Childhood Acute Lymphoblastic Leukemia Treatment (PDQ®)
For acute lymphoblastic leukemia (ALL), the 5-year survival rate has improved significantly since 1975. Get information about risk factors, signs, diagnosis, molecular features, survival, risk-based treatment assignment, and induction and postinduction therapy for children and adolescents with newly diagnosed and recurrent ALL.
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Childhood Acute Lymphoblastic Leukemia Treatment (PDQ®)–Health Professional Version

General Information About Childhood Acute Lymphoblastic Leukemia (ALL)

Cancer in children and adolescents is rare, although the overall incidence of childhood cancer, including ALL, has been slowly increasing since 1975.[1] Dramatic improvements in survival have been achieved in children and adolescents with cancer.[1-3] Between 1975 and 2020, childhood cancer mortality decreased by more than 50%, although cancer remains the leading cause of death by disease past infancy among children in the United States.[1,2,4,5] For ALL, the 5-year survival rate increased over the same time, from 60% to approximately 90% for children younger than 15 years, and from 28% to more than 75% for adolescents aged 15 to 19 years.[2,3,6] Childhood and adolescent cancer survivors require close monitoring because cancer therapy side effects may persist or develop months or years after treatment. For specific information about the incidence, type, and monitoring of late effects in childhood and adolescent cancer survivors, see Late Effects of Treatment for Childhood Cancer.

Incidence

ALL, the most common cancer diagnosed in children, represents approximately 25% of cancer diagnoses among children younger than 15 years.[7] In the United States, ALL occurs at an annual rate of approximately 40 cases per 1 million people aged 0 to 14 years and approximately 21 cases per 1 million people aged 15 to 19 years.[3] Approximately 3,100 children and adolescents younger than 20 years are diagnosed with ALL each year in the United States.[8] Since 1975, there has been a gradual increase in the incidence of ALL.[2,9]

A sharp peak in ALL incidence is observed among children aged 1 to 4 years (81 cases per 1 million per year), with rates decreasing to 24 cases per 1 million by age 10 years.[3] The incidence of ALL among children aged 1 to 4 years is approximately fourfold greater than that for infants and for children aged 10 years and older.[3]

The incidence of ALL appears to be highest in American Indian or Alaska Native children and adolescents (65.9 cases per 1 million) and Hispanic children and adolescents (48 cases per 1 million).[3,10,11] The incidence is substantially higher in White children than in Black children, with a twofold higher incidence of ALL from age 1 to 4 years in White children than in Black children.[3,10]

Anatomy

Childhood ALL originates in the T and B lymphoblasts in tissues with hematopoietic progenitor cells, such as the bone marrow and thymus (see Figure 1).

Marrow involvement of acute leukemia as seen by light microscopy is defined as follows:

M1: Fewer than 5% blast cells.

M2: 5% to 25% blast cells.

M3: Greater than 25% blast cells.

Almost all patients with ALL present with an M3 marrow.

Morphology

In the past, ALL lymphoblasts were classified using the French-American-British (FAB) criteria as having L1, L2, or L3 morphology.[12] However, it is no longer used because of the lack of independent prognostic significance and the subjective nature of this classification system.

Most cases of ALL that show L3 morphology express surface immunoglobulin (Ig) and have a MYC gene translocation identical to those seen in Burkitt lymphoma (i.e., t(8;14)(q24;q32), t(2;8)) that join MYC to one of the Ig genes. Patients with this specific rare form of leukemia (mature B-cell or Burkitt leukemia) should be treated according to protocols for Burkitt lymphoma. For more information about the treatment of mature B-cell lymphoma/leukemia and Burkitt lymphoma/leukemia, see Childhood Non-Hodgkin Lymphoma Treatment. Rarely, blasts with L1/L2 (not L3) morphology will express surface Ig.[13] These patients should be treated in the same way as patients with B-ALL.[13]

Risk Factors for Developing ALL

The primary accepted risk factors for ALL and associated genes (when relevant) include the following:

Prenatal exposure to x-rays.

Postnatal exposure to high doses of radiation (e.g., therapeutic radiation previously used for conditions such as tinea capitis and thymus enlargement).

Previous treatment with chemotherapy.

Genetic conditions that include the following:

Down syndrome. For more information, see the Down syndrome section.

Neurofibromatosis (NF1).[14]

Bloom syndrome (BLM).[15]

Fanconi anemia (multiple genes; ALL is observed much less frequently than acute myeloid leukemia [AML]).[16]

Ataxia telangiectasia (ATM).[17]

Li-Fraumeni syndrome (TP53).[18-20]

Constitutional mismatch repair deficiency (biallelic variant of MLH1, MSH2, MSH6, and PMS2).[21,22]

Low- and high-penetrance inherited genetic variants.[23] For more information, see the Low- and high-penetrance inherited genetic variants section.

Carriers of a constitutional Robertsonian translocation that involves chromosomes 15 and 21 and carriers of constitutional ring chromosome 21 are specifically and highly predisposed to developing intrachromosomal amplification of chromosome 21 (iAMP21) ALL.[24,25]

Down syndrome

Children with Down syndrome have an increased risk of developing both ALL and AML,[26-28] with a cumulative risk of developing leukemia of approximately 2.1% by age 5 years and 2.7% by age 30 years.[26,28] These rates represent a 20- to 30-fold increased risk of ALL and over 100-fold increased risk of AML for children with Down syndrome.[27,28]

A genome-wide association study found that four susceptibility loci associated with B-ALL in the non-Down syndrome population (IKZF1, CDKN2A, ARID5B, and GATA3) were also associated with susceptibility to ALL in children with Down syndrome.[29] CDKN2A risk allele penetrance appeared to be higher for children with Down syndrome.

Approximately one-half to two-thirds of cases of acute leukemia in children with Down syndrome are ALL, and about 2% to 3% of childhood ALL cases occur in children with Down syndrome.[30-33] ALL in children with Down syndrome has an age distribution similar to that of ALL in children without Down syndrome, with a median age of 3 to 4 years.[30,31] In contrast, nearly all cases of AML in children with Down syndrome occur before the age of 4 years (median age, 1 year).[34]

Patients with ALL and Down syndrome have a lower incidence of both favorable (ETV6::RUNX1 fusion and hyperdiploidy [51–65 chromosomes]) and unfavorable (BCR::ABL1 or KMT2A::AFF1 fusions and hypodiploidy [<44 chromosomes]) genomic alterations and a near absence of T-cell phenotype.[30-32,34,35]

Approximately 50% to 60% of cases of ALL in children with Down syndrome have genomic alterations affecting CRLF2 that generally result in overexpression of the protein produced by this gene, which dimerizes with the interleukin-7 receptor alpha to form the receptor for the cytokine thymic stromal lymphopoietin.[36-38] The P2RY8::CRLF2 fusion occurs much more commonly than the IGH::CRLF2 fusion in children with Down syndrome, particularly in those of younger age.[38,39] CRLF2 genomic alterations are observed at a much lower frequency (<10%) in children with B-ALL who do not have Down syndrome; when they do occur, they are more often associated with the BCR::ABL1-like subtype.[38,40,41] In one retrospective study, the frequency of CRLF2 rearrangements was nine times higher in children with Down syndrome and ALL than in children with ALL but without Down syndrome (54.2% vs. 6.0%). In that study, only 25% of the cases with CRLF2 rearrangements and Down syndrome were classified as BCR::ABL1-like, compared with 54% of cases with CRLF2 rearrangements without Down syndrome.[42]

Based on the relatively small number of published series, it does not appear that genomic CRLF2 aberrations in patients with Down syndrome and ALL have prognostic relevance.[35,37] However, among patients with Down syndrome and CRLF2 rearrangements, those with the BCR::ABL1 signature appear to have a worse prognosis than those who do not have the BCR::ABL1 fusion.[42]

Approximately 20% to 30% of ALL cases arising in children with Down syndrome have somatically acquired JAK1 or JAK2 variants,[36,37,42-45] which are strongly associated with the presence of CRLF2 rearrangements.[36-38,42] JAK variants are uncommon among younger children with ALL who do not have Down syndrome but are observed more frequently in older children and adolescents with high-risk B-ALL, particularly in those with the BCR::ABL1-like subtype.[46] Preliminary evidence suggests no correlation between JAK2 variant status and 5-year event-free survival (EFS) in children with Down syndrome and ALL.[37,44]

IKZF1 gene deletions, observed in 20% to 35% of patients with Down syndrome and ALL, have been associated with a significantly worse outcome in this group of patients.[37,47,48]

Approximately 10% of patients with Down syndrome and ALL have genomic alterations leading to overexpression or abnormal activation of the CEBPD, CEBPA, and CEBPE genes.[42] Of the CEBP-activated cases with ALL and Down syndrome, approximately 40% also have FLT3 point variants or insertions/deletions, compared with 4.1% in cases with Down syndrome and other ALL subtypes.

Low- and high-penetrance inherited genetic variants

Genetic predisposition to ALL can be divided into several broad categories, as follows:

Association with genetic syndromes. Increased risk can be associated with the genetic syndromes listed above in which ALL is observed, although it is not the primary manifestation of the condition.

