Pediatric Acute Lymphoblastic Leukemia Workup

Updated: Jul 22, 2022
  • Author: Vikramjit S Kanwar, MBBS, MBA, MRCP(UK); Chief Editor: Jennifer Reikes Willert, MD  more...
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Approach Considerations

Upon initial presentation, ensure the patient is clinically stable and work to establish a diagnosis. Obtain a complete blood cell (CBC) count with peripheral smear to be evaluated by a hematologist or hematopathologist for the presence of blasts. If significant number of blasts are seen, flow cytometry may rapidly confirm whether T-lineage or B-lineage acute lymphoblastic leukemia (ALL) is present. Blood chemistry drawn should include serum levels of uric acid, potassium, phosphorus, calcium, and lactate dehydrogenase (LDH) to determine the level of tumor lysis.

No imaging studies other than chest radiography to evaluate for a mediastinal mass should be required. If the physical examination reveals enlarged testes, perform ultrasonography to evaluate for testicular infiltration.

A bone marrow aspirate and biopsy performed under sedation will confirm the diagnosis with special stains (immunohistochemistry), and immunophenotyping, and allow collection of adequate sample for cytogenetics and molecular studies. A lumbar puncture with cytospin morphologic analysis to assess for central nervous system (CNS) involvement and to administer intrathecal chemotherapy is often performed at the same time.

Bone marrow and CSF studies are performed before any systemic chemotherapy, including oral steroid, is administered. In addition, if anthracyclines are to be administered, obtain a baseline echocardiogram and an electrocardiogram (ECG). Wherever possible, a central venous line should be placed prior to starting chemotherapy.






Acute lymphoblastic leukemia (ALL) cells rearrange their immunoglobulin and T-cell receptor (TCR) genes and express antigen receptor molecules that correspond to such processes in normal developing B and T lymphocytes, so that ALL can be classified as B-lineage or T-lineage ALL.

Mature B-cell ALL should be differentiated from other B-lineage ALL and accounts for only 1-3% of childhood ALL. Diagnosis depends on the detection of surface immunoglobulin on leukemic blasts, as well as distinctive morphology, with deeply basophilic cytoplasm containing prominent vacuoles, designated L3 in the French-American-British (FAB) system (see Histologic Features).

B-lineage ALL accounts for 80% of childhood ALL and involves lymphoblasts that have cell-surface expression of 2 or more B-lineage–associated antigens (ie, CD19, CD20, CD24, CD22, CD21, or CD79). [5] CD10 is commonly expressed, which makes it a useful diagnostic marker, and the presence of aberrant myeloid markers (eg, CD7) is occasionally noted but has little prognostic impact. B-cell precursors of ALL can be further subclassified as early pre–B-cell, pre–B-cell, or transitional pre–B-cell, but distinguishing these subtypes is usually not clinically relevant.

T-lineage ALL accounts for 10-15% of childhood ALL, and is identified by the expression of T-cell–associated surface antigens, of which cytoplasmic CD3 is specific. T-cell ALL cases can be classified by early, mid, or late thymocytes, and 10% of cases are early T-precursor (ETP) ALL characterized by absent CD1a and CD8, weak CD5, and one or more myeloid or stem cell markers (eg, CD117, CD34, CD13, CD33). ETP ALL is thought to have worse prognosis, but this assumption may not be valid on modern T-cell ALL chemotherapy protocols.

The overall outcome for T-lineage and high risk B-lineage ALL is similar, provided intensive chemotherapy is used.


Cytogenetic and Molecular Studies

In 80% of pediatric acute lymphoblastic leukemia (ALL) cases, specific genetic alterations can be found in leukemic blasts using routine karyotype analysis and molecular techniques, such as fluorescence in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR), and Southern blot analysis. Important diagnostic, therapeutic, and prognostic implications are associated with the abnormalities described. [12]

See Acute Lymphoblastic Leukemia Staging for summarized information.

