Acute Myeloid Leukemia

Transcription

1 Acute Myeloid Leukemia Session Chair: Hillard M. Lazarus, MD Speakers: Clara D. Bloomfield, MD; Donald Small, MD, PhD; and Wendy Stock, MD Chromosome Aberrations, Gene Mutations and Expression Changes, and Prognosis in Adult Acute Myeloid Leukemia Krzysztof Mrózek and Clara D. Bloomfield Pretreatment clinical features and prognosis of patients with acute myeloid leukemia (AML) are strongly influenced by acquired genetic alterations in leukemic cells, which include microscopically detectable chromosome aberrations and, increasingly, submicroscopic gene mutations and changes in gene expression. Cytogenetic findings separate AML patients into three broad prognostic categories: favorable, intermediate and adverse. The cytogeneticrisk classifications differ somewhat for younger adult patients and those aged 60 years or older. In many instances, patients with specific cytogenetic findings, e.g., those with a normal karyotype or those with either Division of Hematology and Oncology, Department of Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH Correspondence: Dr. Krzysztof Mrózek, Division of Hematology and Oncology and the Comprehensive Cancer Center, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Room 1248B, The Ohio State University, 300 West Tenth Avenue, Columbus, OH ; Phone ; Fax ; krzysztof.mrozek@osumc.edu Supported in part by National Cancer Institute, Bethesda, MD, grants CA77658, CA101140, and CA16058, and The Coleman Leukemia Research Foundation. t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) [collectively referred to as core-binding factor (CBF) AML] can be further subdivided into prognostic categories based on the presence or absence of particular gene mutations or changes in gene expression. Importantly, many of these molecular genetic alterations constitute potential targets for riskadapted therapies. In this article, we briefly review major cytogenetic prognostic categories and discuss molecular genetic findings of prognostic significance in two of the largest cytogenetic groups of patients with AML, namely AML with a normal karyotype and CBF AML. Adult acute myeloid leukemia (AML) is a very heterogeneous disease with regard to clinical features and acquired genetic alterations, both those detectable microscopically as structural and numerical chromosome aberrations, and those detected as submicroscopic gene mutations and changes in gene expression. At present, cytogenetic aberrations detected at the time of AML diagnosis constitute the most common basis for predicting clinical outcome. 1-4 However, molecular genetic alterations are increasingly being used to refine prognosis further. In this article we briefly review major cytogenetic-risk prognostic categories and then focus on how molecular genetic findings, many of which are or likely will become therapeutic targets, contribute to prognostication of patients belonging to two of the larger cytogenetic groups: AML with a normal karyotype and core-binding factor (CBF) AML. Prognostic Groups Based on Cytogenetic Findings Acquired clonal chromosome abnormalities, i.e., a structural aberration or a trisomy observed in at least 2 and monosomy found in at least 3 metaphase cells, are detected in the pretreatment marrow of 50% to 60% of adults with de novo AML. In 10% to 20% of patients, the abnormal karyotype is complex, i.e., contains at least 3 chromosome aberrations, whereas in 40% to 50% of patients no cytogenetic abnormality can be discerned using standard banding methods. 1-4,8,9 Recent large collaborative studies have proposed Hematology

2 cytogenetic-risk systems classifying patients into favorable, intermediate and adverse risk groups according to the specific karyotypic findings at diagnosis (Table 1). Although there are some differences among these prioritization schemata, pretreatment cytogenetic findings are being used to stratify therapy. 5 Furthermore, for patients whose pretreatment karyotype is abnormal, cytogenetic analysis has been recommended for documenting complete remission (CR), 6 based on data showing that persistence of even 1 metaphase with an abnormality present at diagnosis results in significantly inferior cumulative incidence of relapse (CIR), disease-free survival (DFS) and overall survival (OS). 7 All large cytogenetic studies of AML agree that patients with t(15;17)(q22;q12-21) have an excellent and those with t(8;21)(q22;q22) or inv(16)(p13q22)/ t(16;16)(p13;q22) a relatively favorable prognosis, whereas those with inv(3)(q21q26)/ t(3;3)(q21;q26), 7 and a complex karyotype, defined in some studies as containing at least 3 and in others at least 5 chromosome aberrations, have a poor clinical outcome (Table 1). Notably, the complex karyotype category usually does not include patients with t(8;21), inv(16)/t(16;16), t(15;17), or t(9;11)(p22;q23) because their prognosis has generally not been shown to be affected unfavorably by the presence of 2 or more additional chromosome aberrations. 