Genetics of therapy-related myelodysplasia and acute myeloid leukemia

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1 (2008) 22, & 2008 Nature Publishing Group All rights reserved /08 $ REVIEW Genetics of therapy-related myelodysplasia and acute myeloid leukemia J Pedersen-Bjergaard, MK Andersen, MT Andersen and DH Christiansen Department of Clinical Genetics, The Cytogenetic Laboratory, Rigshospitalet, Copenhagen, Denmark Myelodysplasia (MDS) and acute myeloid leukemia (AML) are heterogeneous, closely associated diseases arising de novo or following chemotherapy with alkylating agents, topoisomerase II inhibitors, or after radiotherapy. Whereas de novo MDS and AML are almost always subclassified according to cytogenetic characteristics, therapy-related MDS (t-mds) and therapyrelated AML (t-aml) are often considered as separate entities and are not subdivided. Alternative genetic pathways were previously proposed in t-mds and t-aml based on cytogenetic characteristics. An increasing number of gene mutations are now observed to cluster differently in these pathways with an identical pattern in de novo and in t-mds and t-aml. An association is observed between activating mutations of genes in the tyrosine kinase RAS BRAF signal-transduction pathway (Class I mutations) and inactivating mutations of genes encoding hematopoietic transcription factors (Class II mutations). Point mutations of AML1 and RAS seem to cooperate and predispose to progression from t-mds to t-aml. Recently, critical genetic effects underlying 5q / 5 and 7q / 7 have been proposed. Their association and cooperation with point mutations of p53 and AML1, respectively, extend the scenario of cooperating genetic abnormalities in MDS and AML. As de novo and t-mds and t-aml are biologically identical diseases, they ought to be subclassified and treated similarly. (2008) 22, ; doi: /sj.leu ; published online 17 January 2008 Keywords: therapy-related myelodysplasia; therapy-related acute myeloid leukemia; 7q / 7; 5q / 5; Class I mutations; Class II mutations Introduction For more than two decades, patients with myelodysplasia (MDS) or acute myeloid leukemia (AML) have been classified and treated according to their cytogenetic characteristics, which represent the most important prognostic factor of these diseases. 1 5 Three major cytogenetic subgroups are considered. The first includes patients with the recurrent unbalanced chromosome aberrations. Loss of whole chromosome 5 or 7 ( 5/ 7) or various parts of the long arms of the two chromosomes (5q /7q ) or gain of a whole chromosome 8 ( þ 8) are the most common unbalanced aberrations. The abnormalities of chromosomes 5 and 7 are often associated with a complex karyotype, including other recurrent and nonrecurrent chromosome aberrations. Such patients are often elderly, present as MDS and have a poor prognosis. The second subgroup comprises patients with recurrent balanced chromosome aberrations, mainly reciprocal translocations. These are often observed as sole abnormalities, in younger patients and in cases presenting as overt AML. Many balanced aberrations confer a favorable prognosis. Finally, the third subgroup Correspondence: Dr J Pedersen-Bjergaard, Department of Clinical Genetics, the Cytogenetic Laboratory, Section 4052, Rigshospitalet, Blegdamsvej 9, Copenhagen 2100, Denmark. chromjpb@rh.dk Received 19 November 2007; accepted 27 November 2007; published online 17 January 2008 includes patients with a normal karyotype. They present as either MDS with a favorable prognosis 5 or as AML with an intermediate prognosis. 1 4 Whereas the critical genetic effects underlying 5q / 5, 7q / 7 and most other recurrent unbalanced aberrations until recently have remained unidentified, the recurrent balanced translocations and inversions have consistently been shown to result in chimeric rearrangement between genes encoding hematopoietic transcription factors, such as AML1, CBFB, MLL, RARA or EVI1 and their many alternative partner genes. 6,7 These changes characteristically result in a dominant loss of function of the transcription factor (Class II mutations) with defects in differentiation and increased self-renewal. Experiments in mice have shown that the chimeric genes may lead to development of MDS- or leukemia-like diseases. 8 Patients with a normal karyotype were previously poorly characterized with respect to genetic changes. More recently, particularly in this subgroup of patients with AML, mutations of the FLT3 gene encoding a receptor tyrosine kinase, 9 14 of the CEBPA gene encoding a hematopoietic transcription factor and of the NPM1 transcription regulating gene have been observed. In leukemogenesis, a cooperation between two general types of gene mutations have been proposed (Figure 1). 