Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer?

Acute myeloid leukaemia (AML) is an uncontrolled clonal proliferation of abnormal myeloid progenitor cells in the bone marrow and blood. Advances in cancer genomics have revealed the spectrum of somatic mutations that give rise to human AML and drawn our attention to its molecular evolution and clonal architecture. It is now evident that most AML genomes harbour small numbers of mutations, which are acquired in a stepwise manner. This characteristic, combined with our ability to identify mutations in individual leukaemic cells and our detailed understanding of normal human and murine haematopoiesis, makes AML an excellent model for understanding the principles of cancer evolution. Furthermore, a better understanding of how AML evolves can help us devise strategies to improve the therapy and prognosis of AML patients. Here, we draw from recent advances in genomics, clinical studies and experimental models to describe the current knowledge of the clonal evolution of AML and its implications for the biology and treatment of leukaemias and other cancers.KEY WORDS: Acute myeloid leukaemia, Cancer, Clonal evolution, In vivo models of leukaemia, Mutation     

Natural and chemotherapy-induced clonal evolution of tumors

Evolution and natural selection of tumoral clones in the process of transformation and the following carcinogenesis can be called natural clonal evolution. Its main driving factors are internal: genetic instability initiated by driver mutations and microenvironment, which enables selective pressure while forming the environment for cell transformation and their survival. We present our overview of contemporary research dealing with mechanisms of carcinogenesis in different localizations from precancerous pathologies to metastasis and relapse. It shows that natural clonal evolution establishes intratumoral heterogeneity and enables tumor progression. Tumors of monoclonal origin are of low-level intratumoral heterogeneity in the initial stages, and this increases with the size of the tumor. Tumors of polyclonal origin are of extremely high-level intratumoral heterogeneity in the initial stages and become more homogeneous when larger due to clonal expansion. In cases of chemotherapy-induced clonal evolution of a tumor, chemotherapy becomes the leading factor in treatment. The latest research shows that the impact of chemotherapy can radically increase the speed of clonal evolution and lead to new malignant and resistant clones that cause tumor metastasis. Another option of chemotherapy-induced clonal evolution is formation of a new dominant clone from a clone that was minor in the initial tumor and obtained free space due to elimination of sensitive clones by chemotherapy. As a result, in ~20% of cases, chemotherapy can stimulate metastasis and relapse of tumors due to clonal evolution. The conclusion of the overview formulates approaches to tumor treatment based on clonal evolution: in particular, precision therapy, prediction of metastasis stimulation in patients treated with chemotherapy, methods of genetic evaluation of chemotherapy efficiency and clonal-oriented treatment, and approaches to manipulating the clonal evolution of tumors are presented.     

Pathogenic pathways in acute myeloid leukemias.

Despite the common clinical, hematological and prognostic features that define acute myeloid leukemia (AML) there is considerable heterogeneity among individual cases, suggesting different pathogenic pathways. Based on a simple theoretical model, according to the vital characteristics of the leukemic clone (proliferative rate and resistance to apoptosis) we propose a classification of AML into two broad categories: a) high leukemic clone vitality (HLV) AML, corresponding roughly to the World Health Organization (WHO) classification group of entities "AML with recurrent cytogenetic abnormalities" and b) low leukemic clone vitality (LLV) or "opportunistic" AML corresponding to the WHO groups "AML with multilineage dysplasia" and "therapy-related AML". HLV-AML leukemic clones are characterized by rate-limiting genomic mutations capable of conferring proliferation/survival advantage over a normal hematopoietic environment while in LLV-AML, the leukemic clones are not particularly proliferative or apoptosis-resistant, but are nevertheless selected against an impaired, previously damaged hematopoietic environment. Such a pathogenesis-oriented classification might have therapeutic and prognostic implications, providing a theoretical basis for a further adaptation of the current standard treatment strategies to the individual characteristics of the AML patients.     

Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells

One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.     

Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Early detection of cancer-associated genomic instability is crucial, particularly in tumour types in which this instability represents the essential underlying mechanism of tumourigenesis. Currently used methods require the presence of already established neoplastic cells because they only detect clonal mutations. In principle, parallel sequencing of single DNA filaments could reveal the early phases of tumour initiation by detecting low-frequency mutations, provided an adequate depth of coverage and an effective control of the experimental error. We applied ultradeep sequencing to estimate the genomic instability of individuals with hereditary non-polyposis colorectal cancer (HNPCC). To overcome the experimental error, we used an ultraconserved region (UCR) of the human genome as an internal control. By comparing the mutability outside and inside the UCR, we observed a tendency of the ultraconserved element to accumulate significantly fewer mutations than the flanking segments in both neoplastic and nonneoplastic HNPCC samples. No difference between the two regions was detectable in cells from healthy donors, indicating that all three HNPCC samples have mutation rates higher than the healthy genome. This is the first, to our knowledge, direct evidence of an intrinsic genomic instability of individuals with heterozygous mutations in mismatch repair genes, and constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability.     