3 On the other hand, the majority of patients with 5, del(7q), 17/17p, 18, or 20, whose prognosis is usually very poor, have a complex karyotype, which precludes evaluation of the prognostic significance of each of these chromosome aberrations individually. Interestingly, the relatively rare AML patients with del(7q) in the absence of 5/ del(5q), 3q abnormalities or a complex karyotype, 1 and those with del(5q) in a non-complex karyotype 3 are classified in the intermediate-risk category. The cytogenetic-risk classification summarized in Table 1 is based on studies that predominantly 1,3 or exclusively 2 included younger AML patients, below 56 or 60 years of age. Two subsequent studies analyzed the prognostic significance for pretreatment cytogenetic findings in outcome of older AML patients, a group whose outcome is generally worse than that of younger patients. Both the United Kingdom Medical Research Council (MRC) study 8 and the recent Cancer and Leukemia Group B (CALGB) study 9 confirmed the value of pretreatment karyotype as an independent prognostic factor in older patients with AML. However, the impact of some specific chromosome aberrations on clinical outcome is different in older as opposed to younger AML patients. An MRC study 8 found the outcome of older adults with fewer than 5 abnormalities, regardless of the presence of abnormalities involving chromosomes 5, 7 and 3q, to be comparable to that of patients with a normal karyotype or intermediate-risk abnormalities, and therefore all these patients were included in the intermediate-risk category, leaving patients with a complex karyotype with 5 or more Table 1. Risk group assignments of younger adults with acute myeloid leukemia (AML) with the more frequent cytogenetic findings. Favorable-Risk Group Balanced structural rearrangements: t(15;17)(q22;q12-21) t(8;21)(q22;q22) inv(16)(p13q22)/t(16;16)(p13;q22) Intermediate-Risk Group Normal karyotype* Balanced structural rearrangement: t(9;11)(p22;q23) Unbalanced structural rearrangements: del(7q) del(9q) del(11q) del(20q) Numerical aberrations: Y Unfavorable-Risk Group Complex karyotype Balanced structural rearrangements: inv(3)(q21q26)/t(3;3)(q21;q26) t(6;9)(p23;q34) # t(6;11)(q27;q23)** t(11;19)(q23;p13.1)** Unbalanced structural rearrangement: del(5q) Numerical aberrations: 5 7 Bold type denotes chromosome aberrations whose prognostic impact is agreed on by major studies. 1-3 * No clonal abnormality detected in 20 or more fully analyzed marrow metaphase cells. Would be included in abn 11q group and classified in the adverse-risk category by Slovak et al. 2 Classified in the adverse-risk category by Slovak et al. 2 Would be included in abn 20q group and classified in the adverse-risk category by Slovak et al. 2 Classified in the adverse-risk category with respect to overall survival by Byrd et al. 3 Defined as the presence of 3 or more abnormalities in karyotypes not including inv(16), t(16;16), t(8;21), t(15;17) or t(9;11). # Classified in the intermediate-risk category by virtue of being other structural abnormality by Grimwade et al. 1 In a study of Byrd et al, 3 included in the intermediate-risk category only with respect to probability of achievement of CR. ** Would be included in abnormal 11q23 group and classified in the intermediate-risk category by Grimwade et al. 1 In a study of Byrd et al, 3 included in the intermediate-risk category only with regard to probability of achievement of CR. Classified in the intermediate-risk category with respect to probability of achievement of CR and survival by Byrd et al, 3 but only if not part of a complex karyotype. 170 American Society of Hematology

3 Table 2. Significant prognostic cytogenetic risk groups in adults aged 60 years or older with acute myeloid leukemia (AML) identified by tree analysis for complete remission, disease-free survival and overall survival in a Cancer and Leukemia Group B study.* Cytogenetic Risk Groups No. Outcome, % (95% CI) Complete remission (CR: CR rate: Complex karyotype with 3 aberrations (17-32) Rare aberrations (15-46) Non-complex 3, non-rare aberrations (51-60) Disease-free survival: 5-year DFS: Complex karyotype with 5 aberrations 22 0 Fewer than 5 aberrations ( ) Overall Survival: 5-year OS: Complex karyotype with 5 aberrations 94 0 CBF AML ( ) Rare aberrations 33 0 Fewer than 5 non-cbf AML, ( ) non-rare aberrations * This research was originally published in Blood. 9 Farag et al. Pretreatment cytogenetics add to other prognostic factors predicting complete remission and long-term outcome in patients 60 years of age or older with acute myeloid leukemia: results from Cancer and Leukemia Group B Blood. 2006;108: the American Society of Hematology. aberrations as the only ones in the adverserisk category. A CALGB study confirmed that older AML patients with a complex karyotype with 5 or more aberrations have particularly poor DFS and OS, with no patient surviving 5 years after diagnosis (Table 2). 9 In contrast, among older patients those with CBF AML were among those who survived the longest in both studies. 8,9 Patients with a normal marrow karyotype are the largest cytogenetic subset of AML and are classified in the intermediate prognostic category. 1-4 Although the risk of relapse and DFS of cytogenetically normal patients in a recent study 10 were improved by postremission therapies that included 4 cycles of high- (HDAC) or intermediate-dose (IDAC) cytarabine or 1 cycle of HDAC/etoposide followed by autologous stem cell transplantation (ASCT) compared with regimens including fewer cytarabine cycles or no ASCT, not all patients benefited equally from these regimens. The likely reason for such variable response to treatment is the striking molecular heterogeneity of cytogenetically normal AML patients. During the last decade several studies have shown that the presence or absence of specific gene mutations and/or changes in gene expression have an effect on the patients prognosis. 