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New Genetic Abnormalities and Treatment Response in Acute Lymphoblastic Leukemia

Jules P.P. Meijerink, Monique L. den Boer, and Rob Pieters


Numerous genetic abnormalities have been identied in acute lymphoblastic leukemia (ALL). Here we review the recurrent abnormalities with emphasis on those recently discovered, and discuss their association with chemotherapy resistance or sensitivity and with clinical response to therapy. Also, the role of genetic abnormalities in leukemogenesis and their potential as therapeutic targets will be discussed. Semin Hematol 46:16 23 2009 Elsevier Inc. All rights reserved.

B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

n general, children with B-lineage acute lymphoblastic leukemia (ALL) have a more favorable clinical outcome than those suffering from T-lineage ALL. B-lineage ALL forms a heterogeneous group and harbors many underlying genetic lesions with variable treatment responses (Table 1).

Genotypes Currently Used for Risk-Adapted Stratication of Pediatric Precursor B-ALL


The two main genetic subtypes TEL-AML1positive and hyperdiploidy with greater than 50 chromosomes together account for 50% of precursor B-ALL cases. Both are associated with a favorable outcome, having a 5-year disease-free survival (DFS) of greater than 85%.1 This favorable prognosis is most likely due to relative sensitivity of TEL-AML1 precursor B ALL to L-asparaginase,whereas hyperdiploid cases respond well to L-asparaginase and antimetabolites like 6-mercaptopurine and methotrexate.2,3 Within the TEL-AML1positive subtype, prognosis is impaired in approximately 10% of the cases bearing two copies of the TEL-AML1translocated gene, due to the high frequency of early relapses and increased resistance to prednisolone.4 The unfavorable genetic subtypes BCR-ABL1positive ALL and MLL-rearranged ALL each account for less than 5% of children with ALL who are older than 1 year of age. In contrast, the portion of MLL-rearranged ALL
Department of Pediatric Oncology/Hematology, Erasmus Medical Center RotterdamSophia Childrens Hospital, Rotterdam, The Netherlands. Address correspondence to Rob Pieters, MD, PhD, Department of Pediatric Oncology/Hematology, Erasmus Medical Center Rotterdam Sophia Childrens Hospital, Dr Molewaterplein 60, 3015GJ Rotterdam, The Netherlands. E-mail: rob.pieters@erasmusmc.nl 0037-1963/09/$ - see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2008.09.006

is greater than 80% in infants up to 12 months of age.5 The dismal prognosis has been linked to resistance to various drugs, L-asparaginase for BCR-ABL1positive ALL and glucocorticoids and L-asparaginase for MLLrearranged ALL.6,7 Investigations have shown that wellknown resistance mechanisms found in (solid) cancers, such as abnormalities in drug efux systems (P-glycoprotein, multidrug resistanceassociated protein, and others), detoxifying systems (glutathione-linked), apoptosis pathways, amino acid metabolism, and glucocorticoid receptor signaling are not the main explanations for drug resistance in pediatric ALL.8,9 In contrast, genome-wide technologies have revealed new insights as to the causes of resistance in pediatric ALL, and as a consequence have opened a new era of potential resistance modifying agents, such as reversing glucocorticoid resistance by glycolysis inhibitors.10-12 The above-mentioned genetic abnormalities are mutually exclusive, although incidentally combinations have been reported, such as hyperdiploidy and TEL-AML1positivity. These genotypes (with the exception of hyperdiploidy) result in fusion genes that affect the self-renewal and differentiation capacity of hematopoietic cells. Drugs that specically target fusion gene products and/or associated pathways are being developed and tested in clinical trials. Small-inhibitory molecules such as imatinib (Gleevec/Glivec, Novartis) and dasatinib (Bristol-Myers Squibb) have relative specicity towards activated tyrosine kinases and may therefore effectively kill cells that have abnormalities in these genes, as in BCR-ABL1positive ALL.13,14

New Recurrent Genetic Abnormalities in Precursor B-ALL


The currently known genotypes used to stratify patients in risk-adapted treatment regimens only comprise about 60% of precursor B-ALL cases. The genetic
Seminars in Hematology, Vol 46, No 1, January 2009, pp 16 23

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Genetic abnormalities and treatment response in ALL

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Table 1. Summary of Genetic Lesions and Outcome in Pediatric Precursor B-ALL (older than 1 year)
Genetic Abnormality Known genotype TEL-AML1 Hyperdiploid E2A-PBX1 BCR-ABL1 MLL New recurrent abnormalities Del9p Del9p iAMP21 Dic(9;20) BCR-ABL1like Mutations FLT3 SHP-2 RAS/MAPK Rearrangement t(12;21)(p13;q22) 50 chromosomes t(1;19)(q23;p13) t(9;22)(q34;q11) 11q23 Gene(s) TEL; AML1 E2A; PBX1 BCR; ABL1 MLL; various fusion partners 5-Year DFS (%) 80-85 95-90 85 25-40 30 Frequency (%) 20-25 25 5 3-5 2 Therapeutic Inhibitor

Imatinib/dasatinib

del(9)(p21) del(9)(p21) 21 or dup(21)(q22q22) dic(9;20)(p11-13;q11) ND

CDKN2A/B PAX5 AML1 ND ND FLT3 PTPN11 kRAS or nRAS

75 29 ND 60 202 ND ND

30-35 (40-45)1 30-35 (40-45)1 2 2 15-20 8 (15-20)3 7 16-584 PKC412/CEP-701 Farnesyl transferase inhibitor

Abbreviation: ND, not determined. 1Remaining group; negative for TEL-AML1, E2A-PBX1, BCR-ABL1, MLL-rearrangements and being non-hyperdiploid. 2For MLL-rearranged or hyperdiploid pre-B-ALL. 3For MLL-rearranged pre-B-ALL. 4For hyperdiploid pre-B-ALL.

abnormalities in the remaining 40% are unknown, rare, or not mutually exclusive. Since the highest absolute number of relapses occurs within this remaining category, progress in overall treatment results in pediatric ALL can only be obtained by the discovery of new genetic markers that can be used to identify those patients who are at high risk of treatment failure.15

Intrachromosomal Amplication of Chromosome 21


A small but prognostic highly unfavorable group, with a 5-year pDFS of less than 30%, harbors an intrachromosomal amplication of chromosome 21 (iAMP21).16 The iAMP21 includes additional copies of the AML1 gene and is found in about 2% of precursor B-ALL cases. An additional copy of the AML1 gene is linked to a favorable outcome of TEL-AML1positive ALL cases, suggesting that the adverse prognosis in iAMP21 cases may not be due to increased activity of AML1-responsive genes.4

Gene Mutations
Mutations in genes may affect the activity of corresponding proteins: activity may increase by mutations that alter the life-time, substrate specicity, binding capacity, or autoregulatory elements of the protein in question. In childhood precursor B-ALL activating mutations have been found in the Fms-like tyrosine kinase receptor gene (FLT3) in approximately 8% of cases,

especially MLL-rearranged and hyperdiploid ALL.17,18 This gene is involved in the early hematopoiesis by activating signal transduction pathways involved in proliferation and survival of progenitor cells. Mutations in FLT3 have been shown to abolish the auto-inhibitory capacity of the juxtamembrane domain (FLT3-ITD mutation) or result in constitutive activity due to single amino acid substitutions in the kinase domain (FLT3835/836 mutations). In addition to mutations, a high expression level of FLT3 has been linked to a poor prognosis of MLL-rearranged ALL in infants; the high expression level itself was sufcient to result in phosphorylated (and hence activated) FLT3 receptor without the need for activating mutations.19 Activated FLT3 has been shown to be a good target for newly developed small-molecule inhibitors that interfere with the catalytic domain of the tyrosine kinase, abolishing the further triggering of the downstream survival (AKTmediated) and proliferation (RAS/MAPK-mediated) signaling cascades, such as the small-molecule inhibitors PKC412 and CEP-701.20-22 Besides FLT3, mutations have also been found in downstream effector genes of tyrosine kinase receptors, such as the SHP-2 protein tyrosine phosphatase encoding gene PTPN11. This gene is mutated in about 7% of precursor B-ALL cases, mainly common ALL cases negative for the TEL-AML1 translocation. Mutations in this gene are often mutually exclusive with other genes that also affect the downstream RAS/MAPK signaling