B-lineage ALL has a variety of chromosomal abnormalities, which in almost half of cases confer favorable outcomes:

  • t(12;21)(p13;q22) or ETV6-RUNX1 (formerly known as TEL-AML1) (20-25% cases)

  • Hyperdiploidy (>50 chromosomes/cell) (25% cases) associated with non-random trisomies X, 4, 6, 10, 14, 17, 18 and 21. Trisomy 4 and 10 alone (and in the past with trisomy 17 or "triple trisomy”) has been found to confer benefit. It is very important to confirm that "hyperdiploidy" does not represent doubling of a near-haploid clone, which has a poor prognosis

Other, less common, chromosomal abnormalities confer a poor outcome:

  • Extreme hypodiploidy with < 44 chromosomes/cell (1% of cases) which can be subdivided into near-haploid ALL (24–31 chromosomes) harboring mutations affecting receptor tyrosine kinase and RAS signaling, and low-hypodiploid ALL (32–39 chromosomes) characterized by alterations in TP53 as a manifestation of Li-Fraumeni syndrome.

  • MLL gene rearrangement eg t(4;11)(q21;q23) (6% cases)

  • Philadelphia chromosome positivity t(9;22)(q34;q11) (3% cases)

  • Internal amplification of chromosome 21 (iAMP 21) (2% cases)

And some chromosomal abnormalities are of uncertain significance:

  • t(1;19)(q23;p13) or TCF3-PBX1 (formerly known as E2A-PBX1) (4% cases) was thought to confer poor prognosis with increased risk of CNS relapse, but this is no longer true with contemporary treatment.

Genome-wide association studies (GWAS) use techniques that are not widely available to define genetic abnormalities in the 20% of B-lineage ALL cases where routine testing is unrevealing, demonstrating changes associated with poor outcome:

  • "Ph-like" ALL (10-15% of cases) with gene expression similar to Ph+ ALL which commonly have IZKF1 alteration, and in half of cases cryptic rearrangements of CRLF2 (cytokine receptor like factor 2). Approximately half of CRLF2 -rearranged cases have point mutations in Janus kinase (JAK) family members JAK1 and JAK2

  • Increased CRLF2 (cytokine receptor like factor 2) expression, which may have an underlying CRLF2 mutation (5-7% of cases). Interestingly, increased CRLF2 expression is also seen in 50% of the patients with Down syndrome and ALL, where its prognostic value is uncertain.

T-lineage ALL also has a variety of chromosomal abnormalities with less well-defined prognostic value:

  • Constitutive activation of NOTCH1 signaling is the most common abnormality and secondary to activating mutations in NOTCH1 (>50% of cases of T-ALL), FBXW7 mutations (15% of cases), and t(7;9)(q34;q34.3) (< 1% of cases), and may convey favorable outcome.

  • Genetic lesions also include homeobox (HOX), LMO, and bHLH family members. Amongst the HOX genes TLX1 rearrangement t(10;14)(q24;q11) was thought to confer a favorable prognosis (5-10% cases) and TLX3 rearrangement t(5;14)(q35;q32) less favorable (20-25% cases).


Minimal Residual Disease Studies

Historically, the response to leukemia treatment was assessed morphologically, which can be challenging when looking for small numbers of leukemic cells, especially in bone marrow specimens recovering from chemotherapy or after transplantation.

Studies of minimal residual disease (MRD) may be based on the detection of chimeric transcripts generated by fusion genes, the detection of clonal TCR or immunoglobulin heavy-chain (IgH) gene rearrangements, or the identification of a immunophenotype specific to the leukemic blasts. [2] All of the methods for detecting MRD have a much higher sensitivity than that of morphology, and all studies using MRD techniques have shown significant correlations between end-of-induction leukemia burden and outcome. [13] As a result, current treatment protocols use MRD measurements for acute lymphoblastic leukemia risk assignment. [14]

For B-lineage ALL patients, the significance of MRD was quantitatively different among genetic subgroups and NCI risk groups, with the time point for measurement varying between studies:

  • On COG P9904/5/6 clinical trials, multivariate analysis found Day 29 (end of induction) bone marrow (BM) MRD measured by flow cytometry with a cut-off of 0.01% was the most significant predictor of outcome in patients with B-ALL. These studies also found that Day 8 peripheral blood MRD level was an independent predictor of outcome in multivariate analysis.