11 At present, probably the most important prognostic factor in cytogenetically normal AML is the internal tandem duplication (ITD) of the FLT3 gene (discussed in detail in this volume by Small). Below we will review other molecular markers known to influence clinical outcome of AML patients with a normal karyotype (Table 3). Table 3. Genetic alterations affecting clinical outcome of cytogenetically normal acute myeloid leukemia (AML) patients. Genetic Alteration Favorable NPM1 mutations CEBPA mutations Prognostic Significance Patients with NPM1 mutations who do not harbor FLT3-ITD have significantly better CR rates, EFS, RFS, DFS, and OS than patients without NPM1 mutations and FLT3-ITD. NPM1 mutations do not have a significant effect on prognosis of patients with FLT3-ITD. Patients with CEBPA mutations have CRD and OS significantly longer than patients with the wild-type CEBPA gene. Unfavorable FLT3-ITD MLL-PTD BAALC overexpression ERG overexpression Patients with FLT3-ITD have significantly shorter CRD, DFS and OS than patients who do not harbor FLT3-ITD. Particularly poor prognosis is conferred by FLT3-ITD coupled with no expression of a FLT3 wild-type allele or a high FLT3 mutant to FLT3 wild-type allele ratio. Patients with MLL-PTD have remission duration significantly shorter than patients without MLL-PTD. Patients with high expression of the BAALC gene in blood have significantly worse CR rates and shorter DFS, EFS and OS than patients with low expression of the BAALC gene. Patients with high expression of the ERG gene in blood have significantly shorter OS and higher CIR than patients with low expression of the ERG gene. Abbreviations: NPM1, nucleophosmin (nucleolar phosphoprotein B23, numatrin); FLT3-ITD, internal tandem duplication of the fmsrelated tyrosine kinase 3 (FLT3) gene; CR, complete remission; EFS, event-free survival, RFS, relapse-free survival; DFS, diseasefree survival; OS, overall survival; CEBPA, CCAAT/enhancer binding protein (C/EBP), alpha; CRD, CR duration; MLL-PTD, partial tandem duplication of the myeloid/lymphoid or mixed-lineage leukemia (MLL) gene; BAALC, brain and acute leukemia gene, cytoplasmic; ERG, v-ets erythroblastosis virus E26 oncogene like (avian); CIR, cumulative incidence of relapse. Hematology

4 Cytogenetically Normal AML Mutations of the nucleophosmin, member 1 gene Heterozygous mutations in exon 12 of the nucleophosmin member 1 (NPM1) gene, resulting in abnormal cytoplasmic expression of its protein product, nucleophosmin, have recently been reported in 46% to 62% of AML patients with a normal karyotype, thus becoming the most frequent genetic alterations in this cytogenetic group of AML The presence of NPM1 mutations has been associated with such pretreatment features as female sex, 13,14,16 increased bone marrow (BM) blast percentages, 13,16 LDH levels, 13 WBC and platelet 13,14,16 counts, and low or absent CD34 + expression ,16 Approximately 40% of patients with NPM1 mutations also harbor FLT3-ITDs, which, together with mutations in the tyrosine kinase domain of FLT3 (FLT3- TKD), are twice as common in NPM1 mutation-positive patients as in patients with wild-type NPM ,16 While CEBPA mutations occur with a similar frequency in patients with and without NPM1 mutations, 13,14 the partial tandem duplication (PTD) of MLL is very rare in the former. 13,14,16 Most, but not all, 15 studies analyzing either exclusively patients with a normal karyotype 13,14,16 or those classified in the intermediate-risk cytogenetic category, 17 which usually contains a large proportion of cytogenetically normal patients, have demonstrated that NPM1 mutations are associated with clinical outcome. In the seminal report by Falini et al, 12 cytoplasmic localization of nucleophosmin, a feature highly correlated with the presence of NPM1 mutation, was an independent favorable prognostic factor for achievement of CR. Later studies evaluating the clinical outcome of patients with and without NPM1 mutations irrespective of the presence of other genetic rearrangements have yielded somewhat inconsistent results. In some analyses, patients with NPM1 mutations had a significantly better CR rate 14 and longer event-free survival (EFS), 14 relapsefree survival (RFS) 13 and DFS 16 than those with wild-type NPM1; in others, no significant difference in CR rate, 15 EFS, 15 RFS 14,15 and OS between the mutated and unmutated groups was found. However, among patients without the FLT3-ITD all studies agreed that the presence of NPM1 mutations was associated with significantly improved CR rates, 13,16 EFS, 14 RFS, 13 DFS, 16 and OS. 13,14,16 In contrast, there was no major effect of NPM1 mutations on the poor outcome conferred by FLT3-ITD in patients carrying this rearrangement. The combined status of mutated NPM1 and wild-type FLT3 was also shown to be an independent favorable prognostic factor for the attainment of CR, 13 DFS, 16 EFS, 14 and survival 13,16 in multivariable analyses. Mutations of the CCAAT/enhancer-binding protein α (CEBPA) gene Mutations of CEBPA, a gene encoding a myeloid transcription factor that plays an important role in normal granulopoiesis, have been detected in 15% to 20% of AML patients with normal cytogenetics. 15,18,19 That CEBPA mutations confer favorable prognosis was initially demonstrated in patients included in the intermediate cytogenetic-risk category 20,21 and later confirmed by studies investigating exclusively patients with a normal karyotype. 