Abstract 170: Development and analytical validation of a novel next-generation DNA sequencing assay, the oncomine lymphoma panel, to detect SNV, insertion, deletion and copy number variants in 25 Lymphoma genes in FFPE samples
Authors: Hyland Fiona , Scafe Charles , Zhu Yun , Yang Chenchen , Tseng Yu-Ting , McKnight Brooke , Farfan Fernando , Bandla Santhoshi , Sadis Seth , Roman Steve 
Published: September 2020

Introduction: Lymphomas, including diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, and other lymphomas, are clinically heterogeneous: some respond well to therapy, but many fail to respond. Much of this variability in response is reported to reflect molecular heterogeneity of the tumor. Identifying somatic variants including SNVs, insertions, deletions, and copy number variations (CNVs) is important in characterizing these samples. Robust detection of variants in multiple genes using fine needle aspirate (FNA) samples, low abundance DNA, and FFPE samples is needed.

Methods: We describe a next-generation sequencing assay with 25 genes, the Oncomine Lymphoma Panel, including ARID1A, ATM, B2M, BCL2, BCL6, BRAF, BTK, CARD11, CD79B, CDKN2A, CREBBP, EZH2, GNA13, HIST1H1E, KMT2D, MTOR, MYC, MYD88, PIM1, SF3B1, SOCS1, TNFAIP3, TNFRSF14, TP53, and XPO1. This panel comprises 976 amplicons in total. The assays for these genes have been optimized, and performance has been tested on control samples and on representative clinical research samples. Another 300 genes, with optimized and verified performance, can be added to customize the panel. This panel is designed to work with 20 ng input DNA from FFPE samples and can also be used with samples having non-degraded DNA. A comprehensive bioinformatics analysis solution was developed to detect SNPs, indels, and CNVs; to perform filtering for the most relevant variants; to annotate these variants with a wide variety of bioinformatics databases; and to report on the interpretation of the selected variants.

Results: We tested this panel on various types of input material, including control samples and FFPE samples. Uniformity, coverage, on target mapping, reproducibility, and sensitivity to detect variants were high in all cases and above established quality criteria (&gt;90% or &gt; 95%). Finally, a coordinated analysis solution uses information about the panel and provides an integrated analysis pipeline with a simple and powerful visual interface, including variant calling, CNV detection, functional annotation, population MAF, predicted protein effect, and annotations including ClinVar, COSMIC, etc. Filtering tools utilizing this information facilitate variant prioritization.

Conclusions: An NGS lymphoma assay with a comprehensive data analysis approach was developed and analytically validated. The system is capable of detecting both small mutations and CNVs simultaneously with high sensitivity in FFPE samples for lymphoma translational research. For research use only. Not for use in diagnostic procedures.

Citation Format: Fiona Hyland, Charles Scafe, Yun Zhu, Chenchen Yang, Yu-Ting Tseng, Brooke McKnight, Fernando Farfan, Santhoshi Bandla, Seth Sadis, Steve Roman. Development and analytical validation of a novel next-generation DNA sequencing assay, the oncomine lymphoma panel, to detect SNV, insertion, deletion and copy number variants in 25 Lymphoma genes in FFPE samples [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 170.