a. Multistep Process
The progression of a pre-malignant clone through intermediate steps to eventually becoming a malignant neoplasm is promoted by an initiating event. The majority of malignancies are thought to be initiated by spontaneous or induced somatic mutations but latency periods between the development of a mutation and the transformation into a malignancy
suggests the need for more than one event (Knudson, 2001). Foulds (1954) described a step-wise development of neoplastic tumours of increasing autonomy due to changes in the cellular phenotype (Foulds, 1954) In vitro models and in vivo mice models of virally-infected leukaemia have helped to observe the multi-step process involved in the transformation of leukaemia. Following a long latency period from infection with a helper-independent Friend Murine Leukemia Virus (F- MuLV), bone marrow cultures and mice developed myeloblastic leukaemia displaying several steps in the progression to malignant transformation. During a “pre-leukaemic” stage the cells in culture displayed an abnormal response to GM-CSF, a promotion in their proliferation of colonies containing both mature granulo-macrophagic and immature myeloblastic cells, and acquired growth autonomy before finally achieving an in vivo tumourigenicity (Heard et al, 1984).
b. Clonality
Normal haematopoiesis is a process of polyclonal expansion; several stem cells proliferating and differentiating to generate a mixed pool of terminally-differentiated end-cells. However, the majority of haematological disorders result from monoclonal expansions. A genetic event resulting in a growth advantage of one cell over others can lead to the development of a clone. The multistep nature of AML can result in the generation of a range of sub-clones differing in their acquired secondary abnormality. One clone will usually dominate and direct the course of the disease but the application of pressure, for example
chemotherapy, can generate a clonal shift allowing a minor clone to dominate the disease. As relapsed leukaemia is often resistant to chemotherapy, it suggests that the event contributing to a leukaemic relapse is not always the original leukaemia present at diagnosis but instead a leukaemia resulting from a clonal shift and therefore relapse is accompanied by clonal evolution (Nakano et al, 1999). Monitoring of both the FLT3 (Fms-like tyrosine kinase 3) gene and the N-ras (neuroblastoma RAS viral (v-ras) oncogene homologue) gene in AML have demonstrated the instability of such aberrations and their role as secondary events with the observation of the acquisition or loss of mutations over the course of the disease in AML patients (Nakano et al, 1999;Shih et al, 2004b;Chen et al, 2005).
Historically, methods used to identify clonality in haematological malignancies have adopted a system of genetic markers. Intrinsic markers, for example cellular markers or somatic cytogenetic aberrations, that have developed during normal cell processes or as part of the disease can act as useful intrinsic markers for clonality studies. The Philadelphia (Ph) chromosome is expressed in the majority of chronic myeloid leukaemic (CML) cells, but in almost no non-CML cells; the fusion product resulting from the translocation of chromosomes 9 and 22. This led to the suggestion that this leukaemia evolved from a single cell in which the aberrant chromosomal event occurred, therefore demonstrating the monoclonality of CML. However, the possibility of CML resulting from a polyclonal expansion can not be
ruled out based on this evidence alone and further analysis with extrinsic markers is required. Extrinsic markers take advantage of cellular mosaicism. The nature of mosaicism allows for markers completely independent of the disease of interest to be studied and to not be restricted to a particular cell lineage (Raskind et al, 1998). Mosaicism results from the inactivation of all but one X chromosome in cells containing two or more X chromosomes and occurs during the early stages of embryogenesis. The selection of either the maternal X chromosome or paternal X chromosome for inactivation is usually a random event and is stably transmitted during mitosis to daughter cells. Beutler et al (1962) demonstrated the mosaicism of cells in females using the X-linked enzyme G6PD as a marker (Beutler et al, 1962). Early studies of X-chromosome inactivation patterns (XCIPs), using isoforms of the G6PD enzyme resulting from a polymorphism, for determining the clonal origin of neoplastic cells were limited to small minority of the female population (Fialkow et al, 1967;Fialkow et al, 1981). Vogelstein et al (1987) were able to extend the use of XCIPs to a wider population using restriction fragment length polymorphisms (RFLP) and methylation patterns of the inactive and active X chromosomes.
c. Target Cells
As previously mentioned, an early study by Fialkow et al (1981) demonstrated the clonality of acute non-lymphocytic leukaemia through chromosomal studies of the x-linked enzyme G6PD. The data indicated
some cases of acute myeloid leukaemia (AML) originated from pleuripotent stem cells whilst others originated from cells already restricted to the granulocyte/monocyte pathway (Fialkow et al, 1981). Later studies by Griffin et al (1986) of surface marker analysis further supported the heterogeneity of AML showing AML clonal cells arose from various points in the haematopoietic hierarchy in different patients (Griffin & Lowenberg, 1986;Griffin et al, 1986). These models predicted that differences in phenotype of the leukaemic stem cells would be observed between AML patients depending on the origin of their disease (Fialkow et al, 1981;Griffin & Lowenberg, 1986). Alternatively,
McCulloch et al (1983) proposed leukaemias originate from stem cells with the ability to differentiate or acquire surface markers from the influence of transformation agents resulting in little variability of stem cell phenotype between patients (McCulloch, 1983). Fluorescence In situ hybridisation (FISH) and flow cytometric analysis studies of MDS and AML samples identified characteristic cytogenetic aberrations in the CD34+/CD38' cell compartment providing evidence of primitive cell involvement in MDS and AML (Haase et al, 1995;Mehrotra et al, 1995).