Common alleles. Another category for genetic predisposition includes common alleles with relatively small effect sizes that are identified by genome-wide association studies. Genome-wide association studies have identified a number of germline (inherited) genetic polymorphisms that are associated with the development of childhood ALL.[23] For example, the risk alleles of ARID5B are associated with the development of hyperdiploid (51–65 chromosomes) B-ALL. ARID5B is a gene that encodes a transcriptional factor important in embryonic development, cell type–specific gene expression, and cell growth regulation.[49,50] Other genes with polymorphisms associated with increased risk of ALL include GATA3,[51] IKZF1,[49,50,52] CDKN2A,[53] CDKN2B,[52,53] CEBPE,[49] PIP4K2A,[51,54] and TP63.[55]

Genetic risk factors for T-ALL share some overlap with the genetic risk factors for B-ALL, but unique risk factors also exist. A genome-wide association study identified a risk allele near USP7 that was associated with an increased risk of developing T-ALL (odds ratio, 1.44) but not B-ALL. The risk allele was shown to be associated with reduced USP7 transcription, which is consistent with the finding that somatic loss-of-function variants in USP7 are observed in patients with T-ALL. USP7 germline and somatic variants are generally mutually exclusive and are most commonly observed in T-ALL patients with TAL1 alterations.[56]

Genetic risk factors that have similar impact for developing both B-ALL and T-ALL include CDKN2A, CDKN2B, and 8q24.21 (cis distal enhancer region variants for MYC).[56]

Rare germline variants with high penetrance. Germline variants that cause pathogenic changes in genes associated with ALL and that are observed in kindreds with familial ALL (i.e., large effect sizes) comprise another category of genetic predisposition to ALL. Many of the genes associated with ALL risk play key roles in B-cell development (e.g., PAX5, ETV6, and IKZF1).[57]

PAX5. A germline variant in PAX5 that substitutes serine for glycine at amino acid 183 and that reduces PAX5 activity has been identified in several families that experienced multiple cases of ALL.[58,59]

ETV6. Several germline ETV6 variants that lead to loss of ETV6 function have been identified in kindreds affected by both thrombocytopenia and ALL.[60-64] Sequencing of ETV6 in remission (i.e., germline) specimens identified variants that were potentially related to ALL in approximately 1% of children with ALL that were evaluated.[60] Most of the germline variants (approximately 75%) were shown to be deleterious for ETV6 function, and 70% of cases with a deleterious germline ETV6 variant had a hyperdiploid karyotype. The remaining cases with a deleterious variant had diploid ALL, with a transcriptional profile similar to that of cases with ETV6::RUNX1 fusion–positive ALL.[64]

TP53. Pathogenic germline TP53 variants are associated with an increased risk of ALL.[65] A study of 3,801 children with ALL observed that 26 patients (0.7%) had a pathogenic TP53 germline variant, with an associated odds ratio of 5.2 for ALL development.[65] Compared with ALL in children with TP53 wild-type status or TP53 variants of unknown significance, ALL in children with pathogenic germline TP53 variants was associated with older age at diagnosis (15.5 years vs. 7.3 years), hypodiploidy (65% vs. 1%), inferior EFS and overall survival, and a higher risk of second cancers.

IKZF1. Germline IKZF1 variants were identified in a kindred with familial ALL and in 43 of 4,963 (0.9%) children with sporadic ALL. Most (22 of 28) IKZF1 variants were shown to adversely affect IKZF1 gene function.[66] Germline variants in IKZF1 have been identified in hereditary hypogammaglobulinemia. In one series, 2 of 29 affected patients developed B-ALL during childhood.[67]

Prenatal origin of childhood ALL

Development of ALL is a multistep process in most cases, with more than one genomic alteration required for frank leukemia to develop. In at least some cases of childhood ALL, the initial genomic alteration occurs in utero. Evidence to support this comes from the observation that the immunoglobulin or T-cell receptor antigen rearrangements that are unique to each patient’s leukemia cells can be detected in blood samples obtained at birth.[68,69] Similarly, in ALL characterized by specific chromosomal abnormalities, some patients have blood cells that carry at least one leukemic genomic abnormality at the time of birth, with additional cooperative genomic changes acquired postnatally.[68-70] Genomic studies of identical twins with concordant leukemia further support the prenatal origin of some leukemias.[68,71]

Evidence also exists that some children who never develop ALL are born with rare blood cells carrying a genomic alteration associated with ALL. Initial studies focused on the ETV6::RUNX1 translocation and used reverse transcriptase–polymerase chain reaction (PCR) to identify RNA transcripts indicating the presence of the gene fusion. For example, in one study, 1% of neonatal blood spots (Guthrie cards) tested positive for the ETV6::RUNX1 translocation.[72] While subsequent reports generally confirmed the presence of the ETV6::RUNX1 translocation at birth in some children, rates and extent of positivity varied widely.

To more definitively address this question, a highly sensitive and specific DNA-based approach (genomic inverse PCR for exploration of ligated breakpoints) was applied to DNA from 1,000 cord blood specimens and found that 5% of specimens had the ETV6::RUNX1 translocation.[73] When the same method was applied to 340 cord blood specimens to detect the TCF3::PBX1 fusion, two cord specimens (0.6%) were positive for its presence.[74] For both ETV6::RUNX1 and TCF3::PBX1, the percentage of cord blood specimens positive for one of the translocations far exceeds the percentage of children who will develop either type of ALL (<0.01%).

Clinical Presentation

The typical and atypical symptoms and clinical findings of childhood ALL have been published.[75-77]

Diagnosis

The evaluation needed to definitively diagnose childhood ALL has been published.[75-79]

Overall Prognosis

Among children with ALL, approximately 98% attain remission. Approximately 85% of patients aged 1 to 18 years with newly diagnosed ALL treated on current regimens are expected to be long-term event-free survivors, with more than 90% of patients alive at 5 years.[80-83] In one study of patients with newly diagnosed ALL, relapses were rare (occurring in fewer than 1% of patients) by 6 to 7 years after diagnosis.[84] In addition, the excess risk of death associated with the leukemia diagnosis had decreased such that the mortality rate of the surviving patients at 6 to 7 years after diagnosis was similar to that of the general population.

Cytogenetic and genomic findings combined with minimal residual disease (MRD) results can define subsets of ALL with EFS rates exceeding 95% and, conversely, subsets with EFS rates of 50% or lower. For more information, see the sections on Cytogenetics/Genomics of Childhood ALL and Prognostic Factors Affecting Risk-Based Treatment.

Despite the treatment advances in childhood ALL, numerous important biological and therapeutic questions remain to be answered before the goal of curing every child with ALL with the least associated toxicity can be achieved. The systematic investigation of these issues requires large clinical trials, and the opportunity to participate in these trials is offered to most patients and families.

Clinical trials for children and adolescents with ALL are generally designed to compare therapy that is currently accepted as standard with investigational regimens that seek to improve cure rates and/or decrease toxicity. In certain trials in which the cure rate for the patient group is very high, therapy reduction questions may be asked. Much of the progress made in identifying curative therapies for childhood ALL and other childhood cancers has been achieved through investigator-driven discovery and tested in carefully randomized, controlled, multi-institutional clinical trials. Information about ongoing clinical trials is available from the NCI website.

Current Clinical Trials

Use our advanced clinical trial search to find NCI-supported cancer clinical trials that are now enrolling patients. The search can be narrowed by location of the trial, type of treatment, name of the drug, and other criteria. General information about clinical trials is also available.

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Greaves MF, Wiemels J: Origins of chromosome translocations in childhood leukaemia. Nat Rev Cancer 3 (9): 639-49, 2003. [PUBMED Abstract]

Taub JW, Konrad MA, Ge Y, et al.: High frequency of leukemic clones in newborn screening blood samples of children with B-precursor acute lymphoblastic leukemia. Blood 99 (8): 2992-6, 2002. [PUBMED Abstract]

Bateman CM, Colman SM, Chaplin T, et al.: Acquisition of genome-wide copy number alterations in monozygotic twins with acute lymphoblastic leukemia. Blood 115 (17): 3553-8, 2010. [PUBMED Abstract]

Greaves MF, Maia AT, Wiemels JL, et al.: Leukemia in twins: lessons in natural history. Blood 102 (7): 2321-33, 2003. [PUBMED Abstract]

Mori H, Colman SM, Xiao Z, et al.: Chromosome translocations and covert leukemic clones are generated during normal fetal development. Proc Natl Acad Sci U S A 99 (12): 8242-7, 2002. [PUBMED Abstract]

Schäfer D, Olsen M, Lähnemann D, et al.: Five percent of healthy newborns have an ETV6-RUNX1 fusion as revealed by DNA-based GIPFEL screening. Blood 131 (7): 821-826, 2018. [PUBMED Abstract]

Hein D, Dreisig K, Metzler M, et al.: The preleukemic TCF3-PBX1 gene fusion can be generated in utero and is present in ≈0.6% of healthy newborns. Blood 134 (16): 1355-1358, 2019. [PUBMED Abstract]

Gramatges MM, O'Brien MM, Rabin KR: Acute lymphoblastic leukemia. In: Blaney SM, Helman LJ, Adamson PC, eds.: Pizzo and Poplack's Pediatric Oncology. 8th ed. Wolters Kluwer, 2020, pp 419-53.

Chessells JM; haemostasis and thrombosis task force, British committee for standards in haematology: Pitfalls in the diagnosis of childhood leukaemia. Br J Haematol 114 (3): 506-11, 2001. [PUBMED Abstract]

Onciu M: Acute lymphoblastic leukemia. Hematol Oncol Clin North Am 23 (4): 655-74, 2009. [PUBMED Abstract]

Margolskee E, Waith Wertheim GB, Harvey RC: Pathology and molecular diagnosis of leukemias and lymphomas. In: Blaney SM, Helman LJ, Adamson PC, eds.: Pizzo and Poplack's Pediatric Oncology. 8th ed. Wolters Kluwer, 2020, pp 117-30.

Cheng J, Klairmont MM, Choi JK: Peripheral blood flow cytometry for the diagnosis of pediatric acute leukemia: Highly reliable with rare exceptions. Pediatr Blood Cancer 66 (1): e27453, 2019. [PUBMED Abstract]

Möricke A, Zimmermann M, Valsecchi MG, et al.: Dexamethasone vs prednisone in induction treatment of pediatric ALL: results of the randomized trial AIEOP-BFM ALL 2000. Blood 127 (17): 2101-12, 2016. [PUBMED Abstract]

Vora A, Goulden N, Wade R, et al.: Treatment reduction for children and young adults with low-risk acute lymphoblastic leukaemia defined by minimal residual disease (UKALL 2003): a randomised controlled trial. Lancet Oncol 14 (3): 199-209, 2013. [PUBMED Abstract]

Place AE, Stevenson KE, Vrooman LM, et al.: Intravenous pegylated asparaginase versus intramuscular native Escherichia coli L-asparaginase in newly diagnosed childhood acute lymphoblastic leukaemia (DFCI 05-001): a randomised, open-label phase 3 trial. Lancet Oncol 16 (16): 1677-90, 2015. [PUBMED Abstract]

Pieters R, de Groot-Kruseman H, Van der Velden V, et al.: Successful Therapy Reduction and Intensification for Childhood Acute Lymphoblastic Leukemia Based on Minimal Residual Disease Monitoring: Study ALL10 From the Dutch Childhood Oncology Group. J Clin Oncol 34 (22): 2591-601, 2016. [PUBMED Abstract]

Moorman AV, Antony G, Wade R, et al.: Time to Cure for Childhood and Young Adult Acute Lymphoblastic Leukemia Is Independent of Early Risk Factors: Long-Term Follow-Up of the UKALL2003 Trial. J Clin Oncol 40 (36): 4228-4239, 2022. [PUBMED Abstract]

World Health Organization (WHO) Classification System for Childhood ALL

The 5th edition of the WHO Classification of Haematolymphoid Tumours lists the following entities for acute lymphoid leukemias:[1]

WHO 5th Edition Classification of B-Cell Lymphoblastic Leukemias/Lymphomas

B-lymphoblastic leukemia/lymphoma, not otherwise specified (NOS).

