7
Myeloid malignancies andchromosome 5 deletions CAROL A. WESTBROOK MAURI J. KEINANEN
CLINICAL SYNDRO~IES ASSOCIATED WITH DELETIONS OF Sq Interstitial deletions of the long arm of chromosome 5 were first described in haematological disorders by van den Berghe et al in 1974, who reported deletions in three patients with long-standing refractory anaemia. Subsequently, many more cases were reported, and a distinct clinical picture has developed of a clinical entity which is known as the 5q- syndrome. These patients typically have refractory anaemia, usually macrocytic, and morphological abnormalities of their megakaryocytes. There is a predominance of older women. usually in their mid 60s. Most cases fit into the categories (Bennett et al, 1982) of refractory anaemia, refractory anaemia with excess blasts (RAEB) or refractory idiopathic sideroblastic anaemia (RISA). Progression to acute leukaemia is uncommon in this disorder, though it has been reported; chronic refractory anaemia is the typical clinical picture. The Sq- syndrome, as described above, is a rare clinical entity. Much more frequently. deletions of chromosome 5 are seen in a variety of haematological disorders with two unifying characteristics: they are myeloid disorders of trilineage involvement, and they are clinically aggressive. In particular. they include myelodysplastic syndrome (MOS), acute myeloid leukaemia (AML), and therapy-related MOS or AML (t-MOS/AML). The clinical aggressiveness of these disorders distinguishes them, as a whole, from the 5q- syndrome, and raises the question as to whether they involve the same gene(s) on chromosome 5. Leukaemia in association with MOS has been recognized to develop after cytotoxic therapy for cancer and .for non-malignant disease. Deletions of chromosome 5, or loss of an entire chromosome 5, are found in a large number of cases of patients who survive cancer treatment. The association was first reported by Rowley et al (1981), who reported that loss of an entire chromosome 5 or 7. or a deletion of the long arm of these chromosomesdel(5q) or del(7q}-was present in 23 of 26 cases of t-AMUMOS. (For the purpose of this ~hapter, we will use the terminology del(5q) and del(7q) to include cases With whole chromosome loss as well as those with interstitial deletions.) This high prevalence has been confirmed by other investigators, Bai/liir~'s Q inicalliatma(ologyVol. s. No.4. October1992 ISBN 0-7020-1691-8
931 . Copyright © 1992.by Bailliere Tindall All rights of reproduction in any form reserved
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C. A. WESTBROOK AND M. 1. KEINANEN
including our own updated series (Le Beau et al, 1986). In particular, chromosome 5 deletion or loss occurs in over 40% of cases, usually with concurrent loss or deletion of chromosome 7. Because of the frequent coexistence of del(5q) and del(7q) in t-MOS/AML, it has not been possible to ascertain whether either chromosomal abnormality has distinct clinical or morphological features; indeed, the syndrome may be related to a combined action of both mutations. In t-MOS, the del(5q) is usually present at diagnosis, and is found in all of the chromosomally abnormal cells examined at the time of diagnosis rather than at progression to AML (PedersenBjergaard et aI, 1990); nonetheless, additional cytogenetic abnormalities are common and it rarely occurs alone. It is interesting that del(5q) and/or del(7q) are highly correlated with exposure to alkylating agents (Pedersen-Bjergaard et al, 1990). Several large studies have examined the incidence of secondary leukaemia in survivors of Hodgkin's disease (Kaldor et al, 1990a) and ovarian cancer (Kaldor et al, 1990b). It was concluded that chemotherapy-but not radiation therapy-e-confers a 9- to 12-fold risk of developing leukaemia, with the worst offenders being the alkylating agents chlorambucil, melphalan, procarbazine and mustine (mechlorethamine USP). t-AML (usually with antecedent MOS) has been described as a single clinical entity. As for de novo AML, it is likely that the clinical picture is more closely related to the chromosomal changes rather than to the aetiologic agent; increasingly the features which were considered typical of t-AML are now being recognized as those associated with del(5q) and/or del(7q), as these are the overwhelmingly predominant cytogenetic abnormalities in t-AML (Pedersen-Bjergaard et ai, 1990). The presentation occurs with a latency of about 4 years after treatment, and usually begins as MOS. t-MDS typically presents with cytopenias, while the marrow is hypercellular or normocellular with characteristic changes in the megakaryocytes and trilineage involvement (Vardiman et ai, 1983). t-MDS is aggressive compared with de novo MDS, and many patients die of the complications of cytopenias-infections or haemorrhage. If the patient survives, the marrow blast count increases until the diagnosis of acute leukaemia is made, usually within 5 months of presentation of MDS. Survival after AML is shorter than it is in most cases of AML (median survival is less than 9 months) due to a low remission rate and short remission duration. Most investigators consider t-MDS and t-AML to be a continuum rather than discrete diseases, and we will use a combined term in this chapter as well. del(5q) isfound in about 10% of patients with AMLarising de novo (Larson et al, 1983; Fourth International Workshop on Chromosomes in Leukemia, 1984). In AML, other cytogenetic abnormalities often accompany the del(5q) (such as +8, -7 and -17), and a history of occupational exposure iscommon (Mitelman et al, 1978; Golomb et al, 1982; Brandt et al, 1983). As with t-AML, trilineage involvement iscommon, and evolution from a pre-existing MDS can often be demonstrated. Clinically these patients tend to be older and poorly responsive to chemotherapy compared with other cytogenetic subtypes (Larson et al, 1983; Fourth International Workshop on Chromosomes in Leukemia, 1984; Samuels et al, 1988; Schiffer et ai, 1989). For
MYELOID MALIGNANCIES AND CHROMOSOME 5 DELETIONS
