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Introduction: Circulating tumour cells (CTCs) are a real-time reflection of the ad hoc relevant subpopulation in patients with progressive disease. The study comprises the clinical application of a semi-automated methodology to determine the PIK3CA mutational status at a single cell level. Methods: Using CellSearch and DEPArray, we purified single (n=172) and groups (n=93, ranging 5–120 cells) of CTCs from peripheral blood in 18 patients with metastatic ER+/PR+/HER2- breast cancer, of whom archival primary tumour (PT) material was available. Isolation of WBCs served as internal negative control. Recovered cells were subjected to whole-genome-amplification (WGA). During CTC blood draw, an extra SST tube was filled for isolation of circulating cell-free DNA (cfDNA) from serum as comparator. All samples from different compartments underwent gene mutation analysis via targeted amplification of exons 9 and 20 of the PIK3CA gene and downstream 454 massive parallel sequencing (MPS) on the GS Junior system. Results: WGA of single and group CTC samples had a success rate of 83±22 % and 96±8%, respectively. MPS resulted in an average throughput of 135.996±24.423 high quality reads with a normal-distributed mean fold coverage depth of 1257±703 and 1494±897 reads for exon 9 and 20, respectively. PIK3CA mutations were present at a relatively high frequency in archival PT (20/29 (68,9%)) and showed poor and moderate agreement with cfDNA (n=24; 33,3% disparity; kappa=0,03) and CTCs (n=14; 11,28% disparity; kappa=0,329), respectively. Comparison of CTCs and temporally matched cfDNA samples revealed substantial agreement (n=14; 14,3% disparity; kappa=0,536). A concordant PIK3CA status across all compartments (PT, cfDNA and CTCs) was observed in 11/15 (73,3%) samples. At the used sequencing depth, cfDNA failed to the detect PIK3CA mutations in 2 cases (13,3%), which were present in the respective PT and corresponding CTCs. CTCs failed to detect the mutant PIK3CA gene in one case (6,7%), which was present in PT and cfDNA. Gain of mutation was observed in 2/15 patients (13,3%), with a wild-type PT and mutant cfDNA and CTCs at progression. A wild-type PIK3CA sequence in recovered WBCs of all patients (n=15) indicates a high specificity and tumorigenic nature of the picked up variants. In depth intra-patient analysis of mutant CTCs on a single cell level reveals PIK3CA mutational heterogeneity with the presence of both mutant and wild-type CTCs (average %MT/%WT ratio of 50/50). Additionally, unique double-mutated CTCs were detected in 5/15 (30%) cases as well. Conclusions: We report the frequent occurrence of PIK3CA hotspot mutations in metastatic HR+ breast cancer. Intra-patient mutational heterogeneity was observed in the single CTC samples of the same patient. Thereby, this study provides evidence of both concordance and discordance of the PIK3CA genotype in the intravascular compartment (down to the single cell level) with comparison to the temporally unmatched PT. The study presents the utilization of a liquid biopsy, thereby paving the way towards the application of a more personalized medicine in the management of patients with metastatic cancer. Citation Format: Bram De Laere, Dieter JE Peeters, Salgado Roberto, Vermeulen B Peter, Van Dam A Peter, Dirix Y Luc, Van Laere J Steven. Exploring the intra-patient PIK3CA mutational heterogeneity of circulating tumour cells by massive parallel sequencing in patients with metastatic hormone receptor-positive breast cancer [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 P4-01-09.