Abstract
BackgroundTissue 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®.MethodsMammaTyper® 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.ResultsCompared 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.ConclusionExpression 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.
Highlights
Tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) breast cancer specimens may affect the accuracy of reverse transcription quantitative real-time PCR (RT-qPCR)
The concordance of the binary categories was 100% for human epidermal growth factor receptor 2 (ERBB2), estrogen receptor 1 (ESR1) and progesterone receptor (PGR) and 90% for marker of proliferation Ki-67 (MKI67) caused by one single case where the initial value was very close to the cut-off (Additional file 1, sample 3)
We investigated the robustness of MammaTyper®, an RT-qPCR-based gene-expression assay for ERBB2, ESR1, PGR and MKI67 against heterogeneity due to various tissue types
Summary
Tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) breast cancer specimens may affect the accuracy of reverse transcription quantitative real-time PCR (RT-qPCR). On hematoxylin and eosin (H&E) stained histological slides, invasive tumor cells are seen in close proximity to other neoplastic or non-neoplastic microanatomical structures such as in situ carcinoma, atypical ductal hyperplasia, non-neoplastic ductulo-lobular structures, and stromal cells, including adipocytes, blood vessels, and other cells of the tumor microenvironment. These morphologically distinct cell types have unique biological and molecular fingerprints [1,2,3,4]. IHC requires interpretation of the chromogen signal and semi-quantitative scoring of intensity or proportion of staining, procedures that are both subject to intra- and inter-observer variability and will result in discordance rates [6,7,8,9]
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