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example, only 28% of del(5q) cases achieved complete remission in the Fourth International Workshop series, with a median survival of only 4 months. These findings prompted the suggestion that del(5q) cells are resistant to standard leukaemia chemotherapy (Samuels et al, 1988). del(5q) is considered to be one of the worst indicators of poor prognosis in AML, and a strong case can be made for the development of molecular markers for diagnosis, prognosis and therapy evaluation. del(5q) is also seen in cases of de novo MDS that are distinct from the 5qsyndrome; these cases have a wide variety of clinical manifestations. del(5q) in this setting is often associated with the more aggressive forms of MDS (RAEB , or refractory anaemia in transition) and may resemble t·MDS in clinical features and poor prognosis. Cytopenias are accompanied by a hypercellular marrow, and characteristic features of megakaryocytic dysplasia are present as well. In these cases, progression to acute leukaemia is rapid. Good cytogenetic and/or molecular descriptions of the various MDS syndromes are still lacking; one reason to study del(5q) is that it willlead to a better understanding of the MDS subtypes and their clinical features, and may help to evaluate prognosis in individual cases. Overall, a picture is beginning to emerge of del(5q) (Nimer and Golde, 1987) as a clonal stem-cell disorder, characterized by trilineage dysplasia, megakaryocytic abnormalities and cytopenias. Progression to leukaemia is common, and may occur prior to presentation, usually accompanied by additional genetic changes such as del(7q) . The relationship of the 5qsyndrome to these other cases is poorly understood. Whether 5qsyndrome involves a different gene on 5q from that involved in t-MDSt AML, or whether the accompanying changes on other chromosomes are important, will probably not be understood until the relevant genes are cloned and molecular markers are available. TUMOUR SUPPRESSOR GENES AND CHROMOSOMAL DELETIONS: DOMINANT AND RECESSIVE ONCOGENES The association of recurring chromosomal translocations with particular leukaemia subtypes led to the hypothesis that a specific gene is involved for each leukaemia subtype. A considerable amount of research effort has been directed toward cloning these translocation breakpoints, identifying the genes involved, and understanding their role in the leukaemic process. There are numerous examples in this volume. This data accumulated over the years had led to .the paradigm t~at chro~oso.mal translocations in leukaemia have a domm~nt effect, leadI~g to activation of an oncogene by transcriptional deregulation or production of a modified protein. Genes which are activat~d in t~i~ manner can be cons!dered 'dominant oncogenes' , because oncogemc activity occurs due to a single mutational event and is effective in spite of th~ presence of a normal copy of the gene. Chromosomal deletions, on the other hand, have received much less attention. This is partly because deletions, even those which occur consistently in a particular leukaemia, vary considerably in extent from patient
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C. A. WESTBROOK AND M. 1. KEINANEN
to patient; it is difficult to postulate a single, dominant-acting oncogene if a discrete chromosomal breakpoint cannot be identified. It is only with the concept of 'recessive oncogenes' evolving from research in solid tumours that a paradigm has been developed which can be applied to these deletions. The recessive oncogene model stipulates that it is the loss of function of a critical gene, rather than the positive activation of an oncogene, that is the determinant of cancer progression or initiation. Genes which lead to cancer in this manner must be considered 'recessive' because mutation is required at both alleles before the effect is fully manifest. The classic example of a recessive oncogene is the RB1 gene in retinoblastoma (Friend et ai, 1989). Genetic evidence postulated a two-step mechanism (Cavenee et ai, 1983), and eventual cloning of the gene confirmed the theory, showing that mutational inactivation or deletion occurs successively at both alleles of the RB1 gene, leading to complete cessation of gene activity. There are now several other recessive oncogenes which have been identified, all from solid tumours. These include the DCC gene in colorectal cancer (Fearon et al, 1990), the p53 gene in a variety of cancers (Nigro et ai, 1989), the Wilms' tumour gene (Call et ai, 1990;Gessler et ai, 1990) and recently the APC gene in familial adenomatous polyposis, a form of hereditary colorectal cancer (Groden et al, 1991). Although homozygous loss has not been confirmed for each of these genes, in every case mutations have been found which demonstrate loss or inactivation of at least one allele. Consistently occurring deletions in the long arm of chromosome 5 in myeloid malignancies postulate the existence of a recessive oncogene or 'antileukaemia' gene (ALG) on 5q which is homozygously inactivated in these malignancies. A molecular hypothesis must be consistent with the clinical and cytogenetic features that are observed. Thus, we expect to find that this gene is located within the visibly deleted region of every case of del(5q) that meets the clinical criteria studied. A two-step mechanism is in accordance with the long latent period of 4 years (varies between 2-5 years) observed in therapy-related leukaemia, in contrast to the relatively short latency observed with the development of t-AML with translocations at llq23 (Pedersen-Bjergaard et ai, 1990), which may be a single-step mechanism (see Chapter 6). Although it is not strictly possible to rule out a one -step mechanism for del(5q), in which transformation is caused by loss of only one allele (gene-dosage effect), there have been no demonstrated examples among the known recessive oncogenes. If the ALG functions as a typical recessive oncogene, it is expected to be inactivated on both chromosomes; in one instance by a visible deletion or chromosomal loss (including allelic deletion), in the other by a smaller event. As with other recessive oncogenes such as RBI (Friend et al, 1989) and Dee (Fearon et al, 1990), this event on the residual, non-deleted homologue might be a point mutation or a larger structural abnormality (homozygous deletion or internal rearrangement); on occasion functional loss will instead be caused by other mechanisms such as translocation. The types of mutations found might be random, but more than likely will have some relevance to the mutagenizing agent (Vogelstein and Kinzler, 1992). It
MYELOID MALIGNANCIES AND CHROMOSOME 5 DELETIONS
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is the smaller event on the non-deleted chromosome that will eventually help to identify the gene. In the remainder of this chapter, we will review the present state of knowledge about the cytogenetic definition of del(5q) and the extent of the deleted regions in relation to the current genome map, and discuss possible candidate genes which lie within this region. CYTOGENETIC DELINEATION OF CHROMOSOME 5 DELETIONS Cytogenetic studies have attempted to define the breakpoints and deletion extent in patients with 5q- syndrome, t-MDS/AML or de novo AML. These deletions are interstitial, that is, two breaks occur in the long arm with loss ofthe intermediate segment. Furthermore, the deletion extent is similar in most cases, with a distal breakpoint usually in q33-35 and a proximal breakpoint in ql3-15. By comparing the cytogenetic overlap in deletions from 17 cases of t-AMUMDS, Le Beau et al (1986) suggested there was a
P
1 2
1
q
2 I HDSIAHL del 5Q31
5Q- syndrome del 5Q22-33
3
Figure 1. Minimal overlapping region for deletions of chromosome 5 in myelo id malignancies. Depicted is an ideogram of chromosome S. Next to the chromosome, the smallest overlapping region is shown for t-MDSlAML and de novo MDS/AML (5q31) and for Sq- syndrome (Sq22-33). Based on Le Beau et al (1989).