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J.P.P. Meijerink, M.L. den Boer, and R. Pieters

pathway, such as mutations in NRAS and KRAS. These RAS mutations have been observed at high (but variable) frequency in hyperdiploid precursor B-ALL and are hypothesized (but not yet proven) to be associated with disease progression.23,24 FLT3, PTPN11, and RAS mutations are mostly mutually exclusive and collectively are present in about 35% of all precursor B-ALL cases. This high percentage of abnormalities implies that the FLT3-RAS signaling pathway should be further explored as a potential target for novel inhibitory drugs.

tor genes that affect their function, including Ikaros, E2A, EBF1, or PAX5.26,29 9p deletions were also found in 84% of BCR-ABL1positive ALL cases and highly correlated with (partial) deletions of the B-cell transcription factor Ikaros/IKZF1 on chromosome 7p.30 A multi-step deciency in the cell differentiation machinery of precursor B-ALL is implicated, although the causal relationship between 9p and 7p deletions remains to be demonstrated.31

BCR-ABL1like ALL
Recent gene expression proling studies have identied a new subtype that includes 15% to 20% of all precursor B-ALL cases and is associated with an unfavorable outcome, with a 5-years pDFS of approximately 60%.32 The gene expression prole of these cases resembles that of BCR-ABL1positive patients, although the latter are negative for this translocation; Additionally, so-called BCR-ABL1like cases are negative for other known genetic abnormalities, including TELAML1, MLL-rearrangements, E2A-PBX1, and hyperdiploidy. Further characterization of this relative large unfavorable prognostic group revealed greater than 70% abnormalities in B-cell differentiation genes, including PAX5, Ikaros, and EBF1, which is signicantly higher compared to 40% observed in the other common/preB-ALL subgroups,26,29 further providing evidence that the BCR-ABL1like cases reect a distinct entity.33 Some of these cases harbor a dicentric chromosome dic(9;20), but this nding presumably can not explain the unfavorable prognosis of BCR-ABL1like cases, since limited data suggest that the prognosis of dic(9; 20)-positive cases is not worse compared to follow-up data reported for other precursor B-ALL cases.15,34 It is intriguing that the newly discovered abnormalities/deletions in B-cell transcription factors occur in high frequency (40%), are often small in size and found in restricted loci (focal deletions), affect normal function by lack of expression or by generating dominant (negative) isoforms, or dysregulate B-cell differentiation similar to fusion genes such as TEL-AML1 and BCR-ABL1. In general, the well-characterized genotypes TEL-AML1, BCR-ABL1, MLL-fusion genes (50 partner genes), and E2A-PBX1 result in aberrant transcription and differentiation factors affecting normal hematopoiesis and cell fate. The recently discovered abnormalities in B-cell transcription factors such as PAX5 and Ikaros may point to a more general mechanism underlying B-lineage leukemia, such as a disturbed pre-B-cell receptor maturation machinery. The activity and/or specicity of recombinase-activating RAG1/2 genes that are normally involved in rearranging V(D)J segments as part of the B-cell receptor maturation process may be altered in these leukemic cells. Recently, RAG enzymes were postulated to be involved in producing isoforms of B-cell transcription factors, as spe-

Chromosome 9p Deletions
A relative large group of common/pre-B-ALL cases harbor deletions in chromosome 9p, including (among others) the cell cycleinhibitory CDKN2 locus encoding p16INK4A and p15INK4B and the B-cell transcription factor PAX5 gene.25,26 However, these 9p deletions were not restricted to the remaining group but were also found in BCRABL-positive, MLL-rearranged, TEL-AML1 positive, and hyperdiploid cases. Chromosome 9p deletions can vary between the loss of the entire 9p chromosomal region and regions of less than 50 kB in size of all precursor B-ALL cases.26 Deletions of the CDKN2 locus at primary diagnosis did not affect the prognosis of common/pre-B cases, and involvement of CDKN2 deletions in tumor progression is unlikely since the incidence of these deletions did not increase at time of relapse in paired initial-relapse samples.25

