Abstract 4315: NGS-based reference materials for fusion and somatic variant detection in myeloid cancers
Authors: Tomson Farol Lovell , Kamineni Praveena , Huang Catherine , Dickens Jessica , Brudzewsky Dan , Clement Omoshile , Santhanam Ram , Anekella Bharathi 
Published: September 2020

Abstract
The large range of genetic aberrations and genes involved Myeloid malignancies, clonal diseases of hematopoietic progenitor cells which can lead to accumulation of immature blast cells in the bone marrow and peripheral blood, make Next Generation Sequencing (NGS) assays a cost effective and sensitive way to determine genetic changes. Understanding the genetic changes which lead to the clonal proliferation can aid in both determining prognosis and therapy selection. Consequently, there are an increasing number of NGS-based oncology tests available for somatic mutation detection in patients with hematological malignancies. However, reference materials that contain the relevant genetic blood cancer mutations are lacking. We designed a purified RNA reference material that contains nine RNA fusions two different ETV6-ABL1 transcripts, including BCR-ABL1, MYST3-CREBBP, RUNX1-RUNX1T1, and PML-RARα which are important in myeloid cancers. Conversely, a purified DNA reference material was designed to include 23 somatic variants included two FLT3 internal tandem duplications. The biosynthetic RNA or DNA constructs were mixed with either total RNA or purified genomic DNA from GM24385 reference human cell line. Digital PCR was used to quantify the variant sequences to determine the abundance of the fusion RNAs and the allele frequencies of the somatic mutations. NGS testing on a variety of targeted NGS assays was then performed to show compatibility. The Seraseq Myeloid RNA Mix was tested by digital PCR to confirm that each of the nine fusions in the reference samples are present at approximately 100 fusion copies per nanogram of total RNA. The FusionPlex Myeloid Kit for Illumina (ArcherDx) as well as the Oncomine Myeloid Panel (Thermo Fisher) showed positive detection for all fusions present in the reference samples. In addition, the Seraseq Myeloid DNA Mix was also tested by digital PCR to confirm the variant allele frequencies were on target at the 5%, 10% or 15% VAF. While, dPCR allele specific assays could not be obtained for CEBPA mutations, NGS testing of the reference samples by VariantPlex Core Myeloid Kit for Illumina (ArcherDx) and Oncomine Myeloid Panel (Thermo Fisher) confirmed the presence of these mutations at the expected levels, as well as all of the other variants in the DNA reference samples. In conclusion, SeraCare has developed highly multiplexed RNA and DNA-based reference materials for evaluating Myelogenous disorders by NGS. The reference materials allow monitoring of a broad range of somatic mutations and gene fusions which can aid optimization and verification of detection limits for NGS-based Myeloid disease assay testing, and provide laboratories greater assurance in their ability to correctly detect and quantify various types of genetic events in diseased patient samples.
Citation Format: Farol Lovell Tomson, Praveena Kamineni, Catherine Huang, Jessica Dickens, Dan Brudzewsky, Omoshile Clement, Ram Santhanam, Bharathi Anekella. NGS-based reference materials for fusion and somatic variant detection in myeloid cancers [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 4315.