Acute Lymphoblastic Leukemia - ALL

Diagnosis

Indications for Testing

  • Abnormal blood count
  • Constitutional symptoms without other etiology

Laboratory Testing

  • CBC with peripheral smear – leukocytosis and blasts are not uncommon
  • Cytochemical staining – used infrequently due to availability of immunophenotyping
    • MPO – usually negative (if used)
  • Immunophenotyping – determine antigen markers for identifying cell lineage and predicting outcome
    • CD expression – helps establish ALL lineage
    • B-ALL characterized by B-cell antigens – PAX5, CD19, CD20, CD22, CD24, CD79a
      • CD20 – only partially expressed in non-mature forms
      • CD10 – frequently expressed
    • T-ALL is characterized by T-cell antigens – CD2, CD3, CD4, CD5, CD7, CD8
      • Others – CD1a, CD10, CD34, CD99, HLA-DR, and TdT
      • Subclassification of T-ALL by stage of normal thymocyte maturation

        Subclassification of T-ALL
        by Stage of Normal Thymocyte Maturation

        T-ALL Subtype

        CD1a

        CD2

        cCD3

        sCD3

        CD4

        CD5

        CD7

        CD8

        CD34

        Pro-T

        -

        -

        +

        -

        -

        -

        +

        -

        +

        Pre-T

        -

        +

        +

        -

        -

        ±

        -

        -

        ±

        Cortical T

        +

        +

        +

        -

        +

        ±

        +

        +

        -

        Medullary T

        -

        +

        +

        +

        ±*

        ±

        +

        ±

        -

        *Medullary stage T-ALL shows either CD4 or CD8 expression

      • May also express myeloid antigens – CD11b, CD13, CD15, CD33
  • Cytogenetic studies – important for diagnostic and prognostic workup
    • Treatment protocols are stratified by age and the presence of t(9;22)
    • Most B-ALL contains genetic abnormalities (usually translocations)
      • Abnormality correlates with prognosis
    • T-ALL/LBL contains TCR gene rearrangements
    • See Genetics section in Clinical Background for specific abnormalities
  • FISH – more sensitive than conventional cytogenetics in detecting genomic aberrations
    • Routine in initial diagnostic workup, prognostic stratification (especially in children), and determination of treatment approach
    • Adult probes – BCR/ABL1, E2A, MLL, IGH, and MYC
    • Childhood probes – BCR-ABL1 t(9;22), TEL/AML (ETV6-RUNX1), CEP4 and CEP10, and MLL/11q23 rearrangements
  • Cytogenomic SNP microarray – detects abnormalities that may not be detected by FISH or cytogenetics including deletions in the regions of CDKN2A/B, BTG1, IKZF1, and EBF1
    • Does not detect BCR-ABL1 t(9;22), TEL/AML (ETV6/RUNX1), or balanced rearrangement of MLL, IGH, MYC, and TCF3
      • BCR-ABL1 by next generation sequencing offers advantages over Sanger sequencing

Histology

  • Bone marrow biopsy – enumerate blasts and collect material for ancillary testing (eg, cytogenetics)
  • Immunohistochemistry – most useful stains include TdT; CD3; CD10 (CALLA); CD20, L26; CD79A

Prognosis

  • Prognosis by age, WBC, and mutation status (adult)

    Prognosis by Age, WBC, and Mutation Status (Adult)

    Prognosis

    Good

    Poor

    Age

    Younger age

    • Especially <25 years when treated with a pediatric protocol

    Older age

    • Individuals >60 years have a particularly poor prognosis

    High WBC

    • >30 x 109/L for B-ALL
    • >100 x 109/L for T-ALL

    Mutations

     
    • t(9;22) positive
      • Most frequent chromosomal abnormality
    • MLL rearrangements
    • t(8;14)
    • Complex karyotype (>5 chromosomal abnormalities)
    • Low hypodiploidy/near triploidy
    Prognosis by age, WBC, and mutation status (pediatric)

    Prognosis by Age, WBC, and Mutation Status (Pediatric)

    Prognosis

    Good

    Poor

    Age

    Younger age

    • Especially <25 years when treated with a pediatric protocol
    • Older age
    • High WBC
      • >30 x 109/L for B-ALL
      • >100 x 109/L for T-ALL

