Abstract
PurposeApproximately 1–2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients.MethodsBCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR).ResultsIn total, 330 blood samples (2–34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios.ConclusionsCharacterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.
Highlights
Materials and methodsChronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph), produced by the balanced reciprocal translocation t(9;22)(q34;q11) leading to the breakpoint cluster region-Abelson (BCR-ABL1) fusion gene (Hehlmann et al 2007)
Since the levels of disease for these cases cannot be expressed on the International Scale (IS), we suggest a new evaluation criterion for molecular monitoring of these patients—the individual molecular response (IMR) level based on a log reduction from pretreatment levels
The small fusion transcript e13a3 with a product size of 128 bp is found in lane 5 of the agarose gel and one sample with the BCR-ABL1 double transcript e13a3 and e14a3 is shown in lane 6
Summary
Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph), produced by the balanced reciprocal translocation t(9;22)(q34;q11) leading to the breakpoint cluster region-Abelson (BCR-ABL1) fusion gene (Hehlmann et al 2007). Each 20 μL reaction mix contained 4 μL LightCycler-FastStart DNA MasterPLUS HybProbe master mix (Roche Diagnostics), 2 μL cDNA template or plasmid dilution, 0.5 μM forward primer (specific for atypical BCR-ABL1 transcript; Table 2), 0.5 μM reverse primer (ABL1 primer NA4-), 0.25 μM of each hybridization probe listed in Table 5 (TIB Molbiol, Berlin, Germany) and 1 μL Uracil-DNA-Glycosidase (UDG; New England Biolabs GmbH, Frankfurt/Main, Germany).
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