Advancements in Rapid NGS for Myeloid and Lymphoid Malignancies

Health Services Laboratories (HSL) is United Kingdom based clinical laboratory that provides a comprehensive range of diagnostic services, including pathology and clinical laboratory testing, to support patient care in hospitals, clinics, and other healthcare settings. Robert Baker, Operations Manager of the molecular hematology lab at HSL, provides comprehensive molecular testing services, including the use of NGS for a broad range of hematologic malignancies. The lab processes hundreds of hematological samples annually. Previously, the lab relied on a combination of PCR assays and NGS for molecular analysis. However, this workflow, while effective, had limitations in terms of labor intensity and throughput.

Prior to adopting Rapid NGS testing, HSL relied on multiple single-analyte tests along with a traditional NGS assay. Comprehensive results for all biomarkers generally takes about two weeks.

• FLT3-ITD PCR and capillary electrophoresis assay: used for rapid detection of significant variants
• Custom real-time PCR panel: targeted the 14 most common RNA and DNA markers associated with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)
• Myeloid NGS and RNA sequencing: provided comprehensive profiling with a 14-day turnaround time

While this method allowed for rapid initial detection, it was labor-intensive, had low throughput, and often required subsequent NGS to uncover additional significant variants.

In response to the need for a more efficient approach, Baker’s team conducted a feasibility study using the Ion Torrent Genexus System with the Oncomine Myeloid Assay GX v2. This system enables rapid turnaround times and comprehensive genomic profiling. By pooling samples and optimizing the workflow, the lab could process up to 2,500 samples annually on a single sequencer, significantly enhancing their capacity.

Key features of the new workflow

• Automated and rapid—minimal hands-on time with results available within 24 to 48 hours
• Comprehensive profiling—detects a wide range of highly relevant variants, including rare fusions and mutations
• Economic viability—efficient sample pooling maximizes the use of sequencing chips, helping to reduce costs 

Building on the success with myeloid malignancies, the laboratory also utilized the Ion AmpliSeq Liverpool Lymphoid Network Panel for rapid genomic profiling of lymphoid malignancies. This panel enabled the laboratory to quickly obtain genomic data within approximately 27 hours, effectively supporting the analysis of low-grade T- and B-cell lymphoma samples. Additionally, the laboratory leveraged a network of laboratories for ongoing analytical validation and updates, enabling the reliability and accuracy of the profiling results.

HSL has also started to explore new NGS applications for B-cell lymphomas. They are evaluating the use of gene expression signatures as a rapid method for detecting single/double/triple hit high-grade B-cell lymphomas. 

Mr. Baker presented preliminary data from an AML sample profiled using the Oncomine Myeloid MRD (RUO) Assay and Ion GeneStudio S5 system. This assay uses AmpliSeq™HD technology to enable a low Limit of Detection (LoD) down to 0.05% variant allele frequency (VAF) for studying measurable residual disease (MRD). In their initial run, the lab was able to detect a low-frequency DNMT3A variant at 0.14% VAF and an IDH2 variant at 0.07%VAF.

Mr. Baker presented data suggesting that overexpression in MYC, BCL2, BCL6 may be a reliable indicator of fusion involving the overexpressed gene(s). In their workflow, a gene expression profile can be rapidly measured using the Oncomine Childhood Cancer Research Assay (OCCRA). Any samples with an overexpression of MYC, BCL2, or BCL6 are then tested using an orthogonal NGS fusion assay to confirm the presence of a translocation.This approach may result in a fast and efficient way to perform a primary analysis without the need to run a separate NGS fusion assay, though Mr. Baker stressed that more research must be done in this area.

Some potential future strategies for lymphoma genomic profiling include subjecting all high-grade lymphomas to both the OCCRA and Liverpool Lymphoid Network Panels, while low-grade lymphomas would only be analyzed using the Liverpool Lymphoid Network Panel.

The advancements presented by Robert Baker highlight the transformative impact of rapid NGS technologies in the field of hematological malignancies. By significantly reducing turnaround times and improving genomic profiling accuracy, these innovations may help enhance future clinical decision-making and streamline laboratory workflows.