Abstract Background: Evaluation of cell-free DNA (cfDNA) is a very attractive tool to serve as "liquid biopsy" to define and establish mutational changes in circulating tumor DNA (ctDNA) non-invasively during therapy. Aim: To identify tumor specific mutations in serum cfDNA associated with resistance against tamoxifen in metastatic breast cancer. Materials & Methods: Ten metastatic ER-positive breast cancer patients treated with first-line tamoxifen of whom blood sera was available at start therapy, during treatment and at disease progression were selected. DNA was isolated from normal (nDNA) and primary tumor (ptDNA) tissue and from sera (cfDNA). DNA was analyzed with next generation sequencing by the ion-PGM system (Life Technologies) for a panel of 45 cancer genes. This panel included the most frequently mutated genes for breast, colon, prostate and ovarian cancer reported in the Catalogue Of Somatic Mutations In Cancer (Cosmic database, Release 67; cancer.sanger.ac.uk/). In total 1242 exons (∼255kb) were sequenced with 200 to 5000x reads depth coverage. The panel analyzed all exons for 39 genes and only hotspot exons for 6 oncogenes. Variant Caller software (Life Technologies; version 4.16) was applied to detect non-synonymous and stop-gain single nucleotide variants (SNVs) within the sequenced DNA. Hotspot mutations detected in PIK3CA exons 9 and 20 were validated with snapshot multiplex assays. Results: Variant Caller analyses revealed in total 252 SNVs within the 40 DNAs from tumor tissue and/or serum analyzed which were not detected in normal tissue. Of these, 229 SNVs were novel and not yet reported in the Cosmic database. Mutations were detected for 10 genes in both ptDNA and cfDNA, which enabled us to characterize ctDNA in serum of nine out of ten patients. The discovered mutations were already reported in Cosmic for PIK3CA, TP53, and NF1, but not for CDH1, APC, SMAD4, MLL, AKAP, CREBBP and MLL2. The PIK3CA, TP53 and APC mutations were observed in tumor and sera for 2, 3 and 2 patients, respectively. Mutations in the other genes, except MLL, were unique for individual patients. The mutation for MLL was seen in almost all patients at low frequencies (1-5%). The hotspot mutations in PIK3CA were confirmed with snapshot assays in the sequenced samples and in additional sera. In four patients, Cosmic reported mutations in BRCA1, KAT6B, MAP3K1, MLL2, PTCH1 and PTEN occurred in ctDNA at disease progression while they were not found in the primary tumor nor in preceding sera. The mutation frequencies ranged from 3% for BRCA1 and MAP3K1 to 6% for MLL2. Moreover, mutations for MED12 (3%) and MLL3 (4%), not yet reported in Cosmic, were each observed in two sera at disease progression for two additional patients. The remaining SNVs are currently verified for their authenticity and occurrence in ptDNA and/or ctDNA. Conclusion: Molecular characterization of tumor and serum derived DNA with targeted next generation sequencing enabled us to identify ctDNA in serum and to detect mutations at disease progression that might play a role in resistance to first-line tamoxifen. Citation Format: Maurice Jansen, Corine Beaufort, Jean Helmijr, Ronald van Marion, Niels Krol, Kim Monkhorst, Marian van Fessem, Ron van Schaik, Marcel Smid, Marion Meijer van Gelder, Maxime Look, Anita Trapman, Diana Ramirez-Ardila, Irene Lurkin, Ellen Zwarthoff, John Martens, John Foekens, Winand Dinjens, Stefan Sleijfer, Els Berns. Targeted molecular characterization of serum derived cell free DNA from metastatic breast cancer patients treated with first-line tamoxifen [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-02.