B-lymphoblastic leukemia/lymphoma with high hyperdiploidy.

B-lymphoblastic leukemia/lymphoma with iAMP21.

B-lymphoblastic leukemia/lymphoma with BCR::ABL1 fusion.

B-lymphoblastic leukemia/lymphoma with BCR::ABL1-like features.

B-lymphoblastic leukemia/lymphoma with KMT2A rearrangement.

B-lymphoblastic leukemia/lymphoma with ETV6::RUNX1 fusion.

B-lymphoblastic leukemia/lymphoma with ETV6::RUNX1-like features.

B-lymphoblastic leukemia/lymphoma with TCF3::PBX1 fusion.

B-lymphoblastic leukemia/lymphoma with IGH::IL3 fusion.

B-lymphoblastic leukemia/lymphoma with TCF3::HLF fusion.

B-lymphoblastic leukemia/lymphoma with other defined genetic abnormalities.

The category of B-ALL with other defined genetic abnormalities includes potential novel entities, including B-ALL with DUX4, MEF2D, ZNF384 or NUTM1 rearrangements; B-ALL with IG::MYC fusions; and B-ALL with PAX5alt or PAX5 p.P80R (NP_057953.1) abnormalities.

WHO 5th Edition Classification of T-Lymphoblastic Leukemia/Lymphoma

T-lymphoblastic leukemia/lymphoma, NOS.

Early T-precursor lymphoblastic leukemia/lymphoma.

2016 WHO Classification of Acute Leukemias of Ambiguous Lineage

For acute leukemias of ambiguous lineage, the group of acute leukemias that have characteristics of both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), the WHO classification system is summarized in Table 1.[2,3] The criteria for lineage assignment for a diagnosis of mixed phenotype acute leukemia (MPAL) are provided in Table 2.[4]

Table 1. Acute Leukemias of Ambiguous Lineage According to the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid TissuesaCondition DefinitionAcute undifferentiated leukemia Acute leukemia that does not express any marker considered specific for either lymphoid or myeloid lineageMPAL with BCR::ABL1 (t(9;22)(q34;q11.2))Acute leukemia meeting the diagnostic criteria for mixed phenotype acute leukemia in which the blasts also have the (9;22) translocation or the BCR::ABL1 rearrangementMPAL with KMT2A rearranged (t(v;11q23))Acute leukemia meeting the diagnostic criteria for mixed phenotype acute leukemia in which the blasts also have a translocation involving the KMT2A geneMPAL, B/myeloid, NOS (B/M MPAL)Acute leukemia meeting the diagnostic criteria for assignment to both B and myeloid lineage, in which the blasts lack genetic abnormalities involving BCR::ABL1 or KMT2AMPAL, T/myeloid, NOS (T/M MPAL)Acute leukemia meeting the diagnostic criteria for assignment to both T and myeloid lineage, in which the blasts lack genetic abnormalities involving BCR::ABL1 or KMT2AMPAL, B/myeloid, NOS—rare typesAcute leukemia meeting the diagnostic criteria for assignment to both B and T lineageOther ambiguous lineage leukemiasNatural killer–cell lymphoblastic leukemia/lymphoma

Table 2. Lineage Assignment Criteria for Mixed Phenotype Acute Leukemia According to the 2016 Revision to the World Health Organization Classification of Myeloid Neoplasms and Acute LeukemiaaLineage CriteriaMyeloid lineageMyeloperoxidase (flow cytometry, immunohistochemistry, or cytochemistry); or monocytic differentiation (at least two of the following: nonspecific esterase cytochemistry, CD11c, CD14, CD64, lysozyme)T lineageStrongb cytoplasmic CD3 (with antibodies to CD3 epsilon chain); or surface CD3B lineageStrongb CD19 with at least one of the following strongly expressed: CD79a, cytoplasmic CD22, or CD10; or weak CD19 with at least two of the following strongly expressed: CD79a, cytoplasmic CD22, or CD10

Leukemias of mixed phenotype may be seen in various presentations, including the following:

Bilineal leukemias in which there are two distinct populations of cells, usually one lymphoid and one myeloid.

Biphenotypic leukemias in which individual blast cells display features of both lymphoid and myeloid lineage.

Biphenotypic cases represent most of the mixed phenotype leukemias.[5] Patients with B-myeloid biphenotypic leukemias lacking the ETV6::RUNX1 fusion have lower rates of complete remission (CR) and significantly worse event-free survival (EFS) rates compared with patients with B-ALL.[5] Cases of MPAL (B/myeloid) that have ZNF384 gene fusions have been reported,[6,7] and a genomic evaluation of MPAL found that ZNF384 gene fusions were present in approximately one-half of B/myeloid cases.[8]

Some studies suggest that patients with biphenotypic leukemia may fare better with a lymphoid, as opposed to a myeloid, treatment regimen.[9-12]; [13][Level of evidence C1] A large retrospective study from the international Berlin-Frankfurt-Münster group demonstrated that initial therapy with an ALL-type regimen was associated with a superior outcome compared with AML-type or combined ALL/AML regimens, particularly in cases with CD19 positivity or other lymphoid antigen expression. In this study, hematopoietic stem cell transplant in first CR was not beneficial, with the possible exception of cases with morphological evidence of persistent marrow disease (≥5% blasts) after the first month of treatment.[12]

For more information about key clinical and biological characteristics, as well as the prognostic significance for these entities, see the Cytogenetics/Genomics of Childhood ALL section.

References

Alaggio R, Amador C, Anagnostopoulos I, et al.: The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia 36 (7): 1720-1748, 2022. [PUBMED Abstract]

Béné MC: Biphenotypic, bilineal, ambiguous or mixed lineage: strange leukemias! Haematologica 94 (7): 891-3, 2009. [PUBMED Abstract]

Borowitz MJ, Béné MC, Harris NL: Acute leukaemias of ambiguous lineage. In: Swerdlow SH, Campo E, Harris NL, et al., eds.: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. International Agency for Research on Cancer, 2008, pp 150-5.

Arber DA, Orazi A, Hasserjian R, et al.: The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 127 (20): 2391-405, 2016. [PUBMED Abstract]

Gerr H, Zimmermann M, Schrappe M, et al.: Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations. Br J Haematol 149 (1): 84-92, 2010. [PUBMED Abstract]

Shago M, Abla O, Hitzler J, et al.: Frequency and outcome of pediatric acute lymphoblastic leukemia with ZNF384 gene rearrangements including a novel translocation resulting in an ARID1B/ZNF384 gene fusion. Pediatr Blood Cancer 63 (11): 1915-21, 2016. [PUBMED Abstract]

Yao L, Cen J, Pan J, et al.: TAF15-ZNF384 fusion gene in childhood mixed phenotype acute leukemia. Cancer Genet 211: 1-4, 2017. [PUBMED Abstract]

Alexander TB, Gu Z, Iacobucci I, et al.: The genetic basis and cell of origin of mixed phenotype acute leukaemia. Nature 562 (7727): 373-379, 2018. [PUBMED Abstract]

Rubnitz JE, Onciu M, Pounds S, et al.: Acute mixed lineage leukemia in children: the experience of St Jude Children's Research Hospital. Blood 113 (21): 5083-9, 2009. [PUBMED Abstract]

Al-Seraihy AS, Owaidah TM, Ayas M, et al.: Clinical characteristics and outcome of children with biphenotypic acute leukemia. Haematologica 94 (12): 1682-90, 2009. [PUBMED Abstract]

Matutes E, Pickl WF, Van't Veer M, et al.: Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood 117 (11): 3163-71, 2011. [PUBMED Abstract]

Hrusak O, de Haas V, Stancikova J, et al.: International cooperative study identifies treatment strategy in childhood ambiguous lineage leukemia. Blood 132 (3): 264-276, 2018. [PUBMED Abstract]

Orgel E, Alexander TB, Wood BL, et al.: Mixed-phenotype acute leukemia: A cohort and consensus research strategy from the Children's Oncology Group Acute Leukemia of Ambiguous Lineage Task Force. Cancer 126 (3): 593-601, 2020. [PUBMED Abstract]

Cytogenetics/Genomics of Childhood ALL

Genomics of childhood ALL

The genomics of childhood acute lymphoblastic leukemia (ALL) has been extensively investigated, and multiple distinctive subtypes have been defined on the basis of cytogenetic and molecular characterizations, each with its own pattern of clinical and prognostic characteristics.[1] The discussion of the genomics of childhood ALL below is divided into three sections: the genomic alterations associated with B-ALL, followed by the genomic alterations associated with T-ALL and mixed phenotype acute leukemia (MPAL). Figures 2, 3, and 5 illustrate the distribution of B-ALL (stratified by National Cancer Institute [NCI] standard- and high-risk B-ALL) and T-ALL cases by cytogenetic/molecular subtypes.[1]

Throughout this section, the percentages of genomic subtypes from among all B-ALL and T-ALL cases are derived primarily from a report describing the genomic characterization of patients treated on several Children's Oncology Group (COG) and St. Jude Children's Research Hospital (SJCRH) clinical trials. Percentages by subtype are presented for NCI standard-risk and NCI high-risk patients with B-ALL (up to age 18 years).[1]

B-ALL cytogenetics/genomics

B-ALL is typified by genomic alterations that include: 1) gene fusions that lead to aberrant activity of transcription factors, 2) chromosomal gains and losses (e.g., hyperdiploidy or hypodiploidy), and 3) alterations leading to activation of tyrosine kinase genes.[1] Figures 2 and 3 illustrate the distribution of NCI standard-risk and high-risk B-ALL cases by 23 cytogenetic/molecular subtypes.[1] The two most common subtypes (hyperdiploid and ETV6::RUNX1 fusion) together account for approximately 60% of NCI standard-risk B-ALL cases, but only approximately 25% of NCI high-risk cases. Most other subtypes are much less common, with most occurring at frequencies less than 2% to 3% of B-ALL cases. The molecular and clinical characteristics of some of the subtypes are discussed below.