24,25 Class I mutations result in constitutive activation of receptor tyrosine kinases or genes downstream in the RAS BRAF MEK ERK signal-transduction pathway, thereby stimulating cell cycling and proliferation. Class II mutations, on the contrary, inactivate hematopoietic transcription factors and result in loss of differentiation. Therapy-related MDS (t-mds) and AML (t-aml) developing after chemotherapy or radiotherapy of a primary disease are of particular interest to study. First, these diseases represent the most serious long-term complication to cancer therapy. Second, as will be discussed below, they share cytogenetic abnormalities and gene mutations with de novo MDS and AML, allowing to extrapolate between observations in the two subtypes of disease. Third, they offer insight into the etiology of MDS and AML, as their cytogenetic abnormalities and gene mutations in many cases are directly related to specific cytotoxic exposures Whereas alkylating agents characteristically induce t-mds with the chromosome defects 5q/ 5 or 7q / 7 and associated genetic abnormalities, topoisomerase II inhibitors induce overt t-aml with one of the recurrent balanced translocations or inversions. 28 Fourth, a close clinical follow-up after intensive therapy for the primary tumor results in a high diagnostic accuracy of t-mds and allows to study the entire evolution from cytotoxic exposure to development of overt leukemia. To obtain further insight into leukemogenesis, we have reevaluated and summarized the results of our previous studies of chromosome abnormalities and mutations of 16 different genes in a cohort of 140 patients with t-mds or t-aml from the Copenhagen series. In particular, associated, possibly cooperating mutations and their relations to stages of the disease are

2 241 Figure 1 Signal-transduction pathways and classification of mutations in MDS and AML. analyzed in the light of recent research disclosing critical genetic consequences of 5q /5 and 7q /7. Finally, the results are compared to those of other studies of therapy-related and de novo MDS and AML. Patients and genes previously studied in the Copenhagen series Cryopreserved bone marrow cells obtained at the time of diagnosis from 89 patients with t-mds and 51 patients with t-aml were studied. The patients were unselected, as they represent all cases with the two diagnoses observed at our institution for more than 20 years, and material still available for examination. The detailed clinical and cytogenetic characteristics of 130 patients have been presented previously, whereas data for the remaining 10 cases are unreported. The genes studied for mutations include the receptor tyrosine kinases FLT3, ckit and cfms and the intracellular non-receptor tyrosine kinase JAK2, together with KRAS, NRAS, BRAF and PTPN11 genes in the RAS BRAF MEK ERK signal-transduction pathway (Figure 1, Table 1). Furthermore, the transcription factors AML1, CBFB, MLL, RARA, EVI1 and CEBPA, the transcription regulatory NPM1 gene and the tumor suppressor gene p53 were studied. Methods used for mutation detection and primary data have been published elsewhere Mutations and internal tandem duplications were all confirmed by sequencing. Chimeric gene rearrangements in cases with the recurrent balanced chromosome aberrations were all confirmed by fluorescent in situ hybridization using breakpoint-specific probes and, in some cases, by supplementary sequencing. Frequencies and associations of gene mutations observed in the Copenhagen series of t-mds and t-aml Point mutations of p53 Point mutation of p53, often with loss of heterozygosity, was the most common genetic abnormality observed in 34/140 cases of t-mds or t-aml (Table 1). 29,36 This mutation was highly significantly related to the cytogenetic defects 5q / 5 (Table 2), as 26/34 patients with p53 mutations presented 5q / 5, to previous therapy with alkylating agents 29 and to complex karyotypes with highly rearranged chromosomes. 37 Duplication or amplification of chromosome bands 11q23 and 21q22, including the unrearranged MLL and AML1 genes, were occasionally observed in t-mds or t-aml 38,39 and closely associated with mutations of p53 (Figure 2). Point mutations of AML1 These mutations were the second most frequent genetic abnormality detected in 22/140 patients with t-mds or t-aml1 (Table 1). 31 Mutations of AML1 were significantly related to previous therapy with alkylating agents, to presentation of the disease as t-mds and to progression from t-mds to t-aml. With high significance, mutations of AML1 were associated with the cytogenetic defects 7q / 7 (Table 2), 31 as 17/22 patients with AML1 point mutations presented 7q / 7. Five patients with AML1 point mutations also presented a RAS mutation (Figure 2) similarly related to progression from t-mds to t-aml. 32 As shown in Table 2, there was no significant overlapping between the pathway defined by 5q / 5 þ p53 mutations and the pathway defined by 7q / 7 þ AML1 point mutations.

