Abstract Background In metastatic breast cancer (MBC) patients ESR1 mutations (mESR1) in cell-free DNA (cfDNA) have been related to endocrine therapy (ET) resistance. Such mutations might also be detectable in circulating tumor cells (CTCs). Mutation detection in small amounts of cfDNA and in CTCs in a background of leukocytes is highly challenging. The current study evaluated how to reliably investigate mESR1 status in such minute amounts of cfDNA and in DNA from CellSearch-enriched CTCs. Materials & Methods Plasma (200 µL) and matched CellSearch-enriched CTC fractions of 7 healthy blood donors (HBD) and 29 MBC patients at baseline and after ET (≥ 5 CTC/7.5 mL) were evaluated. cfDNA was isolated from plasma with the QIAamp CNA kit and CTC-enriched DNA with the AllPrep kit (Qiagen). mESR1 status in both cfDNA and CTC-enriched DNA fractions was compared with or without whole genome amplification (repli-g WGA, Qiagen) or ESR1 target specific amplification. Quantitative PCR (qPCR) for wild type (WT) ESR1 was used to control the number of WT copies loaded into the chips for digital PCR (dPCR) analysis. The variant allele frequencies (VAF) of hotspot mutations for ESR1 (D538G, Y537S, Y537C and Y537N) were evaluated with mutation-specific Taqman assays by chip-based dPCR (QuantStudio 3D, Thermo Fischer Scientific). Results To allow inclusion of as many samples as possible, we successfully downscaled the volume of required plasma from 1 mL to 200 µL as this resulted in the same VAF. Sample-type specific thresholds for mESR1 presence were established (2% for the cell-free plasma samples, at which percentage all HBDs were negative, and 0.5% for the CTCs to allow identification of one mutated CTC-specific copy in a background of ~1,000 leukocytes). WGA was unable to adequately amplify fragmented cfDNA, resulting in a too low DNA yield. However, locus-specific target pre-amplification of a 136 bp fragment covering all 4 different mutations followed by mutant specific dPCR performed well for both cfDNA and CTC DNA, but only if the loading of the pre-amplified product into the dPCR chips was optimized by qPCR for the number of WT ESR1 copies. The most optimal results for dPCR data interpretation were obtained after: 1. including at least one positive sample in each dPCR session; 2. using a “safe loading window”, 3. loading and reading chips at least twice in QuantStudio 3D ; 4. critically evaluating the contribution by a non-specific “comet effect”; and 5. after loading the data in the software, performing at least two independent data analyses to exclude intra-observer variations. Summary Here we describe our workflow to assess mESR1 in a limited amount of plasma cfDNA or CellSearch enriched CTC DNA. This workflow has been successfully used to investigate the mESR1 VAF status in DNA from matched CTC DNA and cfDNA of MBC patients before start of 1st line endocrine therapy and at progression (see also abstract number 851017). Citation Format: Vitale SR, Sieuwerts AM, Helmijr J, Beije N, van der Vlugt – Daane M, Foekens JA, Sleijfer S, Jansen MPHM, Martens JWM. An optimized workflow to analyze ESR1 mutations in both circulating cell-free and circulating tumor cell DNA by digital PCR [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-20.