The Mutation Spectrum of CML Patients Resistant to Imatinib: More Heterogeneous Than Just BCR-ABL Kinase Domain Point Mutations?.

Abstract Cells harboring BCR-ABL kinase domain (KD) mutations are the major source for resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). A second and third generation of TKIs are in development for inhibition of mutated forms of BCR-ABL. At present, clinical treatment strategies are based on molecular response and mutational profiling. Profiling is usually performed using unbiased methods (sequencing and D-HPLC of PCR products), but mutation specific methods are upcoming (ARMS-PCR, ASO-PCR, ligation-PCR). Here, we describe molecular findings resulting from a collaborative Nordic approach for molecular characterization of imatinib resistant Norwegian (N) and Finnish (F) CML patients. Complementary DNA derived from a total of 87 subjects (60 N and 27 F) with suboptimal or failed response to imatinib (less than 3 log reduction in tumor load after 12 months of treatment or loss of any response) were subjected to BCR-ABL KD sequencing of amino acid residues 240 to 420 (N) and 209 to 498 (F). In addition, single samples were analyzed by multiplex PCR for detection of transcript variants as well as cloning and sequencing of single clones. BCR-ABL KD mutations were found in 35 patients (20 N, 15 F, 40% of the total material). The most frequent findings were ABL KD point mutations with M351T and G250E representing the single most frequent mutations, 25 and 16%, respectively. The most frequently mutated cluster was in the catalytic loop (40% of all mutations), followed by P-loop (38%) and other regions (22%). Mutations in the gatekeeper cluster were extremely rare in our material (one case of a F317L). Additional sequence variations were observed in 6 patients: the L248V associated deletion of exon 5 (n=1), smaller insertion or deletion variants (2–4 basepairs) resulting in truncated proteins (n=2) and ABL exon 7 splicing (n=3). Finally, three cases with imatinib resistance expressed rare BCR-ABL transcript types without any evidence for KD mutations (two cases of e6a2 and one with e19a2 transcript). Imatinib resistance due to T315I BCR-ABL is extremely rare in Nordic CML patients. In addition to known hotspot mutations we identified novel sequence irregularities in association to imatinib resistance. We hypothesize that resistance-associated BCR-ABL isoforms are more heterogeneous than previously thought. Consequently, diagnostic procedures aiming in comprehensive molecular verification of TKI resistance should be designed as unbiased, since methods solely targeting known mutated BCR-ABL variants will miss many patients. However, an early detection of low-level mutated clones will require sensitive, mutation-specific analyses.

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High Sensitive Detection of Imatinib-Resistance Associated Mutations Based on ARMS-PCR.

Blood ◽

10.1182/blood.v104.11.2943.2943 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2943-2943

Author(s):

Franz X.E. Gruber ◽

Mikchail Soevershaev ◽

Marita Olsen ◽

Bjoern Skogen

Keyword(s):

Kinase Inhibitors ◽

Point Mutations ◽

High Sensitivity ◽

Kinase Domain ◽

Single Step ◽

Sensitive Detection ◽

Imatinib Resistance ◽

Abl Kinase ◽

Loop Region ◽

Highly Sensitive Detection

Abstract Background: Point mutations in the Abl kinase domain are associated with resistance against imatinib. Strategies to overcome resistance include dose escalation, combination treatment using imatinib with conventional or other developmental agents or, in the future, imatinib may be replaced by other tyrosine kinase inhibitors which work effectively against mutated clones. Mutational profiling of the BCR-ABL kinase domain will in this scenario become an important analysis as a supplement to BCR-ABL quantitation and may provide the rational basis for therapy, once resistance is diagnosed. Our group reported recently a sensitive, single step PCR assay for quantitation of mutated clones based on the ARMS principle. Aim: We describe an optimized, two step analysis for high sensitivity screening of mutated clones associated with resistance against imatinib targeting all P-Loop mutations, the T315I and M351T. Methods: In a first conventional PCR-reaction a cDNA-region spanning the BCR-ABL breakpoint is amplified resulting in an isolation of the BCR-ABL kinase domain for further analysis. An aliquot is then analysed in a second PCR step, conducted on the real time PCR Taqman platform. Selectivity for the mutated clone is conferred by the amplification refractoriness of non complementary primer 3′-ends (ARMS principle). By introducing potent nucleotide-mismatches in position n-2, selectivity of the assay could be further increased. Even in the P-Loop region, which is known to be a difficult PCR template, misannealing could be reduced to an acceptable level. Results: Assays targeting all P-Loop mutations inclusive the T315I and M351T were tested by analysis of patient samples diluted in normal cDNA and non-mutated BCR-ABL and plasmid dilutions, containing the targeted mutation in a background of wildtype plasmids. Generally a 1:1000 dilution of mutated templates could be detected (sensitivity 0.1%). For some mutations even higher sensitivity could be achieved (0.01%). The level of sensitivity is generally higher than reported for other methods described before. The first PCR step can be conducted in parallel to other PCR-based detection strategies. The second step can be run simultanously to Taqman based BCR-ABL quantitation. This makes the described assay the ideal supplement to general mutation detection approaches like D-HPLC or sequencing strategies. Compared to the single step assay we desribed before, the two step approach increases sensitivity with one or two log factors. Conclusion: The described assay may be suitable for highly sensitive detection of mutated clones in resistant CML patients as a supplement to less sensitive general screening approaches and BCR-ABL quantitation.

