C H A P T E R 63
PATHOBIOLOGY OF ACUTE LYMPHOBLASTIC LEUKEMIA
Alejandro Gutierrez, Scott A. Armstrong, and A. Thomas Look
Normal lymphoid precursors undergo somatic recombination at their
immunoglobulin (Ig) or T- cell receptor (TCR) gene loci,1 and the
successful completion of V(D)J recombination, with the resultant
formation of a functional Ig or TCR, is required for the survival of
lymphocyte precursors. Positive and negative selection steps ensure
that only lymphocytes with Ig or TCRs that function appropriately
within the context of an individuals immune microenvironment are
allowed to proceed through the proliferation and differentiation steps
required for the development of mature lymphocytes. This develop-
mental process generates a repertoire of lymphocytes with unique
variations in the antigen-recognition portions of the Ig or TCR genes,
which form the foundation of a fully competent adaptive immune
system that can recognize a countless variety of foreign antigens.
The acquisition of somatic genetic alterations involving oncogenes
or tumor suppressors can lead to the dysregulated proliferation and
clonal expansion of lymphoid precursors that are characteristic of
acute lymphoblastic leukemia (ALL). Many genes that are critical to
leukemogenesis have been identified through the cloning and char-
acterization of genetic alterations induced by recurrent chromosomal
translocations.2-5 Additionally, a number of translocations have prog-
nostic significance and are used in modern ALL treatment protocols
to adjust the intensity of therapy. In many cases, the malignant
transformation of lymphoid cells is the result of altered expression of
transcription factors that play critical roles in normal B- and T-cell
development, although it may also involve the aberrant expression of
otherwise quiescent genes. More recent evidence has implicated
members of signal transduction pathways in leukemogenesis.6-8
Although the incidence of specific genetic alterations in ALL varies
according to patient age, evidence is accumulating that the pathogen-
esis underlying malignant transformation in similar genetic altera-
tions is similar across age groups.9,10
CLONAL ORIGIN OF LEUKEMIC LYMPHOID CELLS
Human ALL arises from a single progenitor cell that has acquired
somatic genetic changes, which lead to dysregulated proliferation and
differentiation arrest. The clonal origin of leukemia was first sug-
gested by the identification of the Philadelphia chromosome in
chronic myeloid leukemia (CML).11 Subsequently, numerous lines of
evidence have provided additional support for this theory, which is
now generally accepted. Uniform structural and numerical chromo-
somal abnormalities are frequently demonstrated in all leukemic lym-
phoblasts from an individual patient. Identical rearrangements of Ig
or TCR genes, which are somatic in origin, have been demonstrated
in ALL cell populations.2,12 Additionally, identical patterns of
X-chromosome inactivation have been demonstrated within all cells
of individual patients with ALL by allelic analysis of the glucose-6-
phosphate dehydrogenase gene on the X chromosome.13 Because
X-inactivation occurs in early embryogenesis before somatically
acquired mutations begin to promote the transformed phenotype,
this is particularly strong evidence for the clonal origin of leukemia.
In addition, the methylation patterns of restriction fragment length
polymorphisms in X-linked genes, as detected by Southern blot
analysis, have been used to show that even rare ALL cases with two
completely different cytogenetic clones probably arise by clonal evo-
lution from a single transformed progenitor.14
LINEAGE-SPECIFIC FEATURES
OF LEUKEMIC LYMPHOBLASTS
An important advance in the understanding and treatment of ALL
was the realization that malignant lymphoblasts share many of the
features of normal lymphoid progenitors.15-17 Thus, ALL cells rear-
range their Ig and TCR genes and express components of antigen
receptor molecules and other differentiation-linked cell-surface gly-
coproteins in ways that correspond to features of developing normal
B and T lymphocytes. In many cases, leukemic cells appear to repre-
sent the clonal expansion of a lymphoid progenitor that has arrested
its development at an early stage of B- or T-cell differentiation.18
Furthermore, research continues with the goal of defining stem cell
populations in lymphoblastic leukemia, such as those that have been
identified in acute myeloid leukemia (AML).19 However, with better
understanding of the normal patterns of antigen-independent lym-
phoid cell development, it has become clear that leukemic lympho-
blasts can show asynchronous gene expression with subtle variations
in phenotype.20 Hence, it should not be surprising that in some cases
of ALL, the blast cell phenotypes differ from those of normal lym-
phocyte progenitors, which is likely a result of aberrant regulation of
gene expression. Still, the general concept that leukemic cells should
be classified according to their normal developmental stage remains
an important one, providing a basis for the study of immunophenotype-
specific genetic changes.
B-Cell Acute Lymphoblastic Leukemia
The diagnosis of mature B-cell ALL is based on the detection of
surface Ig on leukemic blasts. This rare phenotype accounts for only
2% to 3% of ALL cases, and the lymphoblasts generally have distinc-
tive morphology, with deeply basophilic cytoplasm containing promi-
nent vacuoles; this morphologic pattern is designated L3 in the
French-American-British (FAB) system.21-23 Prominent clinical fea-
tures include concomitant extramedullary lymphomatous masses
in the abdomen or head and neck, frequent involvement of the
central nervous system (CNS) and cranial nerves, and tumor
lysis syndrome, often complicated by acute renal failure from uric
acid nephropathy.
Acute B-cell leukemia appears to be a disseminated form of
Burkitt lymphoma because these conditions share common cytoge-
netic, molecular genetic, immunologic, cytologic, and clinical fea-
tures.24 Acute B-cell leukemia does not respond well to chemotherapy
traditionally used for childhood ALL. However, good outcomes have
been obtained with treatments designed for Burkitt lymphoma,
which involve relatively brief but intensive regimens that emphasize
cyclophosphamide and the rapid rotation of antimetabolites in high
dosages.25-29 Thus, B-cell leukemia is the first form of ALL to be
935
 936 Part VII Hematologic Malignancies
recognized as a distinct clinical entity based on immunophenotypic
and cytogenetic features and the first to be treated by separate pro-
tocols designed specifically for the leukemias unique features.
B-Precursor Acute Lymphoblastic Leukemia
Approximately 80% of ALL patients have lymphoblasts with pheno-
types corresponding to those of B-cell progenitors.23,30 These cases can
be identified on the basis of cell surface expression of CD19 and at
least one other recognized B lineageassociated antigen: CD20,
CD24, CD22, CD21, or CD7923,30; most B-lineage ALL cases also
express CD10 (common ALL antigen [CALLA]). The lymphoblasts
may also express nuclear terminal deoxynucleotidyl transferase (TdT)
or CD34. About one-fourth of B-progenitor ALL cases express cyto-
plasmic Ig heavy-chain proteins and are designated preB-cell ALL.
Pre-B cases were originally shown to have a worse long-term response
to therapy compared with early pre-B cases, an observation that was
later attributed to the presence of the t(1;19) translocation that forms
the E2APBX1 fusion gene in about one-fourth of the pre-B cases.31
However, with the introduction of more intensive, risk-adjusted treat-
ment regimens, the prognosis for patients with t(1;19)+ ALL has
improved significantly.32,33
DNA rearrangement of Ig genes occurs before heavy-chain gene
expression in B-cell development, providing a genetic marker of
B-lymphocyte ontogeny. Korsmeyer and coworkers34,35 pioneered the
use of heavy- and light-chain gene rearrangements to support an early
B-lineage origin of most ALL blasts. This work was extended to
establish synchrony between Ig gene rearrangements and the expres-
sion of B lineagerestricted cell surface antigens. However, Ig heavy-
chain gene rearrangements have also been documented in about 15%
of T-cell ALL cases and a similar percentage of AML cases.36-38 Thus,
caution must be exercised when assigning cell lineage on the basis of
studies of Ig gene rearrangement.
The identification of specific immunophenotypic, genetic, and
clinical features that predict response to therapy in patients with
B-lineage ALL and the incorporation of these predictors into clinical
decision making are now widespread in modern ALL treatment pro-
tocols. This ability to predict outcome in these patients has been
closely tied to the remarkable improvements in therapy for children
with this disease, which 50 years ago was universally fatal. However,
many subgroups of pediatric and adult patients face a much poorer
prognosis, and much progress remains to be made.
T-Cell Acute Lymphoblastic Leukemia
Leukemias of T-cell precursors can be identified and classified accord-
ing to the sequence of expression of T-cellassociated surface antigens
during normal thymocyte ontogeny.39,40 In this tightly regulated
process, the earliest T-cell precursors are characterized by the lack of
expression of CD4 and CD8 surface markers. These double-negative
(DN) thymocytes proceed through a series of differentiation stages
(DN1-DN4 in mice; pro-T to pre-T in humans) during which the
cells lose CD34 expression, gain CD1a expression, and the TCR
genes TCR-, TCR-, and TCR- become rearranged. The successful morphology that coexpress myeloid-associated antigen54,55 and those
rearrangement of TCR- drives the production of immature single-
positive cells (ISPs) with a surface CD4+, CD8, sCD3 phenotype
(CD4 CD8+ sCD3 in mice). These cells then differentiate into early
double-positive (CD4+, CD8+) cells, at which point the TCR- gene
rearrangement occurs. Subsequently, these double-positive progeni-
tors differentiate into late cortical thymocytes showing a loss of CD1
and a gain of surface CD3 expression. This thymic T-cell develop-
mental process ends when mature CD4+ or CD8+ single-positive cells
emerge from the thymus,40,41 although further T-cell differentiation
occurs in the periphery upon antigen presentation. Numerous inves-
tigators, using a battery of monoclonal antibodies specific for T-cell
surface glycoproteins, have confirmed the close relationship between
the recognizable patterns of surface antigen expression on leukemic
T cells and the normal stages of thymocyte development.42-45
Lymphoblasts with a T-cell phenotype comprise approximately
15% of cases of ALL. These are often associated with distinctive
clinical features that include high circulating leukocyte counts, a male
predominance, CNS involvement, and a radiographically evident
thymic mass in many cases at presentation. Historically, patients
with T-cell ALL had an adverse prognosis compared with
patients with B-lineage ALL, but this gap has narrowed with the
intensification of therapy for these patients.46-48 In marked contrast to
B-precursor ALL, in which the availability of a wide range of clinical
and genetic prognostic markers has allowed the development of treat-
ment protocols tailored to an individual patients risk of relapse,
robust pretreatment prognostic markers had been lacking in T-ALL49
until the recent identification of the early T-cell progenitor subtype
of T-ALL.
Early T-Cell Progenitor Acute
Lymphoblastic Leukemia
Recent work has identified a very high-risk subset of T-ALL cases
characterized by differentiation arrest at the earliest identifiable stages
of T-cell development, which are defined either by absence of biallelic
TCR deletion (ABD), indicating differentiation arrest before TCR
gene rearrangement, or by expression of a characteristic early T-cell
precursor (ETP) phenotype.50,51 These cases comprise 8% to 15% of
pediatric T-ALL but appear to comprise a much higher fraction of
T-ALL in adults,52 thus providing one possible explanation for the
significantly inferior outcomes of adults versus children with T-ALL.
ABD/ETP T-ALL cases have recently been shown to harbor charac-
teristic genetic alterations that include PTEN deletions and activating
mutations of RAS, IL-7R, and FLT3, thus implicating these kinases,
and the signal transduction pathways they regulate, as novel thera-
peutic targets in high-risk T-ALL.50,52,53 Moreover, ETP T-ALL patients
also harbor oncogenic alterations that are characteristic of AML or
myelodysplastic syndrome (MDS), including DNMT3A, IDH1/2,
and EZH2 mutations,52,53 suggesting that the cell of origin of ABD/
ETP T-ALL may be a multipotent hematopoietic stem or progenitor
cell rather than a committed T-cell progenitor. Indeed, approximately
20% of relapses in T-ALL patients with biallelic TCR rearrange-
ments at diagnosis are characterized by absence of biallelic TCR
deletion (ABD) at relapse.50 It is implausible that a cell with a biallelic
deletion of genomic DNA would reacquire the deleted sequences;
thus, these data further support the hypothesis that, at least in some
cases of high-risk T-ALL, the transformed leukemia-initiating clone
is an immature progenitor characterized by differentiation arrest at
an earlier stage than that of the bulk lymphoblast population at the
time of T-ALL diagnosis.
Mixed-Lineage Leukemia
Acute mixed-lineage leukemias are defined by blast cells that coex-
press markers of both the lymphoid and myeloid lineages. Two dis-
tinct forms of these leukemias are recognized: those with lymphoid
with myeloid morphology and reactivity to myeloperoxidase staining
that coexpress cell-surface antigens normally restricted to lymphoid
cells. The origin of mixed-lineage leukemias has not been established.
One possibility is malignant transformation of pluripotent hemato-
poietic stem or progenitor cells that retain the ability to differentiate
in both the myeloid and lymphoid lineages; another is immortaliza-
tion of rare progenitor cells that normally coexpress features of both
lineages; and a third is aberrant gene expression caused by specific
genetic alterations.56 There was initially considerable controversy over
whether patients with lymphoid leukemia with expression of one or
more myeloid cell surface antigens (e.g., CD13, CD33, or CD14)
had an adverse prognosis. However, more recent data suggest that the
coexpression of myeloid antigens in ALL does not carry an adverse
prognosis in the setting of contemporary treatment regiments.57-59 The
 Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 937

