Mutations of the ABL kinase domain (KD) are common in patients with chronic myelogenous leukemia (CML) who develop resistance to imatinib. We developed an RT-PCR-based denaturing high-performance liquid chromatography (D-HPLC) assay to detect mutations of the ABL KD. Validation experiments using mixtures of wild type and mutant amplicons showed that the D-HPLC assay could detect mutant transcripts when they represented at least 15% of the total, and was thus twice as sensitive as automated sequencing. When D-HPLC was applied to 30 cDNAs from patients with imatinib resistance that had previously been characterized for KD mutations by direct sequencing of BCR-ABL RT-PCR products, there was concordance in 97% of samples. Resequencing confirmed the original mutations in all cases. In addition, sequencing of individual clones detected a mutation in one sample that had been mutation-positive by D-HPLC but wild type by conventional sequencing. In serial samples from the same individuals, D-HPLC detected mutations as early as 260 days before hematological relapse. D-HPLC is suitable for routine clinical monitoring of CML patients for emergence of KD mutations and may be useful for optimizing therapy. Early detection of emerging mutant clones may aid in guiding decisions regarding alternative treatment options.
Imatinib has become the standard drug treatment for chronic myelogenous leukemia (CML) in all phases of the disease.1 While responses are generally durable in chronic phase,2,3 there is a high incidence of relapse in patients treated in accelerated phase4 or blast crisis.5,6 Mutations in the kinase domain (KD) of Bcr-Abl have been detected in up to 90% of patients who relapse after an initial response.7,8,9,10 These mutations affect amino acids involved in imatinib binding or in regulatory regions of the KD, resulting in decreased in vitro sensitivity to imatinib as assessed in assays of tyrosine phosphorylation and cellular proliferation.7,11,12 Detection of mutations may be clinically important for several reasons. First, the degree of imatinib resistance varies among different mutants. For example, the M351T mutant remains sensitive to concentrations of imatinib that may be achieved in patients, whereas the T315I mutant is completely resistant.11 This suggests that dose escalation of imatinib may be associated with clinical responses in patients with M351T but not those with T315I mutations. Second, the presence of certain mutations may offer prognostic information. This is best documented for mutations in the P-loop of the KD that appear to confer an extremely poor prognosis.13 Importantly, mutations have been detected in patients still responding to imatinib13 or even before treatment,14 suggesting that regular screening for mutations may be justified.
For optimal clinical management of CML patients being treated with imatinib, early detection of mutations is crucial, since patients may benefit from alternative therapeutic strategies, such as drug combinations, novel Abl kinase inhibitors or allogeneic stem cell transplantation. Direct sequencing of the Abl KD is laborious, has limited sensitivity,7 and is therefore not suitable as a clinical screening test. In contrast to direct sequencing, denaturing high-performance liquid chromatography (D-HPLC) allows for high throughput mutation screening.15,16 This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplexes. Based on our experience with D-HPLC for the detection of mutations of KIT, platelet-derived growth factor receptors (PDGFR) and BRAF mutations,15,17 we reasoned that this technique may also be useful for the detection of mutations of the ABL KD.
Patients and methods
A total of 29 patients with CML and one patient with Philadelphia-positive ALL were treated with imatinib in Novartis-sponsored phase II protocols. The studies were approved by the Institutional Review Boards of the University of Leipzig, Germany and Oregon Health&Science University and Portland VA Medical Center, Portland, Oregon. Informed consent according to the Declaration of Helsinki was obtained from all patients. Patient demographics are listed in Table 1. The present patient series partially overlaps with a cohort of patients recently described.10
Sequence analysis of BCR-ABL
For initial mutational analysis, total RNA was extracted using the Qiagen RNAEasy kit. cDNA was synthesized using random hexamer primers and the integrity of the cDNA was assessed by amplification of BCR, as described.18 In the case of CML samples, primers for PCR amplification of BCR-ABL were BCR-F4 (5′-IndexTermACAGCATTCCGCTGACCATCAATAAG-3′) corresponding to nucleotides (nt) 3254–3299 of BCR (accession Y00661) and ABL-R6 (5′-IndexTermAGAACTTGTTGTAGGCCA-3′) corresponding to nt 1596–1613 of ABL (accession M14752). In the patients with Ph-positive ALL, BCR-109 (5′-IndexTermACCATCGTGGGCGTCCGCAAGA-3′), corresponding to nt 1803–1824 of BCR, was used as the forward primer. Reaction conditions were as described.18 The PCR products were then subjected to automated sequencing from both directions using primers ABL-F4 (5′-IndexTermTCCCCCAACTACGACAA-3′), corresponding to nt 1049–1065, and ABL-R6. Thus, the sequence analysis covered amino acids 234–410 of Abl. In the case of the ALL samples, only ABL-R6 was used for sequencing.