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C. A. WESTBROOK AND M. J. KEINANEN
much smaller region that was commonly deleted in all patients, which included 5q23-Sq31. This series was later expanded to 80 cases, which included Sq- syndrome as well as de novo and t-MOS/AML (Le Beau et aI, 1989). The inclusion of de novo and t-AML cases with distal (three patients) or proximal (two patients) breakpoints in 5q31, made it possible to define a smaller overlapping region of deletion which comprised the proximal half of band 5q31. The minimal region in the Sq- syndrome was considerably larger, extending from 5q22 through to Sq33, but only 12 cases were included and all had large deletions. The smaller deletion region defined for de novo and t-MOS/AML is completely contained within the larger region defined for the Sq- syndrome (see Figure 1). Thus, it is not possible on cytogenetic grounds to determine if these closely related diseases involve different genes. Such a distinction may not be possible until the involved genes are cloned and the syndromes can be defined on a molecular level. RELATIONSHIP OF THE GENOME MAP TO CHROMOSOME 5 DELETIONS Since the total length of chromosome 5 has been estimated to be about 160Mb, based on the percentage of the total genome measured from ISeN (International System for Human Cytogenetic Nomenclature) idiograms (Stephens et ai, 1990), and band 5q31 is about one-tenth of the chromosome, the minimal deleted region for the ALG is about 16 Mb, and for the 5q - region is about 40 Mb. These are both extremely large regions containing many genes; it will not be easy to find the ALG unless the regions are better defined on a molecular level, and further refined to smaller regions. To date, little has been published attempting to molecularly define these deletions, that is, to demonstrate the inclusions of markers both within and outside of the critically deleted region. What is particularly lacking is a direct comparison, using molecular markers, of cytogenetic deletions seen in these two clinically disparate entities, 5q- syndrome and del(5q) in MOS/AML. It is hoped that additional information will result from the recent increase of probes and markers available for this region of chromosome 5 resulting from human genome efforts (Wasmuth et aI, 1991). One approach is to compare the location of known genes and markers with the deletion extent. Although this is a rough approach, relying as it does on cytogenetic interpretation, it is a rapid way of identifying genes which lie approximately in the critical region and might be considered as candidates for recessive oncogenes. The current map of chromosome 5 contains many genes and markers on 5q. The cloned genes which lie within or near the minimal deleted regions are listed in Table 1; this list includes over 30 genes. There are a number of genes that are of particular interest in this list. Most striking, perhaps, is that two known recessive oncogenes, MCC and APC are located at 5q21-22. These genes were both identified by their involvement in colorectal cancer, APe in familial and sporadic colorectal cancer, and M CC in sporadic cancers only. More recent data position APC, and the closely related MCC, in 5q21, and as such it lies outside of the 5q- region
937
MYELOID MALIGNANCIES AND CHROMOSOME 5 DELETIONS
Table 1. Genes localizedto the distal (q21-qter) long arm of chromosome 5. Symbol
Gene name
Location·
FER APC MCC CAMK4 CD14 IL9 CD49B CSF2 EGRI FBN2 IL3 IL4 IL5 IRFI ADRAIB PDEA FGFA TCF7 ADRB2 GRL DHLAG RPS14 SPARC ARH9 ADRAIAC ANX6 GAPDL16 CSFI CSFIR PDGFRB F12 FGFR4 DRDI GABRAI CHR
Fer (fps/fesrelated) tyrosine kinase Adenomatosis polyposiscoli Mutated in colorectal cancers Ca2+/calmodulin dependent protein kinase IV Antigen CD14 Interleukin 9 Antigen CD49B (a2 subunit of VLA·2 receptor) Colony-stimulating factor 2 (granulocyte-macrophage) Early growth response 1 Fibrillin 2 Interleukin 3 Interleukin 4 Interleukin 5 Interferon regulatory factor 1 Adrenergic, alB receptor Phosphodiesterase, cyclicGMP (rod receptor) a-polypeptide Fibroblast growth factor, acidic (endothelial growth factor) Transcription factor 7 (T-cell specific) Adrenergic.Bs receptor, surface Glucocorticoid receptor Major histocompatibility complex, class II, gamma polypeptide Ribosomal protein S14 Secreted protein, acidic, cysteine-rich(osteonectin) Aplysia ras-related homologue 9 Adrenergic alA receptor Annexin VI (p68) Glyceraldehyde·3-phosphate dehydrogenase-like 16 Colony-stimulating factor 1 (macrophage) CSFl receptor (formerly FMS) Platelet-derived growth factor receptor, ~-polypeptide Coagulation factor XII (Hageman factor) Fibroblast growth factor receptor 4 Dopamine receptor 01 'Y-Aminobutyric acid (GABA) A receptor, al Chromate resistance; sulphate transport
q21 q21-22 q21-22 q21-33 q22-32 q22-32 q23-31 q23-31 q23-31 q23-31 q23-31 q23-31 q23-31 q23-31 q23-32 q31.2-34 q31.3-33.2 q31 q31-32 q31-32 q31-33 q31-33 q31-33 q31-qter q32-34 q32-34 q33.1-35.3 q33 q33-35 q33-35 q33-qter q33-gter q34-35 q34-35 q35
• For complete citations on the genes and their locations, refer to Wasmuth et al (1991).
and is clearly far from the ALG region of 5q31. However, the gene has not been systematically studied for mutations in leukaemias. The long arm of chromosome 5 is particularly rich in growth factor genes and receptor protein genes. These include genes for haematopoietic growth factors such as interleukin (IL) 3, 4, 5 and 9, colony-stimulating factor (CSF) 2 fibroblast/endothelial cell growth factor, and haematopoiesis-related r;ceptors (glucocorticoid receptor, CSFI receptor and platelet-derived growth factor receptor J3). ~ere are a large number of genes for neurotransmitter recepto!s, including the alphaj , alpha; and beta, adrenergic receptors, the 'Y-ammobutyncacid (~ABA) receptor, the vanillylmandelic acid (VMA) receptor and the dopamine receptor. Also of note is the EGRI gene, which encodes a gr~'Yth regulatory protein with DNA-binding transcriptional regulatory activity (Sukhatme et al, 1988) and genes encoding
938
C. A. WESTBROOK AND M. J. KEINANEN
transcription factor 7 (Tvcell-specific), interferon regulatory factor 1, and the myeloid surface protein CD14. Based on their biological activity, many of these genes are good candidates for the ALG; thus, it will be important to determine which of these are consistently present within deletions and, of these, which are frequently mutated in del(5q) cases. There are an additional60-odd anonymous DNA segment markers which also map to this region, including many RFLPs (Restriction Enzyme Length Polymorphism) and CA (dinucleotide) repeats. These DNA markers will be extremely useful in molecular delineation of the deletions. A large number of probes have been cytogenetically localized by our group as a first step in analysing deletions of chromosome 5 (Wasmuth et al, 1991;M. M. Le Beau, W. L. Neuman, M. Dixon et ai, unpublished data; W. L. Neuman, C. A. Westbrook, B. Weiffenbach et ai, unpublished data). These probes will be mapped against the deletions in individual patients to better define the regions involved. CANDIDATE GENES
