Abstract 756: Comprehensive NGS-based reference materials for variant detection in lymphoid cancer
Authors: Watkins Tonya N. , Forson Benedicta , Lowe Dana Ruminski , Konigshofer Yves , Huang Catherine , Clement Omoshile , Anekella Bharathi 
Published: June 2022

Next Generation Sequencing (NGS) is an important technology to identify genetic changes involved in lymphoid malignancies. Genome-level understanding of these changes can aid in diagnosis, prognosis, and therapy selection. Cancer biopsies are often preserved by formalin-fixed, paraffin-embedding (FFPE) procedures which provide long-term preservation but introduce damage to nucleic acids that are present in tissue including double strand breaks, nicks, and oxidation. The gold standard for Lymphoma diagnosis is surgical removal of the lymph node making FFPE the preferred sample format for analysis. To address the need for high quality reference materials for tumor profiling assays for these patients, we developed two multiplexed, biosynthetic reference materials: the Seraseq® FFPE Lymphoma DNA Reference Material and the Seraseq® Lymphoma DNA Mutation Mix. Both contain 26 plasmids containing important SNVs, INDELs, and gene fusions in lymphoid disease. For the FFPE format, plasmids were pooled at equal concentrations and introduced into the GM24385 reference human cell line. Engineered cells were diluted to achieve desired allele frequencies (AFs) as determined by digital PCR (dPCR). The cells were processed with a proprietary FFPE protocol that mimics damage in patient samples. The QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit were used for DNA isolation. DNA was quantified by the Qubit dsDNA HS Assay and quality assessed by the Agilent TapeStation. AFs were determined by dPCR for QIAamp-extracted DNA and a custom ArcherDX VariantPlex NGS panel for DNA extracted by both kits. DNA yield was similar, 193±44 ng (QIAamp) and 124±11 ng (Maxwell); however, a higher DNA integrity number was observed with the QIAamp kit (6.1 vs 2.7). The average % AF by dPCR was 9.6±1.8 (QIAamp) (target ≥5%). The average % AF by NGS was 7.6±2.8 (QIAamp) and 3.3±1.1 (Maxwell). For the DNA mix format, the same plasmids were pooled and blended with total purified genomic DNA from GM24385 cells. The average % AF was 8.4±0.6 by dPCR (target 5-10%) and 8.2±1.6 for the ArcherDX VariantPlex NGS panel. Overall, there was good concordance between AFs measured by dPCR and NGS for the DNA and FFPE formats. We noted that overall sample performance may be impacted by the FFPE DNA extraction method used. In conclusion, these reference materials allow for monitoring of a broad range of somatic mutations and gene fusions which can aid testing laboratories in accurately detecting and quantifying various genetic events in Lymphoid patient samples. The FFPE format can serve as an extraction control in assay development, while the DNA mix format allows laboratories to go directly into NGS library preparation and dPCR. A complementary circulating tumor DNA (ctDNA) version of these reference materials would aid laboratories in detecting minimal residual disease in patient blood resulting in less invasive lymphoid testing and therapeutic response monitoring.

Citation Format: Tonya N. Watkins, Benedicta Forson, Dana Ruminski Lowe, Yves Konigshofer, Catherine Huang, Omoshile Clement, Bharathi Anekella. Comprehensive NGS-based reference materials for variant detection in lymphoid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 756.