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 comprehensive profile provides to diagnostic tests. NGS has high resolution and by targeting RNA we can detect more fusions than traditional DNA approaches. Comparison of FISH and NGS results show that detecting a functional mutation maybe important for characterizing a disease. However, both may need to be combined for a complete picture to improve detection and characterization of various hematologic diseases.

Citation Format: Cynthie Wong, Brad Thomas, Yanglong Mou, Christophe Magnan, Segun Jung, Tibor Gyuris, Francys Alarcon, Eve Shinbrot, Fei Ye, Ryan Bender, Sally Agersborg, Lawrence Weiss, Vincent Funari. A comprehensive genomic profiling approach to detect functional translocations and genomic alterations in a single tube workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2208.