Multiplexed ion beam imaging analysis for quantitation of protein expression in cancer tissue sections.

Part of developing therapeutics is the need to identify patients who will respond to treatment. For HER2-targeted therapies, such as trastuzumab, the expression level of HER2 is used to identify patients likely to receive benefit from therapy. Currently, chromogenic immunohistochemistry on patient tumor tissue is one of the methodologies used to assess the expression level of HER2 to determine eligibility for trastuzumab. However, chromogenic staining is fraught with serious drawbacks that influence scoring, which is additionally flawed due to the subjective nature of human/pathologist bias. Thus, to advance drug development and precision medicine, there is a need to develop technologies that are more objective and quantitative through the collection and integration of larger data sets. In proof of concept experiments, we show multiplexed ion beam imaging (MIBI), a novel imaging technology, can quantitate HER2 expression on breast carcinoma tissue with known HER2 status and those values correlate with pathologist-determined IHC scores. The same type of quantitative analysis using the mean pixel value of five individual cells and total pixel count of the entire image was extended to a blinded study of breast carcinoma samples of unknown HER2 scores. Here, a strong correlation between quantitation of HER2 by MIBI analysis and pathologist-derived HER2 IHC score was identified. In addition, a comparison between MIBI analysis and immunofluorescence-based automated quantitative analysis (AQUA) technology, an industry-accepted quantitation system, showed strong correlation in the same blind study. Further comparison of the two systems determined MIBI was comparable to AQUA analysis when evaluated against pathologist-determined scores. Using HER2 as a model, these data show MIBI analysis can quantitate protein expression with greater sensitivity and objectivity compared to standard pathologist interpretation, demonstrating its potential as a technology capable of advancing cancer and patient diagnostics.

Multiplexed ion beam imaging analysis for quantitation of protein expresssion in cancer tissue sections.

This corrects the article DOI: 10.1038/labinvest.2017.50.

Quantitative in situ measurement of estrogen receptor mRNA predicts response to tamoxifen.

PURPOSE: Quantification 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. METHODS: Messenger 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. CONCLUSION: Quantification 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.