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 ◽  
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.

Point Mutation and Alternative Splicing in the ABL Kinase Domain as a Mechanism for Imatinib Resistance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4814-4814
Author(s):  
Rong Chen ◽  
Steven Potts ◽  
Wanlong Ma ◽  
Hagop Kantarjian ◽  
Francis Giles ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Tyrosine Kinase ◽  
Point Mutations ◽  
Sr Proteins ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Imatinib Resistance ◽  
Exon Junction ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Abstract Missense point mutations in the region encoding the ABL tyrosine kinase domain have been reported in approximately 35% of patients with imatinib-resistant chronic myeloid leukemia (CML). The reported mutations result in reactivation of the BCR-ABL tyrosine kinase. Screening patients with imatinib-resistant CML, we identified 42 different mutations in the ABL tyrosine kinase domain-encoding region, 2 of which were silent (no amino acid change): A864G and G909A. The A864G mutation was associated with a 54-nucleotide reduction in the length of the mRNA transcript, representing a loss of nucleotides 1089-1143; the G909A mutation was associated with a normal-length transcript. The nt1089-1143 transcript deletion represents a partial exon deletion in which the first half of exon 8 is skipped, suggesting that A864G leads to abnormal splicing. Splicing is regulated by 6- to 8-nucleotide exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs recognized by the SR proteins (a family of splicing factors). We therefore used ESEfinder to examine whether A864G or G909A alter ESE motifs, which could block the ability of SR proteins to recognize and bind. This search showed that A864G is at the 7th position of an AGCTGCAG ESE motif, a binding site for SR35, and is within 35 bp of the intron-exon junction. In total, ESEfinder predicted 18 putative SR35-binding ESEs within 50 bp of the intron-exon junction, covering 20% of the kinase domain. The AGCTGCAG motif is conserved in primates (chimpanzees and monkeys) but not in rodents, while A864G is found in mice Although similar links cannot be made with the G909A mutation, these data suggest that imatinib resistance may develop in some patients through alternative splicing and the expression of a truncated (or potentially elongated) protein.

Management of Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL)

Hematology ◽  
2009 ◽  
Vol 2009 (1) ◽  
pp. 371-381 ◽  
Author(s):  
Oliver G. Ottmann ◽  
Heike Pfeifer
Keyword(s):  
Tyrosine Kinase ◽  
Second Generation ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Molecular Response ◽  
Front Line ◽  
Imatinib Resistance ◽  
Term Survival ◽  
Abl Tyrosine Kinase ◽  
Tki Treatment

AbstractThe tyrosine kinase inhibitor (TKI) imatinib has become an integral part of front-line therapy for Ph+ ALL, with remission rates exceeding 90% irrespective of whether imatinib is given alone or combined with chemotherapy. Treatment outcome with imatinib-based regimens has improved compared with historic controls, but most patients who do not undergo allogeneic stem cell transplantation (SCT) eventually relapse. Acquired resistance on TKI treatment is associated with mutations in the bcr-abl tyrosine kinase domain in the majority of patients, and may be detected at low frequency prior to TKI treatment in a subset of patients. Second generation TKIs, eg, dasatinib and nilotinib, show activity against most of the bcr-abl tyrosine kinase domain (TKD) mutations involved in acquired imatinib resistance, but clinical benefit is generally short-lived. Accordingly, SCT in first complete remission (CR) is considered to be the best curative option. Molecular monitoring of minimal residual disease levels appears to have prognostic relevance and should be used to guide treatment. International standardization and quality control efforts are ongoing to ensure comparability of results. Mutation analysis during treatment relies increasingly on highly sensitive PCR techniques or denaturing HPLC and may assist in treatment decisions, eg, in case of molecular relapse. Results from current studies of second-generation TKI as front-line treatment for Ph+ ALL are promising and show high molecular response rates, but follow-up is still too short to determine their impact on remission duration and long-term survival. Strategies to improve outcome after SCT include the pre-emptive use of imatinib, which appears to reduce the relapse rate. In patients ineligible for transplantation, novel concepts for maintenance therapy are needed. These could involve novel immunotherapeutic interventions and combinations of TKI.

