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. 2016 Jun 17;29(10):1143–1154. doi: 10.1038/modpathol.2016.112

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Copyright © 2016 United States & Canadian Academy of Pathology

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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AQUA and Tissue Studio quantification and data-driven dichotomization of nuclear pYStat5 levels reproducibly identify a similar subset of estrogen receptor-positive breast cancer patients with low tumor levels of nuclear pYStat5 at increased risk of disease recurrence. Quantitative fluorescence immunohistochemistry values were computed for Nuc-pYStat5 in 193 estrogen receptor-positive breast cancer specimens using the PM2000/AQUA and ScanScopeFL/TissueStudio platforms. Data-driven, objective cutpoints to identify tumors with low fluorescent immunohistochemistry-detected Nuc-pYStat5 (Nuc-pYStat5 IF^low) or high Nuc-pYStat5 (Nuc-pYStat5 IF^high) were derived from data generated on the (a) PM2000/AQUA or (c) ScanScopeFL/TissueStudio platform, and Kaplan–Meier analysis of recurrence-free survival was performed for each platform. Both platforms identified a similar sub-population of patients whose tumors displayed low Nuc-pYStat5 (≈20%) and were at increased risk of recurrence. The data-driven optimal cutpoint for PM2000/AQUA was linearly calibrated to the (b) ScanScopeFL/TissueStudio data using the equation ScanScopeFL/TissueStudio=0.43+0.95 × PM2000/AQUA and the data-driven optimal cutpoint for ScanScopeFL/TissueStudio was linearly calibrated for the (d) PM2000-AQUA data using the equation PM2000/AQUA=−0.45+1.06 × ScanScopeFL/TissueStudio. Calibrated cutpoints derived from one platform were then applied to the fluorescence immunohistochemistry values of the alternative platform and subjected to a second Kaplan–Meier analysis (b and d).