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Background  Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ ERBB2 ), estrogen receptor (ER/ ESR1 ), progesterone receptor (PgR/ PGR ), and marker of proliferation Ki67 ( MKI67 ) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper® test.    Methods  Ten international pathology institutions participated in this study and determined messenger RNA expression levels of  ERBB2 ,  ESR1 ,  PGR , and  MKI67  in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper® test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss’ kappa statistics, and interclass correlation coefficients (ICCs).    Results  Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980–0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00.    Conclusions  On the basis of these data, the MammaTyper® has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.    Electronic supplementary material  The online version of this article (doi:10.1186/s13058-017-0848-z) contains supplementary material, which is available to authorized users.

An international reproducibility study validating quantitative determination of ERBB2, ESR1, PGR, and MKI67 mRNA in breast cancer using MammaTyper®