoph PhD; Dietel, Manfred MD; Denkert, Carsten MD

Diagnostic Molecular Pathology: March 2011 - Volume 20 - Issue 1 - p 1–10
doi: 10.1097/PDM.0b013e3181e3630c
Original Articles

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.

*Institute of Pathology

Breast Cancer Center, Charité Hospital, Campus Mitte, Berlin

Siemens Healthcare Diagnostics Products GmbH, Molecular Research Germany, Cologne

§Institute of Pathology, University Hospital, Heidelberg

University of Düsseldorf, Universitätsstr. 1, Düsseldorf, Germany

Institute of Pathology, University Hospital, Zürich, Switzerland

Reprints: Berit Maria Müller, MD, Institute of Pathology, Charité Hospital, Campus Mitte, Charitéplatz 1, D-10117 Berlin, Germany (e-mail:

Berit Maria Müller and Ralf Kronenwett have contributed equally to this publication.

Ralf Kronenwett, Guido Hennig, Heike Euting, Karsten Weber, Kerstin Bohmann, Gabriela Altmann, Claudia Roth and Christoph Petry are employees at Siemens Healthcare Diagnostics Products GmbH, Molecular Research Germany, Nattermann Allee 12, 50829 Cologne, Germany.

© 2011 by Lippincott Williams & Wilkins.