3 242 Mutations of genes encoding tyrosine kinases Point mutations or internal duplications of such genes were observed in 15/140 patients (Table 1). 30,32,33 They were mutually exclusive (Figure 2), except for one patient presenting two different FLT3 mutations simultaneously, possibly in different subclones. In our series, mutations of FLT3 were related to previous radiotherapy but not to chemotherapy and were often observed in patients with a normal karyotype. 30,32 FLT3 and ckit mutations were related the to presentation of the disease as overt t-aml, 32 whereas two patients with JAK2 mutations presented as t-mds. 33 Mutations of cfms were not Table 1 Gene mutations observed in 140 patients with t-mds (n ¼ 89) or t-aml (n ¼ 51) (The Copenhagen series) t-mds t-aml Class I mutations Tyrosine kinases FLT3 ITD+point mutations 1 10 ckit point mutations 0 2 cfms point mutations 0 0 JAK2 point mutations 2 0 Genes in the RAS/BRAF pathway KRAS or NRAS point mutations 7 7 a BRAF point mutations 0 3 a PTPN11 point mutations 2 2 Class II mutations Transcription factors AML1/CBFB chimerically rearranged 3 7 AML1 point mutations 20 2 MLL chimerically rearranged 0 11 MLL ITD 1 1 RARA chimerically rearranged 0 2 EVI1 chimerically rearranged 3 1 CEBPA point mutations 0 0 NPM1 point mutations 3 7 Tumor suppressor gene p53 point mutations 25 9 Total 131 mutations observed Abbreviations: t-aml, therapy-related acute myeloid leukemia; t-mds, therapy-related myelodysplasia. a One patient had a mutation of KRAS together with a mutation of BRAF possibly in different subclones. observed. Two patients with ckit mutations presented as t-aml, in one case associated with a t(8;21). The two patients with JAK2 mutations were uncharacteristic cases of t-mds disclosing a myeloproliferative pattern with splenomegaly. Mutations of genes downstream in the RAS/BRAF signal-transduction pathway Such mutations were observed in 20/140 patients (Table 1) 32,34 and were mutually exclusive except for one case presenting a KRAS and a BRAF mutations simultaneously (Figure 2) and another case presenting two different KRAS mutations. 32 In both patients, the two mutations were observed in subclones. Mutations of RAS did not show associations to any specific type of previous therapy and were often observed in atypical cases with a normal karyotype, perhaps representing de novo MDS or AML. 32 All three patients with BRAF mutations and 3/14 patients with RAS mutations presented as overt t-aml of FAB (French American British) subtypes M4 or M5 with chimeric rearrangement of the MLL gene (Figure 2). All four patients with mutations of PTPN11 presented rare balanced translocations with chimeric rearrangement of the EVI1 gene (two cases) or of the AML1 gene (two cases), all with unknown partners. 34 Similar to point mutations of AML1, mutations of RAS were significantly related to progression from t-mds to t-aml. 32 Mutations of transcription factors A total of 61 mutations of genes involved in hematopoietic transcription and differentiation were observed in 59 patients with t-mds or t-aml, if classifying mutations of the NPM1 gene together with the mutations of transcription factors (Class II mutations; Table 1). Two patients presented a balanced t(3;21) with chimeric rearrangement between the AML1 and the EVI1 gene. Except for these two cases, mutations of transcription factors were mutually exclusive (Figure 2). Mutations of CEBPA were not observed. The chimeric rearrangements of AML1, CBFB, MLL, RARA and EVI1 were significantly related to previous therapy with topoisomerase II inhibitors. Mutations of NPM1 were not related to any specific type of previous therapy and often seen in patients with a normal karyotype (7/10 cases). 35 Patients with chimeric rearrangement of AML1, CBFB, MLL or RARA and patients with NPM1 mutations primarily presented as Table 2 Point mutations of p53 and AML1 related to the cytogenetic defects 5q / 5 and 7q / 7 in 140 cases of t-mds or t-aml (The Copenhagen Series) p53-mutated, AML1 germline p53 and AML1 mutated AML1-mutated, p53 germline p53 and AML1 germline Total 5q / 5 normal chromosomes q / 5 and 7q / q / 7 normal chromosomes Normal chromosomes 5 and Total Abbreviations: t-aml, therapy-related acute myeloid leukemia; t-mds, therapy-related myelodysplasia. Mutations of p53 (n ¼ 34) and 5q / 5 (n ¼ 34) associated in 26 cases (Po0.0001*). Mutations of AML1 (n ¼ 22) and 7q / 7 (n ¼ 63) associated in 17 cases (P ¼ 0.002*). 5q / 5 (n ¼ 34) and 7q / 7 (n ¼ 63) associated in 18 cases (P ¼ 0.38*). Mutations of p53 (n ¼ 34) and mutations of AML1 (n ¼ 22) associated in five cases (P ¼ 0.46*). Mutations of p53 (n ¼ 34) and 7q / 7 (n ¼ 63) associated in 19 cases (P ¼ 0.17*). Mutations of AML1 (n ¼ 22) and 5q / 5 (n ¼ 34) associated in four cases (P ¼ 0.59*). *Pearson s w 2 -test with Yates correction.

