Abstract Comprehensive characterization of genetic profiles in clinically actionable genes in each tumor promises to facilitate personalized approaches to cancer treatment. However, development of systematic genetic profiling of cancers in the clinical setting is facing unique technical challenges. More than 90% of tumor samples available for clinical sequencing are formalin-fixed and paraffin-embedded (FFPE). These samples are not only limited by quantities, the DNA quality in these samples is often compromised due to different degree of degradation, cross-links and chemical modifications. Targeted sequencing, in which genomic regions of interests are selectively enriched before sequencing to achieve high coverage and sensitivity, can potentially provide a cost-effective and efficient solution for clinical sequencing. However, very few studies have been reported on the development of methodology that is applicable and robust in analyzing “real-world” clinical FFPE samples. Here we report the development of a novel methodology that integrates microfluidic multiplex PCR-based target enrichment with massively parallel sequencing for high-throughput, high-content targeted sequencing of clinically actionable cancer genes in FFPE tumor tissues. This method enables amplifying 963 regions from 88 common cancer genes in 48 samples and simultaneously sequencing 48 pooled barcoded samples in a single sequencing reaction. Using this method, we sequenced 40 cancer cell lines and a well-defined Latin square study with dilution series of 8 known somatic mutations, to comprehensively assess the sensitivity, specificity, reproducibility in both target enrichment and variant detection. Furthermore, using paired fresh frozen/FFPE tissues obtained from 16 cancer patients, we developed and optimized a FFPE protocol that yields high-quality genetic profiles highly concordant to those derived from parallel fresh frozen samples. To demonstrate the clinical utility of this method, we sequenced a cohort of 73 FFPE tumor samples from endometrial cancer patients. Many clinically actionable mutations were identified in these patients’ samples and confirmed using orthogonal validations. Our results demonstrated the feasibility and clinical utility of our method in clinical sequencing, setting the stage for ultimately translating this method in clinical setting to inform and guide cancer treatment. Citation Format: Yulei Wang, Richard Bourgon, Shan Lu, Yibing Yan, Yinghui Guan, Lisa Ryner, Rajesh Partel, Mark Lackner, Lukas Amler. Comprehensive profiling of actionab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3231. doi:10.1158/1538-7445.AM2013-3231