Abstract LB-227: Targeted next-generation sequencing (NGS) of circulating tumor cells (CTCs) and matched primary tumors.
Authors: Liu Stephen V. , Dempsey Paul W. , Strauss William , Xu Yucheng , Xu Tong , Winer-Jones Jessamine , Triche Tim J. , Quinn David I. , McMurdie Janine , Defusco Andre , Goldkorn Amir 
Published: August 2014

Abstract
Introduction: Personalizing cancer care relies on accurate detection of actionable genomic aberrations in tumor cells. Conventionally, this strategy relies on analysis of primary tumor samples, which are often temporally and biologically distinct from recurrent, metastatic or treatment-resistant disease. As an alternative, CTCs offer real-time cancer tissue for analysis that may more accurately represent the current state of a patient's disease. In this pilot, we used CTCs as source material for targeted NGS across a range of malignancies.
Methods: Under IRB approval, blood samples from patients with advanced cancer were labeled with EpCAM ferrofluid and placed into the LiquidBiopsy® platform (Cynvenio Biosystems, Inc.), an immunoaffinity-based microfluidic device tailored to query genomic events. CTCs were identified by CK, CD45 and DAPI expression. A matched WBC pellet served as a control representing germline sequence. Amplicon libraries were generated using Life Technologies AmpliSeq 2.0 and sequenced with an Ion Torrent sequencer. When available, matched formalin fixed paraffin embedded (FFPE) primary tumor tissue from the same patient was analyzed in parallel. Somatic single nucleotide variants (SNV) present in CTCs or FFPE samples but not in WBC were identified.
Results: CTCs were detected in 18 of 19 patients with advanced prostate (8), breast (6), renal cell (2), bladder (1), lung (1) and rectal (1) cancer (CTC median 54, range 15-421). Germline SNPs were consistently detected across WBC, CTC and FFPE samples. Significant SNVs (occurring in &gt; 1% of DNA in a sample) were found in 6 of 18 patient CTC samples (range 1-6 SNVs/sample, frequency 1.1%-11.9% with 620X-14,422X sequence coverage depth). Numerous SNVs were identified in all 9 matched primary tumor FFPE specimens but did not correlate with the SNVs identified in the CTCs.
Conclusion: This pilot demonstrates the feasibility of using CTCs as a real-time disease relevant substrate for NGS to identify personalized genomic targets. A high number of CTCs were detectable across malignancies, and CTC germline variants correlated with matched WBC controls. Cancer relevant SNVs were detected in a third of patients even using the relatively narrow primary tumor derived AmpliSeq platform. The FFPE specimens generated a high number of SNVs but did not correlate with CTC profiles, likely reflecting biological disparity between early localized tumors and advanced metastatic disease, as well as genomic artifacts introduced into primary tumor specimens by FFPE preservation. These data demonstrate the feasibility and potential biological and technical advantages of CTCs over traditional FFPE samples for genomic analysis in the pursuit of personalized cancer medicine.
Citation Format: Stephen V. Liu, Paul W. Dempsey, William Strauss, Yucheng Xu, Tong Xu, Jessamine Winer-Jones, Tim J. Triche, David I. Quinn, Janine McMurdie, Andre Defusco, Amir Goldkorn. Targeted next-generation sequencing (NGS) of circulating tumor cells (CTCs) and matched primary tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-227. doi:10.1158/1538-7445.AM2013-LB-227