Chronic Myelogenous Leukemia - CML

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disease accounting for 15% of all leukemias. Presumptive diagnosis is made from blood cell counts and examination of blood film. Further laboratory testing methods include cytogenetics, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and next generation sequencing (NGS).

Key Points

Monitoring CML

Chronic myelogenous leukemia is a chronic disease that is managed with tyrosine kinase inhibitor (TKI) therapy. Most patients are able to achieve a sustained response from these agents. Use of TKIs requires serial testing to monitor response. Failure to respond or relapse of disease may be due to nonadherence, subtherapeutic dose, or resistance. Nonadherence or subtherapeutic dose of imatinib may be detected by measuring the concentration of imatinib in plasma.

Resistance develops in a subset of individuals receiving TKI therapy. A 5- to 10-fold increase in the levels of BCR-ABL1 fusion transcripts by quantitative PCR suggests resistance. BCR-ABL1 kinase domain mutation testing is recommended to guide choice of second-line TKI therapy. The BCR-ABL1 T3151 mutation imparts resistance to all currently approved TKIs.

Content in tables below based on Cortes J, 2011; Hochhaus A, 2011; Hughes, 2009; Kantarjian H, 2007; Saglio G, 2012; Yeung, 2011.

Diagnosis

ARUP Tests

  • Chromosome Analysis, Bone Marrow 2002292
  • Chromosome FISH, Interphase 2002298
  • BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010
    • Recommended if diagnosis is uncertain
  • BCR-ABL1, Major (p210), Quantitative 2005017
    • Major transcript characterized by p210 fusion gene product is typical in CML

Cytogenetic Analysis (Bone Marrow)

  • Recommended test; considered gold standard

FISH

  • May be used as alternate test if cytogenetics or molecular testing not available, or if cytogenetics are negative for BCR-ABL transcript
  • Order as baseline test if monitoring cytogenetic complete remission (CCR) with FISH

Quantitative PCR (measures BCR-ABL transcript levels)

  • Most CML patients have p210 fusion; p190 and p230 fusions are rare in CML
  • May be used as alternate to cytogenetic testing

Prior to complete cytological response (CCR)

ARUP Tests

  • Chromosome Analysis, Bone Marrow 2002292
  • BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010
    • Recommended if diagnosis is uncertain
  • BCR-ABL1, Major (p210), Quantitative 2005017
    • Major transcript characterized by p210 fusion gene product is typical in CML
  • Imatinib 3000539
    • Imatinib concentration measured in plasma

Cytogenetic Analysis (Bone Marrow)

  • Recommended test for establishing CCR
    • Perform every 3-6 mos until CCR achieved

Quantitative PCR (measures BCR-ABL transcript levels)

  • Perform every 3-6 mos until CCR achieved

Imatinib (therapeutic drug monitoring)

  • Optimize imatinib dose, detect variable pharmacokinetics, and monitor patient adherence

Confirming CCR

ARUP Tests

  • Chromosome Analysis, Bone Marrow 2002292
  • Chromosome FISH, Interphase 2002298
  • BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010
    • Recommended if diagnosis is uncertain
  • BCR-ABL1, Major (p210), Quantitative 2005017
    • Major transcript characterized by p210 fusion gene product is typical in CML

Cytogenetic Analysis (Bone Marrow)

  • Recommended test

FISH

  • Alternative test to document CCR achieved

Quantitative PCR (measures BCR-ABL transcript levels)

  • Alternative test to document CCR achieved when cytogenetics are not available

Monitoring CCR

ARUP Tests

  • Chromosome Analysis, Bone Marrow 2002292
  • Chromosome FISH, Interphase 2002298
  • BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010
    • Recommended if diagnosis is uncertain
  • BCR-ABL1, Major (p210), Quantitative 2005017
    • Major transcript characterized by p210 fusion gene product is typical in CML
  • Imatinib 3000539
    • Imatinib concentration measured in plasma

Cytogenetic Analysis (Bone Marrow)

  • Every 12-24 mos after CCR achieved
  • PCR has equivalent sensitivity
  • Can detect secondary changes suggestive of transition to blast crisis

FISH

  • Alternative to cytogenetic analysis once CCR achieved
  • Every 6 mos after CCR achieved

Quantitative PCR (measures BCR-ABL transcript levels)

  • Most CML patients have p210 fusion; p190 and p230 fusions are rare in CML
  • Alternative to cytogenetic analysis
  • Every 3-6 mos after CCR achieved

Imatinib (therapeutic drug monitoring)

