Abstract 182: A comprehensive genomic profiling approach to interrogate hematologic malignancies using a novel multimodal next generation sequencing assay in a single-tube
Authors: Jung Segun C. , Yu YongXin , Mou Yanglong , Nam Hyunjun , Wong Cynthie , Koo Samuel , Thomas Brad , Blocker Forrest , Lyle Derek , Bender Ryan , Agersborg Sally , Weiss Lawrence M. , Funari Vincent A. 
Published: September 2020

Abstract
Background: Many new guidelines require a comprehensive genomic profiling approach for diagnosis, risk stratification and therapy decisions. Limitations in sample quantity and throughput may limit the number of single biomarker tests (FISH, karyotyping, sequencing, qRT-PCR, etc.) that can be performed for the patient. There are currently multiple commercial NGS assay options for total nucleic acid, however they involve independent parallel workflows and twice the amount of sample and effort. Here we developed a novel consolidated DNA/RNA workflow in a single-tube assay utilizing custom QIAseq multimodal chemistry. This simplified workflow enables a discovery approach of all critical DNA/RNA abnormalities in hematologic malignancies, extending our NGS capabilities to large structural changes, RNA fusions and expression.
Methods: 297 Heme-focused genes and 14 chromosomes were targeted in the genome, along with 213 RNA genes targeting 712 exons involved in known fusions in the transcriptome using a custom QIAseq workflow. Captured DNA/RNA targets from 135 patients were sequenced with unique dual indices on an Illumina's NovaSeq 6000. Coverage and variant allele frequency from all gene and chromosomal targets in 25 disease free patients was compared to the same genomic targets in 76 patients that were referred for a suspected hematological malignancy (e.g. MDS, CML, AML, ALL, etc.). We compared results from our custom algorithm to karyotyping and FISH. In addition, we assessed the relationship between structural changes and the average mutation load for each indication. Positives gene fusions were confirmed by qRT-PCR or Sanger sequencing.
Results: Cytogenetic abnormalities in 30/32 patients were confirmed by karyotyping and FISH; two cases with abnormalities were missed by NGS. NGS detected additional abnormalities not detected by cytogenetics, including a case of loss of chr17 including deletion of driver genes, NF1 and SUZ12. No significant relationship between chromosome abnormalities and tumor mutation burden was observed. However, patients referred for myeloid disorders with structural abnormalities had a significantly higher mutational burden (p&lt;9.12x10−7). Mutational load in these patients was significantly associated with chromosome 17 abnormalities, primarily loss (p&lt;0.013). qRT-PCR confirmed 100% of BCR-ABL fusions (p210, p190) in all patients (59/59) with International Scale (IS) percentages ranging from 2.4-100%. Notably, we confirmed two clinically significant fusions, SET-NUP214 and RUNX1-RUNX1T1 in two patients not previously interrogated by qRT-PCR. The SET-NUP214 fusion is normally associated with acute lymphoblastic leukemia (ALL) was identified in a patient with suspected CML. We also confirmed PCALM-MLLT10 gene fusion was detected in a commercial universal human RNA reference material, commonly used in expression profiling studies.
Conclusions: This study confirms the validity and utility of simple but efficient comprehensive genomic profiling for use in hematologic malignancies. Coupled with FISH and cytogenetics tests, NGS can offer a better diagnostic and prognostic testing service for patients with hematologic disease to assist in treatment selection as well as precise patient care.
Citation Format: Segun C. Jung, YongXin Yu, Yanglong Mou, Hyunjun Nam, Cynthie Wong, Samuel Koo, Brad Thomas, Forrest Blocker, Derek Lyle, Ryan Bender, Sally Agersborg, Lawrence M. Weiss, Vincent A. Funari. A comprehensive genomic profiling approach to interrogate hematologic malignancies using a novel multimodal next generation sequencing assay in a single-tube [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 182.