Abstract P5-10-13: Low influence of tumor cell content on mRNA expression levels of ESR, PGR, HER2 and KI67 when performing the MammaTyper® RT-PCR kit
Authors: Wirtz Ralph M , Rau Tilman , Laible Mark , Schlombs Kornelia , Lakis Sotirios , Wachter David , Sahin Ugur , Hartmann Arndt 
Published: May 2015

Abstract
Background
Breast cancer patient management relies on approximations of molecular subtypes by immunohistochemical staining (IHC) of ER, PgR, Her2/neu and Ki-67. However, routine application of IHC is subject to important pre-analytical, analytical and interpretational variations which result in significant inaccuracy (Hammond et al. 2010, Polley et al, 2013). In contrast, quantification of biomarker RNA expression by RT-qPCR using the MammaTyper® RTqPCR kit displayed a high concordance of single marker results of 100 % HER2, 96.8 % ESR1, 97.2 % PGR and 97.6 % KI67 based on predefined cutoffs (see Laible et al, abstract submitted). However, varying tumour cell content (TCC) could possibly affect the validity of quantitative assessment of ESR1, PGR, HER2 and KI67. Herein we aimed to investigate the performance of MammaTyper® RT-qPCR kit under extreme scenarios of TCC enrichment.
Materials and Methods
Ten extreme cases with low TCC (10 - 30%) and enriched in DCIS (15 – 70%) were selected. Two RNA samples were prepared for each case using the RNXtract® IVD kit. One sample contained at most 20% TCC whereas its pair contained &gt; 80% after macrodissection by an experienced technician. ESR1, PGR, HER2 and KI67 RNA were determined using the MammaTyper® RT-qPCR kit on a Roche Light Cycler. Differences in DDCT values and coefficient of variation were used to analyze differences between non-macrodissected and macrodissected paired samples. IHC served as a reference for biomarker status evaluation.
Results
Despite the varying TCC content of invasive carcinomas the median 40-DDCT Differences of the mRNA Expression of HER2, ESR1, PGR and HER2 between non-macrodissected and macrodissected samples were 0.34, 0.46, 0.27 and 0.41, respectively. When previously established clinical cut-offs for biomarker positivity were considered, only a single case for KI67 appeared to be affected by low TCC (negation). The concordance with IHC data was 88.9% for ESR1, 100% for PR, 88.9 for HER2 and 44.5% for KI67 in this series.
Relative mRNA expression differences between non-macrodissected and macrodissected tumor specimenSample IDDCIS contentInvasive Carcinoma contentMacrodissectedDifference in ESR1 mRNA [DDCT]Difference in PGR mRNA [DDCT]Difference in HER2 mRNA [DDCT]Difference in KI67 mRNA [DDCT]110%30%&gt;80%0,650,260,650,55270%10%&gt;80%0,430,350,38-0,19340%10%&gt;80%-0,44-0,54-0,170,25430%5%&gt;80%0,46-0,980,30,59510%50%&gt;80%1,320,950,71,16620%10%&gt;80%0,160,32-0,140,58725%10%&gt;80%1,691,321,26-0,56870%15%&gt;80%0,16-0,42-0,07-0,31940%10%&gt;80%0,46-0,130,471,091040%30%&gt;80%0,520,270,280,28
Conclusion
The performance of the MammaTyper® diagnostic assay does not appear to be affected by fluctuations in the TCC of the original FFPE specimens under the presence of increased amounts of DCIS. Similar findings have been previously reported in a research setting (Kotoula, Virchows Arch 2013). Effective RNA extraction and optimal PCR output normalization provide sufficient robustness for tolerating up to 8-fold TCC specimen changes, independent of the presence of DCIS. Our analysis suggests that extra time spent on macro-dissection of specimens for routine RT-qPCR assays could be avoided with safety when using the MammaTyper® RT-qPCR kit.
Citation Format: Ralph M Wirtz, Tilman Rau, Mark Laible, Kornelia Schlombs, Sotirios Lakis, David Wachter, Ugur Sahin, Arndt Hartmann. Low influence of tumor cell content on mRNA expression levels of ESR, PGR, HER2 and KI67 when performing the MammaTyper® RT-PCR kit [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-13.