E01 Biology, cytogenetics and molecular studies in acute myeloid leukemia

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E01 Biology, cytogenetics and molecular studies in acute myeloid leukemia K. Mrózek, P. Paschka, G. Marcussi, S.P. Whitman, C.D. Bloomfield. Division of Hematology and Oncology, Department of Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA Acute myeloid leukemia (AML) is a genetically heterogeneous disease both at the cytogenetic and molecular genetic levels [1–3]. While pretreatment cytogenetic findings are among the most important independent factors for predicting clinical response [4–8], there is increasing data on the prognostic role of molecular genetic alterations within cytogenetically defined groups of AML. Herein, we briefly discuss molecular genetic alterations contributing to prognostication of patients with core-binding factor (CBF) AML, and those with cytogenetically normal (CN) AML. Together these cytogenetic groups account for approximately 60% of adults with AML younger than 60 years [5]. Core-binding factor AML (CBF-AML) CBF-AML comprises two cytogenetic groups, patients with t(8;21)(q22;q22) and those with inv(16)(p13q22)/t(16;16) (p13;q22), hereafter abbreviated inv(16), that constitute, respectively, 7% and 8% of adults with de novo AML [8]. Both chromosome aberrations result in rearrangements involving genes encoding different subunits of CBF, a transcription factor involved in regulation of normal hematopoiesis [9]. In inv(16), the CBFB gene, encoding subunit β of CBF, is fused with the MYH11 gene, and in t(8;21), the RUNX1(AML1) gene, encoding subunit α of CBF, is fused with the RUNX1T1(ETO) gene. Protein products of the CBF fusion genes act as dominant negative inhibitors of normal hematopoiesis and contribute to leukemogenesis [9]. The introduction of consolidation therapy that incorporates repetitive cycles of higher doses of cytarabine (HiDAC) has substantially improved the clinical outcome of CBF-AML patients [10–12]. Because of their favorable response to treatment, patients with inv(16) are often combined with those with t(8;21) into one favorable-risk prognostic category of AML. However, despite similarities, patients with inv(16) differ from those with t(8;21) with respect to many pretreatment features and outcome [13–15]. CR rates of 85% to 89% are similar in both cytogenetic groups, but patients with inv(16) usually have a significantly longer survival after relapse than t(8;21) patients [13–15]. Because approximately one-half of all CBF-AML patients are still not cured with modern chemotherapy [14], it is vital

to identify patients who are likely to fail standard therapy early, so that they can receive novel investigational or more aggressive therapies, e.g., stem cell transplantation (SCT). Mutations in the KIT gene, which encodes a member of the type III receptor tyrosine kinase (RTK) family [9], have been reported in 20 to 45% of CBF-AML patients [16,17]. The KIT mutations cluster mainly in exon 17, encoding the KIT activation loop in the kinase domain, and in exon 8 that encodes an extracellular part of the receptor [16,17]. They represent the first molecular prognostic marker in CBF-AML, predicting a less favorable outcome [16–19]. The abnormal KIT protein encoded by the mutated KIT gene constitutes a potential therapeutic target. KIT mutations in patients with t(8;21) at diagnosis have been associated with inferior event-free survival (EFS) [18,19], relapse incidence [16], relapse-free survival (RFS) [19], cumulative incidence of relapse (CIR) [17] and overall survival (OS) [16,18,19]. The prognostic impact of KIT mutations in AML with inv(16) is less clear [16,19]. A recent study of inv(16) patients treated on Cancer and Leukemia Group B protocols incorporating higher cytarabine doses for consolidation [17], showed that KIT mutations were associated with a higher CIR. The difference in CIR was primarily caused by KIT mutations in exon 17. In multivariable analyses, KIT mutations impacted negatively on OS [17]. Mutations in the KIT gene result in a constitutive activation of the RTK making the abnormal KIT protein a potential target for TK inhibitors. It is necessary to determine the exact type of KIT mutation in each patient because of their differential sensitivity to TK inhibitors [18,20]. In the future, diagnostic testing for the presence of KIT mutations might guide therapeutic decisions. Cytogenetically normal AML (CN-AML) The result of cytogenetic analysis of a bone marrow sample is normal in approximately 45% of adults with AML at diagnosis [5–8]. Outcomes of these patients are varied, with 20–40% of patients being long-term survivors [2,5–8,21]. Using molecular genetic techniques, recurring molecular alterations of prognostic significance are increasingly being identified in CN-AML [22,23]. These include gene mutations and overexpression of single genes. Moreover, global geneexpression profiling has been recently used to identify gene expression signatures associated with important molecular markers [24–26]. Partial tandem duplication of the MLL gene The MLL gene is a homeotic regulator encoding a nearly

