ABL is the main target of several tyrosine kinase inhibitors used in the treatment of chronic myeloid leukemia (CML) patients. ABL tyrosine kinase domain (TKD) point mutations are the main mechanism of acquired tyrosine kinase inhibitor resistance.1, 2, 3 Although approximately 90 ABL TKD point mutations at 57 residues have been described, substitutions at seven amino-acid residues account for a large proportion of point mutations detected. The T315I and the ATP-binding loop (p-loop) mutations have been associated with poor outcomes on imatinib mesylate (IM) therapy in several studies.2, 3 Furthermore, the ‘gatekeeper’ mutation, T315I, is resistant to currently available ABL tyrosine kinase inhibitors.
Direct nucleotide sequencing is the primary method of ABL TKD point mutation detection used in clinical practice. This technique has a sensitivity of 20–25% (that is, to be detected the mutant clone must comprise at least 20% of the sample tested). Sensitivity can be improved slightly by pyrosequencing or denaturing high-performance liquid chromatography methods. Because of this limited sensitivity, ABL TKD mutations are typically detected at relapse, rather than early in the course of the emergence of a mutated ABL clone.2
We have devised a mutation-specific PCR assay using a novel nanofluidic platform to identify and quantify T315I ABL in 34 CML patient samples from 28 patients.4 The Fluidigm BioMark Real-Time PCR System and a nanofluidic chip, the BioMark digital array (Fluidigm, South San Francisco, CA, USA) were used in conjunction with mutation-specific PCR to detect the T315I mutation. The digital array uses nanoscale channels, valves and pumps to partition samples into 12 panels, each panel containing either 765 or 1104 chambers (depending on the platform design). There is improvement in the detection of rare mutations as a consequence of sample partitioning before PCR. For example, if a mixture containing 1 molecule of T315I ABL in 100 000 molecules of unmutated ABL is partitioned into 1000 independent chambers, the chamber containing the single mutant molecule now only contains ∼100 molecules of unmutated ABL. This 1000-fold increase in relative concentration theoretically allows for a 1000-fold improvement in the detection sensitivity of PCR reactions, thereby facilitating rare copy detection in limited patient samples.
Extensive experimental details are available at http://www.fhcrc.org/science/labs/radich/. In general, the experimental protocol first requires quantification of total amplifiable mRNA, specifically total ABL mRNA, and subsequently quantification of T315I mutated ABL using mutation-specific PCR. For mutation-specific primers, the ultimate 3′ base pair was specific for each mutation, and specificity was increased by placing another mismatch 4 base pairs from the 3′ end. ABL, rather than BCR-ABL, was analyzed owing to the product size constraints when using quantitative PCR. A pre-amplification step (Applied Biosystems, Foster City, CA, USA) was performed for all samples where remaining cDNA was extremely limited. Comparisons utilizing model systems demonstrated that no bias was introduced by the use of pre-amplification before mutation detection.
Total ABL was quantified across three panels of 765 reactions per panel (total 2295 reactions) using the average number of positive chambers, the dilution factor and Poisson distribution to calculate the original number of ABL molecules. Subsequently, for each T315I assay, equal numbers of ABL molecules were loaded into three panels (samples 5–34) or one panel (samples 1–4). The number of T315I mutant molecules in each panel was calculated using Digital PCR Analysis software (Fluidigm) as described above. The sensitivity and specificity of the reaction were determined using serially diluted plasmids and cell lines containing the T315I mutation in a background of unmutated ABL. The assay detected as few as three T315I mutated molecules in a total background of 100 000 unmutated ABL molecules (Figure 1).
Samples from CML patients (all phases) and normal patients were obtained from the University of California, Los Angeles (samples 1–4), Oregon Health and Sciences University (samples 5–22) and the Fred Hutchinson Cancer Research Center (samples 23–34). Prior approval for these studies was obtained from the Institutional Review Boards at each center. The negative control for each T315I assay consisted of 5000 molecules per panel of unmutated ABL, and the positive control consisted of a spike-in of 2 molecules per panel (6 molecules in total) of T315I mutated ABL in the background of 5000 molecules of unmutated ABL. Receiver operating characteristic curve analysis (Prism software, version 4, GraphPad Software, San Diego, CA) was used to determine the diagnostic performance of the assays using area under the curve analysis computed using a non-parametric method.5 The negative and positive controls run on each chip were used to calculate the false positive and true positive rates, respectively, and to generate the receiver operating characteristic curves. To ensure specificity, the negative control was used to set the cycle threshold at which patient data would be analyzed. Data were accepted only if the negative control showed no amplification below the established cycle threshold. The area under the receiver operating characteristic curve at cycle 36 was 0.88 (95% CI 0.77–1.0; P<0.0001).
