A Novel Multimodal Next Generation Sequencing Assay with Total Nucleic Acid Input to Provide Comprehensive Genomic Profiling for Hematologic Malignancies

Background: A cost effective and comprehensive genomic profiling (CGP) approach for diagnosis, risk stratification and therapy would be useful for the evaluation of oncologic specimens. Available approaches involving additive testing for DNA and RNA abnormalities through traditional methods (e.g. Sanger, FISH, cytogenetics, qRT-PCR) are not comprehensive, require multiple different workflows and are sample consuming, often resulting in incomplete testing. While there are next generation sequencing (NGS) assays designed for detecting DNA and RNA abnormalities, they have separate workflows that require twice the amount of sample and effort. To address this, we developed a novel total nucleic acid (TNA) extraction method and single tube workflow utilizing TNA and a custom multimodal chemistry designed for hematologic malignancies. This consolidated workflow enables an efficient discovery based approach for both DNA/RNA abnormalities including single nucleotide variants (SNVs), InDels, copy number variants (CNVs), large structural changes from DNA and gene fusions and gene expression levels from RNA. This method maximizes data derived from valuable samples while delivering a comprehensive profile of the patient's tumor which can help guide therapeutic and clinical decisions.

Methods: Total nucleic acid (TNA) was extracted from bone marrow and peripheral blood of 95 patients (CML, CMML, CLL, AML and myeloid disorders). 297 genes that have DNA mutations specific to hematological cancers were targeted, along with 213 genes that were targeted for clinically significant RNA abnormalities. Enriched genomic and transcriptomic regions of interest from 85 patients were successfully sequenced with unique dual indices on an Illumina NovaSeq 6000. DNA variant detection as well as fusion detection from RNA were compared to traditional orthogonal NGS assays that use DNA input or compared to qRT-PCR and Sanger sequencing assays that use RNA as input.

Results: In this study, we developed an efficient and high-quality TNA extraction method that can purify enough total nucleic acid from bone marrow, peripheral blood, cytogenetic pellets, flow suspension, and FFPE samples for the downstream NGS assay. The average OD 260/280 value was 1.9 and the OD 260/230 was 2.18. After sequencing, 256/262 (97.7% accuracy) SNV and Indel variants that were candidate pathogenic mutations were concordant from 38 patients. Meanwhile, 100% (7/7) of all BCR/ABL1 gene fusions which had an international scale (IS) value above 6.4% were concordant. In addition, 69 fusion positive samples containing 20 unique gene fusions which had been previously reported by an independent ArcherDX assay designed specifically for gene fusions were also evaluated with this chemistry. Analysis revealed a 92.5% (64/69) concordance. More importantly, the QIAseq multimodal TNA NGS assay detected both DNA and RNA abnormalities in a single tube. For example, in one myeloid leukemia patient, we not only identified pathogenic variants of ASXL1 and JAK2 which had been previously detected by a DNA NGS assay, but also detected a concurrent BCR-FGFR1 fusion which had been previously reported by a FISH assay. Moreover, we were able to provide more comprehensive genomic profiling by investigating many DNA and RNA abnormalities simultaneously. In our study, for 5 patients that previously been tested for BCR-ABL1 fusion only, we are able to assess BCR-ABL1 fusion status from RNA as well as identify pathogenic DNA variants at the same time, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71, which provides valuable information to assist diagnosis and treatment in a cost effective and efficient way.

Conclusions: We developed a single tube TNA based workflow with a custom multimodal chemistry that simultaneously detects many DNA and RNA abnormalities in a cost effective and efficient way while reducing sample requirements. This unique TNA NGS assay provides comprehensive genomic profiling for hematologic malignancies and improves the diagnostic testing options for precise patient care.