expression of lymphoid markers in predominantly myeloid leukemias
is relatively rare, and although some studies reported an adverse effect
of T-lymphoid markers on outcomes,60 more recent analyses did not
demonstrate a significant prognostic value to lymphoid antigen
expression in AML.61
GENETIC BASIS OF LYMPHOID LEUKEMIA
Multiple acquired genetic abnormalities are responsible for the
aberrant proliferation and differentiation arrest characteristic of
ALL. These include chromosomal rearrangements detectable by
conventional cytogenetics, as well as lesions that are evident only by
molecular analysis. The ability to identify these changes and the
discovery that many of these can be used to predict response to
therapy have led to risk-adapted therapy for patients with ALL. It is
worth noting that the vast majority of patients with leukemia have
normal constitutional karyotypes, indicating that the genetic abnor-
malities in ALL lymphoblasts are usually acquired somatically and
thus are restricted to the malignant clone.
Chromosomal translocations are found in 75% of ALL patients
(Fig. 63-1).22,62 They can be broadly classified as recurrent lineage-
restricted abnormalities, which account for approximately two-thirds
of the translocations in ALL, or as random translocations, which

A
TCR translocations Random Hyperdiploid
7q35/TCR 25% 30%
14q11/TCR

Partner genes in
TCR translocations
TAL1 TAL2
LMO1 LMO2
LYL1 BHLHB1 3% 4% BCR-ABL
HOX11 MYC
HOX11L2 LCK 4% t(19;22)

MYC-lg translocations 2% 22% TEL-AML1

t(8;14), t(2;8), t(8;22) t(12;21)

6% MLL fusions
E2A-PBX1 5%
t(4;11), t(1;11), t(11, 19)
t(1;19) 1% 1%
E2A-HLF Low hypodiploid
t(17;19)

T cell Mature B cell Precursor B cell

Hyperdiploid
B Random
6%
41%
TCR translocations BCR-ABL
7q35/TCR 25% t(19;22)
14q11/TCR

Partner genes in
TCR translocations
2% TEL-AML1
TAL1 TAL2
LMO1 LMO2 2% t(12;21)
LYL1 BHLHB1 6%
HOX11 MYC 7% MLL fusions
HOX11L2 LCK
t(4;11), t(1;11), t(11, 19)

MYC-lg translocations 4% 4% Low hypodiploid

t(8;14), t(2;8), t(8;22) 3% E2A-PBX1

t(1;19)

T cell Mature B cell Precursor B cell

Figure 63-1 FREQUENCY OF THE MAJOR CHROMOSOMAL TRANSLOCATIONS IN PEDIATRIC (A)
AND ADULT (B) ACUTE LYMPHOBLASTIC LEUKEMIA (ALL). The genes affected by chromosomal transloca-
tion are shown in bold. T-cell receptor (TCR) translocations in T-ALL can activate a number of different proto-
oncogenes as shown in the insert, including TAL1, LMO1/2, HOX11, HOX11L2, and MYC.
 938 Part VII Hematologic Malignancies

have been identified only in single or very small numbers of cases.
The characterization of genes that span the breakpoints of recurrent
translocations has allowed the identification of genes that play critical
roles in leukemogenesis.3-5
control and coordinate the expression of large numbers of proteins
required for completion of lymphoid cell differentiation programs.
Disruption of transcription factors in leukemic blasts occurs by at
least two distinct mechanisms: the dysregulated expression of intact
genes and the creation of chimeric transcription factors.

Central Role of Transcription Factors

Molecular studies on recurrent chromosomal translocations in ALL
have demonstrated a central role for the aberrant expression of tran-
scription factors in the pathobiology of leukemia (Table 63-1). The
dysregulated expression of these transcription factors, which are often
functionally normal, leads to abnormal proliferation and differentia-
tion arrest of leukemic lymphoid and myeloid progenitors.3-5 Con-
served amino acid sequence motifs within the sequence-specific DNA
binding domains of these nuclear trans-activating proteins allow
them to be grouped into families that, in many cases, appear to be
involved in similar regulatory processes. Thus, the transcription
factor genes in Table 63-1 are grouped according to shared structural
features of their DNA-binding domains: basic region helixloop
helix (bHLH), cysteine-rich (LIM), homeodomain (HOX), basic
region-leucine zipper (bZIP), zinc-finger, A-T hook minor groove,
ETS-like, or runt homology. Another important association is the
lineage restriction of transcription factor genes affected by specific
chromosomal translocations, suggesting that the proteins they encode
disrupt the differentiation of specific lymphoid progenitors. This
interpretation implicates a central role for transcription factors in the
initiation of leukemia and suggests that the normal developmental
programs of progenitor cells of different lineages are controlled by
different regulatory programs.
Rabbitts63 has aptly described a key group of regulatory transcrip-
tion factors as the products of master genes. In his model, the
nuclear proteins encoded by these genes act positively to upregulate
critical target genes or negatively to interfere with normal regulatory
pathways. The net effect is disruption of gene regulatory cascades that
Developmental Biology of Oncogenic
Transcription Factors
A surprising connection has emerged from studies of oncogenic tran-
scription factors and the developmental proteins regulating segmenta-
tion in Drosophila because the DNA-binding domains of these
proteins often show striking homology. The proteins participating
in leukemogenesis interfere with a highly regulated network of
hematopoietic transcription factors, leading to arrested cell develop-
ment at stages corresponding to those of early lymphoid or myeloid
progenitors. Recent evidence implicating the major HOX genes
in the control of hematopoiesis as well as embryogenesis, together
with the established roles of Drosophila segmentation genes in
controlling HOM-C gene expression, suggests that the HOX loci
may serve as proximal targets of a wide spectrum of hybrid and
dysregulated transcription factors in the acute leukemias.5 In this
model, the oncogenic transcription factors inappropriately activate or
suppress the expression of HOX genes, which in turn regulate gene
programs with pleiotropic effects on normal hematopoietic cell
development.
The mechanisms that lead to the activation of oncogenic tran-
scription factors in ALL tend to vary according to the ALL subtype.
In mature B-cell ALL and in T-cell ALL, genetic lesions that lead to
the aberrant expression of structurally intact genes predominate, but
the chromosomal translocations that often occur in B-precursor ALL
generally lead to the formation of chimeric proteins, which produced
as a result of the translocation-induced fusion of the coding sequence
of two genes.

Table 63-1 Transcription Genes Affected by Chromosomal Breakpoints in the Acute Lymphoblastic Leukemias

Family* Translocation Affected Gene Disease

Basic helix-loop-helix (bHLH) proteins t(8;14)(q24;q32) MYC Burkitt lymphoma and
t(2;8)(p12;q24) MYC B-cell ALL
t(8;22)(q24;q11) MYC T-cell ALL
t(8;14)(q24;q11) MYC T-cell ALL
t(7;19)(q35;p13) LYL1 T-cell ALL
t(1;14)(p32;q11) TAL1 T-cell ALL
t(7;9)(q35;q34) TAL2 T-cell ALL
t(14;21)(q11.2;q22) BHLHB1
Cysteine-rich (LIM) proteins t(11;14)(p15;q11) LMO1 T-cell ALL
t(11;14)(p13;q11) LMO2 T-cell ALL
t(7;11)(q35;p13) LMO2 T-cell ALL
Homeodomain (HOX) proteins t(10;14)(q24;q11) HOX11 T-cell ALL
t(7;10)(q35;q24) HOX11 T-cell ALL
t(5;14)(q35;q32.2) HOX11L2 and BCL11B T-cell ALL
t(1;19)(q23;p13) E2A-PBX1 Pre-B-cell ALL
Basic-region/leucine-zipper (bZIP) proteins t(17;19)(q22;p13) E2A-HLF EPB ALL
A-T hook minor groove binding proteins
t(4;11)(q21;q23) MLL-AF4 EPB ALL
t(11;19)(q23;p13.3) MLL-ENL ALL or AML
ETS-like (TEL, ERG) proteins t(12;21)(p13;q22) TEL-AML1 ALL
Runt homology (AML1) t(12;21)(p13;q22) TEL-AML1 ALL

ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; EPB, early pre-B.
*Based on DNA-binding domain.

Partial list of MLL fusions.
 Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 939

transcriptional activation by MYCMAX complexes promotes prolif-

Dysregulated Expression of Structurally Intact Genes
Activation of MYC in B-Cell Acute
Lymphoblastic Leukemia
The vast majority of cases of mature B-cell ALL and Burkitt lym-
phoma are characterized by a translocation that places one allele of
MYC from chromosome 8 under the control of the regulatory ele-
ments of an Ig gene, either the heavy-chain gene on chromosome
14q32 or the or light-chain genes on chromosomes 2 and 22. This
leads to the aberrant expression of MYC, a prototypical basic helix
loophelix oncogenic transcription factor.64-72 In the predominant
t(8;14) translocation, the involved MYC locus is translocated into the
heavy-chain gene on chromosome 14 adjacent to the coding sequences
of the Ig constant region.64-66 The coding sequences of the Ig variable
region generally are reciprocally translocated to the distal tip of chro-
mosome 8. In variant translocations, the MYC gene remains on chro-
mosome 8, and portions of the respective light-chain genes are
translocated to that chromosome downstream of the MYC locus.67-71
Although the MYC coding region is not structurally altered by
translocation in most cases of B-cell ALL or Burkitt lymphoma, point
mutations in the N-terminal phosphorylation domain of MYC com-
monly arise in these tumors at codons 58 or 62.73-75 These codons are
phosphorylation sites involved in the regulation of the activation and
degradation of the protein,76 and these mutations lead not only to
the aberrant stabilization of MYC protein77-79 but also inhibit the
ability of MYC to activate the proapoptotic BIM gene while its ability
to stimulate proliferation remains intact.80
The mechanisms by which MYC exerts its potent growth-
promoting effects are complex and only partially understood. MYC is
estimated to regulate the expression of 15% of the genome81 and leads
to the transcriptional activation of a large number of genes involved
in cell division, growth, metabolism, adhesion, and motility.82 It also
leads to the transcriptional repression of many other genes, such as the
cell cycle inhibitors p27 and p21. MYC exerts its transcriptional activ-
ity via the formation of heterodimers with its DNA-binding partner
protein MAXMYCMAX heterodimers bind to canonical hexa-
meric E-box DNA sequences (5-CACGTG-3) where they activate
transcription.83 MAX can also heterodimerize with other bHLHZip
proteins, including MAD,84 MXI-1 (MAD2),85 and MNT.86 Whereas
eration, binding by MADMAX and other MAX heterodimers pro-
duces opposite effects; for example, MAD inhibits MYC function
both by competing with MYC for binding to MAX and by directly
inhibiting transcription. Moreover, in addition to its well-established
role as a transcriptional regulator, MYC is also an important regulator
of microRNA expression and ribosome biogenesis,87-90 thus implicat-
ing additional post-transcriptional regulation of protein expression
through which MYC exerts its potent biologic effects.
BHLH, LIM, and HOX Genes in T-Cell Acute
Lymphoblastic Leukemia
In leukemias with a T-cell phenotype, chromosomal breakpoints con-
sistently involve the TCR enhancer (7q34) or the TCRA/D enhancer
(14q11), both of which are highly active in committed T-cell progeni-
tors and can cause dysregulated expression of transcription factor
genes located at the breakpoint on the reciprocal chromosome
involved in these phenotype-specific rearrangements.91 The affected
transcription factors include (2) genes encoding bHLH family
members, such as TAL1,92,93 TAL2,94 LYL1,95 MYC,96-98 and BHLHB199;
(2) LIM-only domain (LMO) genes, such as LMO1 and LMO2100;
and (3) the orphan homeobox genes HOX11 and HOL11L1.37,101-106
The observation that T-ALL oncogenes act as master transcriptional
regulators during the embryologic development of specific organ
systems suggests that their aberrant expression in T-cell precursors
may contribute to the onset of leukemia by disrupting the mecha-
nisms that control cell proliferation, differentiation, and survival
during the discrete steps of normal T-cell development. Gene expres-
sion profiling and mutational analyses have shown that cases of
T-ALL can be separated into five subtypes based on the pattern of
multistep genetic abnormalities that occur (Fig. 63-2).
The best characterized of the oncogenic transcription factors
involved in T-ALL is TAL1 (also known as SCL), which is altered by
the t(1;14) or by site-specific deletions in approximately one-fourth of
childhood T-ALL cases.107-112 TAL1 is aberrantly expressed in the leu-
kemic cells of 60% of children and 45% of adults with T-ALL, and its
expression is required for maintenance of the leukemic phenotype in
human cell culture studies.113 TAL1 acts as a master regulatory protein
during early hematopoietic development and is required for the

p14ARF H2AX,
1. NOTCH-1 + TAL1 + LMO1/2 + CyclinD3 + MYC + p16INK4A + ATM

5q gene H2AX,
2. NOTCH-1 + LYL1 + LMO2 + CyclinD2 + MYCN + 13q gene + ATM

p14ARF
3. NOTCH-1 + HOX11 (TLX1) + NUP214-ABL + p16INK4A

4. p14ARF
NOTCH-1 + HOX11L2 (TLX3) + NUP214-ABL + p16INK4A

5. NOTCH-1 + MLLENL + HOXA9, HOXA10, HOXC6, MEIS1

Figure 63-2 MULTISTEP ONCOGENIC PATHWAYS IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

(ALL). Gene expression profiling and mutation analyses have revealed that five different multistep molecular pathways
can lead to the malignant transformation of developing thymocytes. NOTCH1 mutations are seen in samples from all
five T-ALL subtypes. HOX11, HOX11L2, and TAL1-overexpressing cases show high levels of MYC expression and share
the loss of the tumor suppressor genes p16/INK4A and p14/ARF on chromosome 9p. HOX11 and HOX11L2 are often
associated with a novel NUP214-ABL episomal fusion gene, which may render these T-ALLs sensitive to imatinib.
LYL1+ cases show high levels of expression of N-MYC and frequently have deletions affecting as yet unidentified loci
on chromosomal arms 5q and 13q. Finally, MLLENL+ cases have a clearly distinct gene expression profile character-
ized by low expression of MYC and other genes involved in cell growth and proliferation but high expression of HOXA9,
HOXA10, and HOXC6 in concert with the HOX gene regulator MEIS1. (Adapted from Armstrong SA, Look AT: Molecular
genetics of acute lymphoblastic leukemia. J Clin Oncol 23:6306, 2005, with permission.)
 940 Part VII Hematologic Malignancies
generation of all blood cell lineages.114,115 However, it does not seem to
be required for the generation and function of hematopoietic stem
cells (HSCs) during adult hematopoiesis.116 This class II bHLH tran-
scription factor binds to DNA by forming heterodimers with class I
bHLH factors such as E2A.117 Although TAL1 binding to promoter
sites that are normally occupied by TAL1, E2A or HEB can lead to
either repression or activation of transcription.113 TAL1 appears to
exert its leukemogenic effects largely via the inhibition of E2A activ-
ity.118 The observation that loss of E2A function induces T-cell leuke-
mias in mice119,120 and that the DNA-binding domain of TAL1 is
dispensable for transformation in transgenic mouse models121 gives
additional support to the notion that TAL1-mediated inhibition of
E2A plays a central role in its pathogenesis in T-ALL.
The LIM-only domain genes, LMO1/RBTN1/TTG1 and LMO2/
RBTN2/TTG2,100,122,123 encode proteins that possess duplicated
cysteine-rich LIM domains involved in proteinprotein interactions.
LMO2 interacts with TAL1 in erythroid cells and in T-cell
leukemias.124-126 Moreover, homozygous disruption of LMO2 in mice
causes the same phenotype as described earlier for TAL1 knock-outs,
indicating that a multiprotein complex is required for normal hema-
topoietic development that involves LMO2, TAL1, and other pro-
teins such as GATA1.127-130 In addition, overexpression of LMO1 or
LMO2 in thymocytes of transgenic mice leads to T-cell lymphomas,
recapitulating human T-cell tumors,131-135 and accelerates the onset of
leukemias in TAL1 transgenic mice lines.126
The homeodomain gene HOX11 (also known as TLX1), located
on chromosome 10, band 24, is one of the more interesting proteins
activated by translocation into the vicinity of the TCR loci.37,102,104-106
HOX11 is the founding member of a family of HOX genes that
includes HOX11L1 and HOX11L2106 and whose family members are
characterized by the presence of a threonine in the third helix of the
homeodomain, which confers specific DNA binding properties.
HOX11 was originally isolated from the recurrent t(10;14)(q24;q11)
in T-ALL37,102,104,105 and is aberrantly expressed in 5% of pediatric and
up to 30% of adult T-ALL patients.101,136,137 The relatively low fre-
quency translocations involving the HOX11 locus on 10q24, 2% to
5% in childhood T-ALL105 and 14% in adult cases,138 suggests that
alternative mechanisms leading to HOX11 overexpression account for
the activation of this transcription factor oncogene in a significant
fraction of T-ALL cases.
Similar to other HOX genes, HOX11 plays an important role in
embryonic development, and functions as a master transcriptional
regulator necessary for the genesis of the spleen.139,140 In the mouse
embryo, Hox11 expression can be detected in the branchial arches,
restricted areas of the hindbrain, and the splenic primordium,141,142
where it is required for the survival of early splenic progenitors.140 The
HOX11 oncogenic transcription factor is associated with T-cell leu-
kemogenesis at the early cortical thymocyte stage and relies on spe-
cific homeodomain-DNA interactions for its leukemogenic effects.143
Additionally, HOX11 directly interacts with the catalytic subunits of
the serinethreonine phosphatases PP2A and PPA, which mediates
disruption of the G2/M cell cycle checkpoint.144
A second HOX11 family member, HOX11L2, has also been impli-
cated in the pathogenesis of human T-ALL through characterization
of the t(5;14)(q35;q32), a cryptic chromosomal rearrangement
detectable only by fluorescence in situ hybridization or by chromo-
some painting techniques.103 This translocation leads to the ectopic
expression of HOX11L2, possibly by bringing it under the influence
of regulatory elements in the CTIP2/BCL11B gene, which is highly
expressed during T-lymphoid differentiation. In contrast to the pre-
dominance of HOX11 expression in adult T-ALL cases, both the
t(5;14) and expression of HOX11L2 can be detected in 20% to 25%
of children but in only 5% of adults with T-ALL.101,137,145-147 The role
of HOX11L2 as a master transcriptional regulator upstream of
important pathways involved in cell fate determination is supported
by its importance during embryonic development.148 In mice, Hox11l2
expression is essential for normal development of the ventral medul-
lary respiratory center.148 Mice deficient in this protein die soon after
birth from respiratory failure that resembles congenital central
hypoventilation syndrome in humans.
HOX11 and HOX11L2 are closely related in structure and have
a high degree of homology at the amino acid level, especially in the
homeobox domain, where their sequences differ by only three amino
acids. The high level of structural homology in their DNA-binding
domains supports the hypothesis that HOX11 and HOX11L2 may
induce T-ALL through regulation of the same transcriptional targets.
HOX11 expression has recently been shown to induce T-ALL in mice
and to disrupt the mitotic checkpoint via inhibition of CHK1, thus
accounting for the association of HOX11/HOX11L2 overexpression
with aneuploidy, which is otherwise rare in human T-ALL.149
Recently, the analysis of gene expression profiling using oligonu-
cleotide microarrays has shown that the expression of different tran-
scription factor oncogenes such as TAL1, LYL1, HOX11, and
HOX11L2 is associated with distinct gene expression profiles. These
unique signatures resemble those of thymocytes blocked at discrete
stages of T-cell development (Fig. 63-3),17 suggesting that transcrip-
tion factor oncogenes contribute to the pathogenesis of T-ALL by
interfering with critical regulatory networks that control cell prolif-
eration, survival, and differentiation during T-cell development.17
Based on the patterns of aberrant gene expression, human cases of
T-ALL can be classified into five different subtypes (see Fig. 63-2).
HOXA Cluster Translocations in T-Cell Acute
Lymphoblastic Leukemia
A recurrent inversion on chromosome 7 that places the HOXA cluster
in the vicinity of the TCR gene regulatory elements and that leads
to activation of the entire HOXA cluster was recently discovered in
approximately 5% of cases of T-ALL.150,151 Many of these patients also
carried cooperating oncogenic lesions consisting of NOTCH1 gene
mutations and deletions of 9p21.152 This new translocation that
directly activates HOXA gene expression provides additional evidence
for the role of aberrant HOXA activation in leukemogenesis and in
the pathogenesis of MLL- and CALM-AF10rearranged leukemias.
CD8+
CD4-
MLL-
ENL
LYL1+
HOX11+ TAL1+ CD8-
Late CD4+
CD4- CD8- Early
double negative cortical cortical
thymocytes Mature
CD4+ CD8+ single positive
double positive T cells
thymocytes
Figure 63-3 THYMOCYTE DEVELOPMENT IS A TIGHTLY REGU-
LATED PROCESS IN WHICH PROGENITOR CELLS UNDERGO
SEQUENTIAL STAGES OF DIFFERENTIATION, PROLIFERATION,
LINEAGE COMMITMENT, AND SELECTION THAT RESULT IN
THE PRODUCTION OF FUNCTIONALLY COMPETENT MATURE
T CELLS. Microarray gene expression profiling shows that T-ALL lympho-
blasts expressing high levels of the LYL1 transcription factor oncogene
undergo an early arrest at the double-negative thymocyte (CD4, CD8) stage
of development. In T-ALL cases with aberrant expression of HOX11, the
leukemic cells show a developmental arrest at the double-positive (CD4+,
CD8+) early cortical stage of thymocyte differentiation, but those with aber-
rant expression of TAL1 are arrested at the double positive late cortical stage.
T-ALL cells with expression of the MLL-ENL fusion gene are characterized
by an early arrest of differentiation with a gene expression signature that
indicates commitment to the -delta lineage. (Adapted from Ferrando AA,
Neuberg DS, Staunton J, etal: Gene expression signatures define novel oncogenic
pathways in T cell acute lymphoblastic leukemia. Cancer Cell 1:75, 2002, and Fer-
rando AA, Look AT: Gene expression profiling in T-cell acute lymphoblastic leukemia.
Semin Hematol 40:274, 2003, with permission.)

Other Genes Activated by Translocation
Transcription factors are not the only genes activated by translocation
to the sites of the Ig or TCR genes. In cases of B-precursor ALL car-
rying the t(5;14), for example, the IL-3 gene is activated by juxtaposi-
tion with the Ig heavy-chain locus.153,154 Similarly, relocation to the
TCRB locus activates expression of the LCK tyrosine kinase genes in
T-ALL cases with the t(1;7).155-157
Chimeric Transcription Factor Genes
Formation of chimeric proteins whose functional domains come from
two normally separate genes represents a second mechanism of aber-
rant transcription factor activation, which is more prevalent in
B-precursor ALL. These chromosomal translocations result in the
production of a chimeric protein by fusing the DNA-binding, dimer-
ization, and trans-activation regions of discrete genes. This process is
facilitated by the molecular structure of transcription factors in which
discrete coding regions of each gene encode particular functional
domains; this molecular structure likely arose for evolutionary
reasons.
E2A-PBX1 Fusion Genes in Pre-B Cell Acute
Lymphoblastic Leukemia
A well-known example of a chimeric transcription factor with onco-
genic potential is the E2A-PBX1 rearrangement, which results from
the t(1;19)(q23;p13) chromosomal translocation present in about
5% of all B-lineage ALLs and in 25% of cases with a pre-B (cyto-
plasmic Ig-positive) phenotype.32,91,158 This translocation fuses the
transactivation domain of the E2A transcription factor on chromo-
some 19 to a homeobox gene (PBX1) on chromosome 1, leading to
the expression of several forms of hybrid E2A-PBX1 oncoproteins.159-161
PBX1 is related to the Drosophila exd gene, a homeobox gene that
plays a role in lymphocyte development.162,163 The hybrid proteins
resulting from the t(1;19) retain the amino-terminal trans-activation
domains of E2A (AD1 and AD2) but not the bHLH DNA-binding/
protein interaction domain.160,161,164 The bHLH domain is replaced by
the homeobox DNA-binding domain of PBX1, enabling the fusion
protein to function as a chimeric transcription factor.165-167
The transforming potential of E2APBX1 was first demonstrated
by the rapid induction of AML in lethally irradiated mice repopulated
with bone marrow stem cells that had been infected with recombi-
nant retroviruses containing E2APBX1 genes.168 The fusion has also
been shown to transform NIH-3T3 fibroblasts and induce T-cell
lymphomas in transgenic mice.169,170 Additional studies have shown
that deletion of one of the E2A activation domains diminishes its
transforming activity, but deletion of the PBX1 homeodomain has
no effect.170,171 However, the homeodomain and flanking sequences
are required for interactions with other HOX proteins and for optimal
binding of E2APBX1 to specific DNA sequences.172-176 It thus
appears that complex interactions between E2APBX1 and other
HOX proteins target the fusion protein to specific target genes whose
activation is critical to lymphoid cell transformation. The presence
of the E2APBX1 translocation was originally associated with a poor
prognosis.31 However, this translocation no longer imparts an adverse
prognosis in the setting of modern risk-adjusted protocols for child-
hood ALL.32,177-179
E2AHLF Fusion Genes in Early Pre-B Acute
Lymphoblastic Leukemia
The t(17;19) is a rare recurrent chromosomal translocation that fuses
the amino-terminal transactivation domains of E2A to the C-terminal
DNA binding and dimerization domains of HLF,180,181 which belongs
to the PAR subfamily of bZIP transcription factors. Although E2A
HLF can bind DNA either as a homodimer or as a heterodimer with
Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 941
HLF and related proteins, no other PAR proteins are expressed in
hematopoietic cells, and the E2AHLF fusion binds DNA as a
homodimer in cells harboring the t(17;19). Similar to E2APBX1,
E2AHLF can transform NIH3T3 fibroblasts, a process that
requires the HLF leucine zipper domain and the E2A transactivation
domains.182 E2AHLF can also induce lymphoid tumors in trans-
genic mice.183
A major consequence of the activation of E2AHLF in lymphoid
precursors is dysregulation of the mechanisms that control pro-
grammed cell death in lymphoid progenitors. Expression of a
dominant-negative form of E2AHLF in t(17;19)-carrying cell lines
blocks E2AHLF function and results in apoptosis.184 In normal
pro-B lymphocytes, expression of E2AHLF reversed IL-3-depen-
dent and p53-induced apoptosis. HLF is the mammalian homologue
of the nematode protein ces-2, which regulates the death of specific
nerve cells in Caenorhabditis elegans.184-186 Ces-2 is necessary for the
death of the sister cells of a specific pair of serotonergic neurons
during worm development. Ces-2 induces apoptosis by inhibiting the
expression of ces-1, a prosurvival gene that normally inhibits pro-
grammed cell death by antagonizing the activity of the proapoptotic
factor egl-1. This pathway, which is highly conserved through evolu-
tion, is disrupted by E2AHLF fusion. Thus, in contrast to the
proapoptotic role of ces-2 in the worm via repression of ces-1, E2A
HLF blocks apoptosis by inducing the expression of SLUG, a ces-1
homologue normally responsible for protecting hematopoietic pro-
genitors from DNA-damageinduced apoptosis.187-190
The t(17;19) is seen in fewer than 1% of ALL patients, typically
occurs in adolescents, and is associated with disseminated intravascu-
lar coagulation and hypercalcemia at diagnosis. The t(17;19) seems
to impart unfavorable prognosis because each of seven patients whose
blasts expressed E2AHLF died of leukemia despite aggressive
therapy.91 It seems likely that resistance to chemotherapy in these cases
is mediated by the role of E2AHLF in driving the expression of
SLUG and inhibiting apoptosis.190
MLL Fusion Genes
Translocations involving chromosome 11 band q23 occur in approxi-
mately 80% of infant ALL cases, 5% of AML cases, and 85% of
secondary AML cases that occur in patients treated with topoisom-
erase II inhibitors.91 The gene bisected by 11q23 translocations is
designated MLL (also known as TRX1 or ALL-1), which is the human
ortholog of the trithorax Drosophila gene.191-194 The trithorax group
family of proteins are positive regulators of homeobox gene expres-
sion and act antagonistically to the polycomb group of proteins.195
Wild-type MLL positively regulates HOX gene expression and is
required for both primitive and definitive hematopoiesis.196-198 The
MLL protein undergoes proteolytic processing by Taspase1, a special-
ized protease that cleaves the MLL protein into N-terminal (MLLN)
and C-terminal (MLLC) fragments that remain associated through
intramolecular proteinprotein interaction domains.199-201 MLLN con-
tains several DNA-binding domains, including AT-hook domains
that nonspecifically bind the minor groove of DNA, a methyltrans-
ferase homology region (CxxC domain) that specifically binds
unmethylated DNA, four plant homeodomain zinc fingers with an
embedded bromodomain, and a transcriptional repression domain.195
The C-terminal fragment MLLC contains a transcriptional activation
domain that recruits the histone acetyltransferase cAMP response
element-binding protein (CBP) and a SET domain that is responsible
for its histone 3 lysine 4 (H3K4) methyltransferase activity.201 Recent
studies have revealed that wild-type MLL is a member of a large
multiprotein complex involved in chromatin modification and
remodeling, together with histone deacetylases and members of the
Swi/Snf chromatin-remodeling complex.202
MLL fusion oncogenes result from translocations whose break-
points cluster between exons 5 and 11 of MLL, and the resultant
fusion proteins retain the N-terminal region of MLL, including the
AT-hook and CxxC domains that bind DNA in a sequence-
nonspecific manner.203 By contrast, the C-terminal domains that
 942 Part VII Hematologic Malignancies

mediate the association of wild-type MLL with its endogenous chro-
matin modificationremodeling complex and its H3K4 methyltrans-
ferase activity are invariably lost from oncogenic MLL fusion proteins.
Instead, the C-terminus of MLL fusion oncoproteins is provided by
one of more than 60 different translocation partners, with common
translocations such as the t(4;11), t(9;11), and t(11;19)(q23;p13.3)
resulting in the in-frame fusion of MLL to AF4, AF9, and ENL,
respectively. The unrelated t(11;19)(q23;p13.1) translocation results
in fusion of MLL to ELL, the RNA polymerase II elongation
factor.204,205
Formal proof that MLL fusions play a critical role in the develop-
ment of leukemias has come from the generation of murine models
of MLL-induced leukemias. Chimeric mice harboring a MLLAF9
fusion gene generated by homologous recombination developed leu-
kemias with a latency of 4 to 12 months.206 Retroviral transduction
of MLLENL, MLLELL, and MLLCBP fusion genes in hemato-
poietic precursors induces transformation upon transplantation into
recipient mice.207-209 Similar results were recently obtained with a
model in which chromosomal translocations involving the Mll locus
are induced by directed interchromosomal recombination in mice, a
strategy that reproduces experimentally the initiating events in the
pathogenesis of MLL-rearranged leukemias.210 Interestingly, the intro-
duction of MLLAF9 into committed granulocyte-macrophage pro-
genitors in the mouse leads to the reactivation of a subset of genes
normally expressed only in HSCs and transforms these committed
precursors into AML leukemic stem cells by imparting the properties
of self-renewal,211 suggesting that the leukemogenic lesion in MLL-
rearranged leukemia might occur in a committed progenitor rather
than in a pluripotent HSC.
The analysis of gene expression profiles in MLL-rearranged
B-lineage leukemias has shown that these tumors have a characteristic
gene expression signature that includes the upregulation of several
HOX genes and the expression of numerous myeloid markers.212-214
Both early B- and T-cell ALLs with MLL rearrangements showed a
characteristic upregulation of specific HOX genes, including HOXA9,
HOXA10, and HOXC6 and the HOX gene regulator MEIS1.212-214
These results, together with the demonstration that HOXA9 plays
important roles in the transformation of hematopoietic precursors by
MLL fusion oncogenes in murine leukemia models,215,216 emphasize
the central role of HOX gene dysregulation in the pathogenesis of
MLL-rearranged leukemias. Additionally, as discussed later in this
chapter, overexpression or activating mutations of the FLT3 receptor
tyrosine kinase are frequent in MLL-rearranged leukemias,212,213,217,218
and a model of the multistep pathogenesis of MLL-rearranged ALL
is depicted in Fig. 63-4.
Until recently, the precise mechanisms mediating the oncogenic
activity of MLL fusion oncoproteins was unclear because MLL trans-
location partners have little sequence or functional similarity.
However, recent work has shown that a number of distinct MLL
translocation partners are functionally linked through their associa-
tion in protein complexes that mediate transcriptional elongation.219
Oncogenic MLL-fusion proteins associate with at least three such
complexes, PAFc, DOT1L, and pTEFb (also known as SEC or AEP).
PAFc, the polymerase-associated factor complex, regulates RNA poly-
merase II and associates with the amino-terminus of both wild-type
MLL and MLL fusions.220 The pTEFb complex (CDK9cyclin T)
phosphorylates RNA polymerase II; interacts with several MLL
fusion partners, including ENL, ELL, AF4, and AF5; and co-purifies
with several MLL fusion proteins.221,222 The DOT1L complex consists
of DOT1L; a histone H3 lysine 79 (H3K79) methyltransferase; and
multiple MLL fusion partners, including AF9, ENL, and AF10.223-228
Analysis of H3K79 methylation status has revealed increased
methylation at genes overexpressed in MLL fusion-driven
leukemias, including several direct targets of MLL fusions such as the
5 HOXA cluster genes and MEIS1.229-231 Interestingly, whereas the
DOT1L methyltransferase is required for transformation of murine

