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

Soverini, Simona; Martinelli, Giovanni; Amabile, Marilina; Poerio, Angela; Bianchini, Michele; Rosti, Gianantonio; Pane, Fabrizio; Saglio, Giuseppe; Baccarani, Michele

doi:10.1373/clinchem.2004.031112

Cited by 115 publications

(63 citation statements)

References 34 publications

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“…3 Sequencing for ABL1 kinase domain mutation analyses was performed as previously described 4 with the following forward primer being used to amplify the p190 BCR/ABL1 transcript: 5'-ACCATCGTGGGCGTCCGCAAGA-3'.…”

Section: Real-time Polymerase Chain Reaction and Mutation Analysismentioning

confidence: 99%

Autologous transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia achieves outcomes similar to allogeneic transplantation: results of CALGB Study 10001 (Alliance)

et al. 2013

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Allogeneic stem cell transplantation is the standard approach to Philadelphia chromosome positive acute lymphoblastic leukemia. We hypothesized that imatinib plus sequential chemotherapy will result in significant leukemia cell cytoreduction in patients with Philadelphia chromosome positive acute lymphoblastic leukemia, allowing collection of normal hematopoietic stem cells uncontaminated by residual BCR/ABL1 + lymphoblasts and thus reduce the likelihood of relapse after autologous stem cell transplantation for patients under 60 years of age without sibling donors. We enrolled 58 patients; 19 underwent autologous and 15 underwent allogeneic stem cell transplantation on study. Imatinib plus sequential chemotherapy resulted in reverse-transcriptase polymerase chain reaction-negative stem cells in 9 patients and remained minimally positive in 4 (6 were not evaluable). Overall survival (median 6.0 years vs. not reached) and disease-free survival (median 3.5 vs. 4.1 years) were similar between those who underwent autologous and those who underwent allogeneic stem cell transplantation. We conclude that autologous stem cell transplantation represents a safe and effective alternative for allogeneic stem cell transplantation in Philadelphia chromosome positive acute lymphoblastic leukemia patients without sibling donors (clinicaltrials.gov identifier:00039377).

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Section: Real-time Polymerase Chain Reaction and Mutation Analysismentioning

confidence: 99%

Autologous transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia achieves outcomes similar to allogeneic transplantation: results of CALGB Study 10001 (Alliance)

et al. 2013

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“…The selection for resistance took 10 months. A denaturing highperformance liquid chromatography -based assay was employed for detection of ABL mutations (11). Experiments with resistant cells were carried out under the continuous presence of the compounds.…”

Section: Reagentsmentioning

confidence: 99%

Antileukemia effects of xanthohumol in Bcr/Abl-transformed cells involve nuclear factor-κB and p53 modulation

Monteghirfo

Tosetti

Ambrosini

et al. 2008

Molecular Cancer Therapeutics

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The oncogenic Bcr-Abl tyrosine kinase activates various signaling pathways including phosphoinositide 3-kinase/ Akt and nuclear factor-KB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl(+) myeloid leukemia cells. The hop flavonoid xanthohumol inhibits tumor growth by targeting the nuclear factor-KB and Akt pathways and angiogenesis. Here, we show that xanthohumol has in vitro activity against Bcr-Abl(+) cells and clinical samples and retained its cytotoxicity when imatinib mesylate -resistant K562 cells were examined. Xanthohumol inhibition of K562 cell viability was associated with induction of apoptosis, increased p21 and p53 expression, and decreased survivin levels. We show that xanthohumol strongly inhibited Bcr-Abl expression at both mRNA and protein levels and show that xanthohumol caused elevation of intracellular reactive oxygen species and that the antioxidant N-acetylcysteine blunted xanthohumol-induced events. Further, we observed that xanthohumol inhibits leukemia cell invasion, metalloprotease production, and adhesion to endothelial cells, potentially preventing in vivo life-threatening complications of leukostasis and tissue infiltration by leukemic cells. As structural mutations and/or gene amplification in Bcr-Abl can circumvent an otherwise potent anticancer drug such as imatinib, targeting Bcr-Abl expression as well as its kinase activity could be a novel additional therapeutic approach for the treatment of Bcr-Abl(+) myeloid leukemia. [Mol Cancer Ther 2008;7(9):2692 -702]

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“…7,11 PCR products can also be analysed for mutations using denaturing high-performance liquid chromatography (D-HPLC). 12,13 This method determines the presence of a mutation, but unlike sequencing, does not identify the actual mutation. 13 Assays targeting known mutations are more sensitive, but depend either on existence of restriction sites or allele-specific primer annealing.…”

Section: Introductionmentioning

confidence: 99%

“…12,13 This method determines the presence of a mutation, but unlike sequencing, does not identify the actual mutation. 13 Assays targeting known mutations are more sensitive, but depend either on existence of restriction sites or allele-specific primer annealing. 6,14 Based on the amplification refractory mutation system, 15 we have developed a reverse transcriptase, quantitative PCR assay for detection of mutated clones (qPCR MUT ), a sensitive and practical PCR-based technique for detection and monitoring of selected key mutations associated with imatinib resistance.…”

Section: Introductionmentioning

confidence: 99%

Selecting and deselecting imatinib-resistant clones: observations made by longitudinal, quantitative monitoring of mutated BCR-ABL

et al. 2005

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Resistance to imatinib during the treatment of chronic myeloid leukaemia (CML) is frequently associated with point mutations in the ABL gene encoding the ATP binding region likely to cause disease relapse. Early diagnosis and monitoring of these mutations may be important in order to prevent rapid expansion of resistant clones. We describe a quantitative mutationspecific PCR assay based on the readily available Taqman platform. Selectivity for the mutated target is conferred by mutation-specific primers destabilised by additional mismatches. The assay can be carried out in parallel to standard BCR-ABL quantification and is therefore more quickly compared to standard sequencing procedures. The sensitivity of the assay reaches 0.1%. It also allows for quantitative assessment of mutated clones. By analysing sequential samples of resistant subjects, we show how mutated clones were selected, maintained or deselected depending on the individual treatment setting. The high sensitivity and practical merits of this method makes it a good candidate for prospective molecular surveillance of patients at high risk for imatinib resistance.

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

Cited by 115 publications

References 34 publications

Autologous transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia achieves outcomes similar to allogeneic transplantation: results of CALGB Study 10001 (Alliance)

Autologous transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia achieves outcomes similar to allogeneic transplantation: results of CALGB Study 10001 (Alliance)

Antileukemia effects of xanthohumol in Bcr/Abl-transformed cells involve nuclear factor-κB and p53 modulation

Selecting and deselecting imatinib-resistant clones: observations made by longitudinal, quantitative monitoring of mutated BCR-ABL

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