Acute Lymphoblastic Leukemia - ALL

Dx
Monitor
PGx
Background
Lab Tests

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 is characterized by B-cell antigens – PAX5, CD19, CD20, CD22, CD24, CD79a
- CD20 only partially expressed in non-mature forms
- CD10 also frequently expressed

T-ALL is characterized by T-cell antigens – CD2, CD3, CD4, CD5, CD7, CD8

Also 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
- Most B-ALL contains genetic abnormalities, usually translocations
  - Abnormality highly associated 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
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 9;22 (ABL1/BCR), translocation 12;21 (ETV6/RUNX1) and balanced rearrangement of MLL, IGH, MYC, and TCF3

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

Age – strong prognostic effect
- Children (1-9 years) have a better outcome than infants, adolescents or adults
Immunophenotyping – presence of CD10 or CD95 indicates favorable prognosis; B precursor subtype most favorable
Molecular markers
- Favorable prognosis
  - Hyperploidy (chromosomes 51-67 or DNA index ≥1.1) with triple trisomy of 4, 10, 17
  - TEL-AML1 t(12;21) fusion positive, E2A-PBX1 t(1;19)
- Poor prognosis – 11q23 MLL rearrangement; BCR-ABL1 t(9;22) fusion and hypodiploidy (if <45 chromosomes), near haploidy
  - BCR-ABL1 relapse – typically occurs following short chronic relapse (CR) and correlates with extremely poor prognosis
    - Second CR cannot be induced in many patients
- T-ALL – absence of biallelic T-cell receptor (TCR) gamma cells and distinctive early T-cell precursor immunophenotype is predictive of induction failure or relapse
Leukocytosis is a continuous variable – poor prognosis
- ≥50x10⁹/L increases risk of death
- >100x10⁹/L increases risk of relapse in the central nervous system (CNS)
- >400x10⁹/L – high risk for CNS hemorrhage along with pulmonary and neurologic leukostasis event
Minimal residual disease present at the end of consolidation – poor prognosis in adults
Disease response
- Favorable – good response to steroids
  - Clearance of blasts by day 8 of induction
  - <5% blasts in bone marrow by day 15 of induction

Differential Diagnosis

Acute myeloid leukemia
Lymphoma
- Blastic mantle cell
- Burkitt
- Peripheral T-cell
Mixed-lineage leukemias
Juvenile rheumatoid arthritis
Infection
- Endocarditis
- Myelodysplastic syndrome
Ewing sarcoma
Merkel cell carcinoma
Benign proliferative disease
- Benign precursor B-cells (hematogones)
- Normal cortical thymocytes

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 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 – 3-4/100,000 children; occurs five times more frequently than acute myeloid leukemia
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

Ionizing radiation
Genetic diseases – Down syndrome, Fanconi anemia, Bloom syndrome, ataxia telangiectasia, Nijmegen breakage syndrome
Probable factors – hydrocarbon exposure and pesticides