4 243 Figure 2 Associated, possibly cooperating gene mutations observed in the Copenhagen studies of t-mds and t-aml., tyrosine kinases;, RAS/BRAF signaling pathway;, transcription factors;, p53 tumor suppressor gene; &, number of cases with a Class I þ a Class II mutation; J, number of cases with either two Class I or two Class II mutations; cr, chimeric rearrangement; pm, point mutation; ITD, internal tandem duplication; dup, duplication; ampl, amplification; therapy-related MDS, t-mds; therapy-related AML, t-aml. overt t-aml, whereas rearrangements of EVI1 and point mutations of AML1; as discussed above, were most common in t-mds (Table 1). Cooperation between Class I and Class II mutations An association, possibly indicating cooperation, was observed between Class I and Class II mutations (Figure 2). In total, 35/140 patients with t-mds or t-aml presented a Class I mutation, 59/140 patients presented a Class II mutation, whereas 25 patients disclosed a mutation of both classes of genes simultaneously (P ¼ , Pearson w 2 -test with Yates correction). The associations between point mutations of AML1 and RAS, between mutations of NPM1 and FLT3, between mutations of MLL and RAS and between mutations of MLL and BRAF were most frequently identified. In addition, individual cases with many other combinations between mutated genes of the two classes were observed (Figure 2). Critical genetic effects of 5q / 5, 7q / 7, and p53 mutations New studies of the molecular effects In a recent study, Dr Neal Young and his group at the National Cancer Institute observed a constitutive activation of the STAT1/ STAT5 signal-transduction pathway in bone marrow cells from cases of MDS with monosomy 7 after GCSF stimulation. 40 This effect was related to an overexpression of isotype 4 of the GCSF receptor, which failed to internalize following GCSF binding. No explanation was provided of the association between the cytogenetic defect and the shift in isoform of the receptor. If confirmed also in patients with MDS and 7q-, and in patients with AML and 7q / 7, the critical lesion of 7q / 7 is probably equivalent to a Class I activating mutation of a tyrosine kinase. As far as the important genetic effects of 5q / 5 are concerned, haploinsufficiency of the EGR1 gene was recently emphasized as a candidate abnormality. 41 In another study, haploinsufficiency together with promoter methylation of the CTNNA1 gene was considered. 42 Both genes are located within the commonly deleted region on chromosome band 5q31.2. Whether one of these changes, their combination or abnormalities of other genes located within this region is responsible for the decisive genetic effect of 5q / 5 remains to be determined. In solid tumors, p53 has repeatedly been demonstrated as a tumor suppressor gene of importance for genomic stability. Its roles in normal hematopoiesis and in leukemogenesis were previously almost unknown. A recent study, however, demonstrated p53 as mediating quiescence of normal hematopoietic stem cells. 43 Despite this limited knowledge, the biological effects of p53 seem to differ markedly from those of the genes involved in Class I and Class II mutations in AML. Consequently, p53 mutations most likely represent a new Class III type of mutation in MDS and AML, to which also mutations of WT1 may belong. Re-evaluation of combinations of gene mutations in the Copenhagen Series On the basis of the associations between 7q / 7 and chimeric rearrangement or point mutation of AML1, as observed in many studies, we previously predicted 7q / 7 to represent a Class I mutation-like abnormality in leukemogenesis. 36 This now seems to be supported. 40 In an attempt to characterize the genetic effects of 5q / 5 and mutations of p53 in a similar way, we searched for their partner chromosome aberrations or partner mutations of Class I or Class II. In patients with 5q / 5 plus mutated p53, the only recurrent partner abnormality was 7q / 7, observed in 14/26 cases (Table 3, central column). This association is not significantly different from what could be expected by chance, as in total 63/140 patients with t-mds or t-aml presented defects of chromosome arm 7q. Noteworthy, only one of the 14 cases with the combination 5q / 5 þ p53 mutation þ 7q / 7 presented an additional AML1 point mutation.

