Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific 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.

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

Blood ◽

10.1182/blood.v108.11.4814.4814 ◽

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.

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Analysis of BCR–ABL1 tyrosine kinase domain mutational spectra in primitive chronic myeloid leukemia cells suggests a unique mutator phenotype

Leukemia ◽

10.1038/leu.2010.179 ◽

2010 ◽

Vol 24 (10) ◽

pp. 1817-1821 ◽

Cited By ~ 8

Author(s):

H Grant ◽

X Jiang ◽

J Stebbing ◽

L Foroni ◽

C Craddock ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Leukemia Cells ◽

Tyrosine Kinase Domain ◽

Mutator Phenotype ◽

Mutational Spectra

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Secondary mutations as mediators of resistance to targeted therapy in leukemia

Blood ◽

10.1182/blood-2014-10-605808 ◽

2015 ◽

Vol 125 (21) ◽

pp. 3236-3245 ◽

Cited By ~ 66

Author(s):

Naval Daver ◽

Jorge Cortes ◽

Farhad Ravandi ◽

Keyur P. Patel ◽

Jan A. Burger ◽

...

Keyword(s):

Targeted Therapy ◽

Tyrosine Kinase ◽

Small Molecule ◽

Myeloid Leukemia ◽

Point Mutations ◽

Kinase Domain ◽

Lymphocytic Leukemia ◽

Tyrosine Kinase Domain ◽

Flt3 Inhibitors ◽

Kinase Domain Mutations

AbstractThe advent of small molecule-based targeted therapy has improved the treatment of both acute and chronic leukemias. Resistance to small molecule inhibitors has emerged as a common theme. The most frequent mode of acquired resistance is the acquisition of point mutations in the kinase domain. FLT3 inhibitors have improved response rates in FLT3-mutated acute myeloid leukemia (AML). The occurrence of the ATP-binding site and activation loop mutations confers varying degrees of resistance to the individual FLT3 inhibitors. Second-generation FLT3 inhibitors such as crenolanib may overcome the resistance of these mutations. Furthermore, nonmutational mechanisms of resistance such as prosurvival pathways and bone marrow signaling may be upregulated in FLT3 inhibitor-resistant AML with secondary kinase domain mutations. More recently, point mutations conferring resistance to the Bruton tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia, arsenic trioxide in acute promyelocytic leukemia, and the BH3-mimetic ABT199 in lymphoma have been identified. In chronic myeloid leukemia, the emergence of tyrosine kinase domain mutations has historically been the dominant mechanism of resistance. The early identification of secondary point mutations and their downstream effects along with the development of second- or third-generation inhibitors and rationally designed small molecule combinations are potential strategies to overcome mutation-mediated resistance.

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Absolute Quantitative Detection of Abl Tyrosine Kinase Domain Mutations Using a Novel Microfluidic Platform and Allele-Specific PCR.

Blood ◽

10.1182/blood.v106.11.2859.2859 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2859-2859

Author(s):

Vivian Oehler ◽

Suchitra Ananthnarayan ◽

Frank McCormick ◽

Antoine Daridon ◽

Geoff Facer ◽

...

Keyword(s):

Nucleic Acid ◽

Point Mutations ◽

Kinase Domain ◽

Clinical Samples ◽

Tyrosine Kinase Domain ◽

Abl Tyrosine Kinase ◽

Specific Pcr ◽

Allele Specific ◽

T315i Mutation ◽

Acid Sample

Abstract Point mutations in the Abl tyrosine kinase domain are the main mechanism of secondary resistance to imatinib mesylate (IM) therapy in chronic myeloid leukemia (CML) patients. Current mutation detection approaches including direct sequencing, DHPLC, and SSCP range in sensitivity from 5% to 20%. Detection of mutations in early treatment and possibly pre-treatment samples could allow patient therapy to be altered before resistance is detected cytogenetically or disease progression. A sensitive assay using a novel nanofluidic chip designed for digital isolation and detection (DID) by mutant allele-specific amplification was used to detect rare mutant copies of Abl in a very high background of wild-type (Wt) Abl cDNA. The DID chip partitions a nucleic acid sample into thousands of isolated reaction chambers, thereby enriching rare targets and enabling absolute quantitative, specific and sensitive detection of rare sequences and mutations. Each sample mix of 7.2 mcl containing the necessary reagents for a quantitative PCR 5′-nuclease assay is filled into a nanofluidic network. The sample is partitioned into 1,200 chambers using microfluidic valves fabricated by multilayer-soft-lithography. Each 6 nl reaction chamber contains a fraction of the original nucleic acid sample and reagents for detecting the target of interest. Since the mutant target is rare, the majority of partitions will not contain the mutant, while a small number of partitions will contain just one mutant copy. Single copy detection using the DID chip is reproducible and reliable. For initial proof of concept experiments a set of assays able to detect a group of p-loop mutations and the T315I mutation were optimized using model mixtures. Studies by French centers and Australian groups suggest that these mutations confer worse prognosis and the T315I mutation is refractory to other targeted therapies. Mutation-specific PCR assays were developed for the following mutations: G250E, Q252H (2 substitutions), Y253F, E255K, E255V, and T315I. Initial experiments utilized mutated Bcr-Abl cDNA and plasmids serially diluted into the background of 105 equivalents of Wt Bcr-Abl cDNA from a transformed BaF3 cell line. As few as 9-88 copies were specifically and reliably detected in each of the mutation-specific assays in the background of 105 Wt copies. A non-extendable blocker primer for the Wt and streptavidin beads are also being tested to enrich for mutants prior to PCR. Wt Abl is also quantified to allow for the determination of percent Abl mutated in the assays applied to clinical samples. Conclusion: This method utilizes a novel platform to provide highly sensitive and specific detection of known point mutations and can be applied to early clinical samples. The assay is unique in that it can be extended to a large group of mutations as detection is achieved in very small amounts of rare samples partitioned into thousands of wells prior to PCR. Figure 1 Figure 1.

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