Activated signaling
molecules (FLT3, others)

Program of
self-renewal
Cytoplasm

Survival or
proliferation HOX genes

Meis 1
Fusion
MLL
Nucleus

Figure 63-4 MULTISTEP PATHOGENESIS OF MLL-REARRANGED ACUTE LYMPHOBLASTIC LEUKE-

MIA (ALL). MLL translocations induce self-renewal in hematopoietic progenitors as a first step in leukemogenesis.
The presence of FLT3 mutations in MLL-rearranged ALLs support activation of FLT3 or other kinases as cooperating
events in this disease. Clinical trials designed to assess the efficacy of FLT3 inhibitors in MLL-rearranged ALL are
being developed. (Adapted from Armstrong SA, Look AT: Molecular genetics of acute lymphoblastic leukemia. J Clin Oncol
23:6306, 2005, with permission.)

bone marrow progenitors by MLL fusion oncoproteins, DOT1L
plays a less prominent role in normal hematopoiesis, thus suggesting
a therapeutic window for therapeutic targeting of DOT1L in MLL-
driven leukemias.227,232-235 Indeed, a small molecule inhibitor of
DOT1L has recently been developed and demonstrates promising
activity against MLL-positive leukemia cells in vitro and in vivo, with
little toxicity in murine models.236
The presence of MLL rearrangements is associated with dismal
outcomes despite aggressive chemotherapy in most cases of ALL.23,237-
239
Additionally, bone marrow transplantation (BMT) in first remis-
sion in these patients is not clearly beneficial and may actually be
associated with a decrease in event-free and overall survival compared
with chemotherapy alone.239,240 However, the recent data implicating
DOT1L as a promising therapeutic target in MLL-driven leukemias
have generated considerable enthusiasm for the development of clini-
cal DOT1L inhibitors for the treatment of MLL-rearranged
leukemias.
CALMAF10 Fusion Gene in T-Cell Acute
Lymphoblastic Leukemia
The t(10;11)(p13;q14) detected in approximately 3% to 10% of
T-ALL patients and in occasional AML patients and results in the
fusion of CALM, encoding a protein with high homology to the
murine clathrin assembly protein ap3, with AF10, a gene identified
as an MLL partner in the MLLAF10 fusion resulting from the
t(10;11)(p13;q23).241 Although the mechanism of action of CALM
AF10 remains incompletely understood, the expression of this fusion
transcript has been associated with early arrest in T-cell development
and to differentiation into the -lineage in T-ALL.242 Additionally,
recent evidence suggests that aberrant upregulation of HOX gene
expression appears to be involved in CALMAF10-mediated leuke-
mogenesis, at least in AML cells that carry this translocation.243 Inter-
estingly, analysis of a mouse model of CALMAF10-induced AML
suggests that the leukemia stem cell in this model has lymphoid
characteristics, and cells from human patients with AML can be
identified that have similar characteristics to the disease-propagating
cell in this animal model.244
TELAML1 (ETV6-RUNX1) Fusion Gene in Early
Pre-B Acute Lymphoblastic Leukemia
Although most t(12;21) translocations are not detectable by standard
cytogenetic analysis, this translocation is detectable by molecular
techniques in approximately 25% of childhood B-lineage ALL, which
makes this the most common genetic lesion in pediatric ALL.91 The
TELAML1 translocation often arises prenatally and is probably the
initiating mutation in ALL, as evidenced by the identification of
identical TELAML1 translocations in identical twins with concor-
dant ALL245 and in retrospectively analyzed neonatal blood specimens
of children diagnosed with ALL.246 However, TELAML1 is insuffi-
cient in causing leukemia because the incidence of detectable TEL
AML1 fusions in the blood of normal newborns is about 100-fold
greater than the incidence of leukemia.247
The molecular mechanisms mediating TELAML1-induced leu-
kemogenesis remain poorly understood. This fusion gene encodes a
chimeric protein that contains the helixloophelix (HLH) domain
of TEL fused to nearly all of AML1 (also known as RUNX1 or
CBFA2), including both the transactivation domain and the DNA-
and protein-binding Runt homology domain. Both of these genes
are found in other leukemia-related translocations, and both are
essential for normal hematopoiesis. TEL was first identified in the
t(5;12) in chronic myelomonocytic leukemia, where it is fused to the
platelet derived growth factor receptor gene (PDGFRB), and is also
fused to ABL, MN1, and EVI1 in AML and to JAK2 in T-ALL.248
The role of TEL in hematopoiesis has been demonstrated with a
conditional mouse model in which the absence of TEL in early
development leads to the absence of fetal hematopoiesis. Interestingly,
Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 943
the inactivation of TEL in adult mice leads to the selective loss of
HSCs from adult bone marrow, but hematopoiesis is sustained by
committed precursors.249 Most TELAML1 leukemias show loss of
the normal TEL allele, suggesting that the leukemogenic effect of
TEL-AML1 may be mediated in part by loss of function of the TEL
gene.250-253
AML1 is the DNA-binding component of the AML1CBF tran-
scription factor complex disrupted by the t(8;21), t(3;21), and
inv(16) in AML, making it the most common target of chromosomal
translocations in leukemia. AML1 is a transcription factor that has
been shown to be required for the expression of several hematopoietic
genes involved in myeloid and lymphoid development, including
PU.1 and IL-3, although it can also act as a transcriptional repressor
in some settings.254 Homozygous disruption of the murine AML1
gene or CBFB gene results in the lack of definitive hematopoiesis,
indicating that genes regulated by AML1 are essential for normal
hematopoietic development.255,256 Additionally, rare familial muta-
tions in the AML1 DNA-binding domain lead to the familial platelet
disorder and predisposition to myeloid malignancy syndrome.257
However, the molecular pathways mediating TELAML1-induced
leukemogenesis remain incompletely understood.
The presence of the TELAML1 translocation is associated with
an excellent prognosis, with event-free survival rates of approximately
90% in a variety of studies.91,258,259 However, TELAML1 may not
represent an independent predictor of prognosis when age and white
blood cell count at the time of diagnosis are taken into account in
multivariate analysis.259 Nevertheless, this translocation identifies a
large subset of children with B-precursor ALL who appear to repre-
sent good candidates for less intensive therapy. The TELAML1
translocation is associated with lower expression of the MDR-1 mul-
tidrug resistance gene260 and of genes involved in purine metabo-
lism,261 which might account for the particular efficacy of these cases
to current combination chemotherapy regimens that rely heavily on
methotrexate and mercaptopurine, agents that inhibit de novo purine
synthesis.
Tyrosine Kinase Genes
BCRABL in B-Precursor Acute
Lymphoblastic Leukemia
The 22q chromosomal marker, often called the Philadelphia (Ph)
chromosome, which arises from the t(9;22)(q34;q11), was originally
identified in patients with CML, although it is also found in about
4% of childhood cases and 25% of adult cases of ALL,91 which are
almost always of the B-precursor subtype. The t(9;22) generates a
BCRABL fusion gene, consisting of 5 (upstream) sequences
from BCR and 3 (downstream) sequences of ABL. The t(9;22) break-
points on the distal tip of the long arm of chromosome 9 are scattered
over a distance of nearly 200kb within the first intron of the ABL
proto-oncogene, upstream of the tyrosine kinase domain.262-264 The
breakpoints in the BCR gene on chromosome 22 cluster in two sepa-
rate regions of that gene, known as the major breakpoint cluster
region (M-bcr) or minor breakpoint cluster region (m-bcr). In two-
thirds of cases of Ph-positive ALL, the breakpoint in the BCR gene
occurs in the minor breakpoint cluster region (m-bcr), but in all cases
of CML and about one third of cases of ALL, the breaks occur in the
major breakpoint cluster region (M-bcr).265 The fusion transcript
more commonly present in ALL (m-bcr) encodes a 190-kd protein
(p190), but the transcripts found in CML and some cases of ALL
(M-bcr breakpoint) encodes a 210-kd hybrid protein (p210).266-269
Both types of fusions generate chimeric oncoproteins that are acti-
vated as a tyrosine-specific protein kinase, similar to the v-abl
protein.270-272
The ABL tyrosine kinase is localized both in the nucleus and in
the cytoplasm of proliferating cells. It is normally activated by DNA
damage downstream of ATM and appears to promote p53-mediated
growth arrest.273-276 Mice deficient in ABL develop a wasting syndrome
 944 Part VII Hematologic Malignancies
and die soon after birth.277,278 In contrast to the nuclear and cytoplas-
mic distribution of normal ABL, the BCRABL fusion oncoprotein
has a cytoplasmic location and shows increased tyrosine kinase activ-
ity.279,280 When expressed in murine hematopoietic precursors, both
p190 and p210 transform hematopoietic cells in vitro and induce a
syndrome similar to CML in mice.281-284 Transformation by the BCR
ABL oncoprotein involves activation of the RASMAPK (mitogen-
activated protein kinase) pathway, PI-3 and JUN kinases, c-CBL and
CRKL, JAKSTAT, NFB (nuclear factor kappa-B), Src, and cyclin
D1.285-292 Among these targets, the RAS, JUNkinase, and PI-3 kinase
pathways are involved in the induction of cell proliferation.293 The
BCRABL oncoprotein affects multiple aspects of cell homeostasis,
including apoptosis, differentiation, and cell adhesion. An important
cellular effect of BCRABL is the induction of cellular resistance to
DNA damage agents such as cytostatic drugs and irradiation. After
DNA damage, BCRABL extends the duration of the G2/M cell
cycle checkpoint and facilitates DNA repair. It also upregulates the
antiapoptotic BCLXL gene, contributing to the suppression of apop-
totic cell death.294
The presence of the Philadelphia chromosome has historically
been associated with an extremely poor prognosis in ALL patients
despite treatment with intensified chemotherapeutic regimens.295-297
However, these patients have been shown to have particularly good
responses to allogeneic BMT in first remission, whether from matched
sibling or from unrelated donors.298-302 Moreover, the development of
imatinib mesylate, a pharmacologic tyrosine kinase inhibitor target-
ing the BCRABL oncoprotein, has opened novel therapeutic oppor-
tunities for the management of Ph-positive ALL. The utility of
imatinib as a single agent for BCRABL ALL is limited by the rapid
development of drug resistance.303,304 However, the combination of
imatinib with conventional chemotherapy has led to remarkable
improvements in outcome for patients with BCRABL ALL, with
3-year survival rates in children with this disease rivaling those
obtained with BMT.305
Despite its activity, imatinib resistance remains a barrier to further
therapeutic improvements in BCRABL ALL. Resistance most com-
monly emerges because of point mutations in the kinase domain of
BCRABL, and a series of novel BCRABL1 kinase inhibitors have
been developed that retain activity against many of these mutant
oncoproteins.306-308 Although not all BCRABL mutations that confer
resistance can be overcome with newer agents, the potential of these
newer BCRABL inhibitors with broader specificity to further
improve outcomes is actively being investigated. Additionally, a novel
pathway mediating resistance to BCRABL inhibition has recently
been uncovered in BCRABL ALL lymphoblasts. Upon BCRABL
inhibition, these cells markedly upregulate expression of the BCL6
transcription factor, a well-known oncogene that is often translocated
in diffuse large B-cell lymphomas.309 BCL6 then blocks activation of
the p53 pathway via transcriptional repression of ARF and thus
blocks the therapeutic efficacy of monotherapy with imatinib in
BCRABL ALL cells. Importantly, inhibition of BCL6 activity gener-
ically or using a peptide inhibitor of BCL6 demonstrated marked
activity in patient-derived BCRABL ALL cells grown in immuno-
deficient mice, highlighting the therapeutic relevance of these
findings.310
NUP214ABL1 in T-Cell Acute
Lymphoblastic Leukemia
Although the BCRABL1 translocation is rare in T-cell ALL, ampli-
fied episomes containing NUP214ABL1 fusion genes have recently
been described in approximately 6% of children and adults
with T-cell ALL.311 These episomes appear to arise via a mechanism
in which the genomic region of chromosome 9q34, which contains
both the NUP214 and ABL1 genes, is circularized in a manner
that leads to the fusion of these two genes. The breakpoint in the
ABL1 gene in all of these cases occurs in intron 1, which is the same
breakpoint observed in Philadelphia-positive CML and B-precursor
ALL, but the NUP214 breakpoints are variable. The wild-type
NUP214 protein is a component of the nuclear pore complex and
may contribute oligomerization motifs to the NUP214ABL1 fusion
oncogene. The NUP214-ABL1 fusion protein has constitutively acti-
vated ABL1 tyrosine kinase activity, which is inhibited by the BCR
ABL kinase inhibitor imatinib.311 The therapeutic potential of
imatinib or second-generation tyrosine kinase inhibitors for NUP214
ABL1-positive T-cell ALL is of considerable interest.
FLT3 in MLL-Rearranged Acute
Lymphoblastic Leukemia
FLT3 encodes a receptor tyrosine kinase that is highly expressed in
early hematopoietic precursors, where it plays important functional
roles.312,313 Multiple studies have shown that activating mutations of
FLT3, which lead to constitutive receptor tyrosine kinase activity even
in the absence of ligand, are common in leukemic myeloblasts in
patients with AML but are rare in adults with ALL.314-316 However,
gene expression studies demonstrated high expression of FLT3 in
most cases of ALL that involve MLL gene rearrangements or hyper-
diploidy.212,213,217 Additionally, activating mutations were identified in
18% of infants with MLL-rearranged ALL,218 in 21% to 24% of
hyperdiploid ALL cases,7,218 and in all three cases of the prothymic
CD117/KIT+ subtype of T-cell ALL in adults who were
examined.317
In the absence of FLT3 ligand, wild-type FLT3 receptors are inac-
tive because of autoinhibition mediated by the juxtamembrane
domain of the receptor. Upon binding of FLT3 ligand, normal FLT3
receptors homodimerize, become activated by phosphorylation, and
lead to the activation of signal transduction pathways that promote
proliferation and cell survival.318 Activating mutations of FLT3 found
in leukemias occur in two separate regions of the gene. In-frame
tandem duplications in the juxtamembrane domain lead to loss of
the autoinhibiton mediated by this domain, with subsequent dimer-
ization and receptor activation in the absence of FLT3 ligand.319
Alternatively, point mutations or insertions in the second tyrosine
kinase domain of the FLT3 receptor lead to autophosphorylation and
activation of downstream signaling in the absence of FLT3 ligand.6,316,320
Small molecule inhibitors of the FLT3 kinase lead to apoptosis in
AML cell lines in vitro and are currently undergoing phase I and II
testing in adults with AML and MDS.321 These small molecule inhibi-
tors are also active against MLL-rearranged ALL cell lines217,322 and
hold promise as targeted therapies for cases of ALL that rely on aber-
rant activation of FLT3.
CRLF2 and JAK2 Mutations in B-Precursor
Acute Lymphoblastic Leukemia
The CRLF2 cytokine receptor binds its ligand, thymic stromal lym-
phopoietin (TSLP), as a heterodimeric complex with the IL-7 recep-
tor subunit (IL-7R). TSLPCRLF signaling plays physiologic roles
during normal B-cell development and in inflammation. Genomic
analyses of B-precursor ALL patient samples revealed recurrent dele-
tions within the pseudoautosomal region of Xp22.3/Yp11.3 that
result in overexpression of the entire coding region of CRLF2 under
control of gene regulatory elements of P2RY8, a purinergic receptor
that is expressed at high levels in ALL cells.323-325 In some cases, such
rearrangements are also accompanied by a Phe232Cys point muta-
tion in CRLF2 that promotes constitutive dimerization and cytokine-
independent growth.324,325 CRLF2 rearrangements occur with
especially high frequency in patients with Down syndrome323-325 and
have been found to portend a poor prognosis in adults.324 Interest-
ingly, aberrant CRLF2 expression very frequently co-occurs with
JAK2 activating mutations, and these two genetic lesions collaborate
to induce ligand-independent activation of downstream signal trans-
duction pathways, suggesting that CRLF2 may act as a scaffold that
is required for activation of oncogenic JAK-STAT signaling by JAK2
mutations in B-precursor ALL.323-325 A number of JAK2 inhibitors
have been developed for the treatment of myeloproliferative