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Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC).

Blood ◽

10.1182/blood.v110.11.4580.4580 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4580-4580

Author(s):

Cintia C. Mascarenhas ◽

Anderson F. Cunha ◽

Katia B.B. Pagnano ◽

Rosana A. Silveira ◽

Fernando F. Costa ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Point Mutations ◽

Kinase Domain ◽

Molecular Response ◽

Abl Kinase ◽

Clinical Resistance

Abstract Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. 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 homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

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Complete Molecular Response due to nilotinib as III line treatment in a patient with CML and F317L point mutation of the Bcr-Abl kinase domain

Clinical Management Issues ◽

10.7175/cmi.v5i5s.1129 ◽

2015 ◽

Vol 5 (5S) ◽

pp. 21-25

Author(s):

Maria Iovine ◽

Giuseppe Monaco ◽

Mario Troiano ◽

Antonio Abbadessa

Keyword(s):

Point Mutation ◽

Kinase Inhibitors ◽

Mutational Analysis ◽

Point Mutations ◽

Cytogenetic Response ◽

Kinase Domain ◽

Molecular Response ◽

Abl Kinase ◽

First Line Therapy ◽

Complete Molecular Response

In 1997, a forty-three years old woman was diagnosed with CML and treated with alfa-Interferon, achieving complete haematological response (CHR). Three years later, patient was switched to hydroxiurea due to thyroid toxicity. For logistic reasons, therapy with imatinib 400 mg/die was initiated only in 2003, obtaining complete cytogenetic response (CCyR) and suboptimal molecular response in twelve months. CCyR and CHR were then lost three years later. Doubling imatinib dose to 800 mg/die gave no positive results. Mutational analysis performed in September 2007 showed F317L point mutation of the Bcr-Abl kinase domain. In October 2007 dasatinib was started and in April 2008 CCyR was reached with suboptimal molecular response. In March 2009 Bcr-Abl transcript progressively increased, and in August 2009 cytogenetic analysis showed loss of CCyR. Therapy with nilotinib 800 mg/die was started, and in October 2009 the patient obtained complete molecular response (CMR). Bcr-Abl kinase-domain point mutations, acquired during first line therapy, are a common cause of resistance to tyrosine kinase inhibitors. While several Bcr-Abl mutations have been identified, involvement of codon 317 has been reported in the literature following treatment with imatinib and dasatinib.

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A Cell-Based Screening Method for Resistance of Bcr-Abl Positive Leukemia Identifies the Mutation Pattern for an Alternative Abl Kinase Inhibitor.

Blood ◽

10.1182/blood.v104.11.558.558 ◽

2004 ◽

Vol 104 (11) ◽

pp. 558-558

Author(s):

Nikolas von Bubnoff ◽

Darren R. Veach ◽

Heiko van der Kuip ◽

Walter E. Aulitzky ◽

Jana Saenger ◽

...

Keyword(s):

Kinase Inhibitors ◽

Screening Method ◽

Point Mutations ◽

Kinase Domain ◽

Screening Strategy ◽

Culture Conditions ◽

Cross Resistance ◽

Imatinib Resistance ◽

Abl Kinase ◽

A Cell

Abstract The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.

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Bcr-Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107)

Blood ◽

10.1182/blood-2005-12-010132 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1328-1333 ◽

Cited By ~ 120

Author(s):

Nikolas von Bubnoff ◽

Paul W. Manley ◽

Jurgen Mestan ◽

Jana Sanger ◽

Christian Peschel ◽

...

