Endleukemia Assay v1: Enabling NGS-Based Comprehensive Routine Molecular Profiling of Leukemias in Routine Clinical Care

Abstract Background: Molecular biomarkers provide diagnostic, prognostic, predictive and minimal residual disease (MRD) information and constitute an integral part of diagnostic workup of hematologic neoplasms. Since a variety of different hematologic disorders can often show non-specific clinical symptoms as well as laboratory findings, the initial diagnostic workup needs to be optimized for timely and efficient coverage of a broad range of differential diagnoses. We developed a customized 81-gene panel, EndLeukemia Assay v1, for comprehensive genetic characterization of disorders that include bone marrow (BM) and peripheral blood (PB). The biomarkers on the panel cover genetic alterations in myeloid neoplasms (acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPN), aplastic anemia (AA), acute lymphoblastic leukemia (ALL), hairy cell leukemia, T-large granular lymphocytic leukemia (T-LGL) and T-prolymphocytic leukemia (T-PLL). Materials and Methods: Genomic DNA was isolated from fresh BM aspirate/PB or formalin-fixed paraffin-embedded (FFPE) BM clot specimens. Either entire coding sequences or limited exons/hotspots for 81-gene target genes were captured from 250 ng DNA using the hybridization-based HaloPlexTM target enrichment system (Agilent Technologies, Santa Clara, CA). These genes include A NKRD26, ASXL1, ASXL2, BCOR, BCORL1, BRAF, BRINP3, CALR, CBL, CBLB, CBLC, CEBPA, CREBBP, CRLF2, CSF3R, CUX1, DDX41, DNMT3A, EED, ELANE, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GFI1, GNAS, HNRNPK, HRAS, IDH1, IDH2, IKZF1, IL2RG, IL7R, JAK1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MAP2K1, MPL, NF1, NOTCH1, NPM1, NRAS, PAX5, PHF6, PIGA, PML, PRPF40B, PTEN, PTPN11, RAD21, RARA, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG1, STAG2, STAT3, STAT5A, STAT5B, SUZ12, TERC, TERT, TET2, TP53, U2AF1, U2AF2, WT1, and ZRSR2. The panel design incorporated molecular barcodes to improve accuracy and sensitivity. Bidirectional paired-end sequencing was performed using the Illumina Miseq platform (Illumina, San Diego, CA). Variant calling was performed using Agilent SureCall software v3.5 and an in-house developed bioinformatics pipeline. Incorporation of single nucleotide polymorphisms (SNP) allows detection of sample identification errors and improves patient safety. Results: During the validation studies, high concordance was observed for missense mutations, insertions/deletions (indels), and selected copy number aberrations (CNAs) when compared with orthogonal methods. Under standardized conditions, a minimum average coverage depth of 3000x is achieved across the entire target region. The analytical sensitivity of the assay was established at 5% using a per base coverage cut-off of 250x. Of note, >90% of the CEBPA coding sequence, which is challenging to sequence due to a high GC component, was covered at >250x coverage depth. Copy number alterations including KMT2A partial tandem duplications (KMT2A -PTD) and IKZF1 deletions were detected successfully. Initial validation of FFPE tissue shows 100% concordance for all mutation calls for matched fresh and FFPE sample pairs. The ability to provide critical diagnostic and prognostic information across mutation hotspots from FFPE tissue if a fresh PB/BM sample is not available improves patient care. The top 25 mutations in the first 500 patient samples across different clinical indications are shown in the figure below. In addition, efficient panel design allowed uniform testing of follow-up bone marrow samples for the detection of MRD and clonal evolution. Conclusion: EndLeukemia assay v1 allowed comprehensive molecular profiling of a variety of hematologic neoplasms in routine clinical testing. Routine molecular biomarker profiling can be facilitated by incorporating smart design features in clinical NGS panels. Download : Download high-res image (99KB) Download : Download full-size image Figure . Disclosures Kantarjian: Delta-Fly Pharma: Research Funding; Novartis: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding.