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
|
Cytogenetic Analysis (Bone Marrow)
FISH
Quantitative PCR (measures BCR-ABL transcript levels)
|
Prior to complete cytological response (CCR)
ARUP Tests
|
Cytogenetic Analysis (Bone Marrow)
Quantitative PCR (measures BCR-ABL transcript levels)
Imatinib (therapeutic drug monitoring)
|
Confirming CCR
ARUP Tests
|
Cytogenetic Analysis (Bone Marrow)
FISH
Quantitative PCR (measures BCR-ABL transcript levels)
|
Monitoring CCR
ARUP Tests
|
Cytogenetic Analysis (Bone Marrow)
FISH
Quantitative PCR (measures BCR-ABL transcript levels)
Imatinib (therapeutic drug monitoring)
|
Consideration of major change in therapy
ARUP Tests
|
Cytogenetic Analysis (Bone Marrow)
Quantitative PCR (measures BCR-ABL transcript levels)
Mutational Studies (detects BCR-ABL KD mutations)
Imatinib (therapeutic drug monitoring)
|
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
- White blood cell count shows leukocytosis
- 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
- Identify BCR-ABL1 fusion transcripts – allow for monitoring of response to therapy
- 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
- 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
- Should be reported in standardized format (%) on the BCR-ABL1 international scale (IS)
- Quantitative real-time PCR for BCR-ABL1 t(9;22) (molecular) – p210 fusion most common; p190 and p230 fusions are rare
- 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
- Patients being treated with the TKI imatinib are monitored for the following reasons
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
- T3151 mutation resistant to most TKIs – indicates poor prognosis
- Amplification or overexpression
- Quiescent CML stem cells
- Low bioavailability
- Kinase domain mutations
- 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
- Perform testing for BCR-ABL1 kinase domain mutations
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)
- t(9;22) translocation causes a fusion of the ABL1 gene and BCR gene
- 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
- Resistance to initial therapy may require increased dosing or use of a different TKI
- 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
- Standard of care – treatment with TKI, including imatinib, nilotinib, or dasatinib
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
- Chronic phase
ARUP Laboratory Tests
Recommended when submitting initial diagnostic sample for CML (no previous BCR-ABL1 testing)
If the presence of either the p210 or p190 BCR-ABL1 fusion is detected, then the appropriate quantitative test will be performed
Reverse Transcription Polymerase Chain Reaction
Aids the monitoring of individuals with CML who have e13a2 or e14a2 transcripts (p210)
Does not detect p190 or p230
Reverse Transcription Polymerase Chain Reaction
Aid in diagnosis and monitoring of individuals with CML who have e1a2 transcripts (p190)
Does not detect the p210 or p230 form
Quantitative Reverse Transcription Polymerase Chain Reaction
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)
Massively Parallel Sequencing
Differentially diagnose cases of neutrophilia, including CML and leukemoid reaction
Helpful first-line screening test; however, does not definitively diagnose CML
Cytochemical Stain
Diagnose, prognose, and monitor hematopoietic neoplasms
Giemsa Band
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
Fluorescence in situ Hybridization (FISH)
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
Immunoturbidimetry
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
Fluorescence in situ Hybridization (FISH)
References
19959094
Apperley J. CML in pregnancy and childhood. Best Pract Res Clin Haematol. 2009;22(3):455-474.
23803709
Baccarani M, Deininger MW, Rosti G, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood. 2013;122(6):872-884.
22997458
Baccarani M, Pileri S, Steegmann JL, et al. Chronic myeloid leukemia: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2012;23 Suppl 7:vii72-vii77.
20960522
Cortes J, Quintás-Cardama A, Kantarjian HM. Monitoring molecular response in chronic myeloid leukemia. Cancer. 2011;117(6):1113-1122.
22160024
Hochhaus A. Educational session: managing chronic myeloid leukemia as a chronic disease. Hematology Am Soc Hematol Educ Program. 2011;2011:128-35.
20008233
Hughes TP, Branford S. Monitoring disease response to tyrosine kinase inhibitor therapy in CML. Hematology Am Soc Hematol Educ Program. 2009;477-487.
18055868
Kantarjian H, Schiffer C, Jones D, et al. Monitoring the response and course of chronic myeloid leukemia in the modern era of BCR-ABL tyrosine kinase inhibitors: practical advice on the use and interpretation of monitoring methods. Blood. 2008;111(4):1774-1780.
Practical Diagnosis of Hematologic Disorders - Chronic Myelogenous Leukemia and Related Disorders
Malley D, Vardiman J. Chronic myelogenous leukemia and related disorders. In: Kjeldsberg C, ed. Practical Diagnosis of Hematologic Disorders. 5th ed. American Society for Clinical Pathology; 2006.
NCCN - Chronic Myelogenous Leukemia
National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: chronic myelogenous leukemia. [Accessed: Aug 2018]
CAP - ALL, Bone Marrow
Protocol for the examination of specimens from patients with hematopoietic neoplasms involving the bone marrow. Based on AJCC/UICC TNM, 7th ed. College of American Pathologists (CAP). [Revised: Jun 2012; Accessed: Apr 2018]
Blood - Personalized daily doses of imatinib by therapeutic drug monitoring increase the rates of molecular responses in patients with chronic myeloid leukemia.
Rousselot P, Johnson-Ansah H, Huguet F, et al. Personalized daily doses of imatinib by therapeutic drug monitoring increase the rates of molecular responses in patients with chronic myeloid leukemia. Final results of the randomized OPTIM imatinib study [abstract]. Blood. [Accessed: May 2018]
22223872
Saglio G, Fava C. Practical monitoring of chronic myelogenous leukemia: when to change treatment. J Natl Compr Canc Netw. 2012;10(1):121-129.
21562040
Soverini S, Hochhaus A, Nicolini FE, et al. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. Blood. 2011;118(5):1208-1215.
21419537
Vigil CE, Griffiths EA, Wang ES, et al. Interpretation of cytogenetic and molecular results in patients treated for CML. Blood Rev. 2011;25(3):139-146.
21881536
Yeung DT, Parker WT, Branford S. Molecular methods in diagnosis and monitoring of haematological malignancies. Pathology. 2011;43(6):566-579.
Medical Experts
Lamb

McMillin

Patel
