FRCPath 12 th January 2010

1. Describe the mechanism of leukaemogenesis concentrating primarily on CML but helpful to touch on the other leukaemias as well. FRCPath 12th January 2010

2. Leukaemia – disease affecting blood forming cells originates in bone marrow characterised by an abundance of white cells in the body classified as chronic or acute and named after the progenitor cells involved in the disorder 4 main types chronic myeloid leukaemia (CML) acute myeloid leukaemia (AML) chronic lymphocytic leukaemia (CLL) acute lymphocytic leukaemia (ALL) Leukaemogenesis – induction, development and progression of leukaemic disease multi-step process in which successive transformational events enhance the ability of haematopoietic progenitor cells to proliferate, differentiate and survive.

3. CML studied extensively - good model for leukaemogenesis 15 – 20% of all leukaemias incidence of ~ 1 – 2 per 100,000 occurs in all age groups – incidence increases with age (6th decade) Men > affected women women > survival advantage no obvious geographical or ethnic differences exist characterised – increase in the number of granulocytes mainly neutrophils in different stages of maturation also basophilia and eosinophilia. symptoms can be gradual and non-specific splenomegaly fever tiredness night sweats

4. disease progresses through three distinct phases chronic phase (CML-CP) relatively long lasting, ~3 – 4 years without treatment most patients present during this phase mature granulocytes produced increased number (<10%) of myeloid progenitor cells present in blood accelerated phase (CML-AP) haematopoietic differentiation arrested blasts 10 – 19% blast crisis (CML-BC) WHO criteria blasts > 20% in peripheral blood or bone marrow

5. 1960 – abnormally small chromosome discovered Philidelphia (Ph) chromosome 1st eg – specific cytogenetic abnormality associated with human neoplasm Ph chromosome – modified chromosome 22 translocation long arms chromosome 22 and 9 – t(9;22)(q34;q11) previously thought to be a deletion reciprocal translocation – fusion gene - 3’ region of ABL1 oncogene (9q34) and 5’ region of breakpoint cluster region (BCR) gene (22q11) present in 90 – 95% of patients 5 – 10% – cryptic or complex rearrangement - BCR-ABL fusion gene presence BCR-ABL required for clinical diagnosis of CML reciprocal Abl-Bcr fusion gene is expressed in ~2/3 of patients transcripts are in-frame, no fusion proteins detected

6. function of normal ABL and BCR proteins wild type ABL1 gene encodes nonreceptor tyrosine kinase ubiquitously expressed 2 isoforms – alternative splicing of first exon Abl protein localised in cytoplasm and nucleus specifically binds DNA involved in cell cycle regulation in response to genotoxic stress transmission of information about the cellular environment through integrin signalling also thought to be involved in apoptosis Bcr – phosphoprotein – serine/threonine kinase activity ubiquitously expressed thoutht normal funtion related to maintenance of cytoskeleton

7. Bcr-Abl fusion protein cytoplasm only lacks ability to bind DNA thought that fusion of Bcr to Abl interferes with the negative regulation of Abl results in constitutive activation of tyrosine kinase activity Bcr-Abl excessively phosphorylates multiple cellular proteins not normally available to the Abl protein results in an altered expression profile of the stem cell CML unusual – single genetic lesion – necessary and sufficient for cell transformation

8. ABL gene breakpoints anywhere over large (>300kb) area - 5’ end upstream of first alternative exon 1b downstream of second alternative exon 1a more frequently between the two splicing of primary hybrid transcript yields a transcript - BRC sequences fused to ABL exon 2a

9. BCR gene breakpoints 1 of 3 so-called breakpoint cluster regions M-Bcr – major breakpoint cluster region - 5.8Kb region – exons 12 – 16 alternative splicing - 2 fusion proteins - BCR exons 1 – 13 or 1 – 14 p210Bcr-Abl – most CML patients and ~ 25% ALL patients m-Bcr – minor - 54.4Kb – alternate exons e2’ and e2 p190Bcr-Abl – rest ALL patients, rarely in CML patients ~5 X higher tyrosine kinase activity than p210Bcr-Abl – correlates with more frequent association with acute rather than chronic form of leukaemia and with its greater transforming potency μ-Bcr - downstream of exon 19 p230Bcr-Abl - chronic neutrophilic leukaemia (CML-N) - rare myeloproliferative disorder, more indolent course than classic CML, blastic transformation usually occurs much later if at all lowest tyrosine activity of the three Bcr-Abl fusion proteins inclusion or exclusion of BCR exons is largely responsible for determining the disease phenotypes caused by these fusion proteins

10. CML-CP leukaemic cells have a growth advantage over normal cells proliferate beyond the homeostatic cell density limit retain the capacity to differentiate almost normally morphological, biochemical and functional defects that occur do not prevent the cells from carrying out their essential functions during this stage CML progenitor cells still dependent on external growth factors for survival and proliferation (less than normal progenitors)

11. disease progression to CML-AP and CML-BC transition from mature, terminally differentiated cells to immature, undifferentiated cells 3 major mechanisms implicated in malignant transformation by Bcr-Abl altered adhesion decreased adhesion to bone marrow stroma cells and extracellular matrix adhesion to stroma negatively regulates cell proliferation constitutively active mitogenic signalling Ras/MAPK, Jak/Stat (mainly Stat5), PI3K and Myc all implicated together lead to anti-apoptotic effects, cell proliferation and self renewal of progenitor cells reduced apoptosis 4th possible mechanism proteosome-mediated degration of Abl inhibitory proteins

