Multisite Evaluation of Breast Cancer Assay

This comparative three-site study used replicate breast tumor tissue specimens from the same FFPE block for testing with the MammaTyper (Table 2). A 16-member panel of breast cancer samples (15 ductal carcinomas and 1 lobular carcinoma) obtained from commercial vendors except for one clinical sample and comprising all different tumor subtypes were analyzed. All tissue specimens were sectioned at BioNTech Diagnostics GmbH. The first and last section were analysed with MammaTyper at site 1 and showed nearly identical marker expression. After rearranging, sections were shipped to the other testing sites (Stratifyer Molecular Pathology GmbH, Cologne and Institute of Pathology, University of Erlangen). Each tissue sample was processed during three, 4-day cycles, starting with RNA extraction and aliquoting of eluates (day 1) followed by three RT-qPCR runs on consecutive days (day 2–4). The procedure was performed by one operator per site using a single instrument (Versant kPCR Cycler, Siemens), a single lot of RNXtract and a single lot of the MammaTyper Assay. At BioNTech Diagnostics six additional cycles were performed by the same operator, two with different MammaTyper lots, one with an alternative RNXtract lot and three on a LightCycler 480 II (Roche) qPCR device to account for related effects on precision.
Intra-run precision was estimated based on the variance of triplicate measurements. All calculations for the precision studies were carried out based on CLSI guideline EP 05-A3 [25 ] using a random effects model II ANOVA via PROC mixed in SAS Version 9.2.