BCR/ABL1

**Overview of the BCR/ABL1 Fusion Gene**

Definition: The BCR-ABL1 fusion gene is an abnormal gene formed by a reciprocal translocation between chromosomes 9 and 22, denoted as t(9;22)(q34.1;q11.2)
Historical Context: This translocation results in the formation of the Philadelphia chromosome (Ph chromosome), named after the city where it was discovered
Significance:
- Diagnostic Marker for CML: The presence of BCR-ABL1 is the defining characteristic of Chronic Myeloid Leukemia (CML)
- Prognostic Marker: In CML, the level of BCR-ABL1 transcript is used to monitor treatment response and predict the risk of disease progression
- Diagnostic and Prognostic Marker for ALL: Present in some cases of Acute Lymphoblastic Leukemia (ALL), particularly adult B-ALL, and is associated with a poorer prognosis
- Target for Therapy: The BCR-ABL1 fusion protein is a tyrosine kinase, making it a target for specific tyrosine kinase inhibitors (TKIs)

**Molecular Basis of the BCR-ABL1 Fusion Gene**

Normal Genes:
- ABL1 (Abelson murine leukemia viral oncogene homolog 1): Located on chromosome 9 (9q34.1)
  - Encodes a non-receptor tyrosine kinase involved in cell growth, differentiation, and survival
- BCR (Breakpoint Cluster Region): Located on chromosome 22 (22q11.2)
  - Encodes a serine/threonine kinase involved in signal transduction
Translocation Process:
- A reciprocal translocation occurs between chromosomes 9 and 22:
  - Part of the ABL1 gene from chromosome 9 translocates to the BCR gene on chromosome 22
  - Part of the BCR gene from chromosome 22 translocates to chromosome 9
Resulting Fusion Gene:
- The translocation creates a new fusion gene, BCR-ABL1, on the Philadelphia chromosome (derivative chromosome 22)
- The BCR-ABL1 fusion gene encodes a 210 kDa protein that has constitutive tyrosine kinase activity

Pathophysiology

Constitutive Tyrosine Kinase Activity:
- The BCR-ABL1 fusion protein has uncontrolled tyrosine kinase activity, meaning it is always “switched on”
- This unregulated kinase activity leads to:
  - Uncontrolled cell proliferation
  - Inhibition of apoptosis (programmed cell death)
  - Genomic instability
- These effects result in the clonal expansion of malignant myeloid cells (in CML) or lymphoid cells (in Ph+ ALL)
Downstream Signaling Pathways:
- BCR-ABL1 activates multiple downstream signaling pathways, including:
  - RAS/MAPK pathway
  - PI3K/AKT pathway
  - JAK/STAT pathway
- These pathways regulate cell growth, survival, and differentiation