15,18,19 Compared with patients with wild-type CEBPA, those with CEBPA mutations present with higher percentages of peripheral blood (PB) blasts, lower platelet counts, less lymphadenopathy and extramedullary involvement, and are less likely to also carry FLT3-ITD, FLT3-TKD and MLL-PTD. 18 While there were no significant differences in CR rates between patients with and without CEBPA mutations, 15,18,19 patients with CEBPA mutations had significantly better CRD, 18 DFS, 19 EFS 15 and OS. 18,19 In multivariable analyses, CEBPA mutational status has added prognostic information to that provided by MLL-PTD and FLT3-ITD status, age and resistant disease after the first course of induction therapy with regard to CR duration (CRD) and by FLT3- ITD status, age and WBC count for OS. 18 Similar results were obtained in another study, where CEBPA mutations, BAALC and FLT3-ITD status were prognostic for DFS; CEBPA mutations, age, BAALC and FLT3-ITD status were prognostic for OS. 19 Preliminary data suggest that CRD of patients with N- terminal nonsense CEBPA mutations is the longest, followed by that of patients with other CEBPA mutations, including C-terminal mutations in the bzip domain; CRD of patients with wild-type CEBPA was the shortest. 18 However, pairwise comparisons did not show significant differences suggesting that larger studies are necessary to assess the prognostic role of different CEBPA mutation types. Partial tandem duplication (PTD) of the myeloid/ lymphoid or mixed lineage leukemia (MLL) gene MLL-PTD was the first molecular alteration shown to impact on clinical outcome of cytogenetically normal adults with de novo AML. 22 It is detected in approximately 5% to 10% of these patients and usually involves exons 5 through 11 or, less frequently, exons 5 through 12. In 30% to 40% of patients with the MLL-PTD a FLT3-ITD is also found, 13 whereas CEBPA 18 or NPM1 13,14 mutations are rarely found together with MLL-PTD. Patients with MLL-PTD do not differ significantly from those without this mutation with regard to pretreatment characteristics. However, their CRD 23,24 is significantly shorter than that of patients without the MLL- PTD. A multivariable analysis that did not include other molecular genetic markers revealed MLL-PTD status as the only prognostically significant factor for CRD. 24 Recently, Whitman et al 25 have reported that the MLL wild-type allele is silenced in AML blasts harboring the MLL-PTD, most likely as a result of differential DNA methylation and histone modifications directly or indirectly regulating the promoter of the MLL wild-type allele. Importantly, the transcription of the MLL wild-type allele can be re-activated in vitro by the combination of depsipeptide, a histone deacetylase inhibitor, and decitabine (5'-aza-2'- deoxycytidine), a DNA methyltransferase inhibitor, and this 172 American Society of Hematology

5 leads to enhanced apoptosis. 25 These data suggest that MLL- PTD-positive patients might benefit from therapy that includes DNA methyltransferase and/or histone deacetylase inhibitors. However, until such molecularly targeted therapy is shown to be clinically effective in these patients, allogeneic SCT seems to be the best therapeutic approach. 24 Overexpression of the BAALC gene The BAALC gene is expressed mainly in neuroectodermderived tissues and hematopoietic precursors. 26 However, high BAALC expression in PB at diagnosis was also detected in a subset of karyotypically normal adults under the age of 60 years with de novo AML, and associated with lower WBC and less frequent diagnosis of FAB M5 AML. 27 High BAALC expression was shown to be an independent risk factor unfavorably affecting DFS, EFS and OS. 27 Similar results were obtained in a second study, in which BAALC expression was measured in marrow relative to the median value of BAALC expression in marrow from healthy volunteers. 19 In a subgroup analysis restricted to patients without CEBPA mutations and FLT3-ITD, high BAALC expression was again associated with inferior DFS and OS. 19 The latest and hitherto largest study of 307 adults with AML aged 60 years or younger showed high expression of BAALC in PB to be an independent adverse prognostic factor not only for remission duration and OS, but also for resistance to initial induction chemotherapy. 28 On multivariable analysis, high BAALC expression and a high ratio of FLT3-ITD to wild-type FLT3 allele were the only factors predicting worse CIR and OS. Interestingly, among patients with high BAALC expression, those who underwent allogeneic SCT had a significantly lower CIR compared with patients who received autologous SCT, whereas among all patients undergoing allogeneic SCT, there was no difference in CIR between high and low BAALC expressers. 28 These preliminary data suggest that allogeneic SCT in first CR might overcome the adverse prognostic effect of high BAALC expression. Overexpression of the ETS-related gene (ERG) This is a recently identified molecular marker predicting adverse outcome in karyotypically normal AML patients. First discovered in AML patients with prognostically unfavorable complex karyotypes with cryptic amplification of chromosome, 21,29,30 ERG overexpression was also detected in a subset of cytogenetically normal AML, 30 raising the possibility that it might also be associated with poor prognosis in this cytogenetic subgroup of AML. This has been demonstrated in a study comprising 84 adults with de novo AML below the age of 60 years, which revealed that patients with ERG expression values in the uppermost quartile of ERG expression in pretreatment PB, who aside from having higher percentages of PB blasts did not have distinctive pretreatment characteristics, had a significantly worse CIR and OS compared with patients having lower ERG expression values. 