Emergence of heterogeneity in acute leukemias
 Thomas Stiehl^{1, 2, 3}Email authorView ORCID ID profile,
 Christoph Lutz^{4} and
 Anna MarciniakCzochra^{1, 2, 3}
DOI: 10.1186/s1306201601541
© The Author(s) 2016
Received: 21 June 2016
Accepted: 29 September 2016
Published: 12 October 2016
Abstract
Background
Leukemias are malignant proliferative disorders of the blood forming system. Sequencing studies demonstrate that the leukemic cell population consists of multiple clones. The genetic relationship between the different clones, referred to as the clonal hierarchy, shows high interindividual variability. So far, the source of this heterogeneity and its clinical relevance remain unknown. We propose a mathematical model to study the emergence and evolution of clonal heterogeneity in acute leukemias. The model allows linking properties of leukemic clones in terms of selfrenewal and proliferation rates to the structure of the clonal hierarchy.
Results
Computer simulations imply that the selfrenewal potential of the first emerging leukemic clone has a major impact on the total number of leukemic clones and on the structure of their hierarchy. With increasing depth of the clonal hierarchy the selfrenewal of leukemic clones increases, whereas the proliferation rates do not change significantly. The emergence of deep clonal hierarchies is a complex process that is facilitated by a cooperativity of different mutations.
Conclusion
Comparison of patient data and simulation results suggests that the selfrenewal of leukemic clones increases with the emergence of clonal heterogeneity. The structure of the clonal hierarchy may serve as a marker for patient prognosis.
Reviewers
This article was reviewed by Marek Kimmel, Tommaso Lorenzi and Tomasz Lipniacki.
Keywords
Acute leukemia Heterogeneity Clonal evolution Mathematical modeling Patient prognosis Mutation Selfrenewal Clonal hierarchyBackground
Acute leukemias are clonal diseases of the blood forming (hematopoietic) system. They lead to expansion of malignant cells and resulting impairment of blood cell formation. Over the last years evidence has accumulated that many leukemia subtypes are maintained by a subpopulation of cells with stem celllike properties [1–3]. These cells are referred to as leukemic stem cells (LSCs) or leukemia initiating cells (LICs) and potentially trigger relapse of the disease [4, 5]. Recent sequencing studies have confirmed that the leukemic cell population is composed of different clones [6–8]. The size and number of clones follows a complex evolution over the course of the disease [9–12]. Genetic heterogeneity of different clones seems to result in functional differences, such as a different engraftment potential in mice or different proliferation rates [13, 14]. Nevertheless, a direct link between genotype and cell function is still missing [13].

What is the source of interindividual differences of the clonal hierarchy of leukemias?

What is the functional difference in terms of selfrenewal and proliferation rates between cells at the top of the hierarchy and their descendants which have acquired additional mutations?

How do clones that appear early during the disease differ from clones that appear later?

How do properties of leukemic cells present at one point in time influence the structure of the clonal hierarchy in the future?

How do mutation rates influence the structure of the clonal hierarchy?
These questions are of clinical relevance, since properties of the leukemic stem cells are important determinants of disease dynamics, therapy resistance and relapse [14, 22, 23]. Deciphering of the clonal architecture using genomic methods has become more efficient and less expensive in recent years. Prediction of patient prognosis based on genetic markers alone is not straightforward, since leukemogenetic hits vary considerably among patients and the interplay of the different detected mutations is complex and only partially understood [24, 25]. Therefore, the question arises if the structure of the clonal architecture provides additional insights into cell properties and patient prognosis.
We propose a mathematical modeling approach to provide potential answers to these questions. Mathematical models allow to systematically study the impact of cell parameters such as mutation rates, proliferation rates and selfrenewal probability on the clonal hierarchy of cells. Simulation of the clonal selection process provides hints about which cell properties are linked to selective advantage and how these properties evolve over time. The modeling approach allows linking the position of a clone within the hierarchy to functional properties, such as selfrenewal and proliferation rates, and to compare it to functional properties of clones located at different positions in the hierarchy. The structure of the clonal architecture obtained in model simulations is compared to experimental data from the literature and thus allows linking observations at the level of population dynamics to the cell function in terms of selfrenewal and proliferation rates.
Different mathematical concepts have been proposed to model mutations. Depending on the focus of interest, different approaches can be applied. Moran process [26, 27] is used to describe populations with size that is constant in time. Branching processes are used to describe acquisition of mutations in growing populations. Examples include the accumulation of passenger and driver mutations, interaction among driver mutations or accumulation of mutations during DNA copying [28–31]. In case of a large population and a continuous trait space, diffusion [32, 33] or integral kernels [34–36] have been used to describe the effect of mutations. A rigorous relationship between processes at the level of single cells and limit descriptions in terms of deterministic or probabilistic equations is provided in ref. [37]. Examples for deterministic approaches to study mutations in discrete or continuously structured population models are given in ref. [38–40]. Computer simulations of individualbased models and cellular automata provide a framework to study the impact of cellular processes on the whole population. Examples for individualbased cancer models can be found in ref. [41, 42].
This work is structured as follows. In the “Methods” section, we introduce the mathematical model. In the “Results” section, we present simulation results and their biological interpretation. The “Discussion” section concludes with a discussion of clinical implications of the results obtained.
We adhere to the following terminology. Clonal architecture (clonal hierarchy) is understood as the genetic relationship between different clones. We represent the clonal architecture as a tree. Progeny of a node has acquired one additional mutation compared to its mother node. As a clone we understand all genetically identical stem and nonstem cells. A clone consisting of at least 1 % of the total cell mass is denoted as a significant clone. The threshold of 1 % has been chosen based on the sensitivity of sequencing methods [43].
Methods
Model structure
Based on the classical understanding of the hematopoietic system [48] blood cell formation is considered as a stepwise process, with cells sequentially traversing an ordered sequence of discrete maturation states (compartments). We treat each compartment as a “wellmixed tank” and describe its evolution using an ordinary differential equation. The large count of cells in the hematopoietic system justifies this approach [48].

