Giant strides have been made in the management of childhood acute lymphoblastic leukemia (ALL) over previous decades. Extensive collaborative efforts internationally have played a vital role in the remarkable progress made in not only improving therapeutic outcomes but also deciphering the complex biology of childhood ALL. This review summarizes various insights gained from biological studies of childhood ALL, with a focus on recent studies, and also discusses genomic lesions and epigenetic regulatory mechanisms associated with leukemic transformation. The importance of studying the biology of the host so as to understand additional heterogeneity in treatment response and toxicities is highlighted.
Key points
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Childhood acute lymphoblastic leukemia (ALL) is a heterogeneous disease with multiple distinct biological subtypes.
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High-throughput genomic profiling and next-generation sequencing technologies have identified submicroscopic genomic lesions and sequence mutations that define novel subtypes of ALL.
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The discovery of various oncogenic pathways and candidate genes has led to the development of biologically based targeted therapy.
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Host germline polymorphisms influence susceptibility to ALL, chemotherapy-related toxicities, and response to therapy.
Introduction
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, accounting for 25% of all childhood cancers. In the United States, approximately 3000 children aged 1 to 19 years are diagnosed with ALL annually. Giant strides have been made in the management of childhood ALL over the past 50 years, which has resulted in improvement in cure rates from approximately 10% to approximately 90%. The rational use of multiagent systemic chemotherapy over a prolonged duration (2–3 years) and adequate central nervous system (CNS)-directed therapy, in addition to improved antibiotic and blood product support in the 1960s and 1970s were responsible for the early improvements in outcome. However, insights into the heterogenic biology of ALL and monitoring of minimal residual disease (MRD) have helped to refine therapy based on risk of relapse to maximize cure and minimize toxicities. For example, identification of the Philadelphia chromosome in a subset of patients with ALL has made it possible to incorporate ABL tyrosine kinase inhibitors into chemotherapy regimens. This targeted-therapy approach has improved the cure rate of patients with Philadelphia chromosome–positive ALL from 35% to approximately 70% over the last 10 years, even without stem cell transplantation.
Leukemic cells have, and are being thoroughly investigated by methods ranging from karyotyping, which identifies large chromosomal alterations, to whole-genome sequencing, which identifies cryptic changes in the entire genome. ALL is particularly amenable to biological studies because of the relative ease of obtaining samples, which in most cases is an enriched population of blasts. Moreover, because most children with ALL are treated uniformly on large clinical trials, well-annotated clinical information is available to correlate with biological findings. Extensive collaborative efforts among various study groups internationally have played a vital role in the remarkable progress made in not only improving therapeutic outcomes but also deciphering the complex biology of childhood ALL.
This review summarizes various insights gained from biological studies of childhood ALL, with a focus on recent studies, and also discusses genomic lesions and epigenetic regulatory mechanisms associated with leukemic transformation. Finally, the importance of studying the biology of the host so as to understand additional heterogeneity in treatment response and toxicities is highlighted.
Introduction
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, accounting for 25% of all childhood cancers. In the United States, approximately 3000 children aged 1 to 19 years are diagnosed with ALL annually. Giant strides have been made in the management of childhood ALL over the past 50 years, which has resulted in improvement in cure rates from approximately 10% to approximately 90%. The rational use of multiagent systemic chemotherapy over a prolonged duration (2–3 years) and adequate central nervous system (CNS)-directed therapy, in addition to improved antibiotic and blood product support in the 1960s and 1970s were responsible for the early improvements in outcome. However, insights into the heterogenic biology of ALL and monitoring of minimal residual disease (MRD) have helped to refine therapy based on risk of relapse to maximize cure and minimize toxicities. For example, identification of the Philadelphia chromosome in a subset of patients with ALL has made it possible to incorporate ABL tyrosine kinase inhibitors into chemotherapy regimens. This targeted-therapy approach has improved the cure rate of patients with Philadelphia chromosome–positive ALL from 35% to approximately 70% over the last 10 years, even without stem cell transplantation.