Abnormalities in Transcription Factors Involved in B-Cell Differentiation


PAX5 is a transcription factor involved in the commitment of hematopoietic cells to B-lineage differentiation. PAX5 deletions and translocations [PAX5-TEL as the result of the t(9;12) and PAX5-ELN as the result of the t(7;9)] have been observed in 30% to 35% of pediatric precursor B-ALL cases.26 The prognosis of patients having leukemic cells with PAX5 fused to the Ets transcription factor TEL hypothetically may be inferior due to blockade of B-cell differentiation, increased cellular migration and homing, and a reduced apoptotic potential of affected cells.27 PAX5 deletions deregulate B-cell development by a dominant negative loss-of-function mechanism.28 The prognostic impact of both PAX5 translocations and PAX5 deletions is unclear in pediatric ALL. However, even if PAX5 abnormalities have no prognostic value, the affected genes and pathways still represent highly interesting candidates for targeted therapy, since the defect is leukemia-specic and present in a high percent of cases. In addition to PAX5, a high frequency of abnormalities in other transcription factors involved in B-cell differentiation has been detected. Overall, about 40% of precursor B-ALL cases have deletions, amplications, mutations, or translocations in B-cell transcription fac-

Genetic abnormalities and treatment response in ALL

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cic recombination signal sequences (RSS) recognized by RAG were found near deleted areas involving the Ikaros gene.30

T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA


In contrast to childhood B-lineage ALL, for which the general outcome has improved over the last decades to cure rates reaching nearly 85%, the prognosis for children with T-ALL remains inferior: about 30% of these patients relapse following initiation of current treatment protocols.35 To date, many different types of genetic abnormalities have been identied in T-ALL, including chromosomal translocations as consequence of erroneous Tcell receptor (TCR) rearrangements, nonTCR-driven translocations, amplication, deletions, and point mu-

tations.36,37 Some of these abnormalities, which we denote as type A mutations, occur in a mutually exclusive fashion and are responsible for arrest at specic T-cell development stages (see Table 2). Based on gene expression proling studies using microarrays, growing evidence emerges that T-ALL may comprise at least ve distinct subgroups, each with a unique gene expression signature.38-40 The HOX11/TLX1 and HOX11L2/TLX3 subgroups comprise T-ALL cases with chromosomal translocations affecting the HOX11 or the HOX11L2 oncogenes, respectively. These two subgroups reect distinct entities that may have opposing prognostic relevance. The HOX11 subgroup has been associated with excellent prognosis,41,42 whereas HOX11L2positive T-ALL in various studies has been associated with a poor outcome.38,43,44

Table 2. Frequency of MolecularCytogenetic Aberrations in T-All, Relation to Outcome, and Potential

Therapeutic Targets
Type A Mutations/ T-ALL Subgroups TAL/LMO

Rearrangement t(1;14)(p32;q11)/t(1;7)(p32;q34) 1p32 deletion t(7;9)(q34;q32) t(11;14)(p15;q11)/t(7;11)(q34;p15) t(11;14)(p13;q11)/t(7;11)(q34;p13) 11p13 deletions t(10;14)(q24;q11)/t(7;10)(q34;q24) t(5;14)(q35;q32) inv(7)p15q34)/t(7;7)(p15;q34) t(10;11)(p13;q14) t(11;19)(q23;p13) 9q34 deletions t(7;19)(q34;p13) t(14;21)(q11.2;q22) t(6;7)(q23;q34) Rearrangement 9p21 deletions hypermethylation

Gene(s) TAL1 SIL/TAL1 TAL2 LMO1 LMO2 LMO2 HOX11 HOX11L2 HOXA CALM-AF10 MLL-ENL SET-NUP214 LYL1 BHLHB1 MYB Gene(s)

Outcome Frequency (%) Good? Good? Unknown Unknown Unknown Unknown Good Poor Undened Poor Unknown Unknown Unknown Unknown Unknown 15 4 1 1 7 3 8 24 5 4 1 3 1 1 3

Therapeutic Inhibitor HDAC inhibitor

HOX11 HOX11L2 HOXA

Histone H3K79 methyltransferase inhibitor

Unknown

Type B Mutations Cell cycle

Outcome Frequency (%) Unknown Unknown Unknown Good Good Unknown Unknown Unknown Unknown Unknown Unknown Poor Unknown Unknown Unknown Unknown No impact 70 1 1 50 9-30 1 10 3 1 17 8-15 4 1 1 1 1 3