Proliferation rate, describing the frequency of cell divisions per unit of time. In case of postmitotic cells the proliferation rate is considered equal to zero.

Fraction of selfrenewal (selfrenewal rate), describing the fraction of progeny cells returning to the compartment occupied by the parent cells that gave rise to them.

Death rate, describing the fraction of cells dying per unit of time. For simplicity, we assume that dividing cells do not die and that nondividing cells die at constant rates.
We denote the compartment of dividing healthy cells as c _{1} and that of mature cells as c _{2}. We count the leukemic clones starting from 1. The respective compartments of the ith leukemic clone are denoted as \({l_{1}^{i}}\) and \({l_{2}^{i}}\) resp. The proliferation rate of the healthy cells is denoted as p _{ c } and that of the mitotic cells of the ith leukemic clone \({p^{i}_{l}}\). The respective fractions of selfrenewal are denoted a _{ c } and \({a_{l}^{i}}\). Death rates of the nondividing compartments are d _{ c } and \({d^{l}_{i}}\).
Feedback regulation of healthy hematopoiesis
Formation of healthy blood cells is subject to a tight regulation, mediated by a system of lineage and stagespecific cytokines. If there is a need for more blood cells of a certain type, the concentration of cytokines increases and stimulates formation of mature cells [17, 49]. For simplicity, we consider only one feedback loop. We denote s(t) the value of the feedback signal at time t. We set \(s(t)=\frac {1}{1+{kc}_{2}(t)}\), where k is a positive constant depending on production and elimination of cytokines [44]. This expression can be derived from cytokine kinetics [44]. It takes into account that the concentrations of important cytokines such as EPO and GCSF depend on the concentration of mature cells [49]. The feedback signal assumes values between 0 and 1.
On the basis of our earlier work and compatibility with clinical data [44, 46], we assume feedback inhibition of the fraction of selfrenewal by mature cells. The fraction of selfrenewal of the healthy cells is assumed to be equal to \(a_{c}(t)=\hat a_{c} s(t)\) and that of leukemic cells of clone i to \({a_{l}^{i}}(t)=\hat {a}_{l}^{i} s(t)\). The parameters \(\hat {a}_{c}\) and \(\hat {a}_{l}^{i}\) can be interpreted as the maximal possible fraction of selfrenewal. Numerical solutions of the model of hematopoiesis subject to this feedback were validated on the basis of clinical observations and show good agreement with patient data upon recovery from bone marrow transplantation [46].
Model equations for the hematopoietic system
The flux to mitosis of healthy cells at time t equals p _{ c } c _{1}(t). During mitosis, a parent cell is replaced by two progeny cells. The outflux from mitosis at time t equals 2p _{ c } c _{1}(t), of which the fraction \(2\hat {a}_{c} s(t){p_{c}}c_{1}(t)\) stays in compartment 1 (process referred to as selfrenewal). The fraction \(2\left (1\hat {a}_{c} s(t)\right){p_{c}}c_{1}(t)\) moves to compartment 2 (process referred to as differentiation).
with the initial conditions c _{1}(0), c _{2}(0) given.
Model of leukemia
with the initial conditions c _{1}(0), …, \({l_{2}^{n}}(0)\) given.
Mutations
A similar system of equations has been obtained in [40].
Since \({l^{i}_{2}}\) is considered to be postmitotic, we do not distinguish between cells that acquired a mutation during the divisions and those that did not. The influx α(t) of mutated mitotic cells of all leukemic clones at time t is given by \(\alpha (t)=\sum _{i=1}^{n(t)} \alpha _{i}(t)\), where n(t) is the number of leukemic clones present at time t.
We consider the rate α(t) as the rate of an inhomogeneous Poisson process. Poisson processes describe rare events [50], therefore, they are a suitable framework to describe mutations. We use the Poisson process to determine the time points of the mutations. At the respective time points, one cell acquires a new mutation and gives rise to a new clone. This founder cell is chosen from the present clones according to their contribution α _{ i } to the total rate α. Selfrenewal and proliferation rates of the new clone are chosen based on the parameters of the founder cell. We assume that the traits (selfrenewal and proliferation rates) of the new clone are normally distributed with a predefined variance and the mean value corresponding to the parameters of the founder cell. Since biological parameters are restricted to a predefined interval, we use truncated normal distributions. A suitable interval for proliferation rates is between one division per year and one division per day [46] and the fraction of selfrenewal is by definition between zero and one.
At the time of its birth a new clone consists of one mitotic and zero postmitotic cells. Due to the stochasticity of cell fate decisions or due to cell death it is possible that the new clone becomes extinct. For example, if the newly generated mitotic cell divides and gives rise to two differentiated progeny, the new clone will eventually extinct, since there exist no more mitotic cells. We use the theory of GaltonWatson processes to calculate the probability of extinction of new clones. We adopt the methodology from ref. [51], which is similar to the approach used in ref. [31]. We notice that a clone eventually becomes extinct if it has no mitotic cells. If a mitotic cell divides, with the probability a ^{2} both progeny are mitotic cells, with the probability 2(1−a)a one progeny is a mitotic cell and with probability (1−a)^{2} both progeny are fully differentiated. By a we denote the fraction of selfrenewal of the mitotic cells. The probability generating function for the number of mitotic progeny is f(x)=a ^{2} x ^{2}+2a(1−a)x+(1−a)^{2}. If we assume in addition that the parent cell dies with a probability d during division the probability generating function is f(x)=(1−d)(a ^{2} x ^{2}+2a(1−a)x+(1−a)^{2})+d.
We notice that p _{ e }<1 if a>0.5. For each new clone we calculate the extinction probability based on its selfrenewal rate at the time of emergence. With probability 1−p _{ e } the new clone is introduced to the system by adding two equations to the system describing dynamics of mitotic and postmitotic cells of the new clone. With probability p _{ e } the new clone is not introduced to the system since it becomes extinct.
