Chronic Myeloid Leukemia - CML | Choose the Right Test

Chronic myeloid leukemia (CML) is a malignancy characterized by the presence of the Philadelphia (Ph) chromosome. The Ph chromosome arises from a t(9;22) translocation, which creates a BCR-ABL1 fusion transcript (also referred to as BCR::ABL1). CML can present in a chronic, accelerated, or blast phase.

A presumptive diagnosis of CML is usually made by examining blood cell counts and peripheral smears, often during the chronic phase. Detection of BCR-ABL1 in the blood or bone marrow confirms the diagnosis. Laboratory methods used in the diagnosis, prognosis, and monitoring of CML include:

Chromosome banding analysis (karyotyping)
Fluorescence in situ hybridization (FISH)
Qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR)

Quick Answers for Clinicians

What laboratory test results are associated with each phase of chronic myeloid leukemia?

Multiple organizations provide classification schemes for the chronic, accelerated, and blast phases of chronic myeloid leukemia (CML), which vary with respect to the percentage of blast cells and other laboratory findings that define each phase.^, Refer to the table, Laboratory Findings in Each Phase of CML, for details.

What pediatric-specific guidance is available for chronic myeloid leukemia?

Pediatric cases of chronic myeloid leukemia (CML) are very rare. Refer to the 2019 Recommendations from the Children's Oncology Group CML Working Group for specific guidance on laboratory testing for CML in pediatric patients.

What is the role of minimal residual disease testing in chronic myeloid leukemia?

Minimal residual disease (MRD) testing is used to assess the molecular response to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) and can be used to determine if therapy can be discontinued. Quantitative reverse transcription polymerase chain reaction (RT-PCR) testing for BCR::ABL1 (BCR-ABL1) is used to detect MRD in patients with CML.

Indications for Testing

Testing for CML should be considered in individuals who present with an abnormal CBC (typically showing left-shifted granulocytosis with basophilia), often with splenomegaly.

Laboratory Testing

Diagnosis

Initial Workup

A complete history and physical examination with palpation of the spleen are recommended in an initial workup for CML. A CBC with differential, peripheral smear, and chemistry profile should be performed. A presumptive diagnosis is usually made based on subsequent findings, such as granulocytosis and a left shift (more immature granulocytes in the blood), along with mild to moderate basophilia.

Bone marrow aspiration and core biopsy are recommended at workup for morphology testing and to obtain material for cytogenetic and other tests.

The diagnosis of CML is confirmed by the presence of the Ph chromosome and/or BCR-ABL1 transcripts in blood or bone marrow by chromosome banding analysis, RT-PCR, or FISH. If neither the Ph chromosome nor BCR-ABL1 are detected, evaluation for other myeloproliferative neoplasms should be considered.

Chromosome Banding Analysis

Conventional bone marrow cytogenetics, specifically chromosome banding analysis (karyotyping) of at least 20 Giemsa-stained metaphases from the bone marrow, is recommended at diagnosis. Karyotyping is used for the detection of the Ph chromosome and can identify atypical (ie, three way) translocations resulting in Ph chromosome formation.

Chromosome banding analysis is especially important because it detects other abnormalities, including additional copies of the Ph chromosome, isochromosome 17q, trisomy 8, and trisomy 19, which may indicate disease progression. Chromosome banding analysis is required to detect clonal evolution (ie, new, additional chromosomal abnormalities [ACAs] that are detected over time).

Reverse Transcription PCR

RT-PCR testing should be performed along with chromosomal karyotyping at initial diagnosis for baseline BCR-ABL1 transcript identification and quantification.

Identification of the type of transcript (eg, p210/major breakpoint, p190/minor breakpoint, p230/micro breakpoint) using a qualitative or reflex RT-PCR test should be performed to identify the appropriate breakpoint for future quantitative PCR testing and informs selection of the appropriate test for continued monitoring. Additional qualitative RT-PCR tests are unnecessary and should not be performed once the type of transcript has been established.

A quantitative RT-PCR test for the relevant BCR-ABL1 transcript should be performed at the initial workup and for therapeutic response assessment and monitoring. Quantitative RT-PCR testing is very sensitive and can be performed on peripheral blood or bone marrow. Quantitative RT-PCR tests should use the International Scale (IS) and have a sensitivity of ≥4 log reduction from the standardized baseline.

FISH

FISH testing may be used if chromosome banding analysis cannot be performed or if the breakpoint is cryptic and cannot be detected by chromosome banding analysis. FISH tests can detect nearly all variant and cryptic Ph translocations and can confirm the diagnosis of CML if BCR and ABL1 probes are used. However, depending on the probe and assay, some FISH BCR-ABL1 strategies in peripheral blood show a 1-5% false-positive rate.