Design and optimization of the D-HPLC assay
Primer sequences were designed to separately amplify the 5′ and 3′ regions of the ABL KD. Specifically, we used the primer pair ABL start forward (5′-ACGACAAGTGGGAGATGGAA-3′), corresponding to nt 1059–1078, and ABL start reverse (5′-IndexTermTCTGAGTGGCCATGTACAGC-3′), corresponding to nt 1384–1403, to amplify the 5′ portion and the primer pair ABL end forward (5′-IndexTermTCATGACCTACGGGAACCTC-3′), corresponding to nt 1314–1333, and ABL end reverse (5′-IndexTermATACTCCAAATGCCCAGACG-3′), corresponding to nt 1627–1646, to amplify the 3′ portion (Figure 1). The 5′ KD amplicon included ABL codons 238–339 and the 3′ KD amplicon included ABL codons 323–420. Amino acid and nucleotides of ABL are numbered according to the sequence containing Abl exon Ia (accession M14752).
To optimize the D-HPLC assay, we prepared gDNA from factor-independent BaF3 cells that had been stably transduced with retroviral constructs encoding wild-type or kinase mutant BCR-ABL cDNAs.11,19,20 The ABL KD was amplified using ABL-specific primers and the resultant amplicons were analyzed by D-HPLC under nondenaturing conditions (50°C) using a Transgenomic WAVE instrument (Transgenomics, Omaha, Nebraska) as previously described.15 Wild-type amplicons were mixed in a 1:1 ratio based on the area under the OD260 HPLC peak with amplicons from different mutants and the mixture was analyzed at different partially denaturing conditions to optimize mutation detection. Based on these experiments we chose 62.9 and 61.6°C as the optimal assay temperatures for detection of mutations in the 5′ KD and 3′ KD amplicons, respectively.
To determine the sensitivity of the D-HPLC assay, amplicons of wild-type and the various types of mutant BCR-ABL were mixed together in fixed ratios ranging from 0 to 100% mutant amplicon using the OD260 of the BCR-ABL amplicon peak determined at 50°C to quantify the concentration of the different amplicons. Serial dilutions were calculated using the formula F=(AX)(100)/[(AX+αB−BX)], where F is the desired fraction % mutant ABL KD amplicon, A the mutant ABL amplicon peak area, X the microliters of mutant ABL amplicon to be added, α the total volume of loaded sample and B the wild-type ABL relative peak area. The annealed mutant ABL/wild-type DNA mixtures were then reanalyzed by D-HPLC to provide an estimate of the percent mutant DNA detectable.17
The mixed amplicons were heated to 94°C in a thermocycler and then cooled to 4°C over 2 min to allow heteroduplex formation. The annealed amplicons were analyzed by D-HPLC using the previously optimized gradient and temperature conditions. To directly compare the sensitivity of D-HPLC and direct sequencing, a subset of the annealed amplicons were sequenced in a blinded manner.
The same assay was used to analyze a collection of previously characterized patient samples. Since D-HPLC detection of point mutations is dependent upon the formation of heteroduplexes between mutant and wild-type sequence strands, no mutation may be apparent in cases where the mutant amplicon comprises >85% of the total amplicons. Therefore, all amplicons with a wild-type elution pattern were mixed and annealed with an equal amount of wild-type amplicon before being subjected to repeat D-HPLC analysis. This repeat analysis allows the detection of mutations when the mutant transcript is >85% of the total transcript. When necessary, sequencing was performed on the undiluted original amplicon.
Confirmation of BCR-ABL KD mutations in patient samples
Patient cDNAs were anonymized and amplified to produce amplicons containing either the 5′ or 3′ region of the ABL KD using the ABL-specific primer pairs listed above. The resultant amplicons were analyzed by D-HPLC in a blinded manner. For confirmation of the D-HPLC results and the initial sequencing, both ABL amplicons analyzed by D-HPLC were directly sequenced in a blinded manner using an automated sequence analyzer.
Quantitative RT-PCR for BCR-ABL
Reactions were performed on a DNA Engine Opticon® 2 System (MJ Research, Reno, NV, USA) using Sybr® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), primers at 0.2 μ M, 2 μl of 1:10 diluted cDNA and water to a volume of 20 μl. Primers were as described by Emig et al.21 Amplification conditions were 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 1 min. Standard curves were obtained from amplification of a serial dilution of a BCR-ABL plasmid. BCR-ABL expression is given as the ratio BCR-ABL/ABL.