Bcr-Abl Tyrosine Kinase Domain Mutations Both Pre-Exist and Develop during Imatinib Treatment and Are Responsible for Resistance in Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 639-639 ◽  
Author(s):  
Heike Pfeifer ◽  
Barbara Wassmann ◽  
Anna Pavlova ◽  
Lydia Wunderle ◽  
Patrick Brueck ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
De Novo ◽  
Point Mutations ◽  
Kinase Domain ◽  
Disease Stage ◽  
Tyrosine Kinase Domain ◽  
Imatinib Treatment ◽  
Newly Diagnosed ◽  
Imatinib Resistance ◽  
Prior Chemotherapy

Abstract Background: Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL are an important cause of resistance to imatinib (IM) in pts. with CML and Ph+ ALL. The significantly inferior response to IM in Ph+ALL pts. who failed prior chemotherapy compared to those with de novo Ph+ALL suggests that treatment with cytotoxic drugs may promote the development of TKD mutations. However, it is not known whether the frequency and pattern of TKD mutations at the time of treatment initiation with TK inhibitors are related to disease stage or prior anti-leukemic therapy. Moreover, the potential of combined treatment with IM and multi-agent chemotherapy to influence the development of mutational resistance, as compared to IM alone, has not been determined. Patients and methods: 51 pts. with newly diagnosed Ph+ALL (>55 yrs.) enrolled in a GMALL study of combined IM and chemotherapy, and 68 Ph+ALL pts. who had failed prior chemotherapy and received single-agent IM as salvage therapy were analysed for the occurrence of point mutations within the TKD. Bone marrow samples collected pre-treatment, during therapy and at relapse were examined by denaturing high-performance liquid chromatography (D-HPLC) and cDNA sequencing. Results: The frequency of TKD mutations pre-IM was 44% in newly diagnosed Ph+ALL and 53% (34/64) in pts. with advanced Ph+ALL. At relapse after combination therapy (n=19), the frequency of de novo ALL pts. harbouring a TKD mutation had increased to 89% (P-loop 47%, T315I 29%, A-loop 24%), 2 pts. (11%) showed wild-type BCR-ABL. The frequency of TKD mutations in pts. with advanced disease who relapsed after IM was 55% (P-loop 73%, T315I 23%, A-loop 4 %). In both patient groups, the D-HPLC pattern showed concordance between the mutation detected in pre-therapeutic specimens and the dominant mutation detected at relapse. The CR rate in de novo pts. receiving IM induction was 90 % irrespective of detectable mutations pre-study. Bcr-abl transcripts became undetectable during the course of therapy in 40% of pts. with and 37% of pts. without a mutation. Median remission duration in pts. with a T315I mutation (n=4) was 130 d (range: 53–319d), in contrast to 526 d (range: 504–549d) with activation loop and 411 d (range: 106–745d) with P-loop mutations. To date, 7 pts. with an initially detected mutation remain in CR after median FU of 12.8 mo (range 2.4–24.5 mo.). Conclusions: Bcr-abl TKD mutations are detectable prior to first imatinib exposure in approximately 50% of Ph+ALL patients. Clinical imatinib resistance is in most cases associated with the identical mutation detected pre-IM, which is not eradicated by the combination of chemotherapy and IM. Identification and elimination of TKD mutations during early stages of treatment is essential to improve treatment.

scholarly journals 4558 Investigating the functional consequences of anaplastic lymphoma kinase (ALK) mutations arising upon Lorlatinib treatment

2020 ◽  
Vol 4 (s1) ◽  
pp. 9-10
Author(s):  
Gabriela Maria Witek ◽  
Whelton Miller ◽  
David Slochower ◽  
Esther Berko ◽  
Yael Mossé ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Anaplastic Lymphoma Kinase ◽  
De Novo ◽  
Point Mutations ◽  
Md Simulations ◽  
Competitive Inhibitor ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Anaplastic Lymphoma ◽  
Mechanism Of Resistance

OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.

scholarly journals Denaturing-HPLC-Based Assay for Detection of ABL Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib

2004 ◽  
Vol 50 (7) ◽  
pp. 1205-1213 ◽  
Author(s):  
Simona Soverini ◽  
Giovanni Martinelli ◽  
Marilina Amabile ◽  
Angela Poerio ◽  
Michele Bianchini ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Treatment ◽  
Abl Tyrosine Kinase ◽  
Reverse Transcription Pcr

