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A. Tuval and L.I. Shlush et al.
tually develop AML (approx. 0.1-3%).13,14
Nevertheless, since some of these clones do evolve into
full-blown leukemia, these observations can be exploited to build a model for early detection of AML, at its pre-clin- ical, pre-leukemic phase. In fact, two recently published papers studied large prospective cohorts of healthy individ- uals and compared benign clonal hematopoiesis (one that did not evolve into AML) with malignant, pre-AML, clonal hematopoiesis. These studies used deep targeted sequenc- ing methods to search for mutations in driver genes in a total of 307 cases (individuals that subsequently developed AML) and 626 age- and gender-matched controls (individ- uals that did not develop AML) in blood samples obtained 6-9 years before AML diagnosis. These studies found dis- criminative characteristics between the two cohorts.9,22
Clone size
A pre-leukemic state is manifested by an increased inci- dence of clonal hematopoiesis. Setting a 10% threshold for VAF value significantly discriminated pre-AML from controls. Thirty-nine percent of pre-AML individuals have clones of this size, as opposed to 4% of control individu- als. Although statistically significant, there is a large over- lap between VAF values of driver mutations found in benign and in malignant ARCH (especially for DNMT3A and TET2 mutations) that precludes it from being a single predictor of a rare disease such as AML.
Number of accumulated mutations
Pre-AML individuals have significantly more mutations in driver genes (including a few variants in the same gene, e.g. in DNMT3A22) per individual (not necessarily in the same clone) when compared with controls. This is espe- cially evident among older individuals (>60-65 years of age) underscoring the time frame required for mutations to accumulate. Nevertheless, it is important to note that a substantial number of patients (20-46%) do develop AML without having a mutation in any driver gene prior to the diagnosis. This decreases the negative predictive value of these models.
When measured at a certain time point, these two char- acteristics indirectly reflect increased clonal fitness, as manifested by increased expansion and increased number of replications with the accumulation of mutations over time. These characteristics were also found to be predic- tive of progression to myeloid neoplasms when found during the evaluation of unexplained cytopenias.8
Specific high-risk mutations
Progression to AML was found to be preceded by accu- mulation of specific high-risk mutations. These muta- tions were more prevalent among pre-leukemic clonal hematopoiesis when compared to benign ARCH. Specifically, the presence of spliceosome-machinery mutations in SRSF2 P95R and U2AF1 Q157P as well as in TP53, in IDH1 R132, in IDH2 R140 and in RUNX1 (even at VAF values <10%) confer the highest risk for subse- quent AML development in healthy individuals.9,22 When these clones appear at a relatively young age (>50 years) they tend to evolve into AML. The simple explanation for this could be that there is more time for AML transforma- tion to take place. Another explanation could be that the environment that positively selected this clone continues to exert its selective pressure, eventually leading to AML. Table 1 summarizes the various ARCH-defining events
and the risk that each of them confers for AML progres- sion.
Temporal progression
While some low-risk clones can remain stable over a period of 3-10 years,15 clones that are characterized by high-risk mutations show a more rapid increase in their size, as manifested by an increase in their VAF values over time9,22 (Figure 1). Additional prospective cohorts might better define which clone develops to other hematologic malignancies [e.g. myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN)].
Progression to AML depends on the identity of the ini- tiating mutation and on the identity of additional muta- tions that are subsequently accumulated. It is conceivable that many factors influence the timing of the appearance of the mutation and the positive selection of such a clone, among which are probably the underlying specific germline background.
In addition, specific environmental pressures confer a selective advantage to HSCs carrying specific mutations; clear examples are TP53 and PPM1D mutations that are enriched following exposure to chemotherapy and radio- therapy. Chemotherapy does not increase the number of somatic single nucleotide variants or the percentage of chemotherapy-related transversions. Rather, it positively selects for pre-existing TP53 and PPM1D mutated clones.23-29
Moreover, a third-generation, single-molecule real-time sequencing assay with long-read length of AML and MDS samples exposed different TP53 variants residing on dif- ferent alleles in each sample.30 This emphasizes the impor- tance of the environmental conditions that select a certain phenotype, thus enabling the evolution of a few clones in parallel, all sharing similar driver mechanisms (TP53 muta- tions). It is important to note that chemotherapy exerts a selective pressure regardless of the specific mutation that characterizes the pre-leukemic clone. Following chemotherapy, BM is enriched with pre-leukemic clones and their prevalence increases by 10% or even 30% among younger and elderly individuals, respectively, when compared to their prevalence in the general age- matched population.24,13 The clones that were selected can neither be categorized according to a certain mutation, nor according to a certain chemotherapy (with the exception of topoisomerase II inhibitors, for which see below). However, they can be divided into three groups according to patient age groups, with younger individuals enriched with DNMT3A mutated clones. This holds true also for AML patients in remission that were found to have resid- ual pre-leukemic clones.19,31 This implies that most pre- leukemic clones have an inherent chemoresistance (Figure 2), a phenomenon that was also shown in both in vivo and in vitro models.32
The time frame of evolution from pre-leukemia to AML depends both on the context (extrinsic factors) and the driver mutations (intrinsic factors). Pre-leukemia in healthy individuals usually progresses slowly with a laten- cy period that can sometimes be as long as 20 years. Presence of specific mutations was correlated with a shorter timeframe, as in the case of RUNX1 mutations (associated with a rapid progression to AML of <2 years) and of TP53 mutations.22 Specifically, following chemotherapy, TP53 mutated clones, as well as PPM1D mutated clones, evolve to hematologic malignancies with-
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haematologica | 2019; 104(5)