Laboratory Detection of BCR-ABL1

Cytogenetic Analysis (Karyotyping):
- Principle: Visualizes the Philadelphia chromosome (Ph chromosome) resulting from the t(9;22) translocation
- Procedure:
  - Cells are cultured, arrested in metaphase, and stained to visualize the chromosomes
  - Karyotype is analyzed to identify the Ph chromosome
- Reporting:
  - t(9;22)(q34.1;q11.2)
- Advantages:
  - Can detect other chromosomal abnormalities in addition to the Ph chromosome
- Limitations:
  - Requires viable cells and cell culture
  - Time-consuming
  - Cannot detect cryptic translocations or variant translocations
Fluorescence In Situ Hybridization (FISH):
- Principle: Uses fluorescently labeled DNA probes to detect the BCR and ABL1 genes and the BCR-ABL1 fusion gene
- Procedure:
  - Interphase FISH: Performed on uncultured bone marrow cells
  - Metaphase FISH: Performed on cultured cells
  - Hybridization: Fluorescent probes are hybridized to the chromosomes on a slide
  - Microscopic Examination: The slide is examined under a fluorescence microscope, and the number and location of the fluorescent signals are counted
- Advantages:
  - More rapid than karyotyping
  - Can be performed on non-dividing cells
  - Can detect cryptic translocations
- Limitations:
  - Only detects known chromosomal abnormalities for which probes are available
  - Cannot detect novel translocations or complex rearrangements
Reverse Transcription Polymerase Chain Reaction (RT-PCR):
- Principle: Amplifies and detects the BCR-ABL1 fusion transcript (mRNA)
- Procedure:
  1. RNA Extraction: Extract RNA from bone marrow aspirate or peripheral blood
  2. Reverse Transcription: Convert RNA to complementary DNA (cDNA) using reverse transcriptase
  3. Amplification: Use PCR to amplify the BCR-ABL1 fusion transcript using specific primers
  4. Detection: Detect the amplified DNA product using gel electrophoresis or real-time PCR
- Qualitative RT-PCR:
  - Detects the presence or absence of the BCR-ABL1 transcript
  - Used for initial diagnosis
- Quantitative Real-Time PCR (RQ-PCR):
  - Measures the level of BCR-ABL1 transcript
  - Used for monitoring minimal residual disease (MRD) and assessing treatment response
  - Results are typically reported as a percentage of BCR-ABL1 transcript relative to a control gene (e.g., ABL1 or GUS)
  - The International Scale (IS) is used to standardize BCR-ABL1 transcript levels across different laboratories
- Advantages:
  - High sensitivity and specificity
  - Can quantify the amount of BCR-ABL1 transcript
  - Rapid turnaround time
- Limitations:
  - Only detects known fusion transcripts for which primers are available
  - Requires specialized equipment and expertise
  - Susceptible to contamination
Next-Generation Sequencing (NGS):
- Principle: Massively parallel sequencing technology that can detect the BCR-ABL1 fusion gene and other mutations
- Advantages:
  - Can detect both known and novel fusion genes and mutations
  - Can identify co-occurring mutations that may affect prognosis or treatment response
- Limitations:
  - More complex and expensive than PCR-based methods
  - Requires bioinformatics expertise for data analysis

Clinical Interpretation

CML Diagnosis:
- A diagnosis of CML requires the presence of the BCR-ABL1 fusion gene or the Philadelphia chromosome
- The BCR-ABL1 transcript level is used to monitor treatment response
CML Response to TKI Therapy:
- Hematologic Response: Normalization of blood counts
- Cytogenetic Response: Reduction or elimination of the Ph chromosome
- Molecular Response: Reduction in BCR-ABL1 transcript levels, as measured by RQ-PCR
  - Major Molecular Response (MMR): BCR-ABL1 transcript level ≤ 0.1% on the International Scale (IS)
  - Deep Molecular Response (MR4, MR4.5): Very low or undetectable BCR-ABL1 transcript levels
Prognosis:
- The level of BCR-ABL1 transcript is a key prognostic factor in CML
- Patients who achieve a deep molecular response have a lower risk of disease progression
Resistance to TKI Therapy:
- Mutations in the ABL1 kinase domain can lead to resistance to tyrosine kinase inhibitors (TKIs)
- ABL1 kinase domain mutation testing is performed to identify resistance mutations and guide treatment decisions

Key Terms

BCR-ABL1* Fusion Gene: A fusion gene created by the translocation between chromosomes 9 and 22, resulting in the Philadelphia chromosome
Philadelphia Chromosome (Ph Chromosome): The derivative chromosome 22 that contains the BCR-ABL1 fusion gene
Translocation: The transfer of genetic material from one chromosome to another
Tyrosine Kinase Inhibitor (TKI): A drug that inhibits the activity of tyrosine kinases, such as the BCR-ABL1 protein
Minimal Residual Disease (MRD): Small numbers of residual cancer cells that remain after treatment
Major Molecular Response (MMR): A significant reduction in BCR-ABL1 transcript level
Deep Molecular Response: Very low or undetectable BCR-ABL1 transcript levels
ABL1 Kinase Domain Mutation: Mutations in the ABL1 kinase domain that can cause resistance to TKIs
FISH (Fluorescence In Situ Hybridization): Uses fluorescent probes to identify certain mutations in the body

Recurring Abnormalities

JAK2