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Background  Tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) breast cancer specimens may affect the accuracy of reverse transcription quantitative real-time PCR (RT-qPCR). Herein, we tested the impact of tissue heterogeneity of breast cancer specimen on the RT-qPCR-based gene expression assay MammaTyper®.    Methods  MammaTyper® quantifies the mRNA expression of the four biomarkers  ERBB2 ,  ESR1 ,  PGR , and  MKI67.  Based on pre-defined cut-off values, this molecular in vitro diagnostic assay permits binary marker classification and determination of breast cancer subtypes as defined by St Gallen 2013. In this study, we compared data from whole FFPE sections with data obtained in paired RNA samples after enrichment for invasive carcinoma via macro- or laser-capture micro-dissection.    Results  Compared to whole sections, removal of surrounding adipose tissue by macrodissection generated mean absolute 40-ddCq differences of 0.28–0.32 cycles for all four markers, with ≥90% concordant binary classifications. The mean raw marker Cq values in the adipose tissue were delayed by 6 to 7 cycles compared with the tumor-enriched sections, adding a trivial linear fold change of 1.0078 to 1.0156. Comparison of specimens enriched for invasive tumor with whole sections with as few as 20% tumor cell content resulted in mean absolute differences that remained on average below 0.59 Cq. The mean absolute difference between whole sections containing up to 60% ductal carcinoma in situ (DCIS) and specimens after dissection of DCIS was only 0.16–0.25 cycles, although there was a tendency for higher gene expression in DCIS. Observed variations were related to small size of samples and proximity of values to the limit of detection.    Conclusion  Expression of  ESR1, PGR, ERBB2  and  MKI67  by MammaTyper® is robust in clinical FFPE samples. Assay performance was unaffected by adipose tissue and was stable in samples with as few as 20% tumor cell content and up to 60% DCIS.    Electronic supplementary material  The online version of this article (10.1186/s13000-018-0760-6) contains supplementary material, which is available to authorized users.

Robustness of biomarker determination in breast cancer by RT-qPCR: impact of tumor cell content, DCIS and non-neoplastic breast tissue