Abstract Background: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor Imatinib mesylate for the treatment of chronic myeloid leukemia (CML), resistance has been observed in a proportion of cases, especially those with advanced stages of the disease. Point mutations within the ABL kinase domain are emerging as the most frequent mechanism for reactivation of kinase activity within the leukemic clone. Methods: We developed a denaturing-HPLC (D-HPLC)-based assay for screening for ABL point mutations. For each sample, two partially overlapping fragments of 393 and 482 bp corresponding to the kinase domain were amplified by nested reverse transcription-PCR and analyzed under selected temperature and acetonitrile gradient conditions. Fifty-one bone marrow and/or peripheral blood specimens from 27 CML patients who showed cytogenetic resistance to Imatinib were screened in parallel by D-HPLC and by direct sequencing. Results: In 12 of 27 (44%) patients, D-HPLC showed an abnormal elution profile suggesting the presence of a nucleotide change. Direct sequencing confirmed the presence of a point mutation in all cases. Conversely, all samples scored as wild type by D-HPLC showed no evidence of mutations by direct sequencing. In two cases, novel amino acid substitutions at codons already known for being hot-spots of mutation were identified (F311I and E355D). Conclusions: The proposed D-HPLC-based assay is highly specific and at least as sensitive as sequencing; with respect to the latter, it provides a much faster and less expensive semiautomated system for mutational screening. It may therefore potentially be a valuable tool for regular, large-scale testing of patients undergoing Imatinib treatment.

ABCB1 Overexpression May Predispose Imatinib Treated CML Patients to the Development of Abl Kinase Domain Mutations, and May Be an Important Contributor to Acquired Resistance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2144-2144 ◽  
Author(s):  
Deborah White ◽  
Phuong Dang ◽  
Amity Venables ◽  
Verity Saunders ◽  
Stephanie Zrim ◽  
...  
Keyword(s):  
Protein Expression ◽  
Disease Progression ◽  
Acquired Resistance ◽  
Myeloid Cells ◽  
Kinase Domain ◽  
Molecular Response ◽  
Imatinib Resistance ◽  
Treatment Limitation ◽  
Immature Myeloid Cells ◽  
Kinase Domain Mutations

Abstract Imatinib has resulted in excellent and sustained clinical response in most patients with CML. For a few patients, a treatment limitation has been the development of imatinib resistance, frequently the result of kinase domain mutations. The cause of increased susceptibility to mutation development remains unknown. ABCB1 is a recognized efflux transporter of imatinib. Previous studies have demonstrated high ABCB1 expression in imatinib resistant cell lines. We hypothesize that ABCB1, by facilitating drug efflux and therefore limiting the intracellular concentration of imatinib, may contribute to resistance and mutation development. Using RQ-PCR for ABCB1 expression relative to the control gene BCR, flow cytometric analysis and radioactive drug intracellular uptake and retention studies (IUR) we have assessed 32 imatinib treated chronic phase CML patients pre therapy. 29/32(90%) patients had expression of ABCB1 mRNA less than 65% of control (median 49% range 21–65%). The three patients with higher expression of ABCB1 (73%, 130% and 105%) all subsequently developed kinase domain mutations and disease progression. Only 1/29 patients with low mRNA (<65%) expression subsequently developed a mutation. While the depth of molecular response to imatinib of the remaining 28 patients varied, disease progression was not observed in any patient, and ABCB1 was not predictive of molecular response(p=0.74). We have also analyzed samples from 4 patients after they developed imatinib resistance not associated with kinase domain mutations. We found significantly higher levels of ABCB1 mRNA(median 308% range 178–408% p<0.001), and protein expression (control: median 0.3% range 0.2–7%, non-mutational resistance: median 7% range 2–15% p=0.005). There was also a significantly lower IUR at 2 hrs (control: median 22ng range 11–36ng, non-mutational resistance: median 13ng range 10–16ng p=0.006). Further, in 2 of the 4 patients we performed imatinib IUR in the presence of the ABCB1 inhibitor PSC833. In both cases inhibition of ABCB1 significantly increased IUR (2hr IUR 11.4 and 11.5 ng: 2hr IUR + PSC833 18.8 and 18.9 ng).This finding was not observed in 25 control samples tested (IUR 25.4ng, IUR +PSC833 - 16.56ng/200,000 cells). We have performed longitudinal studies on one patient who developed initial resistance to imatinib, then to dasatinib. An additional copy of the Ph, was reported at the time of resistance, but no kinase domain mutation. Over the disease course we observed a 7 fold rise in the mRNA expression of ABCB1 and 49 fold increase in ABCB1 protein expression (from 0.3 to 14.7%). This rise in expression of ABCB1 cannot be explained by an increase in percentage of immature myeloid cells. Patient with imatinib resistance. %ABCB1 mRNA % BCR-ABL %Immature myeloid cells IUR imatinib (ng) IC50 uM Diagnosis 4/5/01 70 89 32 19 2.25 On imatinib 10/1/02 68 0.06 4 28 NA On imatinib 13/5/03 85 0 0 NA NA Relapse 5/5/05 178 326 12 14 8 Relapse on dasatinib 27/2/06 486 6.2 4 15 NA We conclude that patients with high expression of ABCB1 at diagnosis may be predisposed to mutation development. Furthermore increasing expression of ABCB1 over time may be a valuable contributor to acquired resistance. ABCB1 activation may be an important prognostic marker, and potential target for pharmacological manipulation.