Leukemic cells have, and are being thoroughly investigated by methods ranging from karyotyping, which identifies large chromosomal alterations, to whole-genome sequencing, which identifies cryptic changes in the entire genome. ALL is particularly amenable to biological studies because of the relative ease of obtaining samples, which in most cases is an enriched population of blasts. Moreover, because most children with ALL are treated uniformly on large clinical trials, well-annotated clinical information is available to correlate with biological findings. Extensive collaborative efforts among various study groups internationally have played a vital role in the remarkable progress made in not only improving therapeutic outcomes but also deciphering the complex biology of childhood ALL.
This review summarizes various insights gained from biological studies of childhood ALL, with a focus on recent studies, and also discusses genomic lesions and epigenetic regulatory mechanisms associated with leukemic transformation. Finally, the importance of studying the biology of the host so as to understand additional heterogeneity in treatment response and toxicities is highlighted.
B-cell acute lymphoblastic leukemia
Eighty-five percent of the cases of childhood ALL are of the B-cell lineage (B-ALL). To keep pace with the growing impact of biological findings on treatment outcomes, in 2008 the World Health Organization revised the nomenclature from solitary precursor B-ALL to a classification based on 7 specific, recurring genetic lesions (eg, B-ALL with ETV6-RUNX1 , B-ALL with hyperdiploidy). Of note, the term B-ALL is not used for Burkitt leukemia/lymphoma, which is a mature B-cell malignancy. As newer subtypes are identified, biology-centered classifications need to be continually reviewed and updated ( Fig. 1 ).
Conventional Chromosomal Alterations
ALL commonly arises from a series of genetic alterations and, in most ALL subtypes, the interplay of these alterations. For the last 3 decades, several conventional cytogenetic studies of genetic aberrations that include chromosomal translocations and alterations in chromosome number have provided information on the pathogenesis of ALL. Common translocations in children with B-ALL include t(12;21) [ ETV6-RUNX1] (25%), t(1;19) [ TCF3-PBX1 ] (5%), t(9;11) [ BCR-ABL1 ] (3%), and translocations involving the MLL gene with various partner fusion genes (5%). Gains in whole chromosomes, or high hyperdiploidy (>50 chromosomes), accounts for 25% of childhood ALL, whereas hypodiploidy (<44 chromosomes) accounts for approximately 1% of cases. Several of these genetic changes have prognostic and therapeutic implications and are important in risk-stratification schemes. The overall survival (OS) of patients with ETV6-RUNX1 or high-hyperdiploid ALL is more than 93% ; therefore these patients are treated on less intensive regimens, provided that they have an adequate early response to remission induction therapy (as currently assessed by the measurement of MRD). Contemporary therapy has abolished the previously unfavorable prognostic impact of TCF3-PBX1 (t[1;19]). However, because bone marrow and CNS relapse could be competitive events, patients with TCF3-PBX1 may need more intensive intrathecal therapy to reduce the risk of CNS relapse with improved systemic therapy. Similarly, the addition of tyrosine kinase inhibitors has dramatically improved the bone marrow control of patients with BCR-ABL1– positive ALL, and attention should be paid to optimal intrathecal therapy in this genetic subtype of ALL. Hypodiploidy continues to be a high-risk feature, necessitating further understanding of oncogenic mechanisms and the rational use of targeted therapy (eg, for RAS pathway inhibition). The frequency and prognostic impact of MLL rearrangements differ by age. Approximately 80% of infants younger than 1 year harbor MLL rearrangements, and their overall outcome is poor (5-year survival 50%) despite receiving very intensive therapy.
Fusion gene products that result from chromosomal translocations provide the lymphoid progenitor or stem cell with leukemogenic potential, such as constitutional activation of tyrosine kinases (eg, ABL1 ) or disruption of genes that regulate normal lymphoid development (eg, ETV6 , PAX5 ). Additional genetic hits are often required for the ultimate development of leukemia, and include loss of the tumor-suppressor gene CDKN2A and deletion of the nontranslocated ETV6 allele in ETV6-RUNX1 –positive ALL. High-resolution genomic studies are revealing the entire range of cooperating genetic changes and pathogenic mechanisms.
