Abstract
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.
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References
Deininger MW, O'Brien SG, Ford JM, Druker BJ . Practical management of patients with chronic myeloid leukemia receiving imatinib. J Clin Oncol 2003; 21: 1637–1647.
Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: 645–652.
O'Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994–1004.
Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, Guilhot F et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002; 99: 1928–1937.
Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002; 99: 3530–3539.
Ottmann OG, Druker BJ, Sawyers CL, Goldman JM, Reiffers J, Silver RT et al. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002; 100: 1965–1971.
Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002; 2: 117–125.
Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002; 99: 3472–3475.
Hochhaus A, Kreil S, Corbin AS, La Rosee P, Muller MC, Lahaye T et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 2002; 16: 2190–2196.
Al-Ali H, Heinrich MC, Lange T, Krahl R, Mueller M, Mueller C et al. High incidence of BCR-ABL kinase domain mutations and absence of mutations of the PDGFR and KIT activation loops in CML patients with secondary resistance to imatinib. Hematol J 2004; 5: 55–60.
Corbin AS, Rosee PL, Stoffregen EP, Druker BJ, Deininger MW . Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib. Blood 2003; 101: 4611–4614.
Azam M, Latek RR, Daley GQ . Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 2003; 112: 831–843.
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.
Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Lai JL, Philippe N, Facon T et al. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 2002; 100: 1014–1018.
Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299: 708–710.
Corless CL, McGreevey L, Haley A, Town A, Heinrich MC . KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size. Am J Pathol 2002; 160: 1567–1572.
Cruz F, Rubin BP, Wilson D, Town A, Haley A, Bainbridge T et al. Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res 2003; 63: 5761–5766.
Deininger M, Goldman JM, Lydon NB, Melo JV . The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL positive cells. Blood 1997; 90: 3691–3698.
La Rosee P, Corbin AS, Stoffregen EP, Deininger MW, Druker BJ . Activity of the Bcr-Abl kinase inhibitor PD180970 against clinically relevant Bcr-Abl isoforms that cause resistance to imatinib mesylate (Gleevec, STI571). Cancer Res 2002; 62: 7149–7153.
Corbin AS, Buchdunger E, Pascal F, Druker BJ . Analysis of the structural basis of specificity of inhibition of the Abl kinase by STI571. J Biol Chem 2002; 277: 32214–32219.
Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A et al. Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. Leukemia 1999; 13: 1825–1832.
Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001; 293: 876–880.
von Bubnoff N, Schneller F, Peschel C, Duyster J . BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study. Lancet 2002; 359: 487–491.
Wang L, Pearson K, Ferguson JE, Clark RE . The early molecular response to imatinib predicts cytogenetic and clinical outcome in chronic myeloid leukaemia. Br J Haematol 2003; 120: 990–999.
Wang L, Pearson K, Pillitteri L, Ferguson JE, Clark RE . Serial monitoring of BCR-ABL by peripheral blood real-time polymerase chain reaction predicts the marrow cytogenetic response to imatinib mesylate in chronic myeloid leukaemia. Br J Haematol 2002; 118: 771–777.
Merx K, Muller MC, Kreil S, Lahaye T, Paschka P, Schoch C et al. Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha. Leukemia 2002; 16: 1579–1583.
Kantarjian HM, Talpaz M, Cortes J, O'Brien S, Faderl S, Thomas D et al. Quantitative polymerase chain reaction monitoring of BCR-ABL during therapy with imatinib mesylate (STI571; Gleevec) in chronic-phase chronic myelogenous leukemia. Clin Cancer Res 2003; 9: 160–166.
Acknowledgements
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). https://doi.org/10.1038/sj.leu.2403307
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DOI: https://doi.org/10.1038/sj.leu.2403307
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