5 244 In the subgroup of 16 patients with 5q / 5 but germline p53, or with mutated p53 but normal chromosomes 5 (Table 3, left þ right columns), 8/10 cases with other abnormalities presented combinations between well-defined Class I and Class II mutation-like abnormalities. Taken together, these observations do not support 5q / 5 or mutations of p53 to be classified as either Class I or Class II mutation equivalents in leukemogenesis. In conclusion, two general types of mutations (Class I and Class II) predominate and possibly cooperate in MDS and AML. The cytogenetic defects 7q / 7 seem to participate in leukemogenesis representing Class I-like types of abnormalities. 5q / 5 and mutations of p53 seem to represent an alternative combination not interfering or overlapping with Class I or Class II abnormalities. If one of the partners 5q / 5 or mutated p53 is lacking in the combination, it is often replaced by combined Class I and Class II mutations of other genes. The cooperation between different classes of genes may be much more extensive in MDS and AML than previously believed, if also considering chromosome aberrations such as 5q / 5 and 7q / 7 in the system. Chromosome aberrations and gene mutations in therapy-related vs de novo MDS and AML Spectrum and frequencies of chromosome abnormalities As t-mds and t-aml often develop with a more than 100-fold increased risk after highly mutagenic exposures, the pattern and frequency of chromosome aberrations and gene mutations of these patients could be expected to differ markedly from those of patients with de novo MDS and AML without knowledge of similar exposures. This, however, is not the case, in particular, if considering each cytogenetic subgroup separately. Identical recurrent unbalanced and balanced chromosome aberrations are observed in de novo 1 5 and in therapy-related Table 3 Associations between 5q / 5, p53 mutations and other important genetic abnormalities in 140 patients with t-mds or t-aml 5q / 5, germline p53: 4/8 other abnormalities 5q / 5+p53 point mutations: 15/26 other abnormalities Normal chromosome 5, p53 point mutations: 6/8 other abnormalities n n n AML1+RAS mutation 1 7q / 7, germline 13 AML1 4 AML1 mutations+7q / 7 AML1 mutation+7q / 7 1 7q / 7+AML1 1 t(9;11)+flt3 mutation 1 mutation AML1 mutation 1 c-kit mutation 1 7q / 7 1 NPM1+FLT3 mutation 1 Abbreviations: t-aml, therapy-related acute myeloid leukemia; t-mds, therapy-related myelodysplasia. Table 4 Frequencies of gene mutations in t-mds and t-aml compared with frequencies in de novo MDS and AML with all karyotypes Study Type of patients N p53 point AML1 point NRAS/KRAS point PTPN11 point Copenhagen t-mds/t-aml /4 3 Series 29,31,32,34,36 Ben-Yehuda et al. 48 t-mds/t-aml Harada et al. 49 t-mds-t-aml MDS atomic bomb survivors Bacher et al. 50 t-aml Wattel et al. 51 de novo MDS de novo AML Padua et al. 52 de novo MDS 50/ Stirewalt et al. 11 AML, 450 years t-aml /7 included Niimi et al. 53 MDS-AML t-aml /0 4 included Preudhomme et al. 54 AML all FAB types Subgroup MO Harada et al. 55 de novo RA/RARS 45 2 de novo RAEB/RAEB-t de novo AML after MDS Steensma et al. 56 de novo MDS Chen et al. 57 de novo MDS Tartaglia et al. 58 de novo AML,children 49/ Bowen et al. 59 AMLo60 years 1106/739 11/5 Verhaak et al. 19 Primary AML 275 9/3 Bacher et al., 50 de novo AML Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplasia; t-aml, therapy-related AML; t-mds, therapy-related MDS.

