Mutational analysis of circulating tumor cells in breast cancer patients by targeted clonal sequencing.

Abstract

10516 Background: The molecular signature of circulating tumor cells (CTCs) may serve as a surrogate marker for accurate description of the metastatic tumor of interest, especially in the setting of treatment response and selection. We present a method for mutational analysis of CTCs in metastatic breast cancer (MBC) patients by using an emulsion-formulated, semiconductor-based, targeted clonal sequencing platform. Methods: CTCs in MBC patients were enriched by a microfluidic OncoCEE device using antibodies against both epithelial and mesenchymal markers. Genomic DNA was extracted from enriched CTC samples. Emulsion-based multiplex-PCR targeted for various cancer genes was performed, after which semiconductor-based deep sequencing was completed. The read error rates were analyzed based on quality score and context of sequence including homopolymers. Statistical significance for each mutational analysis was assessed using a method based on beta-binomial distribution. Results: Of the 17 patients samples obtained, we were able to enrich CTC samples in 9 of them (CTC range 1-1063, median=12). Multiplex targeted sequencing was performed on DNA from the enriched CTC patient samples (purity range 0.3-6%). Greater than 3000-fold coverage was accomplished. Missense mutations at E545D on PIK3CA (p= 2.0e-07), F354L on STK11 (p=2.0e-04), and Q61R on NRAS (p=2.0e-07) were detected. Novel mutations of L540F and Q1033K within the hot spot regions of PIK3CA were observed (p=1.3e-04). Genomic DNA from WBCs from a healthy female was analyzed concurrently as a negative control, in which none of the mutations were observed. Conclusions: Mutational analysis of CTCs in MBC patients can be accomplished by deep sequencing. We developed a de novo protocol for clonal mutation analysis on CTCs in MBC and detected various significant and novel mutations. We anticipate reporting sequencing results on CTCs and matched WBCs, as negative controls, from 40 MBC patients. This will provide the foundation for the future studies in which we will compare the mutational profile between CTCs and primary/metastatic tumors. We intend to validate clonal mutational analysis of CTCs as a predictive blood-based biomarker in subsequent trials.