Establishment and optimization of a D-HPLC assay for detection of ABL KD mutations
To determine the sensitivity of the D-HPLC assay for detection of clinically relevant ABL KD mutations in patient specimens, we generated a series of ABL KD amplicons using gDNA purified from factor independent BaF3 cells stably transduced with either wild type or different mutant BCR-ABL cDNAs.11,19,20 These amplicons of pure wild type or mutant ABL KD were mixed in fixed ratios based on amplicon concentration (as assessed by the area under the OD260 D-HPLC peak). The percentage of mutant amplicon in these mixed specimens ranged from 0 to 75%. As shown in Figure 2, D-HPLC detected the Y253F, T315I, M351T and H396P mutations, when the percentage of mutant amplicon was more than 15% of the total ABL KD amplicon.
To compare the sensitivity of direct sequencing of amplicons to our HPLC assay, we sequenced samples from several serial dilutions in a blinded fashion. Direct sequencing failed to detect the Y253K, T315I and H396P mutations when the proportion of mutant amplicon was smaller than 30%. Thus, the D-HPLC assay was at least twice as sensitive as direct sequencing for detection of ABL KD mutations. This level of D-HPLC sensitivity is consistent with our previous results comparing D-HPLC to direct sequencing for detection of point mutations in other kinases.16,17
Validation in patient samples
Initial sequencing was performed at the University of Leipzig, Germany, on 30 cDNAs after amplification with BCR-ABL-specific primers. In 16 samples from 15 different patients, mutations were detected (Table 1). In one patient with Ph-positive ALL, contemporaneous samples from peripheral blood and bone marrow were studied. Mutations were detected in 15 of 22 patients (68%) with hematological (n=21) or cytogenetic (n=1) relapse. This incidence is in line with studies from other groups.7,9 No mutations were found in six initial samples (including three samples from patients with primary resistance to imatinib) and one sample from a patient responding to imatinib.
In order to validate the use of D-HPLC for analysis of clinical specimens, the cDNAs were anonymized and analyzed by D-HPLC and sequenced in a blinded manner. In all 16 samples with mutations originally detected by sequencing of BCR-ABL amplicons, D-HPLC showed an abnormal pattern indicative of a mutation and/or single nucleotide polymorphism. In all cases, the localization of the mutation predicted from D-HPLC (according to an abnormal pattern in either the 5′ and 3′ ABL KD amplicon) corresponded to the site of the mutation originally detected by sequencing. The detection of point mutations by D-HPLC is largely dependent upon the formation of heteroduplexes between wild type and mutant amplicons. Thus, at high proportions of mutant amplicon (>85% of total), D-HPLC will show a homozygous pattern, similar to that seen in samples that are more than 85% wild type (Figure 2). In such cases, mutation detection is enhanced by mixing the PCR products with an equal amount of wild-type ABL KD amplicon. Such dilutions were routinely carried out in all cases lacking a detectable KD mutation upon initial D-HPLC analysis. In one case (#26), dilution allowed the detection of an M351I mutation that was not apparent using the undiluted specimen. In this case, sequencing of the undiluted ABL (and also the initial BCR-ABL) amplicons was consistent with a high percentage of mutant allele (>90%). In one patient (#5), a single-nucleotide polymorphism (L323L) was detected in addition to T315I.
In 13/14 samples (93%) that were wild type by initial sequencing, the D-HPLC assay was concordant. In one patient (#17), an abnormal pattern in the 5′ amplicon of ABL was detected, although initial sequencing had been wild type. Sequencing of the ABL amplicons analyzed by D-HPLC confirmed the original sequencing results in all samples, including #17, which was found to be wild type. However, when ABL PCR products were cloned into a bacterial expression vector for sequence analysis of individual clones, a T315I mutation was detected in two of 10 sequenced ABL amplicon clones (20%). This result is consistent with our prior blinded sequencing studies in which the proportion of mutant amplicon had to exceed 30% for accurate detection of mutations. In one patient (#18), sequencing of the ABL amplicon detected an S417Y mutation in addition to F359I. The S417Y mutation had been missed on initial sequencing of the BCR-ABL amplicon, since this residue was not included in the amplicon.
Serial analysis of patient samples
Analysis of samples from various time points prior to relapse was possible in six patients with mutations (Table 2). In patient #1 (with E255K), an abnormal D-HPLC pattern was apparent in a sample taken on day 85 of imatinib therapy, 43 days prior to relapse in blast crisis (Figure 3). In patient #2 (with Y253H), an abnormal D-HPLC pattern was detected on day 253. At that time, karyotyping showed loss of complete cytogenetic response. On day 309, this patient relapsed in accelerated phase. Patient #7 (with T315I) showed an abnormal pattern on days 161, 244 and 328, while maintaining PHR, the best response that she had achieved on imatinib. On day 422, she relapsed in accelerated phase. In the remaining three patients, samples obtained on day 97, 84 and 79 prior to relapse were wild type.
