Abstract 5363: A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA

Abstract

Abstract There is increasing interest in sequence analysis of circulating tumor cells (CTCs) to identify actionable mutations that may complement circulating tumor DNA (ctDNA) findings. Because the amount of DNA present in a single cell is miniscule (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as replication errors that can lead to false positive results. Targeted sequencing is an alternative approach that does not require WGA. We modified the CleanPlex® OncoZoom® Cancer Hotspot Panel protocol to perform targeted sequencing of single CTCs isolated by the RareCyte platform using A549 cell line spike-in and patient samples. We also used the CleanPlex panel to evaluate ctDNA from plasma isolated by blood collection tubes from two manufacturers (RareCyte and Streck). In both cases we compared OncoZoom results with and without prior WGA. Increased uniformity of coverage with decreased target dropout was observed when CleanPlex NGS libraries were prepared directly from cell lysates without WGA. Median read depth increased 48-fold when compared to the WGA method. On average, 23 out of 29 (79%) variants present in bulk A549 genomic DNA were observed without WGA in single model CTCs, while 15 of 29 (52%) were observed after WGA. Additionally, the false positive error frequency of non-WGA samples was 8-fold lower than in the WGA samples. CTCs and cell-free DNA from two metastatic breast cancer patients were also sequenced using OncoZoom. In one patient, PIK3CA E542K, a well-documented oncogenic mutation, was observed in 3 of 5 CTCs. In the other, ERBB2 L755S, a known HER2-reactivating mutation associated with chemoresistance, was found in 3 of 5 CTCs. Both patients' variants were present at similar allelic frequencies in plasma isolated from RareCyte and Streck cell-free DNA BCT tubes. Single-cell CleanPlex amplicon-based sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. This approach shows promise for cell-based liquid biopsy diagnostic applications. Citation Format: Nolan G. Ericson, Vidushi Kapoor, Guoying Liu, Alisa C. Clein, Daniel E. Sabath, Eric P. Kaldjian, Tad George. A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5363.