Technical Notes

BCR/ABL translocation t (9; 22) for diagnosis

Clinical Significance:

The diagnosis of CML is based on the presence of the BCR/ABL gene or Ph chromosome. A majority (90%) of patients with CML have a characteristic reciprocal translocation of DNA between chromosomes 9 and 22, termed the Philadelphia chromosome (Ph'), which can be identified using standard cytogenetic techniques ⁽¹⁾. Approximately 10% of patients with the clinical and hematological features of CML do not have a demonstrable Ph' chromosome by cytogenetic analysis; however, a majority of these patients carry a BCR-ABL rearrangement that is detectable by molecular techniques ⁽²⁾.

As a result of the t (9; 22), the proto-oncogene c-ABL, normally present on chromosome 9, is translocated to chromosome 22 where it is fused with a gene designated BCR (for "breakpoint cluster region"). The resultant fusion gene directs expression of a novel mRNA species (BCR-ABL). Depending on the breakpoint, different forms of BCR-ABL mRNA, and therefore protein, are generated. In CML, the most common breakpoint results in a protein termed p210 BCR-ABL that has low-level transforming ability and results in a relatively indolent clinical course. In Ph'-positive ALL, the breakpoint in BCR gene occurs upstream of the CML breakpoint, resulting in a protein termed p190 BCR-ABL. This protein has powerful transforming activity, resulting in acute leukemia. The mRNAs encoding these two forms of BCR-ABL can be distinguished by a Reverse Transcriptase PCR assay ⁽³⁾.

The BCR/ABL gene is not exclusive to CML and it is also present in a minority of some other oncohematological diseases, e.g. in about 25% of adult and 5% of children acute lymphoid leukemia (ALL), 1% acute myeloid leukemia (AML) and very rarely in lymphoma, myeloma or myelodysplastic syndromes ⁽⁴⁾.

The presence of the BCR/ABL gene does not have a diagnostic significance in these diseases but, at least in ALL, it has a prognostic importance, i.e. it is a negative prognostic factor ⁽⁵⁾.

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Methodology:

Patient RNA would be isolated, purified, and converted to cDNA by the enzyme reverse transcriptase. The cDNA would be detected by a nested PCR reaction. An additional RT-PCR amplification directed at ABL gene segment would be performed as a control for sample RNA and reverse transcription quality. PCR products would be analyzed by electrophoresis and UV transillumination of ethidium bromide-stained gels.

The assay has been validated by a positive control reaction using RNA from a myelogenous leukemia cell line and from RNA of confirmed CML patients.

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References:

1) Sawyers C. (1999) N. Engl. J. Med. 340, 1330-1340.
2) Cross N.C.P., Lin F., Chase A. et al (1993) Blood 82, 1929-1936.
3) Moravcova J., Nadvornikova S. (1999) Blood 94, 3809-3811.
4) Melo J.V. (1996) Blood 88, 2375-2384.
5) Preudhomamme C., Henic N., Cazin B. et al (1997) Leukemia 11, 294-297.