Abstract Background Immunohistochemical (IHC) loss of expression of the estrogen (ER) and progesterone receptor (PR) from endometrial carcinomas is associated with high tumor grade, and with recurrent disease. However, expression of these receptors is not always indicative of functional pathway activity. A novel approach to predict signaling pathway activity, based on Knowledge-based Bayesian computational models interprets quantitative transcriptome data as the functional output of an active signaling pathway by using expression levels of transcriptional target genes. The objectives of this study were to compare ER pathway activity to ER and PR IHC results with respect to endometrial carcinoma grade and disease recurrences. Materials and Methods Originally for Affymetrix, the pathway analysis was adapted for RT-qPCR enabling use on FFPE tissue. Calibration and threshold definition was performed using tissue with known pathway activity. Pathway analysis was performed on: (1) A public dataset (GSE56026) containing Affymetrix expression microarray data from 62 patients with grade 1 (n=23),2 (n=17) and 3 (n=11) endometrioid and serous (n=12) carcinomas, and (2) formalin fixed, paraffin embedded (FFPE) tissue from endometrioid (n=70) and serous carcinomas (n=15), treated at the Radboud university medical center, which were immunohistochemically stained for ER and PR, and analyzed for pathway activity using qPCR. Results (1) Analysis of the public dataset showed an inverse relation between ER pathway activity score and cancer grade, with the lowest ER activity in serous carcinomas. The Mann-Whitney U test showed significant differences between grade 1 and 2 (p=0.015), grade 1 and 3 (p=0.004), and grade 1 and serous carcinomas (p<0.0001). (2) These results were confirmed in the FFPE tissue. The ER pathway was active in 30% (n=18), and inactive in 70% (n=41) of the samples. In advanced stage endometrioid and all serous carcinomas, ER pathway was active in 8% (n=2) and inactive in 92% (n=24) of the samples (Fisher p=0.03). Endometrial carcinoma related death (ECD) rate was 20% (n=17). While individually, ER pathway activity, ER and PR IHC were all significantly correlated with ECD, (logistic regression odds ratio, OR = 0.53, 0.12, 0.15, and p=0.0004, 0.0005, 0.002, respectively). In a multivariate analysis only ER pathway activity and disease stage remained significantly correlated with ECD (OR=0.52, p=0.03). Conclusion In two independent patient cohorts, the ER pathway model analysis, performed on data from Affymetrix microarrays and qPCR measurements showed an inverse relationship between the pathway activity and increasing tumor grade. While ER activity and ER/PR staining were related, only ER activity and cancer stage remained significant in a multivariate analysis with respect to immunohistochemistry alone when trying to predict recurrent disease. Citation Format: Louis J. van der Putten, Anne van Brussel, Willem Jan van Weelden, Márcia A. de Inda, Leon F. Massuger, Henk van Ooijen, Anja van de Stolpe, Johanna M. Pijnenborg. Estrogen receptor pathway activity in endometrial carcinomas and its relation to tumor grade and recurrence [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 2656.