Keyword(s):

Imatinib Mesylate ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Point Mutations ◽

Treatment Strategies ◽

Kinase Domain ◽

Abl Kinase ◽

Advanced Phase ◽

Clinical Resistance ◽

Limited Spectrum

Abstract In advanced-phase chronic myeloid leukemia (CML), resistance to imatinib mesylate is associated with point mutations in the BCR-ABL kinase domain. A new generation of potent ABL kinase inhibitors is undergoing clinical evaluation. It is important to generate specific resistance profiles for each of these compounds, which could translate into combinatorial and sequential treatment strategies. Having characterized nilotinib (AMN107) against a large panel of imatinib mesylate–resistant Bcr-Abl mutants, we investigated which mutants might arise under nilotinib therapy using a cell-based resistance screen. In contrast to imatinib mesylate, resistance to nilotinib was associated with a limited spectrum of Bcr-Abl kinase mutations. Among these were mutations affecting the P-loop and T315I. Rarely emerging resistant colonies at a concentration of 400 nM nilotinib exclusively expressed the T315I mutation. With the exception of T315I, all of the mutations that were identified were effectively suppressed when the nilotinib concentration was increased to 2000 nM, which falls within the peak-trough range in plasma levels (3.6-1.7 μM) measured in patients treated with 400 mg twice daily. Our findings suggest that nilotinib might be superior to imatinib mesylate in terms of the development of resistance. However, our study indicates that clinical resistance to nilotinib may be associated with the predominant emergence of T315I.

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BCR-ABL P-Loop Deletion Detected in Imatinib-Resistant CML Patients Corresponds to Splice Variant Associated with L248V Point Mutation, Retains BCR-ABL Kinase Activity, and Responds to Dasatinib Treatment.

Blood ◽

10.1182/blood.v108.11.2168.2168 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2168-2168

Author(s):

Nikolas von Bubnoff ◽

Philipp Erben ◽

Martin Müller ◽

Tanja Lahaye ◽

Susanne Schnittger ◽

...

Keyword(s):

Splice Variant ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Point Mutations ◽

Kinase Domain ◽

Imatinib Resistance ◽

Abl Kinase ◽

Imatinib Resistant ◽

Exon 4

Abstract Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

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Bcr-Abl Resistance Screening Predicts a Limited Spectrum of Point Mutations To Be Associated with Clinical Resistance to the Abl Kinase Inhibitor AMN107.

Blood ◽

10.1182/blood.v106.11.1983.1983 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1983-1983 ◽

Cited By ~ 1

Author(s):

Nikolas von Bubnoff ◽

Jana Saenger ◽

Paul W. Manley ◽

Juergen Mestan ◽

Christian Peschel ◽

...

Keyword(s):

Clinical Trials ◽

Imatinib Mesylate ◽

Kinase Inhibitors ◽

Point Mutations ◽

Imatinib Resistance ◽

Resistance Profile ◽

Abl Kinase ◽

Clinical Resistance ◽

Limited Spectrum ◽

Abl Kinase Inhibitors

Abstract In advanced-phase CML, resistance to imatinib mesylate is frequently associated with point mutations in the Bcr-Abl kinase domain. New, highly potent Abl kinase inhibitors such as AMN107 and BMS-354824, have recently entered clinical trials. Data from analyses of resistant patients will be available not before a large number of resistant patients will have been treated within clinical trials. Therefore, it will be important to generate specific resistance profiles for each compound prior to its therapeutic application. Using a cell-based screening method for resistance of Bcr-Abl positive leukemia to Abl kinase inhibitors, we generated a resistance profile for AMN107 and compared it to the resistance profile of imatinib mesylate. In contrast to imatinib, resistance to AMN107 was associated with a very limited spectrum of Bcr-Abl kinase mutations. While 26 exchanges at 21 positions occured with imatinib, the AMN107 screen revealed eight different exchanges at seven amino acid positions, with four exchanges affecting the P-loop. Novel mutations which have never been observed with imatinib, either in vitro or in resistant patients, emerged in the presence of AMN107 including an F359 exchange to isoleucine and a Q252H/S349L double mutant. In contrast to imatinib, the frequency of resistant colonies dramatically decreased with increasing AMN107 concentrations. Rarely emerging resistant colonies at a concentration of 400 nM AMN107 exclusively contained T315I. With the exception of T315I, all mutations that were identified were effectively suppressed when AMN107 was increased to 2000 nM, a concentration which is achieved in plasma in treated patients. Thus, in this system, increasing the AMN107 concentration to 400 nM prevented the emergence of resistant colonies, with the exception of T315I. Our findings suggest that AMN107 might be superior to imatinib in terms of the development of resistance. Also, AMN107 at clinically relevant concentrations may overcome imatinib resistant disease, including cases with expression of P-loop mutations. However, our study indicates that clinical resistance to AMN107 may be associated with the predominant emergence of T315I. Using this or similar approaches, it will be possible to provide information that translates into combinatorial and sequential treatment strategies and to determine critical plasma concentrations for mutations that might occur during treatment.