31 High ERG expression and FLT3-ITD were both independent prognostic factors predicting worse CIR and OS. When the analyses were restricted to the more favorable subset of patients who expressed a FLT3 wildtype allele, high ERG expression and MLL-PTD both impacted unfavorably on remission duration, whereas high ERG expression predicted shorter OS only in patients with low expression of the BAALC gene. 31 These results require confirmation by an independent study. Gene-expression profiling Relatively little information on the clinical usefulness of gene-expression profiling in AML is available at present. A recent gene-expression profiling study revealed that most adults with AML and a normal karyotype segregated into two clusters with significantly different survival. Patients in cluster I were more frequently diagnosed with AML FAB M1 and M2 and less frequently with AML FAB M4 and M5, carried more commonly FLT3 aberrations and had shorter OS. 32 However, these results require corroboration, particularly because in other gene-expression studies cytogenetically normal patients segregated into several different clusters. 33,34 The prognostic significance of the geneexpression signature identified by Bullinger et al 32 has been validated recently in a patient population treated on a different protocol, with a longer follow-up (4.7 versus less than 2 years), and using a different microarray platform. 35 Moreover, this study has increased the clinical applicability of the signature because a class prediction algorithm was created that allows for the prediction of outcome for subsequent individual patients. This signature-based classifier identified groups with differences in DFS and OS. 35 However, the outcome classifier was strongly associated with the FLT3-ITD, which might explain the prognostic significance of the signature. Additionally, the classifier for dichotomized outcome classes had only moderate predictive accuracy, with OS and DFS of around 60% of the patients being correctly predicted. Nevertheless, the classifier showed some ability to identify a subset of patients with wild-type FLT3 whose outcome is poor. 35 Future studies should aim at identifying other classifiers that might predict outcome for individual AML patients with a normal karyotype with greater precision. Core-Binding Factor (CBF) AML CBF AML is a relatively frequent subtype of adult de novo AML, with t(8;21) being detected in 7% and inv(16)/ t(16;16) in 8% of patients. 3 Nevertheless, few studies including over 100 patients have assessed the clinical characteristics of t(8;21) and inv(16) AML, and only three compared the two cytogenetic subsets of CBF AML directly Such a comparison is important because CBF AML patients with t(8;21) are often grouped together with those harboring inv(16) in clinical studies based on similar, relatively favorable prognoses, which can be improved by postremission treatment with 3 or 4 cycles of IDAC or HDAC as opposed to 1 cycle, 39 and the fact that t(8;21) and Hematology

6 inv(16) are related at the molecular level because they disrupt the α and β subunits of CBF, respectively. In addition to a characteristic FAB M4Eo morphology that distinguishes inv(16)/t(16;16)-positive patients from those with other karyotypes, patients with inv(16)/t(16;16) differ from t(8;21) patients with regard to several other pretreatment features. They are more frequently white and less frequently African American, 39,40 have higher WBCs, percentages of PB and BM blasts, 39 more often extramedullary involvement, 38,39 in particular lymphadenopathy, 38,39 splenomegaly, 39 gingival hypertrophy 39 and skin/mucosa involvement, 38 and characteristic cytogenetic features. Approximately two thirds of patients with inv(16)/t(16;16) have this rearrangement as a sole chromosome abnormality, whereas sole t(8;21) is detected in only about 30% of patients with this translocation The pattern of secondary chromosome aberrations is also different, with loss of a sex chromosome ( Y in men and X in women) and del(9q) being the most frequent secondary chromosome aberrations in t(8;21)-positive patients, and +22, +8, del(7q) and +21 in inv(16)/t(16;16)-positive patients Importantly, marked molecular genetic differences between the two cytogenetic groups have been revealed by microarray geneexpression studies that can differentiate patients with inv(16)/t(16;16) from those with t(8;21) with greater than 95% accuracy based on their gene-expression signatures. 41 Both cytogenetic subgroups have very similar, relatively high CR rates in the range of 85% to 89%, and in both, the probability of CR attainment is negatively affected by lower platelet counts. 39 However, lower CR probability was associated with hepatomegaly only in inv(16)/ t(16;16) patients, and with higher BM blasts and, unexpectedly, nonwhite race exclusively in the t(8;21) group. Nonwhite patients with t(8;21) had 5.7 times the odds of not achieving a CR compared with the corresponding white population. The reasons for such a difference are unknown, but because the patients were treated on the same protocols the difference might be related to a true biologic diversity resulting in more resistant disease and/or higher susceptibility to drug toxicity of nonwhite patients with t(8;21). 39 Univariable analyses did not reveal significant differences in relapse risk or OS between inv(16)/t(16;16) and t(8;21) groups However, after adjusting for age, log[wbc], and log[platelets], patients with t(8;21) were found to have a significantly shorter OS than those with inv(16)/t(16;16). 