    Mutations

    • t(12;21) positive
    • Hyperdiploidy with gain of chromosomes 4 and 10
    • t(9;22) positive
    • MLL rearrangements
    • Low hypodiploidy/near triploidy

Differential Diagnosis

Monitoring

  • Repeat PCR or FISH testing using leukemia-associated phenotype defined at diagnosis for detection of minimal residual disease (MRD)
    • MRD by PCR or FISH defined as >10-4 cells or >0.01% blasts
    • Qualitative/quantitative PCR testing is very sensitive and may detect MRD that is not of clinical concern
      •  PCR has lower sensitivity than FISH
    • >10% MRD portends higher relapse rate
    • B-cell lines MRD detected by bone marrow; T-cell lines MRD can be detected by peripheral blood
      • T-ALL – detection of TdT or CD34 confirms MDR
    • Relapse mandates new immunophenotyping and molecular testing – karyotype may change and, rarely, second de novo ALL may be discovered

Pharmacogenetics and Therapeutic Drug Monitoring

  • Thiopurine S-methyltransferase (TPMT)
    • Thiopurine prodrugs are metabolized via TPMT enzymatic activity
    • Deficiency of TPMT predicts hematopoietic toxicity after thiopurine treatment
    • Increased risk of therapy-related acute myeloid leukemia and radiation-induced brain tumors in patients receiving intensive thiopurine therapy
    • Testing to determine activity level may be helpful in dosing thiopurine drugs and also help avert bone marrow suppression
      • For deficient activity – dose reduction of 80-90% may be required
      • For intermediate activity – dose reduction of 20-50% may be required
  • Other pharmacogenetic pathways (no testing currently available)
    • During induction therapy for ALL
      • Predominant drug metabolism pathway is via CYP3A
      • CYP3A5 GG genotype (lower CYP3A5 activity) predicts gastrointestinal (GI) toxicity and rates of infection
    • During consolidation and continuation phase of therapy
      • Reduced folate carrier AA or AG genotype predicts GI toxicity
    • During all phases
      • UGT1A1 promotor repeat polymorphism (UGT1A1 7h) predicts hyperbilirubinemia
    • MTHFR polymorphism
      • Probably affects methotrexate toxicity
      • MTHFR C677T variant associated with higher rate of relapse
    • Therapy molecular targets
      • Investigational use of Imatinib for BCR-ABL1-positive ALL
    • Adverse drug effects
      • Polymorphism in eight genes significantly associated with glucocorticoid-induced hypertension (CNTNAP2, LEPR, CRHR1, NTAN1, SLCI2A3, ALPL, BGLAP, and APOB)
      • PAI-1 polymorphism associated with GA/AA genotype – increased risk of glucocorticoid-induced osteonecrosis

Clinical Background

Acute lymphoblastic leukemia (ALL) is a malignant disease of the lymphoid cell line occurring predominantly in children.

Epidemiology

  • Incidence – 1.6/100,000
    • Most common leukemia in childhood
  • Age – peak incidence 2-5 years; second peak >50 years
  • Sex – M>F
  • Ethnicity
    • <3 years – Caucasian predominance
    • ≥3 years – African Americans have higher risk

Risk Factors

Genetics

  • Recurrent genetic abnormalities associated with B-ALL

    Recurrent genetic abnormalities associated with B-ALL

    TranslocationGene

    Adults

    t(9;22)(q34;q11.2)BCR-ABL1
    t(v;11q23)MLL
    t(1;19)(q23;p13.3)TCF3(E2A)-PBX1
    t(5;14)(q31;q32)IL3-IGH (ALL with eosinophilia)
    8q24MYC rearrangement

    Pediatric

    t(9;22)(q34;q11.2)BCR-ABL1
    t(12;21)(p13;q22)TEL-AML (ETV6-RUNX1)
    t(v;11q23)MLL
    t(1;19)(q23;p13.3)TCF3-PBX1
    Hyperdiploidy in association with trisomy 4, 10, and 17 
    Hyperdiploidy in association with <46 chromosomes 
    Common T-ALL rearrangements

    Common T-ALL Rearrangements

    Translocation

    Gene

    t(1;14)(p32;q11)