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430-kilodalton protein with histone lysine 4 methyltransferase activity. MLL protein regulates HOX gene expression during hematopoietic stem cell development [9]. ALU-mediated recombination within the MLL gene generates a partial tandem duplication (PTD) spanning exons 5 through 11 or, less frequently, exons 5 through 12 [27,28]. MLL-PTD, which occurs in around 8% of adults with de novo CN-AML [28–30], was the first prognostically unfavorable molecular alteration identified in CN-AML [28,31]. It has typically been reported to adversely impact complete remission duration (CRD), but not OS [28–30]. Recent data indicate improved outcome in younger adults treated with autologous SCT in first CR [32]. AML blasts carrying MLL-PTD do not expressed the MLL wild-type (WT) transcript [33]. Transcriptional reactivation of the MLL-WT allele can occur in response to histone deacetylase and/or DNA methyltransferase inhibitors, and is associated with enhanced sensitivity to cell death. Thus, pharmacologic reversal of MLL-WT silencing by histone deacetylase inhibitors and/or demethylating agents warrants investigation in CN-AML patients with the MLL-PTD [33]. Internal tandem duplication of the FLT3 gene The FLT3 gene encodes a membrane-bound protein of the class III RTK family, which is involved in regulation of proliferation, differentiation and apoptosis of hematopoietic cell progenitors [34]. Internal tandem duplications (ITDs) of the FLT3 gene occur within the juxtamembrane domain (exons 14 and 15) and create an in-frame transcript, which is translated into a constitutively activated protein in a ligandindependent manner that promotes the aberrant proliferation and survival of leukemic blasts [34]. FLT3-ITDs are detected in 28–33% of CN-AML patients [22]. Multiple studies have demonstrated the adverse impact of FLT3 mutations on clinical outcome of CN-AML patients [22]. FLT3-ITD constitutes an independent prognostic factor for CRD [35,36], CIR [37], and OS [35,37]. Outcome is particularly adverse for FLT3-ITD-positive patients whose blasts do not express the FLT3-WT allele [38], or have a FLT3 mutant/FLT3-WT allele ratio higher than the median value [39]. Optimal treatment, including the role of SCT in first CR, for CN-AML patients with FLT3-ITD mutations is unclear [38,40,41]. The constitutively activated FLT3 protein is an attractive therapeutic target for small-molecule TK inhibitors (e.g., midostaurin, lestaurtinib or tandutinib) [42]. Other approaches currently tested in pre-clinical studies include FLT3 antibody therapy [43], which is predicted to also target overexpressed FLT3-WT, and inhibitors of downregulatory pathways such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90 [44]. Mutations of the NPM1 gene NPM1 encodes nucleophosmin, a nucleus-cytoplasm shuttling protein, implicated in preventing nucleolar protein aggregation, regulation of ribosomal protein assembly and their