Phase Determination

Most cases of CML initially present in the chronic phase. Determining the CML phase is recommended at presentation because the phase will inform prognosis and treatment. Disease progression from chronic to advanced phases also has prognostic and therapeutic implications.

The criteria for each phase of CML, which can vary between classification systems, are based on peripheral blood and bone marrow findings.

Laboratory Findings in Each Phase of CML
Phase	Laboratory Findings per NCCN	Laboratory Findings per ICC	Laboratory Findings per WHO
Chronic phase	<15% blasts Ph chromosome and/or BCR::ABL1 transcripts	<10% blasts Ph chromosome and/or BCR::ABL1	<20% blasts Ph chromosome and/or BCR::ABL1
Accelerated phase	15 to <30% blasts ≥30% blasts plus promyelocytes ≥20% basophils ≤100 x 10⁹/L platelets (unrelated to therapy) Ph cells with ACAs	10-19% blasts ≥20% basophils Ph cells with ACAs	This phase is no longer included in the 5th edition of the WHO Classification of Tumours: Haematolymphoid Tumours
Blast phase	≥30% blasts (depending on classification system used) Extramedullary blasts	≥20% blasts Extramedullary blasts >5% lymphoblasts	≥20% blasts Extramedullary blasts Lymphoblasts (can be <10%)
ICC, International Consensus Classification; NCCN, National Comprehensive Cancer Network; WHO, World Health Organization Sources: NCCN, 2022; ICC, 2022; WHO, 2023

Flow cytometry should be performed to identify the lineage of blasts in advanced phases of CML (ie, accelerated and blast phases). Mutation analysis is recommended if the chronic phase of CML progresses to an advanced phase. Human leukocyte antigen (HLA) testing may be considered before an allogeneic hematopoietic stem cell transplantation (HSCT) for patients in the advanced phases of CML.

Prognosis and Risk Calculation

CBC findings, such as the platelet count and percentage of blasts, are included in multiple CML risk scoring systems. Risk calculation is recommended if CML is diagnosed in the chronic phase. Findings from cytogenetic testing, including ACAs in Ph chromosome-positive cells and clonal evolution (the accumulation of new abnormalities), may also be considered in the determination of prognosis. Bone marrow fibrosis at diagnosis is also considered to have prognostic significance.

Therapeutic Response Assessment and Monitoring

The results from standard hematology tests, cytogenetic tests, and molecular tests are used to assess the respective hematologic, cytogenetic, and molecular responses to tyrosine kinase inhibitor (TKI) therapy, and these test results may have prognostic significance. Laboratory testing is also used for minimal residual disease (MRD) detection and monitoring.

For recommended assessment points and monitoring intervals for individuals receiving TKI treatment or who have discontinued TKI treatment, refer to the NCCN Clinical Practice Guidelines in Oncology, Chronic Myeloid Leukemia.

Standard Hematology Tests

CBCs and peripheral smears can be used to determine whether blood counts, including leukocytes and platelets, have returned to normal and whether blasts or other immature cells are present.

Cytogenetic Testing

Chromosome banding analysis is used to assess cytogenetic response, specifically, to determine whether there is a decrease in the percentage of Ph chromosome-positive metaphases or whether any clonal evolution has occurred. If no analyzable metaphases are obtained, FISH testing with a dual-color probe may be considered. FISH tests may also be useful in patients with cryptic BCR-ABL1 transcripts. However, FISH testing is not recommended for monitoring response to TKI therapy if quantitative RT-PCR testing can be used.

Because of its lower sensitivity, chromosome banding analysis is not suitable as a standalone test for MRD and should be used in conjunction with quantitative RT-PCR testing.

Quantitative PCR Testing

Due to its high sensitivity, quantitative RT-PCR testing for BCR-ABL1 transcript levels (taking into account the type of BCR-ABL1 transcripts present at diagnosis) is recommended to assess the molecular response to TKI therapy and is the preferred method for response monitoring and detection of MRD. Because sensitivity varies by assay and sample quality, use of the IS is recommended to standardize quantitative PCR results. Tests with a sensitivity of ≥4.5 log reduction from the standardized baseline are recommended.

Mutation Analysis

Mutation analysis of the BCR-ABL1 kinase domain (eg, by massively parallel sequencing) is recommended in cases of relapse or failure during first-line therapy. During second-line therapy, mutation analysis should be performed if there is hematologic or cytogenetic failure. Testing is also recommended if TKIs are resumed after a loss of response following discontinuation and the response cannot be reachieved in 3 months. If no kinase domain mutations are identified, testing with a myeloid mutation panel may be useful because other mutations (ie, not BCR-ABL1) may lead to therapeutic resistance.