Fig 3.

Discordant classification of estrogen receptor (ER) status in the YTMA 130 cohort. (A) ER status was determined by immunofluorescence and automated quantitative analysis (AQUA) in Yale retrospective breast cancer cohort YTMA 130 (diagnosed between 1976 and 2005; clinicopathologic characteristics in Appendix Table A1) and compared with ER status as determined by immunohistochemistry (IHC) using the 10% positive nuclei cutoff guidelines. A distribution of ER by AQUA (picograms per microgram standardized as illustrated in Fig 1) is shown, in which each case is color coded according to its ER status by both AQUA and IHC. (B) Kaplan-Meier curves show 10-year recurrence-free survival (RFS), in which patients are grouped according to the classifications shown in (A). The AQUA-negative/IHC-positive group (n = 1) was excluded from survival analysis on account of its size and insufficient power. (C) The AQUA cut point of 2 pg/μg was further validated in this cohort by examining representative immunofluorescent images of ER staining for patients on either side of the cut point (right panels). Cytokeratin (CK) was used as a mask to define regions of tumor (green, left panels). For ER, we contracted the dynamic range of the grayscale image (adjusted maximum red-green-blue input level from 255 to 16 by using Adobe Photoshop) to visualize low levels of specific nuclear staining as well as nonspecific background. DAPI, 4,6-diamidino-2-phenylindole.