Describe the Molecular Basis Underlying Haematological Malignancy The average human produces many billions of red cells

every day. The haemopoietic system maintains this huge output through the tight control of stem cell proliferation, lineage commitment and differentiation. It is corruption of these processes that results in haematological malignancy. Current models of leukaemogenesis suggest a multiple hit process in which critical cellular pathways are corrupted by accumulating genetic damage, resulting in a single proliferating stem cell clone. Most malignancies are thought to be the result of several mutations, acquired sequentially and randomly, rather than a single catastrophic mutation. In humans, there is evidence that a single mutation is not enough for the full malignant phenotype. First, individuals who inherit a mutated RUNX1 gene in all their cells develop a clonal leukaemia in later life, suggest that additional events are needed to induce leukaemogenesis. The model of clonal evolution also suggests that a population of malignant cells contains subclones that have acquired distinct secondary changes, e.g. drug resistance. In studies of CML patients who initially respond to imatinib, but subsequently become resistant, the resistance is often mediated by point mutations that affect the imatinib binding site on the BCR-ABL protein. A central feature of haematological malignancy is a capacity for malignant cells to self-renew. It has been suggested the malignancy results from transformation of a stem cell, because this would explain how the original transformed cell and its progeny survive long enough to acquire secondary mutations. Studies involving CML support the existence of a tumour stem cell because multiple different haemopoietic lineages carry the BCR-ABL fusion gene, and its protein product. This means that a curative strategy for the malignancy must eliminate all malignant cells with the ability to self-renew and regenerate the malignancy. One of the most striking properties of leukaemias is the close relationship between certain cytogenetic or molecular abnormalities and unique morphological characteristics. For example, there is a strong association between acute myelomonocytic leukaemia with abnormal eosinophil precursors (M4Eo AML) and the inv(16) abnormality. The nature of the chromosome arrangement may determine the phenotype of the resultant leukaemia. The best understood genetic abnormalities in haematologically malignancies are chromosomal translocations. Balanced translocations involve a reciprocal exchange of genetic material between two chromosomes and may result in abnormal function of genes adjacent to the breakpoint. There are two common mechanisms; generation of a fusion gene and structurally intact gene being placed next to regulatory elements. A fusion gene may be generated and encode a fusion protein with oncogenic properties, which is seen in many of the translocations associated with myeloid malignancies and some associated with ALL. Fusion proteins contain a combination of function elements from the two partner proteins, and as a consequence the fusion protein has novel functions. For example, fusion proteins that involve a tyrosine kinase, e.g. BCR-ABL, the protein usually contributes a dimerization domain that allows the fusion protein to become constitutively activated. A fusion gene can also result from an interstitial deletion (such as deletion of chromosome 4 causing a fusion gene in chronic eosinophilic leukaemia) or inversion of part of a chromosome (such as inv(16) in M4Eo AML). The second category of translocations results in a structurally intact gene being placed next to regulatory elements from a gene on the partner chromosome, which is frequently observed in lymphoid malignancies in which the normal process of antigen receptor rearrangement goes out of kilter and results in translocations involving immunoglobulin or TCR loci. In some cases of T cell ALL, the SCL and HOX11 genes are ectopically expressed in T cell precursors when they are moved next to enhancers from the TCR loci. In addition, transcriptional dysregulation can occur as a consequence of deletions, such as chromosome 1 deletions resulting in dysregulation of the SCL gene in T cell ALL. 1