6 disease, although their frequencies and genomic break points vary to some extent. Whereas only 10 15% of patients with de novo MDS or AML present the unbalanced aberrations 5q / 5 or7q / 7, 1 5 the figure rises to 50 60% in patients with t-mds or t-aml As far as the recurrent balanced translocations or inversions are concerned, these abnormalities are observed in 10 20% of patients with therapy-related and 10 20% of patients with de novo AML. In the therapy-related subset, the frequency increases with an increased use of topoisomerase II inhibitors, and in de novo AML with an increasing proportion of younger patients. Finally, the third cytogenetic subgroup, patients with a normal karyotype, is represented by 45 55% of cases of de novo MDS and AML 1 5 as compared with only 10 15% of cases of t-mds and t-aml, often atypical cases. Spectrum and frequencies of gene mutations The spectrum of gene mutations observed in de novo and in t-mds and t-aml is also identical, as shown in Tables 4 and 5. Their frequencies, however, vary in some cases according to the cytogenetic characteristics, and in other cases according to the presentation of the disease as MDS or overt AML. For instance, the frequency of p53 point mutations was higher in three studies of t-mds and t-aml, 29,36,48,49 as compared to studies of de novo MDS and AML (Table 4). 11,51 53 This possibly relates to the large fraction of patients with 5q / 5 and complex karyotypes in therapy-related disease. The frequency of AML1 point mutations was in several studies in the same order of 10 20% in therapy-related as in de novo MDS and AML, 31,54 57 with somewhat higher figures in series of patients with MDS progressing to AML. 49,53,54 The frequencies of RAS and PTPN11 mutations, if considering all karyotypes (Table 4), were almost identical in therapy-related 32,34,50 and in de novo MDS and AML. 11,19,50,53,57 59 Finally, mutations of FLT3 and NPM1 were far less common in t-aml as compared to de novo AML. If considering patients with AML and a normal karyotype only (Table 5), similar or slightly lower frequencies of mutations of FLT3 and NPM1 were observed in therapy-related 13,22,30,32,35,60 as compared to de novo disease. 10,13,17 22 Mutations of cfms and CEBPA were not detected in our study and are also less frequent in de novo AML. 17,19,20 In conclusion, the patterns of chromosome aberrations and gene mutations are identical in therapy-related and de novo MDS and AML. Also, the frequencies of gene mutations are rather similar, if taking the cytogenetic differences between the two types of disease into consideration. Classification of mutations and their pattern of combinations Classification of gene mutations in MDS and AML according to the function may seem problematic because of the complicated and partly unknown cellular effects of many of these abnormalities. In the present study, mutations were grouped as previously proposed 24,25 and shown in Table 1 and Figures 1 and 2, with mutations of NPM1 being considered as Class II mutations. The fact that specific combinations between Class I and Class II mutations, such as the ckit-aml1, 14,61 the FLT3- RARA 13 and the FLT3-NPM1, predominate in de novo AML suggests a preference for these specific combinations. The present study, however, for the first time demonstrates a wide fan of less common or unique combinations between Class I and Class II mutations in t-mds and t-aml. This suggests a more extensive cooperation between these two classes of gene mutations than previously believed. The high degree of mutual exclusiveness within Class I and Class II mutations in therapy-related and de novo disease is striking and so far unexplained. If a gene is mutated, there may be no further proliferative advantage from additional mutations of genes within the same class. Alternatively, mutations of two genes of the same class in a cell could result in apoptosis rather than leukemic transformation. The order of Class I and Class II mutations in leukemogenesis So far, it has not been firmly established which mutations are primary or early lesions and which mutations represent later events in the development of MDS and AML, if indeed such an order exists. As previously mentioned, the introduction of activated genes encoding rearranged AML transcription factors in murine bone marrow cells have repeatedly been shown to serve as primary lesions. 8 Furthermore, the recurrent balanced translocations, if present in MDS or AML, are characteristically observed early at diagnosis of overt leukemia and in all abnormal mitoses; and are only rarely detected as evolutionary 245 Table 5 Frequencies of gene mutations in t-mds and t-aml compared with frequencies in de novo AML with a normal karyotype Study Type of patients N FLT3 ITD and point NPM1 point CEBPA point Copenhagen series ,32,35 t-mds/t-aml Schnittger et al. 13 t-aml F F Thiede et al. 22 t-aml a 55 a 13 a 7 a F Fröhling et al. 17 t-aml 179 F F 17 Bacher et al. 60 t-aml Kottaridis et al. 10 de novo AML+7% secondary F F Schnittger et al. 13 de novo AML F F Fröhling et al. 17 de novo AML F 15 Falini et al. 18 Primary AML F Suzuki et al. 21 de novo AML a 47 F Döhner et al. 20 o60 years, de novo AML Verhaak et al. 19 Primary AML a 64 6 Thiede et al. 22 de novo AMLo60 years F Abbreviations: AML, acute myeloid leukemia; t-aml, therapy-related AML; t-mds, therapy-related myelodysplasia. a All karyotypes studied.