    TAL1

    t(10;14)(q24;q11.2)

    TLX1 (formerly known as HOX11)

    t(5;14)(p35;q32)

    HOX11/L2

    t(11;14)

    LMO1

    t(7:11)

    LMO2

Clinical Presentation

  • Vague constitutional symptoms – fever, anemia, pallor, failure to thrive, bruising

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003
Method: Flow Cytometry

Aid in evaluation of hematopoietic neoplasms (ie, leukemia, lymphoma)

Monitor therapy in patients with established diagnosis of hematopoietic neoplasms

Specimens include peripheral blood, bone marrow, and tissue

Markers selected based on clinical history, previous flow studies, and pathologist interpretation

Available markers:

T-cell: CD1, CD2, CD3, CD4, CD5, CD7, CD8, TCR alpha-beta, TCR gamma-delta, cytoplasmic CD3

B-cell: CD10, CD19, CD20, CD22, CD23, CD103, Kappa, Lambda, FMC7, cytoplasmic kappa, cytoplasmic lambda

Myelo/Mono: CD11b, CD13, CD14 (Mo2), CD14 (MY4), CD15, CD33, CD64, CD117, myeloperoxidase

Misc: CD11c, CD16, CD25, CD30, CD34, CD38, CD41, CD42b, CD45, CD56, CD57, CD61, HLA-DR, glycophorin, TdT, bcl-2, ALK-1, CD123, CD138, CD200, CD26, CD45

   
Chromosome FISH, Interphase 2002298
Method: Fluorescence in situ Hybridization

Translocations detected include:  t(12;21) TEL/AML (ETV6-RUNX1) fusion; t(9;22) BCR-ABL1 fusion; 14q32 IGH rearrangement; E2A rearrangement, 11q23 MLL rearrangement, and hyperdiploidy; CDNK2 (p16) deletions

ARUP Oncology FISH Probes menu

   
Chromosome Analysis, Bone Marrow with Reflex to Genomic Microarray 2007130
Method: Giemsa Band/Genomic Microarray (Oligo-SNP array)

Detect chromosome abnormalities in bone marrow aspirate consistent with the diagnosis of ALL, some of which also have classification and prognostic significance

Includes reflex to genomic microarray

   
Chromosome Analysis, Leukemic Blood with Reflex to Genomic Microarray 2007131
Method: Giemsa Band/Genomic Microarray (Oligo-SNP array)

Detect chromosome abnormalities in leukemic blood consistent with the diagnosis of ALL, some of which also have classification and prognostic significance

Includes reflex to genomic microarray

   
Cytogenomic SNP Microarray - Oncology 2006325
Method: Genomic Microarray (Oligo-SNP Array)
Preferred test at time of diagnosis to detect prognostically important genomic abnormalities (loss/gain of DNA and/or loss of heterozygosity) in hematologic malignancies

Low-level mosaicism (<15%-20%) may not be detected; test may not be appropriate for individuals with expected lower levels of malignant cells

Does not detect balanced rearrangements; FISH should be used to evaluate specific balanced rearrangements in the following

  • ALL – balanced t(9;22) (BCR-ABL1), t(12;21)(ETV6-RUNX1), and balanced rearrangement of MLL, IGH, MYC, and TCF3
  • AML – balanced t(15;17) (PML-RARA),  t(8;21) (RUNX1-RUNX1T1), and balanced rearrangement of the MLL or the CBFB gene

Does not detect base pair mutations and very small deletions/duplications; imbalances of the mitochondrial genome; low level clones (not recommended for MRD)

 
Acute Lymphocytic Leukemia (ALL) Panel by FISH, Adult 2002647
Method: Fluorescence in situ Hybridization

Recommended FISH panel for adults with newly diagnosed ALL

FISH probes detect BCR-ABL1 t(9;22); E2A; MLL 11q23 rearrangement (partner not determined); t(1;19) translocation; IGH rearrangement (partner not determined); and MYC rearrangement (partner not determined)

Chromosome alterations outside the regions complementary to these FISH probes will not be detected (IL3 probe)  
Acute Lymphocytic Leukemia (ALL) Panel by FISH, Pediatric 2002719
Method: Fluorescence in situ Hybridization