All balanced translocations given rise to two abnormal chromosomal productions, one of which is usually implicated as the pathogenetic culprit. However, it is increasingly recognized that the other chromosomal product may also influence leukaemogenesis. For example, in CML associated with the t(9;22) translocation it is clear that the Philadelphia chromosome (derivative 22) carries the BCR-ABL fusion gene, however in a subset of patients the derivative 9 chromosome also carries a deletion which is thought to arise at the same time of the Ph translocation, and is associated with a particularly poor prognosis. For myeloid cells, the mechanism of translocation is poorly understood. It is thought that a double stranded break in DNA, which occurs spontaneously or can be increased in frequency by exposure to ionizing radiation or DNA damaging agents, has a key role. Normal repair mechanisms, such as homologous recombination or nonhomologous end joining, then attempt to join the broken ends of DNA. However, these mechanisms are not perfect, and they could repair two simultaneous breaks on different chromosomes to generate a translocation. Germline mutations in DNA repair genes (such as the ataxia teleangiectasia gene) increase the frequency of chromosomal rearrangements and leukaemia. Topoisomerase inhibitors are well-recognized causes of leukaemia, and are often associated with translocations involving the MLL gene on chromosome 11q23. Topoisomerase II creates dsDNA breaks during its function, and Topoisomerase inhibitors may stabilize complexes that are formed between the enzyme and free DNA ends, and increase the likelihood that those ends might participate in translocation. Chromosome deletions and disorders of chromosome number are among the most frequent karyotypic abnormalities seen in haematological malignancies, resulting in an increase or decrease in the copy number of many genes. Increases in chromosome number are frequently seen in haematological malignancy, for example hyperdiploidy is the most frequent cytogenetic abnormality of childhood ALL, and any chromosome can be duplicated. Trisomy 8 is the most common abnormality and can be seen in AML. Quantitative chromosomal changes give risk to altered expression levels of oncogenes or tumour suppressor genes that contribute to leukaemogenesis. There is mounting evidence that in addition to genetic changes, leukaemogenesis also involves epigenetic alterations that affect gene function without altering the nucleotide sequence. For example, epigenetic mechanisms control transcription, DNA replication, imprinting and X-inactivation. DNA methylation is associated with transcriptional silencing of the neighbouring gene, and disorder patterns of DNA methylation have been found in a wide range of haematological malignancies, such as CLL, and AML blasts. DNA hypomethylation has been found at a number of loci implicated in leukaemogenesis, e.g. TNF- in CML and AML, H-RAS in CLL, and FMS in AML. Hypomethylation promotes transcriptional activity, and so can cause overexpression of these oncogenes. DNA hypermethylation of tumour-suppressor genes can also occur. Histone modification is a further epigenetic mechanism that regulates transcription, and disorders of histone acetylation are found in several types of leukaemia including relapsed ALL. Acquired genetic changes result in malignant transformation by corruption of cellular processes, e.g. tyrosine kinase signalling and regulation of gene transcription. Tyrosine kinases are critical for the response of haemopoietic progenitors cells to external growth stimuli. The binding of a ligand to the extracellular surface of a tyrosine kinase receptor causes receptor dimerization, which increases kinase activity. In haemopoietic malignancy, the formation of a fusion protein can cause tyrosine kinases to become constitutively activated by spontaneous dimerization. Tyrosine kinase activity can also be increased by more subtle mutations, e.g. c-KIT. As a consequence, the corresponding signalling pathway (e.g. RAS, STAT, MAP kinase) is activated and this provides the transformed cell with a proliferative or survival advantage.

Genes encoding transcription factors are common targets for rearrangements of mutations in acute leukaemia. Normal transcription programmes have an important role in regulating the behaviour of normal haemopoietic stem or progenitor cells, mean that alterations of normal these transcription programmes can be leukaemogenic. The genes encoding components of the CBF transcription factor complex are frequent targets in AML. The CBF complex is made up of RUNX1 and CBF. The genes for the two CBF subunits represent the most commonly involved genes in acute leukaemia translocations with TEL-RUNX1 found in 25% of childhood ALL, RUNX1-ETO in 15% of AML and SMMHC-CBF in 10% AML. These translocations cause dominant negative inhibition which mediates their leukaemogenic effects. It is thought that a multi-hit theory is responsible for haematological malignancy. Translocations, inversions and deletions form the initiating lesion and then further sequential alterations lead to leukaemogenesis. Epigenetic mechanisms are widespread but are unlikely to represent initiating lesions, but probably reflect downstream consequences of primary lesions. Translocations exert their leukaemogenic effects by corrupting cellular processes, such as tyrosine kinase activation and regulation of transcription factors.