Analyses of 34 samples from 28 patients, taken both before and at the time of relapse, were performed in a blinded manner. As shown in Table 1 and Figure 2, the T315I mutation was detected in all eight patients in whom it was ultimately detected at the time of relapse. The T315I mutation was not detected in eight CP CML patients who maintained complete cytogenetic responses (CCRs) on IM, in three patients who failed IM without evidence of point mutations, in two patients with no clinical information available and no evidence of point mutations or in three CML patients who developed other ABL TKD point mutations. Lastly, the T315I was not detected in one bone marrow sample and two peripheral blood samples from healthy volunteers without leukemia. We then compared directly the performance characteristics of our method with those of allele-specific oligonucleotide PCR (ASO-PCR), where detection is qualitative (yes vs no) rather than quantitative.6 Samples 6–22 comprised archival residual material from the same patient samples examined by Willis et al.6 A pre-amplification step before total ABL quantification was necessary, as the remaining sample quantities were very limited. Within this group, the five patients who ultimately developed a T315I mutation were correctly identified, including three patients in whom the T315I mutation was detected while they maintained complete cytogenetic responses. Overall, the assays were concordant in 14 of 17 cases. One sample (sample 6) was found to be positive for the T315I mutation by ASO-PCR, but negative by the Fluidigm assay. This archival sample was the diagnostic bone marrow sample from a patient in whom the T315I mutation was ultimately detected by direct nucleotide sequencing at the time of relapse on day 158. The Fluidigm assay detected the T315I mutation in two patients (samples 11 and 13) in whom it was not detected by direct nucleotide sequencing or by ASO-PCR. Explanations for these discordant results include very limited sample quantities, differences in sensitivity and limited patient follow-up after the original studies were completed. Serial monitoring for the T315I mutation was possible in eight samples from three patients (Table 1 and Figure 2). The T315I mutation was detected 99, 102 and 144 days before it was detected by direct nucleotide sequencing in these three patients. As shown in Figure 2f, each of the three patients demonstrated a similar 2–3-log10 increase in mutated T315I ABL from the time of detection until overt clinical relapse.
ABL TKD point mutation detection is an important component of therapeutic management in patients, particularly in patients who do not achieve cytogenetic and molecular milestones. Typically, ABL TKD mutations are detected at the time of cytogenetic relapse or at the time of disease progression, although increases in BCR-ABL copy number may indicate the presence of a mutation.7 Although the significance of early ABL TKD mutation detection remains unclear,6 the early identification of the T315I mutation would seem to have clinical relevance, particularly as patients with the T315I mutation cannot be salvaged by nilotinib or dasatinib therapy. Thus, there may be a clinical niche for sensitive methods of ABL mutation detection. Recently, a very sensitive method using a polymerase colony (or ‘polony’) assay was used to quantify ABL TKD mutations in three patients with assay sensitivities of up to 1:10 000.8 The nanofluidic-based mutation-specific PCR assays described here have similar sensitivities and have several unique features. First, the ability to partition the sample into thousands of independent reaction chambers increases the target-to-background ratio, and thus sensitivity increases as increased partitioning capacity is utilized. Second, small sample requirements enable a single sample to be screened for a battery of mutations even if sample amounts are limited. Third, this approach, unlike most other sensitive detection strategies, allows for the assessment of kinetics of ABL TKD mutation development; thus, making it possible to address whether the kinetics of mutation development, rather than the presence or absence of a mutation, correlates more closely with outcome. Fourth, the potential flexibility of this platform allows for thousands of different reactions to be performed on a single sample or multiple samples using only one digital array, and we have designed assays for the simultaneous detection of other ABL TKD mutations. Such sensitive detection of the T315I mutation and other mutations may allow investigators to study the biology of clonal selection and evolution in the context of tyrosine kinase inhibitor therapy, resistance and progression in CML.
Nicolini FE, Corm S, Le QH, Sorel N, Hayette S, Bories D et al. Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(phi)-LMC GROUP). Leukemia 2006; 20: 1061–1066.
Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003; 102: 276–283.
Soverini S, Martinelli G, Rosti G, Bassi S, Amabile M, Poerio A et al. ABL mutations in late chronic phase chronic myeloid leukemia patients with up-front cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival: a study by the GIMEMA Working Party on Chronic Myeloid Leukemia. J Clin Oncol 2005; 23: 4100–4109.
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Willis SG, Lange T, Demehri S, Otto S, Crossman L, Niederwieser D et al. High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy. Blood 2005; 106: 2128–2137.
Branford S, Rudzki Z, Parkinson I, Grigg A, Taylor K, Seymour JF et al. Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations. Blood 2004; 104: 2926–2932.
Nardi V, Raz T, Cao X, Wu CJ, Stone RM, Cortes J et al. Quantitative monitoring by polymerase colony assay of known mutations resistant to ABL kinase inhibitors. Oncogene 2008; 27: 775–782.
This work was supported by NCI CA106796, Leukemia and Lymphoma Society Translational Research Grant and V Foundation for Cancer Research V Scholar Grant (VGO), NCI CA18029 (JPR) and the Canary Foundation (SA and FM).
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