Abstract 219: Single-tube NGS profiling allows identification of molecular signature in ALL patients
Authors: Zelinger Lina , Koo Samuel , Ko Charmeine , Alarcon Francys , Thomas Kenneth B. , Jung Segun C. , Diaz Fernando J. Lopez , Ye Fei , Kulkarni Shashikant 
Published: April 2023

Abstract
Background: Acute lymphoblastic leukemia (ALL) can be classified to subtypes by molecular signatures correlating to prognosis and treatment regimen, making accurate molecular profiling of interest for both clinical and research communities. NGS technology allow for identification of a wide array of molecular events (e.g. structural variants and single nucleotide variants (SNVs)), and integrate these with global profiles of gene expression. Most tests screen either DNA or RNA to provide a piece of a complex molecular puzzle, resulting in higher cost, processing time and amounts of input material. We report a single tube NGS assay that uses total nucleic acid as input for simultaneous screening of DNA and RNA (allowing us to profile gene expression, fusions and multiple variants), establishing a work frame for comprehensive molecular profiling in a single test.
Methods: 243 clinical ALL samples (206 bone marrow and 37 peripheral blood) were used in this study, split in to two cohorts, 102 for initial investigation and 141 for reproducibility testing. Libraries were done using a custom Qiagen Multimodal NGS panel of 302 DNA and 229 RNA targets. Amplicon libraries were sequenced with UDI on Illumina NovaSeq 6000. Data were analyzed by in-house bioinformatic pipeline. Expression values were normalized to GUSB. Previous in-house studies were used to establish cut-offs for upregulation of 31 genes associated with ALL. Variant enrichment for genes was done by normalizing the number of variants to the size of the gene and genes with elevated variant loads were identified. Pathway enrichment analysis was done using gProfiler2.
Results: A set of 102 ALL samples were used to study expression patterns within the cohort. Principal component analysis (PCA) on the 229 RNA targets was used to identify clusters. We then investigated whether the predetermined 31 over-expressed (OE) genes could be driving the clusters. 3 clusters were observed based on OE quantification, low OE degree (0-1 genes), medium (8-11), and high (&gt;17 genes). EPOR OE is characteristic of the high-degree cluster, while Ph+ samples are most common in the medium cluster. Pathway enrichment on the OE genes suggest that the medium cluster is enriched for genes linked with missregulation of transcription, and the high cluster shows enrichment of kinase activation. We then repeated the analysis with a second set of 141 samples, to assess the reproducibility of these findings and confirming our results. Finally, we investigated SNVs’ role in our data. We observed that KMT2D was enriched in variants in our cohort, underscoring that epigenetics is an important component of the disease landscape.
Conclusions: We demonstrate the use of a single tube multimodal NGS assay for comprehensive genomic profiling. Our analysis shows that this powerful and cost-effective tool can help identify molecular signatures, guiding diagnostic and therapeutic management for ALL patients.
Citation Format: Lina Zelinger, Samuel Koo, Charmeine Ko, Francys Alarcon, Kenneth B. Thomas, Segun C. Jung, Fernando J. Lopez Diaz, Fei Ye, Shashikant Kulkarni. Single-tube NGS profiling allows identification of molecular signature in ALL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 219.