Clinical Studies Quantitative Whole Genome Sequencing of Circulating Tumor Cells Enables Personalized Combination Therapy of Metastatic Cancer Natali Gulbahce 1 , Mark Jesus M. Magbanua 2 , Robert Chin 1 , Misha R. Agarwal 1 , Xuhao Luo 1 , Jia Liu 1 , Daniel M. Hayden 1 , Qing Mao 1 , Serban Ciotlos 1 , Zhenyu Li 3 , Yanxiang Chen 3 , Xingpeng Chen 3 , Yuxiang Li 3 , Rebecca Yu Zhang 1 , Katharine Lee 1 , Rick Tearle 1 , Emily Park 4 , Snezana Drmanac 1,3 , Hope S. Rugo 2 , John W. Park 2 , Radoje Drmanac 1,3 , and Brock A. Peters 1,3 Abstract Much effort has been dedicated to developing circulating tumor cells (CTC) as a noninvasive cancer biopsy, but with limited success as yet. In this study, we combine a method for isolation of highly pure CTCs using immunomagnetic enrich- ment/fluorescence-activated cell sorting with advanced whole genome sequencing (WGS), based on long fragment read tech- nology, to illustrate the utility of an accurate, comprehensive, phased, and quantitative genomic analysis platform for CTCs. Whole genomes of 34 CTCs from a patient with metastatic breast cancer were analyzed as 3,072 barcoded subgenomic compart- ments of long DNA. WGS resulted in a read coverage of 23 per cell and an ensemble call rate of >95%. These barcoded reads enabled accurate detection of somatic mutations present in as few as 12% of CTCs. We found in CTCs a total of 2,766 somatic single-nucleotide variants and 543 indels and multi-base sub- stitutions, 23 of which altered amino acid sequences. Another 16,961 somatic single nucleotide variant and 8,408 indels and multi-base substitutions, 77 of which were nonsynonymous, were detected with varying degrees of prevalence across the 34 CTCs. On the basis of our whole genome data of mutations found in all CTCs, we identified driver mutations and the tissue of origin of these cells, suggesting personalized combination therapies beyond the scope of most gene panels. Taken together, our results show how advanced WGS of CTCs can lead to high- resolution analyses of cancers that can reliably guide personal- ized therapy. Cancer Res; 77(16); 4530–41. Ó2017 AACR. Introduction Emerging evidence point to the potential role of circulating tumor cells (CTC) as biomarkers for patient stratification and selection of targeted therapy (1). Given their easy accessibility via blood draws, CTCs could be an ideal source of cancer cells for genomic analysis, versus more invasive tissue-based biopsy. In patients with advanced cancer, molecular profiling of CTCs have shown that these cells provide a better representation of tumor diversity than a single biopsy (2); this finding is most relevant in patients with multisite metastases where collection of tumor material from each location is not feasible. In addi- tion, higher levels of CTCs in metastatic cancers can provide more cells for in-depth molecular analyses to discover common and lineage-specific genomic alterations that can help guide personalized treatment (3). In contrast to circulating tumor DNA analysis, which is mainly limited to interrogating small panels of genes (4), CTCs allow complete genomic and gene expression analysis providing opportunities for evaluation of clinically relevant mutations and gene expression–based molecular subtypes, and the identification of novel therapeutic targets. Over the past decade, technologies for isolating CTCs have dramatically improved (5). For example, the develop- ment of an EPCAM-based immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) approach has enabled the isolation of highly pure CTCs that are amenable to complex molecular analyses (6–11). At the same time methods for analyzing the entire genomic content of small numbers of cells, such as long fragment read (LFR) technol- ogy, are now available (12–14). LFR is a novel sequencing technology that allows for comprehensive, phased, accurate, and quantitative analysis of genomic variations, from large structural changes to single-base variants (12, 13). In this study, these two powerful approaches (IE/FACS and LFR) are combined to generate high-accuracy, high-coverage whole genome sequencing (WGS) data from CTCs isolated from a female patient diagnosed with ER-positive/HER2-negative metastatic breast cancer. On the basis of both driver and passenger mutations, possible personalized combination ther- apies were selected that could potentially be effective for 1 Complete Genomics, Inc, San Jose, California. 2 Division of Hematology/Oncol- ogy, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California. 3 BGI-Shenzhen, Shenzhen, China. 4 Advanced Cell Diagnostics, Inc, Hayward, California. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). N. Gulbahce and M.J.M. Magbanua contributed equally to this article. Corresponding Authors: Brock A. Peters, Complete Genomics, Inc., 2904 Orchard Parkway, San Jose, CA 95134. Phone: 408-648-2560, ext. 3043; E-mail: bpeters@completegenomics.com; and Radoje Drmanac, rdrmanac@completegenomics.com doi: 10.1158/0008-5472.CAN-17-0688 Ó2017 American Association for Cancer Research. 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