Undoubtedly, many genes are under active investigation as candidates for the ALG, but little has been published to date. Among the first genes suggested as candidates for the ALG were the interleukin genes located at 5q23-31 (Nienhuis et ai, 1985; Le Beau et ai, 1986, 1989). We examined some of these genes for structural mutations, as our hypothesis suggests that the 'normal' chromosome 5 homologue will contain an inactivating mutation within the ALG when the other chromosome contains a del(5q). At least some of these deletions should be large enough to be seen by pulsed field gel electrophoresis or Southern blots. In a small study, pulsed field gel electrophoresis was used to examine the leukaemic cells of cases with del(5q) or translocations at 5, compared with their control Iymphoblastoid cell lines. This included three patients with MDS and del(5q), and one with t·AML and an inversion involving a breakpoint at 5q31. With probes for IL4 and csn we did not observe any rearrangements in this group with the enzymes NotI and NruI (Le Beau et ai, 1989). EGR1 and eD14 were also applied to these blots, but no rearrangements were seen (unpublished data). These preliminary studies do not rule out any of these genes as candidates, and it will be necessary to investigate many more cases to find a gross rearrangement visible by Southern blot methods. Based on experience with retinoblastoma, this may be up to 30% of cases (Friend et ai, 1989); on the other hand, with the Dee gene the number with structural abnormalities within the DeC gene visible on Southern blot was lower, at only about 15% (Fearon et ai, 1990). Such studies are still warranted with potential candidates. However, a necessary selection for these candidate genes is that they be located within the minimal deleted region. It is difficult to postulate a role for the interleukins as tumour suppressor genes, since they are growth factors for myeloid cells, and would more likely function as dominantly acting oncogenes. This is, in fact, the case with the
MYELOID MAUGNANCIES AND CHROMOSOME 5 DELETIONS
939
t(5;14)(q31 ;q32) translocation occurring in B-lineage acute lymphoblastic leukaemia with eosinophilia (Grimaldi and Meeker, 1989; Meeker et ai, 1990). Here, the juxtaposition of the immunoglobulin gene enhancer from chromosome 14 to the IL3 gene results in excess production of this growth factor and consequent myeloid hyperproliferation. More intriguing is recent work examining a possible role for the gene CSFlR (formerly EMS, v-fms homologue) in 5q- syndrome. This gene codes for the receptor for the growth factor CSF1, and a role might be postulated as a tumour suppressor gene, although v-fms is dominantly acting in its role as a viral oncogene (Roussel et ai, 1984). Boultwood et al (1991) examined ten patients with Sq- syndrome or MDS and del(Sq) with deletions inclusive of 5q33. All cases had lost at least one allele of the gene FMS, presumably within the deletion; however, five cases had evidence for loss of the other copy of the gene in at least some myeloid cells. This finding was based on densitometry and in situ hybridization to cell specimens containing mixed normal and malignant cells, and will require confirmation on pure populations of cells. Nonetheless, this is the first report of a gene which might satisfy two of the criteria for a tumour suppressor gene: that is, it is located within the visible deletion, and it is inactivated on the nondeleted homologue. Arguing against a role for CSF1R in del(5q) is the cytogenetic analyses by Le Beau (1989) showing that a fraction of deletions have their distal boundary proximal to 5q33, demonstrating that CSFlR is not consistently lost when deletions are present. One such case was studied by in situ hybridization using the CSFlR probe, which showed that the gene was present on both chromosomes. This case represented a 'classical' t-AML, and the karyotype showed a majority of cells that had -7 in addition to a del(S)(q22q33.1). One possible explanation for these discrepant findings is that these studies are addressing two different clinical populations, and that two distinct chromosomal loci might be involved. Boultwood et al (1991) have confined their analysis in refractory anaemia and Sq- cases. Le Beau's population with smaller deletions is predominantly del(5q) associated with de novo or t-AML, whereas a1l5q- cases have deletions which extend through to Sq33 or 34. Clearly it will be necessary to examine many more cases to specify the deletion extent relative to CSFlR in both syndromes, and to attempt to define the clinical populations in terms of molecular markers.
CLINICAL IMPLICATIONS AND POTENTIAL USES FOR MOLECULAR MARKERS OF DEL(Sq) Cloning the genes related to structural chromosomal abnormalities has invariably provided useful clinical markers and contributed to the understanding o~ the. b~sic processes o~ leukaemogenesis. The practical implications of identifying the Ieukaemia-related genes on 5q are considerable. First of all, the relationship of 5q - syndrome to other malignancies would
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C. A. WESTBROOK AND M. J. KEINANEN
be clarified. Useful DNA probes might become available that would complement cytogenetic detection in allowing the diagnosis of poor prognosis AML, monitoring the effects of treatment, and the detection of residual disease after remission or bone marrow transplantation. Similar considerations apply in MDS. Studies of larger clinicalpopulations would give an idea of the prevalence and clinical importance of del(5q) in myeloid malignancies, as it is likely that a number of cases have submicroscopic deletions of 5q that would only be detectable by molecular analysis. Early detection of individuals at risk for developing t·MDS/AML (post-chemotherapy or after mutagen exposure) might permit the early treatment and cure of some of these individuals. Lastly, the leukaemia suppressor gene might prove to be an ideal choice for 'gene therapy' approaches to treat an otherwise aggressive and incurable disease.
SUMMARY Deletions of chromosome 5 were initially reported as a consistently occurring chromosomal abnormality in 5q- syndrome. They have since been recognized to occur in other myeloid malignancies such as therapyrelated leukaemia and de novo AML as well. The variability of the deletions, and the heterogeneity of the clinical syndromes, have made it difficult to describe a single clinical-molecular entity such as we see with chromosomal translocations described elsewhere in this volume. Translocations in leukaemogenesis often have a dominant effect leading to activation of oncogenes or the production of a modified protein. Consistently occurring chromosomal deletions in human tumours, however, have been regarded as evidence that the affected regions contain tumour suppressor genes. Loss of function of these tumour suppressor genes or 'recessive oncogenes' leads to cancer. Deletions in the long arm of chromosome 5 in myeloid malignancies are thought to signal the existence of a recessive oncogene on 5q, which is homozygously inactivated in these malignancies. Here we describe the clinical and molecular features of the diseases associated with deletions of chromosome 5 in an attempt to propose a unified approach to identifying the genes on 5q which are involved in leukaemogenesis. It is likely that the clinical heterogeneity of these disorders will not be understood until the relevant genes are cloned and their role in the initiation or progression of leukaemia is known.
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SchifferCA, Lee EJ, Tomiyasu T et al (1989) Prognostic impact of cytogenetic abnormalities in patients with de novo acute nonlymphocytic leukemia. Blood 73: 26l-270. Stephens JC, Cavanaugh ML, Gradie MI ct al (1990) Mapping the human genome: Current status. Scienc« 250: 237-244. Sukhatrne VP, Cao X, Chang LD et al (1988) Azine finger-encoding gene coregulated v.ith e-fo« during growth and differentiation, and after cellular depolarization . CeU 53: 37-43. van den Berghe H, Cassiman JJ, David 0 et al (1974) Distinct haematological disorder with deletion of long arm of no. 5 chromosome, Nature 151: 437-438 . Vardiman JW, Coelho A, Golomb 11M & Rowley J (l9K3) Morphologic and cytochemical observations on the overt leukemia phase or therapy-related leukemia. AmericQII JoU11Ull
of Clinical Pathology 79: S2.S-5JO. Vogelstein B & Kinzler KW (1992) Carcinogens leave fingerprints. Nature355: 209-210. Wasmuth JJ, Bishop DT & Westbrook CA (1991) lIuman gene mapping: Report of the committee on the genet ic constitution of chromosome 5. C)'togenetia and CellGenetia 58:
1-34.
