Abstract Uterine carcinosarcoma (UCS) is a rare and aggressive form of uterine cancer. It is bi-phasic, exhibiting histological features of both malignant epithelial (carcinomatous) and mesenchymal (sarcomatous) elements. Studies have indicated that UCS arises from sarcomatous differentiation of high-grade carcinoma while others have suggested a bi-clonal nature. Given these differences, we sought to separate the carcinoma and sarcoma elements of UCS to try to understand their molecular differences and gain further insights into how these tumors develop. We macrodissected carcinomatous, sarcomatous, and normal cells from formalin fixed paraffin embedded (FFPE) uterine samples of 10 UCS patients. DNA and RNA were isolated and extracted using the Qiagen AllPrep DNA/RNA FFPE kit. Whole-genome SNP microarrays and deep sequencing of 26 cancer genes was performed, using the Illumina Infinium OmniExpressExome array and the TruSight Tumor panel respectively. Illumina HiSeq mRNA sequencing was also performed to quantify gene expression. The genomic allelic imbalance (AI) profiling, called from the SNP data by hapLOH, showed that sarcoma samples were more aberrant than their carcinoma counterparts (abstract 131, AACR 2016). From the targeted sequencing, the Illumina Amplicon-DS Somatic Variant Caller was employed to call somatic mutations. Mutations were identified in TP53 in both the sarcoma and carcinoma samples of all 10 patients. Frequently mutated genes included APC, EGFR, MET and MSH6 which were found in 60-80% of the patients. Genes mutated in less than 50% of the patients included PTEN, KRAS, KIT, FBXW7, PIK3CA, FGFR2, and CTNNB1. Current results showed no association of a mutated gene to either the sarcoma or carcinoma component of UCS. RSEM, STAR and EBSeq were applied to the RNA-seq data for gene expression quantification. Approximately 2500 genes were identified as being differentially expressed (DE) between normal and carcinoma samples. Just over 4000 genes were identified as being DE between normal and sarcoma samples. 75% of the DE genes in the carcinoma were also identified in the sarcoma. Using DAVID functional annotation tool, we characterized these gene sets with KEGG pathways. Deregulated pathways identified in both carcinoma and sarcoma include: cell cycle, transcriptional regulation, Ras and p53 signaling. Some additional pathways are putatively associated with sarcoma only, including MAPK and PI3K-Akt signaling. We report here the differences between sarcoma and carcinoma components of UCS from multiple molecular perspectives. From the genomic AI and DE analysis, the carcinoma aberrations appear to be mostly a subset of the sarcoma tumor profiles, where sarcoma samples appear to be more highly aberrant compared to the carcinoma samples. One possible inference from this is that the sarcoma originated and evolved from the carcinoma cells. Citation Format: Yihua Liu, Zachary Weber, F. Anthony San Lucas, Aditya Deshpande, Raed Sulaiman, Mary Fagerness, Natasha Flier, Joseph Sulaiman, Christel M. Davis, Jerry Fowler, Gareth E. Davies, David Starks, Luis Rojas-Espaillat, Paul Scheet, Erik A. Ehli. Tumor profiling of separated carcinomatous and sarcomatous components from uterine carcinosarcoma biopsies provides insights into their development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2017-2463