Acute Lymphoblastic Leukemia - ALL

Acute lymphoblastic leukemia (ALL) is an aggressive type of leukemia of either B- or T-lineage immature lymphoid cells. ALL is primarily a childhood disease (ALL represents 75-80% of all acute leukemias in children ) and follows a bimodal distribution; the first peak occurs between 3 and 5 years of age and the second in adults older than 50 years.  Prognosis can be inferred from age and white blood cell (WBC) count at diagnosis. Adults and those with high WBC counts typically have a poorer prognosis.  An initial ALL workup generally involves CBC testing, coagulation studies, liver function testing, and chemistry profiles; definitive diagnosis requires demonstration of ≥20% bone marrow lymphoblasts in bone marrow aspirate or biopsy.   Morphology, cytogenetics, and immunophenotyping allow for identification of recurrent genetic abnormalities that inform risk stratification, treatment planning, and monitoring.  

Quick Answers for Clinicians

What is the testing strategy for acute lymphoblastic leukemia?

At diagnosis, the minimum acute lymphoblastic leukemia (ALL) workup includes a bone marrow aspirate for morphology, immunophenotyping, cytogenetics, and fluorescence in situ hybridization (FISH) or molecular genetic testing. These studies allow for diagnosis and characterization of the type of ALL based on combined cytogenetic and immunophenotypic characteristics of lymphoblasts in the bone marrow. Results of these tests also determine appropriate treatment protocols and provide a basis for subsequent detection of minimal residual disease (MRD).    

What role does next generation sequencing play in acute lymphoblastic leukemia?

Next generation sequencing (NGS)-based assays may detect fusion genes (eg, BCR-ABL1), clonal rearrangements, and/or T-cell receptor genes that can provide information to direct therapy and aid in the detection of minimal/measurable residual disease (MRD).

What pediatric-specific guidance is available?

ALL is the most common pediatric cancer, and due to the emergence of targeted therapies and immunotherapy, current cure rates are about 90%. The National Comprehensive Cancer Network (NCCN) recently released its first version of the Pediatric Acute Lymphoblastic Leukemia guidelines; these recommendations are categorized by risk level, which can be age related. For example, the highest risk is linked to those diagnosed as infants or between 10 and 21 years of age. The guidelines also focus on long-term supportive care. Refer to the NCCN guidelines for specific guidance. 

Indications for Testing

ALL testing should be considered for patients who present with signs of bone marrow failure (eg, anemia, thrombocytopenia, leukopenia) and constitutional symptoms (eg, fever, lethargy, weight loss).  In children, joint or extremity pain may be the only presenting symptom.  

Laboratory Testing

Diagnosis

Bone marrow aspirate and biopsy allow bone marrow blasts to be enumerated and provide material for ancillary testing, including immunophenotyping, morphology, cytogenetics, and fluorescence in situ hybridization (FISH) or molecular testing. These studies enable characterization, prognostication, and treatment planning.     For patients with diagnosed ALL, it is also important to obtain a cerebrospinal fluid (CSF) sample; ALL may involve the central nervous system (CNS) both at diagnosis and at relapse. Knowledge of CSF status at diagnosis directs appropriate therapy. 

Immunophenotyping

Flow cytometric immunophenotyping is performed to identify certain cell surface markers associated with leukemia subtypes and predict outcome.   ALL is broadly classified into two immunophenotypic groups: B-cell ALL and T-cell ALL.  B-cell ALL is predominantly seen in children (roughly 88% of cases).  Refer to the tables below for markers commonly associated with B-cell and T-cell ALL.

Cytogenetics

Cytogenetics are an important part of the diagnosis, prognosis, and treatment of ALL in pediatric and adult patients.  These studies further characterize ALL subtypes and provide prognostic stratification, especially in children. The majority of B-cell ALLs involve chromosomal abnormalities; high hyperdiploidy and the ETV6-RUNX1 subtype (which results from chromosomal translocation t[12;21]) are among the subtypes that most commonly occur in children.    Both have favorable prognoses.  In adults, Philadelphia chromosome-positive (Ph positive) ALL is the most common subtype and is associated with a poor prognosis.   BCR-ABL1-like ALL, often referred to as Ph-like ALL, a recently identified subtype, is also associated with a poor prognosis.     Refer to the tables below for more information about recurrent genetic abnormalities.