Genetic evolution of nevus of Ota reveals clonal heterogeneity acquiring BAP1 and TP53 mutations

Melanoma presents molecular alterations based on its anatomical location and exposure to environmental factors. Due to its intrinsic genetic heterogeneity, a simple snapshot of a tumor's genetic alterations does not reflect the tumor clonal complexity or specific gene–gene cooperation. Here, we studied the genetic alterations and clonal evolution of a unique patient with a Nevus of Ota that developed into a recurring uveal‐like dermal melanoma. The Nevus of Ota and ulterior lesions contained GNAQ mutations were c‐KIT positive, and tumors showed an increased RAS pathway activity during progression. Whole‐exome sequencing of these lesions revealed the acquisition of BAP1 and TP53 mutations during tumor evolution, thereby unmasking clonal heterogeneity and allowing the identification of cooperating genes within the same tumor. Our results highlight the importance of studying tumor genetic evolution to identify cooperating mechanisms and delineate effective therapies.     

Myelodysplastic syndromes: their history, evolution and relation to acute myeloid leukaemia

The myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal disorders arising from a multipotent haemopoietic progenitor which share a leukaemic propensity, 30% of cases culminating in acute myeloid leukaemia (AML). Their pathogenesis probably entails multiple steps, phenotypic progression being determined by either expansion or evolution of the abnormal clone. The clonal origin of certain cases of de novo AML is analogous to that of MDS and evidence that they share a common pathogenesis and distinct biological characteristics is beginning to emerge.     

Clonal and non-clonal chromosome aberrations and genome variation and aberration.

The theoretical view that genome aberrations rather than gene mutations cause a majority of cancers has gained increasing support from recent experimental data. Genetic aberration at the chromosome level is a key aspect of genome aberration and the systematic definition of chromosomal aberrations with their impact on genome variation and cancer genome evolution is of great importance. However, traditionally, efforts have focused on recurrent clonal chromosome aberrations (CCAs). The significance of stochastic non-clonal chromosome aberrations (NCCAs) is discussed in this paper with emphasis on the simple types of NCCAs that have until recently been considered "non-significant background". Comparison of various subtypes of transitional and late-stage CCAs with simple and complex types of NCCAs has uncovered a dynamic relationship among NCCAs, CCAs, overall genomic instability, and karyotypic evolution, as well as the stochastic nature of cancer evolution. Here, we review concepts and methodologies to measure NCCAs and discuss the possible causative mechanism and consequences of NCCAs. This study raises challenging questions regarding the concept of cancer evolution driven by stochastic chromosomal aberration mediated genome irregularities that could have repercussions reaching far beyond cancer and organismal genomes.     

The Relative Timing of Mutations in a Breast Cancer Genome

Many tumors have highly rearranged genomes, but a major unknown is the relative importance and timing of genome rearrangements compared to sequence-level mutation. Chromosome instability might arise early, be a late event contributing little to cancer development, or happen as a single catastrophic event. Another unknown is which of the point mutations and rearrangements are selected. To address these questions we show, using the breast cancer cell line HCC1187 as a model, that we can reconstruct the likely history of a breast cancer genome. We assembled probably the most complete map to date of a cancer genome, by combining molecular cytogenetic analysis with sequence data. In particular, we assigned most sequence-level mutations to individual chromosomes by sequencing of flow sorted chromosomes. The parent of origin of each chromosome was assigned from SNP arrays. We were then able to classify most of the mutations as earlier or later according to whether they occurred before or after a landmark event in the evolution of the genome, endoreduplication (duplication of its entire genome). Genome rearrangements and sequence-level mutations were fairly evenly divided earlier and later, suggesting that genetic instability was relatively constant throughout the life of this tumor, and chromosome instability was not a late event. Mutations that caused chromosome instability would be in the earlier set. Strikingly, the great majority of inactivating mutations and in-frame gene fusions happened earlier. The non-random timing of some of the mutations may be evidence that they were selected.     