7 246 events or in subclones. These observations suggest that Class II mutations often represent primary events. RAS and FLT3 mutations, although often observed throughout the whole course of AML, are sometimes present only at diagnosis but not at relapse, and in other cases first seen at relapse of the disease. 9,57 Furthermore, two different Class I mutations are sometimes observed simultaneously, possibly representing different subclones. In our study, 32 1 of the 14 patients with RAS mutations at diagnosis presented two different KRAS mutations. Another patient presented a t(9;11) in all mitoses examined, with mutations of KRAS and BRAF being observed in subclones. In approximately 25% of patients with de novo AML and mutations of FLT3, two, three or even four different mutations are observed in the same case. 10 One of our 11 patients with FLT3 mutations 32 presented an internal tandem duplication as well as the I836 deletion of the gene simultaneously. Recently, patients with AML and simultaneous mutations of NPM1 and FLT3 were shown always to present only a single NPM1 mutation, but in several cases different FLT3 mutations. 22 Finally, cooperating, so far unknown, mutations seem to predate mutations of JAK2 in the development of myeloproliferative diseases. 62 Although sporadic, these observations all suggest that Class II mutations often represent primary or early events, and Class I mutations represent later lesions in a multistep development of MDS and AML. Other genetic abnormalities in leukemogenesis In this review, we have focused on gene mutations and chromosome abnormalities in MDS and AML, and identification of novel genetic abnormalities will possibly in the future increase our understanding of leukemic transformation. Other phenomena, however, such as an abnormally high or low dose of normal regulatory proteins may play an important role. Particularly in t-mds and t-aml, a decreased dose of the p15 INK4B - 63, the EGR1-41 or the CTNNA1-42 encoded proteins as a result of promoter methylation or haploinsufficiency must be considered. Also, an increased dose of the unrearranged MLL and AML1 gene products caused by chromosome duplication or amplification 38,39,64 66 may represent examples of gene dose effects in AML. Conclusion The identical pattern of cytogenetic abnormalities and gene mutations in de novo and in t-mds and t-aml supports that these are indeed identical diseases. Consequently, they should be subclassified and treated similarly. Their biological similarity allows to extrapolate experience from one to the other subgroup of disease. Thus, three types of etiology must be considered in de novo MDS and AML. These include exposures to exogenous or endogenous metabolites with alkylating properties, illegitimate gene recombinations related to activities of topoisomerase II and spontaneous mutations or mutations induced by ionizing radiation. The pathogenesis of MDS and AML with cooperating mutations of at least two general classes of genes and chromosome abnormalities may preferentially be studied in therapy-related disease with follow-up from leukemogenic exposures to subsequent development of leukemia. Finally, response to various types of therapy is best studied in de novo MDS and AML. Thus, patients with therapy-related disease must, in many cases, be treated individually with less intensive regimens, as they are often elderly, present a poor performance status with decreased tolerance to intensive therapy and suffer from a primary malignancy, which is still active. Acknowledgements The study was supported by grants from the Danish Cancer Society. The authors are indebted to Cand. 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