Abstract 5899: Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons
Authors: Lodato Nicholas , Liu Zonghan , Kontor Akuah , Wu Xiaoxi , Hillmer Lukas , Polvino Sean , Ke Yue , Petrilli Erin , Labonte Adam , Richman Geoffrey , Wang Zhaohui 
Published: September 2020

Abstract
Introduction: SLIMamp technology allows multiplex-PCR of tiled amplicons in a single tube, which enables targeting of large exons for NGS analysis with a streamlined process. Copy number variants (CNVs) are DNA segments that are present with variation in the number of copies compared to a normal genome. CNVs have a high prevalence in the pathogenesis of cancer and their characterization is important to acquire a more comprehensive picture of the mutations present in a patient sample. Pillar developed a proprietary CNV algorithm and combined it with SLIMamp to develop an integrated multi-cancer plus CNV detection NGS panel which identifies CNVs in the ERBB2, EGFR, MET, and MYC genes.
Methods: To assess the ability of the ONCO/Reveal Multi-Cancer with CNV Panel to detect ERBB2 CNV compared to DISH (dual in situ hybridization), libraries were created from 44 well-characterized FFPE breast cancer samples plus 3 Genome in a Bottle (GIAB) samples. 23 tumor samples had known ERBB2 amplification (20 with matched normal tissue), 21 were known non-amplified tumors, and 3 GIAB samples were used as negative controls. Libraries were sequenced on an Illumina MiSeq and data was analyzed by the Pillar Variant Analysis Toolkit (PiVAT). The previous DISH results were not known to Pillar. Results were provided to an independent collaborator who unblinded the study and compared the NGS results to DISH.
Results: Overall the assay performed well with a mapping rate of 99.4% ± 2.1%, on-target rate of 99.1% ± 0.4%, and coverage uniformity &gt;0.2x mean coverage of 93.5% ± 4.1%. With respect to CNV calling, the assay and software detected 100% (21/21) of ERBB2 amplification negative samples as confirmed by DISH. For amplification positive samples, the NGS assay detected 100% (23/23) of positive samples. The software correctly identified 91% (21/23) of amplification positive samples. The two samples that were not called by PiVAT had normalized genes counts of 1.2 and 1.4, and also had the lowest DISH scores. All samples with a normalized gene count &gt;1.5 were correctly called by PiVAT. The NGS assay was able to accurately call CNV independent of tumor cellularity with tumor content of 20%-90% not impacting the CNV call.
Conclusions: The ONCO/Reveal Multi-Cancer with CNV Panel is a robust assay for the detection of CNVs, SNVs, and Indels of interest across multiple solid tumor cancer types. The workflow is streamlined, with same day loading of finished libraries when starting from as little as 5ng of isolated input DNA. The assay and software demonstrate detection of low CNV with the recommended cutoff set at 1.5. With further optimization of the calling algorithm accurate calling of even lower CNVs may be possible. The detection and identification of CNVs along with other mutations gives a more comprehensive overview of the mutations present in a sample, supporting better clinical management of patients.
Citation Format: Nicholas Lodato, Zonghan Liu, Akuah Kontor, Xiaoxi Wu, Lukas Hillmer, Sean Polvino, Yue Ke, Erin Petrilli, Adam Labonte, Geoffrey Richman, Zhaohui Wang. Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons [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 5899.