According to biological data suggesting that all leukemic cells are derived from one leukemic or preleukemic clone [12], we neglect mutations in the healthy cells. Instead we introduce one leukemic founder clone at t=0 to the system and study the dynamics of the founder clone and the clones arising from it. Parameters of the founder clone are assumed to be normally distributed with the means equal to parameters of healthy hematopoietic cells.
Parametrization
The parameters of the hematopoietic system are adopted from ref. [22]. In the following we shortly describe the calibration. For details see ref. [22] and the references therein. The numbers of myeloid mitotic (\(\bar {c}_{1}\)) and postmitotic (\(\bar {c}_{2}\)) cells and the neutrophil clearance (d _{2}) are taken from literature. Analytical expressions of the steady state cell counts allow to calculate p _{ c } based on \(\bar {c}_{1}\), \(\bar {c}_{2}\) and d _{2}. The parameter k can be expressed as a function of known quantities and \(\hat {a}_{c}\). To obtain an appropriate value for \(\hat {a}_{c}\), we simulate stem cell transplantation. We know that after transplantation of a dose of 3 to 5·10^{6} cells per kg body weight, patients recover to 5·10^{8} neutrophils per liter of blood within 15 days. To set the initial condition we assume that the ratio of myeloid to erythroid cells in the transplant is as in the healthy marrow between 2:1 and 4:1. We choose a _{1} such that we observe recovery after 23 weeks. This results in the following parameters: \({\hat {a}_{c}}=0.87\), p _{ c }=0.45/d a y, d _{ c }=2.3/d a y, k=1.85·10^{−9}, d _{ l }=0.5/d a y. These parameters refer to healthy cells. Parameters of the leukemic cells are chosen randomly according to normal distributions.
For the simulations the rate ν is varied between 5·10^{−8} and 10^{−11}. Standard deviations for the normal distributions are varied between 0.001 and 0.1. The standard deviations and mutation rates used to obtain the figures are specified in the figure captions. Stopping criteria for simulations are either decline of healthy blood cells to less than 5 % of the healthy steady state value or a simulated time span that exceeds 100 years of age for a given patient. An example simulation is depicted in Fig. 2 b.
Results
Simulations over wide parameter ranges, including variation of mutation rates over several orders of magnitude, show that the phenomena presented below are robust with respect to the parameter choice.
Selfrenewal rate of significant clones increases during the course of the disease
Properties of the first clone determine if there is no outbreak of the disease, a monoclonal disease, or clonal diversity
Selfrenewal rate increases with an increasing depth of clones
In leukemia patients, clonal hierarchies show considerable interindividual variations [12]. We ask how the properties of clones influence the depth of the clonal hierarchy. We assume that the founder clone has depth 1. Clones that have acquired k additional mutations in comparison to the founder clone have depth 1+k. Depth of a clonal hierarchy is understood as the maximal depth of its clones. Simulations indicate that the selfrenewal rate of the clones increases with their depths. This is plausible: To be able to give rise to new clones, a given clone has to achieve a critical mass of cells. Only then it is probable that cells of this clone mutate and give rise to offspring clones. To achieve the critical mass, a clone needs properties favorable for efficient expansion. This occurs if and only if its selfrenewal rate is increased compared to its mother clone, since otherwise the mother clone outgrows its offspring.
Simulations show that a deep clonal hierarchy requires a stepwise increase of selfrenewal rate with each mutation. The step size determines how deep the hierarchy will be. If the step size is too small, offspring clones grow slowly and it takes long time until they and their offspring grow to a significant size. In this case the parent clone remains dominant in size and is responsible for disease progression. If the step size is large, the offspring clones grow fast and the patient dies before potential new offspring achieve a significant size. In both cases the hierarchy is flat. Only if selfrenewal rate increases by steps of intermediate size, deep hierarchies are observed. In this case, the offspring clones have a sufficient growth advantage compared to their parents but they grow slow enough for their offspring to achieve a significant size and to give rise to new clones.
Selfrenewal rate of the significant clone which appears first has a major impact on the depth of the hierarchy. If it has a high selfrenewal rate, the disease progresses fast and the patient dies before offspring achieve a critical mass. In case of small selfrenewal rate of the first clone, deep hierarchies can emerge, supposed that it gives rise to offspring with higher selfrenewal rates. Analogously the properties of a clone of depth 2 determine whether a clone of depth 3 can emerge. The proliferation rate has no impact on the depth of the hierarchy.
Cooperativity of mutations might explain emergence of deep hierarchies
In the patients investigated in ref. [12], hierarchies of depths between 3 and 5 have been detected. Due to the findings described in the previous paragraph, emergence of such hierarchies requires a coordinated increase of the selfrenewal rate with each acquired mutation. Appearance of clones with too high selfrenewal rates leads to fast progression and death before deep hierarchies can be established. Therefore, the existence of deep hierarchies is not compatible with mutations that lead to large changes of cell properties. Indeed, if we assume that traits of mutated clones are uniformly distributed in the trait space, deep hierarchies are never observed in simulations.
