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Cytogenetics in acute myeloid leukaemia
Cytogenetics in Acute Myeloid Leukaemia
H. Walker, F. J. Smith, D. R. Betts S UMMA R Y. A wealth of literature spanning 20 years describing cytogenetic abnormalities in acute myeloid leukaemia ( AML) already exists. It ranges from single case reports of unusual abnormalities to large multicentre studies of lumdredsof cases. A landmark publication was the Fourth International Workshop on Chromosomes in Acute Leukaemia which established a base line for diagnosis, prognosis and frequency of chromosome abnormalities in AML. Two large sources of information are a book, ‘The Chromosomes in Human Cancer and Leukemia’ and a catalogue of chromosome abnormalities, which aims to list all chromosome abnormalities described in the scientific and medical literature from 1973, when the widespread use of banding techniques, enabled the precise definition of the chromosome breakpoints. In this review the common cytogenetic abnormalities seen in AML with reference to associations with the French-American-British (FAB) classification, their possible prognostic signiscance and their associated molecular biology are summarized.
In England and Wales, acute myeloid leukaemia (AML) accounts for 77% of acute leukaemias and the incidence is 3.4 per 100000 of population. In children age 0 to 15 years AML accounts for only 19% of acute leukaemia while, in adults age 16 to 79 years AML accounts for 89% of acute 1eukaemia.l The classification of AML, is based on the clinical presentation, cellular morphology, cytochemistry and immunology.’ The French-American-British (FAB) type serves as an important basis for classification and in some cases prognosis and therapy of the individual leukaemias and can act as a pointer to the likely chromosome abnormality which may be found. The frequency of the observation of chromosome abnormalities is related to the occurence of the FAB types. The incidence of the different FAB types is reported as Ml 18%, M2 28%, M3 8%, M4 27%, M5 lo%, M6 4% and M7 5°h.3 Some chromosome abnormalities are specific to certain FAB types and H. Walker, F. J. Smith, D. R. Retts, Department of Haematology, University College Hospital, Gower St. London WClE 6AU, UK. Correspondence to: Mrs H. Walker. Blood Reviews (1994) 8, 0 1994 GroupUK
indeed the t( 15; 17) seen in FAB type M3 has not been reported in any other malignancy.4 Cytogenetic abnormalities seen in AML are numerical or structural and may occur alone or together. Single abnormalities are described as primary in nature and are thought to be related to a causal event with the secondary abnormalities occuring as associations in some cases. The frequency of chromosome abnormalities detected in AML at diagnosis, is variously reported as between 60 to 90%5 but is dependant on factors such as length of time in culture, banding techniques and referral patterns of reporting centres.6 Some leukaemic cells may not have an abnormal karyotype. The most frequently observed numerical abnormalities in all FAB types of AML are trisomy 8 and monosomy 7. ‘,* Rarely observed are trisomies of chromosomes 4, 11, 13, 21 and 22. There are a number of common specific chromosome rearrangements which include translocations, inversions and deletions namely t (8;2 1) (q22;q22), t ( 15; 17) (q22;q2 1) and inv( 16)(pl3q22), which are associated with
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particular FAB types. Less commonly seen are 1lq23 and chromosome abnormalities of t (6;9) (p23;q34). Rarely seen, but nevertheless reported in a few dozen cases each, are abnormalities of chromosome 3 involving bands q21-q26, t(8;16)(pll;p13) and t(9;22)(q34;qll). Chromosome abnormalities seen at diagnosis in AML are not detectable cytogenetically when complete remission (CR) is achieved, but can reappear at relapse, sometimes with additional changes or even with a different abnormality. (personal observations)
Numerical Abnormalities Trisomy 8 Trisomy 8 is the most common chromosome abnormality seen in AML,(j occuring as either the sole aberration or as an additional change. In M3 approximately 17% of patients at diagnosis are found to have +8 in conjunction with the t(15;17).’ Trisomy 8 is not associated with a specific FAB type but is found most frequently in Ml, M4 and M5. A finding of +8 as the sole abnormality is generally associated with a poor or intermediate prognosis”*” but, there are studies where a relatively high CR is achieved.12,‘3 Fluorescence in situ-hybridization (FISH) techniques can be applied to detect +8.14 This has its benefits and may prove to be more sensitive in samples where the +8 is cytogenetically nonclonal, but it does not detect additional abnormalities and in good preparations is no more sensitive.”
Non Random Trisomies In addition to trisomy 8, trisomies of chromosomes 4, 11, 13, 21 and 22 occur as non-random sole abnormalities in AML. They are relatively rare events, with each trisomy accounting for between 25 to 60 cases. Trisomy 4 has been described in both de novo and secondary AML”j with a significant proportion of cases occuring in M4 and M2.17,18 As yet the prognostic significance is unclear but it is possibly poor. Additional common chromosome changes seen with trisomy 4 include double minutes and other trisomies. Trisomy 4 has only recently been reported which has led to speculation that there may be influencing factors including geographical and unknown environmental factors7 but at present there is no evidence to support either of these theories. Trisomy 11 in AML appears to be associated with previous myelodysplastic syndrome (MDS), old age and karyotypic evolution is a feature. The MDS is often secondary in nature. The prognosis is poor but it is associated with poor prognostic factors.‘**” Trisomy 13 has the most distinct phenotype of these rare non-random trisomies, it may also occur
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as tetrasomy. The majority of patients are elderly males, there is a low CR rate and if remission is obtained it is usually brief.20*21 The AML is often undifferentiated or biphenotypic, expressing lymphoid as well as myeloid markers.” This has led to the suggestion that the leukaemia arises from an early stem cell which retains the potential for both myeloid and lymphoid differentiation. It is unclear why trisomy 13 should have this effect but Cuneo et al” have suggested that 13q13 may be the critical region. Trisomy 21 occurs in a number of neoplasia and is often seen in secondary AML. When it occurs as the sole abnormality there are no specific associations.7 Trisomy 22 has no known prognostic significance. It is associated with M4, often with eosinophiliaz3 and may arise as a secondary event to inv( 16). However, there have been reports where it is the sole abnormality with eosinophilia, which raises the question whether an inv( 16) is present at the molecular level. There is a suggestion that it may be associated with a younger age and the leukaemia may be characterised by the expression of immature differentiation antigens. It is not totally appropriate to group these trisomies together but they are generally associated with undifferentiated or secondary AML.
Structural Abnormalities Abnormalities of Chromosome 3 Though rare, abnormalities of chromosome 3 are interesting as they have a marked clinical association with platelet abnormalities with micromegakaryocytes in the marrow and thrombocytosis.24 The specific cytogenetic abnormalities associated with thrombocytosis involve bands 3q21 and 3q26 either as inversions, translocations or insertions. Abnormalities of chromosome 3q are associated with a high expression of the Evi-1 gene which is not normally detectable in haemopoetic cell~.~~
Abnormalities of Chromosomes 5 and 7 Much of the data relating to -5/de1(5q) and -7/de1(7q) is obtained from cases of secondary or therapy related MDS and AML with abnormalities found in up to 80% of patients.2G28 In the majority of studies of de novo AML patients with abnormalities of chromosomes 5 and/or 7 whether deletions, translocations or monosomies tend to be pooled for analysis of data. lo Patients with these abnormalities tend to be older and do not respond to chemotherapy. They often fail to achieve CR and therefore have a poor prognosis. In de novo AML abnormalities of 5 and/or 7 are common2g,30 but neither are specifically associated with a single FAB type. However, a recent study by Olopade et a13’ suggests that there is a subgroup of
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CYTOGENETICS IN ACUTE MYELOID LEUKAEMIA
M6 with complex karyotypes, including non random abnormalities of chromosomes 5 and 7. The important band in de novo AML is thought to be 5q31. It is suggested that there is a recessive oncogene or ‘antileukaemia gene’ within the deleted band.31 However, within this band there are a number of candidate genes such as granulocytemacrophage clonony stimulating factor (GM-CSF), interleukin 3 (IL-3), IL-4, IL-5 and others. Recent studies by Wilhnan et a13’ suggest that the critical tumour-suppressor gene may be IRF-1 located at 5q31.1, amidst to the haemopoietic growth factor genes. In the 5q- syndrome of refactory anaemia the important region appears to be larger spanning bands q22-q33.31 More work needs to be done on both de novo AML and 5q- syndrome patients to define whether the gene of interest is common to the two diseases.
