Evaluation of the New NGS Liverpool Lymphoid Network Panel for Deployment in a Clinical Laboratory

Introduction:

This study evaluated the Liverpool Lymphoid Network Panel using the Ion AmpliSeq technology for potential deployment in a clinical testing laboratory. The lymphoid NGS panel intended for hemato-oncology assessment, is a targeted NGS panel designed by clinical researchers at Liverpool Clinical Labs, UK. This targeted panel covers 60 genes, providing comprehensive assessment of key biomarkers of multiple lymphoid disorders in a single test and is applicable for the study of a range of lymphoid neoplasms. Available specimen inputs are blood, buffy coat, bone marrow, and formalin-fixed paraffin embedded (FFPE) tissue. Our study was intended to evaluate the user experiences with the system, as well as panel performance in the molecular characterization of a cohort of commercially available lymphoid neoplasms.

Methods:

Lymphoid specimens (buffy coat, n=30) representing chronic lymphocytic leukemia (CLL; n=2), lymphomas including Hodgkins (n=6), non-Hodgkins (n=18), and lymphoplasmacytic (n=4), were procured from a commercial biorepository as examples of typically collected clinical specimens. The demographics of the 30-specimen cohort were a range of 23-78 years of age (mean=56), 18 females and 12 males, and all were Caucasian. DNA extraction was performed using the QIAamp DNA Blood Mini Kit per manufacturer instructions. DNA quantification was conducted on the Qubit using the DNA HS kit. Eight specimens were extracted twice to recover sufficient DNA required for sequencing. The range of DNA quantification was 1.36-60 ng/µl due to variability in cellularity. For concentrations above 13 ng/µl, a 1:10 dilution was done before loading 25 µl of each DNA onto the sequencer. Specimens were loaded onto the sequencer over two chips with 15 specimens and one no template control (NTC) per chip. Each batch was completed within 14 hours on the instrument and analysis was completed on sequencer. To assess usability, four operators were trained on the system, specimen preparation and run protocols.

Results:

All specimens were successfully processed to yield analytic results. The average total reads per specimen were 3.5 million with an average mean read depth of 2,646. Variants were analyzed using a high stringency filter (15%) to call positives. Twelve of the thirty retrospectively analyzed specimens contained at least one mutation regardless of clinical diagnosis. A total of 18 mutations were detected over the cohort of samples, with several being considered of high clinical relevance. Hotspot mutations were common in the KMT2D (8/30) gene. Additional targets of interest included DNMT3A, GNA13, TET2, FOX01, PRDM1 and EP300. User training was successful for multiple users that were new to the system. In terms of user experiences, all operators reported minimal hand on times in specimen handling.

Conclusion:

The newly available Lymphoid Liverpool panel can provide clinicians new and expanded molecular insights into lymphoid and lymphoproliferative disorders. The panel, coupled with a low user intervention, highly sensitive chemistry, and system that can generate results with rapid turnaround time was very amenable to evaluation by our laboratory. We successfully processed all 30 clinical specimens in our test cohort with the panel and system performing as expected. Additional studies are planned to support the performance of the bioinformatics pipeline, panel analytic/clinical verification and validation, prior to implementation in the clinical laboratory.

Pestano:Biodesix: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties; Thermo Fisher Scientific: Honoraria; Bio-Rad: Honoraria. Weaver:Biodesix: Current Employment. Diercks:Biodesix: Current Employment. Pestano:Biodesix: Current Employment. Pestano:Biodesix: Current Employment. Sathyanarayana:Biodesix: Consultancy.