The genomic landscape of B-ALL is characterized by a range of genomic alterations that disrupt normal B-cell development and, in some cases, by variants in genes that provide a proliferation signal (e.g., activating variants in RAS family genes or variants/translocations leading to kinase pathway signaling). Genomic alterations leading to blockage of B-cell development include translocations (e.g., TCF3::PBX1 and ETV6::RUNX1 fusions), point variants (e.g., IKZF1 and PAX5), and intragenic/intergenic deletions (e.g., IKZF1, PAX5, EBF, and ERG).[2]

The genomic alterations in B-ALL tend not to occur at random, but rather to cluster within subtypes that can be delineated by biological characteristics such as their gene expression profiles. Cases with recurring chromosomal translocations (e.g., TCF3::PBX1 and ETV6::RUNX1 fusions and KMT2A-rearranged ALL) have distinctive biological features and illustrate this point, as do the examples below of specific genomic alterations within unique biological subtypes:

IKZF1 deletions and variants are most commonly observed within cases of BCR::ABL1 ALL and BCR::ABL1-like ALL.[3,4]

Intragenic ERG deletions occur within a distinctive subtype characterized by gene rearrangements involving DUX4.[5,6]

TP53 variants, often germline, occur at high frequency in patients with low hypodiploid ALL with 32 to 39 chromosomes.[7] TP53 variants are uncommon in other patients with B-ALL.

Activating point variants in kinase genes are uncommon in high-risk B-ALL. JAK genes are the primary kinases that are found to be altered. These variants are generally observed in patients with BCR::ABL1-like ALL who have CRLF2 abnormalities, although JAK2 variants are also observed in approximately 25% of children with Down syndrome and ALL, occurring exclusively in cases with CRLF2 gene rearrangements.[4,8-10] Several kinase genes and cytokine receptor genes are activated by translocations, as described below in the discussion of BCR::ABL1 ALL and BCR::ABL1-like ALL. FLT3 variants occur in a minority of cases (approximately 10%) of hyperdiploid ALL and KMT2A-rearranged ALL, and are rare in other subtypes.[11]

Understanding of the genomics of B-ALL at relapse is less advanced than the understanding of ALL genomics at diagnosis. Childhood ALL is often polyclonal at diagnosis and under the selective influence of therapy, some clones may be extinguished and new clones with distinctive genomic profiles may arise.[12] However, molecular subtype–defining lesions such as translocations and aneuploidy are almost always retained at relapse.[1,12] Of particular importance are new variants that arise at relapse that may be selected by specific components of therapy. As an example, variants in NT5C2 are not found at diagnosis, whereas specific variants in NT5C2 were observed in 7 of 44 (16%) and 9 of 20 (45%) cases of B-ALL with early relapse that were evaluated for this variant in two studies.[12,13] NT5C2 variants are uncommon in patients with late relapse, and they appear to induce resistance to mercaptopurine and thioguanine.[13] Another gene that is found altered only at relapse is PRSP1, a gene involved in purine biosynthesis.[14] Variants were observed in 13.0% of a Chinese cohort and 2.7% of a German cohort, and were observed in patients with on-treatment relapses. The PRSP1 variants observed in relapsed cases induce resistance to thiopurines in leukemia cell lines. CREBBP variants are also enriched at relapse and appear to be associated with increased resistance to glucocorticoids.[12,15] With increased understanding of the genomics of relapse, it may be possible to tailor upfront therapy to avoid relapse or detect resistance-inducing variants early and intervene before a frank relapse.

Several recurrent chromosomal abnormalities have been shown to have prognostic significance, especially in B-ALL. Some chromosomal alterations are associated with more favorable outcomes, such as favorable trisomies (51–65 chromosomes) and the ETV6::RUNX1 fusion.[16][Level of evidence B4] Other alterations historically have been associated with a poorer prognosis, including the BCR::ABL1 fusion (Philadelphia chromosome–positive [Ph+]; t(9;22)(q34;q11.2)), rearrangements of the KMT2A gene, hypodiploidy, and intrachromosomal amplification of the RUNX1 gene (iAMP21).[17]

In recognition of the clinical significance of many of these genomic alterations, the 5th edition revision of the World Health Organization Classification of Haematolymphoid Tumours lists the following entities for B-ALL:[18]

B-lymphoblastic leukemia/lymphoma, NOS.

B-lymphoblastic leukemia/lymphoma with high hyperdiploidy.

B-lymphoblastic leukemia/lymphoma with hypodiploidy.

B-lymphoblastic leukemia/lymphoma with iAMP21.

B-lymphoblastic leukemia/lymphoma with BCR::ABL1 fusion.

B-lymphoblastic leukemia/lymphoma with BCR::ABL1-like features.

B-lymphoblastic leukemia/lymphoma with KMT2A rearrangement.

B-lymphoblastic leukemia/lymphoma with ETV6::RUNX1 fusion.

B-lymphoblastic leukemia/lymphoma with ETV6::RUNX1-like features.

B-lymphoblastic leukemia/lymphoma with TCF3::PBX1 fusion.

B-lymphoblastic leukemia/lymphoma with IGH::IL3 fusion.

B-lymphoblastic leukemia/lymphoma with TCF3::HLF fusion.

B-lymphoblastic leukemia/lymphoma with other defined genetic abnormalities.

The category of B-ALL with other defined genetic abnormalities includes potential novel entities, including B-ALL with DUX4, MEF2D, ZNF384 or NUTM1 rearrangements; B-ALL with IG::MYC fusions; and B-ALL with PAX5alt or PAX5 p.P80R (NP_057953.1) abnormalities.

These and other chromosomal and genomic abnormalities for childhood ALL are described below.

Chromosome number.

High hyperdiploidy (51–65 chromosomes).

High hyperdiploidy, defined as 51 to 65 chromosomes per cell or a DNA index greater than 1.16, occurs in approximately 33% of NCI standard-risk and 14% of NCI high-risk pediatric B-ALL cases.[1,19] Hyperdiploidy can be evaluated by measuring the DNA content of cells (DNA index) or by karyotyping. In cases with a normal karyotype or in which standard cytogenetic analysis was unsuccessful, interphase fluorescence in situ hybridization (FISH) may detect hidden hyperdiploidy.

High hyperdiploidy generally occurs in cases with clinically favorable prognostic factors (patients aged 1 to <10 years with a low white blood cell [WBC] count) and is an independent favorable prognostic factor.[19-21] Within the hyperdiploid range of 51 to 65 chromosomes, patients with higher modal numbers (58–66) appeared to have a better prognosis in one study.[21] Hyperdiploid leukemia cells are particularly susceptible to undergoing apoptosis and accumulate higher levels of methotrexate and its active polyglutamate metabolites,[22] which may explain the favorable outcome commonly observed in these cases.

While the overall outcome of patients with high hyperdiploidy is considered to be favorable, factors such as age, WBC count, specific trisomies, and early response to treatment have been shown to modify its prognostic significance.[23,24]

Multiple reports have described the prognostic significance of specific chromosome trisomies among children with hyperdiploid B-ALL.

A study combining experience from the Children's Cancer Group and the Pediatric Oncology Group (POG) found that patients with trisomies of chromosomes 4, 10, and 17 (triple trisomies) have a particularly favorable outcome.[25]; [16][Level of evidence B4]

A report using POG data found that NCI standard-risk patients with trisomies of 4 and 10, without regard to chromosome 17 status, have an excellent prognosis.[26] COG protocols currently use double trisomies of chromosomes 4 and 10 to define favorable hyperdiploidy.

A retrospective analysis evaluated patients treated on two consecutive UKALL trials to identify and validate a profile to predict outcome in high hyperdiploid B-ALL. The investigators defined a good-risk group (approximately 80% of high hyperdiploidy patients) that was associated with a more favorable prognosis. Good-risk patients had either trisomies of both chromosomes 17 and 18 or trisomy of one of these two chromosomes along with absence of trisomies of chromosomes 5 and 20. All other patients were defined as poor risk and had a less favorable outcome. End-induction MRD and copy number alterations (such as IKZF1 deletion) were prognostically significant within each hyperdiploid risk group.[27]

Chromosomal translocations may be seen with high hyperdiploidy, and in those cases, patients are more appropriately risk-classified on the basis of the prognostic significance of the translocation. For instance, in one study, 8% of patients with the BCR::ABL1 fusion also had high hyperdiploidy,[28] and the outcome of these patients (treated without tyrosine kinase inhibitors) was inferior to that observed in non-BCR::ABL1 high hyperdiploid patients.

Certain patients with hyperdiploid ALL may have a hypodiploid clone that has doubled (masked hypodiploidy).[29] Molecular technologies, such as single nucleotide polymorphism microarrays to detect widespread loss of heterozygosity, can be used to identify patients with masked hypodiploidy.[29] These cases may be interpretable based on the pattern of gains and losses of specific chromosomes (hyperdiploidy with two and four copies of chromosomes rather than three copies). These patients have an unfavorable outcome, similar to those with hypodiploidy.[30]

Near triploidy (68–80 chromosomes) and near tetraploidy (>80 chromosomes) are much less common and appear to be biologically distinct from high hyperdiploidy.[31] Unlike high hyperdiploidy, a high proportion of near tetraploid cases harbor a cryptic ETV6::RUNX1 fusion.[31-33] Near triploidy and tetraploidy were previously thought to be associated with an unfavorable prognosis, but later studies suggest that this may not be the case.[31,33]

The genomic landscape of hyperdiploid ALL is characterized by variants in genes of the receptor tyrosine kinase (RTK)/RAS pathway in approximately one-half of cases. Genes encoding histone modifiers are also present in a recurring manner in a minority of cases. Analysis of variant profiles demonstrates that chromosomal gains are early events in the pathogenesis of hyperdiploid ALL and may occur in utero, while variants in RTK/RAS pathway genes are late events in leukemogenesis and are often subclonal.[1,34]

Hypodiploidy (<44 chromosomes).

B-ALL cases with fewer than the normal number of chromosomes have been subdivided in various ways, with one report stratifying on the basis of modal chromosome number into the following four groups:[30]

Near-haploid: 24 to 29 chromosomes (n = 46).

Low-hypodiploid: 33 to 39 chromosomes (n = 26).

High-hypodiploid: 40 to 43 chromosomes (n = 13).

Near-diploid: 44 chromosomes (n = 54).