Cytogenetic testing methodologies include karyotyping, FISH, and single nucleotide polymorphism (SNP) microarray. Karyotyping, or chromosome analysis, is used to identify recurrent cytogenetic abnormalities. FISH is more sensitive than karyotyping in detecting cytogenetic aberrations, such as the ETV6-RUNX1 fusion.  Microarray is capable of detecting some abnormalities that may be missed by chromosome analysis or FISH, such as focal deletions, but it will not detect balanced translocations.

Recurrent Genetic Abnormalities Associated with B-Cell ALL
Translocation/Gene Frequency Immunophenotype Prognosis/Predictive Factors

t(9;22)(q34.1;q11.2)

BCR-ABL1

Children, 2-4%

Adults, 25%

CD10, CD19, TdT, CD25 (in adults) positivity

CD13, CD33 frequently expressed

Considered worst prognosis of all major ALL subtypes

BCR-ABL1-like

10-25% of all ALL cases

CD19, CD10 positivity

Poor prognosis

Higher risk of MRD

t(v;11q23.3)

KMT2A (MLL) rearranged

Most common in infants <1 yr

Becomes increasingly common in adulthood

CD19, CD15 positivity

CD10, CD24 absent

Poor prognosis

t(1;19)(q23;p13.3)

TCF3-PBX1

Children, 6%

Adults, rare

CD19, CD10 positivity

Cytoplasmic mu heavy chain

Poor prognosis; may be improved with intensive therapy

Increased risk of CNS relapse

t(5;14)(q31.1;q32.1)

IGH/IL3

<1% of all cases of ALL

CD19, CD10 positivity

No different than that of other types of ALL

iAMP21

2% of all cases of B-cell ALL

More common in children

Unknown

Poor prognosis; may be improved with intensive therapy

t(12;21)(p13.2;q22.1)

ETV6-RUNX1

Children, 25%

Adults, rare

CD19, CD10, CD34 positivity

Favorable prognosis, with cure seen in >90% of children

Hyperdiploidy (>50 chromosomes)

Children, 25%

Adults, rare

CD19, CD10, CD34 positivity

CD45 often absent

Favorable prognosis, with cure seen in >90% of children

Hypodiploidy (<46 chromosomes)

5% of all cases of ALL

CD19, CD10 positivity

Poor prognosis

DUX4 rearranged 7% of BCP-ALL High CD2 expression Favorable prognosis
ETV6-RUNX1-like

Children, 1-3%

Adults, unknown

CD27 positivity

Low to negative CD44 expression

Few relapses

Additional studies are needed to determine the prognosis
ZNF384 rearranged

Children, 3-4%

Adults, 6-7%

CD13, CD33 positivity

Low CD10 expression
Intermediate prognosis
MEF2D rearranged

Children, 3-4%

Adults, 6-7%

CD38 positivity

CD10 absent
Poor prognosis

BCP-ALL, B-cell precursor acute lymphoblastic leukemia; MRD, minimal residual disease

Sources: Borowitz, 2017 ; Arber, 2017 ; Lilljebjörn, 2017 ; Iacobucci, 2017 

Common T-Cell ALL Rearrangements
Translocation/Gene Frequency Immunophenotype Prognosis/Predictive Factors

10q24

TLX1 (HOX11)

Children, 7%

Adults, 30%

TdT positive

Variable expression of CD1a, CD2, CD3, CD4, CD5, CD7, and CD8

Worse prognosis in childhood than B-cell ALL

Associated with higher risk of induction failure, early relapse, and isolated CNS relapse

t(5;14)

TLX3 (HOX11L2)

Children, 20%

Adults, 10-15%

CD1a, CD10 positivity

Poor prognosis

Source: Borowitz, 2017 

In addition to the gene fusions and numerical abnormalities above, other gene alterations that have been shown to carry prognostic importance in ALL include PAX5, JAK1/2, IKZF1, CRLF2, and NOTCH1. 

