Abstract P2-02-15: Discovery of putative circulating tumor cells through somatic mutation profile of epithelial cell adhesion molecule positive single cells from blood of metastatic breast cancer patients

Background: Circulating tumor cell (CTC) enumeration provides prognostic information for chemotherapy in metastatic breast cancer. However, due to its rarity and heterogeneity, it is difficult to distinguish true CTCs from normal blood cells and perform genomic analysis on them for use in therapeutic strategies. The main application of most currently available CTC detection systems consists of an enumeration of putative CTCs without further analysis. The aim of this study was to evaluate the feasibility of single cell picking and target sequencing of epithelial cell adhesion molecule (EpCAM)-positive cells for detecting CTCs. Methods: Whole blood sampled from metastatic breast cancer patients who were newly diagnosed with metastasis or who had disease progression during palliative treatment were used for this study. After applying IsoFlux Circulating Tumor Cell Enrichment Kit (Fluxion, South San Francisco, CA, USA), single CTC candidates were picked from a pool of EpCAM-positive cells. Genomic DNA from the picked cells was whole genome amplified and target sequencing was performed using Ion AmpliSeq™ Cancer Hotspot Panel (Life Technologies, Carlsbad, CA, USA). Target sequencing reads were mapped to human genome reference (hg19) using BWA-MEM (0.7.10). Single nucleotide variants (SNVs) were annotated using dbSNP, Variome Data 0.2, and COSMIC databases. Results: A total of 172 EpCAM-positive cells were selected according to size and EpCAM status from whole blood of 11 patients. The remaining cells were grouped into a pooled sample for each patient. The mean read depth of the target genes was 13455×. A mean 7.82 mutations as determined by SNVs listed in the COSMIC database but not in dbSNP and Variome Data 0.2 were detected in each patient. Cells with multiple mutated genes, or those with a mutated gene repeatedly observed in another cell from the same patient were judged to be putative CTCs. At least 2 putative CTCs were detected in 7 patients while no CTCs were detected in 2 patients. Mutated genes observed in the putative CTCs were ABL1, AKT1, APC, CDH1, CDKN2A, ERBB2, FGFR3, HRAS, IDH1, JAK2, KDR, NPM1, RB1, RET, SMARCB1, STK11, and TP53. Conclusions: Potential CTCs were successfully identified by single cell picking and target sequencing of EpCAM-positive cells from whole blood of metastatic breast cancer patients. Unique mutations not detected in other single cells and pooled samples can be used to distinguish putative CTCs from normal cells. Genomic profiling of corresponding primary tumor and metastatic site biopsy is warranted to verify the CTCs and investigate their role in disease progression. Citation Format: Lee H-B, Jeon S, Kim BC, Jho S, Kim J, Kang YJ, Yoo T-K, Han JH, Kim Y, Im S-A, Moon H-G, Noh D-Y, Han W. Discovery of putative circulating tumor cells through somatic mutation profile of epithelial cell adhesion molecule positive single cells from blood of metastatic breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-15.