The translocation t(6;9)(p23;q34) is a relatively rare finding confined to patients with AML or refactory anaemia with excess of blasts (RAEB).33 It is however of interest because of its association with a specific group of patients and more recently due to the identification of the genes involved. The t( 6;9) was first described in 1976 by Rowley et a134and to date 54 patients with this abnormality have been reported. 35 It is associated with bone marrow basophilia, 36 although it has been suggested that this is related to an underlying MDS3’ Patients with this sub-group are usually young (median age 30 years) and have a poor prognosis.36 The translocation t(6;9) is associated with FAB types Ml or M4 (90%) although there have been several reports of patients with M2 or MDS. Additional abnormalities at diagnosis are rare but have been reported during disease progression and relapse.37 The genes which are involved in the t(6;9) translocation are dek at 6~23 and can at 9q3438*3g and the translocation can be identified using Southern blotting and polymerase chain reaction (PCR) techniques. 33 At the molecular level the translocation leads to the fusion of the can gene on chromosome 9q with the dek gene on chromosome 6p resulting in a chimaeric dek-can gene on the derived chromosome 6p [der (6p)]. This fusion gene is consistently transcribed into a leukaemia specific 5.5 Kb mRNA. It has been suggested that dek-can may be involved in transcriptional events altering the activity or abundance of certain transcriptional factors leading to inappropriate cell growth and differentiation4’
erythrophagocytosis41 a finding since seen in the majority of reported cases. The prognosis is poor. The breakpoint on chromosome 8 at band 8~11 involved in this translocation is the location of the spherocytosis (sph) gene.42 t(8;21) (q22;q22) The t (8;2 1) (q22;q22) translocation was tist identified in 1973.43 Over 90% of patients with t(8;21) have a diagnosis of M2, while approximately 40% of patients with M2 have a t( 8;21).’ A proportion of these cases have reactive eosinophilia. The prognostic significance of the translocation is debatable as patients have a good CR rate but it is generally believed that the overall survival is no better than for AML as a whole. In most series there is a predominance of younger patients. Cytogenetically over 50% of patients with t(8;21) have additional changes, the majority losing a sex chromosome. In a French collabarative study of 146 patients with t (8;21), 44 loss of Y was observed in 61% of males and loss of X in 41% of females. In adults with t(8;21) there is a male predominance which has not been observed in children. Other cytogenetic changes include + 8, del(9q) and deletions or translocations of 7q. No prognostic difference has been found between patients with additional changes and those without. The translocation at the molecular level has recently been described with the genes involved being eto on chr 8 and All on chr 21. The translocation results in the fusion of these genes on the der(8) resulting in a novel chimaeric gene and message. The translocation can be detected using PCR or Southern blotting using AMLl probes.45*46 t(9;22)(q34;qll) The Philadelphia translocation t (9;22) (q34;q 11) (Ph) is rarely found in AML, but is usually associated with FAB type M2. 47 The prognosis of Ph+ve AML is generally regarded as poor, though it can transform to chronic phase chronic granulocyte leukaemia (CGL).13 Cytogenetically the Philadelphia chromosome appears identical to that seen in CGL and Ph +ve acute lymphoblastic leukaemia (ALL), but at the molecular level the chimaeric bcr/abl gene may be different from that seen in CGL. However, the 190 kD c-ABL protein as seen in Ph+ve ALL is expressed.48 Abnormalities of Chromosome 11
t(8;M) (pll;p13) This rare translocation has been reported in M4 and M5 in both adults and children.’ It was first described in 1983 in association with a marked marrow
Abnormalities of chromosome 11 are deletions or translocations, involving band q23, the latter occuring with a variety of partner chromosomes. Common including translocations t(6;ll)(q26;q23), t(9;11)(p21;q23) and t(11;19)(q23;p13) are found in
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M4 and M5.4g Abnormalities of llq23 are found in therapy related leukaemias, particularly in patients treated with VP16, epipodophyllotoxin. These patients commonly have AML with monocytic or myelomonocytic features. 5o Adults presenting with any rearrangement of chromosome llq23 have a poor CR and poor survival.‘3 Children with t( 1l;V) (q23;V) are reported as having a poor prognosis with early relapse occuring, whereas children with t( 9; 11), achieving CR, are reported as having a disease free survival of 2 years.51 Recent molecular studies have identified a human homologue of the Drosophila trithorax (trx) gene, which is a developmental regulator and is structurally altered in translocations in acute leukaemia with an abnormality at band llq23. The human trithorax gene (hrx) is interrupted by the breakpoint involved in the translocation on chromosome llq.52 As there is also a transcription unit or gene designated myeloid/lymphoid leukaemia (MLL)53 at 1lq23, it is hypothesised that the rearrangement within this band may affect an early progenitor cell capable of differentiation along both myeloid and lymphoid pathways.54 t(15;17)(q21;q21)
AML M3 and M3v is a good example of a disease with a specific chromosome abnormality, t(15;17)(q21;q21), Ilrst described by Rowley in 1977.55 The classic presenting feature of M3, is the coagulopathy which can cause early death. If this is controlled patients can achieve CR and a large percentage demonstrate a 5 year disease free surviva1.56 The t( 15;17) has not been described in any other malignancy and is thought to occur in all cases of M3, sometimes as a variant translocation.57 A feature of the translocation is that it is not detected in direct preparations and cells need to be cultured. The molecular basis of the t( 15; 17) is the fusion of the retinoic acid receptor rara gene on chromosome 17 with the pm1 gene on chromosome 15 forming a chimaeric pmllrara gene. 58 This rearrangement can be detected by Southern blotting, PCR and FISH. These methods can be used to assist diagnosis, where the cytogenetics is inadequate, to monitor the disease and predict relapse.5g The treatment of M3 with all-trans retinoic acid (ATRA) is based on the premise that the promyelocytes are induced to differentiate and die rather than to proliferate, and was first reported from China in 1986.60 Several large clinical trials have been undertaken to assess the efficacy of ATRA, alone or in combination with standard chemotheraphy.61 inv(16)(p13q22)
AML with inversion of chromosome 16 [inv( 16)] is characteristic of M4Eo. In a number of reported series with inv( 16) up to 90% of patients have a
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diagnosis of M4Eo. 62,63Other patients are described as having classical morphologically abnormal eosinophils but do not have M4 morphology.64 There is general agreement that inv(16) has a good CR rate65,51 although the long term outlook may be less clear. In most cases the inv( 16) occurs as the sole abnormality but when secondary changes are present they are frequently either + 8, +22 or del(7q). The molecular basis of the inversion is still unclear, but recently it has been demonstrated that the inversion can be detected by in-situ hybridisation using probes specific for sequences on the p arm.@j In some studies del( 16)(q22) has been included with inv( 16) but this is probably erroneous.67 Patients may share similar clinical features, but del( 16) is less common and more often characterised by a high incidence of MDS, older age and complex karyotypes. Secondary AML Secondary AML (sAML) is a general term defined here as AML resulting from treatment with cytotoxic drugs and/or radiotherapy, or from exposure to environmental toxic agents. Cytogenetically the abnormality rate is higher than in de novo AML ranging from 75 to 90% and is often characterised by quite distinct abnormalities.68*6g*70There are over 500 cases reported in the literature with fewer than half having a single cytogenetic abnormality. The most common abnormalities are - 5/de1(5q) and - 7/del( 7q) which are often present in the same clone. Other common cytogenetic changes are + 8, -17, -18, $21, -21 and abnormalities of 3q, 6p, 1lq, 12p, 17p and 21q. Complex and hypodiploid karyotypes are a common feature. The typical abnormalities seen in de novo AML are rare. Cytogenetically sAML is very similar to secondary MDS (sMDS), although del(5q) is more common in sMDS whereas abn( 3q) and abn( 1lq) are more common in sAML. Interestingly +8 occurs less frequently in sAML than in de novo AML. There is some correlation between cytogenetic abnormalities in sAML and the type of exposure. Monosomy 7 is more common in patients exposed to chemotherapy, particularly cytostatics. Del( 5q) is associated with exposure to ionizing radiation but appears to be restricted to sMDS. Etoposide induced sAML has a high incidence of 1lq23 abnormalities. Where exposure to pesticides and organic solvents is documented abnormalities of 17p, 16q22 and + 1lq are common.71 The presence of a 17p abnormality has led to the suggestion that ~53, the protein product of a gene located on 17p, is involved in leukaemogenesis. The prognosis of sAML patients is poor, as CR may not be achieved or has a short duration. However, it has been proposed that the abnormalities t(8;21), t( 15;17) and inv( 16), when present in sAML confer a prognosis similar to that observed in de novo AML.” Two groups of patients who share the clinical and
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CYTOGENETICS IN ACUTE MYELOID LEUKAEMIA
biological features of sAML are those who develop AML after de novo MDS73 and a sub-group of patients who have AML M6.30 Patients with apparently de novo AML who have cytogenetic findings more typical of sAML have a poor prognosis and do as poorly as patients with genuine sAML. Therefore the question is asked, are they the same disease and should more effort be made to establish whether exposure to environmental or toxic agents has occured. Relapse A feature of AML is the disappearance of chromosome abnormalities in complete remission and their reappearance in relapse. The changes seen at relapse can generally be classified into three categories, relapse with the same karyotype as at diagnosis, relapse with a more complex karyotype, relapse with a karyotype either normal or less complicated.74 The significance of the appearance of an abnormal clone in a patient at relapse who was apparently cytogenetically normal at diagnosis is still open to debate. This may be explained by a failure of detection at diagnosis, especially in cases with a t (15;17) or inv( 16) or the presence of a subclone derived from a common ‘cytogenetically normal’ progenitor ce11.75 Both numerical and structural changes are seen at relapse. Trisomy of either chromosome 8 or 21 is the most common numerical change. Structural changes and multiple subclonal changes are seen more commonly in relapse and can be more complex, possibly due to treatment. Relapse with chromosome abnormalities associated with sAML such as del(5q) and del(7q) are again associated with a poor prognosis. A number of studies have compared the survival of patients with and without karyotypic changes at relapse. Testa et al”j reported a shorter survival rate whereas a number of other series disagree.” Other groups report no statistical significance.78~75To clarify the situation further large coordinated studies are needed. The Future It is some 10 years since an already well defmed cytogenetic abnormality was found to be characterised by a molecular rearrangement. This was the Philadelphia translocation t (9;22) (q34;qll) which at the molecular level is the fusion of the c-abl oncogene located on chromosome 9q to the bcr on chromosome 22q, to form the chimaeric gene bcrfabl. During these 10 years cytogeneticists and molecular geneticists have worked together and localization of genes at or near breakpoints in chromosomes involved in haematological malignancies have led to the analysis of these consistent chromosome rearrangements. Indeed, several translocations such as the t (6;9), t (8;2 1) and
t ( 15;17) have associated aberrant chimaeric protein products associated with them. As more reports of apparently rare but consistent chromosome translocations with specific clinical associations are identified, such as the t(1;22)(p13;q13) seen in M7,” there is a potential for identifying new genes at these breakpoints and determining whether their altered function has a role in leukaemogenesis. Cytogenetics will continue to play a major role in the study of AML, as it is clear that cytogenetics can provide an independant prognostic indicator. In the future, specific treatment regimes could be tailored for treatment of patients with common abnormalities such as t( 8;21) and inv( 16) and currently rare abnormalities may emerge to be associated with specific phenotypes and prognosis and will be treated accordingly. New techniques such as PCR and in-situ hybridisation will have an increasing importance especially with respect to the detection of minimal residual disease. However, the most cost effective and accurate answer to the use of cytogenetics in the diagnosis of AML, will be the use of traditional methods at presentation in conjunction with PCR and in-situ hybridization. This combination of cytogenetics and molecular genetics at diagnosis will give a broader picture of the disease allowing one or more of these methods to be used subsequently in disease monitoring.