Myeloid malignancies andchromosome 5 deletions CAROL A. WESTBROOK MAURI J. KEINANEN
CLINICAL SYNDRO~IES ASSOCIATED WITH DELETIONS OF Sq Interstitial deletions of the long arm of chromosome 5 were first described in haematological disorders by van den Berghe et al in 1974, who reported deletions in three patients with long-standing refractory anaemia. Subsequently, many more cases were reported, and a distinct clinical picture has developed of a clinical entity which is known as the 5q- syndrome. These patients typically have refractory anaemia, usually macrocytic, and morphological abnormalities of their megakaryocytes. There is a predominance of older women. usually in their mid 60s. Most cases fit into the categories (Bennett et al, 1982) of refractory anaemia, refractory anaemia with excess blasts (RAEB) or refractory idiopathic sideroblastic anaemia (RISA). Progression to acute leukaemia is uncommon in this disorder, though it has been reported; chronic refractory anaemia is the typical clinical picture. The Sq- syndrome, as described above, is a rare clinical entity. Much more frequently. deletions of chromosome 5 are seen in a variety of haematological disorders with two unifying characteristics: they are myeloid disorders of trilineage involvement, and they are clinically aggressive. In particular. they include myelodysplastic syndrome (MOS), acute myeloid leukaemia (AML), and therapy-related MOS or AML (t-MOS/AML). The clinical aggressiveness of these disorders distinguishes them, as a whole, from the 5q- syndrome, and raises the question as to whether they involve the same gene(s) on chromosome 5. Leukaemia in association with MOS has been recognized to develop after cytotoxic therapy for cancer and .for non-malignant disease. Deletions of chromosome 5, or loss of an entire chromosome 5, are found in a large number of cases of patients who survive cancer treatment. The association was first reported by Rowley et al (1981), who reported that loss of an entire chromosome 5 or 7. or a deletion of the long arm of these chromosomesdel(5q) or del(7q}-was present in 23 of 26 cases of t-AMUMOS. (For the purpose of this ~hapter, we will use the terminology del(5q) and del(7q) to include cases With whole chromosome loss as well as those with interstitial deletions.) This high prevalence has been confirmed by other investigators, Bai/liir~'s Q inicalliatma(ologyVol. s. No.4. October1992 ISBN 0-7020-1691-8
931 . Copyright © 1992.by Bailliere Tindall All rights of reproduction in any form reserved
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C. A. WESTBROOK AND M. 1. KEINANEN
including our own updated series (Le Beau et al, 1986). In particular, chromosome 5 deletion or loss occurs in over 40% of cases, usually with concurrent loss or deletion of chromosome 7. Because of the frequent coexistence of del(5q) and del(7q) in t-MOS/AML, it has not been possible to ascertain whether either chromosomal abnormality has distinct clinical or morphological features; indeed, the syndrome may be related to a combined action of both mutations. In t-MOS, the del(5q) is usually present at diagnosis, and is found in all of the chromosomally abnormal cells examined at the time of diagnosis rather than at progression to AML (PedersenBjergaard et aI, 1990); nonetheless, additional cytogenetic abnormalities are common and it rarely occurs alone. It is interesting that del(5q) and/or del(7q) are highly correlated with exposure to alkylating agents (Pedersen-Bjergaard et al, 1990). Several large studies have examined the incidence of secondary leukaemia in survivors of Hodgkin's disease (Kaldor et al, 1990a) and ovarian cancer (Kaldor et al, 1990b). It was concluded that chemotherapy-but not radiation therapy-e-confers a 9- to 12-fold risk of developing leukaemia, with the worst offenders being the alkylating agents chlorambucil, melphalan, procarbazine and mustine (mechlorethamine USP). t-AML (usually with antecedent MOS) has been described as a single clinical entity. As for de novo AML, it is likely that the clinical picture is more closely related to the chromosomal changes rather than to the aetiologic agent; increasingly the features which were considered typical of t-AML are now being recognized as those associated with del(5q) and/or del(7q), as these are the overwhelmingly predominant cytogenetic abnormalities in t-AML (Pedersen-Bjergaard et ai, 1990). The presentation occurs with a latency of about 4 years after treatment, and usually begins as MOS. t-MDS typically presents with cytopenias, while the marrow is hypercellular or normocellular with characteristic changes in the megakaryocytes and trilineage involvement (Vardiman et ai, 1983). t-MDS is aggressive compared with de novo MDS, and many patients die of the complications of cytopenias-infections or haemorrhage. If the patient survives, the marrow blast count increases until the diagnosis of acute leukaemia is made, usually within 5 months of presentation of MDS. Survival after AML is shorter than it is in most cases of AML (median survival is less than 9 months) due to a low remission rate and short remission duration. Most investigators consider t-MDS and t-AML to be a continuum rather than discrete diseases, and we will use a combined term in this chapter as well. del(5q) isfound in about 10% of patients with AMLarising de novo (Larson et al, 1983; Fourth International Workshop on Chromosomes in Leukemia, 1984). In AML, other cytogenetic abnormalities often accompany the del(5q) (such as +8, -7 and -17), and a history of occupational exposure iscommon (Mitelman et al, 1978; Golomb et al, 1982; Brandt et al, 1983). As with t-AML, trilineage involvement iscommon, and evolution from a pre-existing MDS can often be demonstrated. Clinically these patients tend to be older and poorly responsive to chemotherapy compared with other cytogenetic subtypes (Larson et al, 1983; Fourth International Workshop on Chromosomes in Leukemia, 1984; Samuels et al, 1988; Schiffer et ai, 1989). For
