Figure 3.
Sequencing-based tumor burden monitoring in myeloid neoplasms. (A) In this AML example, sequencing identifies NPM1 and DNMT3A mutations at diagnosis with the NPM1 mutation representing the founding clone (green: based on higher mutation VAF) and DNMT3A representing a subclone (gray: based on lower mutation VAF). Estimated sensitivity to detect mutations for different sequencing approaches is shown below. As the patient enters remission, the NPM1-mutated clone is partially cleared, becoming undetectable by panel-based sequencing and WGS but remains detectable by high-sensitivity MRD sequencing. In this example, the DNMT3A-mutated clone remains without significant change in VAF, consistent with persistent clonal hematopoiesis. Finally, the patient relapses with the same NPM1-mutated founding clone plus a newly acquired NRAS mutation. (B) A comparison of sequencing methods for MRD monitoring. Although WGS offers the greatest sequencing breadth and is capable of detecting structural variants such as CNAs and chromosomal translocations, standard coverage is generally only ∼60×, limiting detection to mutations with VAFs > 10%. Targeted sequencing is generally limited to 50 to 500 genes, providing minimum sequencing breadth but can achieve high coverages (1000×) at a lower cost than WGS and provides adequate sensitivity (2% VAF) for initial diagnostic evaluation. MRD panels are similar to targeted panels but use much higher sequencing depths and use UMIs to achieve sensitivities of ∼0.1% VAF allowing for MRD monitoring. MRD panels are generally easy to implement but may be of limited clinical utility for patients with few mutations covered by the panel. In patient-specific MRD sequencing, mutations are identified at diagnosis using broad methods such as exome sequencing or WGS, ensuring an adequate number of mutations to track. These mutations are then individually targeted using custom probes at subsequent time points. By focusing sequencing on known mutations, patient-specific MRD can achieve extremely high detection sensitivities for nearly all patients. Patient-specific MRD, however, can be logistically challenging and expensive to implement because it requires custom probe designs and validation for every patient. Patient-specific methods also cannot detect newly acquired mutations that were not targeted by probes at diagnosis. Professional illustration by Patrick Lane, ScEYEnce Studios.

Sequencing-based tumor burden monitoring in myeloid neoplasms. (A) In this AML example, sequencing identifies NPM1 and DNMT3A mutations at diagnosis with the NPM1 mutation representing the founding clone (green: based on higher mutation VAF) and DNMT3A representing a subclone (gray: based on lower mutation VAF). Estimated sensitivity to detect mutations for different sequencing approaches is shown below. As the patient enters remission, the NPM1-mutated clone is partially cleared, becoming undetectable by panel-based sequencing and WGS but remains detectable by high-sensitivity MRD sequencing. In this example, the DNMT3A-mutated clone remains without significant change in VAF, consistent with persistent clonal hematopoiesis. Finally, the patient relapses with the same NPM1-mutated founding clone plus a newly acquired NRAS mutation. (B) A comparison of sequencing methods for MRD monitoring. Although WGS offers the greatest sequencing breadth and is capable of detecting structural variants such as CNAs and chromosomal translocations, standard coverage is generally only ∼60×, limiting detection to mutations with VAFs > 10%. Targeted sequencing is generally limited to 50 to 500 genes, providing minimum sequencing breadth but can achieve high coverages (1000×) at a lower cost than WGS and provides adequate sensitivity (2% VAF) for initial diagnostic evaluation. MRD panels are similar to targeted panels but use much higher sequencing depths and use UMIs to achieve sensitivities of ∼0.1% VAF allowing for MRD monitoring. MRD panels are generally easy to implement but may be of limited clinical utility for patients with few mutations covered by the panel. In patient-specific MRD sequencing, mutations are identified at diagnosis using broad methods such as exome sequencing or WGS, ensuring an adequate number of mutations to track. These mutations are then individually targeted using custom probes at subsequent time points. By focusing sequencing on known mutations, patient-specific MRD can achieve extremely high detection sensitivities for nearly all patients. Patient-specific MRD, however, can be logistically challenging and expensive to implement because it requires custom probe designs and validation for every patient. Patient-specific methods also cannot detect newly acquired mutations that were not targeted by probes at diagnosis. Professional illustration by Patrick Lane, ScEYEnce Studios.

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