Therapeutic Inhibitor DNA methyltransferase inhibitor

NOTCH1

(pre)TCR

Differentiation Tyrosine kinases

CDKN2A/2B CDKN2A/2B t(7;12)(q34;p13)/t(12;14)(p13;q11) CCND2 t(7;9)(q34;q34) NOTCH1 Mutations NOTCH1 Mutations FBXW7 t(1;7)(p34;q34) LCK Mutations RAS 17q11.2 deletion NF1 10q23.31 deletion PTEN Mutations PTEN 6q23 duplication MYB 9q34 amplication NUP214ABL1 t(9;14)(q34;q32) EML1-ABL1 t(9;12)(q34;p13) ETV6-ABL1 t(9;22)(q34;q11) BCR-ABL1 t(9;12)(p24;p13) ETV6-JAK2 Mutations FLT3

-secretase inhibitors

SRC kinase inhibitor Farnesyltransferase inhibitor PI3K/AKT inhibitors

ABL kinase inhibitor

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Other subgroups, including the TAL/LMO and the HOXA subgroups, may comprise various molecular cytogenetic abnormalities affecting many different oncogenes. The TAL/LMO subgroup includes T-ALL cases with chromosomal aberrations affecting one of the homologous basic helix-loop-helix genes (bHLH) TAL1 or TAL2, and/or one of the homologous LIM-domain only (LMO) genes LMO1 or LMO2. These bHLH and LMO genes encode for cofactors that form a multifactor transcription complex, possibly explaining why these abnormalities share a similar expression prole.37,40 The oncogenic role of this complex has been hypothesized to reect inhibition of the E2A/HEB transcription factors.45 As transcriptional repression requires recruitment of histone deacetylases (HDACs), patients with TAL/LMO abnormalities might benet from the addition of HDAC inhibitors to combination treatment. Whether or not this subgroup has prognostic signicance needs to be established. The HOXA subgroup includes T-ALL cases with aberrant activation of various members of the HOXA gene cluster including HOXA9 and HOXA10.39,40,46 HOXA activation can be due to rearrangements of the TCR locus directly into the HOXA gene cluster due to an inversion or translocations on chromosome 7 [Inv(7)(p15q34) or t(7;7)(p15;q34)],39,46 translocations resulting in CALM-AF1039,47 or MLL fusion products,48 or due to a deletion on chromosome 9 [del(9) (q34.11q34.13)] giving rise to a SET-NUP214 fusion product.40 The CALM-AF10 and the MLL and the SETNUP214 fusion products bind in the promoter regions of specic members of the HOXA gene cluster, and recruit the histone H3-Lysine79 methyltransferase hDOT1L that promotes further epigenetic chromatine modications and HOXA genes activation.40,49,50 Patients in this subgroup may therefore benet from histone H3-K79 methyltransferase inhibitors. CALMAF10positive T-ALL has been associated with a poor outcome,51,52 but further investigation is necessary to study whether this prognosis applies to the entire HOXA subgroup. In contrast to type A mutations, type B mutations are present in T-ALL irrespective of the T-ALL subgrouping (see Table 2). Type B abnormalities therefore mirror common abnormalities and affect various cellular processes, including cell cycle, T-cell commitment and self-renewal, TCR signaling processes, or they result in the aberrant activation of tyrosine kinases. In relation to loss of cell cycle regulators, the most important abnormalities observed in T-ALL are homoor heterozygous deletions of the cyclin-D/cyclin-dependent kinase-4 (CDK4) inhibitors p15/CDKN2B, p16/ CDKN2A in about 65% of pediatric T-ALL cases. The CDKN2A locus also encodes for the alternative p14ARF gene, which is part of the p53-regulated cell cycle and apoptosis machinery. The true proportion may be un-

