Abstract With the advent for technology of next generation sequencing, targeted sequencing is now becoming part of decision of chemotherapeutics in cancer patients, especially in refractory and metastatic cancer. We performed targeted sequencing to develop patient-centered trials to find therapeutic target based on molecular alternations. We used cancer panel for capturing of target region covering 83 cancer-related genes and validated it using 3 cases with ALK-fusion, ERBB2 amplification, or KRAS mutation. A total of 25 refractory metastatic cancer patients enrolled on this study so far. The majority of alterations were identified in ATM, FBXW7, NOTCH1, TP53, STK11, ARID1B, GNAS, FGFR3, SMAD4, and HNF1A. Among 25 patients, 12 had FDA-approved drug responsive or resistant alterations. However, targeted therapy was guided in 3 patients (12 %) on the basis of sequencing results because of lack of access to clinical trials, declining performance status and/or stable disease with previous treatment; crizotinib to ALK-EML4 fusion in malignancy of undefined origin (MUO) patient, pazopanib to FGFR amplification in malignant peripheral nerve sheath tumor (MPNST) patient, and everolimus to AKT3 amplification in uterine endometrial stromal sarcoma patient. All 3 patients showed partial response after targeted therapy. In addition, we identified KRAS codon 146 mutation (A146V), associated with resistance to anti-EGFR, in a cetuximab resistant colon cancer patient with wild-type KRAS exon 2 and 12 and EGFR. Taken together, this study suggests that identifying targets associated with response or resistance to specific targeted therapies using targeted sequencing can show great promise for improving the care of patients with refractory and metastatic cancer patients. Citation Format: Chae Hwa Kwon, Young Mi Seol, Young Keum Kim, Yuri Choi, Yeo Jin Won, Do Youn Park. Use of targeted sequencing of cancer genome (cancer panel) to develop patient-centered therapeutic target based on molecular alternations: A pilot prospective study for 25 patients with refractory and metastatic cancer and treatment response [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 CT112. doi:10.1158/1538-7445.AM2017-CT112