Abstract 5099: Evaluation of two next-generation sequencing platforms for genomic analysis in circulating tumor cells
Authors: Lam Chi Shan Candy , Dai Wei , Ko Josephine Mun Yee , Lung Maria 
Published: August 2019

Abstract
Background: Circulating tumor cells (CTCs) are released from primary or metastatic tumors into the cancer patient’s bloodstream and may establish secondary tumors in distant body sites. Their frequencies are thought to have great prognostic impact and their mutation signatures may yield pointers in treatment decisions. However, CTCs are extremely rare in the blood and are challenging to sequence. Current next-generation-sequencing (NGS) technologies and analysis procedures must be carefully strategized to maximize sensitivity, while maintaining specificity and controlling false discovery rates.
Aim of the study: This study aims to establish a robust and sensitive NGS bench work and analysis pipeline for genetic analysis of CTCs with good positive predictive value (PPV) and low false discovery rate (FDR), which can ultimately be translated into clinical application. We will compare and evaluate the performance of two NGS platforms for CTC analysis in the Hong Kong cancer patients.
Methods: This study will streamline the sequencing and analyses of two NGS approaches, i.e. amplicon-based or hybridization capture-based, for their application for CTC analyses. Firstly, a cancer cell line with known mutations will be spiked into normal blood cells at different percentages and sequenced with both approaches independently. The amplicon-based platform covers 60 cancer relevant and actionable genes, while the customized capture kit for the hybridization-based platform includes 245 cancer relevant genes curated in our group. Both platform’s sensitivity, specificity, PPV and FDR will be evaluated. Secondly, CTCs from several cancer types with high incidence in Asia will be enriched by size, enumerated for comparative analysis, and sequenced. Sequencing results will be used to refine the analysis pipelines for both platforms. Results of sequencing analyses, including potential druggable targets and prognostic markers identified, will be determined.
Results: We successfully detected over 80% of known mutations at as low as 1.25% CTC purity, and with FDRs below 10% at 2.5% CTC purity by the hybridization-based method. We detected damaging missense, stopgain, or splicing mutations with the amplification-based platform in all 19 samples sequenced so far. More than 80% of blood samples from cancer patients had at least 5 CTCs detected. Preliminary comparison of the sequencing results of one high CTC purity sample showed that more than 50% of variants detected by this platform was also identified by hybridization-based sequencing.
Conclusion: Our study sheds light on studying the genomic profiles of the rare CTCs for clinical application and identifying the potential actionable targets in Asian cancer patients.
Acknowledgements: This study is supported by the HKU Platform Technology Fund and the Hong Kong Research Grants Council Area of Excellence grant AoE/M-06/08 to MLL.
Citation Format: Chi Shan Candy Lam, Wei Dai, Josephine Mun Yee Ko, Maria Lung. Evaluation of two next-generation sequencing platforms for genomic analysis in circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5099.