This observation raises the question which probability distributions are suitable choices to model the effect of mutation in the trait space. We have investigated the assumption that the traits of the new clone follow normal distributions with means equal to the traits of the cell that gave rise to the new clone. Depending on the assumed standard deviations of the normal distributions we can observe hierarchies of varying depths. If the standard deviations are too large, the hierarchies remain flat, since aggressive clones that lead to fast progression and death emerge early in the disease. If the standard deviations are too small, the traits of the offspring clones are very close to the traits of the parent clones. Therefore, the offspring clones have little growth advantage compared to their parent clones and consequently need long time to grow to a significant size and to produce offspring growing to a significant size. In these cases the hierarchy of significant clones remains flat. Only if the standard deviation is within a limited range, a considerable number of patients with deep hierarchies is observed in the simulations. In acute leukemias, where genetic instability is rare, generation of large numbers of mutated cells and selection of those which exactly fit the properties required to establish deep hierarchies is not a realistic scenario, since mutation rates are relatively low, compared to other cancers. Leukemias show high interindividual genetic variability. The assumption that all described mutations will lead to exactly those changes in the selfrenewal rates that are required to establish deep hierarchies seems also improbable.
If we assume that the standard deviation of the normal distribution according to which the traits of the offspring are chosen increases with each mutation, deep hierarchies become a more frequent event. This is plausible since small standard deviations for the first mutation avoid emergence of clones that show fast expansion and subsequent death of the patient. Stepwise increase of standard deviation with each mutation allows the offspring to gain sufficient growth advantage compared to the parent clones that they can grow to a significant size. At the same time large jumps leading to aggressive clones remain rare.
The assumption that jump sizes in the trait space increase with the number of mutated genes in a cell seems plausible from a biological point of view. Cells are known to have redundant pathways for regulation of important functions. Perturbation of one pathway by a mutation might therefore lead to only small jumps in the trait space, whereas subsequent perturbation of multiple pathways may lead to larger jumps in the trait space. This means that the presence of a mutation facilitates the occurrence of large effects due to an additional mutation. In this sense the different mutations are cooperative.
Impact of the mutation rates and probability distributions on the clonal hierarchies
We studied the architecture of clonal hierarchies for several mutation rates. For increased mutation rates the total number of clones increases. Interestingly, the number of significant clones increases only moderately if the mutation rates are varied over several orders of magnitude; for example, if the rate increases from 5·10^{−10} to 5·10^{−9}, the mean number of all clones increases by a factor of 8, whereas the mean number of significant clones increases only by 1. In all cases the number of significant clones was smaller than 15 and for 80 % of the patients smaller than 10. This is in line with the observation of clone numbers in experimental studies [11, 12]. This finding underlines the role of competition between the different clones. The competition selects among an increasing total number of clones always a small number of significant clones.
Simulation results imply that patients with less aggressive clones and without disease outbreak are overrepresented in case of small mutation rates. Patients with highly aggressive clones and fast disease progression are overrepresented in case of high mutation rates. This is plausible: The higher the mutation rate, the more clones are generated per unit of time. The probability that at least one clone per patient has favorable growth properties increases with the number of generated clones. Similarly the probability that highly aggressive clones and fast disease progression occur increases with increasing mutation rate. For all mutation rates we observed that clonal hierarchies are flat in case of fast disease progression and in case of very slow disease progression compared to cases with intermediate disease progression.
Increased mutation rates act in favor of deep hierarchies. Nevertheless this effect is mild and the mean depth increases by 1 if the mutation rate increases by a factor of 10. This observation can be explained by the fact that high mutation rates lead to increased numbers of leukemic clones. Therefore, the probability that a clone gives rise to at least one offspring with favorable growth properties increases. As discussed above, probability distributions according to which the traits of new clones are determined have an important effect on the depth of the hierarchy. If uniform distributions over the possible parameter range are chosen, deep hierarchies are very rarely observed. Also the total number of significant clones is decreased. Similarly, if standard deviations of normal distributions increase over a certain threshold, the mean number of significant clones slightly decreases, e.g., the number of significant clones decreases by 1 if the standard deviations are increased from 0.01 to 0.05.
Comparison to data
Discussion
We propose a mathematical model to study the emergence of clonal heterogeneity in acute leukemias. The model considers the interactions of multiple leukemic clones with healthy hematopoiesis and the emergence of new clones due to mutations. We use computer simulations to study the impact of leukemic cell selfrenewal and proliferation rates on the structure of the clonal hierarchy. At the same time, the model provides insights into how the properties of clones at different positions in the clonal hierarchy differ. These questions are clinically relevant, since the patients’ prognosis and the treatment response may depend on the properties of the leukemic cells [23].
Model simulations suggest that the selfrenewal rate of leukemic clones has a major impact on the structure of the clonal hierarchy, whereas proliferation rates show no significant influence. The selfrenewal rate of the emerging clones increases during the course of the disease. There is evidence that a high selfrenewal rate of clones may be linked to poor prognosis [23]. In this sense, clones appearing later during the disease are more aggressive than those present at the beginning of the disease. Similarly, simulations suggest that the selfrenewal rates of clones increase with increasing depth of the hierarchy, whereas proliferation rates do not depend significantly on the depth of clones in the hierarchy. Simulations of large patient groups suggest that there might exist a significant relationship between the depth of the clonal hierarchy and the maximal selfrenewal rate. This finding suggests to evaluate the depth of the clonal hierarchy as a potential marker for patient prognosis.