Submicroscopic Alterations
The advent of genome-wide profiling of RNA and DNA and next-generation sequencing (NGS) technologies has greatly increased our ability to identify and catalog submicroscopic genetic alterations and sequence mutations in ALL, which in turn help define new molecular subtypes. Some novel genomic lesions have prognostic and therapeutic significance, and may be used to refine risk-stratification schemes in the near future; for example, IKZF1 deletion predicts poor prognosis in children with B-ALL. In addition, the recognition of specific molecular lesions and critical oncogenic pathways paves the way for developing novel targeted approaches to therapy (eg, inhibition of the ABL tyrosine kinase or JAK-STAT pathways). Single-nucleotide polymorphism (SNP) array analyses revealed that gross genomic instability is not present in most children with ALL. A mean of 6.4 genomic lesions were present per case, with wide variability within the genetic subtypes of ALL. Structural alterations in genes encoding transcriptional regulators of B-lymphoid development and differentiation occur in more than 40% of patients with B-ALL. PAX5 is the most common target; other targets include IKZF1 and EBF1 . Several additional lesions have also been identified in lymphoid signaling, transcription factors, and tumor suppressors. Several of these alterations cooperate in leukemogenesis. For example, the deletion of IKZF1 accelerates the onset of ALL in murine models of BCR-ABL1 ALL. The assortment and accumulation of “driver” and “passenger” mutations, and the sequence of events in leukemia development and progression, continue to be investigated.
High-risk subtypes of B-cell acute lymphoblastic leukemia
BCR-ABL –Like Acute Lymphoblastic Leukemia
IKZF1 deletions are a hallmark of BCR-ABL1– positive ALL, but these deletions also occur in a subset of patients with poor-response, high-risk ALL without any known chromosomal rearrangement. Using genome-wide analyses, 2 groups of investigators independently identified a subgroup of B-ALL, which has a gene-expression profile similar to that of BCR-ABL1– positive ALL including a high frequency of IKZF1 alterations, but lacks the BCR-ABL1 fusion protein; they termed this genetic subtypes BCR-ABL1– like or Philadelphia chromosome–like ALL. This subtype comprises 10% of the cases of B-ALL in children and 25% of the cases of ALL in adolescents and young adults. NGS techniques and downstream functional experiments show that BCR-ABL1– like ALL is characterized by genetic changes that result in constitutive activation of cytokine receptor and/or tyrosine kinase signaling. The spectrum of genetic alterations is extremely diverse; however, several rearrangements involve tyrosine kinases such as ABL and PDGFR , which respond to imatinib and dasatinib in vitro and in vivo. Even though risk-directed therapy including intensive chemotherapy, with or without transplant based on MRD level during remission induction therapy may abolish the poor prognosis of this group of patients, it is important to look for genetic lesions responsive to ABL tyrosine kinase inhibitor so that some patients can be spared transplantation. Several other rearrangements target JAK and EPOR , which are sensitive to JAK inhibitors in preclinical models. In addition, rearrangements involving the cytokine receptor gene CRLF2 have been identified in 50% of patients with BCR-ABL1– like ALL, with frequent coexisting JAK mutations, also potentially sensitive to JAK inhibition. In view of the therapeutic implications for this high-risk subset of patients, array-based and sequencing-based methodologies are being developed for rapid classification of patients with BCR-ABL1– like ALL and identification of targetable lesions; the incorporation of tyrosine kinase inhibitors in frontline therapy is also planned.
Acute Lymphoblastic Leukemia with Intrachromosomal Amplification of Chromosome 21
ALL with intrachromosomal amplification of chromosome 21 (iAMP21) was originally discovered by the observation of multiple copies of the RUNX1 gene during routine screening for ETV6-RUNX1 by fluorescent in situ hybridization. This particular ALL subtype is characterized by the instability of chromosome 21. The incidence of iAMP21 is approximately 2%, and the median age of patients is 9 to 11 years. Intensification of chemotherapy has abolished the poor prognosis once associated with this ALL subtype.