39 This difference is likely related to a dissimilar response to salvage therapy, since patients with t(8;21) had a significantly shorter survival after relapse compared with inv(16)/t(16;16) patients in three large, independent studies These studies identified additional cytogenetic prognostic factors differentiating the two cytogenetic subsets of CBF AML. Among patients with inv(16)/t(16;16), those who harbored +22 as a secondary abnormality were found to have a significantly lower CIR compared with patients with inv(16)/t(16;16) as a sole abnormality in the CALGB study 39 and longer RFS than patients without +22 in the German Acute Myeloid Leukemia Intergroup study. 38 In the latter study, 38 loss of the Y chromosome ( Y) in male patients with t(8;21) was associated with a shorter OS, although this result could not be corroborated in the CALGB series. 39 Instead, Marcucci et al 39 observed a possible interaction between secondary chromosome abnormalities and race in patients with t(8;21). Nonwhites with secondary cytogenetic abnormalities other than del(9q) had shorter OS than nonwhites who had an isolated t(8;21) or a secondary del(9q). In contrast, among white patients, neither del(9q) nor other secondary aberrations, including Y in men, influenced the patients survival. Because a substantial proportion of patients with CBF AML carry mutations in the KIT gene (Table 4), recent studies examined prognostic significance of these mutations. As shown in Table 4, various KIT mutations analyzed as one group and, more specifically, mutations in exon 17, which encodes the activation loop in the kinase domain of KIT, have been associated with inferior OS, EFS, 43,44 RFS, 44 relapse incidence 42 and CIR 45 in patients with t(8;21). The prognostic significance of KIT mutations in patients with inv(16)/t(16;16) has been less firmly established. Care et al 46 found KIT mutations in exon 8 to unfavorably affect the relapse rate (but not OS), whereas two smaller studies did not detect any prognostic impact of KIT mutations. 42,44 In a recent CALGB study, 45 performed on a relatively large number of similarly treated patients with inv(16), the presence of all KIT mutations bestowed a higher CIR. Notably, subset analyses revealed that the difference in CIR was mainly caused by the presence of mutations in exon 17 of KIT. Patients with these mutations had over six times higher CIR than those with wild-type KIT. In multivariable analyses, KIT mutations, both those in exon 17 and exon 8, impacted adversely on OS after adjusting for sex. 45 These results await independent confirmation. The discovery of prognostically relevant KIT mutations in CBF AML is important because it has potential therapeutic significance. Mutations in the KIT gene have been previously shown to constitute potential targets for tyrosine kinase (TK) inhibitors. Importantly, specific TK inhibitors are active against particular KIT mutations, e.g., imatinib is active against exon 17 mutations involving N822 in the activation loop, or variants of mutations in exon 8, but not against D816 mutations in the exon 17 activation loop. On the other hand, exon 17 D816 mutations can be successfully targeted with other TK inhibitors, such as PKC412 and dasatinib. Hence, it is critical to establish the exact type of KIT mutation in each case. 45 The efficacy of TK inhibitors as part of therapy administered to patients with CBF AML should be investigated in future clinical trials. Conclusions and Future Directions It has been well established that AML is a very heterogeneous disease at both the cytogenetic and molecular genetic levels. Because cytogenetic findings are among the 174 American Society of Hematology

7 Table 4. Prognostic significance of mutations in the KIT gene in CBF AML patients. Age, Range Chromosome (Median) No. Aberration Years Patients Total Exon 8 Exon 17 Other Prognostic Significance Ref. t(8;21) (40.5) (47%) 5 (14%) 12 (33%) 2 (6%) OS: Significantly shorter for pts with any KIT mutation 42 (24 mo OS rates: 42% vs 77%, P = 0.017) and for pts with exon 17 mutation (24 mo OS rates: 25% vs 77%, P = 0.006) compared with pts without KIT mutations (44%) 5 (15%) 10 (29%) 2 (6%) RI: Significantly higher for pts with any KIT mutation (24 mo RI rates: 77% vs 35%, P = 0.005) and for pts with exon 17 mutation (24 mo OS rates: 90% vs 35%, P = 0.002) compared with pts without KIT mutations. t(8;21) (13%) NA 8 (13%) NA OS: Significantly shorter for pts with exon KIT mutation (median: 304 d vs 1836 d, P < 0.001). EFS: Significantly shorter for pts with exon 17 KIT mutation (median: 244 d vs 744 d, P = 0.003). t(8;21) 1-75 (33) 50 6 (12%) 3 (6%) 3 (6%) NA OS: Significantly shorter for pts with any KIT mutation (P = 0.03). 