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example, only 28% of del(5q) cases achieved complete remission in the Fourth International Workshop series, with a median survival of only 4 months. These findings prompted the suggestion that del(5q) cells are resistant to standard leukaemia chemotherapy (Samuels et al, 1988). del(5q) is considered to be one of the worst indicators of poor prognosis in AML, and a strong case can be made for the development of molecular markers for diagnosis, prognosis and therapy evaluation. del(5q) is also seen in cases of de novo MDS that are distinct from the 5qsyndrome; these cases have a wide variety of clinical manifestations. del(5q) in this setting is often associated with the more aggressive forms of MDS (RAEB , or refractory anaemia in transition) and may resemble t·MDS in clinical features and poor prognosis. Cytopenias are accompanied by a hypercellular marrow, and characteristic features of megakaryocytic dysplasia are present as well. In these cases, progression to acute leukaemia is rapid. Good cytogenetic and/or molecular descriptions of the various MDS syndromes are still lacking; one reason to study del(5q) is that it willlead to a better understanding of the MDS subtypes and their clinical features, and may help to evaluate prognosis in individual cases. Overall, a picture is beginning to emerge of del(5q) (Nimer and Golde, 1987) as a clonal stem-cell disorder, characterized by trilineage dysplasia, megakaryocytic abnormalities and cytopenias. Progression to leukaemia is common, and may occur prior to presentation, usually accompanied by additional genetic changes such as del(7q) . The relationship of the 5qsyndrome to these other cases is poorly understood. Whether 5qsyndrome involves a different gene on 5q from that involved in t-MDSt AML, or whether the accompanying changes on other chromosomes are important, will probably not be understood until the relevant genes are cloned and molecular markers are available. TUMOUR SUPPRESSOR GENES AND CHROMOSOMAL DELETIONS: DOMINANT AND RECESSIVE ONCOGENES The association of recurring chromosomal translocations with particular leukaemia subtypes led to the hypothesis that a specific gene is involved for each leukaemia subtype. A considerable amount of research effort has been directed toward cloning these translocation breakpoints, identifying the genes involved, and understanding their role in the leukaemic process. There are numerous examples in this volume. This data accumulated over the years had led to .the paradigm t~at chro~oso.mal translocations in leukaemia have a domm~nt effect, leadI~g to activation of an oncogene by transcriptional deregulation or production of a modified protein. Genes which are activat~d in t~i~ manner can be cons!dered 'dominant oncogenes' , because oncogemc activity occurs due to a single mutational event and is effective in spite of th~ presence of a normal copy of the gene. Chromosomal deletions, on the other hand, have received much less attention. This is partly because deletions, even those which occur consistently in a particular leukaemia, vary considerably in extent from patient
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C. A. WESTBROOK AND M. 1. KEINANEN
to patient; it is difficult to postulate a single, dominant-acting oncogene if a discrete chromosomal breakpoint cannot be identified. It is only with the concept of 'recessive oncogenes' evolving from research in solid tumours that a paradigm has been developed which can be applied to these deletions. The recessive oncogene model stipulates that it is the loss of function of a critical gene, rather than the positive activation of an oncogene, that is the determinant of cancer progression or initiation. Genes which lead to cancer in this manner must be considered 'recessive' because mutation is required at both alleles before the effect is fully manifest. The classic example of a recessive oncogene is the RB1 gene in retinoblastoma (Friend et ai, 1989). Genetic evidence postulated a two-step mechanism (Cavenee et ai, 1983), and eventual cloning of the gene confirmed the theory, showing that mutational inactivation or deletion occurs successively at both alleles of the RB1 gene, leading to complete cessation of gene activity. There are now several other recessive oncogenes which have been identified, all from solid tumours. These include the DCC gene in colorectal cancer (Fearon et al, 1990), the p53 gene in a variety of cancers (Nigro et ai, 1989), the Wilms' tumour gene (Call et ai, 1990;Gessler et ai, 1990) and recently the APC gene in familial adenomatous polyposis, a form of hereditary colorectal cancer (Groden et al, 1991). Although homozygous loss has not been confirmed for each of these genes, in every case mutations have been found which demonstrate loss or inactivation of at least one allele. Consistently occurring deletions in the long arm of chromosome 5 in myeloid malignancies postulate the existence of a recessive oncogene or 'antileukaemia' gene (ALG) on 5q which is homozygously inactivated in these malignancies. A molecular hypothesis must be consistent with the clinical and cytogenetic features that are observed. Thus, we expect to find that this gene is located within the visibly deleted region of every case of del(5q) that meets the clinical criteria studied. A two-step mechanism is in accordance with the long latent period of 4 years (varies between 2-5 years) observed in therapy-related leukaemia, in contrast to the relatively short latency observed with the development of t-AML with translocations at llq23 (Pedersen-Bjergaard et ai, 1990), which may be a single-step mechanism (see Chapter 6). Although it is not strictly possible to rule out a one -step mechanism for del(5q), in which transformation is caused by loss of only one allele (gene-dosage effect), there have been no demonstrated examples among the known recessive oncogenes. If the ALG functions as a typical recessive oncogene, it is expected to be inactivated on both chromosomes; in one instance by a visible deletion or chromosomal loss (including allelic deletion), in the other by a smaller event. As with other recessive oncogenes such as RBI (Friend et al, 1989) and Dee (Fearon et al, 1990), this event on the residual, non-deleted homologue might be a point mutation or a larger structural abnormality (homozygous deletion or internal rearrangement); on occasion functional loss will instead be caused by other mechanisms such as translocation. The types of mutations found might be random, but more than likely will have some relevance to the mutagenizing agent (Vogelstein and Kinzler, 1992). It
MYELOID MALIGNANCIES AND CHROMOSOME 5 DELETIONS
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is the smaller event on the non-deleted chromosome that will eventually help to identify the gene. In the remainder of this chapter, we will review the present state of knowledge about the cytogenetic definition of del(5q) and the extent of the deleted regions in relation to the current genome map, and discuss possible candidate genes which lie within this region. CYTOGENETIC DELINEATION OF CHROMOSOME 5 DELETIONS Cytogenetic studies have attempted to define the breakpoints and deletion extent in patients with 5q- syndrome, t-MDS/AML or de novo AML. These deletions are interstitial, that is, two breaks occur in the long arm with loss ofthe intermediate segment. Furthermore, the deletion extent is similar in most cases, with a distal breakpoint usually in q33-35 and a proximal breakpoint in ql3-15. By comparing the cytogenetic overlap in deletions from 17 cases of t-AMUMDS, Le Beau et al (1986) suggested there was a
P
1 2
1
q
2 I HDSIAHL del 5Q31
5Q- syndrome del 5Q22-33
3
Figure 1. Minimal overlapping region for deletions of chromosome 5 in myelo id malignancies. Depicted is an ideogram of chromosome S. Next to the chromosome, the smallest overlapping region is shown for t-MDSlAML and de novo MDS/AML (5q31) and for Sq- syndrome (Sq22-33). Based on Le Beau et al (1989).
