BCR-ABL1 Gene Rearrangement, Quantitative PCR

For new patients, Quest Diagnostics will test for both the P210 isoform (e13a2 and e14a2 transcripts) and the P190 isoform (e1a2 fusion). Once a BCR-ABL1 fusion is detected, subsequent samples from the patient will be tested for the indicated isoform(s) only.

BCR-ABL1 fusion testing can be performed using either peripheral blood or bone marrow aspirate samples. While lavender-top EDTA collection tubes are preferred, yellow-top citrate or green-top heparin tubes will be accepted.

The level of the BCR-ABL1 fusion transcript is expressed as a percent of the ABL1 gene transcript. For standardization purposes, the P210 transcript ratio is further normalized to the international scale (IS) and reported as BCR-ABL1/ABL1%. The IS provides a standardized correction factor that enables direct comparison of results from different laboratories, regardless of differences in methodology.^1,2 No IS currently exists for the P190 isoform, so P190-positive cases are reported as BCR-ABL/ABL1% with no further normalization.

To assess molecular response to therapy, results are compared against a “universal baseline” BCR-ABL1 ratio. A value of 100% indicates no response. Values of 10%, 1%, 0.1%, 0.01%, 0.0032%, and 0.001% correspond to decreases of 1, 2, 3, 4, 4.5, and 5 logs, respectively, relative to the universal baseline of 100%. A BCR-ABL1 expression of ≤0.1% corresponds to a major molecular response (MMR).³

The test is designed to routinely detect a minimum of 4.5 log reduction of the p210 and p190 isoforms from the baseline levels (BCR-ABL to ABL ratio of 0.0032%). However, the sensitivity is eventually dependent on the quality and quantity of RNA specimens tested.

Cases that are PCR-positive and FISH-negative are common, since FISH is a less sensitive technique. However, approximately 1% to 3% of CML and Ph-positive ALL cases will have an altered fusion transcript (usually missing exon A2 of ABL1) that cannot be detected using this PCR assay. In those cases, FISH can be used to monitor patients. FISH can also be used to monitor patients with the very rare P230 BCR-ABL1 isoform, which is not reliably detected by this PCR assay.

Lack of initial response to tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (Gleevec^®), is called primary resistance. Primary resistance is most commonly related to inadequate drug levels or pharmacodynamic factors, although not all causes are known.

The most frequent cause of secondary resistance to TKIs is a mutation in the ABL1 kinase domain. Such mutations can be detected using the ABL kinase domain mutation test (test code 16029). Another cause of secondary resistance is acquisition of additional cytogenetic changes; these can be detected using a bone marrow aspirate chromosome study (test code 14600).

Extra copies of the Ph chromosome can mediate both primary and secondary partial TKI resistance. Extra copies can be detected using the BCR-ABL1 FISH test (test code 12070).

Therapeutic response to initial treatment is indicated by a drop in the BCR-ABL1 level. A subsequent 5-to 10-fold increase in the % (IS) level suggests developing drug resistance, which is usually due to an ABL1 mutation or clonal evolution. Other indicators of secondary resistance include overt hematologic or cytogenetic relapse and emergence of blast phase.

For patients in the first year of treatment, a repeat PCR test is indicated to confirm the increase in BCR-ABL1 transcript prior to a drug-resistance workup.

When a logarithmic increase in BCR-ABL1 level is confirmed by repeat testing, resistance testing can be performed using the ABL Kinase Sequencing Test (test code 16029).