Near-haploid cases represent approximately 2% of NCI standard-risk and 2% of NCI high-risk pediatric B-ALL.[1]

Low-hypodiploid cases represent approximately 0.5% of NCI standard-risk and 2.6% of NCI high-risk pediatric B-ALL cases.[1]

Most patients with hypodiploidy are in the near-haploid and low-hypodiploid groups, and both of these groups have an elevated risk of treatment failure compared with nonhypodiploid cases.[30,35] Patients with fewer than 44 chromosomes have a worse outcome than do patients with 44 or 45 chromosomes in their leukemic cells.[30] Several studies have shown that patients with high minimal residual disease (MRD) (≥0.01%) after induction do very poorly, with 5-year event-free survival (EFS) rates ranging from 25% to 47%. Although hypodiploid patients with low MRD after induction fare better (5-year EFS rates, 64%–75%), their outcomes are still inferior to most children with other types of ALL.[36-38]

The recurring genomic alterations of near-haploid and low-hypodiploid ALL appear to be distinctive from each other and from other types of ALL.[7] In near-haploid ALL, alterations targeting RTK signaling, RAS signaling, and IKZF3 are common.[39] In low-hypodiploid ALL, genetic alterations involving TP53, RB1, and IKZF2 are common. Importantly, the TP53 alterations observed in low-hypodiploid ALL are also present in nontumor cells in approximately 40% of cases, suggesting that these variants are germline and that low-hypodiploid ALL represents, in some cases, a manifestation of Li-Fraumeni syndrome.[7] Approximately two-thirds of patients with ALL and germline pathogenic TP53 variants have hypodiploid ALL.[40]

Chromosomal translocations and gains/deletions of chromosomal segments.

ETV6::RUNX1 fusion (t(12;21)(p13.2;q22.1)).

Fusion of the ETV6 gene on chromosome 12 to the RUNX1 gene on chromosome 21 is present in approximately 27% of NCI standard-risk and 10% of NCI high-risk pediatric B-ALL cases.[1,32]

The ETV6::RUNX1 fusion produces a cryptic translocation that is detected by methods such as FISH, rather than conventional cytogenetics, and it occurs most commonly in children aged 2 to 9 years.[41,42] Hispanic children with ALL have a lower incidence of ETV6::RUNX1 fusions than do White children.[43]

Reports generally indicate favorable EFS and overall survival (OS) rates in children with the ETV6::RUNX1 fusion; however, the prognostic impact of this genetic feature is modified by the following factors:[44-48]; [16][Level of evidence B4]

Early response to treatment.

NCI risk category (age and WBC count at diagnosis).

Treatment regimen.

In one study of the treatment of newly diagnosed children with ALL, multivariate analysis of prognostic factors found age and leukocyte count, but not ETV6::RUNX1 fusion status, to be independent prognostic factors.[44] However, another large trial only enrolled patients classified as having favorable-risk B-ALL, with low-risk clinical features, either trisomies of 4, 10, and 17 or ETV6::RUNX1 fusion, and end induction MRD less than 0.01%. Patients had a 5-year continuous complete remission rate of 93.7% and a 6-year OS rate of 98.2% for patients with ETV6::RUNX1.[16] It does not appear that the presence of secondary cytogenetic abnormalities, such as deletion of ETV6 (12p) or CDKN2A/B (9p), impacts the outcome of patients with the ETV6::RUNX1 fusion.[48,49]

There is a higher frequency of late relapses in patients with ETV6::RUNX1 fusions compared with other relapsed B-ALL patients.[44,50] Patients with the ETV6::RUNX1 fusion who relapse seem to have a better outcome than other relapse patients,[51] with an especially favorable prognosis for patients who relapse more than 36 months from diagnosis.[52] Some relapses in patients with ETV6::RUNX1 fusions may represent a new independent second hit in a persistent preleukemic clone (with the first hit being the ETV6::RUNX1 translocation).[53,54]

BCR::ABL1 fusion (t(9;22)(q34.1;q11.2); Ph+).

The BCR::ABL1 fusion leads to production of a BCR::ABL1 fusion protein with tyrosine kinase activity (see Figure 4).[1] The BCR::ABL1 fusion occurs in approximately 2% of NCI standard-risk and 5% of NCI high-risk pediatric B-ALL cases.[1] The BCR::ABL1 fusion is also the leukemogenic driver for chronic myeloid leukemia (CML). The most common BCR breakpoint in CML is different from the most common BCR breakpoint in ALL. The breakpoint that typifies CML produces a larger fusion protein (termed p210) than the breakpoint most commonly observed for ALL (termed p190, a smaller fusion protein).

Ph+ ALL is more common in older children with B-ALL and high WBC counts, with the incidence of the BCR::ABL1 fusions increasing to about 25% in young adults with ALL.

Historically, the BCR::ABL1 fusion was associated with an extremely poor prognosis (especially in those who presented with a high WBC count or had a slow early response to initial therapy), and its presence had been considered an indication for allogeneic hematopoietic stem cell transplant (HSCT) in patients in first remission.[28,55-57] Inhibitors of the BCR::ABL1 tyrosine kinase, such as imatinib mesylate, are effective in patients with BCR::ABL1 ALL.[58] A study by the Children's Oncology Group (COG), which used intensive chemotherapy and concurrent imatinib mesylate given daily, demonstrated a 5-year EFS rate of 70% (± 12%), which was superior to the EFS rate of historical controls in the pre-tyrosine kinase inhibitor (imatinib mesylate) era. This result eliminated the recommendation of HSCT for patients with a good early response to chemotherapy using a tyrosine kinase inhibitor.[59,60]

The International Consensus Classification of acute lymphoblastic leukemia/lymphoma from 2022 divides BCR::ABL1–positive B-ALL into two subtypes: cases with lymphoid-only involvement and cases with multilineage involvement.[61] These subtypes differ in the timing of their transformation event. A multipotent progenitor serves as the target cell of origin for BCR::ABL1–positive B-ALL with multilineage involvement, and a later progenitor is the target cell of origin for BCR::ABL1–positive B-ALL with lymphoid-only involvement.

BCR::ABL1–positive B-ALL with lymphoid-only involvement is the predominate subtype. Only a minority of cases in children and adults have multilineage involvement (estimated at 15%–30%).[62]

BCR::ABL1–positive B-ALL cases with lymphoid-only involvement and cases with multilineage involvement have similar clinical presentations and immunophenotypes. In addition, both subtypes commonly have the p190 fusion protein.[62,63]

One way of distinguishing between patients with lymphoid-only and multilineage involvement is to detect the BCR::ABL1 fusion in normal non-ALL B cells, T cells, and myeloid cells.[63]

A second way of distinguishing between patients with lymphoid-only and multilineage involvement is to detect quantitative differences in MRD levels (typically 1 log) using measures that quantify BCR::ABL1 DNA or RNA, compared with measures based on flow cytometry, real-time quantitative polymerase chain reaction (PCR), or next-generation sequencing (NGS) quantitation of leukemia-specific immunoglobulin (IG) or T-cell receptor (TCR) rearrangements.[62-64]

For patients with lymphoid-only BCR::ABL1–positive B-ALL, MRD estimates for these methods will be correlated with each other.

For patients with multilineage involvement BCR::ABL1–positive B-ALL, posttreatment MRD estimates based on detection of BCR::ABL1 DNA or RNA will often be higher than estimates based on flow cytometry or quantitation of leukemia-specific IG/TCR rearrangements.

For patients with BCR::ABL1–positive B-ALL and multilineage involvement, levels of BCR::ABL1 transcripts and DNA may remain stable over time despite continued treatment with chemotherapy and tyrosine kinase inhibitors. In these situations, the persisting BCR::ABL1 DNA or RNA likely represents evidence of a residual preleukemic clone and not leukemia cells. Therefore, the term MRD is a misnomer.

A corollary of the difference in MRD detection by methods based on BCR::ABL1 DNA or RNA detection versus MRD detection based on flow cytometry or IG/TCR rearrangements is that the latter methods provide more reliable prognostication.[62,64,65] For example, the presence of MRD by BCR::ABL1 DNA or RNA detection in the absence of MRD detection by IG/TCR rearrangements does not confer inferior prognosis.

Based on the limited numbers of patients studied to date, prognosis appears similar in both adults and children with lymphoid-only versus multilineage involvement BCR::ABL1–positive B-ALL.[62,64]

There are case reports of patients with multilineage involvement BCR::ABL1–positive B-ALL who relapse years from their initial diagnosis. In addition, their relapsed leukemia has the same BCR::ABL1 breakpoint as their initial leukemia, but it has a different IG/TCR rearrangement.[64] These case reports suggest that patients with multilineage BCR::ABL1–positive B-ALL are at risk of a second leukemogenic event, leading to a second BCR::ABL1 leukemia.

There is no evidence that a specific monitoring schedule or prolonged treatment with a tyrosine kinase inhibitor provides clinical benefit for patients with multilineage involvement BCR::ABL1–positive B-ALL who have maintained presence of BCR::ABL1 transcripts or DNA at the completion of a standard-duration course of leukemia therapy.

KMT2A-rearranged ALL (t(v;11q23.3)).

Rearrangements involving the KMT2A gene with more than 100 translocation partner genes result in the production of fusion oncoproteins. KMT2A gene rearrangements occur in up to 80% of infants with ALL. Beyond infancy, approximately 1% of NCI standard-risk and 4% of NCI high-risk pediatric B-ALL cases have KMT2A rearrangements.[1]

These rearrangements are generally associated with an increased risk of treatment failure, particularly in infants.[66-69] The KMT2A::AFF1 fusion (t(4;11)(q21;q23)) is the most common rearrangement involving the KMT2A gene in children with ALL and occurs in approximately 1% to 2% of childhood ALL.[67,70]

Patients with KMT2A::AFF1 fusions are usually infants with high WBC counts. These patients are more likely than other children with ALL to have central nervous system (CNS) disease and to have a poor response to initial therapy.[71] While both infants and adults with the KMT2A::AFF1 fusion are at high risk of treatment failure, children with the KMT2A::AFF1 fusion appear to have a better outcome.[66,67,72] Irrespective of the type of KMT2A gene rearrangement, infants with KMT2A-rearranged ALL have much worse event-free survival rates than non-infant pediatric patients with KMT2A-rearranged ALL.[66,67,72]

Whole-genome sequencing has determined that cases of infant ALL with KMT2A gene rearrangements have frequent subclonal NRAS or KRAS variants and few additional genomic alterations, none of which have clear clinical significance.[11,73] Deletion of the KMT2A gene has not been associated with an adverse prognosis.[74]

Of interest, the KMT2A::MLLT1 fusion (t(11;19)(q23;p13.3)) occurs in approximately 1% of ALL cases and occurs in both early B-lineage ALL and T-ALL.[75] Outcome for infants with the KMT2A::MLLT1 fusion is poor, but outcome appears relatively favorable in older children with T-ALL and the KMT2A::MLLT1 fusion.[75]

TCF3::PBX1 fusion (t(1;19)(q23;p13.3)) and TCF3::HLF fusion (t(17;19)(q22;p13)).