44 EFS: Significantly shorter for pts with any KIT mutation (P = 0.006). RFS: Significantly shorter for pts with any KIT mutation (P = 0.005). t(8;21) (37) (22%) 2 (4%) 9 (18%) NA OS: No significant difference between pts with or without 45 KIT mutations. CIR: Significantly higher for patients with any KIT mutation (5-year CIR 70% vs 36%, P = 0.017) compared with those without KIT mutations. inv(16) (44) (32%) 15 (24%) 5 (8%) NA OS: No significant difference between pts with and without 46 exon 8 mutations. Mutations in exon 17 not analyzed. RR: Significantly higher for pts with exon 8 mutations compared with pts without exon 8 mutations. Mutations in exon 17 not analyzed. inv(16)/ (51) 17 8 (47%) NR 7 (41%) NR OS: No significant differences between pts with any KIT mutation 42 t(16;16) or with exon 17 mutation and those without KIT mutations. RI: No significant difference between pts with any KIT mutation or with exon 17 mutation and those without KIT mutations. inv(16)/ 1-75 (33) (22%) 9 (20%) 1 (2%) NA OS: No significant difference between pts with any KIT mutation 44 (16;16) and those without KIT mutations. inv(16)/ (40) (30%) 8 (13%) 10 (16%) NA OS: No significant differences between pts without KIT mutations 45 t(16;16) and those with any KIT mutation, with exon 17 mutation and exon 8 sole mutation. In multivariable analyses, any KIT mutation predicted worse overall survival (OS) after adjusting for sex. CIR: Significantly higher for patients with any KIT mutation (5-year CIR 56% vs 29%, P = 0.05) and pts with exon 17 mutation (5-year CIR 80% vs 29%, P = 0.002) compared with those without KIT mutations. No significant difference between pts with exon 8 sole mutation and those without KIT mutations. Abbreviations: KIT, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; pts, patients; OS, overall survival; RI, relapse incidence; EFS, eventfree survival; RFS, relapse-free survival; CIR, cumulative incidence of relapse; RR, relapse rate; NA, not analyzed; NR, not reported. * Numbers of patients for whom clinical data were available. Median age and range of all 1940 patients analyzed for KIT mutations. Age range and median provided for all patients with CBF AML, including 56 with t(8;21) and 47 with inv(16)/t(16;16). Mean age. One patient with inv(16) whose clinical outcome was analyzed had a non-exon 17 mutation, but it is unclear if this was an exon 8 or other KIT mutation. most important prognostic factors, cytogenetic analysis of BM is now mandatory in the diagnostic workup of newly diagnosed patients with AML. 47 Results of this analysis are also used for selecting therapy. The presence of fusion genes characteristic of CBF AML, acute promyelocytic leukemia with t(15;17) and AML with rearrangements of band 11q23 can also be detected by RT-PCR and fluorescence in situ hybridization (FISH). These methods are especially valuable in patients with variants of typical cytogenetic aberrations and in patients suspected of carrying cryptic rearrangements, e.g., those with FAB M4Eo, M3 or M3v marrow morphology but without, respectively, inv(16)/t(16;16) and t(15;17) on standard cytogenetic examination. Cytogenetic, FISH and molecular analyses for the more common recurring translocations are readily available. 48 The clinical significance of some of the molecular genetic tests discussed in this review, e.g., microarray geneexpression profiling and testing for ERG gene expression, is not yet firmly established and requires further study before such testing can be translated into clinical practice. Moreover, although testing for FLT3 mutations in younger patients with de novo AML is now recommended by the Practice Guidelines in Oncology, 47 testing for FLT3-ITD and for the other molecular markers is available mostly at the large university centers and performed as part of clinical trials. Because two or more genetic alterations can be Hematology

8 present simultaneously in leukemic blasts of the same patient, studies that would test all currently known molecular alterations simultaneously to determine the relative impact of each of them are in progress, as are clinical trials of FLT3 inhibitors used in combination with chemotherapy. 11 References 1. Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. Blood. 1998;92: Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group study. Blood. 2000;96: Byrd JC, Mrózek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002;100: Mrózek K, Heerema NA, Bloomfield CD. Cytogenetics in acute leukemia. Blood Rev. 2004;18: Kolitz JE, George SL, Dodge RK, et al. Dose escalation studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer and Leukemia Group B study J Clin Oncol. 2004;22: Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol. 2003;21: Marcucci G, Mrózek K, Ruppert AS, et al. Abnormal cytogenetics at date of morphologic complete remission predicts short overall and disease-free survival, and higher relapse rate in adult acute myeloid leukemia: results from Cancer and Leukemia Group B study J Clin Oncol. 2004;22: Grimwade D, Walker H, Harrison G, et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood. 2001;98: Farag SS, Archer KJ, Mrózek K, et al. Pretreatment cytogenetics add to other prognostic factors predicting complete remission and long-term outcome in patients 60 years of age or older with acute myeloid leukemia: results from Cancer and Leukemia Group B Blood. 2006;108: Farag SS, Ruppert AS, Mrózek K, et al. Outcome of induction and postremission therapy in younger adults with acute myeloid leukemia with normal karyotype: a Cancer and Leukemia Group B study. J Clin Oncol. 2005;23: Mrózek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD. Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification? Blood Sep 7 [epub ahead of print]. 12. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype [Erratum in: N Engl J Med. 2005;352:740]. N Engl J Med. 2005;352: Döhner K, Schlenk RF, Habdank M, et al. Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics - interaction with other gene mutations. Blood. 2005;106: Schnittger S, Schoch C, Kern W, et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood. 2005;106: Boissel N, Renneville A, Biggio V, et al. Prevalence, clinical profile and prognosis of NPM mutations in AML with normal karyotype. Blood. 