Abstract Background: Circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in bone marrow are being studied to monitor disease and guide therapy, but the relationship between CTCs and DTCs is weak and may confound clinical decision-making. Because blood sampling is easier than sampling bone marrow, CTC analyses are used more frequently than DTC analyses, although the relationship between CTCs and DTCs and the mutational heterogeneity within both populations at the single cell level are not usually examined simultaneously. Methods: We used the MagSweeper to immunomagnetically isolate single tumor cells from blood and bone marrow samples in breast cancer patients. Isolated tumor cells were used for immunohistochemical identification, PIK3CA gene mutation analysis, and to propagate cells in culture. In one patient, CTC and DTC single cell genotypes were compared during multiple treatment courses as the disease course progressed. Results: 242 individual tumor cells were isolated from 17 breast cancer patients. All tumor cells were assayed for single nucleotide mutations on exons 9 and 20 of the PIK3CA gene, and 48 mutated cells were identified in three patients. Heterogeneity and temporal discordance were observed in and between CTCs and DTCs in the same patient. All DTCs from bone marrow overgrown by tumor cells in a metastatic breast cancer patient carried the same PIK3CA single nucleotide mutation even though CTCs isolated within the same time period were wild type or heterogeneous for the mutation, providing evidence of both concordance and discordance of single cell PIK3CA genotype between CTCs and DTCs at different blood sampling time points. DTCs isolated by the MagSweeper could be directly cultured and consistently maintained the mutant PIK3CA genotype despite morphological changes over time in cell culture. Conclusions: DTCs isolated live by the MagSweeper can be propagated in culture, and a DNA single nucleotide mutation was maintained as a stable marker during cell culture multiple passages. This same mutation was used to monitor CTCs and DTCs at the single cell level. Although others have shown variable correlation between presence of CTCs and DTCs in the same patients, we show here potential discordance at the genotype level of single CTCs isolated from the same patient at different time points and between individual CTCs and DTCs. Our data support that CTCs and DTCs can have independent clinical value and suggest that it may be necessary to independently sample both during overall treatment course. Citation Format: Glenn Deng, Sujatha Krishnakumar, Marc A. Coram, Ashley A. Powell, Haiyu Zhang, Michael N. Mindrinos, Melinda L. Telli, Katharina E. Effenberger, Michael Herrler, Klaus Pantel, Ronald W. Davis, Stefanie S. Jeffrey. Genotype discordance between circulating tumor cells in blood and disseminated tumor cells in bone marrow at single cell level in breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2014-3528