Custom designed array

Gene expression data were independently generated using custom-designed arrays, Agilent Technologies (Santa Clara, CA, USA), containing approximately 32.1K probes, representing approximately 21.5K unique genes from FFPE breast cancer tumor tissue. A total of 652 of 727 breast cancer tumors passed the RNA quality check according to the diagnostic quality model and were used in the analysis. Gene expression data were normalized with quantile normalization: gene expression values were log2-scaled and quantile normalized before analysis [33 (link)]. Microdissection was performed before mRNA extraction as a part of the pipeline in the microarray assay. Gene expression levels of CD4, CD8A (hereafter CD8), CD19, FOXP3, programmed cell death protein 1 (PDC1 or PD1), and programmed death ligand-1 (PD-L1) were analyzed using the microarray platform (Agilent Technologies) [33 (link)]. The definition of a high expression was set at the median for CD4, CD19, FOXP3, PD-L1, PD1, and the immune gene modules IMMUNE1 and IMMUNE2 [26 (link),27 (link)] for all analyses. CD8 was separated in quartiles where CD8 q1–3 = low expression and CD8 q4 = high expression.

Agilent microarray profiling was performed in 2014.² Gene expression data were independently generated using custom‐designed arrays, Agilent Technologies (Santa Clara, CA), containing approximately 32.1K probes, representing approximately 21.5K unique genes from FFPE breast cancer tumor tissue. Out of 727, 652 breast cancer tumors passed the RNA quality check according to the diagnostic quality model and were used in the analysis, of which 538 were ER‐positive (Figure 1).

Gene expression data were independently generated using custom-designed arrays, Agilent Technologies (CA, USA), containing approximately 32.1K probes, representing approximately 21.5K unique genes from FFPE breast cancer tumor tissue. Approximately 90% (or 652 of 727 breast cancer tumors) passed the RNA quality check (according to the diagnostic quality model) and were used in the analysis. The 70-gene (MammaPrint) signature was performed according to standard protocols as previously described, including the use of 465 normalization genes and over 250 probes for hybridization and printing quality control. Patient tumor samples were classified into high or low risk by the 0.00 threshold in the MammaPrint index (high up to and low above 0.00 index, respectively). The subgroup of ultralow tumors is defined by MammaPrint index > +0.355 [17 (link)–19 ].