Fusion of the TCF3 gene on chromosome 19 to the PBX1 gene on chromosome 1 is present in approximately 4% of NCI standard-risk and 5% of NCI high-risk pediatric B-ALL cases.[1,76,77] The TCF3::PBX1 fusion may occur as either a balanced translocation or as an unbalanced translocation and is the primary recurring genomic alteration of the pre-B–ALL immunophenotype (cytoplasmic immunoglobulin positive).[78] Black children are relatively more likely than White children to have pre-B–ALL with the TCF3::PBX1 fusion.[79]

The TCF3::PBX1 fusion had been associated with inferior outcome in the context of antimetabolite-based therapy,[80] but the adverse prognostic significance was largely negated by more aggressive multiagent therapies.[77,81] More specifically, in a trial conducted by St. Jude Children's Research Hospital (SJCRH) in which all patients were treated without cranial radiation, patients with the TCF3::PBX1 fusion had an overall outcome comparable to children lacking this translocation, but with a higher risk of CNS relapse and a lower rate of bone marrow relapse, suggesting that more intensive CNS therapy may be needed for these patients.[82,83]

The TCF3::HLF fusion occurs in less than 1% of pediatric ALL cases. ALL with the TCF3::HLF fusion is associated with disseminated intravascular coagulation and hypercalcemia at diagnosis. Outcome is very poor for children with the TCF3::HLF fusion, with a literature review noting mortality for 20 of 21 cases reported.[84] In addition to the TCF3::HLF fusion, the genomic landscape of this ALL subtype was characterized by deletions in genes involved in B-cell development (PAX5, BTG1, and VPREB1) and by variants in RAS pathway genes (NRAS, KRAS, and PTPN11).[78]

DUX4-rearranged ALL with frequent ERG deletions.

Approximately 3% of NCI standard-risk and 6% of NCI high-risk pediatric B-ALL patients have a rearrangement involving DUX4 that leads to its overexpression.[1,5,6] East Asian ancestry was linked to an increased prevalence of DUX4-rearranged ALL (favorable).[85] The most common rearrangement produces IGH::DUX4 fusions, with ERG::DUX4 fusions also observed.[86] DUX4-rearranged cases show a distinctive gene expression pattern that was initially identified as being associated with focal deletions in ERG,[86-89] and one-half to more than two-thirds of these cases have focal intragenic deletions involving ERG that are not observed in other ALL subtypes.[5,86] ERG deletions often appear to be clonal, but using sensitive detection methodology, it appears that most cases are polyclonal.[86] IKZF1 alterations are observed in 20% to 40% of DUX4-rearranged ALL.[5,6]

ERG deletion connotes an excellent prognosis, with OS rates exceeding 90%. Even when the IZKF1 deletion is present, prognosis remains highly favorable.[87-90] While patients with DUX4-rearranged ALL have an overall favorable prognosis, there is uncertainty as to whether this applies to both ERG-deleted and ERG-intact cases. In a study of 50 patients with DUX4-rearranged ALL, patients with an ERG deletion detected by genomic PCR (n = 33) had a more favorable EFS rate of approximately 90% than did patients with intact ERG (n = 17), with an EFS rate of approximately 70%.[88]

MEF2D-rearranged ALL.

Gene fusions involving MEF2D, a transcription factor that is expressed during B-cell development, are observed in approximately 0.3% of NCI standard-risk and 3% of NCI high-risk pediatric B-ALL cases.[1,91,92]

Although multiple fusion partners may occur, most cases involve BCL9, which is located on chromosome 1q21, as is MEF2D.[91,93] The interstitial deletion producing the MEF2D::BCL9 fusion is too small to be detected by conventional cytogenetic methods. Cases with MEF2D gene fusions show a distinctive gene expression profile, except for rare cases with MEF2D::CSFR1 that have a BCR::ABL1-like gene expression profile.[91,94]

The median age at diagnosis for cases of MEF2D-rearranged ALL in studies that included both adult and pediatric patients was 12 to 14 years.[91,92] For 22 children with MEF2D-rearranged ALL enrolled in a high-risk ALL clinical trial, the 5-year EFS rate was 72% (standard error, ± 10%), which was inferior to that for other patients.[91]

ZNF384-rearranged ALL.

ZNF384 is a transcription factor that is rearranged in approximately 0.3% of NCI standard-risk and 2.7% of NCI high-risk pediatric B-ALL cases.[1,91,95,96]

East Asian ancestry was associated with an increased prevalence of ZNF384.[85] Multiple fusion partners for ZNF384 have been reported, including ARID1B, CREBBP, EP300, SMARCA2, TAF15, and TCF3. Regardless of the fusion partner, ZNF384-rearranged ALL cases show a distinctive gene expression profile.[91,95,96] ZNF384 rearrangement does not appear to confer independent prognostic significance.[91,95,96] However, within the subset of patients with ZNF384 rearrangements, patients with EP300::ZNF384 fusions have lower relapse rates than patients with other ZNF384 fusion partners.[97] The immunophenotype of B-ALL with ZNF384 rearrangement is characterized by weak or negative CD10 expression, with expression of CD13 and/or CD33 commonly observed.[95,96] Cases of mixed phenotype acute leukemia (MPAL) (B/myeloid) that have ZNF384 gene fusions have been reported,[98,99] and a genomic evaluation of MPAL found that ZNF384 gene fusions were present in approximately one-half of B/myeloid cases.[100]

NUTM1-rearranged B-ALL.

NUTM1-rearranged B-ALL is most commonly observed in infants, representing 3% to 5% of overall cases of B-ALL in this age group and approximately 20% of infant B-ALL cases lacking the KMT2A rearrangement.[101] The frequency of NUTM1 rearrangement is lower in children after infancy (<1% of cases).[1,101]

The NUTM1 gene is located on chromosome 15q14, and some cases of B-ALL with NUTM1 rearrangements show chromosome 15q aberrations, but other cases are cryptic and have no cytogenetic abnormalities.[102] RNA sequencing, as well as break-apart FISH, can be used to detect the presence of the NUTM1 rearrangement.[101]

The NUTM1 rearrangement appears to be associated with a favorable outcome.[101,103] Among 35 infants with NUTM1-rearranged B-ALL who were treated on Interfant protocols, all patients achieved remission and no relapses were observed.[101] For the 32 children older than 12 months with NUTM1-rearranged B-ALL, the 4-year EFS and OS rates were 92% and 100%, respectively.

IGH::IL3 fusion (t(5;14)(q31.1;q32.3)).

This entity is included in the 2016 revision of the World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues.[104] The finding of t(5;14)(q31.1;q32.3) in patients with ALL and hypereosinophilia in the 1980s was followed by the identification of the IGH::IL3 fusion as the underlying genetic basis for the condition.[105,106] The joining of the IGH locus to the promoter region of the IL3 gene leads to dysregulation of IL3 expression.[107] Cytogenetic abnormalities in children with ALL and eosinophilia are variable, with only a subset resulting from the IGH::IL3 fusion.[108]

The number of cases of IGH::IL3 ALL described in the published literature is too small to assess the prognostic significance of the IGH::IL3 fusion. Diagnosis of cases of IGH::IL3 ALL may be delayed because the ALL clone in the bone marrow may be small, and because it can present with hypereosinophilia in the absence of cytopenias and circulating blasts.[104]

Intrachromosomal amplification of chromosome 21 (iAMP21).

iAMP21 occurs in approximately 5% of NCI standard-risk and 7% of NCI high-risk pediatric B-ALL cases.[1] iAMP21 is generally diagnosed using FISH and is defined by the presence of greater than or equal to five RUNX1 signals per cell (or ≥3 extra copies of RUNX1 on a single abnormal chromosome).[104] iAMP21 can also be identified by chromosomal microarray analysis. Uncommonly, iAMP21 with an atypical genomic pattern (e.g., amplification of the genomic region but with less than 5 RUNX1 signals or having at least 5 RUNX1 signals with some located apart from the abnormal iAMP21-chromosome) is identified by microarray but not RUNX1 FISH.[109] The prognostic significance of iAMP21 defined only by microarray has not been characterized.

iAMP21 is associated with older age (median, approximately 10 years), presenting WBC count of less than 50 × 109/L, a slight female preponderance, and high end-induction MRD.[110-112] Analysis of variant signatures indicates that gene amplifications in iAMP21 occur later in leukemogenesis, which is in contrast to those of hyperdiploid ALL that can arise early in life and even in utero.[1]

The United Kingdom Acute Lymphoblastic Leukaemia (UKALL) clinical trials group initially reported that the presence of iAMP21 conferred a poor prognosis in patients treated in the MRC ALL 97/99 trial (5-year EFS rate, 29%).[17] In their subsequent trial (UKALL2003 [NCT00222612]), patients with iAMP21 were assigned to a more intensive chemotherapy regimen and had a markedly better outcome (5-year EFS rate, 78%).[111] Similarly, the COG has reported that iAMP21 was associated with a significantly inferior outcome in NCI standard-risk patients (4-year EFS rate, 73% for iAMP21 vs. 92% in others), but not in NCI high-risk patients (4-year EFS rate, 73% vs. 80%).[110] On multivariate analysis, iAMP21 was an independent predictor of inferior outcome only in NCI standard-risk patients.[110] The results of the UKALL2003 and COG studies suggest that treatment of iAMP21 patients with high-risk chemotherapy regimens abrogates its adverse prognostic significance and obviates the need for HSCT in first remission.[112]

PAX5 alterations.

Gene expression analysis identified two distinctive ALL subsets with PAX5 genomic alterations, called PAX5alt and PAX5 p.P80R (NP_057953.1).[113] The alterations in the PAX5alt subtype included rearrangements, sequence variants, and focal intragenic amplifications.

PAX5alt. PAX5 rearrangements have been reported to represent approximately 3% of NCI standard-risk and 11% of NCI high-risk pediatric B-ALL cases.[1] More than 20 partner genes for PAX5 have been described,[113] with PAX5::ETV6, the primary genomic alteration in dic(9;12)(p13;p13),[114] being the most common gene fusion.[113]

Intragenic amplification of PAX5 was identified in approximately 1% of B-ALL cases, and it was usually detected in cases lacking known leukemia-driver genomic alterations.[115] Cases with PAX5 amplification show male predominance (66%), with most (55%) having NCI high-risk status. For a cohort of patients with PAX5 amplification diagnosed between 1993 and 2015, the 5-year EFS rate was 49% (95% confidence interval [CI], 36%–61%), and the OS rate was 67% (95% CI, 54%–77%), suggesting a relatively poor prognosis for patients with this B-ALL subtype.