Recommended FISH panel for children with newly diagnosed ALL

Probes include TEL/AML (ETV6/RUNX1), BCR/ABL1, CEP4, CEP10, MLL

   
TdT by Immunohistochemistry 2004142
Method: Immunohistochemistry

Aid in histologic diagnosis of ALL

Stained and returned to client pathologist; consultation available if needed

   
CD10 (CALLA) by Immunohistochemistry 2003523
Method: Immunohistochemistry

Aid in histologic diagnosis of ALL

Stained and returned to client pathologist; consultation available if needed

   
CD19 by Immunohistochemistry 2005114
Method: Immunohistochemistry

Aid in histologic diagnosis of B-cell leukemia/lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

   
CD20, L26 by Immunohistochemistry 2003532
Method: Immunohistochemistry

Aid in histologic diagnosis of ALL

Stained and returned to client pathologist; consultation available if needed

   
CD79A by Immunohistochemistry 2003800
Method: Immunohistochemistry

Aid in histologic diagnosis of ALL

Stained and returned to client pathologist; consultation available if needed

   
CD3 by Immunohistochemistry 2003508
Method: Immunohistochemistry

Aid in histologic diagnosis of ALL

Stained and returned to client pathologist; consultation available if needed

   
ETV6-RUNX1 (TEL-AML1) Translocation, t(12;21) by RT-PCR 0056008
Method: Reverse Transcription Polymerase Chain Reaction

Use for detection of t(12;21) in B cell acute lymphoblastic leukemia (B-ALL)

Not designed to detect minimal residual disease

Limit of detection is 1/100 cells

Bone marrow samples preferred for maximum sensitivity

Poor RNA yield will lead to false negatives – more common in peripheral blood

 
MLL-AFF1 (MLL-AF4) Translocation, t(4;11) by RT-PCR 0050446
Method: Reverse Transcription Polymerase Chain Reaction

Prognosticator for ALL

Negative result does not exclude the presence of t(4;11) translocation

Not designed to detect minimal residual disease

Limit of detection is 1/100 cells

 
TCF3-PBX1 (E2A-PBX1) Translocation, t(1;19) by RT-PCR 0055346
Method: Reverse Transcription Polymerase Chain Reaction/Polymerase Chain Reaction

Use for detection of t(1;19) in B-ALL

Analytical sensitivity – 1/102

Analytical specificity – 100%

Negative result does not exclude the presence of t(1;19) translocation

Do not use for monitoring of minimal residual disease

 
BCR-ABL1 Mutation Analysis by Next Generation Sequencing 2008420
Method: Massively Parallel Sequencing

Detects mutations in the BCR-ABL1 gene which may impact tyrosine kinase inhibitor (TKI) resistance in Ph+ acute lymphoblastic leukemia (ALL)

Higher sensitivity than traditional Sanger sequencing techniques

Offers coverage of SH2, SH3, and kinase domain

If 2 mutations are present, this test can potentially determine if they are in cis or in trans

Analytical sensitivity limit of detection is 4% BCR-ABL1 mRNA molecules

A negative result does not exclude mutations below the level of detection or mutations outside the sequenced region of this test

 
BCR-ABL1, Major (p210), Quantitative 2005017
Method: Quantitative Reverse Transcription Polymerase Chain Reaction

Aids in diagnosis and monitoring of individuals with CML or Ph+ ALL who have e13a2 or e14a2 transcripts (p210)

Diagnose and monitor CML/Ph+ ALL

Does not detect the p190 or p230 form  
BCR-ABL1, Minor (p190), Quantitative 2005016
Method: Quantitative Reverse Transcription Polymerase Chain Reaction

Aids in diagnosis and monitoring of individuals with CML or Ph+ ALL who have e1a2 transcripts (p190)

Does not detect the p210 or p230 form  
BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010
Method: Reverse Transcription Polymerase Chain Reaction

Recommended when submitting initial diagnostic sample for CML or Ph+ ALL (no previous BCR-ABL1 testing)

If the qualitative test is positive, the appropriate corresponding quantitative test is performed

   
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Chromosome Analysis, Bone Marrow 2002292
Method: Giemsa Band

Detect chromosome abnormalities in bone marrow aspirate consistent with the diagnosis of ALL, some of which also have classification and prognostic significance