syndromes,326 and these findings have thus generated considerable
excitement to the application of such inhibitors to CRLF/JAK2-
mutant B-precursor ALL.
Interleukin-7 Receptor Mutations in T-Cell
Acute Lymphoblastic Leukemia
The IL-7R is required for normal T-cell development, with inacti-
vating IL7R mutations leading to severe combined immunodefi-
ciency.327 The IL-7R protein can heterodimerize with either IL2R,
resulting in the receptor for IL-7, or with CRLF2 to form the receptor
for thymic stromal lymphopoietin.328,329 Recently, point mutations in
the IL-7R have been described in approximately 10% of T-ALL
cases.53,330,331 These point mutations often introduce a novel cysteine
residue in the transmembrane domain of the protein that induces
ligand-independent receptor dimerization and activation of down-
stream oncogenic signal transduction pathways, including JAK
STAT and PI3KAKT. IL-7R mutations occur in approximately 10%
of T-ALL cases and are enriched in the TLX3-rearranged and HOXA-
overexpressing cases, thus suggesting the therapeutic utility of target-
ing this signal transduction pathway in T-ALL.
Mutated Genes
The activation of cellular proto-oncogenes by point mutation is dif-
ficult to detect because such lesions lack the cytogenetic abnormalities
that signal other forms of transforming alterations. Genes of this type
must first be identified in experimental systems so that investigators
know in advance the types of activating point mutations that are
likely to occur in human tumors. The prototypic genes of this class
are NOTCH1 and members of the RAS family, with mutations that
affect defined functional domains of the corresponding proteins.
Signaling
cell -Secretase
Cleavage 2
NOTCH
DSL
Cleavage 1
Metalloprotease
Figure 63-5 ACTIVATION OF NOTCH SIGNALING VIA EXTRACELLULAR AND INTRACELLULAR PROTEO-
LYTIC CLEAVAGE AND NUCLEAR TRANSLOCATION OF THE INTRACELLULAR NOTCH DOMAIN (ICN).
Interaction with serrate ligand (DSL) stimulates proteolytic cleavage of NOTCH by metalloproteases and -secretase. This
leads to the release of the intracellular ICN domain, which translocates to the nucleus, where it acts as a transcription factor
to regulate gene expression. (Adapted from Armstrong SA, Look AT: Molecular genetics of acute lymphoblastic leukemia. J Clin Oncol
23:6306, 2005, with permission.)
Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 945
NOTCH1 in T-Cell Acute Lymphoblastic Leukemia
The NOTCH1 gene was discovered as a partner gene in a t(7;9)
chromosomal translocation that is found in exceedingly rare cases of
T-ALL in which NOTCH1 is truncated and placed under the control
of the TCR locus.332 NOTCH1 plays several critical roles in promot-
ing T-cell development40,333-335 and is highly leukemogenic when
expressed in murine T-cell precursors.336 Despite the rarity of trans-
locations involving this gene, a search for activating NOTCH1 muta-
tions demonstrated that these are present in more than 50% of cases
of T-ALL, and these occur in all molecular subtypes of T-ALL.8
NOTCH1 is a transmembrane protein that is proteolytically
processed during its transit to the cell surface, where it exists as a
heterodimer consisting of extracellular and transmembrane subunits
(Fig. 63-5). Upon ligand binding, the transmembrane subunit
undergoes additional proteolytic cleavage within the plasma mem-
brane, which leads to the release of its intracellular domain, known
as ICN1 (intracellular domain of NOTCH1), into the cytosol. ICN1
subsequently translocates into the nucleus, where it is active as
a transcription factor. Activating NOTCH1 mutations in T-ALL
can occur as either missense mutations in the heterodimerization
domain, which allow constitutive proteolytic activation of the ICN1
domain,8,337 or as frameshift mutations or stop codons that lead to
truncation of the PEST domain.8,338 PEST domains are protein
sequences rich in proline (P), glutamate (E), serine (S), and threonine
(T) that regulate the proteasomal degradation of proteins with short
half-lives, thus these PEST-inactivating mutations impair the degra-
dation of ICN1. Mutations in both regions are often found on the
same allele in cases of T-ALL, and in experimental systems, mutations
in both domains of the same gene indeed are synergistic.8 Recent
work has demonstrated that MYC is an important transcriptional
target of NOTCH1, and it mediates many of the leukemogenic prop-
erties of MYC in human T-ALL cell lines.339-341
Nicastrin
Receiving cell
Nucleus
ICN
MAM1
ICN
CSL
 946 Part VII Hematologic Malignancies
The proteolytic activation of the ICN domain of NOTCH1 upon
ligand binding is mediated by -secretase, and -secretase inhibitors
have previously been developed because of the role of this enzyme in
the pathogenesis of Alzheimer disease. These inhibitors inhibit the
growth of many T-ALL cell lines,8,342 and trials of -secretase inhibitors
in patients with T-ALL are currently underway.
RAS Gene Mutations
Human tumor DNAs were initially found to contain activated homo-
logues of either HRAS or KRAS,343,344 proto-oncogenes that were iden-
tified on the basis of their homology with viral oncogenes. Gene
transfer methods identified an additional member of the RAS gene
family, called NRAS,345,346 that had not been observed as a component
of a transforming retrovirus. Proto-oncogenes of the RAS family
(HRAS, KRAS, and NRAS) encode 21-kDa proteins that are associ-
ated with the inner surface of the cytoplasmic membrane and are
involved in growth factor receptor signaling.347 The RAS proto-
oncogenes are activated to the status of transforming oncogenes by
somatic mutations that alter the amino acids specified by codons 12,
13, or 61. Mutated RAS genes lose their intrinsic GTPase activity and
become insensitive to GAP proteins, thus accumulating in their
active, GTP-bound conformation even in the absence of growth
factor binding to surface receptors. Aberrant RAS-mediated signaling
contributes to transformation through activation of the PI3K (phos-
phoinositide-3-kinase) and MAPK (mitogen-activated protein kinase)
pathways.347
Activated NRAS genes appear to be preferentially involved in
hematopoietic malignancies. They have been detected in myeloid cell
lines,348,349 in fresh leukemic cell samples from patients with AML or
CML,350-352 and in patients with MDS.353 In ALL, mutations of codons
12, 13, or 61 of NRAS have been found in approximately 10% of
patients, and KRAS mutations have been identified in 5% to 10%
of patients.354,355 RAS mutations are particularly common in cases of
MLL-rearranged B-precursor ALL, in which 40% of cases have KRAS
mutations and an additional 10% have NRAS mutations.355 Addition-
ally, RAS mutations have also been associated with high hyperdip-
loidy.356 The presence of RAS mutations does not appear to have
prognostic significance in the setting of contemporary treatment
regimens.354
PTENPI3KAKT Mutations in T-Cell Acute
Lymphoblastic Leukemia
The PI3KAKT signal transduction pathway, which is negatively
regulated by the PTEN tumor suppressor, induces cellular growth
and proliferation while inhibiting apoptosis and is aberrantly acti-
vated in a range of human cancers.357-359 In T-ALL, recent work has
identified a very high frequency of mutational activation of oncogenic
signaling through this pathway, most often via deletions or truncating
mutations of PTEN, but activating mutations of PI3K and AKT
genes also occur.360-363 Moreover, deletions of PTEN have been found
to predict treatment resistance in clinical specimens,53,360 and PTEN
inactivation has been shown to induce resistance to inhibition of
MYC and of NOTCH1 in zebrafish and murine models of T-ALL,
further implicating PTEN loss in T-ALL treatment resistance.362,364,365
Taken together, these findings thus suggest the potential clinical
utility of PI3K-AKT pathway inhibitors in high-risk T-ALL, many
of which are currently in human clinical trials.366
Gene Amplification
Gene amplification at the DNA level provides the cell a means to
increase expression of critical genes whose products are ordinarily
tightly controlled. Clinically important examples of proto-oncogene
amplification have been documented in solid tumors of both adults
and children. The MYCN gene, for example, is amplified from 10- to
300-fold in tumor cells from about one-third of cases of childhood
neuroblastoma; such amplification has been linked to an advanced
stage of disease and a poor prognosis.367 However, the cytogenetic
hallmarks of gene amplificationdouble-minute chromatin bodies
and homogeneously staining regionsare rarely found in karyotypes
of human leukemia cells, making it unlikely that high-level amplifica-
tion of cellular proto-oncogenes is widespread in ALL. However, as
noted earlier, the NUP214ABL1 fusion oncogene is amplified as a
small, cytogenetically undetectable episome in T-ALL.
MYB Duplication in T-Cell Acute
Lymphoblastic Leukemia
A duplication of the c-MYB gene was recently discovered in cases of
T-ALL,368-370a and a chromosomal translocation that juxtaposes c-MYB
to the TCR gene regulatory elements was also identified in a small
number of patients.369 The discovery of this gene duplication involv-
ing a small region of the chromosome was made possible by recent
technologic advances that now allow the high-resolution detection of
small regions of focal amplifications and deletions. The MYB tran-
scription factor is the cellular counterpart of the avian v-MYB avian
myeloblastosis virus, which causes a rapidly fatal monoblastic leuke-
mia in chickens and is essential for normal hematopoiesis, including
T-cell development. The MYB duplication is mediated by homolo-
gous recombination between identical sequence regions that flank the
gene,370a and its expression appears to be important in preventing
T-lymphoblast differentiation because knock-down of MYB expres-
sion in T-ALL cell lines induces T-cell differentiation.368
Tumor Suppressor Genes
Much attention has been focused on tumor suppressors, whose loss
of function via deletion or mutational inactivation leads to malignant
transformation. Knudson first proposed that inactivation of both
alleles of a single locus is needed to initiate the development of reti-
noblastoma, basing his hypothesis on the observed frequencies of
hereditary and sporadic forms of this disease.370b Allelic loss of defined
regions of many different chromosomes has been linked to specific
types of human tumors. By analogy with the findings in retinoblas-
toma, a reasonable hypothesis is that each of these regions harbors a
tumor suppressor gene whose product is uniquely involved in the
inhibition of cell cycle progression and promotion of terminal dif-
ferentiation of the normal cells that give rise to these different types
of tumors. Tumor suppressor genes that play an important role in
ALL include p53 and the p16 locus.
The p53 Tumor Suppressor
p53, located on chromosome 17, band p13, is mutated or lost
through chromosomal deletion in a wide variety of human tumors,371
including colon cancer, lung cancer, breast cancer, and osteosarcoma.
Heritable cancer-associated changes of the p53 tumor suppressor gene
occur in families with Li-Fraumeni syndrome, an unusual aggregation
of sarcomas, brain tumors, leukemias, adrenocortical carcinomas, and
premenopausal breast cancers.372-375 p53 encodes a 53-kDa transcrip-
tion factor that functions as a cell cycle and apoptosis checkpoint reg-
ulator.371,376-380 The p53 protein is increased by DNA damage, blocks
cell division at G1 to allow DNA repair, and activates apoptosis in
cells that have sustained DNA damage.381-386 The mechanism of p53
activation is triggered by the loss of activity of MDM2 after DNA
damage (via ATM) or oncogenic stress (via p14/ARF). As a negative
regulator of p53, MDM2 induces the ubiqitination of p53 and its
degradation by the proteasome. Hence, when MDM2 activity is abol-
ished, p53 accumulates and certain cell cycle regulatory genes such as
p21(WAF1/CIP1/SDI1/CAP20) and proapoptotic factor genes such
as BAX, PUMA, and NOXA are transcriptionally activated.
p53 is also inactivated in a variety of hematopoietic malignancies,
including B-cell ALL and Burkitt lymphoma, but is mutated or

