Abstract Background Treatment with targeted drugs is increasingly used in breast cancer aiming to block the tumor driving pathway(s). Drug choice is often based on a single primary tumor biopsy. It is important to ascertain that biopsied tumor material is representative for the tumor or metastases to treat with respect to tumor driving signaling pathway. Little is known about pathway heterogeneity within primary breast cancer, and between primary 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 which infer a pathway activity score from transcription factor target gene mRNA levels (Cancer Res 2014 Jun 1;74(11):2936-45). Methods Originally for AffymetrixU133Plus2.0, the pathway analysis was adapted for RT-qPCR enabling use on FFPE tissue. Calibration was performed using tissue samples with known pathway activity. Functional pathway activity of ER, AR, PI3K-FOXO, Hedgehog (HH), TGFbeta, and Wnt pathways was analyzed to assess (1) intra-tumor heterogeneity: 2-5 FFPE blocks of 17 primary breast cancers (9 luminal A, 4 luminal B, 1 HER2, 3 triple negative (TN), as defined by surrogate immunohistochemistry); (2) heterogeneity between primary and associated metastases: 9 patients with FFPE blocks from primary and 12 metastases. Results Intra-tumor heterogeneity: In 11 out of 13 Luminal A- and B-like tumors the ER pathway was active in all samples, 1 showed ER pathway heterogeneity; 11 had an active TGFbeta pathway, heterogeneic in 3; in one ER inactive tumor one (out of 5) samples was AR active. In 2 TN tumors TGFbeta and PI3K pathways were combined active, in one TN tumor heterogeneic TGFbeta activity was observed. PI3K pathway activity increased with malignancy grade and showed most intra-tumor variation. Overall, pathway heterogeneity was detected in over one third of tumors, least in Luminal A-like (2/9) compared to Luminal B-like (3/4) and triple negative (1/3) tumors. Extensive heterogeneity was found between primary breast cancer and metastases, and between metastatic locations of the same patient. ER pathway activity decreased in 5/9 patients and increased in 4/9 patients in one or more metastatic tumors; PI3K became active in metastasis of 2 patients and inactive in 1; the Wnt pathway became active in 3 patients in bone and ileum metastases; TGFbeta was lost in 5 patients, and became active in 1 bone metastasis; HH became active in 1 ovarian and AR in 1 bone metastasis. Conclusion Intra-tumor heterogeneity was lower in ER active compared to TN breast cancer, suggesting a need for multiple biopsies to adequately characterize TN for neoadjuvant therapy. Between primary tumor and metastases, heterogeneity was extensive indicating the need for pathway analysis on metastatic tumors prior to targeted treatment. Citation Format: Anja Van De Stolpe, Anne van Brussel, Cathy Moelans, Marcia A. Inda, Wim Verhaegh, Eveline den Biezen, Paul van Diest. Measuring functional signal transduction pathway activity on breast cancer tissue samples to determine intra-tumor heterogeneity and heterogeneity between primary and metastatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3690.