Abstract
PurposeQuantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein.MethodsMessenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody.Results ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin.ConclusionQuantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.
Highlights
Despite the usefulness of Estrogen Receptor a (ER) as a predictive marker for endocrine therapy 50% of ER positive patients still recur, indicating a need for additional predictive biomarkers for endocrine therapy [1]
We modified the AQUA method for quantitative measurement of protein to combine it with the RNAscope method to quantify ER messenger RNA (mRNA) (ESR1) in situ and to compare to ER protein levels determined by quantitative immunofluorescence (QIF) on two breast cancer cohorts
RNAscope Assay Validation The RNAscopeH assay for ESR1, Ubiquitin C (UbC), and DapB was first performed on serial sections of a control tissue microarray (TMA) containing a panel of ER positive and ER negative breast cancer cell lines in 2-fold redundancy and quantified using AQUA (Fig. 1a)
Summary
Despite the usefulness of ER as a predictive marker for endocrine therapy 50% of ER positive patients still recur, indicating a need for additional predictive biomarkers for endocrine therapy [1]. Genomic technologies have allowed the search for new potential biomarkers beyond the traditional protein-based Immunohistochemistry (IHC) markers to gene expression signatures using messenger RNA (mRNA) to provide prognostic or predictive information [2]. One such example is the Oncotype DX assay that uses 21 genes to determine a recurrence score to quantify the risk of distant recurrence in tamoxifentreated, lymph node negative, ER positive breast cancer [3,4,5]. We modified the AQUA method for quantitative measurement of protein to combine it with the RNAscope method to quantify ER mRNA (ESR1) in situ and to compare to ER protein levels determined by quantitative immunofluorescence (QIF) on two breast cancer cohorts
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