deleted in fewer than 3% of pediatric B-precursor or T-cell ALL cases
at diagnosis.387-389 It thus appears to play a limited role in the etiology
of pediatric leukemia. However, p53 mutations are seen in approxi-
mately 25% of relapsed T-cell ALL cases, suggesting a role for p53
inactivation in the development of resistant disease.387,388 In addition,
p53 mutations were detected in three of 10 ALL patients who failed
on induction therapy or suffered early relapse, further supporting a
role for p53 inactivation in disease progression.390,391
The Cyclin-Dependent Kinase Inhibitors
The cyclin-dependent kinase (CDK) inhibitors, which include p15
(INK4B/MTS2), p16 (INK4A/MTS1/CDKN2), p18 (INK4C), p19
(INK4D), p21 (WAF1/CIP1/SDI1/CAP20), p27 (KIP1), and p57
(KIP2)m constitute a family of tumor suppressors that negatively
regulate the cell cycle by inhibiting CDK phosphorylation of pRB.392
The INK4A locus, located on the short arm of chromosome band
9q21, contains two different tumor suppressor genes, p16INK4A
and p14ARF (p19ARF in mice),393,394 each with a distinct promoter
and first exon but common second and third exons. Despite this
close relationship at the genomic level, p16 and p14 have totally
unrelated amino acid sequences because they use different reading
frames in their common second and third exons.395 A third tumor
suppressor gene, the CDK inhibitor p15INK4B, also resides in this
region.396,397 p16INK4A and p15INK4B directly inhibit cyclin
DCDK4/6 complexes and interfere with cell cycle progression.
Cyclin DCDK4/6 complexes promote entry into S phase through
phosphorylation of the retinoblastoma protein, PRB, leading to
the release of transcription factors, such as E2F, that promote entry
into S phase. By contrast, p14ARF lacks a direct effect on the cell
cycle machinery, acting instead to stabilize and upregulate p53
through the inhibition of MDM2.396-400 The role of the INK4a
locus in tumorigenesis was substantiated when mice with targeted
disruption of exon 2 in p16 developed tumors (primarily lymphomas
and fibrosarcomas) whose induction was enhanced by the topical
application of carcinogens and ultraviolet light.401,402 The phenotype
of the p16/p19/ mice could be duplicated by selective mutation of
the p19ARF gene (by targeting the first exon),393 although mice defi-
cient in p16INK4A with intact p19ARF also showed increased sus-
ceptibility to cancer.401,402
The short arm of chromosome 9 is the most frequent target of
chromosomal alterations in human cancer. In particular, the human
leukemias and lymphomas show a high frequency of 9p21 deletions
involving both the p16INK4A/p14ARF and the p15INK4B loci. Epi-
genetic silencing of these tumor suppressor genes through hyper-
methylation of their promoter sequences represents an alternative
mechanism of gene inactivation. Although p16INK4A/p14ARF and
p15INK4B are homozygously deleted in 20% to 30% of B-precursor
ALL cases and in 70% to 80% of T-cell ALL cases, epigenetic silenc-
ing of the p15INK4B promoter has been observed in 44% of primary
B-lineage ALLs.403-415
FBW7 in T-Cell Acute Lymphoblastic Leukemia
FBW7 is an E3 ubiquitin ligase that targets the transcriptionally
active intracellular form of NOTCH (ICN), MYC, and cyclin E for
degradation, and this gene is inactivated by mutation or deletion in
approximately 10% of T-ALL cases.416 In T-ALL cell lines, FBW7
mutation or homozygous deletion leads to resistance of NOTCH
pathway inhibition by -secretase inhibitor therapy, likely because
intracellular NOTCH protein levels remain high in the absence of
FBW7-mediated degradation despite inhibition of -secretase activ-
ity. Additionally, tumor-derived FBW7 mutations maintain their
ability to bind MYC but do not lead to its degradation, and these
may act as dominant negative mutants that protect MYC from deg-
radation.416 More recent work has shown that FBW7 also targets the
antiapoptotic protein MCL1 for proteasomal degradation in T-ALL,
providing an additional oncogenic consequence resulting from inac-
tivation of the FBW7 tumor suppressor.417
Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 947
PAX5 and Other B-Cell Developmental
Gene Alterations in B-Precursor Acute
Lymphoblastic Leukemia
A recent high-resolution genome-wide analysis of B-precursor ALL
cases using single nucleotide polymorphism arrays identified copy
number alterations in a number of genes that play important roles in
B-cell development.418 Genes involved in B-cell development were
found to be altered by deletion, amplification, mutation, or rear-
rangement in 40% of cases of B-precursor ALL. The most common
abnormalities identified were deletions of PAX5, and, upon further
analysis in other cases, other mechanisms that led to inactivation of
PAX5 were identified. These included a number of translocations that
led to fusion proteins that maintained the ability to bind to PAX5
transcriptional targets but lost regulatory ability, thus having domi-
nant negative activity, and inactivating point mutations that altered
the transcriptional activity of PAX5. Deletions were also detected in
the TCF3, EBF1, LEF1, IKZF1, and IKZF3 genes, all of which play
important roles in B-cell development.
Ikaros Mutations in High-Risk B-Precursor Acute
Lymphoblastic Leukemia
Ikaros is a DNA-binding transcription factor that is required for the
development of all lymphoid lineages, and expression of a dominant
negative Ikaros mutation in mice was shown to lead to T-cell lym-
phomas.419,420 However, the role of Ikaros in human leukemias was
not appreciated until genomic analyses of B-precursor ALL patient
samples revealed Ikaros (IKZF1) deletions in 29% of pediatric
samples.418 Ikaros deletions are strongly associated with BCRABL-
positive ALL, and these are characteristically acquired at transforma-
tion of CML to ALL (lymphoid blast crisis).421 Ikaros deletions
predict a very high risk of treatment failure that appears to be inde-
pendent of BCRABL, with patients with Ikaros-deleted, BCRABL
negative ALL faring similarly poorly to those with BCRABL-
positive ALL.422 Interestingly, recent work has implicated mutational
activation of CRLF2-JAK2 signaling in Ikaros-deleted, BCRABL-
negative ALL.423 Given the remarkable clinical activity of imatinib in
high-risk BCRABL ALL, these findings thus suggest the need for
clinical trials of small molecule inhibitors of JAK-STAT signaling in
this high-risk subtype of B-precursor ALL.
Inactivation of LEF1 in T-Cell Acute
Lymphoblastic Leukemia
The LEF1 transcription factor, a member of the LEF/TCF (lymphoid
enhancer binding factor/T-cell specific transcription factor) family of
DNA-binding transcription factors, is best known for its role as a
positive mediator -catenin transcriptional activity, a well-established
oncogene.424 Recent genomic analyses have identified mono- and
biallelic deletions and truncating mutations of LEF1 in 18% of
primary T-ALL patient samples, unexpectedly implicating LEF1 as a
T-ALL tumor suppressor.425 LEF1 inactivation is associated with a
very young age at diagnosis, T-cell differentiation arrest at an early
cortical stage of T-cell development, and activating NOTCH1 muta-
tions. Moreover, T-ALL cases with LEF1 inactivation are character-
ized by the highest expression of MYC mRNA of any T-ALL subtype,
a surprising finding given that LEF/TCF transcription factors trans-
activate MYC expression when bound to -catenin.426,427 In addition
to their well-established functions as transcriptional activators, LEF/
TCF transcription factors can also act as transcriptional repressors
when bound to Groucho/TLE family members in the absence of
-catenin activation.428 Although the precise mechanisms responsible
for the pathogenic effect of LEF1 inactivation in T-ALL have not yet
been established, these findings thus suggest the intriguing possibility
that LEF1 may actively repress MYC in T-ALL cells lacking -catenin
 948 Part VII Hematologic Malignancies
activation, and that LEF1 inactivation in this context may promote
maximal MYC overexpression downstream of other oncogenic lesions
that drive MYC overexpression, such as NOTCH1 mutations.
BCL11B Inactivation in T-Cell Acute
Lymphoblastic Leukemia
The BCL11B transcription factor is required for normal T-cell devel-
opment. In murine T-cell progenitors, inactivation of BCL11B leads
to developmental arrest at DN stages, acquisition of NK-like features,
and aberrant self-renewal activity.429-432 Monoallelic BCL11B deletions
or point mutations have recently been identified in 9% to 16% of
primary T-ALL patient samples.149,433 The point mutations identified
typically occur within the DNA-binding domains of BCL11B and
are predicted to disrupt their ability to bind DNA. Moreover, previ-
ous work in murine models has shown that BCL11B suppresses
T-lymphoblastic malignancies induced by p53 haploinsufficiency,
radiation, or the BCRABL oncogene,434,435 and BCL11B inactivation
is a particularly common cooperating lesion in murine T-ALL
induced by the TLX1 oncogene or by ATM deficiency.149,436 In human
T-ALL, recurrent cryptic t(5;14)(q35;q32) translocations juxtaposing
BCL11B and TLX3 have been described, which result in BCL11B
gene regulatory elements driving overexpression of TLX3.103,437,438
These translocations were long thought to be pathogenic because of
the resultant overexpression of the TLX3 oncogene. However, these
recent findings indicate that both BCL11B inactivation and TLX
oncogene overexpression are important pathogenic consequences of
this translocation, thus representing two oncogenic events from a
single genomic lesion.
PHF6 Mutations in T-Cell Acute
Lymphoblastic Leukemia
T-ALL has a conspicuous male predominance, suggesting the poten-
tial involvement of tumor suppressors on the X-chromosome. Tar-
geted mutational analysis of the X chromosome revealed one such
candidate tumor suppressor, PHF6, which encodes a nucleolar
protein of unknown function whose germline mutation leads to the
Borjeson-Forssman-Lehmann syndrome (OMIM 301900), which is
associated with severe mental retardation and facial dysmorphisms
but is not known to be associated with T-ALL. Somatic PHF6 muta-
tions were identified in 16% of pediatric and in 38% of adult T-ALL
patient samples, were most often nonsense or frameshift mutations,
and were associated with overexpression of the TLX1 or TLX3 onco-
genic transcription factors.439 The pathogenic consequences of PHF6
inactivation in T-ALL are presently unknown but are a matter of
active investigation.
Abnormalities of Leukemia Cell Ploidy
Abnormalities of chromosome number, which generally occur in the
absence of specific chromosomal translocations, have important prog-
nostic implications in childhood ALL. Found in 25% to 30% of
childhood ALL cases, hyperdiploidy of more than 50 chromosomes
in the leukemic clone is one of the most powerful means of identify-
ing patients with a very good prognosis.91,258 In particular, trisomies
of chromosomes 4, 10, and 17 impart a particularly good prognosis,
and hyperdiploidy in the absence of these trisomies is less of a favor-
able prognostic factor.440 As mentioned previously, activating muta-
tions of FLT3, a receptor tyrosine kinase, are common in these
patients. Patients with hyperdiploidy typically present with favorable
prognostic indicators, such as age between 2 and 10 years, a low white
blood cell count, and an early pre-B immunophenotype, and can
expect cure rates that approach 90%.441-443 The mechanism accounting
for the favorable outcome of patients with hyperdiploid ALL
remains elusive but may reflect an increased sensitivity to antimetabo-
lite therapy444 and a greater propensity to undergo apoptosis.445
Conversely, hypodiploidy (<45 chromosomes) carries an extremely
poor prognosis.91,258,446 Overall, adults with ALL have significantly
fewer numeric chromosomal abnormalities than do children; however,
abnormalities of ploidy do also appear to have prognostic significance
in adults because one large series demonstrated a favorable prognostic
impact of hyperdiploidy, but adults with hypodiploidy had extremely
poor outcomes.33
CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA:
A MODEL FOR GENE-BASED RISK ASSESSMENT
Many of the chromosomal and molecular genetic abnormalities in
the leukemic blasts of patients with B-precursor ALL are important
predictors of response to currently available chemotherapy. Lympho-
blasts from each new case of childhood ALL should be examined for
molecular prognostic markers, including leukemia cell ploidy, MLL
gene rearrangements, and the presence of BCRABL and TELAML1
fusion transcripts. Additional clinical criteria that have prognostic
significance, such as the age of the patient, the peripheral blood white
blood cell count at diagnosis, and the presence of CNS involvement,
are used by modern pediatric ALL treatment regimens to further
adjust the intensity of therapy to an individual patients risk of
relapse. It is also worth noting that B-precursor ALL in patients
younger than 1 year of age is associated with an extremely poor
prognosis secondary to an exceedingly high rate of relapse, which is
at least partly attributable to the very high frequency of MLL trans-
locations in this population, and these patients are typically treated
with unique treatment regimens designed specifically for infant ALL.
In T-ALL, the paucity of clinically useful prognostic markers has
greatly hindered progress toward personalized therapy, but the recent
identification of differentiation arrest at the earliest stages of T-cell
development as a marker of very poor prognosis,50,51 together with the
identification of mutations and gene expression signatures that closely
resemble those of AML and HSCs,52,53 has generated considerable
excitement for the application of intensified, AML-like treatment
regimens to this high-risk subset of T-ALL. Finally, response to treat-
ment has independent prognostic significance. Induction failure,
often defined in pediatric protocols as any morphologic evidence of
leukemia at the end of the initial month of induction chemotherapy,
represents a very poor prognostic factor and is an indication for BMT
after remission is achieved in most centers. Additionally, sensitive
flow cytometric and polymerase chain reactionbased methodologies
for the detection of minimal residual disease (MRD) are now avail-
able, and the level of MRD after induction chemotherapy has recently
been shown to provide an additional means of identifying patients at
low or high risk of relapse.447,448 With the availability of these prog-
nostic indicators, there remains little justification for uniform treat-
ment of newly diagnosed cases of ALL. Rather, modern trials
emphasize risk-based therapy to reduce toxicity in patients likely to
become long-term responders and to intensify therapy for those at
high risk of relapse.
A risk classification scheme based on a patients clinical features
and on the genetic features of leukemic blasts has been proposed for
patients with B-precursor or T-cell ALL who are 1 year of age or older
(Table 63-2). Patients with TELAML1 positivity or with simultane-
ous trisomies of chromosomes 4, 10, and 17 constitute the lower risk
group and are candidates for reduced-intensity treatment regimens.
The intermediate-risk group includes patients between the ages of 1
and 9 years with standard-risk age and leukocyte count, as defined
by criteria of the National Cancer Institute, and whose leukemic
lymphoblasts lack prognostically important genetic features.
The high-risk group includes patients who meet any of the following
criteria: age 10 years or older, high leukocyte counts at diagnosis,
a T-cell immunophenotype, or a slow early response to therapy.
Patients in the very high-risk group, defined by extreme hypodip-
loidy, MLL gene rearrangements, the BCRABL translocation, high
MRD at the end of induction chemotherapy, or T-ALL blasts harbor-
ing the high-risk early T-cell progenitor (ETP) or absence of biallelic
TCR deletion (ABD) markers are eligible for BMT in first remis-
 Chapter 63 Pathobiology of Acute Lymphoblastic Leukemia 949

