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Technical Briefs |
Laboratoire d’Hématologie et EA 3805, CHU de Poitiers, France;
aaddress correspondence to this author at: Laboratoire d’Hématologie, CHU de Poitiers, 2 rue de la Milétrie, 86021 Poitiers Cedex, France; fax 335-49444095, e-mail j.c.chomel{at}chu-poitiers.fr
Chronic myeloid leukemia (CML) is a rare hematopoietic stem cell disorder characterized by the t(9;22) translocation. This somatic event leads to the formation of the BCR-ABL fusion gene, which is translated in a functional protein (1). The Bcr-Abl oncoprotein differs from the endogenous c-Abl protein in both its subcellular localization and its tyrosine kinase (TK) activity. The deregulated Abl TK activity of the Bcr-Abl protein initiates the oncogenic process and represents a target to TK inhibitors.
Among a series of compounds exhibiting TK inhibition, STI 571 (imatinib) was found to be highly effective against Abl and its derivative Bcr-Abl (2)(3). This molecule targets the inactive conformation of the kinase, which leads to stabilization of the protein in its inactive form and impairs ATP binding (4). Approved for the treatment of CML, imatinib induces hematologic and cytogenetic remissions in the chronic phase as well as in the blast phase (5)(6)(7).
A significant proportion of patients, however, become resistant to the treatment. Different mechanisms of imatinib resistance have been described, in particular point mutations within the sequence of the BCR-ABL gene coding for the TK domain (8)(9)(10). Approximately 30 missense mutations have been identified in CML patients. Within the Bcr-Abl TK domain, these mutations are located in the nucleotide binding loop (P-loop), in the active site (imatinib contact site), or in the activation loop (A-loop; Fig. 1A
). Among these mutations, some prevent the binding of imatinib, and others make this binding more difficult. In the latter case, a dose increase can overcome the resistance. Recently, a novel TK inhibitor was described (11). In vitro, this molecule remains active for the majority of Bcr-Abl mutants, with the exception of T315I.
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Molecular monitoring of CML patients is based on the quantification of BCR-ABL transcripts in peripheral blood (or bone marrow) samples by real-time reverse transcription-PCR at regular intervals. It has recently been proposed that during imatinib therapy, a more than 2-fold increase in the BCR-ABL transcript concentration is a primary indicator for an emerging mutation within the Bcr-Abl kinase domain (12).
Detection of mutations in the ABL TK domain of the BCR-ABL oncogene by several methods has been reported. Some of these focus on known mutations (e.g., allele-specific oligonucleotide PCR and PCR with restriction fragment length polymorphism analysis). Others, such as denaturing HPLC (13)(14) can identify unknown single-base mutations in a given sequence. We describe a mutation screening method based on double-gradient–denaturing-gradient gel electrophoresis (DG-DGGE) (15)(16), which relies on wild-type (WT)/mutant heteroduplex formation. The entire BCR-ABL TK domain covers 4 exons of the ABL gene; therefore, the analysis was performed at the RNA level by means of 2 overlapping PCR fragments (5' and 3' in Fig. 1A
). DG-DGGE is generally performed on the ABL sequence by a single-step PCR. In this case, both BCR-ABL and ABL tyrosine kinase domains were amplified. Nested PCR was used when the analysis was restricted to BCR-ABL.
Total RNA was extracted from blood or bone marrow samples with RNABle reagent (Eurobio) according to the manufacturer’s instructions. RNA pellets were resuspended in 40 µL of RNase-free water. cDNA was synthesized from 1 µg of total RNA in a 20-µL reaction mixture by use of Moloney murine leukemia virus reverse transcriptase (Invitrogen).
For a nested PCR analysis, 2 µL of cDNA was subjected to a first round of PCR (25 cycles) with the forward primer located in exon 13 of the BCR gene (5'-ACAGCATTCCGCTGACCATC-3') and the reverse primer located in exon 8 of the ABL gene (5'-GAACGGTCAATTCCCGGG-3') under the following conditions: denaturation at 95 °C for 15 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 90 s. Amplifications were carried out in 50 µL with 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 µM each deoxynucleotide triphosphate, 0.5 µM each primer, and 2.5 U of Sure Prime DNA polymerase (Q-Biogene).
An aliquot of the first PCR product (5 µL of a 1:100 dilution) or 5 µL of cDNA (in the case of a single-step PCR) was used for the ABL TK amplification. For this purpose, 2 pairs of primers, 5F (5'-CTGTCTATGGTGTGTCCCCC-3') and 5R [5'-(40GC)-GCAGTTTCGGGCAGCAAGAT-3'] and 3F (5'-AAAGAGATCAAACACCCTAACC-3') and 3R [5'-(40GC)-TCCCAAAGCAATACTCCAAATG-3'] were designed using the MELT94 program (http://web.mit.edu/osp/www/melt.html). A 40-base GC-rich fragment (40GC; 5'-GCCCGCCGTCCCGGCCCGACCCCCGCGCGTCCGGCGCCCG-3') was added to the 5' end of each reverse primer (5R and 3R). The 2 amplicons, of 437 and 422 bp, respectively, overlapped on more than 200 bp. However, MELT programs predict the detection of any mutations in a sequence corresponding to amino acids 236–417 with little redundancy between the 2 amplicons. PCR was performed with the following conditions: denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 30 s. PCR was terminated by a final extension at 72 °C for 7 min. Amplifications were carried out as described above. The heteroduplexes were generated during the last cycles of PCR.
