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Next-generation sequencing in AML
of sensitive and specific custom-made FLT3 ITD NGS- based mutation detection assays have been published.4,5
Sequencing
A selected number of fusion transcripts have been incorporated in the 2017 ELN recommendations and clas- sify AML patients into various risk categories.1 Although the majority of these gene rearrangements are revealed by cytogenetic analyses, molecular approaches are often complementary. Fusion transcript detection was long lim- ited to those transcripts for which standardized assays, generally real-time quantitative PCR, were available. However, all fusion transcripts relevant for risk stratifica- tion of AML can now be detected in a single assay with RNA-based NGS (RNA sequencing). Given that at least one of the partner genes involved in all clinically relevant fusions is known, RNA sequencing analysis can focus on these specific genes, leaving the possibility of revealing novel fusion partners. Such targeted RNA sequencing- based assays are commercially available. However, since the number of clinically relevant fusion transcripts is lim- ited, one could also consider developing customized methods in which the AML-associated transcripts are amplified by (multiplex) PCR and subsequently sequenced by NGS.6 Again, proper validation of these assays at the local site is essential.
Currently, library preparation and sequencing with amplicon-based NGS assays usually require several days, whereas analyses of the limited number of 2017 ELN genes can be done rather quickly. The introduction of novel NGS machines with faster turnaround times, such as the Illumina iSeq100, and the development of cu- stomized assays now enable fast library preparation and overnight sequencing, thus allowing for a quick return of test results to the clinic. This is of particular interest when targetable mutations, such as those in FLT3, IDH1 or IDH2, are needed for selection of the appropriate drug for front-line AML therapy or for relapsed patients with a high disease burden.
Since most of the clinically relevant mutations in myeloid malignancies are known, targeted sequencing is currently the method of choice. However, it can be fore- seen, when turnaround times and costs are reduced, that whole exome or whole genome sequencing will become the standard approach to genomic characterization of AML at diagnosis. The use of whole exome or whole genome sequencing will allow identification of all somat- ic coding mutations, including those that are targetable but less frequently present in AML. Moreover, one can prioritize analysis of key AML genes first, such that initial results regarding the clinically most relevant genes can be obtained with a short turnaround and more comprehen- sive genomic profiling can follow later. Furthermore, whole genome sequencing allows identification of novel biomarkers located outside of protein coding regions, which may be useful not only for proper assessment of the prognosis but also for detection of minimal residual disease (MRD) in AML as they can be used to identify and follow leukemic clones regardless of their role in AML initiation and maintenance.
Minimal residual disease
Our improved understanding of the molecular land- scape of AML has resulted in better treatment decisions at the time of complete remission after induction treatment.
Although the majority of AML patients achieve complete remission, many eventually relapse. Thus, there is still a great need for adequate prediction for subsequent relapse to adapt treatment accordingly and improve the out- comes of patients at high risk of relapse. MRD detection has already proven to have substantial value in predicting relapse and overall survival when applied to AML in com- plete remission but the use of molecular enumeration of MRD has been limited to only specific, molecularly defined subtypes of AML.7-9 By contrast, flow cytometric analysis of MRD can be done in nearly all AML patients, but is operator- and center-dependent and there is no cen- trally agreed approach to enumerate flow-based MRD in AML. NGS enables MRD detection by measuring all mutations, including patient-specific persistent muta- tions, in complete remission. In fact, it has recently been shown that molecular MRD detection by NGS is applica- ble to virtually every newly diagnosed AML patient because of the frequent prevalence of multiple molecular aberrations among patients with AML.10-13 However, MRD detection based on NGS must overcome several challenges before it can be reliably introduced into clini- cal practice.
The known oligoclonality of the disease at diagnosis has a clear impact on MRD detection. Molecular markers in small AML subclones at diagnosis could easily be missed by panel-based NGS at lower depth. However, these small populations of cells may be selected during therapy and ultimately result in AML relapse. This issue could be overcome by sensitive detection of all possible mutations frequently present in myeloid malignancies. However, because of the relatively high error rates of current standard NGS technologies, reliable detection of a multitude of mutations at high sensitivities (<0.01%) is not yet easily achieved. In fact, the currently high intrin- sic error rates (1 to 0.1%) impede sensitive MRD moni- toring at later time points during therapy as well. At these time points certain targets present at diagnosis can be sequenced individually with single amplicons at high depth, but true residual mutations may still not be reli- ably discriminated from noise at levels below 0.1%. Attempts should be made to improve the signal-to-noise ratios in order to detect low-level variants accurately.14 Genomic DNA isolated from bone marrow, peripheral blood or mononuclear cells is generally of high quality, but noise in NGS is subsequently introduced at different levels during library preparation and sequencing.14 The rate of sequencing artefacts can be reduced biochemical- ly, e.g., by using proof-reading polymerases, or compu- tationally; however, these corrections are only modest and cannot attenuate errors/artefacts entirely. Alternative strategies should be explored. Recently, vari- ous error-corrected NGS methodologies using molecular barcoding have been introduced.14 Error-corrected sequencing is based on barcoding the individual DNA molecules used for NGS library preparation. Using the unique sequence tag all derivative reads, which arise from a common founder, can be recognized after com- putational NGS, which enables removal of PCR dupli- cates and false mutation calls. These approaches and protocols15 have been shown to increase the specificity of low-frequency mutation detection.14 However, whether error-corrected sequencing will improve MRD detection by NGS in AML remains to be demonstrated in large cohorts of AML.
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