What is a BCR-ABL (Breakpoint Cluster Region-Abelson) test?

What is a BCR-ABL Test?

The BCR-ABL test is a molecular diagnostic assay that detects and quantifies the BCR-ABL1 fusion gene—the hallmark genetic abnormality of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). 1

Biological Basis

The BCR-ABL1 fusion gene results from a reciprocal translocation between chromosomes 9 and 22, t(9;22), known as the Philadelphia chromosome. 1 This translocation creates an abnormal fusion gene that produces a protein with deregulated tyrosine kinase activity, which drives the development of CML. 1

Common Transcript Types

• p210 protein (e13a2/e14a2 transcripts): Found in >90% of CML patients, with breakpoints in the major breakpoint cluster region (M-BCR) of the BCR gene. 1
• p190 protein (e1a2 transcript): Typically associated with Ph+ ALL, found in only 1% of CML cases, involving the minor breakpoint cluster region (m-BCR). 1
• p230 protein (e19a2 transcript): Rare variant involving the micro breakpoint cluster region (μ-BCR), associated with enhanced neutrophil differentiation. 1
• Atypical transcripts (e.g., e13a3, e14a3, e6a2): Detected in approximately 1-2% of CML patients. 1

Testing Methodologies

Quantitative RT-PCR (RT-qPCR)

RT-qPCR is the most sensitive assay available for measuring BCR-ABL1 mRNA, capable of detecting one CML cell in a background of 100,000 or more normal cells. 1

Key features include:

• Sample source: Can use peripheral blood or bone marrow, with strong correlation between results from both sources, eliminating the need for routine bone marrow aspirations. 1
• Results expression: Reported as the ratio of BCR-ABL1 transcript numbers to control gene transcripts (BCR, ABL, or GUSB). 1
• International Scale (IS) standardization: Results are converted to the IS, where 100% represents the standardized baseline at diagnosis, and major molecular response (MMR) is defined as 0.1% IS (3-log reduction). 1

RT-Digital PCR (RT-dPCR)

RT-dPCR is an acceptable alternative to RT-qPCR and may offer technical advantages, particularly for assessing very low-level disease (deep molecular response). 1

Qualitative RT-PCR

This assay reports results as simply positive or negative and is rarely used for monitoring purposes. 1

Cytogenetics and FISH

• Conventional cytogenetics: Detects the Philadelphia chromosome and additional chromosomal abnormalities. 1
• FISH (Fluorescence In Situ Hybridization): Can detect BCR-ABL1 fusion genes in both major and minor breakpoint cluster regions, but has very limited sensitivity compared to molecular monitoring and is not recommended for routine disease monitoring. 1, 2

Clinical Applications

Diagnosis

• Initial workup: Bone marrow aspirate with RT-PCR should be performed to establish the presence of quantifiable BCR-ABL1 mRNA transcripts at baseline before initiating tyrosine kinase inhibitor (TKI) therapy. 1
• Transcript identification: Determining the exact transcript type (e13a2, e14a2, or atypical variants) is essential before starting treatment to facilitate accurate monitoring. 1

Monitoring Treatment Response

RT-qPCR is the only tool capable of monitoring responses after patients achieve complete cytogenetic response (CCyR), as BCR-ABL1 mRNA transcripts typically remain detectable even after CCyR. 1

Monitoring schedule per NCCN guidelines:

• Baseline: Before TKI initiation. 1
• During treatment: Regular intervals to assess molecular response milestones (e.g., BCR-ABL1 ≤10% IS at 3 months, ≤1% IS at 6 months, ≤0.1% IS for MMR). 1
• After treatment discontinuation: Monthly for one year, then every 6 weeks for the second year, and every 12 weeks thereafter. 3

Mutation Analysis

BCR-ABL1 kinase domain mutation testing should be performed when patients show suboptimal response or rising transcript levels, as point mutations are a frequent mechanism of TKI resistance. 1

• Next-generation sequencing (NGS) is preferred over Sanger sequencing due to higher sensitivity (3% vs. 20% threshold), allowing early detection of resistance-mediating mutations. 1
• Specific mutations (e.g., T315I, F317L, Y253H, E255K) confer resistance to different TKIs and guide selection of subsequent therapy. 1

Critical Considerations

Atypical Transcript Detection

Laboratories using RT-PCR as the primary screening method must employ methodologies that detect both typical and atypical BCR-ABL1 variants, as screening only for p210 or p210 plus p190 may result in up to 2% of CML cases being misdiagnosed. 1

Standardization Requirements

• Tests should achieve a limit of detection of MR4.5 (≤0.0032% IS) or better for assessing deep molecular response. 1
• Laboratory-specific conversion factors must be established through sample exchange with reference laboratories to report results on the International Scale. 1
• The same FDA-authorized test should be used consistently to monitor molecular response levels while on and off treatment. 3

Quality Assurance

Ongoing internal quality control is essential to monitor assay stability over time, and laboratories should participate in external quality assessment programs. 1