Abstract 1384: nCounter for detection of clinically relevant alterations in liquid biopsies of solid tumor patients
Authors: Giménez-Capitán Ana A. , Huang Chung-Ying , Bracht Jill , Boykin Rich , Mayo-de-las-Casas Clara , Beechem Joseph M. , Teixidó Cristina , Balada-Bel Ariadna , Garcia- Beatriz , Villatoro Sergio , Garzón Monica , Jordana-Ariza Nuria , Aguado Cristina , Viteri Santiago , García Juan José , Rosell Rafael , Gerlach Jay , Reguart Noemi , Molina-Vila Miguel Angel 
Published: August 2019

Abstract
Background: With the advent of precision medicine, screening for clinically relevant mutations and gene fusions is mandatory in many tumor types. However, in a significant number of cancer patients, the tumor tissue available is insufficient for genetic analysis. In addition, repeated tissue biopsies for monitoring the course of the disease and the emergence of mechanisms of resistance to targeted therapies are not feasible. Liquid biopsies constitute the only alternative available in these cases. The nCounter technology has been adapted to detect mutations and gene fusions in FFPE biopsies from cancer patients with a minimum requirement of tumor material and sample handling, a short turnaround time and a straightforward data analysis. However, nCounter has not been tested in liquid biopsy samples.
Methods: For mutation analysis, the SNV Solid Tumor Panel was used, which allows for detection of 97 driver mutations in 24 genes. For fusions, a customized panel for ALK, ROS1, RET, and NTRK1 fusion transcripts was used with a 14-cycles preamp step. First, proof-of-concept experiments were run by spiking plasma samples with a mixture of genomic DNAs or RNAs from positive cell lines. Next, 65 circulating-free DNA (cfDNA) samples from advanced cancer patients, previously genotyped by other techniques, were analyzed using the SNV panel. Of those, 60 had been purified from plasma, 4 from ascites and 1 from the pleural effusion. Nineteen were positive for EGFR mutations, 20 for KRAS, 13 for BRAF, 5 for PIK3CA, 2 for NRAS, and 6 were pan-negative. Finally, 8 circulating cell-free RNA samples isolated from plasma were tested with the nCounter Low RNA Input Kit and the lung fusion panel. Of those, 6 corresponded to lung cancer patients harboring ALK or ROS1 rearrangements in tumor tissue, but previous RT-PCR only detected fusions transcripts in 2.
Results: Spiking experiments revealed that the nCounter SNV Panel was able to detect mutations at allelic fractions as low as 0.2% for most of the drivers. When testing liquid biopsies, 63/65 cfDNA samples from cancer patients were evaluable, despite having DNA concentrations lower than 1 ng/µL. The SNV Panel successfully detected EGFR, KRAS, BRAF, PIK3CA and NRAS mutations with a concordance rate of 97.5% with previous genotyping by NGS, Therascreen® or Taqman® with PNA, corresponding to a Cohen’s kappa of 0,913. In the case of the lung fusion panel, ALK, ROS1 and RET fusion transcripts were detected in all spiked plasma cfRNA. Two samples from lung cancer patients with positive RT-PCR results were also detected by the nCounter low-input lung fusion panel. Research is ongoing to further improve the performance of the nCounter low-input fusion panel in liquid biopsy samples.
Conclusions: Our results demonstrate the feasibility of mutation analysis in the cfDNA of advanced cancer patients using nCounter. The nCounter technology also shows promise for the detection of gene fusions in cfRNA
Citation Format: Ana A. Giménez-Capitán, Chung-Ying Huang, Jill Bracht, Rich Boykin, Clara Mayo-de-las-Casas, Joseph M. Beechem, Cristina Teixidó, Ariadna Balada-Bel, Beatriz Garcia-, Sergio Villatoro, Monica Garzón, Nuria Jordana-Ariza, Cristina Aguado, Santiago Viteri, Juan José García, Rafael Rosell, Jay Gerlach, Noemi Reguart, Miguel Angel Molina-Vila. nCounter for detection of clinically relevant alterations in liquid biopsies of solid tumor patients [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 1384.