Abstract 2208: A comprehensive genomic profiling approach to detect functional translocations and genomic alterations in a single tube workflow

Background: To comprehensively profile mutations in hematologic malignancies, we developed a targeted multimodal NGS assay that can detect SNVs, InDels, fusions, gene expression, and copy number variations from total nucleic acid (TNA) in a single tube workflow. Methods: TNA was extracted from peripheral blood and bone marrow specimens from patients with hematologic cancers. TNA was used to prepare libraries then sequenced on a NovaSeq 6000. DNA variants were compared to results from DNA NGS assays and RNA fusions were compared to FISH and RT-PCR. Results: Our multimodal NGS assay can efficiently use TNA to detect mutations simultaneously within the DNA and RNA in a single tube workflow. From 100 fusion positive samples, we detected fusions in all samples and >25 different fusions were detected. Our NGS assay was 100% concordant with the BCR-ABL1 qRT-PCR assay in samples with an IS value of >0.5, 92.7% concordant with the ArcherDX Heme NGS assay, and 100% sensitive in detecting high-confidence fusions. In 5 previously tested BCR-ABL1 positive samples, we confirmed the RNA expression as well as detected pathogenic DNA variants, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71. In another patient, we found multiple pathogenic mutations (ASXL1 and JAK2), in addition to a BCR-FGFR1 fusion. Two PML(e4)-RARA and PML(e6)-RARA isoforms were detected and confirmed in one sample, illustrating the high resolution that could be used to help monitor the patient. Three fusions involving CXCR4 (CXCR4-FOSL2, CXCR4-DDX5, and ARID5A-CXCR4), a receptor known to promote proliferation, migration and resistance to chemotherapy were also detected in addition to CXCR4 over-expression in three patients. In another patient, we confirmed a KMKT2A-ARHGEF12/del(11)(q23q23) aberration by NGS that was missed by cytogenetics. In one sample, we confirmed expression of 3 out of 4 different MYC fusions (MYC-BCL6, MYC-IgH, IgH-MYC); only NGS could identify the fusion orientation, illustrating the high resolution of NGS over FISH. In several patients with IgH-BCL1 translocations, we expected BCL1 overexpression. Although DNA PCR results were mostly negative, we discovered increased expression in a subset of these samples. This suggests that despite the detection of the fusion by FISH, a subset may lack gene expression which may suggest a different biological or clinical significance. Conclusion: Our findings demonstrate the value that a com