References 1. Leukaemia and lymphoma. Leukaemia Research Fund: Data collection study 1984-1988. London: Leukaemia Research Fund, 1990. 2. Bemiett J M, Catovsky D, Daniel M T, et al. Proposals for the classification of the acute leukaemias. Br J Haematol 1976; 33: 451-458. 3. Blood. Textbook of haematology. J H Jandl, ed. Boston: Little Brown & Co, 1987. 4. Borrow J, Solomon E. Molecular analysis of the t( 15;17) translocation in acute promyelocytic leukaemia. Balliere’s Clin Haematol 1992; 5: 833-856. 5. Rowley J D. Recurring chromosome abnormalities in leukemia and lymphoma. Semin Haematol1990; 27: 122-136. 6. Yunis J J. Recurrent chromosomal defects are found in most patients with acute nonlymphocytic leukemia. Cancer Genet Cytogenet 1984; 11: 125-137. I. Sandberg A A. The chromosomes in human cancer and leukemia. 2nd ed. New York: Elsevier, 1990. 8. Mitelman F. Catalog of chromosome aberrations in cancer. 4th ed. New York Wiley-Liss, 1991. 9. Berger R, Le Coniat M, Derre J, Vecchione D, Jonveaux P. Cytogenetic studies in acute promyelocytic leukemia: a survey of secondary abnormalities. Genes Chromosom Cancer 1991; 3: 332-337. 10. Schiffer C A, Lee E J, Tomiyasu T, Wiernik P H, Testa J R. Prognostic impact of cytogenetic abnormalities in patients with de novo acute nonlymphocytic leukemia. Blood 1989; 73: 263-270. 11. Keating M J, Smith T L, Kantajian H, et al. Cytogenetic pattern in acute myelogenous leukemia: A major reproducible determinant of outcome. Leukemia 1988; 2: 403412. 12. Berger R. Bemheim A. Ochoa-Noauera M E, et al. Prognostic sigr&ance of chromosomal abnormalities in acute nonlymphocytic leukemia a study of 343 patients. Cancer Genet Cytogenet 1987; 28: 293-299.
BLOOD REVIEWS 13. Stasi R, Poeta G, Masi M, et al. Incidence of chromosome abnormalities and clinical significance of karyotype in de novo acute myeloid leukemia. Cancer Genet Cytogenet 1993; 67: 28-34. 14. Jenkins R B, Le Beau M M, Kraker W J, et al. Fluorescence in situ hybridization: A sensitive method for trisomy 8 detection in bone marrow specimens. Blood 1992; 79: 3307-3315. 15. Kibbelaar R E, Muldar J, Dreet E J, et al. Detection of monosmy 7 and trisomy 8 in myeloid neoplasia: A comparison of banding and fluorescence in situ hybridization. Blood 1993; 82: 904913. 16. Weber E, Nowotny H, Haas 0 A, et al. Trisomy 4: A specific karyotype anomaly in primary and secondary acute myeloid leukemia. Leukemia 1990; 4: 219-221. 17. Ribeiro E M S F B, Cavali I J, Tokutake A S. Trisomy 4 in acute nonlymphocytic leukemia: The first Brazilian case. Cancer Genet Cytogenet 1993; 68: 82-83. 18. United Kingdom Cancer Cytogenetics Group (UKCCG). Primary, single, autosomal trisomies associated with haematological disorders. Leukemia Res 1992; 16: 841-851. 19. Avanzi G C, Giovinazzo B, Rosso A, et al. Trisomy 11 in myelodysplastic syndrome-derived acute myeloblastic leukaemias. Eur J Haematoll989; 43: 173-177. 20. Sreekantaiah C, Baer M R, Morgan S, et al. Trisomy/tetrasomy 13 in seven cases of acute leukemia. Leukemia 1990; 4: 781-785. 21. Baer M R, Bloomfield C D. Trisomy 13 in acute leukemia. Leuk Lymphoma 1992; 7: l-6. 22. Cuneo A, Michaux J-L, Ferrant A, et al. Correlation of cytogenetic patterns and clinicobiological features in adult myeloid leukemia expressing lymphoid markers. Blood 1992; 79: 720-726. 23. Najfeld V, Seremetis S, Troy K, et al. Trisomy 22-A new abnormality found in acute leukemia characterized by eosinophilia and monocytoid blasts expressing immature differentiation antigens. Cancer Genet Cytogenet 1986; 23: 105-l 14. 24. Bitter M A, Leilly M E, Le Beau M M, Pearson M G, Rowley J D. Rearrangements of chromosome 3 involving bands 3q21 and 3q26 are associated with normal or elevated platelet counts in acute nonlymphocytic leukemia. Blood 1985; 66: 1362-1370. 25. Fichelson S, Dreyfus F, Berger R, et al. EVI-1 expression in leukemic patients with rearrangements of the 3q25-q28 chromosomal region. Leukemia 1992; 6: 93-99. 26. Rowley J D, Golomb H M, Vardiman J W. Nonrandom chromosome abnormalities in acute leukemia and dysmyelopoietic syndromes in patients with previously treated malignant disease. Blood 1981; 58: 759-767. 27. Le Beau M M, Albain K S, Larson R A, et al. Clinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia: Further evidence for characteristic abnormalities of chromosomes no 5 and 7. J Clin Oncoll986; 4: 3255345. 28. Neuman W L, Rubin C M, Rios R B, et al. Chromosomal loss and deletion are the most common mechanisms for loss of heterozygosity from chromosomes 5 and 7 in malignant myeloid disorders. Blood 1992; 79: 1501-1510. 29. Fourth International Workshop on Chromosomes in Leukemia 1982 1984 Cancer Genet Cytogenet 11: 249-360. 30. Olopade 0 I, Thangavelu M T, Larson R A, et al. Clinical, morphologic, and cytogenetic characteristics of 26 patients with acute erythroblastic leukemia. Blood 1992; 11: 2873-2882. 31. Westbrook C A, Keinanen M J. Myeloid malignancies and chromosome 5 deletions. Bailliere’s Clin Haematol 1992; 5: 931-942. 32. Willman C L, Sever C E, Pallavicim M G et al. Deletion of IRF-1, mapping to chromosome 5q31, in human leukemia and preleukemic myelodysplasia. Science 1993; 259: 968-971. 33. Soekarman D, von Lindern M, Daenen S, et al. The translocation (6;9)(p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features. Blood 1992; 79: 2990-2997. 34. Rowley J D, Potter A. Chromosomal banding patterns in acute nonlymphocytic leukemia. Blood 1976; 47: 705-721. 35. Lillington D M, MacCullum P H, Lister A T, Gibbons B. Translocation t(6;9)(p23;q34) in acute myeloid leukemia without myelodysplasia or basophilia: two cases and a review of the literature. Leukemia 1993; 7: 527-531.