Rapid Identification Of Compound Mutations In Patients With Ph-Positive Leukemias By Long-Range Next Generation Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1373-1373
Author(s):  
Sandra Preuner ◽  
Renate Kastner ◽  
Agnes Zopf ◽  
Proell Johannes ◽  
Pierre Foskett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Long Range ◽  
Sanger Sequencing ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Insertions And Deletions ◽  
Dna Molecule ◽  
Generation Sequencing

Abstract Mutations in the BCR-ABL1 tyrosine kinase domain (TKD) are regarded as the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in patients with Ph-positive leukemias. The occurrence of two or more mutations on the same DNA molecule, the so-called compound mutations, can be associated with particularly high resistance to multiple TKIs. Recent reports indicate that the frequency of compound mutations is rather high, thus rendering their reliable detection an important diagnostic challenge 1,2. Analysis of PCR amplicons of the BCR-ABL1 TKD by next generation sequencing (NGS) has become the method of choice for sensitive detection of compound mutations. This approach is, however, hampered by the requirement of 3-4 overlapping amplicons to cover the entire TKD due to the limited read length offered by most current NGS technologies. This prevents the assignment of nucleotide substitutions located on different amplicons to the same TKD/DNA molecule, and therefore requires additional laborious steps to facilitate unequivocal identification of such constellations. To overcome this limitation, we have established a long-range NGS approach on the FLX instrument (Roche) permitting the coverage of the entire TKD length of ∼0.9 kb in a single read. By testing a series of individual and consecutive specimens derived from five patients with chronic myeloid leukemia, we demonstrate that long-range NGS analysis readily permits the identification of mutations and their assignment to the same or to separate subclones at a limit of sensitivity comparable to NGS-based sequencing of shorter amplicons. In addition to the detection of individual and compound mutations, this approach also facilitates an interpretable documentation of insertions and deletions in the TKD. To address the possibility of artifacts inherent in the technique that could lead to incorrect identification of single and compound mutations, the NGS findings were reevaluated by independent technical approaches. Point mutations were confirmed by Sanger sequencing, LD-PCR 3 and pyrosequencing 4. In select cases, PCR amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®T easy plasmids, and >100 clones were subjected to analysis by Sanger sequencing. The observations made by NGS analysis including various single mutations (e.g. G250E, Y253H, T315A, F317I, Q252H, T315I), compound mutations (e.g. G250E/Y253H, G250E/T315A, G250E/F317I), and combinations of point mutations with small insertions or deletions (e.g. E459K/C475fs, Q252H/R362fs, T315I/R362fs) as well as large deletions involving multiple exons, could be confirmed in individual clones by Sanger sequencing, thereby documenting the reliability of the long-range NGS technology. The technical advancement presented therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, thus extending the diagnostic armamentarium for rapid assessment of impending resistant disease. 1. Khorashad JS, Kelley TW, Szankasi P, et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood. 2013;121(3):489-498. 2. Soverini S, De Benedittis C, Machova Polakova K, et al. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. Blood. 2013. 3. Preuner S, Denk D, Frommlet F, Nesslboeck M, Lion T. Quantitative monitoring of cell clones carrying point mutations in the BCR-ABL tyrosine kinase domain by ligation-dependent polymerase chain reaction (LD-PCR). Leukemia. 2008;22(10):1956-1961. 4. Alikian M, Gerrard G, Subramanian PG, et al. BCR-ABL1 kinase domain mutations: methodology and clinical evaluation. Am J Hematol. 2012;87(3):298-304. Figure Strategy of long-range NGS analysis for the detection of single and compound mutations, insertions and deletions in the BCR-ABL1 TKD. Figure. Strategy of long-range NGS analysis for the detection of single and compound mutations, insertions and deletions in the BCR-ABL1 TKD. Disclosures: Valent: Novartis: Honoraria, Research Funding. Lion:Novartis, Bristol-Myers- Squibb, Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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