Acute Lymphoblastic Leukemia - ALL

Indications for Testing

ALL testing should be considered in 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

Initial Workup

The recommended laboratory testing in the initial workup of suspected ALL includes a CBC with a platelet count and automated differential, peripheral blood smear, chemistry profiles, infectious disease testing, liver function tests, tests for disseminated intravascular coagulation (DIC), tests for tumor lysis syndrome, infectious disease testing, and pregnancy testing. ^, 

Diagnosis

Required testing for the diagnosis of ALL includes morphologic evaluation of bone marrow to determine blast counts, flow cytometric immunophenotyping to define the type of leukemia, and molecular characterization of leukemic cells (eg, cytogenetic evaluation, FISH analysis, and/or molecular genetic testing) to define risk-associated biologic drivers of leukemia.  In clinical scenarios that prohibit bone marrow examination, these studies may be performed on peripheral blood samples, provided there is a sufficient blast count.  Cumulatively, these studies enable characterization, prognostication, and treatment planning. ^, , ,  In 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 on bone marrow aspirate material is performed to identify certain cell surface markers that define leukemia subtypes and to predict patient outcomes. ^,  If the bone marrow aspirate cannot be obtained or used, a core biopsy or peripheral blood (if there are enough blasts) may be tested. 

ALL is broadly classified into two immunophenotypic groups, B-cell ALL and T-cell ALL, based on flow cytometric immunophenotyping. Rare acute leukemias (acute leukemias of ambiguous lineage) may express an ambiguous phenotype, which precludes definitive lineage assignment. 

Cytogenetics and Molecular Testing

Cytogenetic and molecular tests are an important part of the diagnosis, prognosis, and treatment of ALL in pediatric and adult patients.  These tests further characterize ALL subtypes and provide prognostic stratification, especially in children.

Testing methods include karyotyping, FISH, and molecular tests such as single nucleotide polymorphism (SNP) microarray and next-generation sequencing (NGS, also referred to as massively parallel sequencing).

Karyotyping, or chromosome analysis, is used to identify recurrent cytogenetic abnormalities and is recommended in all cases of diagnosed or suspected ALL. ^,  Karyotyping detects important abnormalities such as aneuploidy or chromosomal rearrangements. ^, 

FISH is more sensitive than karyotyping in detecting cytogenetic abnormalities and is recommended (with appropriate probes) for the detection of the most common cytogenetic abnormalities.  Abnormalities that cannot be detected by karyotyping, but can be detected by FISH, include ETV6-RUNX1 and iAMP21. 

Microarray testing can detect some abnormalities that may be missed by chromosome analysis or FISH, such as focal deletions, but it will not detect balanced translocations. Microarray tests may be considered in addition to conventional cytogenetics, especially if karyotyping is not successful or if there is aneuploidy. 

In B-ALL, the National Comprehensive Cancer Network (NCCN) recommends testing for all relevant fusion genes and pathogenic variants via NGS to determine prognosis and plan treatment.  Either bone marrow or peripheral blood (with sufficient lymphoblasts) may be used. 

The BCR-ABL1 fusion gene [t(9;22)(q34.1;q11.2), also referred to as BCR::ABL1, the Philadelphia chromosome, or Ph] is an especially important abnormality in B-ALL. In addition to karyotyping, qualitative or quantitative reverse transcription polymerase chain reaction (RT-PCR) testing that includes transcript size (p190 or p210) determination is recommended. 

Monitoring During Treatment

Therapeutic response monitoring is performed to assess treatment efficacy and determine risk stratification. Minimal residual disease (MRD) evaluation is performed for nearly all pediatric and many adult patients with ALL. MRD assessment may include flow cytometry, quantitative RT-PCR (such as for the BCR-ABL1 p190 or p210 transcript), or NGS-based evaluation for B-cell and/or T-cell clonality. 

Pharmacogenetics

Thiopurine S-methyltransferase (TPMT) activity testing, TPMT 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 Thiopurines and TPMT and NUDT15.