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A. Tuval and L.I. Shlush et al.
of phase I trials, using IDH1/2-targeted monotherapy, need to be repeated in larger cohorts of treatment-naïve patients before conclusions can be drawn. On the other hand, the use of early, pre-leukemic, mutations as markers of the malignant clone should be handled with caution since following chemotherapy, the presence of most resid- ual pre-leukemic clones (carrying DNMT3A, TET2 or ASXL1 mutations) do not increase risk for early (4-year) relapse unless accompanied by other mutations.19,31
In addition, one should bear in mind that, in contrast to the pre-leukemic stage, once overt leukemia evolves, tar- geting pre-leukemic somatic mutations might not prove effective. This can be inferred from the fact that some patients develop relapsed NPM1-mutated leukemia, lack- ing their DNMT3A pre-leukemic mutations.65 This can represent a biological phenomenon in which the relative contribution of these early driver mutations to clonal fit- ness diminishes as the clone evolves to overt leukemia. Indeed, IDH1/2 inhibition induced differentiation of the malignant clone in only 5-7% of the patients and clearance of IDH1 mutated clone was noted only in 21% of clinical- ly responding patients.63 In patients treated with an IDH2 inhibitor, differentiation of the blasts without elimination of the malignant clone was documented,64 in line with pre- vious reports about IDH2 being acquired following AML transformation (as a late event).34 Improving efficacy might be achieved by combining drugs that target both leukemic and late events.
Relapse
Most AML patients experience relapse originating in leukemic stem cells (LSC) that belong to the leukemic clone and that can already be identified at the time of diagnosis. It is, therefore, imperative to identify and target these cells. Two main subtypes of AML were identified. The first AML subtype contains rare stem cells that have a stem/progenitor-like immunophenotype. In the other subtype, relapse originates from the major CD33+ blast population and is more dependent on growth factors when studied in vivo.34,61 Studying gene expression profiles of these subtypes revealed that this division is correlated with French-American-British (FAB) classification. The first subtype is enriched for FAB M4/M5 subtypes and the other is enriched for the less differentiated AML subtypes (M0/M1/M2). Nevertheless, relapse-initiating LSC in both groups had similar gene expression profiles and, as expect- ed, the relapsing clone was found to be characterized by an increased number of LSC.34 Such a "leukemic stemness" transcriptional signature can be used to predict prognosis and to monitor patients in remission.66 Targeting LSC has been studied extensively in xenograft models but less so in clinical trials. A recent study suggests that the combina- tion of Azacitidine and Venetoclax target LSCs, as identi- fied both immune-phenotypically and by their transcrip- tomics signature.67 The number of participants in this trial was small, and, although this therapy does not induce remission in all patients (only in approx. 67%68), and some relapse while on therapy, this is an important step towards LSC-targeted therapy.
Survivorship
Following the elimination of the leukemic clone, chemotherapy-resistant clones, which can sometimes be detected in low VAF values at the time of AML diagnosis,29 expand and re-populate the BM.29,40 Some of these clones
harbor ARCH mutations,29,31 and some of these clones are truly pre-malignant since they go on and evolve into MDS69 or a second AML, albeit following a more pro- longed latency than the relapse of the original leukemia (median 33.7-43 months vs. 8.6-14 months, respectively). Second AML should be diagnosed as a distinct entity whenever leukemic mutations that characterize the pri- mary (diagnosis) AML are not identified at relapse (Table 1). Second AML was described to occur in 10-14% of the patients experiencing relapse.40,65 However, second AML should not be considered as a relapse. Sometimes, second AML does not share its pre-leukemic mutations with the primary AML (Table 1). This is underlined by the identifi- cation of pre-leukemic clones of second AML that lack the primary AML pre-leukemic DNMT3A, TET2, SRSF2 or RUNX1 mutations.40,70 Nevertheless, most of these second- AML-initiating clones share the same early, pre-leukemic mutations as the primary AML clone,40,65 and some even evolve similarly to the primary clone and acquire a differ- ent mutation in the same gene,70,71 emphasizing the role of an environmental selective pressure (Figure 2).
In this regard, environmental influence is best demon- strated when patients that undergo allogeneic stem cell transplantation develop an AML that originates in the donor hematopoietic cells. Two main reasons can lead to this very rare outcome (estimated to occur following 0.08% of transplants72): 1) a pre-existing pre-leukemic clone in the stem cell donation; and 2) evolution of a new leukemic clone in the recipient following the transplanta- tion. Although the stem cell source (BM vs. peripheral blood) did not influence the risk for donor cell leukemia, environmental factors seem to be crucial in promoting the malignant clone. Multivariate analysis revealed three risk factors associated with development of donor cell leukemia: 1) the use of growth factors; 2) in vivo T-cell depletion; and 3) having a previous allograft. These risk factors imply that a reduced immune surveillance and increased replication signals create a more permissive environment that allows the development of the malig- nant clone. As an emphasis, two different trajectory leukemic evolutions were described following a DNMT3A-mutated pre-leukemic clone donation. While the donor developed NPM1-mutated, FLT3-ITD AML, the recipient developed NPM1 SMC1A-mutated AML.73 Therefore, when monitoring AML patients in remission, predicting a rare, second AML becomes somewhat analo- gous to predicting transformation from pre-leukemia to AML. Here, too, some residual or newly evolving pre- leukemic clones confer increased risk for second AML development, heralded by clonal expansion. The exact risk stratification still needs to be validated by appropri- ately designed studies. These studies need to use broad sequencing panels instead of a panel dictated only by the mutations found at diagnosis.
Conclusions
Recently published studies reveal that the evolutionary trajectory of AML begins many years before the patient is actually diagnosed. It is a multistep process character- ized by Darwinian evolution with clonal selection and expansion. Much is still unknown regarding the various factors that influence the path that clones in the hematopoietic system follow. They consist of both clon-
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haematologica | 2019; 104(5)