DG-DGGE analysis was performed on a DGGE-2001 system (CBS Scientific). An aliquot (15 µL) of PCR sample was electrophoresed in a colinearly increasing double gradient of 50% to 95% denaturant (100% denaturant: 7 mol/L urea, 400 mL/L formamide) and 6.5% to 12% polyacrylamide (acrylamide/bisacrylamide, 37.5/1). Electrophoresis was performed in 1x Tris-acetate-EDTA buffer (40 mmol/L Tris-acetate, 20 mmol/L sodium acetate, 1 mmol/L EDTA, pH 8.0) at 1800 V-h (e.g., 15 h at 120 V) and at a constant temperature of 60 °C. Gels were stained with ethidium bromide and visualized through ultraviolet transillumination.
In the presence of a shift in the acrylamide gel, the nucleotide change was characterized by direct sequencing of the PCR product carried out in both directions, using the BigDye Terminator (Ver. 1.1) Cycle Sequencing Kit and the ABI PRISM 310 sequencer (Applied Biosystems) according to the manufacturer’s recommendations. To avoid false-negative results, PCR products that showed no mobility shift in DG-DGGE were mixed with an equal amount of WT amplicon, denatured (5 min at 95 °C), annealed to form heteroduplexes (15 min at 56 °C), and analyzed again.
To evaluate the efficiency of the DG-DGGE methods, we analyzed cDNA samples from patients carrying mutations in the BCR-ABL TK domain that had already been characterized by direct sequencing. Compared with the WT controls, all samples showed an abnormal DGGE pattern (Fig. 1, B and C
). In the presence of a mutation, up to 4 bands were visualized: the WT homoduplex, the mutant homoduplex, and the 2 heteroduplexes (M244V, G250E, E255K, Y253H, M351T, T315I, F317L, and V379I). In these cases, the difference in the intensities of the homoduplex bands reflected the ratio between mutated and WT sequences. In a few samples (Q252E, Q252H, and H396P), the 2 homoduplexes were not separated. A double mutant with a dominant (E255V) and a minority mutation (Y253H) was detected by DG-DGGE. The E255V mutation, but not the minority mutation, was identified by direct sequencing of the corresponding PCR product. The 2 faint bands of high molecular weight were excised from the gel, purified, and amplified for direct sequencing. The 2 mutations (E255V and Y253H) were then characterized. To confirm these results, amplified products from E255V and Y253H mutants were mixed and subjected to DG-DGGE. Three homoduplexes (Y253H, E255V, and WT) and 5 bands corresponding to the WT/Y253H, WT/E255V, and Y253H/E255V heteroduplexes were clearly distinguishable in this assay. Thus, all previously identified mutations were unambiguously detected within the entire ABL TK domain.
Different point mutations leading to amino acid substitution were detected in 26 of 59 imatinib-resistant CML patients (M351T in 6; M244V and T315I in 4; G250E in 3; Q252E in 2; and Q252H, Y253H, E255K, E255V, F317L, V379I, and H396P in 1 each).
To define the detection threshold of the method, we serially diluted cDNAs carrying the M244V, E255K, or T315I mutations to concentrations ranging from 50% to 1% in a WT cDNA (Fig. 1D
). The heteroduplex bands remained clearly detectable until the 5% (M244V and T315I) or the 2% dilution (E255K). Mutated sequences can thus be detected when they account for at least 5% of total cDNA.
When correctly designed and applied, the percentage of point mutations detectable by DGGE is theoretically 100% in a given sequence. The method described here appears to be robust, inexpensive, and sensitive enough to detect a small proportion of mutant with an excess of WT sequences. Moreover, the detection of minority mutations is possible.
Screening of the entire TK domain of the BCR-ABL gene enables the classification of imatinib-resistant CML patients into 3 groups based on the amino acid change. The first group consists of patients for whom an imatinib dose increase can overcome the resistance (e.g., M244V and F359V mutants). The second group includes patients with a permanent resistance to imatinib (e.g., G250E, E255V, and H396P mutants); they could benefit from second-generation TK inhibitors. Finally, CML patients carrying the T315I mutation (found in 15%–20% of imatinib-resistant cases) seem to show a universal resistance toward all known TK inhibitors. Alternative treatments are currently in evaluation for patients carrying this mutation. Thus, a screening procedure such as DG-DGGE can be used to ensure the best therapeutic strategy as targeted therapies are developed for diseases such as CML.
Acknowledgments
This work was supported by grants from the Ligue Contre le Cancer (Comité de la Vienne et de la Charente).
Footnotes
References
The following articles in journals at HighWire Press have cited this article:
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  M. Baccarani, F. Pane, and G. Saglio Monitoring treatment of chronic myeloid leukemia Haematologica, February 1, 2008; 93(2): 161 - 169. [Full Text] [PDF]
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T. Ernst, P. Erben, M. C. Muller, P. Paschka, T. Schenk, J. Hoffmann, S. Kreil, P. La Rosee, R. Hehlmann, and A. Hochhaus Dynamics of BCR-ABL mutated clones prior to hematologic or cytogenetic resistance to imatinib Haematologica, February 1, 2008; 93(2): 186 - 192. [Abstract] [Full Text] [PDF]
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 N. Sorel, L. Roy, G. Martineau, F. Guilhot, A. G. Turhan, and J.-C. Chomel Sequential emergence of ABL-kinase mutations with loss of unmutated BCR-ABL allele during targeted therapies of CML. Blood, September 1, 2006; 108(5): 1782 - 1783. [Full Text] [PDF]
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T. Hughes, M. Deininger, A. Hochhaus, S. Branford, J. Radich, J. Kaeda, M. Baccarani, J. Cortes, N. C. P. Cross, B. J. Druker, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results Blood, July 1, 2006; 108(1): 28 - 37. [Abstract] [Full Text] [PDF]
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