derestimated, as inactivation of these loci in T-ALL may occur from silencing, especially by promoter hypermethylation, which may provide a rationale for clinical utlization of DNA methyltransferase inhibitors. Also, inactivation by point-mutations or post-transcriptional modications has been described.53 Loss of p16 and/or ARF in mouse models promoted T-cell leukemogenesis,54 whereas reintroduction of these loci delayed oncogenesis.55 The transmembrane receptor NOTCH1 is important during hematopoiesis; it promotes self-renewal of stemcells and T-lineage commitment of early lymphoid progenitor cells.56 For a long time, NOTCH1 has been implicated in T-ALL leukemogenesis due to its involvement in the rare translocation t(7;9).57 More recently, NOTCH1 was found to be mutated in more than 50% of T-ALL cases. Mutations are located in the heterodimerization (HD) or adjacent juxtamembrane domains. Other mutations disrupt the C-terminal domain rich in proline, glutamate, serine and threonine amino acids also denoted as the PEST domain, which normally functions as a target for the F-box protein FBXW7 as part of the E3-ubiquitin ligase complex that targets intracellular NOTCH1 (ICN) for proteolytic degradation.58 PEST mutations can occur in combination with HD mutations. NOTCH1 mutations promote ligand-independent NOTCH1 cleavage by proteases such as -secretase,58,59 resulting in the release of ICN, which functions as a transcription factor. Therefore, treatment of T-ALL using -secretase inhibitors seemed promising. However, a phase I/II clinical study using -secretase inhibitors in children with T-ALL has been unsuccessful to date, due to low antitumor effectiveness and severe gastrointestinal toxicity.60 The FBXW7 gene is inactivated by mutations in 8% to 30% of T-ALL patients, occasionally in combination with NOTCH1 HD mutations, and provides an alternative mechanism for NOTCH1 activation in T-ALL.61,62 The presence of NOTCH1 mutations and/or FBXW7 mutations has been correlated to good initial treatment response and good outcome.63,64 During normal T-cell development, NOTCH1 is an important transcription factor that activates a variety of genes. NOTCH1 also controls the assembly of the preTCR complex during T-cell development by regulating the expression of the pre-TCR alpha gene (pT). For various T-ALL oncogenes, a pivotal synergistic role for this pre-TCR complex has been demonstrated in T-cell leukemogenesis. An important oncogenic role of this complex was further supported by the nding of rearrangements or (in)activating point mutations in direct downstream signaling components of this pathway, or in the closely associated RAS-MAPK and the PI3K-AKT pathways. These include aberrant expression of SRCkinase LCK due to the t(1;7) translocation (1%),65 activating RAS mutations (8%10%),66 inactivating deletions/mutations of the RAS regulator NF1 (3%),67 or

Genetic abnormalities and treatment response in ALL inactivating mutations of PTEN (17%) resulting in constitutive activation of the AKT survival pathway.68 These ndings also may provide new rationales for experimental protocols to treat T-ALL with either SRCkinase inhibitors, farnesyltransferase inhibitors, or PI3K-AKT inhibitors. Finally, some mutations involve the formation of fusion products with potent tyrosine kinase activity. Several of these fusion products affect the tyrosine kinase domain of ABL1 due to rare translocations including BCR-ABL1, EML1-ABL1, and ETV6-ABL1. The NUP214-ABL1 fusion product due to an extra chromosomal amplication has been identied in about 6% of T-ALL cases. To date, this abnormality has predominantly been identied in T-ALL subclones of the HOX11L2, HOX11, and HOXA subgroups, suggesting that it represents an important mechanism for disease progression, as a relative late event in T-ALL that synergizes with deregulated HOX genes.69 Activation of the tyrosine kinase activity of FLT3 due to tandem duplications in the juxtamembrane domain have been identied in leukemic subclones in less than 3% of the T-ALL cases.70 Although NUP214-ABL or mutant FLT3 positive T-ALL may respond to potent tyrosine kinase inhibitors, including imatinib69 or PKC412 (Novartis), such treatment may only be effective against these leukemic subclones, leaving residual T-ALL cells from the original clone unaffected.

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CONCLUSIONS
Genetic classication of ALL has already become very important for daily practice in treating children with ALL. In the last decade, the application of new genome-wide screening techniques, such as microarray-based gene expression studies and array-comparative genomic hybridization (array-CGH) studies, have led to the discovery of many new genetic abnormalities in childhood B- and T-lineage ALL. The exact functional role of these abnormalities in the development of ALL remains to be elucidated, as well as their roles as prognostic factors or as potential therapeutic targets. Knowledge gained from current and future studies will lead to a better diagnostic classication and to improved patient-directed or individualized therapy for every child with ALL in the coming decades.
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