Lapidot et al (1994) identified SCID (severe combined
immunodeficiency) leukaemia-initiating cells (SL-IC) as AML-initiating cells that could establish human leukaemia in SCID mice. The SL-ICs displayed an expression pattern of CD34+/CD38' similar to that observed in normal haematopoietic stem cells and when transplanted into SCID mice they initiated leukaemia, unlike the transplanted CD34+/CD38+ cells, an expression pattern observed in more
differentiated cells, even when in the presence of AML-CFUs (Lapidot et al, 1994). Bonnet et al (1997) observed similar results when they transplanted SL-ICs into NOD/SCID (non-obese diabetic-severe
combined immunodeficiency) mice. When transplanted, the
CD34+/CD38* SL-ICs generated large numbers of AML-CFUs and leukaemic blasts expressing the irregular combinations of surface antigens observed in the patient samples from which the cells originated (Bonnet & Dick, 1997). However, In favour of the hypothesis of Fialkow et al (1981), recent studies of mouse bone marrow transduced with oncogenic fusion genes have demonstrated the ability of MLL-ENL (mixed-lineage leukaemia - eleven nineteen leukaemia) and MOZ-TIF2 (monocytic leukaemia zinc finger - TGF-beta induced factor-2) to transform not only HSCs but committed myeloid progenitors (Cozzio et al, 2003;Huntly et al, 2004). Kirstetter et al (2008) demonstrated the potential of leukaemic transformation to occur in progenitors with limited self-renewal capacity. Approximately 9% of de
novo AML cases present with mutations of the C/EBPa
(CCAAT/enhancer binding protein (C/EBP), alpha) gene. C/EBPa is produced of two polypeptides of 30 kDa (p30) and 42 kDa (p42), with most C/EBPa mutations in AML resulting in the loss of p42. Knock-in mice studies demonstrated that whilst GMPs were still generated, a loss of p42 resulted in myeloid progenitors with a vastly increased self renewal capacity and progression to AML and ultimately death from liver and bone marrow failure. These mouse models demonstrated the role of C/EBPa mutations as AML-initiating events that result in the
generation of leukaemia-initiating cells (LIC) displaying an immunophenotype observed in myeloid-committed cells that generate only myeloid cells in irradiated recipients (Kirstetter et al, 2008). Therefore mutations occurring in HSCs or more committed progenitors may both give rise to the leukaemic stem cell.
d. Leukaemic Stem Cell
Early in vitro colony assays observed only a minority of proliferative leukaemic blasts (AML-CFU) were able to give rise to colonies. Several studies also identified similar properties of active proliferation, self renewal and the ability to undergo differentiation of the normal haematopoietic progenitor cells in AML-CFUs in vitro. This led to the suggestion that a leukaemic clone exists in a hierarchy of proliferating progenitors differentiating into a population of non-cycling leukaemic blasts similar to that seen in normal haematopoiesis (Minden et al, 1978;Buick et al, 1979;Pessano et al, 1984;Griffin & Lowenberg, 1986;Bonnet, 2005). Evidence of a leukaemic stem cell (LSC) with the ability to initiate and sustain the growth of a leukaemic clone in vivo combined with their self-renewal capacity supports the concept of a hierarchical organisation of AML sustained by a small number of leukaemic stem cells transformed from normal haematopoietic cells. Their increased resistance to chemotherapeutic drugs have made it difficult to completely eradicate leukaemic stem cells and relapse often occurs. Similar to normal HSCs, a number of LSCs spend a large proportion of their time resting in the Go phase, unlike the AML-CFUs
they generate. Whilst cell cycle-specific chemotherapeutic drugs targeting the leukaemic blasts are able to eradicate the majority of blasts, the LSCs quiescent state protects them allowing relapse to occur due to their self-renewal capacity and generation of new progeny (Guan et al, 2003;Ravandi & Estrov, 2006;Misaghian et al, 2009). Unfortunately the concept of a hierarchical organisation in leukaemia similar to that in normal haematopoiesis is still a very controversial area. Data supporting the hierarchical organisation hypothesis was obtained through studies performing xenografts. It is argued that cells with a potential to be tumourigenic may require extrinsic factors from the surrounding microenvironment to engraft. Therefore when performing a xenograft between mouse and human, there may be a lack of appropriate microenvironment factors required for engraftment due to species-differences. When assessing the capacity of the grafted cells to initiate the growth of tumours, what may appear non-tumourigenic in the host may actually be tumourigenic under the correct microenvironment conditions in the donor (Rosen & Jordan, 2009).
1.3 Acute Myeloid Leukaemia
Acute myeloid leukaemia (AML) is an extremely heterogeneous disorder characterised by an accumulation of immature haematopoietic cells that have lost the ability to differentiate. The classification of myeloid neoplasms provides useful information regarding a patient’s disease particularly with regard to prognosis and the approach to
treatment required. As our knowledge of AML increases it is important to change and add to these classifications to reflect this (Arber, 2001).