2005;106: Thiede C, Koch S, Creutzig E, et al. Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood. 2006;107: Verhaak RGW, Goudswaard CS, van Putten W, et al. Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood. 2005;106: Fröhling S, Schlenk RF, Stolze I, et al. CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations. J Clin Oncol. 2004;22: Bienz M, Ludwig M, Oppliger Leibundgut E, et al. Risk assessment in patients with acute myeloid leukemia and a normal karyotype [Erratum in: Clin Cancer Res 2005;11:5659]. Clin Cancer Res. 2005;11: Preudhomme C, Sagot C, Boissel N, et al. Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood. 2002;100: Barjesteh van Waalwijk van Doorn-Khosrovani S, Erpelinck C, Meijer J, et al. Biallelic mutations in the CEBPA gene and low CEBPA expression levels as prognostic markers in intermediate-risk AML. Hematol J. 2003;4: Bloomfield CD, Mrózek K, Caligiuri MA. Cancer and Leukemia Group B Leukemia Correlative Science Committee: major accomplishments and future directions. Clin Cancer Res. 2006;12:3564s-3571s. 23. Caligiuri MA, Strout MP, Lawrence D, et al. Rearrangement of ALL1 (MLL) in acute myeloid leukemia with normal cytogenetics. Cancer Res. 1998;58: Döhner K, Tobis K, Ulrich R, et al. Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16 to 60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol. 2002;20: Whitman SP, Liu S, Vukosavljevic T, et al. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy. Blood. 2005;106: Tanner SM, Austin JL, Leone G, et al. BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia. Proc Natl Acad Sci USA. 2001;98: Baldus CD, Tanner SM, Ruppert AS, et al. BAALC expression predicts clinical outcome of de novo acute myeloid leukemia patients with normal cytogenetics: a Cancer and Leukemia Group B study. Blood. 2003;102: Baldus CD, Thiede C, Soucek S, Bloomfield CD, Thiel E, Ehninger G. BAALC expression and FLT3 internal tandem duplication mutations in acute myeloid leukemia patients with normal cytogenetics: prognostic implications. J Clin Oncol. 2006;24: Mrózek K, Heinonen K, Theil KS, Bloomfield CD. Spectral karyotyping in patients with acute myeloid leukemia and a 176 American Society of Hematology

9 complex karyotype shows hidden aberrations, including recurrent overrepresentation of 21q, 11q, and 22q. Genes Chromosomes Cancer. 2002;34: Baldus CD, Liyanarachchi S, Mrózek K, et al. Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: amplification discloses overexpression of APP, ETS2, and ERG genes. Proc Natl Acad Sci USA. 2004;101: Marcucci G, Baldus CD, Ruppert AS, et al. Overexpression of the ETS-related gene, ERG, predicts a worse outcome in acute myeloid leukemia with normal karyotype: a Cancer and Leukemia Group B study. J Clin Oncol. 2005;23: Bullinger L, Döhner K, Bair E, et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med. 2004;350: Valk PJM, Verhaak RGW, Beijen MA, et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med. 2004;350: Wilson CS, Davidson GS, Martin SB, et al. Gene expression profiling of adult acute myeloid leukemia identifies novel biologic clusters for risk classification and outcome prediction. Blood. 2006;108: Radmacher MD, Marcucci G, Ruppert AS, et al. Independent confirmation of a prognostic gene-expression signature in adult acute myeloid leukemia with a normal karyotype: a Cancer and Leukemia Group B study. Blood. 2006;108: Nguyen S, Leblanc T, Fenaux P, et al. A white blood cell index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML): a survey of 161 cases from the French AML Intergroup. Blood. 2002;99: Delaunay J, Vey N, Leblanc T, et al. Prognosis of inv(16)/ t(16;16) acute myeloid leukemia (AML): a survey of 110 cases from the French AML Intergroup. Blood. 2003;102: Schlenk RF, Benner A, Krauter J, et al. Individual patient data-based meta-analysis of patients aged 16 to 60 years with core binding factor acute myeloid leukemia: a survey of the German Acute Myeloid Leukemia Intergroup. J Clin Oncol. 2004;22: Marcucci G, Mrózek K, Ruppert AS, et al. Prognostic factors and outcome of core binding factor acute myeloid leukemia patients with t(8;21) differ from those of patients with inv(16): a Cancer and Leukemia Group B study. J Clin Oncol. 2005;23: Appelbaum FR, Kopecky KJ, Tallman MS, et al. The clinical spectrum of adult acute myeloid leukaemia associated with core binding factor translocations. Br J Haematol. Prepublished on August 25, 2006 as doi: /j x 41. Haferlach T, Kohlmann A, Schnittger S, et al. Global approach to the diagnosis of leukemia using gene expression profiling. Blood. 2005;106: Cairoli R, Beghini A, Grillo G, et al. Prognostic impact of c- KIT mutations in core binding factor leukemias. An Italian retrospective study. Blood. 2006;107: Schnittger S, Kohl TM, Haferlach T, et al. KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival. Blood. 2006;107: Boissel N, Leroy H, Brethon B, et al. Incidence and prognostic impact of c-kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia. 2006;20: Paschka P, Marcucci G, Ruppert AS, et al. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): A Cancer and Leukemia Group B study. J Clin Oncol. 2006;24: Care RS, Valk PJM, Goodeve AC, et al. Incidence and prognosis of c-kit and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol. 2003;121: National Comprehensive Cancer Network. Clinical Practice Guidelines in Oncology v Acute Myeloid Leukemia. aml.pdf. Accessed August 29, Association for Molecular Pathology. AMP Test Directory. Accessed September 2, Hematology