Mutations detected in acute leukemias act at different regulatory levels. There is evidence that many of them lead to increased selfrenewal. Important examples for genes where mutations lead to increased selfrenewal are the chromatin modifiers TET2 [53], DNMT3A [54] and MLL [55] or the transcription factors C/EBP α [56], RUNX1/CBF β [57, 58] and factors encoded by the HOX genes, e.g., as NUP98HOXA9 [59]. Other examples include the isocitrate dehydrogenase IDH1 [60], the NRAS gene [61] or the multifunctional protein NPM1 [62]. Importantly, more than one of these mutations can occur in the same cell [63, 64]. This is in line with the stepwise increase in selfrenewal observed in the model simulations.
Emergence of the clonal hierarchy is a dynamic process. Model simulations show that the properties of the existing clones have an impact on the structure of the clonal hierarchy in the future. Presence of aggressive clones with high selfrenewal rates leads to fast progression of the disease. The short remaining lifespan of the patient limits the number of new clones that can emerge and grow to a significant size. Therefore in presence of aggressive clones, the clonal hierarchies consist of a relatively small number of clones. On the other hand, if the selfrenewal rates of new clones is very close to the selfrenewal rate of the parent clone, the new clone expands slowly and takes a long time to reach a significant size. Therefore, mutations causing only small changes in selfrenewal rates also lead to small numbers of significant clones and flat hierarchies.
The model simulations suggest that the emergence of deep clonal hierarchies is a complex process. To give rise to offspring, a clone requires a critical mass, otherwise it is unlikely that a clone acquires new mutations. A deep hierarchy is created if new clones have high enough selfrenewal rates to grow to a critical mass before the patient dies, but not too high selfrenewal rates to avoid fast progression and death before the new clones can produce their own offspring. Simulations imply that these constraints are rarely met if one assumes that different mutations act independently, i.e., the properties of a new clone compared to the parent clone follow the same random distribution for each mutation. This limitation can be overcome if cooperativity of mutations is assumed, i.e., if the probability that a new mutation leads to large changes of cell properties increases with the number of mutations that the cell has experienced in the past. Since deep hierarchies are frequently observed in patients (ref. [12]) it seems very plausible that cooperativity of mutations plays a role during evolution of the acute leukemias. In the presence of cooperativity we observe a significant increase in the emergence of deep hierarchies and also in the interindividual heterogeneity of clonal hierarchies. Assuming cooperativity allows to reproduce many of the clonal hierarchies detected in patients from ref. [12] by model simulations.
Computer simulations of the proposed model suggest that increasing mutation rates have only a limited impact on the number of significant clones. Although the total number of clones increases with increasing mutation rates, the number of significant clones remains approximately constant. This observation can be explained by the strong competition among leukemic clones. This is in line with experimental studies [11, 12] which report similar numbers of clones in different patients.
Genetic and epigenetic mechanisms are closely intertwined in leukemogenesis. Genes involved in epigenetic encoding are often mutated in leukemias [65, 66]. In addition, epigenetic changes can be driven by mutations which are not directly linked to the epigenetic machinery such as IDH1/2 or several transcription factors [67]. In principle, epigenetic mechanisms can be included in the proposed modeling framework. It has to be clarified whether epigenetic effects can be modeled similar to mutations as random, independent, discrete jumps in the trait space. This may be true for epigenetic changes that mimic genetic mutations such as in the case of DNMT3A [68]. In other cases it might be more appropriate to model epigenetic modifications as drift or diffusion in a continuous trait space as done in [33]. Plasticity and potential reversibility are important hallmarks of epigenetic changes. The possibility that clones readopt the traits of their ancestors can be included easily into the model. The same holds for the potentially different time scales of genetic and epigenetic modifications.
Mathematical modeling of clonal hierarchies can be a helpful tool, since it allows to link cell properties to a position in the clonal hierarchy. This may have a clinical relevance. Model simulations imply that the clonal hierarchy allows drawing conclusions about the course of the disease, even if the latter is known only at one time point. This work has the following clinical implications which could be discussed and evaluated in clinical trials: (i) A small number of clones detected at diagnosis may argue in favor of a rapidly progressing disease with aggressive clones. These patients may have poor prognosis and potentially a rapid progression after standard treatment or a poor treatment response. (ii) Deep hierarchies e.g., of order 5 or more, may argue for a long course of the disease before diagnosis. The probability for the presence of aggressive clones is high, which may limit prognosis. Nevertheless clones at the top of the hierarchy may respond to the treatment. (iii) Hierarchies of intermediate depth, e.g., 3 or 4 may argue in favor of long courses and limited aggressiveness of the clones. Patients with good prognosis may be overrepresented in this group.
Conclusion
