Molecular Diagnostics in Chronic Myeloid and Acute Myeloid Leukemias

Detection of BCR-ABL Fusion Gene Transcripts

The presence of the characteristic Philadelphia (Ph) chromosome due to a reciprocal translocation between chromosome 9 and 22 { t(9;22)(q34;q11) } is the hallmark cytogenetic event in about 90% of the cases of chronic myeloid leukemia (CML). Another 5% of cases of CML show cytogenetic variants of the classic Ph chromosome. About one half of the remaining CML patients are Ph chromosome negative but do have cryptic BCR-ABL gene fusions detectable on molecular analysis. Hence, only about 2.5% of CML patients are negative for both the Ph chromosome and the BCR-ABL fusion gene. At the molecular level, the translocation involves the disruption of both the normal BCR ("breakpoint cluster region") gene on chromosome 22 and the ABL (Abelson oncogene) gene on chromosome 9. The resulting translocation chromosomes each carry fusion genes which are often functional; a BCR-ABL fusion gene whose gene product has transforming abilities and an ABL-BCR fusion gene that is also expressed. The BCR-ABL fusion gene products have been characterized as a 210 kD (p210) protein. This gene product is characteristic of CML and typically results from the fusion of either BCR exon b2 or BCR exon b3 to ABL exon a2, a b3a2 or a b2a2 fusion, respectively. A significant proportion (5 -20%) of cases of acute myeloid leukemia (ALL) also carry the Philadelphia chromosome. In about 70% of these Ph+ ALL cases, a BCR-ABL fusion gene is found involving BCR exon e1 and ABL exon a2, an e1a2 fusion, whose gene product is a 190 kD (p190) protein. The remaining 30% of Ph+ ALL cases carry the p210 gene fusions as found in CML.

Southern blot analysis can be used to evaluate the genomic structure of these rearrangements. However, this methodology is time-consuming and, in comparison to PCR-based methods, insensitive. A variety of PCR-based methods permitting the detection of the transcripts produced by the characteristic BCR-ABL gene fusions have been developed (RT-PCR assays). The method used by Celtek is based on that described by Cross et al (1994; Leukemia 8:186-189) and is diagrammed in the figure below. The arrows indicate the relative positions of various PCR primers used in the assay. The upper section of the figure shows the 808 bp PCR product generated from the normal BCR gene. This internal control PCR product provides a direct evaluation of the quality of the cDNA synthesis step critical to the success of the procedure. The section of the figure below shows the possible PCR products that will be detected if a BCR-ABL fusion gene transcript is present in the total RNA isolated from the patient's peripheral blood or bone marrow sample. These PCR products include the p210 b3a2 fusion, the p210 b2a2 fusion, and the p190 e1a2 fusion.

The figure below shows samples of RT-PCR results for two different CML patients. Lane 1 contains DNA size standards while lane 2 contains the PCR products that result from a no cDNA control. The RT-PCR products shown in lanes 3 - 5 are from, respectively, a CML patient who had the classic Ph+ chromosome and has the BCR-ABL b2a2 fusion, a normal individual having only the BCR product, and a patient who carried an unusual cytogenetic variant Ph chromosome 46,XX,t(9;22;22)(q34;q11.2;q13) and has the BCR-ABL b3a2 fusion.