Abstract 2517: Validation of a targeted sequencing workflow for sequence variants and focal copy number alterations (CNAs) in single circulating tumor cells (CTCs)
Authors: Tononi Paola , Monaco Valentina del , Ferrarini Alberto , Buson Genny , Garonzi Marianna , Forcato Claudio , Raspadori Andrea , Manaresi Nicolò 
Published: August 2019

Abstract
Background
Characterization of individual CTCs helps investigating intra-tumour heterogeneity, and provides longitudinal information about temporal evolution of genomic lesions following therapeutic evolutionary pressure, missed by one-time, bulk, single-biopsy analysis. Here we present the validation of a complete workflow to detect hotspot mutations and CNAs in single cells. It combines ligation-mediated PCR (LM-PCR) Whole Genome Amplification (Ampli1™ WGA kit), best-in-class in terms of low allelic drop-out (ADO) and reproducibility of amplification bias, with a tailored, WGA-aware, next generation sequencing (NGS) targeted cancer panel (Ampli1™ OncoSeek) and a fully-automated cloud-based platform for bioinformatic analysis (MSBiosuite).
Methods
Single-cells (n=24) of breast, prostate, lung and neuroblastoma cancer cell lines spiked in healthy-donor blood, alongside 15 single White Blood Cells (WBCs) from 5 healthy donors and CTCs from prostate, lung and breast patients were enriched with CellSearch® System, sorted with DEPArray™ NxT technology and WGA’ed with Ampli1™ WGA kit (Menarini Silicon Biosystems). NGS reference materials (Seraseq™ Breast CNV and Seraseq™ Lung & Brain CNV) with known CNAs were also WGA’ed. WGA products were used as input for the Ampli1™ OncoSeek Panel assay (Menarini Silicon Biosystems), a single-tube, Illumina®-compatible kit that covers 60 clinically relevant genes including more than 1500 mutation hotspots and CNAs for a subset of 19 genes. WGA-tailored primer pairs design and their concentrations were optimized so that targeted amplicons for sequence variants are sufficiently represented. Redundant amplicons were included for robustness of focal CNA detection. Bioinformatic analysis was performed with an assay-specific, cloud-based pipeline (MSBiosuite, Menarini Silicon Biosystems).
Results
Results on Seraseq NGS reference materials with known copy number gains (3, 6, 12) on 6 genes showed accurate detection of expected CNAs and high linearity of response (R2 = 0.97 ± 0.04). We observed low ADO rate (12.7% ± 4.2%). The Ampli1™ OncoSeek Panel assay detected known mutations and CNAs from cell lines at high sensitivity and the analysis of polymorphic variants in WBCs showed high agreement between biological replicates (overall agreement = 0.94 ± 0.06). Sequencing of CTCs from patients is ongoing and will be presented at the conference.
Conclusions
Here we presented a complete solution to detect hotspot mutations and focal CNAs that meets the need for accurate tumour characterization at single-cell level, suitable for individual CTC analysis.
Citation Format: Paola Tononi, Valentina del Monaco, Alberto Ferrarini, Genny Buson, Marianna Garonzi, Claudio Forcato, Andrea Raspadori, Nicolò Manaresi. Validation of a targeted sequencing workflow for sequence variants and focal copy number alterations (CNAs) in single circulating tumor cells (CTCs) [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 2517.