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Optimal Treatment for Patients after Dasatinib or Nilotinib: Comparison of Preclinical Activities of Approved and Promising Investigational Kinase Inhibitors Against BCR-ABL Kinase Domain Mutations That Confer Clinical Resistance to Dasatinib or Nilotinib.

Blood ◽

10.1182/blood.v110.11.4589.4589 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4589-4589

Author(s):

Corynn Kasap ◽

Christopher Weier ◽

Neil P. Shah

Keyword(s):

Second Generation ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Kinase Domain ◽

Inhibitor Therapy ◽

Drug Resistant ◽

Imatinib Resistance ◽

Abl Kinase ◽

Clinical Resistance ◽

Kinase Domain Mutations

Abstract The optimal management of patients with chronic myeloid leukemia (CML) is increasingly reliant upon molecular studies. Loss of response to imatinib in CML is most commonly associated with selection for a limited number of BCR-ABL kinase domain mutations that impair the ability of imatinib to effectively bind to BCR-ABL Molecular understanding of imatinib resistance mechanisms has led to the development of effective “second generation” BCR-ABL kinase inhibitors, such as dasatinib and nilotinib, which have clinical activity against most, but not all, drug-resistant mutations. Analysis of the BCR-ABL kinase domain in patients who develop resistance to second-generation inhibitors has implicated further selection of drug-resistant BCR-ABL kinase domain mutants in nearly all cases reported to date. Encouragingly, the number of resistant mutations capable of conferring clinical resistance to the most clinically-advanced second-generation agents, dasatinib (approved by the US FDA and EMEA) and nilotinib (approved in Mexico and Switzerland), appears to be restricted to a relatively small number of amino acid substitutions. As clinical experience with dasatinib and nilotinib grows, an understanding of the relative sensitivities of dasatinib- and nilotinib-resistant BCR-ABL mutants to other kinase inhibitors, both approved and investigational, is critical to optimize clinical outcomes in patients with resistance to dasatinib or nilotinib. At the present time, kinase inhibitor therapy options for patients with resistance to one of these agents include the investigational options bosutinib and MK-0457 (VX-680), as well as dasatinib and nilotinib (for patients not yet exposed to one of these agents) and re-exposure imatinib. It is likely that the success of therapeutic intervention in these cases can be predicted based upon the preclinical sensitivity of the mutation(s) involved with the agent chosen. We have therefore conducted a thorough biochemical and biological cross-analysis of the activities of each of these clinically-useful kinase inhibitors against mutations that confer clinical resistance to dasatinib or nilotinib. These studies provide clinicians with a useful reference for choosing an appropriate kinase inhibitor based upon the identity of the resistant BCR-ABL kinase domain mutation(s) detected at the time of relapse when faced with a patient who has lost response to dasatinib or nilotinib. It is hoped that the application of such “personalized medicine” strategies to the clinical management of CML cases will further improve outcomes in patients treated with kinase inhibitor therapy.

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Overcoming Resistance in Chronic Myelogenous Leukemia

American Society of Clinical Oncology Educational Book ◽

10.14694/edbook_am.2013.33.306 ◽

2013 ◽

pp. 306-312

Author(s):

Michael J. Mauro

Keyword(s):

Chronic Myelogenous Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Point Mutations ◽

