Abstract Identifying the unique molecular signature of tumors is essential for rational design of personalized treatments tailored to specific genomic alterations. It is now well accepted that the non-malignant microenvironment plays a critical role in tumor behavior and response to therapy. However, to date, multi-compartmental comprehensive genomic and transcriptomic analyses of malignant and nonmalignant compartments remain rare, are highly unstructured, and difficult to translate for practical treatment decisions. Here, we present a new analytic platform that integrates whole exome sequence (WES) and whole transcriptome (RNASeq) data to provide comprehensive tumor portraits. The portrait is based on the analysis of 28 newly curated gene sets capturing selective and specific molecular and functional properties of nonmalignant components of tumor microenvironment (such as fibroblasts, macrophages, dendritic cells, lymphocytes, extracellular matrices and blood and lymphatic networks), as well as intrinsic properties and genomic profile of malignant cells, including mutational and neoantigenic load, oncogenic and clinically actionable alterations. Analysis of tumor RNASeq data from over 8500 patients using unsupervised louvain dense clustering analysis based on detection of tightly connected networks revealed four unique and consistent microenvironment types characterized by distinct immune, stromal and cancer cell composition. Type A (immune “inflamed” cancers) were characterized by increased fibrosis and low mutational burden; Type B was characterized by increased infiltrating cytotoxic T lymphocytes and high mutations. Type C and D were characterized by negligible immune infiltration and minimal non-malignant cellular populations, while type C had increased vascularization and fibrosis. Remarkably, the four microenvironment types have high prognostic significance and are differently associated with immunotherapy response. In over 400 patients with skin and bladder cancers, type B was associated with a response rate (CR/PR) of >38% following immunotherapy (anti-PD1 /CTLA4) vs <10% in patients with type C. Importantly, dynamic changes in the microenvironment types correlated with response to immunotherapy. By identifying the unique combination of driver genomic alterations, immune processes and stromal components present in each tumor, this novel multicompartmental analysis provides a robust tool for better prediction of response to immunotherapy and for future tailoring of personalized therapeutic combinations. This comprehensive model, summarized and visualized as a Molecular-Functional portrait of the tumor (MF-portrait), could ultimately lead to better clinical outcomes. Citation Format: Alexander Bagaev, Nikita Kotlov, Felix Frenkel, Viktor Svekolkin, Nava Almog, Ravshan Ataullakhanov, Nathan Fowler. Integrated whole exome and transcriptome analyses of the tumor and microenvironment provide new opportunities for rational design of cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4418.