Table 63-2 Clinical Risk Assignment in Childhood Acute are assigned to treatment according to their initial clinical features
Lymphoblastic Leukemia and, increasingly, the genetic and biologic properties of their leuke-
mic cells. We are now in a position to view ALL as a group of het-
Risk Group Features Recommended Therapy erogeneous diseases defined by discrete molecular lesions. As these
lesions have been systematically analyzed in larger numbers of
Low risk Simultaneous trisomies of Less intensive
patients, it has been possible to devise new classification schemes for
chromosomes 4, 10, 17 antimetabolite-based
ALL that reflect prognosis with exquisite precision. The development
TELAML1 fusion chemotherapy
of new drugs based on the molecular biology of ALL, whose promise
Intermediate Standard-risk age or Intermediate is highlighted by the early success of imatinib in BCRABLpositive
risk leukocyte count without antimetabolite-based ALL, is clearly a priority for the future and will likely take the form
other genetic risk features chemotherapy of compounds developed to specifically interfere with oncoproteins
High risk Presence of any of the Intensified multiagent expressed by each patients leukemic blasts. Additionally, the discov-
following: chemotherapy with ery that several kinases play important roles in ALL pathogenesis has
Age 10 years or older cranial radiation provided new opportunities for targeted drug development. The
T-cell immunophenotype opportunity is now at hand to improve therapy through randomized
WBC count >50,000/mm3 trials coordinated on a nationwide or even worldwide scale that focus
CNS leukemia on key subsets of acute leukemia patients whose lymphoblasts harbor
specific genetic abnormalities.
Very high BCRABL translocation* Allogeneic bone
risk MLL translocations marrow
Hypodiploidy <45 transplantation in
chromosomes first remission SUGGESTED READINGS
Induction failure or Comprehensive Reviews
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of induction chemotherapy Armstrong SA, Look AT: Molecular genetics of acute lymphoblastic leukemia.
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Pui CH, Robison LL, Look AT: Acute lymphoblastic leukaemia. Lancet
ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia;
CNS, central nervous system; MRD, minimal residual disease; TCR, T-cell
371:1030, 2008.
receptor; WBC, white blood cell.
*Recent data also indicate excellent 3-year survival rates with the use of
imatinib, a targeted BCR-ABL inhibitor, in combination with conventional Specific Genetic Lesions in Acute
chemotherapy. Lymphoblastic Leukemia

Current evidence suggests that bone marrow transplantation in first remission
may not improve outcomes for children with MLL-rearranged ALL. Bernt KM, Zhu N, Sinha AU, et al: MLL-rearranged leukemia is

Although MLL translocations are almost always associated with very poor
outcomes, patients with T-cell ALL who have the t(11;19) MLL-ENL dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell
translocation appear to have excellent outcomes with conventional 20:66, 2011.
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aneuploidy in T cell transformation. Nat Med 16:1321, 2010.
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Mullighan CG, Collins-Underwood JR, Phillips LA, et al: Rearrangement of
of the BCRABL translocation, although its benefit in MLL-
CRLF2 in B-progenitor- and Down syndrome-associated acute lympho-
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