Our results demonstrate that D-HPLC is useful tool to screen for mutations in the KD of ABL. The results of conventional sequencing of PCR products and D-HPLC patterns were concordant in 29/30 samples (97%). Only one discordant result was observed, where the D-HPLC pattern suggested a mutation, while direct sequencing of BCR-ABL or ABL PCR products showed consistently wild-type sequence. However, analysis of individual clones revealed a T315I mutation in two of 10 sequenced ABL clones. Thus, there were no false-positive results, and the D-HPLC assay was able to detect a mutation that had been missed with conventional sequencing, consistent with its higher sensitivity. Importantly, our series includes most mutations that are commonly found in patients with secondary imatinib resistance.8,9,10,22,23 Based on these studies, and our prior experience in using D-HPLC for detection of KIT, PDGFRA, N-RAS or B-RAF mutations, it can be assumed that other ABL KD mutations, including uncharacterized ones, can also be detected with equal sensitivity.15,16,17 In contrast to those studies, however, the assay described here is based on cDNA rather than gDNA. We chose this strategy, since quantitative RT-PCR for BCR-ABL transcripts has become part of routine monitoring of patients on imatinib,24,25,26,27 and thus a cDNA-based D-HPLC assay can easily be incorporated into existing follow-up protocols. Given that ABL-specific primers are used to generate the amplicons, the sensitivity of this assay is influenced by the level of expression of ABL compared to BCR-ABL. In our series of patients, the BCR-ABL/ABL ratio was uniformly high (13–134, median 70%), reflecting the fact that all patients had active disease at the time of the analysis. Additional sensitivity for detection of emergence of mutations in the setting of minimal residual disease (where the BCR-ABL/ABL ratio may be much lower) could be obtained by two-step RT-PCR, using a forward BCR primer and a reverse primer located at the 3′ end of the ABL kinase for the first and the primer pairs described in this study for the second step.
False-negative (homozygous) D-HPLC results may occur, when the proportion of mutant cDNA exceeds 85%, and the BCR-ABL/ABL ratio is high. To avoid this problem, we added wild-type amplicon to samples with a homozygous pattern. In one case (#26), a mutation became apparent only after this manipulation. Consistent with our predictions, the BCR-ABL/ABL ratio in this case was high, and sequencing of the ABL and BCR-ABL amplicons showed almost exclusively mutant.
Abl KD mutations are the most frequent mechanism of resistance to imatinib.7 Most of the mutations published thus far were detected at the time of relapse, usually hematological relapse. However, mutations have also been found in patients who were still responding to imatinib,13 and even prior to treatment.14 From the clinical point of view, early detection of mutations, before the manifestation of relapse, is desirable, since it allows for timely adjustments to the overall treatment strategy. For example, mutations within the P-loop of the KD appear to confer such a poor prognosis13 that a decision may be made to proceed to an allograft, while the patient is still responding to imatinib.
Prospective trials are needed to determine the level of sensitivity required for optimal patient management. Thus, it is not yet clear if the slightly increased sensitivity of D-HPLC compared to conventional sequencing will translate into a clinical benefit. It is also possible that even more sensitive techniques, such as allele-specific PCR, will be needed for optimal monitoring of patients. These assays may be able to detect as little as 0.01% of mutated allele.14 On the other hand, a major disadvantage of allele-specific PCR is that multiple individual assays are necessary to screen for all the known mutations. Obviously, positive D-HPLC results still need to be confirmed by direct sequencing, in order to precisely characterize the mutation. Given that the individual mutations differ greatly with respect to their sensitivity to imatinib11 and prognosis,13 this information is crucial for patient management. Nonetheless, the use of D-HPLC for screening allows for a significant reduction of the number of samples that need to be sequenced. Irrespective of the precise methodology used for screening, a high throughput assay will be needed for screening patients for entry into clinical trials of experimental therapies to overcome imatinib resistance, including trials of new Abl kinase inhibitors.
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This work was supported by grants from the National Institutes of Health (Grants K24 CA82445 and 1R01 CA65823) (BJD), the Leukemia and Lymphoma Society (BJD) and the VA Merit Review System (MCH).
We thank Gerlinde Patzer and Dr Haifa-Kathrin Al-Ali, University of Leipzig, Germany, for help with sequencing and dedicated patient care, respectively.
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Deininger, M., McGreevey, L., Willis, S. et al. Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography. Leukemia 18, 864–871 (2004) doi:10.1038/sj.leu.2403307
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