No evidence for accumulation of deleterious mutations and fitness degradation in clonal fish hybrids: Abandoning sex without regrets

Despite its inherent costs, sexual reproduction is ubiquitous in nature, and the mechanisms to protect it from a competitive displacement by asexuality remain unclear. Popular mutation‐based explanations, like the Muller's ratchet and the Kondrashov's hatchet, assume that purifying selection may not halt the accumulation of deleterious mutations in the nonrecombining genomes, ultimately leading to their degeneration. However, empirical evidence is scarce and it remains particularly unclear whether mutational degradation proceeds fast enough to ensure the decay of clonal organisms and to prevent them from outcompeting their sexual counterparts. To test this hypothesis, we jointly analysed the exome sequences and the fitness‐related phenotypic traits of the sexually reproducing fish species and their clonal hybrids, whose evolutionary ages ranged from F1 generations to 300 ky. As expected, mutations tended to accumulate in the clonal genomes in a time‐dependent manner. However, contrary to the predictions, we found no trend towards increased nonsynonymity of mutations acquired by clones, nor higher radicality of their amino acid substitutions. Moreover, there was no evidence for fitness degeneration in the old clones compared with that in the younger ones. In summary, although an efficacy of purifying selection may still be reduced in the asexual genomes, our data indicate that its efficiency is not drastically decreased. Even the oldest investigated clone was found to be too young to suffer fitness consequences from a mutation accumulation. This suggests that mechanisms other than mutation accumulation may be needed to explain the competitive advantage of sex in the short term.     

Clonally expanded T-cell populations in atomic bomb survivors do not show excess levels of chromosome instability

Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.     

Mutation Patterns of 16 Genes in Primary and Secondary Acute Myeloid Leukemia (AML) with Normal Cytogenetics

Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.     

Radiation-induced genomic instability: a role for secreted soluble factors in communicating the radiation response to non-irradiated cells

Radiation induced genomic instability can be described as the increased rate of genomic alterations occurring in the progeny of an irradiated cell. Its manifestations are the dynamic ongoing production of chromosomal rearrangements, mutations, gene amplifications, transformation, microsatellite instability, and/or cell killing. In this prospectus, we present the hypothesis that cellular exposure to ionizing radiation can result in the secretion of soluble factors by irradiated cells and/or their progeny, and that these factors can elicit responses in other cells thereby initiating and perpetuating ongoing genomic instability.     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

Global chromosomal structural instability in a subpopulation of starving Escherichia coli cells

Copy-number variations (CNVs) constitute very common differences between individual humans and possibly all genomes and may therefore be important fuel for evolution, yet how they form remains elusive. In starving Escherichia coli, gene amplification is induced by stress, controlled by the general stress response. Amplification has been detected only encompassing genes that confer a growth advantage when amplified. We studied the structure of stress-induced gene amplification in starving cells in the Lac assay in Escherichia coli by array comparative genomic hybridization (aCGH), with polymerase chain reaction (pcr) and DNA sequencing to establish the structures generated. About 10% of 300 amplified isolates carried other chromosomal structural change in addition to amplification. Most of these were inversions and duplications associated with the amplification event. This complexity supports a mechanism similar to that seen in human non-recurrent copy number variants. We interpret these complex events in terms of repeated template switching during DNA replication. Importantly, we found a significant occurrence (6 out of 300) of chromosomal structural changes that were apparently not involved in the amplification event. These secondary changes were absent from 240 samples derived from starved cells not carrying amplification, suggesting that amplification happens in a differentiated subpopulation of stressed cells licensed for global chromosomal structural change and genomic instability. These data imply that chromosomal structural changes occur in bursts or showers of instability that may have the potential to drive rapid evolution.     

Genetic instability and clonal expansion

Inactivation of tumor suppressor genes can lead to clonal expansion. We study the evolutionary dynamics of this process and calculate the probability that inactivation of a tumor suppressor gene is preceded by mutations in genes that confer genetic instability. Unstable cells might have a slower rate of clonal expansion than stable cells because of an increased probability of generating lethal mutations or inducing apoptosis. We show that the different growth rates of genetically stable and unstable cells during clonal expansion represent, in general, only a small disadvantage for genetic instability. The intuitive reason for this conclusion is that robust clonal expansion, where cellular birth rates are significantly greater than death rates, occurs on a much faster time scale than waiting for those mutations that allow clonal expansion. Moreover, in special cases where clonal expansion is very slow, genetically unstable cells have a higher probability to accumulate additional mutations during clonal expansion that confer a selective advantage. Clonal expansion represents a major disadvantage for genetic instability only when inactivation of the tumor suppressor gene leads to a very small increase of the cellular reproductive rate that is cancelled by the increased mortality of unstable cells.