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

Zsuzsanna Varga,Fraser Symmans,Claudia Gürtler,Uğur Şahin ,Peter Schraml,Thomas Keller,Mark Laible,Heikki Joensuu,Annette Lebeau,Hans‐Peter Sinn ,Xiaodong Teng,John M.s Bartlett ,Arndt Hartmann,Andreas Turzynski,Kornelia Schlombs,Elena Guerini‐Rocco ,Robert Stoehr,Hong Bu,Reinhard Von Wasielewski,Frédérique Penault‐Llorca ,Giuseppe Viale

doi:10.1186/s13058-017-0848-z

Abstract

BackgroundAccurate 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.MethodsTen 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).ResultsTotal 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.ConclusionsOn 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.

Highlights

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 PGR Progesterone receptor (Ki67) (MKI67) is indispensable for therapeutic decision making in early breast cancer
Intermediate precision On the basis of MammaTyper® measurements of study arm 1 (Fig. 1), quantitative single-marker results were obtained as 40−ΔΔCq values for ERBB2, ESR1, PGR, and marker of proliferation Ki67 (MKI67) and are presented in Fig. 2 as box plots for each marker, sample, and study site
The intersite reproducibility was again computed on the whole sample set, leading to a similar approximation of the total variability of single-marker 40−ΔΔCq assessments with SDs between 0.21 and 0.44 Quantification cycle (Cq) (Table 1, lower panel)

Summary

Introduction

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. In contemporary clinical management of patients with breast cancer, prognostications and therapeutic decisions are based on the assessment of clinicopathological factors as well as on the expression status of biomarkers with established clinical validity (i.e., human epidermal growth factor receptor 2 [HER2]; estrogen receptor [ER]; progesterone receptor [PgR]; and Ki67, a marker of cell proliferation) [1, 2]. There remains an urgent need for alternative, more robust, standardized, and precise assays with proven analytical and clinical validity for Ki67, HER2, ER, and PgR in routine breast cancer diagnostics [17, 18]

Methods

Results

Conclusion