  • The UK ALL 2003 clinical trial also confirmed MRD as the single most important predictor of relapse, and patients with day 29 BM MRD ≥ 0.01% had a threefold higher relapse rate (5-year EFS 79%) compared with low-risk patients (5-year EFS 94-95%).

  • The St Jude Total XV trials confirmed that BM MRD on Day 19 and Day 46 of induction therapy were important predictors of relapse with Day 19 BM MRD of < 1% detecting a favorable risk group (10-year EFS 95-100%) and day 19 BM MRD of >1% who still had >0.01% at day 46 having poor outcomes (10-year EFS 25-69%)

For T-lineage ALL patients:

  • The Italian Association of Pediatric Haematology-Oncology (AIEOP)-Berlin-Frankfurt-Muenster (BFM) 2000 study used PCR to measure MRD on Day 33 (end induction) and Day 78 (end of consolidation, EOC) and found EOC MRD a better predictor of adverse outcome in T-lineage ALL. Regardless of Day 33 MRD, patients who had EOC MRD < .01% had a 7-year EFS of at least 80%, in contrast to patients with EOC MRD >.01% who had a 7-year EFS of 49%.



Perform testicular ultrasonography if the testes are enlarged upon physical examination.

Some clinicians use renal ultrasonography to evaluate for leukemic kidney involvement as an assessment of risk for tumor lysis syndrome.


Bone Marrow Aspiration and Biopsy

Bone marrow aspirate and biopsy results confirm the diagnosis of acute lymphoblastic leukemia (ALL). In addition, special stains (immunohistochemistry), immunophenotyping, cytogenetic analysis, and molecular analysis help in classifying each case. See the images below for examples of bone marrow aspirate findings.

Bone marrow aspirate from a child with B-precursor Bone marrow aspirate from a child with B-precursor acute lymphoblastic leukemia. The marrow is replaced primarily with small, immature lymphoblasts that show open chromatin, scant cytoplasm, and a high nuclear-cytoplasmic ratio.
Bone marrow aspirate from a child with T-cell acut Bone marrow aspirate from a child with T-cell acute lymphoblastic leukemia. The marrow is replaced with lymphoblasts of various sizes. No myeloid or erythroid precursors are seen. Megakaryocytes are absent.
Bone marrow aspirate from a child with B-cell acut Bone marrow aspirate from a child with B-cell acute lymphoblastic leukemia. The lymphoblasts are large and have basophilic cytoplasm with prominent vacuoles.

Histologic Features

Historically, the French-American-British (FAB) classification system allocated acute lymphoblastic leukemia (ALL) into 3 groups based on morphology, as described below. Only L3 morphology retains diagnostic relevance.

  • L1: The lymphoblast cells are usually small, with scant cytoplasm and inconspicuous nucleoli. L1 accounts for 85% of all cases of childhood acute lymphoblastic leukemia.

  • L2: The lymphoblast cells are larger than in L1. The cells demonstrate considerable heterogeneity in size, with prominent nucleoli, and abundant cytoplasm. L2 accounts for 14% of all childhood acute lymphoblastic leukemia.

  • L3: The lymphoblast cells are large with deep cytoplasmic basophilia. They frequently have prominent cytoplasmic vacuolation and are morphologically identical to Burkitt lymphoma cells. L3 accounts for 1% of childhood acute lymphoblastic leukemia cases.

See Acute Lymphoblastic Leukemia Staging for more complete information.


Laboratory Studies

An elevated leukocyte count of more than 10 × 109/L (>10 × 103/µL) occurs in only half of patients with pediatric acute lymphoblastic leukemia (ALL). Neutropenia, anemia, and thrombocytopenia are common in ALL but are often observed secondary to inhibition of normal hematopoiesis by leukemic infiltration. It is important to recognize that 20% of patients with ALL initially present with pancytopenia and no evidence of peripheral blasts. [15]

Various metabolic abnormalities may include increased serum levels of uric acid, potassium, phosphorus, calcium, and lactate dehydrogenase (LDH). The degree of abnormality reflects the leukemic cell burden and destruction (lysis). Coagulation studies can be helpful in patients with T-lineage ALL and should include tests of the prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen level, and D-dimer level to assess for disseminated intravascular coagulation (DIC).