Chronic Myelogenous Leukemia - CML

Key Points

Monitoring Chronic Myelogenous Leukemia

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 and to detect relapse of disease and/or the development of drug resistance. 

Resistance develops in a subset of individuals receiving TKI therapy.  A 5- to 10fold 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.  

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 BCR-ABL, t(9;22) Translocation Qualitative by RT-PCR 0055553

  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 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 BCR-ABL, t(9;22) Translocation Qualitative by RT-PCR  0055553

  Cytogenetic Analysis (Bone Marrow) Recommended test for establishing CCR Perform every 3-6 months until CCR achieved Quantitative PCR (measures BCR-ABL transcript levels) Perform every 3-6 months until CCR achieved

  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 BCR-ABL, t(9;22) Translocation Qualitative by RT-PCR  0055553

  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 BCR-ABL, t(9;22) Translocation Qualitative by RT-PCR  0055553

  Cytogenetic Analysis (Bone Marrow) Every 12-24 months 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 months after CCR achieved Quantitative PCR (measures BCR-ABL transcript levels) Most CML patients have p210 fusion; p190 fusions are rare in CML Alternative to cytogenetic analysis Every 3-6 months after CCR achieved

  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-ABL, t(9;22) Translocation Qualitative by RT-PCR  0055553 BCR-ABL1 Kinase Domain Mutation Analysis  0040138 BCR-ABL1, T315I Mutation Detection, Quantitative  2004924

  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 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 10fold increase in values for PCR test Evidence lacking that use of baseline mutation analysis and subsequent monitoring improves outcome

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 complete cytogenetic response (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 transcript and determine levels to allow for monitoring of response to therapy
    • Considered standard of care for detection of minimal residual disease (MRD)
    • Quantitative testing for p210 fusion; p190 fusion rare
    • Used in determination of CCR
• 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

• Myelodysplastic syndromes
• Myeloproliferative neoplasms
  • Polycythemia vera
  • Idiopathic myelofibrosis
  • Essential thrombocythemia
• Leukemoid reactions
  • Infection
  • Pancreatitis
  • Other cancers
• Acute leukemia (AML or ALL)
• Mixed myeloproliferative/myelodysplastic syndromes
• Chronic myelomonocytic leukemia (CMML)

Monitoring

Pharmacogenetics and Therapeutic Drug Monitoring

• Mutations in the BCR-ABL1 gene lead to tyrosine kinase inhibitor (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 T3151 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 10fold 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

Clinical Background

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disease accounting for 15% of all leukemias.

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 tyrosine kinase inhibitors (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 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

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

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

Test Name and Number	Comments
CBC with Platelet Count 0040002 Method: Automated Cell Count	Initial evaluation for presence of leukocytosis
BCR-ABL, t(9;22) Translocation Qualitative by RT-PCR 0055553 Method: Reverse Transcription Polymerase Chain Reaction	Screen patients suspected of CML or acute lymphoblastic leukemia (ALL) for BCR-ABL1 transcripts Distinguish between major and minor transcripts Major transcript characterized by p210 fusion gene product is typical in CML Minor transcript characterized by p190 fusion gene product is typical in ALL Results alone should not be used for diagnosis of malignancy but correlated with blood or bone marrow findings Assay is qualitative and not intended to detect MRD or monitor patients for MRD