Page 24 - 2019_05-HaematologicaMondo-web

R.L. Levine and P.J.M. Valk et al.
Recently, several studies addressed NGS-based MRD detection in relatively large AML cohorts from clinical tri- als, all demonstrating that NGS-based MRD has a pro- found prognostic impact in patients with AML.11-13,16 In these studies persistent mutations in complete remission were measured with gene panels,11 capture-based deep sequencing10,12,16 or targeted sequencing.13 The amplicon- based approach was specifically designed for MRD detec- tion by including error-correction,13 which suggests that previous NGS-based MRD studies were not yet optimal. The results of these initial studies do not allow any firm conclusions to be drawn with regard to the superiority of error-corrected NGS for MRD detection in AML. However, the fact that NGS MRD has consistent prog- nostic value implies that technological improvements should be made in order to further optimize relapse pre- diction in AML, assuming that in these initial studies minor AML MRD clones were missed in complete remis- sion. The ELN MRD Working Group is currently aiming to improve and harmonize methodologies for NGS-based detection of MRD in AML.
Another successful approach to correct for noise is to use a site-specific error model with a sufficiently large set of reference samples without mutations.11 In such a model the distribution of variants is determined in a reference set without mutations, for example, remission samples. MRD is subsequently defined by those mutations, such as the ones present at diagnosis, which are statistically signifi- cantly different from the distribution of variants in the ref- erence set. In this case the detection sensitivity of muta- tions is variable and dependent on the average coverage for that specific locus for all samples, the observed error vari- ance of the site-specific variant in the reference set (a high variance results in decreased detection sensitivity) and the number of control samples available. A major drawback of this approach is that a set of reference samples to deter- mine MRD has to be available. MRD measurement in a single sample without the dependence of a large reference set is obviously a preferred method since it will be more easily implemented in clinical practice.
Clonal hematopoiesis
In the initial NGS-based MRD studies11-13,16 it became clear that gene mutations persisting in complete remis- sion that are well-known to be associated with clonal hematopoiesis17,18 do not have an impact on the risk of relapse, despite the fact that they are among the most common disease-initiating drivers of AML. As a result of high-dose chemotherapy, AML patients with these muta- tions are apparently brought back into a state of clonal hematopoiesis, in which mutations occurring late in leukemogenesis are irradiated, but mutations also found in clonal hematopoiesis persist. It is clear that these per- sisting mutations, also known as clonal mutations of indeterminate potential, add another layer of complexity to MRD detection in AML.
In studies of molecular MRD, clonal hematopoiesis- related mutations in DNMT3A, TET2 and ASXL1 (DTA) were considered clonal hematopoiesis rather than resid- ual leukemia. Besides acquired mutations in DTA, other well-known pathogenic mutations such as those in TP53, PPM1D, JAK2, CBL, SRSF2 and SF3B1 are involved in clonal hematopoiesis, however, at lower incidence.17,18 In fact, many of these mutations also persist in complete remission with high variant allele frequencies. It needs to
be determined whether and, if so, to what extent persist- ing mutations other than those in DTA represent true residual leukemia or clonal hematopoiesis, respectively with and without an increased risk of relapse. In a disease as heterogeneous as AML these analyses will require large cohorts of patients. Thus, while the recent develop- ments in NGS-based MRD detection represent major steps forward in predicting relapse, they remain imper- fect. It is expected that a better distinction between clonal hematopoiesis and residual leukemia will improve pre- diction of AML patients at higher risk of relapse.
How can the discrimination between true residual leukemia and clonal populations of cells be improved? The numbers of AML patients included in the initial stud- ies precluded detailed analyses of rare mutations and in- depth analysis of common non-DTA mutations. It is con- ceivable that non-DTA mutations are a mixture of muta- tions representative of either true leukemia or clonal hematopoiesis. Improved discrimination of these two conditions by means of types of persisting mutations may have significant value with regards to relapse prediction. Along the same lines it has recently been shown that it may be feasible to discriminate clonal hematopoiesis from pre-AML in healthy individuals.19 Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating clonal expansion, and showed enrichment in specific genes.19 Similar approaches could possibly better differentiate clonal hematopoiesis from true leukemia after induction treatment.
Sensitivity, timing and tissues for next-generation sequencing
How does NGS-based MRD detection perform as com- pared to the ‘golden standard’, multiparameter flow cyto- metric MRD detection? There are only limited studies with a rigorous comparison between NGS- and multipa- rameter flow cytometric MRD detection.11,16 These stud- ies demonstrate that there is a 70% concordance with regard to MRD detection using the two technologies, and that those patients who are MRD-positive according to both techniques have the highest risk of developing an AML relapse.11,16 Interestingly, however, those AML cases with discordant results from NGS and flow cytometry have adverse outcome, such that MRD positivity has value whether determined by flow cytometry, molecular techniques, or both.11,16 We need to improve both the sen- sitivity of our NGS assays and our understanding of the biology of clonal hematopoiesis after high-dose chemotherapy to resolve the discordant cases and deter- mine whether we require both technologies or only one to enumerate MRD.
At which time point(s) should NGS-based MRD detec- tion be carried out? In the majority of AML studies NGS- based MRD detection was performed after high-dose induction treatment. Although this time point may be most suitable for choosing the proper consolidation treat- ment, it is not known whether MRD assessment at other time points may be better prognostic indicators. Few studies have shown that MRD before and after consolida- tion, such as in the setting of allogenic transplantation, affects clinical outcome.12,16,20
A bone marrow biopsy is an invasive procedure that gives stress and physical discomfort to a patient and cre- ates a risk of infection. Patients with chronic myeloid
870
haematologica | 2019; 104(5)