PAX5 p.P80R (NP_057953.1). PAX5 with a p.P80R variant shows a gene expression profile distinctive from that of other cases with PAX5 alterations.[113] Cases with PAX5 p.P80R represent approximately 0.3% of NCI standard-risk and 1.8% of NCI high-risk pediatric B-ALL.[1] PAX5 p.P80R B-ALL appears to occur more frequently in the adolescent and young adult (AYA) and adult populations (3.1% and 4.2%, respectively).[113]

Outcome for the pediatric patients with PAX5 p.P80R and PAX5alt treated in a COG clinical trial appears to be intermediate (5-year EFS rate, approximately 75%).[113] PAX5alt rearrangements have also been detected in infant patients with ALL, with a reported outcome similar to KMT2A-rearranged infant ALL.[103]

BCR::ABL1-like (Ph-like).

BCR::ABL1-negative patients with a gene expression profile similar to BCR::ABL1-positive patients have been referred to as Ph-like,[116-118] and are now referred to as BCR::ABL1-like.[18] This occurs in 10% to 20% of pediatric B-ALL patients, increasing in frequency with age, and has been associated with an IKZF1 deletion or variant.[1,8,116,117,119,120]

Retrospective analyses have indicated that patients with BCR::ABL1-like ALL have a poor prognosis.[4,116] In one series, the 5-year EFS rate for NCI high-risk children and adolescents with BCR::ABL1-like ALL was 58% and 41%, respectively.[4] While it is more frequent in older and higher-risk patients, the BCR::ABL1-like subtype has also been identified in NCI standard-risk patients. In a COG study, 13.6% of 1,023 NCI standard-risk B-ALL patients were found to have BCR::ABL1-like ALL; these patients had an inferior EFS rate compared with non–BCR::ABL1-like standard-risk patients (82% vs. 91%), although no difference in OS rate (93% vs. 96%) was noted.[121] In one study of 40 BCR::ABL1-like patients, the adverse prognostic significance of this subtype appeared to be abrogated when patients were treated with risk-directed therapy on the basis of MRD levels.[122]

The hallmark of BCR::ABL1-like ALL is activated kinase signaling, with approximately 35% to 50% containing CRLF2 genomic alterations [1,118,123] and half of those cases containing concomitant JAK variants.[124]

Many of the remaining cases of BCR::ABL1-like ALL have been noted to have a series of translocations involving tyrosine-kinase encoding ABL-class fusion genes, including ABL1, ABL2, CSF1R, and PDGFRB.[4,119,125] Fusion proteins from these gene combinations have been noted in some cases to be transformative and have responded to tyrosine kinase inhibitors both in vitro and in vivo,[119] suggesting potential therapeutic strategies for these patients.

BCR::ABL1-like ALL cases with non-CRLF2 genomic alterations represent approximately 3% of NCI standard-risk and 8% of NCI high-risk pediatric B-ALL cases.[1] In a retrospective study of 122 pediatric patients (aged 1–18 years) with ABL-class fusions (all treated without tyrosine kinase inhibitors), the 5-year EFS rate was 59%, and the OS rate was 76%.[126]

Approximately 9% of BCR::ABL1-like ALL cases result from rearrangements that lead to overexpression of a truncated erythropoietin receptor (EPOR).[127] The C-terminal region of the receptor that is lost is the region that is altered in primary familial congenital polycythemia and that controls stability of the EPOR. The portion of the EPOR remaining is sufficient for JAK-STAT activation and for driving leukemia development. Point variants in kinase genes, aside from those in JAK1 and JAK2, are uncommon in patients with BCR::ABL1-like ALL.[8]

CRLF2. Genomic alterations in CRLF2, a cytokine receptor gene located on the pseudoautosomal regions of the sex chromosomes, have been identified in 5% to 10% of cases of B-ALL. These alterations represent approximately 50% of cases of BCR::ABL1-like ALL.[128-130] The chromosomal abnormalities that commonly lead to CRLF2 overexpression include translocations of the IGH locus (chromosome 14) to CRLF2 and interstitial deletions in pseudoautosomal regions of the sex chromosomes, resulting in a P2RY8::CRLF2 fusion.[8,123,128,129] These two genomic alterations are associated with distinctive clinical and biological characteristics.

BCR::ABL1-like B-ALL with CRLF2 genomic alterations is observed in approximately 2% of NCI standard-risk and 5% of NCI high-risk pediatric B-ALL cases.[1]

ALL with genomic alterations in CRLF2 occurs at a higher incidence in children with Hispanic or Latino genetic ancestry [123,131] and American Indian genetic ancestry.[85] In a study of 205 children with high-risk B-ALL, 18 of 51 (35.3%) Hispanic or Latino patients had CRLF2 rearrangements, compared with 11 of 154 (7.1%) cases of other declared ethnicity.[123] In a second study, only the frequency of IGH::CRLF2 fusions was increased in Hispanic or Latino children compared with non-Hispanic or non-Latino children with B-ALL (12% vs. 2.7%).[131] In this study, the percentage of B-ALL with P2RY8::CRLF2 fusions was approximately 6% and was not affected by ethnicity.

The P2RY8::CRLF2 fusion is observed in 70% to 75% of pediatric patients with CRLF2 genomic alterations, and it occurs in younger patients (median age, approximately 4 years vs. 14 years for patients with IGH::CRLF2).[132,133] P2RY8::CRLF2 occurs not infrequently with established chromosomal abnormalities (e.g., hyperdiploidy, iAMP21, dic(9;20)), while IGH::CRLF2 is generally mutually exclusive with known cytogenetic subgroups. CRLF2 genomic alterations are observed in approximately 60% of patients with Down syndrome and ALL, with P2RY8::CRLF2 fusions being more common than IGH::CRLF2 (approximately 80%–85% vs. 15%–20%).[129,132]

IGH::CRLF2 and P2RY8::CRLF2 commonly occur as an early event in B-ALL development and show clonal prevalence.[134] However, in some cases they appear to be a late event and show subclonal prevalence.[134] Loss of the CRLF2 genomic abnormality in some cases at relapse confirms the subclonal nature of the alteration in these cases.[132,135]

CRLF2 abnormalities are strongly associated with the presence of IKZF1 deletions. Deletions of IKZF1 are more common in cases with IGH::CRLF2 fusions than in cases with P2RY8::CRLF2 fusions.[133] Other recurring genomic alterations found in association with CRLF2 alterations include deletions in genes associated with B-cell differentiation (e.g., PAX5, BTG1, EBF1, etc.) and cell cycle control (CDKN2A), as well as genomic alterations activating JAK-STAT pathway signaling (e.g., IL7R and JAK variants).[4,123,124,129,136]

Although the results of several retrospective studies suggest that CRLF2 abnormalities may have adverse prognostic significance in univariate analyses, most do not find this abnormality to be an independent predictor of outcome.[123,128,129,137,138] For example, in a large European study, increased expression of CRLF2 was not associated with unfavorable outcome in multivariate analysis, while IKZF1 deletion and BCR::ABL1-like expression signatures were associated with unfavorable outcome.[120] Controversy exists about whether the prognostic significance of CRLF2 abnormalities should be analyzed on the basis of CRLF2 overexpression or on the presence of CRLF2 genomic alterations.[137,138]

IKZF1 deletions.

IKZF1 deletions, including deletions of the entire gene and deletions of specific exons, are present in approximately 15% of B-ALL cases. Less commonly, IKZF1 can be inactivated by deleterious point variants.[117]

Cases with IKZF1 deletions tend to occur in older children, have a higher WBC count at diagnosis, and are therefore more common in NCI high-risk patients than in NCI standard-risk patients.[2,117,136,139,140] A high proportion of BCR::ABL1-positive cases have a deletion of IKZF1,[3,136] and ALL arising in children with Down syndrome appears to have elevated rates of IKZF1 deletions.[141] IKZF1 deletions are also common in cases with CRLF2 genomic alterations and in BCR::ABL1-like ALL cases.[87,116,136]

Multiple reports have documented the adverse prognostic significance of an IKZF1 deletion, and most studies have reported that this deletion is an independent predictor of poor outcome in multivariate analyses.[87,116,117,120,136,142-149]; [150][Level of evidence B4] However, the prognostic significance of IKZF1 may not apply equally across ALL biological subtypes, as illustrated by the apparent lack of prognostic significance in patients with ERG deletions.[87-89] Similarly, the prognostic significance of the IKZF1 deletion also appeared to be minimized in a cohort of COG patients with DUX4-rearranged ALL and with ERG transcriptional dysregulation that frequently occurred by ERG deletion.[6] The Associazione Italiana di Ematologia e Oncologia Pediatrica–Berlin-Frankfurt-Münster group reported that IKZF1 deletions were significant adverse prognostic factors only in B-ALL patients with high end-induction MRD and in whom co-occurrence of deletions of CDKN2A, CDKN2B, PAX5, or PAR1 (in the absence of ERG deletion) were identified.[151] The poor prognosis associated with IKZF1 alterations appears to be enhanced by the concomitant finding of deletion of 22q11.22. In a study of 1,310 patients with B-ALL, approximately one-half of the patients with IKZF1 alterations also had deletion of 22q11.22. The 5-year EFS rate was 43.3% for those with both abnormalities, compared with 68.5% for patients with IKZF1 alterations and wild-type 22q11.22 (P < .001).[152]

There are few published results of changing therapy on the basis of IKZF1 gene status. The Malaysia-Singapore group published results of two consecutive trials. In the first trial (MS2003), IKZF1 status was not considered in risk stratification, while in the subsequent trial (MS2010), IKZF1-deleted patients were excluded from the standard-risk group. Thus, more IKZF1-deleted patients in the MS2010 trial received intensified therapy. Patients with IKZF1-deleted ALL had improved outcomes in MS2010 compared with patients in MS2003, but interpretation of this observation is limited by other changes in risk stratification and therapeutic differences between the two trials.[153][Level of evidence B4]

In the Dutch ALL11 study, patients with IKZF1 deletions had maintenance therapy extended by 1 year, with the goal of improving outcomes.[154] The landmark analysis demonstrated an almost threefold reduction in relapse rate and an improvement in the 2-year EFS rate (from 74.4% to 91.2%), compared with historical controls.

MYC-rearranged ALL (8q24).