  • Optimize imatinib dose, detect variable pharmacokinetics, and monitor patient adherence

Consideration of major change in therapy

ARUP Tests

  • Chromosome Analysis, Bone Marrow 2002292
  • BCR-ABL1, Qualitative with Reflex to BCR-ABL1 Quantitative 2005010
    • Recommended if diagnosis is uncertain
  • BCR-ABL1, Major (p210), Quantitative 2005017
    • Major transcript characterized by p210 fusion gene product is typical in CML
  • BCR-ABL1 Mutation Analysis for Tyrosine Kinase Inhibitor Resistance by Next Generation Sequencing 2008420
  • Imatinib 3000539
    • Imatinib concentration measured in plasma

Cytogenetic Analysis (Bone Marrow)

  • At time of suspected relapse or suspected resistance to TKI

Quantitative PCR (measures BCR-ABL transcript levels)

  • Most CML patients have the p210 fusion; p190 and p230 fusions are rare in CML
  • Use in combination with kinase domain mutation testing

Mutational Studies (detects BCR-ABL KD mutations)

  • Recommended only when patient has 5- to 10-fold increase in values for PCR test
  • Evidence lacking that use of baseline mutation analysis and subsequent monitoring improves outcome
  • Next generation sequencing
    • Offers higher sensitivity over Sanger sequencing techniques
    • Offers additional testing of SH2 and SH3 domains

Imatinib (therapeutic drug monitoring)

  • Optimize imatinib dose, detect variable pharmacokinetics, and monitor patient adherence

Features of Test Types

Cytogenetic analysis (bone marrow)

  • Marrow analyzed for at least 20 evaluable metaphases
  • CCR: 0% Ph-positive metaphases
    • Equivalent to quantitative PCR value of 1%
  • Time consuming
  • Painful procedure to obtain marrow
  • Wide confidence interval
  • Will detect translocation, but not insertion in BCR-ABL fusion gene (occurs in ~5% cases)

FISH

  • Blood specimens analyzed for 200 interphase cells
  • Values not always interchangeable with cytogenetic values
  • Not usually used to confirm CCR
  • Detects both insertions and translocations resulting in BCR-ABL fusion

Quantitative PCR

  • Reports ratio of BCR-ABL1 to reference gene with conversion to international scale (% IS) in blood or bone marrow specimen
    • Good correlation between blood and bone marrow samples
  • Major molecular response: reduction in transcript levels by at least 3-logs (<0.1% IS)
  • Complete molecular response: transcripts not detectable (in assay with at least 4-5 log range of detection)

Mutational studies

  • Good correlation between bone marrow and blood samples

Diagnosis

Indications for Testing

Abnormal CBC, splenomegaly

Laboratory Testing

  • Presumptive diagnosis from blood cell counts and examination of blood film
    • White blood cell count shows leukocytosis
      • Median 100 K/μL (range 12-1,000 K/μL)
      • Blasts rare; basophilia and eosinophilia common
      • Platelet count ranges from normal to 1x1013
    • Cytochemical staining for leukocyte alkaline phosphatase useful in differentiating CML from leukemoid reaction – low score is consistent with CML
  • Karyotyping (using cytogenetics or FISH)
    • Requires cells from bone marrow aspirate
    • Use to determine CCR
    • t(9;22) (q34; q11.2) Philadelphia (Ph) chromosome testing (BCR-ABL1)
      • t(9;22) translocation may occasionally be found in acute lymphoblastic leukemia (ALL)
      • Translocation found in acute myeloid leukemia (AML) may reflect a blast crisis in previously unrecognized CML
      • Patients without Ph chromosome still carry a BCR-ABL1 fusion due to a cytogenetically cryptic insertion identifiable by FISH or PCR
      • Provides information about additional genetic abnormalities in addition to t(9;22)
  • Quantitative PCR (performed simultaneously with karyotyping)
    • Identify BCR-ABL1 fusion transcripts – allow for monitoring of response to therapy
      • Considered standard of care for detection of minimal residual disease (MRD)
      • Quantitative testing – p210 fusion is most frequent (p190 and p230 fusions are rare)
      • Used in determination of CCR
      • Helpful in determining relapse
    • Next generation sequencing increases sensitivity over Sanger sequencing
      • Covers SH2, SH3, and kinase domains
      • Can detect >130 different mutations associated with TKI resistance
  • Immunophenotyping
    • Not required for diagnosis in chronic or accelerated phase CML
    • Used in patients presenting with acute leukemia to identify disease lineage
      • Generally 2/3 myeloid, 1/3 B-lymphoid
      • T-cell lineage rare

