Introduction:

Molecular profiling of tumor samples is becoming routine practice in the clinic, particularly for therapy selection. However, in some cases, tumor tissue is not available for testing. Liquid biopsy, which enables the analysis of circulating tumor DNA (ctDNA) shed from the tumor into the blood, can be used as a surrogate for conventional tissue based testing to detect somatic mutations. Furthermore, ctDNA profiling can have potential applications in tumor burden monitoring. Some technical challenges must be overcome to accurately and sensitively detect the often low amount of ctDNA present in plasma. The AVENIO ctDNA Analysis Kits (Targeted, Expanded and Surveillance Kits; Research Use Only) were developed to sensitively detect all 4 major mutation classes: single nucleotide variants, insertions/deletions, fusions, and copy number variations, using a hybrid capture target enrichment workflow, molecular barcoding, and next generation sequencing. We previously presented a comprehensive study of 370+ samples demonstrating analytical performance of the assays on the Illumina NextSeq 500. As ctDNA based analysis becomes more prevalent in the clinical research community, there is great desire for high performing and cost-effective assays that are easy to implement and available across multiple sequencing platforms.

Methods:

We used the commercially available AVENIO ctDNA Analysis Kits (Expanded, Target and Surveillance Kits; Research Use Only) pre-sequencing workflow to process a panel of cell line blends and plasma-derived ctDNA samples in order to compare the performance of the assays on multiple Illumina sequencing platforms: NextSeq 500, HiSeq 4000 and HiSeq 2500. Ninety samples were tested across all platforms. The samples contain SNVs at 0.5% minor allele frequency (MAF), deletions at 1% MAF, selected fusions at 1% MAF, and MET amplification at 2.3 copies. Analysis was done using the AVENIO ctDNA analysis pipeline for all sequencing runs across the sequencing platforms.

Results: On the NextSeq 500, the AVENIO ctDNA Analysis Kits achieve sensitivities of >99% for 0.5% SNVs, >99% for 1% deletions, >96% for 1% fusions, and >99% for 2.3 copies of MET amplification, with 96% to >99% specificity for all mutations. Similar performance is observed across additional Illumina platforms, HighSeq 4000 and HiSeq 2500. Across all platforms, key sequencing metrics are consistent to achieve required sequencing depths, on-target rates, and uniformity. Also, it is noted that in order to achieve required depths, each platform requires different levels of sample multiplexing. We demonstrated that the AVENIO ctDNA Analysis Kits achieve high sensitivity and specificity across multiple high throughput sequencing platforms. We also will highlight the key performance differences and considerations when performing the assay across these sequencing platforms.

Citation Format: Jonathan Choi, Richard Dannebaum, Ashla Singh, Rob Foley, Jorge Dinis, Cindy Choi, Bosun Min, Jingchuan Li, Liang Feng, Fergal Casey, Janet Jin. Performance of the AVENIO ctDNA assays across multiple high-throughput next-generation sequencing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3648.