nucleocytoplasmic transport, the initiation of centrosome duplication and the regulation of the p53 and Arf tumorsuppressor pathways [45]. Its precise role in oncogenesis is controversial. Whereas nucleophosmin is most prominent in the nucleus, in patients with mutated NPM1, nucleophosmin shows cytoplasmic expression that may interfere with its normal functions [45]. NPM1 mutations occur in 45–64% of CN-AML patients [22,40,46,47], and usually predict outcome only in the context of other markers. Coexistence of NPM1 mutations with CEBPA mutations and MLL-PTD is uncommon, but roughly 40% of patients with NPM1 mutations are also FLT3-ITDpositive. The poor outcome of patients with FLT3-ITD is relatively unaffected by the presence or absence of NPM1 mutations. However, patients with NPM1 mutations who do not harbor FLT3-ITD have a significantly better response to induction therapy, disease-free survival (DFS), RFS, EFS and OS [40,46,47]. Mutations of the CEBPA gene The CEBPA gene encodes the C/EBPα protein, a member of the family of basic region leucine zipper (Bzip) transcription regulators involved in granulopoiesis [9]. CEBPA mutations occur in 15–19% of CN-AML [22,35,36]. They confer significantly longer CRD and OS [22,35,36]. Overexpression of the BAALC gene The BAALC gene encodes a protein with no homology to known proteins or functional domains. BAALC expression is mostly detected in hematopoietic precursors and neuroectoderm-derived tissues. High BAALC expression has been detected in AML, acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia (CML) in blast crisis. It was not found in chronic-phase CML or chronic lymphocytic leukemia (CLL) [48]. High expression of BAALC Mrna in CN-AML is predictive of poor clinical outcome, including primary resistant disease, shorter DFS, OS, EFS and higher CIR [35,37,49]. BAALC expression appears to be especially useful in prognostication of CN-AML patients lacking FLT3-ITD and CEBPA mutations [35]. Baldus et al [37]. suggested that patients with high BAALC expression might benefit from allogeneic SCT. Overexpression of the ERG gene ERG is one of over 30 members of the ETS gene family, most of which are down-stream nuclear targets of signal transduction pathways regulating and promoting cell differentiation, proliferation and tissue invasion [9]. In CN-AML, high ERG expression adversely impacts on CIR and EFS [50,51]. For OS, an interaction between expression of ERG and BAALC has been observed; the adverse impact of high ERG expression on OS was only observed in patients with low BAALC expression [50,51]. Overexpression of the MN1 gene The MN1 gene is a transcriptional co-activator, found

Oral Presentations initially to be rearranged in patients with AML with t(12;22)(p13;q11∼12) [9]. In a recent study of CN-AML, overexpression of MN1 constituted an independent unfavorable prognostic factor for OS and RFS [52]. These results await confirmation. Interrelation of molecular genetic markers It has been proposed that at least two somatic mutations with different consequences cooperate to cause AML, with one mutation alone not being sufficient to transform a normal cell into a leukemic blast. Class I mutations affect genes in the signal transduction pathways (e.g., FLT3-ITD) that confer a proliferation stimulus, and class II mutations occur in genes encoding hematopoietic transcription factors (e.g., CEBPA) that impair cell differentiation [53]. Since distinct mutations and gene expression changes can occur in the same patient, it is important to evaluate the prognostic impact of the interaction between molecular alterations. For instance, NPM1 mutations predict better outcome mainly in the absence of FLT3-ITD [40,46,47], and the level of ERG expression identifies subsets of patients with differing prognoses within the subset of patients harboring NPM1 mutations but not FLT3-ITD [51]. Clearly, prospective investigation of prognostic interactions among many genetic lesions is needed, with the purpose of designing a clinically relevant prognostic classification of CN-AML. Gene-expression profiling in AML Gene-expression profiling (GEP) has been demonstrated to be a useful tool for the classification of leukemias. Initially, it was shown that AML and ALL could be distinguished on the basis of characteristic gene-expression signatures [54]. A more recent study demonstrated the ability of GEP to correctly identify cytogenetic subsets of AML with t(8;21), inv(16) and t(15;17), CLL, and pro-B-cell ALL (pro-B-ALL) with translocations involving 11q23 with 100% specificity and 100% sensitivity, while a similar specificity (i.e., 99.7%) albeit with a lower degree of sensitivity was achieved for the diagnosis of CN-AML, AML with translocations involving 11q23, AML with complex karyotype, pre-B- and T-ALL and CML [55]. Within specific cytogenetic subsets, GEP has also allowed identification of novel biologic and prognostic subgroups. In the study by Bullinger et al. [24], CN-AML patients predominantly clustered into two distinct subclasses differing with regard to the presence or absence of FLT3 mutations and FAB morphologic subtypes (M1/M2 versus M4/M5). Patients in these subclasses had a significantly different OS. Prognostic significance of these clusters for OS and DFS has recently been confirmed by Radmacher et al [26]. in an independent set of patients, using a different microarray platform. Additionally, Radmacher et al [26]. developed a class prediction algorithm that identified a signature-based classifier for outcome prediction. Subgroups of patients with significantly different OS and DFS were identified based on