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C. A. WESTBROOK AND M. J. KEINANEN
much smaller region that was commonly deleted in all patients, which included 5q23-Sq31. This series was later expanded to 80 cases, which included Sq- syndrome as well as de novo and t-MOS/AML (Le Beau et aI, 1989). The inclusion of de novo and t-AML cases with distal (three patients) or proximal (two patients) breakpoints in 5q31, made it possible to define a smaller overlapping region of deletion which comprised the proximal half of band 5q31. The minimal region in the Sq- syndrome was considerably larger, extending from 5q22 through to Sq33, but only 12 cases were included and all had large deletions. The smaller deletion region defined for de novo and t-MOS/AML is completely contained within the larger region defined for the Sq- syndrome (see Figure 1). Thus, it is not possible on cytogenetic grounds to determine if these closely related diseases involve different genes. Such a distinction may not be possible until the involved genes are cloned and the syndromes can be defined on a molecular level. RELATIONSHIP OF THE GENOME MAP TO CHROMOSOME 5 DELETIONS Since the total length of chromosome 5 has been estimated to be about 160Mb, based on the percentage of the total genome measured from ISeN (International System for Human Cytogenetic Nomenclature) idiograms (Stephens et ai, 1990), and band 5q31 is about one-tenth of the chromosome, the minimal deleted region for the ALG is about 16 Mb, and for the 5q - region is about 40 Mb. These are both extremely large regions containing many genes; it will not be easy to find the ALG unless the regions are better defined on a molecular level, and further refined to smaller regions. To date, little has been published attempting to molecularly define these deletions, that is, to demonstrate the inclusions of markers both within and outside of the critically deleted region. What is particularly lacking is a direct comparison, using molecular markers, of cytogenetic deletions seen in these two clinically disparate entities, 5q- syndrome and del(5q) in MOS/AML. It is hoped that additional information will result from the recent increase of probes and markers available for this region of chromosome 5 resulting from human genome efforts (Wasmuth et aI, 1991). One approach is to compare the location of known genes and markers with the deletion extent. Although this is a rough approach, relying as it does on cytogenetic interpretation, it is a rapid way of identifying genes which lie approximately in the critical region and might be considered as candidates for recessive oncogenes. The current map of chromosome 5 contains many genes and markers on 5q. The cloned genes which lie within or near the minimal deleted regions are listed in Table 1; this list includes over 30 genes. There are a number of genes that are of particular interest in this list. Most striking, perhaps, is that two known recessive oncogenes, MCC and APC are located at 5q21-22. These genes were both identified by their involvement in colorectal cancer, APe in familial and sporadic colorectal cancer, and M CC in sporadic cancers only. More recent data position APC, and the closely related MCC, in 5q21, and as such it lies outside of the 5q- region
937
MYELOID MALIGNANCIES AND CHROMOSOME 5 DELETIONS
Table 1. Genes localizedto the distal (q21-qter) long arm of chromosome 5. Symbol
Gene name
Location·
FER APC MCC CAMK4 CD14 IL9 CD49B CSF2 EGRI FBN2 IL3 IL4 IL5 IRFI ADRAIB PDEA FGFA TCF7 ADRB2 GRL DHLAG RPS14 SPARC ARH9 ADRAIAC ANX6 GAPDL16 CSFI CSFIR PDGFRB F12 FGFR4 DRDI GABRAI CHR
Fer (fps/fesrelated) tyrosine kinase Adenomatosis polyposiscoli Mutated in colorectal cancers Ca2+/calmodulin dependent protein kinase IV Antigen CD14 Interleukin 9 Antigen CD49B (a2 subunit of VLA·2 receptor) Colony-stimulating factor 2 (granulocyte-macrophage) Early growth response 1 Fibrillin 2 Interleukin 3 Interleukin 4 Interleukin 5 Interferon regulatory factor 1 Adrenergic, alB receptor Phosphodiesterase, cyclicGMP (rod receptor) a-polypeptide Fibroblast growth factor, acidic (endothelial growth factor) Transcription factor 7 (T-cell specific) Adrenergic.Bs receptor, surface Glucocorticoid receptor Major histocompatibility complex, class II, gamma polypeptide Ribosomal protein S14 Secreted protein, acidic, cysteine-rich(osteonectin) Aplysia ras-related homologue 9 Adrenergic alA receptor Annexin VI (p68) Glyceraldehyde·3-phosphate dehydrogenase-like 16 Colony-stimulating factor 1 (macrophage) CSFl receptor (formerly FMS) Platelet-derived growth factor receptor, ~-polypeptide Coagulation factor XII (Hageman factor) Fibroblast growth factor receptor 4 Dopamine receptor 01 'Y-Aminobutyric acid (GABA) A receptor, al Chromate resistance; sulphate transport
q21 q21-22 q21-22 q21-33 q22-32 q22-32 q23-31 q23-31 q23-31 q23-31 q23-31 q23-31 q23-31 q23-31 q23-32 q31.2-34 q31.3-33.2 q31 q31-32 q31-32 q31-33 q31-33 q31-33 q31-qter q32-34 q32-34 q33.1-35.3 q33 q33-35 q33-35 q33-qter q33-gter q34-35 q34-35 q35
• For complete citations on the genes and their locations, refer to Wasmuth et al (1991).
and is clearly far from the ALG region of 5q31. However, the gene has not been systematically studied for mutations in leukaemias. The long arm of chromosome 5 is particularly rich in growth factor genes and receptor protein genes. These include genes for haematopoietic growth factors such as interleukin (IL) 3, 4, 5 and 9, colony-stimulating factor (CSF) 2 fibroblast/endothelial cell growth factor, and haematopoiesis-related r;ceptors (glucocorticoid receptor, CSFI receptor and platelet-derived growth factor receptor J3). ~ere are a large number of genes for neurotransmitter recepto!s, including the alphaj , alpha; and beta, adrenergic receptors, the 'Y-ammobutyncacid (~ABA) receptor, the vanillylmandelic acid (VMA) receptor and the dopamine receptor. Also of note is the EGRI gene, which encodes a gr~'Yth regulatory protein with DNA-binding transcriptional regulatory activity (Sukhatme et al, 1988) and genes encoding
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C. A. WESTBROOK AND M. J. KEINANEN
transcription factor 7 (Tvcell-specific), interferon regulatory factor 1, and the myeloid surface protein CD14. Based on their biological activity, many of these genes are good candidates for the ALG; thus, it will be important to determine which of these are consistently present within deletions and, of these, which are frequently mutated in del(5q) cases. There are an additional60-odd anonymous DNA segment markers which also map to this region, including many RFLPs (Restriction Enzyme Length Polymorphism) and CA (dinucleotide) repeats. These DNA markers will be extremely useful in molecular delineation of the deletions. A large number of probes have been cytogenetically localized by our group as a first step in analysing deletions of chromosome 5 (Wasmuth et al, 1991;M. M. Le Beau, W. L. Neuman, M. Dixon et ai, unpublished data; W. L. Neuman, C. A. Westbrook, B. Weiffenbach et ai, unpublished data). These probes will be mapped against the deletions in individual patients to better define the regions involved. CANDIDATE GENES
