Abstract 1381: Technical validation of Roche AVENIO sequencing platform for liquid biopsies
Authors: Vessies Daan C. , Linders Theodora C. , Ramkisoensing Kalpana L. , Nederlof Petra M. , Meijer Gerrit A. , Monkhorst Kim , Broek Daan van den 
Published: August 2019

Abstract
Purpose
Circulating cell-free tumor DNA (ctDNA) is rapidly gaining attention as a valuable biosource for the detection and diagnosis of multiple types of cancer. ctDNA can be easily obtained through a minimally invasive liquid biopsy. Multiple NGS-based methods have been developed specifically for application in liquid biopsies.
Before such methods can be implemented in clinical care, they need to be technically validated through measurement of independent reference standards. In the current project we technically validated the Roche AVENIO ctDNA Assay, which is based on the CAPP-seq technology [Newman AM, et al. Nat Biotechnol. 2016;34:547-55]. Its Targeted Kit interrogates 17 genes for Single Nucleotide Variants (SNVs), Insertions and Deletions (InDels), Copy Number Variations (CNVs) and gene fusions in a single workflow.
Methods
The detection of SNVs, CNVs, Indels and gene fusions was evaluated as follows:
- SeraCare Seraseq ctDNA Complete was analyzed in four replicates at 2.5%, 0.5% and 0.1% mutant allele frequency (AF), and at 50ng and 10ng input. This material contains SNVs, InDels, CNVs and gene fusions.
- An in-house characterized reference pool of patient plasma, containing SNVs and InDels at various AF (0.07% - 5.51%), was analyzed in four replicates at 50ng and 10ng input.
- Plasma and serum samples obtained from patients with tissue confirmed presence of CNV were used to validate the robustness of CNV detection. Twelve samples were selected to represent a range of background DNA (serum had 2-4 times higher wildtype DNA levels compared to plasma), amplification levels (CNV levels 1.4-fold to 4.2-fold by ddPCR) and total input (10-50ng cfDNA input).
- Plasma obtained from five patients with EML4-ALK gene fusion confirmed on tissue was used to further validate fusion detection.
Sequencing was performed on an Illumina NextSeq on High Output mode with sixteen samples per run (20-33M read pairs per sample).
Results
Sensitivity for detection of SNVs and InDels was 100% for all variants down to 0.18% AF when using 50ng input, and 50% for variants at 0.10% AF. For 10ng input the variants were detected down to 0.60% AF with sensitivity of 92%. For variants with AF between 0.07% and 0.53%, sensitivity was 67%.
Results from twelve plasma and serum samples obtained from patients with EGFR CNV were 100% concordant with ddPCR results. These results were confirmed in SeraCare samples at 10ng and 50ng input.
Sensitivity of fusion detection in SeraCare material was 100% for AF of 0.5% and 50ng input, and 38% for input of 10ng. No fusions were detected at 50ng or 10ng input for AF of 0.1%. We furthermore confirmed 3 out of the 5 fusions found in tissue.
Conclusions
We have determined the lower limits of detection for SNVs, CNVs and gene fusions. Sensitivity was dependent on DNA input with performance optimized at 50ng.
Our results confirm the feasibility of in-house use of the AVENIO ctDNA Targeted Kit for broad molecular profiling in liquid biopsies.
Citation Format: Daan C. Vessies, Theodora C. Linders, Kalpana L. Ramkisoensing, Petra M. Nederlof, Gerrit A. Meijer, Kim Monkhorst, Daan van den Broek. Technical validation of Roche AVENIO sequencing platform for liquid biopsies [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 1381.