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BCR-ABL1 DNA monitoring of CML
BCR-ABL1 DNA values were significantly higher than the corresponding BCR-ABL1IS values. After three months, the reduction in BCR-ABL1IS levels (2.05-log) was primarily due to depletion of CML cells (1.75-log), with only a small contribution from expression changes (0.3-log reduction; 2-fold decrease). A proportionally greater decline in expression than in cell number is likely due to the early depletion of higher expressing cells. From six months of treatment onwards, there was generally excellent agree- ment between the level of MRD measured by BCR-ABL1 DNA and by RQ-PCR, indicating that the decline in BCR-ABL1IS is closely paralleled by declining numbers of BCR-ABL1-positive cells, as has been predicted using mathematical models based on RQ-PCR data.21,22
Several studies dating back at least two decades have reported inferior treatment responses among CML patients with e13a2 BCR-ABL1 transcripts.17,23 Differences in molecularly-defined end points might simply reflect dif- fering amplification efficiency in the BCR-ABL1 assay, especially in those systems that use a common forward primer in BCR e13, resulting in a 76 bp difference in ampli- con length between the two transcripts.24 In BCR-ABL1 DNA PCR, every patient-specific assay will have differing properties due to varying amplicon size and nucleotide composition. These differences are determined by factors related to the precise intronic location of the breakpoints, and therefore independent of the transcript type (Online Supplementary Figure S3). We took advantage of this to compare the relative expression of e13a2 and e14a2 BCR-ABL1 transcripts by normalizing the BCR-ABL1IS value against BCR-ABL1 DNA. Patients expressing both e13a2 and e14a2 transcripts were excluded from this analysis; in those cases the genomic BCR breakpoint is after exon 14, so e14a2 is the dominant transcript with a fraction of e13a2 expressed due to alternative splicing.25 Despite the small number of patients with each transcript type, we were able to show a significant difference in expression per cell at multiple time points during treat- ment. These findings require independent confirmation in a larger cohort. When treatment decisions are made according to molecular landmark responses, this may lead to incorrect classification of some e13a2 patients as opti- mal responders, and could contribute to adverse out- comes. Intriguingly, we identified 3 e13a2 patients who at diagnosis had discordant low BCR-ABL1 mRNA values (<10%) despite having close to 100% BCR-ABL1I-positive cells by DNA PCR and metaphase karyotyping. All 3 of these patients experienced treatment failure or warning by ELN criteria. The significance of this finding is unclear, given the small number of patients in this subgroup. It is, however, consistent with the experimental observation that imatinib sensitivity was reduced in BCR-ABL1-trans-
duced murine cells selected for low BCR-ABL1 expression.26
The median limit of detection of BCR-ABL1 DNA was MR5.2 versus MR4.6 for conventional RQ-PCR. This improvement in sensitivity led to around half of the sam- ples with undetectable BCR-ABL1 mRNA having measur- able MRD and extended the period of time in which there was detectable BCR-ABL1 by around a year. The median limit of detection for dPCR was MR5.2. These results are similar to those obtained by Alikian et al., who used dPCR for both BCR-ABL1 DNA and mRNA, and found higher sensitivity with the DNA-based assay.6 Whilst the com- parison of PCR methods was not the aim of this study, we found that dPCR was more precise, especially at diagno- sis, but the Fluidigm® system has the disadvantage that more than 80% of the input DNA is lost in the dead space of the microfluidic circuit.
More sensitive mRNA-based methods have also been developed,6,17,27 and comparisons using different technolo- gy may yield different results. Nevertheless, genomic DNA-based methods have the advantage of greater speci- ficity since cross-contamination between samples from different patients cannot cause false-positive results when the assays are patient-specific. A previous study showed that some patients with undetectable BCR-ABL1 mRNA by RQ-PCR had MRD detected by dPCR, and that these patients had a lower probability of successful treatment- free remission (TFR) after imatinib discontinuation.27 In the ENESTfreedom study of discontinuation of first-line nilotinib, patients with MR4.5 on every measurement for 12 months prior to stopping nilotinib were more likely to maintain TFR at 12 months than patients with one or more results above the MR4.0 threshold.28 More sensitive PCR methods might, therefore, have clinical utility as a means of refining estimates of the probability of TFR.
Miminal residual disease measurement by genomic DNA PCR provides insights into the kinetics of molecular response that are not provided by conventional RQ-PCR. The strong correlation between RQ-PCR and DNA-based PCR in follow-up samples beyond three months indicates that the major determinant of RQ-PCR values is the num- ber of circulating leukemic cells, rather than variable expression of BCR-ABL1.
Acknowledgments
The authors wish to thank the patients, investigators, study co- ordinators, and ALLG staff who provided the samples and clini- cal data for this study, and acknowledge the ALLG as the spon- sor of the TIDEL-II clinical trial. Novartis Pharmaceuticals pro- vided a research grant for this project and additional funding was obtained from the South Australian Health Services Charitable Gifts Board.
References
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gene and fusion gene transcripts over time may hamper accurate quantification of minimal residual disease--a study within the Europe Against Cancer Program. Leukemia. 2004;18(4):884-886.
3. Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmo- nizing current methodology for detecting BCR-ABL transcripts and kinase domain
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