Emergence of heterogeneity in acute leukemias is a complex process. Multiclonality and deep hierarchies emerge only from leukemic clones with intermediate selfrenewal. Emergence of deep hierarchies is facilitated by a cooperativity of different mutations. There is no correlation between the position of a clone within the hierarchy and its proliferation rate. Selfrenewal rates of leukemic clones increase with the depth of mutation hierarchy. Therefore, the structure of the hierarchy may serve as a marker for patient prognosis.
Reviewers’ comments
Reviewer’s report 1: Marek Kimmel, Rice University, Houston, TX, USA
Reviewer summary:
The paper is focused on modeling the clonal structure of driver mutations in leukemias. It is based on a previous model by the same group, which is augmented by new mutations appearing at random. The topology of the resulting clonal pedigrees is compared to literature data, notably ref. [12]. The results are plausible in that the collection of structures generated by the model matches that in ref. [12]. I enjoyed reading the paper, which is certainly sufficiently novel to be published.
Reviewer recommendations to authors:
1. The main difficulty I see at the current stage of development of the model is that the simulated mutations are not identified with any particular “real” drivers. At least some comments on the subject will be helpful.
Authors’ Response: We provide an overview of leukemic (driver) mutations that lead to increased selfrenewal rates. Serial acquisition of such mutations could explain the stepwise increase in selfrenewal rates observed in the model. We added the following paragraph to the discussion (p. 14, lines 23–34): Mutations detected in acute leukemias act at different regulatory levels. There is evidence that many of them lead to increased selfrenewal. Important examples for genes where mutations lead to increased selfrenewal are the chromatin modifiers TET2 [53], DNMT3A [54] and MLL [55] or the transcription factors C/EBP α [56], RUNX1/CBF β [57, 58] and factors encoded by the HOX genes, e.g., as NUP98HOXA9 [59]. Other examples include the isocitrate dehydrogenase IDH1 [60], the NRAS gene [61] or the multifunctional protein NPM1 [62]. Importantly, more than one of these mutations can occur in the same cell [63, 64]. This is in line with the stepwise increase in selfrenewal observed in the model simulations.
2. The authors identified a plausible mechanism of selection of clones by assuming that faster clones are not seen in the patient population, since they lead to a sooner death, while on the other hand the slowest clones do not show since the patient may die of competing risks before he/she is diagnosed. It would be nice to see the sensitivity of this mechanism to changes in clone mutation rate distribution, for example.
Authors’ Response: We run simulations for different mutation rates. The simulations imply that this mechanism is preserved. We added a paragraph to the Results section (p. 12 line 53  p. 13 line 9): Simulation results imply that patients with less aggressive clones and without disease outbreak are overrepresented in case of small mutation rates. Patients with highly aggressive clones and fast disease progression are overrepresented in case of high mutation rates. This is plausible: The higher the mutation rate, the more clones are generated per unit of time. The probability that at least one clone per patient has favorable growth properties increases with the number of generated clones. Similarly the probability that highly aggressive clones and fast disease progression occur increases with increasing mutation rate. For all mutation rates we observed that clonal hierarchies are flat in case of fast disease progression and in case of very slow disease progression compared to cases with intermediate disease progression.
3. I understand that once a mutation (= driver mutation?) arises, the clone it initiates survives. This is of course true only of some of the clones. The problem can be fixed by adjusting the rate of the Poisson process by the probability of nonextinction of the clone, as it was done for example in the models in Bozic et al. (2010) or Kimmel and Corey (2013). It would be interesting to know how such adjustment might change the effective mutation rates.
Authors’ Response: We have adopted the approach described in Kimmel and Corey (2013) to take into account extinction probabilities. The introduction of extinction probabilities leaves our main results (increase of selfrenewal over time, increase of selfrenewal with increasing depth of the hierarchy) unchanged. Effective mutation rates decrease if the extinction of clones is modeled. This leads to a later disease outbreak. The frequency of deep hierarchies is reduced in the model with extinction probabilities. This can be explained by the fact that the extinction probability is higher if selfrenewal of the new clone is small. Therefore clones with high selfrenewal probability which lead to a fast course of the disease and patient death are favored. Corresponding explanations have been added to the Methods section on page 7 (lines 25–63).
Reviewer’s report 2: Tommaso Lorenzi, University of St Andrews, Scotland, UK
Reviewer summary:
Stiehl et al. present an in silico study of possible mechanisms which underpin the emergence and evolution of clonal heterogeneity in acute leukaemia. In line with previous work by the same authors, this study relies on a mathematical model consisting of a system of ordinary differential equations describing the evolution of hematopoietic cells and leukemic clones. From the modelling point of view, the main novelty lies in the fact that the authors incorporate here the effects of mutations occurring during genome replication. The manuscript is well organised and the results are presented in a clear and wellstructured way. The work is based on computer simulations alone as the structure of the model would make it very hard to perform any qualitative analyses. The numerical results obtained are interesting and potentially relevant for the cancer research community. In particular, the comparison of numerical results with clinical data presented by Anderson et al. [Nature 469, 356361, 2011] shades light on possible connections between clonal heterogeneity and the disease progression of leukaemia.
Reviewer recommendations to authors:
1. I would recommend that the authors discuss in more detail the originality of their model compared with the models presented in ref. [Werner et al., J. R. Soc. Interface 10, 20130349, 2013] and in the appendix of ref. [Stiehl et al., J. Royal Society Interface 11, 20140079, 2014].

