BCR-ABL Quantitation

BCR-ABL1 is the fusion gene created by the Philadelphia chromosome translocation t(9;22). It makes a constantly active tyrosine kinase, which drives chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. Measuring the amount of BCR-ABL1 transcript in blood is the key way of monitoring response to tyrosine kinase inhibitors.

The test is a quantitative reverse transcription PCR (RT-qPCR) on RNA from peripheral blood. The result is expressed as a ratio of BCR-ABL1 to a control gene (ABL1), converted by a laboratory specific factor to the international reporting scale (IS), so that results from different laboratories can be compared.

A baseline test at diagnosis identifies the transcript type, and regular follow up tests show the depth of response. Blood is collected in EDTA and processed within 24 to 48 hours, because RNA degrades. The result is ready in 2 to 5 days.

Most CML patients on a tyrosine kinase inhibitor reach a major molecular response, and about half reach a deep response. Patients with sustained deep response may try stopping treatment under specialist supervision, and about half of them remain in remission without drugs.

Indications.

• Diagnosis of CML, with the transcript type, in a patient with leucocytosis, splenomegaly, basophilia and a positive karyotype or fluorescence in situ hybridisation.
• Monitoring of response to treatment: every 3 months until major molecular response, then every 3 to 6 months.
• Philadelphia positive acute lymphoblastic leukaemia: diagnosis, and minimal residual disease monitoring.
• Suspected failure or loss of response, rising counts or non-adherence.
• Treatment free remission programmes, with frequent monitoring (monthly for the first 6 months).
• Assessment after stem cell transplantation.

Not indicated for screening of healthy people. A negative result in a patient with a suspected acute leukaemia does not exclude other fusions.

Specimen requirements.

• 10 to 20 mL of peripheral blood in EDTA (or an RNA stabilising tube), sent at room temperature and processed within 24 to 48 hours (some tubes allow up to 72 hours).
• Bone marrow is acceptable at diagnosis, and blood is preferred for follow up.
• The same laboratory and method are used for follow up, since the conversion to the international scale is laboratory specific.
• Clinical details: date of diagnosis, treatment, dose changes and previous results.

Normal appearances.

A normal person has no BCR-ABL1 transcript. Very low levels can be found in some healthy people with a sensitive method, which has no clinical meaning.

Interpretation.

• Result: reported as BCR-ABL1 to ABL1 percent on the international scale, with the control gene copy number and the sensitivity of the assay.
• Transcript types: e13a2 (b3a2) and e14a2 (b2a2) encode the p210 protein in CML. e1a2 encodes p190, typical of Philadelphia positive acute lymphoblastic leukaemia and rare in CML. Atypical transcripts are missed by some primers.
• Early molecular response: 10% or under at 3 months, which predicts better outcomes.
• At 6 months, under 1% (optimal), 1 to 10% (warning), and over 10% (failure).
• At 12 months, 0.1% or under is a major molecular response (a fall of 3 logs or more), 0.1 to 1% is a warning, and over 1% is failure.
• Deep molecular response: MR4 (0.01% or under), MR4.5 (0.0032% or under) and MR5. Treatment free remission needs sustained deep response, usually MR4 for 2 years or more.
• Loss of major molecular response, or a rise of 1 log or more, is confirmed with a repeat test and prompts a check of adherence and drug interactions, and mutation analysis.
• Philadelphia positive acute lymphoblastic leukaemia: results track minimal residual disease, and persistent or rising levels predict relapse.

Critical values and findings needing immediate action.

• A rising transcript level with rising white cell count, new basophilia, splenomegaly or blasts: urgent assessment for progression to accelerated or blast phase.
• Failure to reach milestones: review of adherence, dose and interacting drugs, then mutation testing of the kinase domain (including T315I) and a change of treatment.
• A positive result in a patient with acute leukaemia: start of tyrosine kinase inhibitor with chemotherapy, in a specialist unit.
• Pregnancy in a patient on treatment: urgent counselling, since several drugs harm the foetus.

Limitations and artefacts.

• Variability between laboratories: the conversion to the international scale and the quality control of each laboratory affect results, so serial values come from the same laboratory.
• RNA degradation from delayed or poor sample handling gives falsely low values.
• Variation of 0.5 log (about 3-fold) is within the analytical noise, so a single small rise is not acted upon without repeat testing.
• The test has limited sensitivity at very low levels, and detection of MR4.5 needs a high quality assay and enough control gene copies.
• Atypical transcripts and rare fusions may be missed by routine primers.
• Non-adherence, malabsorption and interacting drugs (enzyme inducers, acid reducing drugs for some inhibitors) are common reasons for rising levels.
• Transcript levels do not show clonal changes (additional chromosomal abnormalities), which are checked by karyotype.
• Blood levels may not reflect marrow disease at an early stage of relapse.

Special groups.

• Pregnant women and those planning pregnancy: most tyrosine kinase inhibitors are harmful to the foetus, and treatment plans are individual.
• Children: growth effects of tyrosine kinase inhibitors, and long term monitoring.
• Older adults: drug interactions and cardiovascular risk influence the choice of drug.
• Patients in treatment free remission: frequent monitoring and quick restart on loss of response.
• Patients after stem cell transplantation: monitoring of relapse.
• Regions: generic imatinib has made treatment widely affordable, but molecular monitoring is limited. Where RT-qPCR is not available, blood counts, spleen size, and cytogenetic or fluorescence in situ hybridisation tests are used, and testing in a regional reference laboratory with shipped blood samples is arranged. Treatment interruptions from drug stock problems are a major cause of failure.

Follow up tests.

• Kinase domain mutation analysis for failure or warning responses.
• Karyotype or fluorescence in situ hybridisation for additional chromosomal abnormalities.
• Full blood count with differential, and bone marrow examination at diagnosis and with suspected progression.
• Liver function, lipids, glucose and cardiovascular risk assessment on treatment.
• Pharmacy and adherence review, and drug level measurement where available.
• HLA typing and transplant review for advanced disease.
• Specialist haematology review.