Undoubtedly, many genes are under active investigation as candidates for the ALG, but little has been published to date. Among the first genes suggested as candidates for the ALG were the interleukin genes located at 5q23-31 (Nienhuis et ai, 1985; Le Beau et ai, 1986, 1989). We examined some of these genes for structural mutations, as our hypothesis suggests that the 'normal' chromosome 5 homologue will contain an inactivating mutation within the ALG when the other chromosome contains a del(5q). At least some of these deletions should be large enough to be seen by pulsed field gel electrophoresis or Southern blots. In a small study, pulsed field gel electrophoresis was used to examine the leukaemic cells of cases with del(5q) or translocations at 5, compared with their control Iymphoblastoid cell lines. This included three patients with MDS and del(5q), and one with t·AML and an inversion involving a breakpoint at 5q31. With probes for IL4 and csn we did not observe any rearrangements in this group with the enzymes NotI and NruI (Le Beau et ai, 1989). EGR1 and eD14 were also applied to these blots, but no rearrangements were seen (unpublished data). These preliminary studies do not rule out any of these genes as candidates, and it will be necessary to investigate many more cases to find a gross rearrangement visible by Southern blot methods. Based on experience with retinoblastoma, this may be up to 30% of cases (Friend et ai, 1989); on the other hand, with the Dee gene the number with structural abnormalities within the DeC gene visible on Southern blot was lower, at only about 15% (Fearon et ai, 1990). Such studies are still warranted with potential candidates. However, a necessary selection for these candidate genes is that they be located within the minimal deleted region. It is difficult to postulate a role for the interleukins as tumour suppressor genes, since they are growth factors for myeloid cells, and would more likely function as dominantly acting oncogenes. This is, in fact, the case with the
MYELOID MAUGNANCIES AND CHROMOSOME 5 DELETIONS
939
t(5;14)(q31 ;q32) translocation occurring in B-lineage acute lymphoblastic leukaemia with eosinophilia (Grimaldi and Meeker, 1989; Meeker et ai, 1990). Here, the juxtaposition of the immunoglobulin gene enhancer from chromosome 14 to the IL3 gene results in excess production of this growth factor and consequent myeloid hyperproliferation. More intriguing is recent work examining a possible role for the gene CSFlR (formerly EMS, v-fms homologue) in 5q- syndrome. This gene codes for the receptor for the growth factor CSF1, and a role might be postulated as a tumour suppressor gene, although v-fms is dominantly acting in its role as a viral oncogene (Roussel et ai, 1984). Boultwood et al (1991) examined ten patients with Sq- syndrome or MDS and del(Sq) with deletions inclusive of 5q33. All cases had lost at least one allele of the gene FMS, presumably within the deletion; however, five cases had evidence for loss of the other copy of the gene in at least some myeloid cells. This finding was based on densitometry and in situ hybridization to cell specimens containing mixed normal and malignant cells, and will require confirmation on pure populations of cells. Nonetheless, this is the first report of a gene which might satisfy two of the criteria for a tumour suppressor gene: that is, it is located within the visible deletion, and it is inactivated on the nondeleted homologue. Arguing against a role for CSF1R in del(5q) is the cytogenetic analyses by Le Beau (1989) showing that a fraction of deletions have their distal boundary proximal to 5q33, demonstrating that CSFlR is not consistently lost when deletions are present. One such case was studied by in situ hybridization using the CSFlR probe, which showed that the gene was present on both chromosomes. This case represented a 'classical' t-AML, and the karyotype showed a majority of cells that had -7 in addition to a del(S)(q22q33.1). One possible explanation for these discrepant findings is that these studies are addressing two different clinical populations, and that two distinct chromosomal loci might be involved. Boultwood et al (1991) have confined their analysis in refractory anaemia and Sq- cases. Le Beau's population with smaller deletions is predominantly del(5q) associated with de novo or t-AML, whereas a1l5q- cases have deletions which extend through to Sq33 or 34. Clearly it will be necessary to examine many more cases to specify the deletion extent relative to CSFlR in both syndromes, and to attempt to define the clinical populations in terms of molecular markers.
CLINICAL IMPLICATIONS AND POTENTIAL USES FOR MOLECULAR MARKERS OF DEL(Sq) Cloning the genes related to structural chromosomal abnormalities has invariably provided useful clinical markers and contributed to the understanding o~ the. b~sic processes o~ leukaemogenesis. The practical implications of identifying the Ieukaemia-related genes on 5q are considerable. First of all, the relationship of 5q - syndrome to other malignancies would
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C. A. WESTBROOK AND M. J. KEINANEN
be clarified. Useful DNA probes might become available that would complement cytogenetic detection in allowing the diagnosis of poor prognosis AML, monitoring the effects of treatment, and the detection of residual disease after remission or bone marrow transplantation. Similar considerations apply in MDS. Studies of larger clinicalpopulations would give an idea of the prevalence and clinical importance of del(5q) in myeloid malignancies, as it is likely that a number of cases have submicroscopic deletions of 5q that would only be detectable by molecular analysis. Early detection of individuals at risk for developing t·MDS/AML (post-chemotherapy or after mutagen exposure) might permit the early treatment and cure of some of these individuals. Lastly, the leukaemia suppressor gene might prove to be an ideal choice for 'gene therapy' approaches to treat an otherwise aggressive and incurable disease.
SUMMARY Deletions of chromosome 5 were initially reported as a consistently occurring chromosomal abnormality in 5q- syndrome. They have since been recognized to occur in other myeloid malignancies such as therapyrelated leukaemia and de novo AML as well. The variability of the deletions, and the heterogeneity of the clinical syndromes, have made it difficult to describe a single clinical-molecular entity such as we see with chromosomal translocations described elsewhere in this volume. Translocations in leukaemogenesis often have a dominant effect leading to activation of oncogenes or the production of a modified protein. Consistently occurring chromosomal deletions in human tumours, however, have been regarded as evidence that the affected regions contain tumour suppressor genes. Loss of function of these tumour suppressor genes or 'recessive oncogenes' leads to cancer. Deletions in the long arm of chromosome 5 in myeloid malignancies are thought to signal the existence of a recessive oncogene on 5q, which is homozygously inactivated in these malignancies. Here we describe the clinical and molecular features of the diseases associated with deletions of chromosome 5 in an attempt to propose a unified approach to identifying the genes on 5q which are involved in leukaemogenesis. It is likely that the clinical heterogeneity of these disorders will not be understood until the relevant genes are cloned and their role in the initiation or progression of leukaemia is known.
REFERENCES Bennett JM, Catovsky D, Daniel MT et al (1982) Proposals for classification of the myelodysplastic syndrome. British Journal of Haematology 51: 189-199. Boultwood J, Rack K, Kelly S et al (1991) Loss of both CSFlR (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion. Proceedings of the National Academy of Sciences ofthe USA 88: 617Cr6180. Brandt L, Mitelman F & Nilsson PG (1983) Chromosome pattern and survival in acute non-lymphocytic leukemia in relation to age and occupational exposure to potential mutagenidcarcinogenic agents. Scandinavian Journal of Hematology 30: 227.
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