Page 3, lines 61–63: The main novelty lies in considering a time dependent number of leukemic clones and in tracking the structure of the clonal hierarchies.

Page 4, lines 8–15: To model stochastic extinction of clones with favorable properties, we take into account their extinction probabilities using the theory of branching processes. Compared to the work presented in [40], which focuses on neutral mutations in nonstem cells without feedback regulation or competition, we are interested in the evolution of nonneutral stem cell mutations under competitive pressure of a nonlinear feedback mechanism.
2. Although I agree that a careful description of the model parametrisation is presented in ref. [22] of the manuscript, I think it would be worthwhile to justify in more detail the choice of the parameter values in the subsection ‘Parametrization’.
Authors’ Response: We added an additional paragraph to the section describing parametrization (p. 8, lines 15–33): In the following we shortly describe the calibration. For details see ref. [22] and the references therein. The numbers of myeloid mitotic (\(\bar c_{1}\)) and postmitotic (\(\bar c_{2}\)) cells and the neutrophil clearance (d _{2}) are taken from literature. Analytical expressions of the steady state cell counts allow to calculate p _{1} based on \(\bar c_{1}\), \(\bar c_{2}\) and d _{2}. The parameter k can be expressed as a function of known quantities and a _{1}. To obtain an appropriate value for a _{1}, we simulate stem cell transplantation. We know that after transplantation of a dose of 3 to 5·10^{6} cells per kg body weight, patients recover to 5·10^{8} neutrophils per liter of blood within 15 days. To set the initial condition we assume that the ratio of myeloid to erythroid cells in the transplant is as in the healthy marrow between 2:1 and 4:1. We choose a _{1} such that we observe recovery after 2–3 weeks.
3. I would suggest that Section 5 is removed with contents moved to Section 4; accordingly, I propose renaming Section 3 ‘Results and Discussion’ and Section 4 ‘Conclusions’.
Authors’ Response: The titles of the sections are prescribed by the journal.
4. I would recommend the authors indicate foreseeable extensions of the present work and future research perspectives. For instance, as recent experimental evidence suggests that epigenetic mechanisms can be implicated in the development of acute myeloid leukaemia [e.g., Jost et al., Leukemia 28, 12271234, 2014], the authors may want to discuss possible ways of extending their model to include the effects of epimutations.
Authors’ Response: We added a paragraph to the Discussion (p. 15, lines 24–41): Genetic and epigenetic mechanisms are closely intertwined in leukemogenesis. Genes involved in epigenetic encoding are often mutated in leukemias [65, 66]. In addition, epigenetic changes can be driven by mutations which are not directly linked to the epigenetic machinery such as IDH1/2 or several transcription factors [67]. In principle, epigenetic mechanisms can be included in the proposed modeling framework. It has to be clarified whether epigenetic effects can be modeled similar to mutations as random, independent, discrete jumps in the trait space. This may be true for epigenetic changes that mimic genetic mutations such as in the case of DNMT3A [68]. In other cases it might be more appropriate to model epigenetic modifications as drift or diffusion in a continuous trait space as done in [33]. Plasticity and potential reversibility are important hallmarks of epigenetic changes. The possibility that clones readopt the traits of their ancestors can be included easily into the model. The same holds for the potentially different time scales of genetic and epigenetic modifications.
Reviewer’s report 3: Tomasz Lipniacki, Institute of Fundamental Technological Research, Polish Academy of Sciences,Warsaw, Poland
Reviewer summary:
Thomas Stiehl et al. propose a simple model of hematopoiesis of healthy and leukemic cell populations. The novelty is in inclusion of mutations that lead to time varying number of leukemic clones. The aim is to link the number of mutations that characterize a given clone with the selfrenewal rate (fraction of divisions that do not lead to differentiation). Overall this is a nice study worth publishing, however some aspects require some clarification or more direct formulation.
Reviewer recommendations to authors:
1. The authors found that the selfrenewal rate depend of the depth of clone in the hierarchy. It is not clear whether it is a simple consequence of assumption that clones pass selfrenewal rate to the new clone emerging due to mutation. If so the clones with higher selfrenewal rate that are more abundant have higher chance to give rise to new clones, and these clones will be characterized with higher selfrenewal rate. The Authors write that traits of the new clone are normally or uniformly distributed (page 7), but no details are given.