MYC gene rearrangements are a rare but recurrent finding in pediatric patients with B-ALL. Patients with rearrangements of the MYC gene and the IGH2, IGK, and IGL genes at 14q32, 2p12, and 22q11.2, respectively, have been reported.[155-157] The lymphoblasts typically exhibit a precursor B-cell immunophenotype, with a French-American-British (FAB) L2 or L3 morphology, with no expression of surface immunoglobulin and kappa or lambda light chains. Concurrent MYC gene rearrangements have been observed along with additional cytogenetic rearrangements such as IGH::BCL2 or KMT2A.[157] Patients reported in the literature have been variably treated with ALL therapy or with mature B leukemia/lymphoma treatment protocols, and the optimal treatment for this patient group remains uncertain.[157]

T-ALL cytogenetics/genomics

T-ALL is characterized by genomic alterations leading to activation of transcriptional programs related to T-cell development and by a high frequency of cases (approximately 60%) with variants in NOTCH1 and/or FBXW7 that result in activation of the NOTCH1 pathway.[158] Cytogenetic abnormalities common in B-ALL (e.g., hyperdiploidy, 51–65 chromosomes) are rare in T-ALL.[159,160]

In Figure 5 below, pediatric T-ALL cases are divided into 10 molecular subtypes based on their RNA expression and gene variant status. These cases were derived from patients enrolled in SJCRH and COG clinical trials.[1] Each subtype is associated with dysregulation of specific genes involved in T-cell development. Within a subtype, multiple mechanisms may drive expression of the dysregulated gene. For example, for the largest subtype, TAL1, overexpression of TAL1 can result from the STIL::TAL1 fusion and a noncoding insertion variant upstream of the TAL1 locus that creates a MYB-binding site.[158,161] As another example, within the HOXA group, overexpression of HOXA9 can result from multiple gene fusions, including KMT2A rearrangements, MLLT10 rearrangements, and SET::NUP214 fusions.[1,158,162] In contrast to the molecular subtypes of B-ALL, the molecular subtypes of T-ALL are not used to define treatment interventions based on their prognostic significance or therapeutic implications.

Notch pathway signaling.

Notch pathway signaling is commonly activated by NOTCH1 and FBXW7 gene variants in T-ALL, and these are the most commonly altered genes in pediatric T-ALL.[158,163] NOTCH1-activating gene variants occur in approximately 50% to 60% of T-ALL cases, and FBXW7-inactivating gene variants occur in approximately 15% of cases. Approximately 60% of T-ALL cases have Notch pathway activation by variants in at least one of these genes.[164,165]

The prognostic significance of NOTCH1 and FBXW7 variants may be modulated by genomic alterations in RAS and PTEN. The French Acute Lymphoblastic Leukaemia Study Group (FRALLE) and the Group for Research on Adult Acute Lymphoblastic Leukemia reported that patients having altered NOTCH1 or FBXW7 and wild-type PTEN and RAS constituted a favorable-risk group (i.e., low-risk group), while patients with PTEN or RAS variants, regardless of NOTCH1 and FBXW7 status, have a significantly higher risk of treatment failure (i.e., high-risk group).[166,167] In the FRALLE study, the 5-year disease-free survival rate was 88% for the genetic low-risk group of patients and 60% for the genetic high-risk group of patients.[166] However, using the same criteria to define the genetic risk group, the Dana-Farber Cancer Institute consortium was unable to replicate these results. They reported a 5-year EFS rate of 86% for genetic low-risk patients and 79% for the genetic high-risk patients, a difference that was not statistically significant (P = .26).[165]

Chromosomal translocations.

Multiple chromosomal translocations have been identified in T-ALL that lead to deregulated expression of the target genes. These chromosome rearrangements fuse genes encoding transcription factors (e.g., TAL1, TAL2, LMO1, LMO2, LYL1, TLX1, TLX3, NKX2-I, HOXA, and MYB) to one of the T-cell receptor loci (or to other genes) and result in deregulated expression of these transcription factors in leukemia cells.[158,159,168-172] These translocations are often not apparent by examining a standard karyotype, but can be identified using more sensitive screening techniques, including FISH or PCR.[159] Variants in a noncoding region near the TAL1 gene that produce a super-enhancer upstream of TAL1 represent nontranslocation genomic alterations that can also activate TAL1 transcription to induce T-ALL.[161]

Translocations resulting in chimeric fusion proteins are also observed in T-ALL.[166]

A NUP214::ABL1 fusion has been noted in 4% to 6% of T-ALL cases and is observed in both adults and children, with a male predominance.[173-175] The fusion is cytogenetically cryptic and is seen in FISH on amplified episomes or, more rarely, as a small homogeneous staining region.[175] T-ALL may also uncommonly show ABL1 fusion proteins with other gene partners (e.g., ETV6, BCR, and EML1).[175] ABL tyrosine kinase inhibitors, such as imatinib or dasatinib, may demonstrate therapeutic benefits in this T-ALL subtype,[173,174,176] although clinical experience with this strategy is very limited.[177-179]

Gene fusions involving SPI1 (encoding the transcription factor PU.1) were reported in 4% of Japanese children with T-ALL.[180] Fusion partners included STMN1 and TCF7. T-ALL cases with SPI1 fusions had a particularly poor prognosis; six of seven affected individuals died within 3 years of diagnosis of early relapse.

BCL11B is a zinc finger transcription factor that plays a dual role as a transcription activator and repressor. It is known to play a critical role in T-cell differentiation. In T-ALL, the BCL11B gene is involved in a t(5;14)(q35;q32) translocation where a distal BCL11B enhancer drives aberrant expression of TLX3 (or NKX2-5).[181] In the process of donating its enhancer, one allele of BCL11B is inactivated. However, the resulting haploinsufficient state itself may also play a role in tumor pathogenesis. The role of BCL11B as a tumor suppressor gene is supported by the finding that about 16% of patients have T-ALL that harbors deletions or missense variants.[158,182] As described in the sections for early T-cell precursor (ETP) and T/myeloid mixed phenotype acute leukemia (T/M MPAL), BCL11B may also be leukemogenic through overexpression.

Other recurring gene fusions in T-ALL patients include those involving MLLT10, KMT2A, NUP214, and NUP98.[158,162]

Ploidy.

Recurrent abnormalities in chromosome number are much less common in T-ALL than in B-ALL. One study included 2,250 pediatric patients with T-ALL who were treated in Associazione Italiana di Ematologia e Oncologia Pediatrica/Berlin-Frankfurt-Münster protocols. The study found that near tetraploidy (DNA index, 1.79–2.28 or 81–103 chromosomes), observed in 1.4% of patients, was associated with favorable disease features and outcomes.[183]

Early T-cell precursor (ETP) ALL cytogenetics/genomics

Detailed molecular characterization of ETP ALL showed this entity to be highly heterogeneous at the molecular level, with no single gene affected by variant or copy number alteration in more than one-third of cases.[184] Compared with other T-ALL cases, the ETP group had a lower rate of NOTCH1 variants and significantly higher frequencies of alterations in genes regulating cytokine receptors and RAS signaling, hematopoietic development, and histone modification. The transcriptional profile of ETP ALL shows similarities to that of normal hematopoietic stem cells and myeloid leukemia stem cells.[184]

Studies have found that the absence of biallelic deletion of the TCR-gamma locus (ABD), as detected by comparative genomic hybridization and/or quantitative DNA-PCR, was associated with early treatment failure in patients with T-ALL.[185,186] ABD is characteristic of early thymic precursor cells, and many of the T-ALL patients with ABD have an immunophenotype consistent with the diagnosis of ETP phenotype.

Allele-specific, generally high expression of BCL11B plays an oncogenic role in a subset of cases identified as ETP ALL (7 of 58 in one study) as well as in up to 30% to 40% of lineage ambiguous leukemia T/M mixed phenotype acute leukemia (T/M MPAL).[187,188] The dysregulated expression of BCL11B can occur by multiple mechanisms.

One such alteration is t(2;14)(q22;q32), which produces an in-frame ZEB2::BCL11B fusion gene.

Other structural variants leading to allele-specific deregulated BCL11B expression include structural variants that juxtapose regulatory sequences of active genes (e.g., ARID1B [chromosome 6], BENC [chromosome 7], and CDK6 [chromosome 7]) upstream or downstream of the BCL11B locus leading to aberrant expression in a process called enhancer hijacking.

Finally, in about 20% of cases with deregulated BCL11B expression, a translocation cannot be identified. In many such cases, amplification of a downstream enhancer, BCL11B enhancer tandem amplification (BETA), leads to BCL11B promoter driven transcription.

There is a high prevalence of FLT3 alterations and JAK/STAT activation in acute leukemias driven by genomic alterations leading to BCL11B expression.[187]

Mixed phenotype acute leukemia (MPAL) cytogenetics/genomics

For acute leukemias of ambiguous lineage, the WHO classification system is summarized in Table 3.[189,190] The criteria for lineage assignment for a diagnosis of MPAL are provided in Table 4.[104]

Table 3. Acute Leukemias of Ambiguous Lineage According to the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid TissuesaCondition DefinitionAcute undifferentiated leukemia Acute leukemia that does not express any marker considered specific for either lymphoid or myeloid lineageMPAL with BCR::ABL1 (t(9;22)(q34;q11.2))Acute leukemia meeting the diagnostic criteria for MPAL in which the blasts also have the (9;22) translocation or the BCR::ABL1 rearrangementMPAL with KMT2A (t(v;11q23))Acute leukemia meeting the diagnostic criteria for MPAL in which the blasts also have a translocation involving the KMT2A geneMPAL, B/myeloid, NOS (B/M MPAL)Acute leukemia meeting the diagnostic criteria for assignment to both B and myeloid lineage, in which the blasts lack genetic abnormalities involving BCR::ABL1 or KMT2AMPAL, T/myeloid, NOS (T/M MPAL)Acute leukemia meeting the diagnostic criteria for assignment to both T and myeloid lineage, in which the blasts lack genetic abnormalities involving BCR::ABL1 or KMT2AMPAL, B/myeloid, NOS—rare typesAcute leukemia meeting the diagnostic criteria for assignment to both B and T lineageOther ambiguous lineage leukemiasNatural killer–cell lymphoblastic leukemia/lymphoma

Table 4. Lineage Assignment Criteria for Mixed Phenotype Acute Leukemia According to the 2016 Revision to the World Health Organization Classification of Myeloid Neoplasms and Acute LeukemiaaLineage CriteriaMyeloid lineageMyeloperoxidase (flow cytometry, immunohistochemistry, or cytochemistry); or mono