589 Background: Treatment with targeted drugs aims to block tumor driving signaling pathway(s). Drug choice is often based on a single preoperative primary breast cancer biopsy. It is important that biopsied cancer tissue is representative for the primary tumor (PT) or metastases to treat. Little is known about pathway heterogeneity within the PT, and between PT and metastatic tumors. A novel analysis method was developed to identify and quantify activity of signal transduction pathways in cancer tissue, based on Bayesian models that infer a pathway activity score from transcription factor target gene mRNA levels (Cancer Res 2014;74:2936-45). Methods: Pathway analysis, originally developed for AffymetrixU133Plus2.0, was adapted to RT-qPCR for use on formalin fixed paraffin embedded tissue. Estrogen (ER) and androgen (AR) receptor, PI3K, Hedgehog (HH), TGFβ and Wnt pathway activities were analyzed. Samples were from multiple locations (“quadrants”) in 15 luminal A, 9 luminal B, and 8 ER-negative primary breast cancers; from subdivided quadrant samples (4 “subquadrants”) of respectively 9, 4, and 4 PTs; and from 13 distant and 24 lymph node (LN) metastases of respectively 9 and 7 matched luminal PTs. Analysis of pathway activity score (PAS) variance was performed with linear mixed models with subgroup-dependent standard deviations. Results: In primary breast cancer intra-tumor PAS variance was not larger at macroscale (“quadrant”) than at microscale (“subquadrant”). For ER, AR, HH, and Wnt pathways, PAS variation was higher between distant metastases and PT than within the PT (p < 0.0002). For HH and Wnt pathways, PAS variation was higher between LN metastases than within the PT (p < 0.002). Correlation between primary and metastatic pathway activities ranged from -0.34 for ER to 0.47/0.50 for TGFβ/HH pathways. Conclusions: A single location tissue sample was representative for the whole primary tumor with respect to signaling pathway activity, suggesting one biopsy as generally sufficient for (neo)adjuvant therapy choice. Pathway activities varied between primary cancer and metastases, indicating the necessity of metastatic sample analysis (biopsies or liquid biopsy) to improve therapy choice.