12. Bcr-Abl causes differentiation arrest - translational suppression CEBPαtranscription factor - promotes differentiation in myeloid cells expressed in CML-CP cells - undetectable in CML-BC cells inactivating mutations are found in AML - none found in CML expression levels BCR-ABL, mRNA and protein, increase with disease progression - translocation retained in most patients in CML-AP/BC secondary changes occur - associated with malignant progressiongenomic surveillance for DNA damage and DNA repair are compromised Bcr-Abl may induce mutations in genes responsible for maintaining genomic integrity - such mutations contribute to genomic instability observed could explain the occurrence of the non-random chromosomal abnormalities that characterise CML progression trisomy 8 (33%) additional Ph chromosome (30%) isochromosome 17 (20%)

13. Telomere shortening associated with increased genomic instability and disease progression in human malignancies CML - rate of telomere loss increases with disease progression expression of TERT is significantly lower - consistent with accelerated rate of telomere shortening Bcr-Abl is a potent oncogene - role of inactivated tumour suppressor genes in CML is unclear protein phosphatase 2A (PP2A) acts as a tumour suppressor in CML dephosphorylates and down-regulates Bcr-Abl Bcr-Abl inhibits PP2A activity - dose-dependent increased Bcr-Abl expression is seen in advanced phase disease loss of tumour suppressor function may be involved in the progression

14. treatment imatinib mesylate - oral therapy extremely effective in inducing remission toxicity low inhibits the Bcr-Abl tyrosine kinase - competitive binding at the ATP-binding site high rates of cytogenetic response (92%) progression free survival (84%) - 5 year follow up highly resistant mutants (point mutations - tyrosine kinase domain) found in patients who develop resistance alternative treatment strategy required mildly resistant mutations (point mutations) also found increase in imatinib dose can overcome resistance imatinib particularly useful in CML-CP treatment response and survival is low - in advanced phases of disease second generation tyrosine kinase inhibitors developed dasatinib and nilotinib - more potent inhibitors than imatinib effective in imatinib resistance patients CML-AP and CML-BC

15. similar to CML - one genetic lesion primary causal event one form of ALL - single translocation also thought to be initiating event ~25% of childhood ALL - t(12;21)(p13;q22) translocation TEL-AML1 fusion gene represents unique subset of B-precursor ALL TEL-AML1 translocation demonstrated in neonatal blood spots shows present at birth 5-10 years before the development of leukaemia evidence that translocation is initiating event in this form of ALL fusion protein - transcription factor and functions as a corepressor at AML1 target genes lack of expression of genes normally activated by AML1 may be important for leukaemogensis in this form of ALL loss of normal TEL allele frequently accompanies the fusion suggests that subsequent genetic events are necessary for full malignant transformation

16. FLT3 overexpression found in most AML patients also in subset of ALL patients and in CML-BC FLT3 - receptor tyrosine kinase normally expressed in haematopoietic stem/progenitor cells - expression lost as cells differentiate FLT3 ligand (FL) causes expansion of these cells when combined with other growth factors somatic FLT3 mutations found in ~1/3 of AML patients 2 main types – internal tandem duplications (ITD) of 3 – 400 bp and point mutations ITDs always in-frame - present in the juxtamembrane domain found in ~23% of patients deletion and deletion/insertion mutations also reported in same domain point mutations most frequently involve aspartic acid 835 of kinase domain present in 8 – 12% of patients result in the constitutive activation of FLT3 ITD mutations - poor prognosis - larger ITDs have even worse outcome conflicting evidence on prognostic outcome for patients with kinase domain mutations

17. constitutive activation of FLT3 - phosphorylation of many proteins - ultimately through PI3K, Ras/MAPK and STAT5 to altered expression of many genes constitutively activated FLT3, both wild type and those with mutations have been shown to contribute to leukaemogenesis evidence that constitutive activation of FLT3 occurs early in leukaemogenesis - FLT3 mutations found in stem cells FLT3 mutations occur in a setting of acute leukaemia where several somatic genetic alterations occur early in disease to fully transform cells difficult to elucidate if single genetic lesion sufficient to initiate leukaemogenesis - what order subsequent secondary changes occur

18. Armstrong and Look (2005) Molecular genetics of acute lymphoblastic leukaemia. Journal of Clinical Oncology 23:6306-6315 Clarkson, et al. (2003) Chronic myelogenous leukaemia as a paradigm of early cancer and possible curative strategies. Leukemia17:1211-1262 Deininger, et al. (2000) The molecular biology of chronic myeloid leukaemia. Blood96:3343-3356 Hehlmann, et al. (2007) Chronic myeloid leukaemia The Lancet370:342-350 Kantarjian, et al. (2007) Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philiadelphia chromosome-positive chronic myelogenous leukaemia in chronic phase following imatinib resistance and intolerance. Blood 110:3540-3546 Melo and Barnes (2007) Chronic myeloid leukaemia as a model of disease evolution in human cancer Nature Reviews Cancer7:441-453