Histology

  • Bone marrow biopsy
    • Increased cellularity with myeloid hyperplasia, small megakaryocytes
    • Usually <5% blasts
    • Reticulin fibrosis in 30%

Prognosis

  • Karyotype
    • Evolution of cytogenetic abnormalities associated with poor prognosis
    • Early cytogenetic response associated with good prognosis
  • Recent literature (Valent, 2008) suggests histamine and tryptase levels may be used in prognosis
  • Molecular response – quantitative PCR for BCR-ABL1 is essential for detecting major molecular response (MMR) and for assessing prognosis
  • CML calculator of relative risk – assess prognosis; based on Sokal and Hasford scoring systems

Differential Diagnosis

Monitoring

  • Serial hematologic, cytogenetic, and molecular testing should be performed throughout the course of therapy
    • Quantitative real-time PCR for BCR-ABL1 t(9;22) (molecular) – p210 fusion most common; p190 and p230 fusions are rare
      • Should be reported in standardized format (%) on the BCR-ABL1 international scale (IS)
        • 100% on the IS corresponds to standardized baseline value at diagnosis as determined by original trial of imatinib therapy in chronic phase CML patients (IRIS trial)
        • A 3-log reduction from baseline (0.1% IS) constitutes an MMR to TKI therapy
        • When IS>10%, recommend karyotyping for Ph evolution
  • Cytogenetics – karyotyping
    • Examination of at least 20 bone marrow metaphases
    • Cytogenetic evolution in Ph positive clone associated with poor prognosis
      • Complete response – no detectable abnormal metaphases
      • Major response – 1-35% abnormal metaphases
      • Minor response – >35% abnormal metaphases
    • Repeat every 3-6 months from initiation of therapy until CCR achieved; after CCR achieved, repeat every 12-24 months
      • Also repeat if patient appears to have rising BCR-ABL1 transcript levels (1 log increase)
  • Imatinib therapeutic drug monitoring (TDM)
    • Patients being treated with the TKI imatinib are monitored for the following reasons
      • Imatinib TDM – when dose is determined according to TDM, imatinib yields MMR similar to that of second-generation TKIs (Rousselot, 2015)
      • Patient adherence
      • Variable pharmacokinetics​

Pharmacogenetics

  • Mutations in the BCR-ABL1 gene lead to TKI resistance of varying degrees
  • Resistance develops secondary to
    • Kinase domain mutations
      • T3151 mutation resistant to most TKIs – indicates poor prognosis
        • Ponatinib – new pan BCR-ABL1 inhibitor which includes T315I mutations
    • Amplification or overexpression
    • Quiescent CML stem cells
    • Low bioavailability
  • Resistance may be overcome with
    • Dose adjustments
    • Change in therapy
  • Indications for testing
    • Perform testing for BCR-ABL1 kinase domain mutations
      • If patient is unresponsive to treatment
      • If there is a 5- to 10-fold increase in BCR-ABL1 fusion transcripts detected by PCR
    • For management of CML, see recommendations from European LeukemiaNet
    • Initial treatment resistance testing – performed only in patients with accelerated phase disease and multiple mutations
      • Not routinely performed prior to first therapy in other patients
      • Low incidence of mutations found prior to TKI therapy in chronic phase CML
      • Next generation sequencing offers benefits over Sanger sequencing techniques

Background

Epidemiology

  • Incidence – 1-2/100,000 annually in U.S.
  • Age – median is 67 years, but may be seen in all age groups
  • Sex – M>F, 1.5:1
    • Females may have a survival advantage

Risk Factors

  • Exposure to significant quantities of ionizing radiation
  • Benzene or alkylating agents

Pathophysiology

  • Clonal expansion of stem cells characterized by reciprocal translocation between chromosomes 9 and 22 that form the Philadelphia chromosome (Ph)
    • t(9;22) translocation causes a fusion of the ABL1 gene and BCR gene
      • BCR-ABL1 fusion codes for an abnormal protein possessing constitutive tyrosine kinase activity
      • Patients with CML typically have the p210 form of the BCR-ABL1 fusion resulting from translocations between BCR exon 13 or 14 and ABL1 exon 2 (e13a2, e14a2)
      • Very rarely, patients with CML may have the p190 form of the BCR-ABL1 fusion resulting from a translocation between BCR exon 1 and ABL1 exon 2 (e1a2)
  • Mechanism of treatment resistance and relapse
    • Standard of care – treatment with TKI, including imatinib, nilotinib, or dasatinib
      • Resistance to initial therapy may require increased dosing or use of a different TKI
        • Second-generation TKIs include dasatinib, nilotinib, bosutinib, ponatinib
    • Relapse after effective chemotherapy mainly a result of outgrowth of leukemic subclones resistant to current TKIs
      • Mutations in BCR-ABL1 kinase are the most common cause of relapse due to imatinib resistance, but are not the only cause