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36. Pearson M G, Vardiman J W, Le Beau M M, et al. Increased numbers of marrow basophils may be associated with a t(6;9) in ANLL. Am J Haematoll985; 18: 393-403. 37. Berger R, Daniel M-T, Jonveaux P, Baruchel A. Relapse as acute monoblastic leukemia (AML-MS) of t(6;9) acute myeloblastic leukemia (AML-M2). Leukemia 1992; 6: 1227-1228. 38. von Lindem M, Poustka A, Lerach H, Grosfeld G. The t(6;9) translocation associated with a specific subtype of acute nonlymphocytic leukemia leads to aberrant transcription of a target gene on 9q34. Mol Cell Biol 1990; 10: 4016-4026. 39. von Lindem M, Fornerod M, van Baa1 S, et al. The translocation t(6;9) associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimaeric leukemia specific dekcan mRNA. Mol Cell Bioll992; 12: 1687-1697. 40. Nichols J, Nimer S D. Transcription factors, translocations, and leukemia. Blood 1992; 80: 2953-2963. 41. Schouten T J, Hustinx T W J, Scheres J M J C, Holland R, de Vaan G A M. Malignant histiocytosis. Clinical and cytogenetic studies in a newborn and a child. Cancer 1983; 52: 1229-1236. 42. Cohen H, Walker H, Delhanty J D A, Lucas S B, Huehns E R. Congenital spherocytosis, B19 parvovirus infection and inherited interstitial deletion of the short arm of chromosome 8. Br J Haematol 1991; 78: 251-257. 43. Rowley J D. Identification of a translocation with quinacrine mustard fluorescence in a patient with acute leukemia. Ann Genet 1973: 16: 109-l 12. 44. Groupe Francais de Cytogenetique Haematologique. Acute myelogenous leukemia with an 8;21 translocation. Cancer Genet Cytogenet 1990; 44: 169-179. 45. Downing J R, Head D R, Curcio-Brint A M. An AMLl/ETO fusion transcript is consistently detected by RNA-based polymerase chain reaction in acute myelogenous leukemia containing the t(8:21 )(q22;q22) translocation. Blood 1993; 81: 2860-2865. 46. Maseki N, Miyoshi H. Shimizu K, et al. The 8;21 chromosome translocation in acute myeloid leukemia is always detectable by molecular analysis using AMLI. Blood 1993; 8 1: 1573-1579. 47. Hirsch-Ginsberg C, Childs C, Chang K-S, et al. Phenotypic and molecular heterogeneity in Philadelphia chromosomepositive acute leukemia. Blood 1988; 71: 186-195. 48. Kursrock R, Shtalrid M, Talpaz M, Kloetzer W S, Gutterman J V. Expression of c-abl in Philadelphia -positive acute myelogenous leukemia. Blood 1987; 70: 1584-1588. 49. Raimondi S C, Kalwinsky D K, Hayashi Y, et al. Cytogenetics of childhood acute nonlymphocytic leukemia. Cancer Genet Cytogenet 1989; 30: 13-27. 50. Whitlock J A, Greer J P, Lukens J N. Epipodophyllotoxinrelated leukemia. Identification of a new subset of secondary leukemia. Cancer 1991; 68: 600-604. 51. Kalwinsky D K, Raimondi S C, Schell M J, et al. Prognostic importance of cytogenetic subgroups in de novo pediatric acute nonlymphocytic leukemia. J Clin Oncol 1990; 8: 75-83. 52. Tkachuk D C, Kohler S, Cleary M L. Involvement of a homolog of Drosophila Trithorax by 1lq23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691-700. 53. Ziemin-van der Poe1 S, McCabe N R, Gill H J, et al. Identification of a gene, MLL, that spans the breakpoint in 1lq23 translocations associated with human leukemias. Proc Nat Acad Sci USA 1991; 88: 10735510739. 54. Djabali M, Selleri L, Perry P. A trithorax-like gene is interrupted by chromosome 1lq23 translocations in acute leukaemias. Nature Genet 2: 113-l 18. 55 Rowley J D, Golomb H M, Vardiman J, et al. Further evidence for a non-random chromosomal abnormality in acute promyelocytic leukemia. Int J Cancer 1977; 20: 869-872. 56. Stone R M, Mayer R J. The unique aspects of acute promyelocytic leukemia. J Clin Oncol 1990: 8: 1913-1921 57. Larson R A, Kondo K, Vardiman J W, et al. Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia. Am J Med 1984; 76: 827-841. 58. Borrow J, Goddard A D. Gibbons B, et al. Diagnosis of acute promyelocytic leukaemia by RT-PCR: detection of PML/RARA and RARAPML fusion transcripts. Br J Haematol 1992; 82: 529-540. 59. Biondi A, Rambaldi A. Alcalay M, et al. RAR 1 rearrangements as a genetic marker for diagnosis and
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60.
61. 62.
63.
64.
65.
66.
67.
68.
69.
CYTOGENETICS IN ACUTE MYELOID LEUKAEMIA monitoring in acute promyelocytic leukemia. Blood 1991; 77: 1418-1422. Huang M E, Ye Y C, Chen S R, et al. All tram-retinoic acid with or without low dose cytosine arabinoside in acute promyelocytic leukemia: report of six cases. Chinese Med J (English) 1987; 100: 949-953. Warrel R P, de The H, Wang Z-Y, Dagos L. Acute promyelocytic leukemia. New Engl J Med 1993; 329: 177-189. Larson R A, Williams S F, Le Beau M M, et al. Acute myelomonocytic leukemia with abnormal eosinophils and inv( 16) or t( 16;16) has a favorable prognosis. Blood 1986; 68: 1242-1249. Campbell L J, Challis J, Fok T, Garson 0 M. Chromosome 16 abnormalities associated with myeloid malignancies. Genes Chromosom Cancer 1991; 3: 55-61. Betts D R, Rohatiner A Z, Evans M L, et al. Abnormalities of chromosome 16q in myeloid malignancy: 14 new cases and a review of the literature. Leukemia 1992; 6: 1250-1256. Bernard P, Dachary D, Reiffers J, et al. Acute nonlymphocytic leukemia with marrow eosinophilia and chromosome 16 abnormality: a report of 18 cases. Leukemia 1989; 3: 740-745. Dauwerse J G, Kievits T, Beverstock G C, et al. Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16~. Cytogenet Cell Genet 1990; 53: 126-128. Ohyashiki K, Ohyashiki J H, Kondo M, Ito H, Toyama K. Chromosome change at 16q22 in nonlymphocytic leukemia: clinical implication on leukemia patients with inv( 16) versus del( 16). Leukemia 1988; 2: 35-40. Mitehnan F, Nilsson P G, Brandt L, et al. Chromosome pattern, occupation, and clinical features in patients with acute nonlymphocytic leukemia. Cancer Genet Cytogenet 1981; 4: 197-214. Johansson B, Mertens F, Heim S, Kristoffersson V, Mitelman F. Cytogenetics of secondary myelodysplasia
70.
71.
72.
73.
74. 75.
76.
77.
78.
79.
(sMDS) and acute nonlymphocytic leukemia (sANLL). Eur J Haematol 1991; 47: 17-27. Pedersen-Bjergaard J, Philip P, Pedersen N T, et al. Acute nonlymphocytic leukemia, preleukemia, and acute myeloproliferative syndrome secondary to treatment of other malignant disease. Cancer 1984; 54: 4522462. Cuneo A, Fagioli F, Pazzi I, et al. Morphologic, immunologic and cytogenetic studies in acute myeloid leukemia following occupational exposure to pesticides and organic solvents. Leuk Res 1992; 16: 789-796. Fenaux P, Lai J L, Quiquandon I, et al. Therapy related myelodysplastic syndrome and leukemia with no ‘unfavourable’ cytogenetic findings have a good response to intensive chemotherapy: a report on 15 cases. Leuk Lymphoma 1991; 5: 117-125. Weh H J, Kuse R, Seeger D, Suciu S, Hossfeld D K. Cytogenetic studies in patients with acute myeloid leukemia following a myelodysplastic syndrome. Leuk Lymphoma 1991; 3: 423429. Sixth International Workshop on chromosomes in leukemia, 1987. Cancer Genet Cytogenet 1989; 40: 187-202. Shikano T, Ishikawa Y, Ohkawa M, et al. Karyotypic changes from initial diagnosis to relapse in childhood acute leukemia. Leukemia 1990; 4: 419-422. Testa J R, Mintz U, Rowley J D, Vardiian J W, Golomb H M. Evolution of karyotypes in acute nonlymphocytic leukemia. Cancer Res 1979; 39: 3619-3627. Morris C M, Fitzgerald P H. Karyotypic evolution in patients with acute myeloid leukemia. Cancer Genet Cytogenet 1984; 11: 143-152. Berger R, Le Coniat M, Derre J, et al. Cytogenetic studies on acute nonlymphocytic leukemia in relapse. Cancer Genet Cytogenet 34: 11-18. Lu G, Altman A J, Benn P A. Review of the cytogenetic changes in acute megakaryoblastic leukemia. One disease or several? Cancer Genet Cytogenet 1993; 67: 81-89.