Genetics

Recurrent genetic abnormalities associated with B-ALL
- Adults
  - B-ALL with t(9;22)(q34;q11.2)(BCR-ABL1)
  - B-ALL with rearrangement of 11q23 (MLL)
  - B-ALL with t(1;19)(q23;p13.3) (E2A/PBX1 [TCF3-PBX1])
  - B-ALL with t(5;14)(q31;q32)(IL3-IGH) – ALL with eosinophilia
  - B-ALL with MYC rearrangement (8q24)
- Pediatric
  - B-ALL with t(9;22)(q34;q11.2)(BCR-ABL1)
  - B-ALL with t(12;21)(p13;q22) (ETV6-RUNX1)
  - B-ALL with rearrangement of 11q23 (AF4-MLL, ENL-MLL)
  - B-ALL with t(1;19)(q23;p13.3) (E2A-PBX1 [TCF3-PBX1])
  - Hyperdiploidy in association with trisomy 4, 10, and 17
  - Hypoploidy in association with <46 chromosomes
T-ALL
- No recurrent abnormalities identified
- Abnormalities typically reveal T-cell receptor rearrangements and abnormal karyotype in 50-70% of cases, often involving rearrangements to T-cell receptor loci (TCRα/β) at 14q11.2
  - Common examples include
    - t(1;14) (p32;q11) (TAL-SCL1)
    - t(10;14) (q24;q11.2) (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
CBC with Platelet Count and Automated Differential 0040003 Method: Automated Cell Count/Differential	Initial evaluation of suspected leukemia
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
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
Acute Lymphocytic Leukemia (ALL) Panel by FISH, Adult 2002647 Method: Fluorescence in situ Hybridization	Provides prognostic information and supplements diagnosis in adult patients with B-ALL Detects the following genetic abnormalities: t(9;22) (BCR-ABL1) and rearrangements of MLL, TCF3(E2A), and IGH Rearrangement of MYC rarely present in pre-B-ALL adults; probe detects this abnormality	Chromosome alterations outside regions complementary to these probes are not detected Other recurrent aberrations, such as hyperdiploidy and t(12;21), are rarely present in adults with B-ALL but may warrant additional FISH tests/chromosome analysis
Acute Lymphocytic Leukemia (ALL) Panel by FISH, Pediatric 2002719 Method: Fluorescence in situ Hybridization	Provides prognostic information and supplements diagnosis in pediatric patients with B-ALL Probes include ETV6/RUNX1, BCR/ABL, CEP4, CEP10, MLL
Chromosome FISH, Interphase 2002298 Method: Fluorescence in situ Hybridization	Use to order individual probes rather than adult or pediatric ALL panels Translocations detected include: t(12;21) TEL-AML1(ETV6-RUNX1) fusion; t(9;22) BCR-ABL1 fusion; 14q32 IGH rearrangement; E2A rearrangement, 11q23 MLL rearrangement, and hyperdiploidy; CDNK2 (p16) deletions Indicate names of probes needed for testing 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)	Detect unbalanced chromosomal abnormalities and loss of heterozygosity in patients with hematologic malignancies
ETV6-RUNX1 (TEL-AML1) Translocation, t(12;21) by RT-PCR 0056008 Method: Reverse Transcription Polymerase Chain Reaction	Prognosticator for ALL	Not designed to detect minimal residual disease Limit of detection is 1/100 cells
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	Follow up minimal residual disease Prognosticator for ALL	Negative result does not exclude the presence of t(1;19) translocation
BCR-ABL1, Major (p210), Quantitative 2005017 Method: Quantitative Reverse Transcription Polymerase Chain Reaction	Identify and monitor p210 BCR-ABL1 mRNA fusion in a subset of ALL with confirmed p210 Ph+ Assess treatment milestones and detect early signs of resistance to TKI therapy	Results must always be interpreted in context of morphologic and other relevant data and should not be used alone for diagnosis of malignancy Samples identified as negative may still harbor BCR-ABL1-positive cells at levels below limit of detection BCR-ABL1 mRNA with minor breakpoint (e1a2; p190) not detected
BCR-ABL1, Minor (p190), Quantitative 2005016 Method: Quantitative Reverse Transcription Polymerase Chain Reaction	Detect and monitor p190 BCR-ABL1 fusion found in an ALL subset Diagnosis, prognosis, and therapeutic monitoring in patients with confirmed p190 Ph+ leukemia	Results must always be interpreted in context of morphologic and other relevant data and should not be used alone for diagnosis of malignancy Samples identified as negative may still harbor BCR-ABL1-positive cells at levels below limit of detection BCR-ABL1 mRNA with major breakpoint (e13a2, e14a2; p210) not detected
BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010 Method: Reverse Transcription Polymerase Chain Reaction	Detect presence of BCR-ABL1 fusion when form is unknown or unclear If either p210 or p190 fusion detected, appropriate quantitative test will be performed
BCR-ABL1, T315I Mutation Detection, Quantitative 2004924 Method: Reverse Transcription Polymerase Chain Reaction/Pyrosequencing	Identify T315I mutation in patients using TKI-directed therapy (Gleevec) who have a known BCR-ABL1 translocation to determine therapy options	Only the T315I mutation will be identified

Additional Tests Available

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
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