BCR/ABL Translocation Probe Dual Fusion

The presence of the Philadelphia chromosome (Ph') has important diagnostic and prognostic implications in a number of Haematological disorders. The abnormality is characteristic of Chronic Myeloid Leukaemia (CML), found in around 90% of cases but also represents a significant abnormality in 30% of adult and 2 to 10% of childhood Acute Lymphoblastic Leukaemia, (ALL) cases. This rearrangement is also seen in rare cases of Acute Myelogenous Leukemia (AML).

As a result of the Philadelphia translocation, t(9;22)(q34;q11), The 3' sequences of the ABL(Abelson) proto-oncogene at 9q34 are joined to the 5' sequences of the BCR (Breakpoint cluster region) gene at 22q11, giving rise to the BCR/ABL hybrid or ‘fusion’ gene. Prakash and Yunis (1984) located the breakpoints in CML to subbands 22q11.21 and 9q34.1. Although the position of the breakpoint in chromosome 9 is quite variable, almost all breaks are located within a 200 kb region covering exons 1b to 1a. In BCR there are two breakpoint regions, m-BCR and MBCR. In CML most translocations involve the Major Breakpoint Cluster Region (M-BCR) located approximately between exons 12 to 16. In ALL most fall in the Minor Breakpoint Cluster Region (m-BCR) located between exons 1 and 2 though the resulting translocation is cytogenetically identical to that of CML.

These alternative breakpoints join different exon sets of BCR to a common subset of the exons of the ABL resulting in 2 alternative chimeric oncogene products, p210(BCR-ABL) and p185(BCRABL). Both of these fusion proteins possess enhanced tyrosine kinase activity, through the activation of ABL tyrosine kinase activity by the direct physical binding of sequences within the first exon of BCR. It is thought that this kinase activity is necessary for the oncogenic potential of the chimeric oncogene.

To block BCR/ABL function, Lim et al. (2000) created a unique tyrosine phosphatase which binds to BCR/ABL in cells and effectively suppressed BCR/ABL function. In ALL, the rearrangement associated with an extremely poor outcome, with an event-free survival (EFS) of 15% or less at 5 years in adult and childhood patients treated with chemotherapy alone. There are no reports of long-term survivors. Allogeneic bone marrow transplantation is the only curative therapy for these patients. Children with this rearrangement are treated on a high-risk protocol and adults are recommended for immediate bone marrow transplantation. Philadelphia (Ph) chromosome-positive Acute Myeloid Leukemia (AML) is characterized by its resistance to conventional standard chemotherapy and poor prognosis. Accurate and rapid identification of this chromosomal abnormality is therefore vital.

In a small number of cases of ALL, the translocation does not result in a cytogenetically visible Philadelphia chromosome. In these cases, FISH is essential for highlighting the fusion gene.

The BCR probe mix contains a probe 3’ of BCR covering a region extending 171 kb 3’, covering the genes GNAZ and RAB36 and a second probe covering a region 262 kb 5’ of the gene extending 148 kb. Both are labelled in green and are orientated such that breakpoints in either mBCR or MBCR will result in a fusion signal. For ABL a probe contig. covers 349 kb from the middle of the FUB3 gene to a point 64 kb from the 5’ end of ABL and it is labelled in red, whilst there is an additional red probe covering a region 212 kb 3’ of ABL, incorporating the ASS gene. This additional probe is 193 kb long and spans the whole of the ASS gene.