From each TMA block, a 5-micron section was cut and used for immunohistochemical (IHC) staining for VDR with a validated monoclonal antibody 9A7 (ThermoFisher catalog # MA1-710, Waltham, MA) and a Dako automated slide stainer following standardized protocols established by PRN. The antibody showed an exclusive nuclear staining without cytoplasmic reactivity in breast tissues (Figure 1), which is consistent with the literature (10 (link), 24 (link)). After staining, whole-slide digital images were captured by the Aperio ScanScope CS Slide Scanner, and a computer-assisted image analysis algorithm optimized for the VDR antibody was used for automated quantitative assessment of staining intensity and percent of positive staining area. Any tumor with a total nuclei count less than 15 was excluded (n=18) from the analysis. An immunoreactive score (IRS) was computed as the product of intensity score (0-3) and percent of positive nuclei score (0-4) for each core and scores across multiple cores of each tumor block were compiled into a final score by the average score weighted by the total count of nuclei of each core. The resultant score thus ranged from 0-12. Based on the distribution of the IRS, VDR expression was classified into three levels with largely similar number of cases in each level: low (0-2), moderate (3-5), and strong (6-12) (see Figure 1 for representative stains).