H. Walker, F. J. Smith, D. R. Betts S UMMA R Y. A wealth of literature spanning 20 years describing cytogenetic abnormalities in acute myeloid leukaemia ( AML) already exists. It ranges from single case reports of unusual abnormalities to large multicentre studies of lumdredsof cases. A landmark publication was the Fourth International Workshop on Chromosomes in Acute Leukaemia which established a base line for diagnosis, prognosis and frequency of chromosome abnormalities in AML. Two large sources of information are a book, ‘The Chromosomes in Human Cancer and Leukemia’ and a catalogue of chromosome abnormalities, which aims to list all chromosome abnormalities described in the scientific and medical literature from 1973, when the widespread use of banding techniques, enabled the precise definition of the chromosome breakpoints. In this review the common cytogenetic abnormalities seen in AML with reference to associations with the French-American-British (FAB) classification, their possible prognostic signiscance and their associated molecular biology are summarized.
In England and Wales, acute myeloid leukaemia (AML) accounts for 77% of acute leukaemias and the incidence is 3.4 per 100000 of population. In children age 0 to 15 years AML accounts for only 19% of acute leukaemia while, in adults age 16 to 79 years AML accounts for 89% of acute 1eukaemia.l The classification of AML, is based on the clinical presentation, cellular morphology, cytochemistry and immunology.’ The French-American-British (FAB) type serves as an important basis for classification and in some cases prognosis and therapy of the individual leukaemias and can act as a pointer to the likely chromosome abnormality which may be found. The frequency of the observation of chromosome abnormalities is related to the occurence of the FAB types. The incidence of the different FAB types is reported as Ml 18%, M2 28%, M3 8%, M4 27%, M5 lo%, M6 4% and M7 5°h.3 Some chromosome abnormalities are specific to certain FAB types and H. Walker, F. J. Smith, D. R. Retts, Department of Haematology, University College Hospital, Gower St. London WClE 6AU, UK. Correspondence to: Mrs H. Walker. Blood Reviews (1994) 8, 0 1994 GroupUK
indeed the t( 15; 17) seen in FAB type M3 has not been reported in any other malignancy.4 Cytogenetic abnormalities seen in AML are numerical or structural and may occur alone or together. Single abnormalities are described as primary in nature and are thought to be related to a causal event with the secondary abnormalities occuring as associations in some cases. The frequency of chromosome abnormalities detected in AML at diagnosis, is variously reported as between 60 to 90%5 but is dependant on factors such as length of time in culture, banding techniques and referral patterns of reporting centres.6 Some leukaemic cells may not have an abnormal karyotype. The most frequently observed numerical abnormalities in all FAB types of AML are trisomy 8 and monosomy 7. ‘,* Rarely observed are trisomies of chromosomes 4, 11, 13, 21 and 22. There are a number of common specific chromosome rearrangements which include translocations, inversions and deletions namely t (8;2 1) (q22;q22), t ( 15; 17) (q22;q2 1) and inv( 16)(pl3q22), which are associated with
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particular FAB types. Less commonly seen are 1lq23 and chromosome abnormalities of t (6;9) (p23;q34). Rarely seen, but nevertheless reported in a few dozen cases each, are abnormalities of chromosome 3 involving bands q21-q26, t(8;16)(pll;p13) and t(9;22)(q34;qll). Chromosome abnormalities seen at diagnosis in AML are not detectable cytogenetically when complete remission (CR) is achieved, but can reappear at relapse, sometimes with additional changes or even with a different abnormality. (personal observations)
Numerical Abnormalities Trisomy 8 Trisomy 8 is the most common chromosome abnormality seen in AML,(j occuring as either the sole aberration or as an additional change. In M3 approximately 17% of patients at diagnosis are found to have +8 in conjunction with the t(15;17).’ Trisomy 8 is not associated with a specific FAB type but is found most frequently in Ml, M4 and M5. A finding of +8 as the sole abnormality is generally associated with a poor or intermediate prognosis”*” but, there are studies where a relatively high CR is achieved.12,‘3 Fluorescence in situ-hybridization (FISH) techniques can be applied to detect +8.14 This has its benefits and may prove to be more sensitive in samples where the +8 is cytogenetically nonclonal, but it does not detect additional abnormalities and in good preparations is no more sensitive.”
Non Random Trisomies In addition to trisomy 8, trisomies of chromosomes 4, 11, 13, 21 and 22 occur as non-random sole abnormalities in AML. They are relatively rare events, with each trisomy accounting for between 25 to 60 cases. Trisomy 4 has been described in both de novo and secondary AML”j with a significant proportion of cases occuring in M4 and M2.17,18 As yet the prognostic significance is unclear but it is possibly poor. Additional common chromosome changes seen with trisomy 4 include double minutes and other trisomies. Trisomy 4 has only recently been reported which has led to speculation that there may be influencing factors including geographical and unknown environmental factors7 but at present there is no evidence to support either of these theories. Trisomy 11 in AML appears to be associated with previous myelodysplastic syndrome (MDS), old age and karyotypic evolution is a feature. The MDS is often secondary in nature. The prognosis is poor but it is associated with poor prognostic factors.‘**” Trisomy 13 has the most distinct phenotype of these rare non-random trisomies, it may also occur
31
as tetrasomy. The majority of patients are elderly males, there is a low CR rate and if remission is obtained it is usually brief.20*21 The AML is often undifferentiated or biphenotypic, expressing lymphoid as well as myeloid markers.” This has led to the suggestion that the leukaemia arises from an early stem cell which retains the potential for both myeloid and lymphoid differentiation. It is unclear why trisomy 13 should have this effect but Cuneo et al” have suggested that 13q13 may be the critical region. Trisomy 21 occurs in a number of neoplasia and is often seen in secondary AML. When it occurs as the sole abnormality there are no specific associations.7 Trisomy 22 has no known prognostic significance. It is associated with M4, often with eosinophiliaz3 and may arise as a secondary event to inv( 16). However, there have been reports where it is the sole abnormality with eosinophilia, which raises the question whether an inv( 16) is present at the molecular level. There is a suggestion that it may be associated with a younger age and the leukaemia may be characterised by the expression of immature differentiation antigens. It is not totally appropriate to group these trisomies together but they are generally associated with undifferentiated or secondary AML.
Structural Abnormalities Abnormalities of Chromosome 3 Though rare, abnormalities of chromosome 3 are interesting as they have a marked clinical association with platelet abnormalities with micromegakaryocytes in the marrow and thrombocytosis.24 The specific cytogenetic abnormalities associated with thrombocytosis involve bands 3q21 and 3q26 either as inversions, translocations or insertions. Abnormalities of chromosome 3q are associated with a high expression of the Evi-1 gene which is not normally detectable in haemopoetic cell~.~~
Abnormalities of Chromosomes 5 and 7 Much of the data relating to -5/de1(5q) and -7/de1(7q) is obtained from cases of secondary or therapy related MDS and AML with abnormalities found in up to 80% of patients.2G28 In the majority of studies of de novo AML patients with abnormalities of chromosomes 5 and/or 7 whether deletions, translocations or monosomies tend to be pooled for analysis of data. lo Patients with these abnormalities tend to be older and do not respond to chemotherapy. They often fail to achieve CR and therefore have a poor prognosis. In de novo AML abnormalities of 5 and/or 7 are common2g,30 but neither are specifically associated with a single FAB type. However, a recent study by Olopade et a13’ suggests that there is a subgroup of
32
CYTOGENETICS IN ACUTE MYELOID LEUKAEMIA
M6 with complex karyotypes, including non random abnormalities of chromosomes 5 and 7. The important band in de novo AML is thought to be 5q31. It is suggested that there is a recessive oncogene or ‘antileukaemia gene’ within the deleted band.31 However, within this band there are a number of candidate genes such as granulocytemacrophage clonony stimulating factor (GM-CSF), interleukin 3 (IL-3), IL-4, IL-5 and others. Recent studies by Wilhnan et a13’ suggest that the critical tumour-suppressor gene may be IRF-1 located at 5q31.1, amidst to the haemopoietic growth factor genes. In the 5q- syndrome of refactory anaemia the important region appears to be larger spanning bands q22-q33.31 More work needs to be done on both de novo AML and 5q- syndrome patients to define whether the gene of interest is common to the two diseases.