Down Syndrome Acute Lymphoblastic Leukemia
Patients with Down syndrome are at an approximately 20-fold increased risk of developing ALL, although the precise role of the extra chromosome 21 in leukemogenesis is unknown. These patients have low frequencies of T-cell ALL (T-ALL) and common ALL translocations such as ETV6-RUNX1 . Patients with Down syndrome ALL have inferior outcome owing to their increased risk of relapse and high rate of treatment-related mortality. High-resolution SNP profiling has identified a submicroscopic deletion of the pseudoautosomal regions of chromosomes X and Y, which leads to the P2RY8-CRLF2 fusion in approximately 50% of patients with Down syndrome ALL. These fusions and other CRLF2 alterations were associated with JAK mutations. Together, these lesions activate the JAK-STAT pathway and promote cytokine-independent growth. Therefore, the inhibition of JAK tyrosine kinase is a potentially useful therapeutic strategy in patients with Down syndrome ALL.
T-cell acute lymphoblastic leukemia
T-ALL accounts for 10% to 15% of the cases of childhood ALL. The outcome of children with T-ALL, which has been historically poor, has improved gradually with the use of intensified therapy, including dexamethasone, asparaginase, and high-dose methotrexate. However, children who relapse have a dismal outcome even with hematopoietic stem cell transplantation. Therefore, it is critical to identify aberrant molecular pathways and targets for therapeutic intervention for T-ALL. Genetic lesions in T-ALL are diverse and complex, and a multitude of alterations contribute in the pathogenesis of various subtypes of T-ALL. Chromosomal translocations are present in approximately 50% of patients with T-ALL cases, but unlike in B-ALL their prognostic impact is not well defined and they are not used for risk stratification. Some translocations result in the juxtaposition of oncogenes to T-cell receptor (TCR) genes, leading to overexpression of the oncogene in T-cell progenitor cells (eg, TLX1-TCRδ ), whereas others result in the fusion of 2 transcription factor oncogenes (eg, STIL-TAL1 ). In addition, rearrangements of the MLL gene occur in 5% to 10% of patients with T-ALL. Gene-expression profiling studies have identified 4 major subtypes of T-ALL on the basis of the predominant oncogenic pathway activation ( TLX1 , LYL1 , TAL/LMO2 , and TLX3 ).
NOTCH Activation in T-Cell Acute Lymphoblastic Leukemia
Constitutive activation of NOTCH signaling, primarily via somatic mutations, is seen in more than 50% of patients with T-ALL, a finding that is not restricted to specific subtypes of T-ALL. In general, the presence of NOTCH1 mutations indicates a favorable prognosis. NOTCH1 is a transmembrane receptor crucial for T-cell development, lineage commitment, cell growth, and survival. Activation of NOTCH1 and the presence of cooperating lesions, such as deletion of the tumor suppressor CDKN2A (found in 70% of patients with T-ALL), can lead to leukemic transformation. In addition, mutations in FBXW7 , which encodes a ubiquitin protein ligase (found in 8%–10% of patients with T-ALL), attenuate the degradation of activated NOTCH1 , further enhancing its downstream signaling. Thus, the inhibition of NOTCH1 , either by small-molecule inhibitors of γ-secretase (which impede the release of activated NOTCH1) or by anti- NOTCH1 antibodies, is being actively pursued as a therapeutic strategy for T-ALL.
Early T-Cell Precursor Acute Lymphoblastic Leukemia
Early T-cell precursors (ETPs) are a subset of immature thymocytes that retain stem cell–like features and can differentiate into multiple lineages, including lymphoid and myeloid lineage. Complementary studies of flow cytometry, gene expression, and DNA copy number showed that the genetic profile of approximately 12% of patients with T-ALL is similar to that of these immature thymocytes. A whole-genome study showed that ETP ALL has frequent mutations of genes involved in hematopoietic development, cytokine receptor and RAS signaling, and chromatin modification. The incidence of activating NOTCH1 mutations is low in ETP ALL, which also lacks a unifying chromosomal abnormality. In general, the outcome of patients with ETP ALL is poor, but myeloid-directed and epigenetic therapies may be beneficial for these patients. A recent, small study suggested that patients with ETP ALL have an intermediate outcome when treated with intensive chemotherapy that includes pegylated asparaginase and dexamethasone (5-year event-free survival of 76.7%), a finding that requires confirmation.
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