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Evolutionary trajectory of AML
in as few as 6 months to up to 10 years.23,24 Another exam- ple is MLL-rearranged AML; this usually develops within 6-18 months following exposure to topoisomerase II inhibitors. In contrast to therapy-related AML with somatic mutations in TP53 or PPM1D, where chemother- apy selects pre-existing mutated clones, MLL-rearrange- ment is assumed to be induced directly by topoisomerase II inhibitors.19,33
Although IDH mutations can be viewed as high-risk mutations, their presence was not found to be correlated with a shortened AML latency.22 This can be explained by the fact that some of the high-risk mutations occur early along the evolutionary trajectory of the clone; they can be considered as the initiating event (U2AF2, SRSF2, TP53 and RUNX1). Other high-risk mutations (IDH1 and IDH2) tend to appear later and require additional, co-operating, driver mutations in order to progress into AML. Indeed, certain combinations of mutations (when found in the same individual) can shorten the time to AML diagnosis, such as when DNMT3A is found with spliceosomal machinery mutations.22
Importantly, the size of the clone9 and the number of mutations identified9,22 mean a shortened interval for AML progression. Clones with increased fitness, as manifested
A
B
C
by their size (VAF) and replication rate (number of muta- tions), are prone to acquire additional mutations until transforming, ‘leukemic’, mutations occur.
Leukemic stage
Molecular analysis of AML reveals a set of somatic mutations that are only present in the leukemic blasts but are absent in normal hematopoietic progenitors, in non- myeloid, and in mature cells. These mutations were not found in healthy individuals.9,13,14,22
These mutations can be further divided into leukemic mutations, which are shared by all the leukemic blasts, and late events. While the former represent the leukemic transformation, the latter represent subclones, as can be inferred from their lower VAF value (Figure 1).
Since there are already subclones at the time of diagno- sis, determining the order of acquisition of the mutations is limited by the sensitivity of the sequencing method used to detect rare clones. It can be achieved by recon- structing the phylogenetic tree of the leukemia with single cell or sub-population sequencing.34 However, there is a general consensus that one mutation can be considered to
Figure 1. High-risk and low-risk pre-leukemic somatic mutations. X: an acquired somatic mutation. Clone size [as manifested by variant allele frequency (VAF) value] is represented by the size of the oval shape. Clonal expansion is represented by the rising curve. (A) Low-risk age-related clonal hematopoiesis (ARCH) mutations, such as DNMT3A or TET2 muta- tions, are acquired at a relatively young age (marked in white). Most of these clones will not progress to acute myeloid leukemia (AML). (B) Pre-leukemic clones, characterized by sim- ilar low-risk mutations have an increased fit- ness (as manifested by an increased VAF). They acquire additional pre-leukemic muta- tions (marked in yellow and red), not necessar- ily in the same clone. These cells are hematopoietic stem and progenitor cells (HSPCs), still capable of differentiation and sustain hematopoiesis. Once a clone acquires a leukemic, transforming, mutation (NPM1, for instance, marked in green) it will progress rap- idly to an overt AML with loss of differentiation capacity and uncontrolled proliferation. Retrospectively, its preceding clones are referred to as pre-leukemic. Leukemic muta- tions are shared by all the leukemic blasts, hence they have a high VAF (50%) in the leukemic clone. Late events (e.g. FLT3-ITD, marked in purple) appear later along the AML evolutionary trajectory, are shared by sub- clones, and represent the clonal heterogeneity of the leukemia; they have VAFs ≤50%. The exact timing of AML diagnosis can vary. Therefore, late events are usually already pres- ent when the actual diagnosis is made. Single cells or sub-populations have to be sequenced in order to accurately determine the order of acquisition of the mutations. (C) Spliceosomal machinery, and other high-risk mutations (such as SRSF2, U2AF1, IDH1, IDH2 and TP53 that are marked in pink) are usually acquired at a more advanced age. These clones expand more rapidly (as manifested by the rate of increase of their VAF value) and most will lead to AML.
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