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this outcome classifier, which appeared strongly associated with the FLT3-ITD. However, the classifier for dichotomized outcome classes had only modest predictive accuracy, with DFS and OS of approximately 60% of the patients being accurately predicted. Moreover, although the classifier showed some ability to identify a subset of patients with adverse outcome among patients without FLT3-ITD [26], other classifiers capable of predicting outcome of CN-AML patients more precisely are required. Gene-expression profiling has shown promise in the classification of AML, but this approach also has limitations. Several studies have shown that the differentiation stage of the lineage, reflected by the FAB classification, might direct the unsupervised clustering, which may obscure proper analyses [56]. Moreover, inconsistencies between data obtained using different microarray platforms have been observed [56,57]. Finally, at least in CN-AML, only a moderate predictive accuracy for outcome prediction has thus far been reported [26]. Nevertheless, by capturing a global view of the molecular heterogeneity of AML, gene-expression profiling may help to dissect pathogenetic mechanisms and thereby provide new insights into tumor biology and identify novel therapeutic targets. Gene-expression profiling by itself will likely not be sufficient to unravel the entire pathobiologic nature of AML. The integration with other genomic technologies, such as high-throughput mutational analyses and proteomic approaches, will be necessary to take on this important challenge [56]. Conclusions A number of molecular markers with prognostic relevance within distinct cytogenetic groups of AML patients have been and continue to be discovered. These molecular markers will likely guide future therapies both as prognostic factors and targets for specific therapeutic intervention. However, it is important to understand intricate interactions among various mutations and gene expression changes. Studies examining all known prognostic molecular alterations concomitantly to determine their relative influence on patients’ prognosis are ongoing, particularly in CN-AML. It is expected that cytogenetic and molecular genetic analyses will eventually allow accurate prediction of the response to treatment and the tailoring of therapy to specific genetic alterations present in the leukemic blasts, and that this will improve clinical outcome of patients with AML. References [1] Fröhling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malignancies: pathogenetic and clinical implications. J Clin Oncol 2005; 23:6285-95. [2] Mrózek K, Heinonen K Bloomfield CD. Clinical importance of cytogenetics in acute myeloid leukaemia. Best Pract Res Clin Haematol 2001; 14:19-47. [3] Estey E, Döhner H. Acute myeloid leukaemia. Lancet 2006; 368;1894-1907.

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E02 Autologous haematopoetic cell transplantation for acute myeloblastic leukaemia J. Holowiecki. Dept. of Haematology and BMT Silesian Medical University, Katowice The therapeutic strategy of acute myeloblastic leukaemia (AML), over the past four decades, has consequently evolved toward maximally aggressive remission induction therapy followed by post-remission treatment necessary to decrease the minimal residual disease (MRD) to a range potentially controllable by the immune system. In malignancies revealing dose dependant response to cytostatics there are two alternative strategies to obtain effective eradication of leukaemic cells: 1) Repeatedly administered high dose consolidation, optionally followed by maintenance treatment. 2) Myeloablative consolidation supported with autotransplantation of haematopoetic cells harvested in post consolidation phase from bone marrow (ABMT) or from peripheral blood (APBSCT). More resistant types of leukemia require allogeneic transplantation providing immunologic graft versus leukemia mechanism. The bone marrow transplantation registry data reflect the usefulness of autologous transplantation in AML. According to the EBMT activity survey of HSCT in 2005 autotransplantation was performed in 871 (24.4%) out of 3573 AML patients treated with HSCT. The corresponding CIBMTR data 2003 are 455/2723 (20,1%). The role of AHCT in AML patients with AML where a suitable donor is not available remains controversial. Major co-operative group trials have attempted to evaluate the contribution of autograft to therapy of AML in CR1. The trials performed before 2001 (EORTC-GIEMEMA, GOELAM, UK MRC, and US Intergroup) randomized over 1200 patients to AHCT versus, or in addition to, chemotherapy. The reduced relapse risk was observed in all studies but overall survival was not better in three of the trials. Only the MRC studt demonstrated certain survival benefit, but