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Technical Briefs |
1 Leukaemia Research Fund Molecular Pharmacology Laboratory and2 Department of Haematology, School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle, Newcastle, UK;
aaddress correspondence to this author at: Northern Institute for Cancer Research, Cookson Building, Medical School, Newcastle NE2 4HH, UK; fax 44-191-222-7556, e-mail j.a.e.irving{at}ncl.ac.uk
The ABL tyrosine kinase inhibitor imatinib (STI571, Glivec, Gleevec) has produced dramatic clinical responses in most patients with chronic myeloid leukemia (1)(2)(3)(4). However, drug resistance has arisen as a result of increased expression of the BCR-ABL oncogene or the emergence of clones of cells harboring mutations in the ABL portion of the gene that reduce drug binding while retaining aberrant kinase activity (5). Original observations suggested that one specific substitution, T315I, was responsible (6). However, subsequent reports have demonstrated that this is not the case (7)(8)(9)(10)(11)(12)(13).
To monitor the emergence of drug resistance in patients treated with imatinib, there is a need to develop a reliable method for the screening of mutations in the BCR-ABL oncogene (5)(7). We report here the use of denaturing HPLC (DHPLC) as a method to screen for mutations in exons 4 and 6 of the ABL gene. Although originally described more than 6 years ago, the widespread application of this technique has only recently become possible with the introduction of commercially available specialized HPLC instrumentation dedicated to the performance of mutation analysis (14). Previous reports have demonstrated that the sensitivity and specificity of the technique are consistently high (15)(16).
Peripheral blood samples were obtained, after receipt of informed consent, from 22 patients with accelerated phase or blast crisis chronic myeloid leukemia. Genomic DNA from control samples was obtained from the Westlakes Research Institute at Cumbria after appropriate ethical consent. Genomic DNA was extracted by a standard phenol-chloroform method. PCR was performed with 50–100 ng of DNA, 2.5 mM MgCl2, 100 µM deoxynucleotide triphosphates, 0.2 µM each of forward and reverse primer, 1.25 U of Taq polymerase (AmpliTaq Gold; Perkin-Elmer), and 1x buffer. Primer pairs were designed with use of Omiga Software (Accelyrs Inc) to flank exons 4 and 6 of the ABL gene, based on criteria recommended by Transgenomic. The primer sequences were as follows: exon 4 forward, 5'-CTG TCT CTG TGG GCT GAA-3', and exon 4 reverse, 5'-AAA CAC ACT CGG ACT ATG AGA A-3'; exon 6 forward, 5'-GAC TGA GGA GCA GAG TCA GA-3' and exon 6 reverse 5'-GCC AGC ACT GAG GTT AGA A-3'. PCR conditions were as follows: 10 min of denaturation at 95 °C; a touchdown protocol of 20 s at 94 °C, 1 min at 66 °C (exon 4) or 63 °C (exon 6), and 1 min at 72 °C (–0.5 °C for 14 cycles); and 20 cycles of 20 s at 94 °C, 1 min at 59 °C (exon 4) or 56 °C (exon 6), and 1 min at 72 °C, with a final extension for 7 min at 72 °C. Heteroduplexes were formed in a thermal cycler by denaturation (95 °C for 5 min) and annealing/stabilization (starting at 94 °C for 2 min with a –1 °C touchdown until 4 °C). DHPLC was performed in a Trangenomic WAVE mutation detection apparatus. Samples (10 µL) were eluted from a DNASep column by a gradient of 250 mL/L acetonitrile in 0.1 mol/L triethylammonium acetate (buffer B) against 0.1 mol/L triethylammonium acetate (buffer A). The gradients used for elution were as calculated by Wavemaker software, and temperatures of 62 °C (exon 6) and 64 °C (exon 4) were found to be optimal for analysis. For direct sequencing, PCR products were purified by use of a PCR Clean up reagent set (Qiagen) and sequenced by use of BigDye Terminators chemistry on an ABI automated DNA sequencer. To characterize minority PCR species, PCR products were subcloned into the Pgem-T-Easy plasmid according to the manufacturer’s instructions and inserted in Escherichia coli (JM109), and isolated plasmid DNA from positive transformants was then sequenced with M13 forward and reverse primers.