The translocation t(6;9)(p23;q34) is a relatively rare finding confined to patients with AML or refactory anaemia with excess of blasts (RAEB).33 It is however of interest because of its association with a specific group of patients and more recently due to the identification of the genes involved. The t( 6;9) was first described in 1976 by Rowley et a134and to date 54 patients with this abnormality have been reported. 35 It is associated with bone marrow basophilia, 36 although it has been suggested that this is related to an underlying MDS3’ Patients with this sub-group are usually young (median age 30 years) and have a poor prognosis.36 The translocation t(6;9) is associated with FAB types Ml or M4 (90%) although there have been several reports of patients with M2 or MDS. Additional abnormalities at diagnosis are rare but have been reported during disease progression and relapse.37 The genes which are involved in the t(6;9) translocation are dek at 6~23 and can at 9q3438*3g and the translocation can be identified using Southern blotting and polymerase chain reaction (PCR) techniques. 33 At the molecular level the translocation leads to the fusion of the can gene on chromosome 9q with the dek gene on chromosome 6p resulting in a chimaeric dek-can gene on the derived chromosome 6p [der (6p)]. This fusion gene is consistently transcribed into a leukaemia specific 5.5 Kb mRNA. It has been suggested that dek-can may be involved in transcriptional events altering the activity or abundance of certain transcriptional factors leading to inappropriate cell growth and differentiation4’
erythrophagocytosis41 a finding since seen in the majority of reported cases. The prognosis is poor. The breakpoint on chromosome 8 at band 8~11 involved in this translocation is the location of the spherocytosis (sph) gene.42 t(8;21) (q22;q22) The t (8;2 1) (q22;q22) translocation was tist identified in 1973.43 Over 90% of patients with t(8;21) have a diagnosis of M2, while approximately 40% of patients with M2 have a t( 8;21).’ A proportion of these cases have reactive eosinophilia. The prognostic significance of the translocation is debatable as patients have a good CR rate but it is generally believed that the overall survival is no better than for AML as a whole. In most series there is a predominance of younger patients. Cytogenetically over 50% of patients with t(8;21) have additional changes, the majority losing a sex chromosome. In a French collabarative study of 146 patients with t (8;21), 44 loss of Y was observed in 61% of males and loss of X in 41% of females. In adults with t(8;21) there is a male predominance which has not been observed in children. Other cytogenetic changes include + 8, del(9q) and deletions or translocations of 7q. No prognostic difference has been found between patients with additional changes and those without. The translocation at the molecular level has recently been described with the genes involved being eto on chr 8 and All on chr 21. The translocation results in the fusion of these genes on the der(8) resulting in a novel chimaeric gene and message. The translocation can be detected using PCR or Southern blotting using AMLl probes.45*46 t(9;22)(q34;qll) The Philadelphia translocation t (9;22) (q34;q 11) (Ph) is rarely found in AML, but is usually associated with FAB type M2. 47 The prognosis of Ph+ve AML is generally regarded as poor, though it can transform to chronic phase chronic granulocyte leukaemia (CGL).13 Cytogenetically the Philadelphia chromosome appears identical to that seen in CGL and Ph +ve acute lymphoblastic leukaemia (ALL), but at the molecular level the chimaeric bcr/abl gene may be different from that seen in CGL. However, the 190 kD c-ABL protein as seen in Ph+ve ALL is expressed.48 Abnormalities of Chromosome 11
t(8;M) (pll;p13) This rare translocation has been reported in M4 and M5 in both adults and children.’ It was first described in 1983 in association with a marked marrow
Abnormalities of chromosome 11 are deletions or translocations, involving band q23, the latter occuring with a variety of partner chromosomes. Common including translocations t(6;ll)(q26;q23), t(9;11)(p21;q23) and t(11;19)(q23;p13) are found in
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M4 and M5.4g Abnormalities of llq23 are found in therapy related leukaemias, particularly in patients treated with VP16, epipodophyllotoxin. These patients commonly have AML with monocytic or myelomonocytic features. 5o Adults presenting with any rearrangement of chromosome llq23 have a poor CR and poor survival.‘3 Children with t( 1l;V) (q23;V) are reported as having a poor prognosis with early relapse occuring, whereas children with t( 9; 11), achieving CR, are reported as having a disease free survival of 2 years.51 Recent molecular studies have identified a human homologue of the Drosophila trithorax (trx) gene, which is a developmental regulator and is structurally altered in translocations in acute leukaemia with an abnormality at band llq23. The human trithorax gene (hrx) is interrupted by the breakpoint involved in the translocation on chromosome llq.52 As there is also a transcription unit or gene designated myeloid/lymphoid leukaemia (MLL)53 at 1lq23, it is hypothesised that the rearrangement within this band may affect an early progenitor cell capable of differentiation along both myeloid and lymphoid pathways.54 t(15;17)(q21;q21)
AML M3 and M3v is a good example of a disease with a specific chromosome abnormality, t(15;17)(q21;q21), Ilrst described by Rowley in 1977.55 The classic presenting feature of M3, is the coagulopathy which can cause early death. If this is controlled patients can achieve CR and a large percentage demonstrate a 5 year disease free surviva1.56 The t( 15;17) has not been described in any other malignancy and is thought to occur in all cases of M3, sometimes as a variant translocation.57 A feature of the translocation is that it is not detected in direct preparations and cells need to be cultured. The molecular basis of the t( 15; 17) is the fusion of the retinoic acid receptor rara gene on chromosome 17 with the pm1 gene on chromosome 15 forming a chimaeric pmllrara gene. 58 This rearrangement can be detected by Southern blotting, PCR and FISH. These methods can be used to assist diagnosis, where the cytogenetics is inadequate, to monitor the disease and predict relapse.5g The treatment of M3 with all-trans retinoic acid (ATRA) is based on the premise that the promyelocytes are induced to differentiate and die rather than to proliferate, and was first reported from China in 1986.60 Several large clinical trials have been undertaken to assess the efficacy of ATRA, alone or in combination with standard chemotheraphy.61 inv(16)(p13q22)
AML with inversion of chromosome 16 [inv( 16)] is characteristic of M4Eo. In a number of reported series with inv( 16) up to 90% of patients have a
33
diagnosis of M4Eo. 62,63Other patients are described as having classical morphologically abnormal eosinophils but do not have M4 morphology.64 There is general agreement that inv(16) has a good CR rate65,51 although the long term outlook may be less clear. In most cases the inv( 16) occurs as the sole abnormality but when secondary changes are present they are frequently either + 8, +22 or del(7q). The molecular basis of the inversion is still unclear, but recently it has been demonstrated that the inversion can be detected by in-situ hybridisation using probes specific for sequences on the p arm.@j In some studies del( 16)(q22) has been included with inv( 16) but this is probably erroneous.67 Patients may share similar clinical features, but del( 16) is less common and more often characterised by a high incidence of MDS, older age and complex karyotypes. Secondary AML Secondary AML (sAML) is a general term defined here as AML resulting from treatment with cytotoxic drugs and/or radiotherapy, or from exposure to environmental toxic agents. Cytogenetically the abnormality rate is higher than in de novo AML ranging from 75 to 90% and is often characterised by quite distinct abnormalities.68*6g*70There are over 500 cases reported in the literature with fewer than half having a single cytogenetic abnormality. The most common abnormalities are - 5/de1(5q) and - 7/del( 7q) which are often present in the same clone. Other common cytogenetic changes are + 8, -17, -18, $21, -21 and abnormalities of 3q, 6p, 1lq, 12p, 17p and 21q. Complex and hypodiploid karyotypes are a common feature. The typical abnormalities seen in de novo AML are rare. Cytogenetically sAML is very similar to secondary MDS (sMDS), although del(5q) is more common in sMDS whereas abn( 3q) and abn( 1lq) are more common in sAML. Interestingly +8 occurs less frequently in sAML than in de novo AML. There is some correlation between cytogenetic abnormalities in sAML and the type of exposure. Monosomy 7 is more common in patients exposed to chemotherapy, particularly cytostatics. Del( 5q) is associated with exposure to ionizing radiation but appears to be restricted to sMDS. Etoposide induced sAML has a high incidence of 1lq23 abnormalities. Where exposure to pesticides and organic solvents is documented abnormalities of 17p, 16q22 and + 1lq are common.71 The presence of a 17p abnormality has led to the suggestion that ~53, the protein product of a gene located on 17p, is involved in leukaemogenesis. The prognosis of sAML patients is poor, as CR may not be achieved or has a short duration. However, it has been proposed that the abnormalities t(8;21), t( 15;17) and inv( 16), when present in sAML confer a prognosis similar to that observed in de novo AML.” Two groups of patients who share the clinical and
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CYTOGENETICS IN ACUTE MYELOID LEUKAEMIA
biological features of sAML are those who develop AML after de novo MDS73 and a sub-group of patients who have AML M6.30 Patients with apparently de novo AML who have cytogenetic findings more typical of sAML have a poor prognosis and do as poorly as patients with genuine sAML. Therefore the question is asked, are they the same disease and should more effort be made to establish whether exposure to environmental or toxic agents has occured. Relapse A feature of AML is the disappearance of chromosome abnormalities in complete remission and their reappearance in relapse. The changes seen at relapse can generally be classified into three categories, relapse with the same karyotype as at diagnosis, relapse with a more complex karyotype, relapse with a karyotype either normal or less complicated.74 The significance of the appearance of an abnormal clone in a patient at relapse who was apparently cytogenetically normal at diagnosis is still open to debate. This may be explained by a failure of detection at diagnosis, especially in cases with a t (15;17) or inv( 16) or the presence of a subclone derived from a common ‘cytogenetically normal’ progenitor ce11.75 Both numerical and structural changes are seen at relapse. Trisomy of either chromosome 8 or 21 is the most common numerical change. Structural changes and multiple subclonal changes are seen more commonly in relapse and can be more complex, possibly due to treatment. Relapse with chromosome abnormalities associated with sAML such as del(5q) and del(7q) are again associated with a poor prognosis. A number of studies have compared the survival of patients with and without karyotypic changes at relapse. Testa et al”j reported a shorter survival rate whereas a number of other series disagree.” Other groups report no statistical significance.78~75To clarify the situation further large coordinated studies are needed. The Future It is some 10 years since an already well defmed cytogenetic abnormality was found to be characterised by a molecular rearrangement. This was the Philadelphia translocation t (9;22) (q34;qll) which at the molecular level is the fusion of the c-abl oncogene located on chromosome 9q to the bcr on chromosome 22q, to form the chimaeric gene bcrfabl. During these 10 years cytogeneticists and molecular geneticists have worked together and localization of genes at or near breakpoints in chromosomes involved in haematological malignancies have led to the analysis of these consistent chromosome rearrangements. Indeed, several translocations such as the t (6;9), t (8;2 1) and
t ( 15;17) have associated aberrant chimaeric protein products associated with them. As more reports of apparently rare but consistent chromosome translocations with specific clinical associations are identified, such as the t(1;22)(p13;q13) seen in M7,” there is a potential for identifying new genes at these breakpoints and determining whether their altered function has a role in leukaemogenesis. Cytogenetics will continue to play a major role in the study of AML, as it is clear that cytogenetics can provide an independant prognostic indicator. In the future, specific treatment regimes could be tailored for treatment of patients with common abnormalities such as t( 8;21) and inv( 16) and currently rare abnormalities may emerge to be associated with specific phenotypes and prognosis and will be treated accordingly. New techniques such as PCR and in-situ hybridisation will have an increasing importance especially with respect to the detection of minimal residual disease. However, the most cost effective and accurate answer to the use of cytogenetics in the diagnosis of AML, will be the use of traditional methods at presentation in conjunction with PCR and in-situ hybridization. This combination of cytogenetics and molecular genetics at diagnosis will give a broader picture of the disease allowing one or more of these methods to be used subsequently in disease monitoring.
