Rapid Clinical Mutation Screening for AML Using the Genexus Platform
Authors: Jiwani Shahanawaz , Radich Jerald P. , Saeed Maria , Aicher Lauri , Chang Ting-Chia , Narava Srikrishna , Holman Zaneta , Beppu Lan , Sala-Torra Olga , Majano Marvin , Taylor Erin , Yee Laura , Li Jessica , McShane Lisa , Sanduria Pooja CS , Williams Paul Mickey M. , Yeung Cecilia C.S. 
Published: December 2023

Introduction: The promise of “precision medicine” is using therapeutic agents matched to the inferred biology of a patient's particular disease. In acute myeloid leukemia this approach demands fast and accurate assessment of the mutation and cytogenetic landscape of each AML case. The NCI Myeloid Malignancies Molecular Analysis for Therapy Choice program (“myeloMATCH”) is a precision medicine clinical trial initiative across the NCI National Clinical Trials Network for patients with newly diagnosed acute myeloid leukemia and myelodysplasia. In myeloMATCH the goal is to have cytogenetic and mutation assays performed within 72 hours of central lab receipt, allowing for a rapid assignment of the appropriate clinical trial from the myeloMATCH trial portfolio. Here we report analytic performance of rapid genomic profiling laboratory developed test based on the Thermo Fisher Genexus Oncomine Myeloid Panel GXv2 (NCI Myeloid Assay version 2, or “NMAv2”) which performs rapid sequencing of SNVs, indels, and gene fusions, capturing 1,661 hotspot variants (45 genes) and 779 fusions (35 genes) with a turnaround time of &lt;48 hours from specimen receipt to clinical reporting.
Methods: The validation testing was performed by two harmonized laboratories; NCI Molecular Characterization laboratory (MoCha) and Fred Hutch Molecular Oncology (MO). We conducted analytical validation and harmonization experiments for CLIA/CAP and FDA regulatory compliant use in this trial across both accredited clinical laboratories with harmonized workflows. Samples are extracted with AllPrep per manufacture protocol. Library preparation with the Oncomine Myeloid Assay GX v2 and the sequencing reactions are performed on the automated Ion Torrent Genexus System. Sequencing data generated by NMAv2 was analyzed using Ion Torrent Genexus system version 6.6.2.1. The default manufacturer workflow has been customized using a custom designed hotspot browser extensible data (BED) file, sequence variant baseline (SVB) file, fusion target file, torrent variant caller (TVC) version 5.18 and Oncomine extended filter chain (OEFC) version 5.16. In NMA, the default 2.5%, and 3% VAFs were set up for calling hotspot and non-hotspots SNV/indel, 1% VAF for FLT3-ITD and 100 read counts for all fusion, except 2000 read counts for KMT2A-PTD.
Results: The validation set included 148 unique samples and comprised of 76 AML/MDS patients (49 peripheral blood, 27 bone marrow aspirate), 51 healthy donor blood, 6 healthy donor bone marrow, 7 cancer cell lines, 5 normal HapMap cell lines, and 3 contrived materials encompassing 364 known mutations (143 SNPs, 132 indels, 27 fusions). 180 runs were completed for this validation (106 at FH and 74 at MoCha) with 89.44% success rate in passing run level metrics. Results of the validation testing are detailed in Table 1. High reproducibility in detecting all reportable variants was observed, with overall &gt;99% mean positive percentage agreement (PPA) across both labs (Figure 1). The limit of detection for each variant type was &lt;0.06% for hotspot SNVs, 0.07% for non-hotspot SNVs, 0.51% for hotspot indels, ~1% for non-hotspot indels, 0.23% for FLT3-ITDs, and 50 minimum reads at 0.1% tumor content for fusions. 99.39% concordance was observed by testing 86 blinded validation sample set with analytically validated orthogonal assays.
Conclusions: The NMAv2 has high specificity, sensitivity, accuracy and reproducibility (inter-lab and intra-lab) with sequencing results generated within 48 hours of assay initiation. As such, the assay is well suited for use to rapidly categorize the genomic alteration of AML to support clinical care in patients with active myeloid malignancies, including those patients potentially enrolled in the myeloMATCH program.
Table 1: Results of validation experiment results for the NMAv2
Figure 1: Harmonization results for the NMAv2 between MoCha and MO labs, Right Panel = Indel; Left panel = SNV