Kinase Domain ◽

Drug Binding ◽

Myelogenous Leukemia ◽

Novel Therapy ◽

Abl Kinase ◽

Clinical Resistance

Resistance in chronic myelogenous leukemia is an issue that has developed in parallel to the availability of rationally designed small molecule tyrosine kinase inhibitors to treat the disease. A significant fraction of patients with clinical resistance are recognized to harbor point mutations/substitutions in the Abl kinase domain, which limit or preclude drug binding and activity. Recent data suggest that compound mutations may develop as well. Proper identification of clinical resistance and prudent screening for all causes of resistance, ranging from adherence to therapy to Abl kinase mutations, is crucial to success with kinase inhibitor therapy. There is currently an array of Abl kinase inhibitors with unique toxicity and activity profiles available, allowing for individualizing therapy beginning with initial choice at diagnosis and as well informed choice of subsequent therapy in the face of toxicity or resistance, with or without Abl kinase domain mutations. Recent studies continue to highlight the merits of increasingly aggressive initial therapy to subvert resistance and importance of early response to identify need for change in therapy. Proper knowledge and navigation amongst novel therapy options and consideration of drug toxicities, individual patient characteristics, disease response, and vigilance for development of resistance are necessary elements of optimized care for the patient with chronic myelogenous leukemia.

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The K247R Substitution in ABL Tyrosine Kinase Domain Is Not a Mutation Leading to Imatinib Resistance but a Polymorphism Rarely Present in CML Patients and in Normal Subjects.

Blood ◽

10.1182/blood.v106.11.4831.4831 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4831-4831

Author(s):

Kaddour Chabane ◽

Franck Nicolini ◽

Jean-Michel Cayuela ◽

Philippe Rousselot ◽

Xavier Thomas ◽

...

Keyword(s):

Epithelial Cells ◽

Tyrosine Kinase ◽

Disease Progression ◽

Direct Sequencing ◽

Point Mutations ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Molecular Response ◽

Imatinib Resistance ◽

Abl Tyrosine Kinase

Abstract The major mechanism for resistance to imatinib mesylate (IM) is the onset of ABL point mutations altering functional inhibition of the tyrosine kinase activity by IM. Biochemical, cellular assays and clinical studies have demonstrated that different BCR-ABL mutations exhibit various degrees of resistance, and mutations occurring in the ATP-binding loop may be correlated with subsequent disease progression. In this study, we investigated the status of the K247R ABL polymorphism and correlated it to disease outcome. Patients and methods: Two patients (P1 and P2) were diagnosed with CML based on peripheral blood findings, karyotyping analysis (showing t(9;22)(q34;q11)) and molecular analysis for BCR-ABL (indicating M-BCR fusion transcripts) in 2,000 and 1,994, respectively. P1, a 54 years old male, enrolled in the Novartis IRIS study, was randomly assigned to the interferon + AraC arm after informed consent. The patient achieved a complete hematologic remission (CHR) at 3 months and a complete cytogenetic response (CCR) at 24 months. At time of cytogenetic relapse (2 years later), P1 had never received IM which was introduced in April 2,005. P2 received IM (400mg to 600mg/day) during 82 months and had shown only CHR during 14 months with no MCR. Epithelial cells were collected in the mouth through sterile foam tipped applicators for both patients. DNA was obtained from blood samples of 232 individuals, including 124 patients with CML, 72 patients with acute myeloid leukemias (AML) and 36 normal healthy donors. The K247R change was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique (AluI restriction enzymatic site abolished) and was confirmed by direct sequencing. Results: Both patients, showing the K247R substitution located close to the P-loop, were retrospectively investigated during disease progression. We found that this change accounted for 100% of the BCR-ABL transcripts at CML diagnosis (and in all the further samples) for P1 (no material was yet available from P2 at this time) and for 50% of the ABL gene in extracted DNA from epithelial cells in the 2 patients. P1, despite of the K247R substitution received IM (400 mg/day) and achieved a CHR, a CCR and a good molecular response (one log reduction after two months of IM). IM resistance observed in P2 was probably due to the presence of an additional F317L mutation, known to induce a very strong resistance to IM. The F317L mutation accounted for 100% of the BCR-ABL transcripts at the resistance, was absent in DNA from epithelial cells. The screening showed the K247R exchange in 1 normal subject (heterozygote), in 2 CML patients (P1 and P2: at heterozygous state in normal cells and at homozygous state in BCR-ABL transcripts) and in none AML samples. According to that, the incidence of this polymorphism seems to reach nearly 1 to 1.5%. Conclusion: This study demonstrates that the K247R substitution in ABL tyrosine kinase domain is not a mutation leading to IM resistance but only a rare polymorphism. Detailed analysis of this polymorphism status will be reported.

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