References 1. Leukaemia and lymphoma. Leukaemia Research Fund: Data collection study 1984-1988. London: Leukaemia Research Fund, 1990. 2. Bemiett J M, Catovsky D, Daniel M T, et al. Proposals for the classification of the acute leukaemias. Br J Haematol 1976; 33: 451-458. 3. Blood. Textbook of haematology. J H Jandl, ed. Boston: Little Brown & Co, 1987. 4. Borrow J, Solomon E. Molecular analysis of the t( 15;17) translocation in acute promyelocytic leukaemia. Balliere’s Clin Haematol 1992; 5: 833-856. 5. Rowley J D. Recurring chromosome abnormalities in leukemia and lymphoma. Semin Haematol1990; 27: 122-136. 6. Yunis J J. Recurrent chromosomal defects are found in most patients with acute nonlymphocytic leukemia. Cancer Genet Cytogenet 1984; 11: 125-137. I. Sandberg A A. The chromosomes in human cancer and leukemia. 2nd ed. New York: Elsevier, 1990. 8. Mitelman F. Catalog of chromosome aberrations in cancer. 4th ed. New York Wiley-Liss, 1991. 9. Berger R, Le Coniat M, Derre J, Vecchione D, Jonveaux P. Cytogenetic studies in acute promyelocytic leukemia: a survey of secondary abnormalities. Genes Chromosom Cancer 1991; 3: 332-337. 10. Schiffer C A, Lee E J, Tomiyasu T, Wiernik P H, Testa J R. Prognostic impact of cytogenetic abnormalities in patients with de novo acute nonlymphocytic leukemia. Blood 1989; 73: 263-270. 11. Keating M J, Smith T L, Kantajian H, et al. Cytogenetic pattern in acute myelogenous leukemia: A major reproducible determinant of outcome. Leukemia 1988; 2: 403412. 12. Berger R. Bemheim A. Ochoa-Noauera M E, et al. Prognostic sigr&ance of chromosomal abnormalities in acute nonlymphocytic leukemia a study of 343 patients. Cancer Genet Cytogenet 1987; 28: 293-299.
BLOOD REVIEWS 13. Stasi R, Poeta G, Masi M, et al. Incidence of chromosome abnormalities and clinical significance of karyotype in de novo acute myeloid leukemia. Cancer Genet Cytogenet 1993; 67: 28-34. 14. Jenkins R B, Le Beau M M, Kraker W J, et al. Fluorescence in situ hybridization: A sensitive method for trisomy 8 detection in bone marrow specimens. Blood 1992; 79: 3307-3315. 15. Kibbelaar R E, Muldar J, Dreet E J, et al. Detection of monosmy 7 and trisomy 8 in myeloid neoplasia: A comparison of banding and fluorescence in situ hybridization. Blood 1993; 82: 904913. 16. Weber E, Nowotny H, Haas 0 A, et al. Trisomy 4: A specific karyotype anomaly in primary and secondary acute myeloid leukemia. Leukemia 1990; 4: 219-221. 17. Ribeiro E M S F B, Cavali I J, Tokutake A S. Trisomy 4 in acute nonlymphocytic leukemia: The first Brazilian case. Cancer Genet Cytogenet 1993; 68: 82-83. 18. United Kingdom Cancer Cytogenetics Group (UKCCG). Primary, single, autosomal trisomies associated with haematological disorders. Leukemia Res 1992; 16: 841-851. 19. Avanzi G C, Giovinazzo B, Rosso A, et al. Trisomy 11 in myelodysplastic syndrome-derived acute myeloblastic leukaemias. Eur J Haematoll989; 43: 173-177. 20. Sreekantaiah C, Baer M R, Morgan S, et al. Trisomy/tetrasomy 13 in seven cases of acute leukemia. Leukemia 1990; 4: 781-785. 21. Baer M R, Bloomfield C D. Trisomy 13 in acute leukemia. Leuk Lymphoma 1992; 7: l-6. 22. Cuneo A, Michaux J-L, Ferrant A, et al. Correlation of cytogenetic patterns and clinicobiological features in adult myeloid leukemia expressing lymphoid markers. Blood 1992; 79: 720-726. 23. Najfeld V, Seremetis S, Troy K, et al. Trisomy 22-A new abnormality found in acute leukemia characterized by eosinophilia and monocytoid blasts expressing immature differentiation antigens. Cancer Genet Cytogenet 1986; 23: 105-l 14. 24. Bitter M A, Leilly M E, Le Beau M M, Pearson M G, Rowley J D. Rearrangements of chromosome 3 involving bands 3q21 and 3q26 are associated with normal or elevated platelet counts in acute nonlymphocytic leukemia. Blood 1985; 66: 1362-1370. 25. Fichelson S, Dreyfus F, Berger R, et al. EVI-1 expression in leukemic patients with rearrangements of the 3q25-q28 chromosomal region. Leukemia 1992; 6: 93-99. 26. Rowley J D, Golomb H M, Vardiman J W. Nonrandom chromosome abnormalities in acute leukemia and dysmyelopoietic syndromes in patients with previously treated malignant disease. Blood 1981; 58: 759-767. 27. Le Beau M M, Albain K S, Larson R A, et al. Clinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia: Further evidence for characteristic abnormalities of chromosomes no 5 and 7. J Clin Oncoll986; 4: 3255345. 28. Neuman W L, Rubin C M, Rios R B, et al. Chromosomal loss and deletion are the most common mechanisms for loss of heterozygosity from chromosomes 5 and 7 in malignant myeloid disorders. Blood 1992; 79: 1501-1510. 29. Fourth International Workshop on Chromosomes in Leukemia 1982 1984 Cancer Genet Cytogenet 11: 249-360. 30. Olopade 0 I, Thangavelu M T, Larson R A, et al. Clinical, morphologic, and cytogenetic characteristics of 26 patients with acute erythroblastic leukemia. Blood 1992; 11: 2873-2882. 31. Westbrook C A, Keinanen M J. Myeloid malignancies and chromosome 5 deletions. Bailliere’s Clin Haematol 1992; 5: 931-942. 32. Willman C L, Sever C E, Pallavicim M G et al. Deletion of IRF-1, mapping to chromosome 5q31, in human leukemia and preleukemic myelodysplasia. Science 1993; 259: 968-971. 33. Soekarman D, von Lindern M, Daenen S, et al. The translocation (6;9)(p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features. Blood 1992; 79: 2990-2997. 34. Rowley J D, Potter A. Chromosomal banding patterns in acute nonlymphocytic leukemia. Blood 1976; 47: 705-721. 35. Lillington D M, MacCullum P H, Lister A T, Gibbons B. Translocation t(6;9)(p23;q34) in acute myeloid leukemia without myelodysplasia or basophilia: two cases and a review of the literature. Leukemia 1993; 7: 527-531.
