Page 103 - 2018_12-Haematologica-web

BCR-ABL1 DNA monitoring of CML
An alternative approach to overcome the variability in RQ-PCR is to measure BCR-ABL1 genomic DNA, since the overwhelming majority of chronic phase CML patients will have a single copy of BCR-ABL1 and two copies of an autosomal control gene in each leukemic cell. In the past, this approach was not practical due to the complexity of sequencing individual genomic break- points. Almost all CML patients express one or both of the two common BCR-ABL1 mRNA transcripts (e13a2, e14a2), whereas the genomic fusion sequences involve introns that are spliced out from the mRNA, and are essentially unique to each individual patient.5 Advances in sequencing technology have made it relatively simple to detect BCR-ABL1 genomic breakpoints, and several meth- ods have been published.6,7
It should be emphasized that DNA PCR and RQ-PCR are not expected to yield identical results. This is perhaps best exemplified by the comparison of RQ-PCR with metaphase karyotyping in CML, which shows that a par- tial cytogenetic response [≤35% Philadelphia-positive (Ph+) cells] is roughly equivalent to BCR-ABL1IS ≤10%.8 Whereas both techniques are clinically useful, measures of the size of the CML clone the end point of each assay is qualitatively different. BCR-ABL1 DNA PCR is analogous to fluorescence in situ hybridization, in that both methods measure the simple proportion of cells in a sample that carry the Philadelphia rearrangement.
We used quantitative BCR-ABL1 DNA techniques, Q- PCR and digital PCR (dPCR), to monitor a cohort of patients in the Australasian Leukaemia and Lymphoma Group (ALLG) CML9 study (TIDEL-II).9 These results were compared with routine RQ-PCR monitoring. Since the number of copies of BCR-ABL1 DNA is directly related to the number of leukemic cells in a sample, we used DNA and mRNA-based methods in order to determine the rela- tive contribution of cell number and expression changes to molecular response in CML. Secondly, where there were differences between RQ-PCR and DNA PCR, we explored whether these differences might provide additional pre- dictive information concerning treatment response.
Methods
Patients’ characteristics and samples
Fifty-nine newly diagnosed chronic phase CML patients from the TIDEL-II clinical trial9 were included in our study. Details of these patients and of the samples analyzed are presented in the Online Supplementary Appendix and Online Supplementary Tables S1 and S2. The overall clinical characteristics and treatment responses of the selected cohort were not significantly different from those of the overall study population. The subset of patients included here were selected in three categories: undetectable MRD (UMRD) achieved within the first 2 years (n=26); treatment failure (n=9); and 24 additional patients not falling into either of the first two categories. Treatment failure was defined following the European LeukemiaNet (ELN) criteria as loss of complete hemato- logic response, loss of complete cytogenetic response, loss of major molecular response (MMR; BCR-ABL1IS ≤0.1%), kinase domain mutations, or progression to accelerated phase/blast cri- sis).10
Peripheral blood samples for molecular analysis were collected prior to commencing TKI treatment (baseline); at one, two, and three months; and every three months thereafter up to 24 months. RQ-PCR was performed centrally in the diagnostic laboratory of
SA Pathology, Adelaide, Australia, using the BCR control gene.11 The results were reported as BCR-ABL1/BCR% applying an IS conversion factor (Online Supplementary Appendix).4 Chromosome banding analysis was routinely performed at diagnosis in the respective local laboratories. Samples with fewer than 10 metaphases were excluded from this analysis.
All samples were collected with informed consent in accor- dance with the Institutional Ethics-approved protocols and with reference to the Declaration of Helsinki.
Breakpoint detection
The BCR-ABL1 genomic DNA breakpoint was determined, as previously described, in blood samples collected at diagnosis using long range PCR with a single forward primer in BCR and multiple reverse primers in ABL1 to amplify the breakpoint (Online Supplementary Appendix).12,13
Quantification of BCR-ABL1 DNA
Genomic DNA was extracted from peripheral blood leukocytes.
The amount of amplifiable DNA in each sample was measured using the GUSB control gene. The earlier assays were performed using real-time Q-PCR with standard curves for both BCR-ABL1 (patient’s diagnostic DNA assigned a value of 100%) and GUSB (plasmid) diluted in non-human DNA. Later assays used digital PCR (dPCR) for both BCR-ABL1 and GUSB with the aim of improving precision. Results were reported as BCR-ABL1/GUSB% (corrected for the two copies of GUSB per cell) normalized against the individual patient’s diagnostic sample. Further details are provided in the Online Supplementary Appendix and Online Supplementary Figures S1-S3.
Statistical analysis
Statistical analysis was performed using the GraphPad Prism 7 statistical software (GraphPad Prism Inc., La Jolla, CA, USA). Agreement between assays was assessed using the method of Bland and Altman.14 Correlation between non-parametric values was assessed using Spearman rank coefficient. Differences between BCR-ABL1 DNA and mRNA measurements were com- pared using a Mann-Whitney test. The cumulative incidence of MMR and MR4.5 was calculated using the Fine and Gray regres- sion method in R. Any event leading to the permanent discontin- uation of imatinib/nilotinib (including treatment failure, intoler- ance, and death) was treated as a competing risk. P<0.05 was con- sidered statistically significant.
Results
Comparison between DNA and mRNA before treatment
Since DNA Q-PCR quantifies BCR-ABL1 relative to the diagnostic DNA, we considered only the absolute dPCR values at diagnosis (n=29) and compared these values with the corresponding mRNA levels and the percentage of Ph+ bone marrow metaphase cells. The median value of BCR-ABL1 DNA prior to TKI treatment was 100% by karyotyping (range, 85-100%) and 84% (range, 45-164%) by dPCR. The corresponding median BCR-ABL1IS value was 70%, with values ranging from 3.7% to 425% (Figure 1A). Two of the 3 patients (#1 and #3) with low BCR- ABL1IS mRNA had stored peripheral blood cells available for interphase fluorescence in situ hybridization, which showed excellent agreement with the BCR-ABL1 DNA values obtained by dPCR (Table 1). Two of these 3 patients experienced treatment failure (blast crisis/sec- ondary resistance with a kinase domain mutation) and the third patient had ELN warning features at baseline (high
haematologica | 2018; 103(12)
2027