Clinical Presentation

  • CML categorized into three phases
    • Chronic phase
      • 20-40% of patients are asymptomatic and identified through routine blood count
      • Untreated chronic phase CML will progress to accelerated phase within 3-5 years
    • Accelerated phase
      • Insidious onset with vague signs and symptoms – weight loss, fatigue, abdominal discomfort due to splenomegaly, fever
      • May demonstrate increased myeloblasts (10-19%) in bone marrow
      • Basophilia (>20% in blood of bone marrow) may also occur
      • Thromboses with vaso-occlusive crisis – cerebral vascular accident (stroke), myocardial infarction (heart attack), visual disturbances
    • Blast phase
      • 10% present with de novo blast crisis (aggressive advanced phase)
      • Splenomegaly, leukocytosis with 20% or more myeloid blasts in blood and/or bone marrow, but full range of myeloid maturation, normochromic normocytic anemia, thrombocytosis
      • Lymphadenopathy is unusual

ARUP Lab Tests

Primary Tests

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

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

Aid in diagnosis and monitoring of individuals with CML who have e13a2 or e14a2 transcripts (p210)

Does not detect p190 or p230

Aid in diagnosis and monitoring of individuals with CML who have e1a2 transcripts (p190)

Does not detect the p210 or p230 form

Order only for patients with an established diagnosis of a BCR-ABL1 positive leukemia

Use to determine if a mutation is present that would interfere with response to TKI therapy in Ph+ lymphoblastic leukemia or CML

Detects all common mutations, including T315I

Higher sensitivity than traditional Sanger sequencing techniques

Offers coverage of SH2, SH3, and kinase domain

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

The sensitivity of this assay may be limited and sequencing may not be possible in patient samples containing low tumor burden (ie, low levels of BCR-ABL1 fusion transcript by IS% or NCN)

Differentially diagnose cases of neutrophilia, including CML and leukemoid reaction

Helpful first-line screening test; however, does not definitively diagnose CML

Diagnose, prognose, and monitor hematopoietic neoplasms

Use to order individual or multiple oncology FISH probes if standard FISH panels are not desired

Alternate test if cytogenetics or PCR not available

Order as a baseline test if monitoring CCR with FISH

May be ordered concurrently with PCR qualitative BCR-ABL1 testing

Specific FISH probes related to CML must be requested and include BCR-ABL1

ARUP Oncology FISH Probes menu

Optimize imatinib dose, detect variable pharmacokinetics, and monitor patient adherence

Therapeutic range based on plasma predose (trough) draw at steady-state concentration

Concentrations >1,000 ng/mL in patients with CML are statistically associated with an improved response

Limited role in the workup of MPN in the setting of otherwise optimal cytogenetic study

​Aid in exclusion of cryptic BCR-ABL1 rearrangement in CML and in the exclusion of a PDGFRA abnormality in cases of neoplastic eosinophilia

Probes target BCR-ABL1 fusion, FGFR1 translocations, FIP1L1-PDGFRA region rearrangements, and PDGFRBtranslocations

Only detects rearrangements targeted by the probes

The translocation partners of the PDGFRB gene on 5q33 and FGFR1 gene on 8p11 have multiple translocation partners; these translocation partners are not identified by this test

Related Test

Initial evaluation for presence of leukocytosis

Medical Experts

Contributor

Lamb

Allen N. Lamb, PhD, FACMG
Allen N. Lamb, PhD, FACMG
Retired Former Professor of Clinical Pathology, University of Utah
Retired Former Laboratory Section Chief, Cytogenetics and Genomic Microarray, ARUP Laboratories
Contributor

McMillin

Gwendolyn A. McMillin, PhD
Professor of Clinical Pathology, University of Utah
Scientific Director, Mass Spectrometry Platform; Medical Director, Clinical Toxicology and Pharmacogenomics, ARUP Laboratories
Contributor

Patel

Jay L. Patel, MD, MBA
Associate Professor of Clinical Pathology, University of Utah
Medical Director, Molecular Oncology and Hematopathology, ARUP Laboratories
Contributor

References

Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®