35
36. Pearson M G, Vardiman J W, Le Beau M M, et al. Increased numbers of marrow basophils may be associated with a t(6;9) in ANLL. Am J Haematoll985; 18: 393-403. 37. Berger R, Daniel M-T, Jonveaux P, Baruchel A. Relapse as acute monoblastic leukemia (AML-MS) of t(6;9) acute myeloblastic leukemia (AML-M2). Leukemia 1992; 6: 1227-1228. 38. von Lindem M, Poustka A, Lerach H, Grosfeld G. The t(6;9) translocation associated with a specific subtype of acute nonlymphocytic leukemia leads to aberrant transcription of a target gene on 9q34. Mol Cell Biol 1990; 10: 4016-4026. 39. von Lindem M, Fornerod M, van Baa1 S, et al. The translocation t(6;9) associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimaeric leukemia specific dekcan mRNA. Mol Cell Bioll992; 12: 1687-1697. 40. Nichols J, Nimer S D. Transcription factors, translocations, and leukemia. Blood 1992; 80: 2953-2963. 41. Schouten T J, Hustinx T W J, Scheres J M J C, Holland R, de Vaan G A M. Malignant histiocytosis. Clinical and cytogenetic studies in a newborn and a child. Cancer 1983; 52: 1229-1236. 42. Cohen H, Walker H, Delhanty J D A, Lucas S B, Huehns E R. Congenital spherocytosis, B19 parvovirus infection and inherited interstitial deletion of the short arm of chromosome 8. Br J Haematol 1991; 78: 251-257. 43. Rowley J D. Identification of a translocation with quinacrine mustard fluorescence in a patient with acute leukemia. Ann Genet 1973: 16: 109-l 12. 44. Groupe Francais de Cytogenetique Haematologique. Acute myelogenous leukemia with an 8;21 translocation. Cancer Genet Cytogenet 1990; 44: 169-179. 45. Downing J R, Head D R, Curcio-Brint A M. An AMLl/ETO fusion transcript is consistently detected by RNA-based polymerase chain reaction in acute myelogenous leukemia containing the t(8:21 )(q22;q22) translocation. Blood 1993; 81: 2860-2865. 46. Maseki N, Miyoshi H. Shimizu K, et al. The 8;21 chromosome translocation in acute myeloid leukemia is always detectable by molecular analysis using AMLI. Blood 1993; 8 1: 1573-1579. 47. Hirsch-Ginsberg C, Childs C, Chang K-S, et al. Phenotypic and molecular heterogeneity in Philadelphia chromosomepositive acute leukemia. Blood 1988; 71: 186-195. 48. Kursrock R, Shtalrid M, Talpaz M, Kloetzer W S, Gutterman J V. Expression of c-abl in Philadelphia -positive acute myelogenous leukemia. Blood 1987; 70: 1584-1588. 49. Raimondi S C, Kalwinsky D K, Hayashi Y, et al. Cytogenetics of childhood acute nonlymphocytic leukemia. Cancer Genet Cytogenet 1989; 30: 13-27. 50. Whitlock J A, Greer J P, Lukens J N. Epipodophyllotoxinrelated leukemia. Identification of a new subset of secondary leukemia. Cancer 1991; 68: 600-604. 51. Kalwinsky D K, Raimondi S C, Schell M J, et al. Prognostic importance of cytogenetic subgroups in de novo pediatric acute nonlymphocytic leukemia. J Clin Oncol 1990; 8: 75-83. 52. Tkachuk D C, Kohler S, Cleary M L. Involvement of a homolog of Drosophila Trithorax by 1lq23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691-700. 53. Ziemin-van der Poe1 S, McCabe N R, Gill H J, et al. Identification of a gene, MLL, that spans the breakpoint in 1lq23 translocations associated with human leukemias. Proc Nat Acad Sci USA 1991; 88: 10735510739. 54. Djabali M, Selleri L, Perry P. A trithorax-like gene is interrupted by chromosome 1lq23 translocations in acute leukaemias. Nature Genet 2: 113-l 18. 55 Rowley J D, Golomb H M, Vardiman J, et al. Further evidence for a non-random chromosomal abnormality in acute promyelocytic leukemia. Int J Cancer 1977; 20: 869-872. 56. Stone R M, Mayer R J. The unique aspects of acute promyelocytic leukemia. J Clin Oncol 1990: 8: 1913-1921 57. Larson R A, Kondo K, Vardiman J W, et al. Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia. Am J Med 1984; 76: 827-841. 58. Borrow J, Goddard A D. Gibbons B, et al. Diagnosis of acute promyelocytic leukaemia by RT-PCR: detection of PML/RARA and RARAPML fusion transcripts. Br J Haematol 1992; 82: 529-540. 59. Biondi A, Rambaldi A. Alcalay M, et al. RAR 1 rearrangements as a genetic marker for diagnosis and
36
60.
61. 62.
63.
64.
65.
66.
67.
68.
69.
CYTOGENETICS IN ACUTE MYELOID LEUKAEMIA monitoring in acute promyelocytic leukemia. Blood 1991; 77: 1418-1422. Huang M E, Ye Y C, Chen S R, et al. All tram-retinoic acid with or without low dose cytosine arabinoside in acute promyelocytic leukemia: report of six cases. Chinese Med J (English) 1987; 100: 949-953. Warrel R P, de The H, Wang Z-Y, Dagos L. Acute promyelocytic leukemia. New Engl J Med 1993; 329: 177-189. Larson R A, Williams S F, Le Beau M M, et al. Acute myelomonocytic leukemia with abnormal eosinophils and inv( 16) or t( 16;16) has a favorable prognosis. Blood 1986; 68: 1242-1249. Campbell L J, Challis J, Fok T, Garson 0 M. Chromosome 16 abnormalities associated with myeloid malignancies. Genes Chromosom Cancer 1991; 3: 55-61. Betts D R, Rohatiner A Z, Evans M L, et al. Abnormalities of chromosome 16q in myeloid malignancy: 14 new cases and a review of the literature. Leukemia 1992; 6: 1250-1256. Bernard P, Dachary D, Reiffers J, et al. Acute nonlymphocytic leukemia with marrow eosinophilia and chromosome 16 abnormality: a report of 18 cases. Leukemia 1989; 3: 740-745. Dauwerse J G, Kievits T, Beverstock G C, et al. Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16~. Cytogenet Cell Genet 1990; 53: 126-128. Ohyashiki K, Ohyashiki J H, Kondo M, Ito H, Toyama K. Chromosome change at 16q22 in nonlymphocytic leukemia: clinical implication on leukemia patients with inv( 16) versus del( 16). Leukemia 1988; 2: 35-40. Mitehnan F, Nilsson P G, Brandt L, et al. Chromosome pattern, occupation, and clinical features in patients with acute nonlymphocytic leukemia. Cancer Genet Cytogenet 1981; 4: 197-214. Johansson B, Mertens F, Heim S, Kristoffersson V, Mitelman F. Cytogenetics of secondary myelodysplasia
70.
71.
72.
73.
74. 75.
76.
77.
78.
79.
(sMDS) and acute nonlymphocytic leukemia (sANLL). Eur J Haematol 1991; 47: 17-27. Pedersen-Bjergaard J, Philip P, Pedersen N T, et al. Acute nonlymphocytic leukemia, preleukemia, and acute myeloproliferative syndrome secondary to treatment of other malignant disease. Cancer 1984; 54: 4522462. Cuneo A, Fagioli F, Pazzi I, et al. Morphologic, immunologic and cytogenetic studies in acute myeloid leukemia following occupational exposure to pesticides and organic solvents. Leuk Res 1992; 16: 789-796. Fenaux P, Lai J L, Quiquandon I, et al. Therapy related myelodysplastic syndrome and leukemia with no ‘unfavourable’ cytogenetic findings have a good response to intensive chemotherapy: a report on 15 cases. Leuk Lymphoma 1991; 5: 117-125. Weh H J, Kuse R, Seeger D, Suciu S, Hossfeld D K. Cytogenetic studies in patients with acute myeloid leukemia following a myelodysplastic syndrome. Leuk Lymphoma 1991; 3: 423429. Sixth International Workshop on chromosomes in leukemia, 1987. Cancer Genet Cytogenet 1989; 40: 187-202. Shikano T, Ishikawa Y, Ohkawa M, et al. Karyotypic changes from initial diagnosis to relapse in childhood acute leukemia. Leukemia 1990; 4: 419-422. Testa J R, Mintz U, Rowley J D, Vardiian J W, Golomb H M. Evolution of karyotypes in acute nonlymphocytic leukemia. Cancer Res 1979; 39: 3619-3627. Morris C M, Fitzgerald P H. Karyotypic evolution in patients with acute myeloid leukemia. Cancer Genet Cytogenet 1984; 11: 143-152. Berger R, Le Coniat M, Derre J, et al. Cytogenetic studies on acute nonlymphocytic leukemia in relapse. Cancer Genet Cytogenet 34: 11-18. Lu G, Altman A J, Benn P A. Review of the cytogenetic changes in acute megakaryoblastic leukemia. One disease or several? Cancer Genet Cytogenet 1993; 67: 81-89.