Abstract Poorly differentiated thyroid carcinoma (PDTC) represents a rare subtype of thyroid cancer with aggressive clinical course and peculiar clinical and pathological characteristics. Recent genetic studies highlighted the main molecular pathways involved in its pathogenesis but data are affected by heterogeneity of case selection and relatively limited sample size. Using a multimodal molecular approach, aim of this project was to implement the PDTC molecular characterization and to identify novel prognostic biomarkers and potential therapeutic targets. A total of 84 cases of PDTC classified according to the Turin proposal were selected from a multi-Institutional series. Fifty-nine samples underwent DNA, RNA and protein analyses. Mismatch repair (MMR) defects were tested using immunohistochemistry for MLH1, MSH2, MSH6 and PMS2. DNA and RNA analyses were performed by means of next generation sequencing (NGS) using Oncomine Comprehensive assay V3. Due to the high failure rate in RNA analysis, gene fusion analysis was enriched by 25 additional samples. MMR protein loss was observed in 7/59 samples (11.9%). In NGS DNA analyses, 51/59 cases were adequate. The most prevalent mutations were in NRAS (13/51, 25%) and TP53 (13/51, 25%), all mutually exclusive each other. TERT promoter (TERTp) mutations were detected in 11/51 of cases (21.6%). Other genes with a relevant prevalence were PTEN (15.7%), NF1 (13.7%), ATM (13.7%), NOTCH3 (11.8%) and BAP1 (11.8%). Mean number of alterations was higher in TP53-mutated cases (5.8 mutations/case) than in RAS-mutated cases (2.8 mutations/case). TP53-mutated samples lacked TERTp co-mutations but were associated with mutations in PTEN and in genes related to MMR system and/or loss of MMR proteins. PIK3CA was the most prevalent co-mutated gene (three samples) in RAS-mutated cases. A third group (25 cases) lacked RAS or TP53 mutations, had a low mean number of alterations (2.7 mutations/case) and was enriched for TERTp mutations (up to 32%). Copy number variations were not detected. Among the 43 cases adequate for NGS RNA analysis, gene fusions in PAX8-PPARG (one case) and TBL1XR1-PIK3CA (two cases) were detected. This latter fusion has never been reported in thyroid cancer, and was validated by fluorescence in situ hybridization (FISH). In conclusion, PDTC are genomically clustered into RAS-mutated tumors (with low mutational burden and co-mutations affecting genes involved in the same pathway), TP53-mutated cancers (with high mutational burden, absence of TERTp mutations and strong association with MMR defects) and a third “double negative” group enriched for TERTp mutations. Overall, targetable gene fusions have a prevalence of 7%. Moreover, 38% of overall cases harbor mutations in genes coding for tyrosine kinases potentially targetable and/or have defects in the MMR that claim a potential role for immunotherapy. Citation Format: Vanessa Zambelli, Marta Fornaro, Giulia Orlando, Ida Rapa, Francesca Napoli, Susanna Cappia, Lorenzo Daniele, Simonetta Piana, Mauro Papotti, Marco Volante. Identification of therapeutic targets in poorly differentiated carcinoma through a multimodal molecular analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5285.