Monitoring

Monitoring is performed using the leukemia-associated phenotype defined at diagnosis for the detection of MRD. Virtually all pediatric and many adult patients with ALL are monitored with MRD techniques to assess treatment effectiveness and determine risk stratification.  The most frequently used methods include flow cytometry for abnormal immunophenotypes, polymerase chain reaction (PCR), and next generation sequencing (NGS)-based assays to detect fusion genes (eg, BCR-ABL1), clonal rearrangements, and/or T-cell receptor genes.   B-cell lineage MRD can be detected using bone marrow, whereas T-cell lineage MRD can be detected using peripheral blood.  Relapse mandates new immunophenotyping and molecular testing. Karyotyping may change and, rarely, a second de novo ALL may be discovered. Ph-positive cases should be tested for BCR-ABL1 tyrosine kinase inhibitor (TKI) mutations. Refer to current National Comprehensive Cancer Network (NCCN) guidelines for specific guidance for adult and pediatric patients.

Pharmacogenetics

The increased survival rates of patients with ALL are due in large part to the identification of effective regimens, treatment durations, and chemotherapeutic agents.  For example, combining imatinib with chemotherapy has shown to be effective in cases of Ph-positive ALL.   Thiopurine S-methyltransferase (TPMT) activity and genetic testing and NUDT15 genetic testing are available to guide appropriate thiopurine dosing and mitigate the risk of toxicity.   Refer to the Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for thiopurine dosing based on TPMT and NUDT15 genotypes. 

ARUP Lab Tests

Immunophenotyping

Flow Cytometry

Diagnosis and monitoring

Specimens: bone marrow, whole blood, tissue, fluid

Detect MRD in patients previously diagnosed with B-cell ALL

Cytogenetics

Karyotyping

Diagnosis, prognosis, and monitoring

Specimen: bone marrow

Specimen: bone marrow

Specimen: whole blood

Specimen: whole blood

FISH

Order for risk stratification and therapeutic management in newly diagnosed ALL

Use if FISH probes other than those included in the standard panels below are desired

Specimens: bone marrow, whole blood

Order for risk stratification and therapeutic management in newly diagnosed ALL

Probes detect BCR/ABL1 t(9;22), IGH 14q32 rearrangement (partner not determined), MLL11q23 rearrangement (partner not determined), MYC 8q24 rearrangement (partner not determined), and TCF3 (E2A) t(1;19) translocation

Specimens: bone marrow, whole blood

Probes detect BCR/ABL1 t(9;22), CEP4, CEP10, RUNX1 (gain of signals), MLL 11q23 rearrangement (partner not determined),TEL/AML (ETV6/RUNX1) t(12;21)

Specimens: bone marrow, whole blood

Order if BCR-ABL-like (Ph-like) B-cell ALL is suspected

Probes detect CRLF2, JAK2, EPOR, CSF1R, ABL1, ABL2, PDGFRB

Specimens: bone marrow, whole blood

SNP Microarray

Detect prognostically important genomic abnormalities involving loss/gain of DNA or loss of heterozygosity (LOH)

Also used for monitoring disease progression

Specimens: bone marrow, peripheral blood

Molecular Testing for BCR-ABL1 (Ph+) Leukemia

PCR

Recommended when submitting initial diagnostic sample for Ph+ ALL when no previous BCR-ABL1 testing has been performed

Specimens: bone marrow, whole blood, extracted RNA

Diagnosis and monitoring of Ph+ ALL with e13a2 or e14a2 transcripts (p210)

Specimens: bone marrow, whole blood, extracted RNA

Diagnosis and monitoring of Ph+ ALL with e1a2 transcripts (p190)

Specimens: bone marrow, whole blood, extracted RNA

NGS

Determine if a mutation is present that would interfere with response to TKI therapy in Ph+ ALL

Detects all common mutations, including T315I

Medical Experts

Contributor

Hong

Assistant Professor of Clinical Pathology, University of Utah
Medical Director, Cytogenetics and Genomic Microarray, ARUP Laboratories
Contributor

McMillin

Gwendolyn A. McMillin, PhD
Professor of Clinical Pathology, University of Utah
Medical Director, Toxicology
Medical Director, Pharmacogenetics,
and Scientific Director, Mass Spectrometry, at ARUP Laboratories
Contributor

Miles

Rodney R. Miles, MD, PhD

Assistant Professor of Clinical Pathology, University of Utah

Laboratory Section Chief, Hematopathology, at ARUP Laboratories

References

  1. T-lymphoblastic leukaemia/lymphoma

    Borowitz MJ, et al. T-lymphoblastic leukaemia/lymphoma. In Swerdlow, SH, et al. WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues, 4th. Lyon, France: IARC Press, 2017.

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