Authors (including presenting author) :
Lau KN, Wong TF, Yip SF
Affiliation :
Department of Clinical Pathology, Tuen Mun Hospital
Introduction :
Risk stratification and management for acute leukaemia relies heavily on genomic profiling. Comprehensive genomic profiling of AML currently requires plethora of genetics and genomics (GG) tests, namely cytogenetic study, targeted gene panels and RNA-sequencing by Next Generation Sequencing (NGS) for selected cases, which are tedious and could take one month to complete. Rapid turnaround time of GG tests results within 5 days for AML is an unmet need. Oxford Nanopore Sequencing (NS) is a third generation long read sequencing technology which enables real-time DNA sequencing. It allows identification of structural variations (SVs), copy number variations (CNVs) and single nucleotide variants (SNVs) simultaneously; it potentially enables rapid comprehensive genomic profiling as a single-assay.
Objectives :
Comprehensive evaluation of NS for adult AML genomic profiling was not published before.
This is a single institution pilot study to evaluate the clinical utility of NS in AML patients.
Methodology :
12 newly diagnosed adult AML patients were recruited. All patients had routine cytogenetic study, fragment analysis for FLT3-ITD and myeloid panel NGS. 3 patients had additional RNA-sequencing by NGS.
NS with adaptive sampling approach using Promethion P2 Solo device was performed. Comprehensive genomic profiling was done using in-house bioinformatics pipeline. Results were compared with the above conventional assays.
Result & Outcome :
Time from DNA extraction to NS profiling took only 1-4 days. Mean coverage at the target regions were 23.3X.
Comparing to cytogenetic, all 4 AML cases with clinically significant chromosomal translocations were detected. 7/8 CNVs (>5Mb) were detected and NS has missed a clonal evolution event (del(13)(q12q14)).
Comparing to myeloid panel, the sensitivity and specificity of SNVs detection were 95.8% (23/24) and 100%. A CEBPA b-ZIP domain variant was missed by NS.
4 FLT3-ITD variants were detected by fragment analysis. Myeloid NGS detected all 4 variants but could not quantify 3 while NS detected and quantified only 2 variants.
However, 3 clinically significant findings (NUP98::NSD1 fusion, KMT2A-PTD and Xp11.4 microdeletion) that were missed by routine CG and myeloid NGS were detected by NS. These would lead to revision of WHO leukaemia classification and risk re-stratification by ELN 2022 for 2 cases with implication on therapy.
Conclusion:
NS is a rapid single-assay that allows comprehensive genomic profiling within 5 days at a competitive cost. Additional important cytogenomic findings can be revealed compared to existing platform. Further assay optimization to reach ~60x coverage might reduce false negative results. Similar to myeloid NGS, FLT3-ITD detection is best complemented by fragment analysis. A larger evaluation study is ongoing.
