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Evolutionary trajectory of AML
mutations”. Therefore, it might seem paradoxical that, in some cases, an increased number of somatic mutations predict a worse prognosis and a more rapid evolution.8 It is, therefore, important to stress that it is not only the number of mutations, but also the identity of the specific mutation acquired, that determines progression.9 Only these true “driver” mutations confer an advantageous phe- notype.
Over recent years, studies aimed at depicting the clonal structure of AML have been published. These studies per- formed deep sequencing of primary AML samples taken from patients, patient-derived xenografts, and from in vitro cultures, and used the various somatic variant allele fre- quencies (VAF) as measures of clonal sizes [assuming no copy number variability (CNV) or loss of heterozygosities (LOH)]. These studies shed light on the selection and expansion that these clones undergo during their evolu- tion and following therapy. Combining this information with clinical data from different time points improves our ability to predict treatment outcomes, enabling us to per- sonalize therapy. Moreover, this approach raises the hope that healthy individuals can be screened for AML, and maybe even preventing the disease; something that was once considered unachievable.
The clonal evolution of most AML subtypes can be viewed as a multistep process. It is now well accepted that this process can be schematically divided into three stages according to the clinical presentation. Each evolutionary stage has a different time frame and is characterized by typical somatic mutations that are, therefore, categorized into three groups according to the timing of their appear- ance: pre-leukemic, leukemic, and late events (Table 1). Although acute promyelocytic peukemia (APL), AML with KDM2A translocations, and the core-binding factor (CBF) leukemias do not have a clear pre-leukemic stage, they too develop over time and acquire late events, as dis- cussed below.
This review summarizes our current knowledge regard- ing somatic mutations in AML, their contribution to clonal fitness under different selective environmental conditions, and their correlation with patients’ clinical outcomes.
Pre-leukemic stage
Pre-leukemic mutations are somatic mutations that are found in leukemic blasts as well as in hematopoietic pro- genitors and mature cells from different lineages that share a common ancestral stem cell. This stem cell, which is still capable of differentiation, is defined as a pre-leukemic hematopoietic stem and progenitor cell (preL-HSPC).10,11 Such preL-HSPCs were isolated from AML patients at diagnosis, remission and relapse.10-12 By definition, the term pre-leukemic can only be inferred retrospectively, after the diagnosis of AML has been made.
Following these findings, somatic mutations were iden- tified in the hematopoietic system of healthy individuals in various allele frequencies increasing with age, a phe- nomenon that was termed age-related clonal hematopoiesis (ARCH).13,14 In fact, deep sequencing tech- niques revealed the presence of DNMT3A and TET2 mutations in nearly all individuals; however, most of them were at lower VAF, in comparison to the original reports (median VAF 0.0024), and remained stable over time.15 Such a ubiquitous phenomenon probably represents the
general structure of the aging human hematopoietic sys- tem, as the same findings could not be replicated among young individuals (aged 20-29 years).16 The large number of human hematopoietic stem cells (HSC) (estimated to be within the range of 50,000-200,000)17 and the number of somatic mutations in each adult single HSC (approx. 1000 mutations) suggest an estimated HSC pool mutation bur- den of 108,17 and explain why somatic mutations can be present at low VAF in every individual.14 However, with age, an exponential increase in the prevalence of ARCH13,14,16 occurs. In addition, this correlation with age is specific to mutations found in DNMT3A, TET2 and a few additional candidate driver genes,14 suggesting that muta- tions in these genes confer a selective advantage to HSPCs. The selective advantage that mutations in DNMT3A and TET2 confer is probably introduced during the third or fourth decade of life. A second selective advantage is introduced later, during the fifth decade and onward, reflecting the aging BM selecting for HSC-carry- ing spliceosome machinery mutations (SRSF2, U2AF1, SF3B1, ZRSR2, DDX41, EZH2, ASXL1, etc.).18,19 The fact that different individuals can carry clones of different sizes at the same age suggests differences in risk factors for clonal expansion.
The presence of such a mutated clone in allele frequency of more than 2% is termed clonal hematopoiesis of inde- terminate potential (CHIP).20 It was found to be associated with development of hematologic cancers, cardiovascular morbidity, chronic obstructive pulmonary disease, and with an increase in all-cause mortality.13,14,21 While these clones predict a somewhat dismal prognosis, only a small number of individuals that harbor such a clone will even-
Table 1. Age-related clonal hematopoiesis defining events and the risk that they confer for acute myeloid leukemia progression.9,22
Pre-leukemic / ARCH-defining events
Leukemic mutations
NPM1c#
"Late events"
FLT3-ITD#
CEBPA (bi-allelic) KIT#
KRAS# NRAS# PTPN11
WT1
Low risk
ASXL1
BCOR CALR# CBL# DNMT3A KIT# KMT2D KRAS# NF1 RAD21 SF3B1# TET2$
High risk
IDH1#
IDH2# JAK2# PHF6 PPM1D$* RUNX1 SRSF2# TP53*
# U2AF1
Unclear risk
BRAF#
CEBPA (mono-allelic) EZH2@ FLT3-TKD# GATA2 KDM6A KMT2C
NRAS#
PAX5
PTPN11 SMC1A
SMC3
STAG2 ZRSR2@
haematologica | 2019; 104(5)
Unless specified, genes that harbor recurrent driver mutations are mentioned (as opposed to specific variants). Some mutations are rarely found, therefore, it is difficult to determine with certainty the risk that they confer.These were designated as "Unclear Risk".Some mutations can appear as an early evolutionary event as well as a "late event" (e.g. KIT, NRAS). Translocations, such as t(8;21) are not mentioned since they can be missed by targeted- sequencing as well as by exome-sequencing methods. Examples for "leukemic mutations" and for "late events" are also presented. *Enriched following chemotherapy for a non-related cancer [other than acute myeloid leukemia (AML)]. @Probably represent myelodysplastic syndromes. $Truncating events only (frameshift and missense mutations). #Only specific hotspots in the gene. ARCH: age-related clonal hematopoiesis.
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