The elution profiles for control samples for ABL exon 6 amplicons (n = 29) all showed a single peak representative of homoduplex DNA. Six of 22 patient samples had profiles containing one or more additional peaks, indicating the presence of a mismatch (Fig. 1A
). Similarly, exon 4 amplicons gave elution profiles with a single homoduplex peak in 47 of 48 control samples, but with a distinct double peak in 1 case, whereas 5 patient samples also showed abnormal profiles (Fig. 1B
). In two patient samples, abnormal elution profiles were detected in both exons 4 and 6.
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The degree of resolution varied greatly from an obvious extra peak (e.g., Fig. 1A
, patient 4) to a more subtle shouldering of the homoduplex peak (e.g., Fig. 1A
, patient 1). However, these more subtle patterns were highly reproducible in repeated analyses using fresh PCR products and were readily detectable when analyzed alongside products from cases without mutations. In cases 2 and 3, serial samples were available from patients who developed imatinib resistance. In these, the aberrant pattern became more obvious with the evolution of resistance, presumably as the proportion of the resistant clones increased.
Direct sequencing of PCR products demonstrated a heterozygote signal representative of the T315I mutation in patient 4, the G250E mutation in patient 8, and the E255K mutation in patient 9, but was not sufficiently sensitive to conclusively identify mutations in the other samples. However, sequencing of cloned PCR products revealed mutations in a minority of clones in all of the remaining cases (range, 5–47%; mean, 17%). Direct sequencing also identified the normal sample associated with an altered DHPLC profile as being a K247R heterozygote, which to our knowledge is not listed in any of the major single-nucleotide polymorphism databases and is presumably a rare polymorphism. Interestingly, three of the nine patients had a mutation at position 359, one with the substitution of phenylalanine for valine (F359V) and two for cysteine (F359C), the latter not having been reported previously. The detection of multiple independent mutant clones in two patients has been observed in other studies (7).
To date, 19 different amino acid mutations at 14 residues of the ABL protein have been described in clinical samples (Table 1
), including the new mutation reported here. Kinase domain mutations can occur in the chronic phase before the emergence of overt resistance (7), and there is evidence that such mutations may even predate the initiation of imatinib therapy (12)(17)(18). In vitro studies have suggested that mutations outside the kinase domain may also confer resistance (19) and that there may be "super mutants" that can hyperactivate autophosphorylation of the BCR-ABL kinase (19)(20). Studies of clinical samples have not yet investigated these non-kinase domain mutants, but it seems likely that their identification may well drive progress in drug development and clinical management in the future. DHPLC could be a useful technique to screen samples for such mutations.
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Mutational analysis may be performed with a variety of gel-based techniques, but these are difficult to perform, are not readily automated, and are prone to high false-positive and -negative detection rates. Automated sequencing is frequently described as the "gold standard" for this form of analysis, but mutations may readily be missed in minority populations of cells unless multiple PCR clones are analyzed. This point was emphasized in the study by Shah et al. (7). The relatively low rate of detection of ABL mutations in some other studies (13)(21) may be partly attributable to the use of direct sequencing without cloning to assess samples. In our study, mutations were detected in only a minority of PCR clones in five of the nine patients studied. Other, more recently described mutational screening methods, with sensitivity superior to that of sequencing, include an endonuclease/ligase-based technique and an enhanced PCR-restriction fragment length polymorphism analysis method (22)(23). However, both techniques are multistep and not easily amenable to routine screening of clinical samples.
We used genomic DNA, rather than cDNA, to screen for mutations. Although this allows retrospective analysis of samples where mRNA may not be available, it will decrease the sensitivity of the technique because alleles from normal cells will be amplified along with normal and chimeric ABL genes in Ph-positive cells. DHPLC could be applied to cDNA samples reverse-transcribed from BCR-ABL mRNA, although such amplicons would have to be mixed with PCR products from nonmutated cases to form heteroduplexes. We found no mutations in patients demonstrating primary resistance, which is in keeping with most other studies, although the recent report by Shah et al. (7) demonstrates that this can occur.
In summary, we show that DHPLC can detect previously characterized and novel mutations associated with acquired resistance to imatinib. Each PCR product analysis is performed in less than 10 min in an automated instrumentation platform that has increased sensitivity over direct sequencing methods with considerably reduced labor and consumable costs.
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Acknowledgments
This work was supported by the Leukemia Research Fund.
References
The following articles in journals at HighWire Press have cited this article:
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