Page 7, lines 15–19: We assume that the traits (selfrenewal and proliferation rates) of the new clone are normally distributed with a predefined variance and the mean value corresponding to the parameters of the founder cell.

Page 8, lines 37–39: Standard deviations for the normal distributions are varied between 0.001 and 0.1. The standard deviations and mutation rates used to obtain the figures are specified in the figure captions.
2. In “Parametrization” subsection Authors write that a _{ c }=0.87 (I think that authors mean \(\hat {a}_{c}\) since a _{ c } depends to the strength of the negative feedback i.e. number of cells), then they write that for Fig. 4 selfrenewal is between 0.872 and 0.875?

Page 8, lines 31–33: \({\hat {a}_{c}}=0.87\), p _{ c }=0.45/d a y, d _{ c }=2.3/d a y, k=1.85·10^{−9}, d _{ l }=0.5/d a y. These parameters refer to healthy cells. Parameters of the leukemic cells are chosen randomly according to normal distributions

Page 10, line 12: Simulations show that if we restrict selfrenewal rate of the leukemic founder clone to intermediate values, e.g., between 0.872 and 0.875, the number of clones per patient increases.
3. It is not clear how long are the simulations? When selfrenewal rate multiplied by feedback strength s(t) is larger than 1/2, the cell subpopulations are growing. It is not clear whether the authors run the simulations long enough to reach the constant population size limit. A figure showing number of cells in each clone would be helpful.
Authors’ Response: We added a panel showing an example for the time course of the leukemic clones to Fig. 2. As shown in [22, 52], the cell count in each clone converges asymptotically to an equilibrium, which is nonzero only in the case of clones with maximal fitness, i.e., the largest selfrenewal. In clinical scenarios, patients die before the equilibrium is reached. Allowing mutations with increasing selfrenewal not approaching one would lead to a dynamics that does not converge to an equilibrium. The latter is however an artificial example as far as biological applications are concerned.
4. Since the clone starts from single founder cell, in the beginning of each clone ODE formalism is not adequate – some comment is needed here.
Authors’ Response: Following the advice of Reviewer 1 (point 3.) we extended the model to take into account extinction probabilities of newly generated clones. For each new clone we calculate the extinction probability based on the theory of GaltonWatson Processes. The probability that a new clone is introduced to the system is equal to its probability of nonextinction.
5. The conclusion in abstract that “clonal heterogeneity might impact the course of the disease” is very vogue.
Authors’ Response: We have replaced this formulation by a more specific statement: Comparison of patient data and simulation results suggests that the selfrenewal of leukemic clones increases with the emergence of clonal heterogeneity.
6. Technical: Page 5 “Model equations for hematopoietic system” there is once p _{ c } once p ^{ c } – I think it denotes the same thing.
Authors’ Response: Thank you. We corrected it.
Abbreviation
 Resp.:

Respectively
Declarations
Acknowledgements
Not applicable.
Funding
This work was funded by research funding from the German Research Foundation DFG (SFB 873; subprojects A13 to C.L and B08 to A.M.C. and T.S.)
Availability of data and materials
Not applicable.
Authors’ contributions
TS, CL and AMC planned the research and designed the model. TS run the simulations and compared them to the data. TS and AMC prepared the manuscript. All authors read and approved the manuscript.
Authors’ information
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
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