A pyrosequencing-based test for detection and relative quantification of the<i>BCR-ABL1</i>T315I point mutation

Schumacher, Jonathan A.; Szankasi, Philippe; Bahler, David W.; Ho, Albert K.; Kelley, Todd W.

doi:10.1136/jcp.2011.089193

Cited by 6 publications

(1 citation statement)

References 26 publications

(29 reference statements)

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“…Methods with sensitivities ranging between 1% and 10% have been set up and proposed by several authors. [37][38][39][40] Nevertheless, the optimal lower detection limit allowing for identification of only those mutants that will outgrow and trigger relapse has not been established yet, 41,42 although we believe that prospective application of UDS in a large series of patients might ultimately clarify this issue. Another "warning" from our observations is that sensitivity of a single or a compound mutant to a TKI in vivo might be dictated by more complex factors than the mere in vitro IC 50 (half maximal inhibitory concentration) value.…”

Section: Discussionmentioning

confidence: 97%

Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

Soverini

Benedittis

Poláková

et al. 2013

Blood

149

162

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Key Points• UDS demonstrated that BCR-ABL KD mutations detectable with conventional methods may just be the tip of the iceberg.• The information provided by conventional Sanger sequencing may not always be sufficient to predict responsiveness to a given TKI.In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted. (Blood. 2013;122(9):1634-1648

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Section: Discussionmentioning

confidence: 97%

Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

Soverini

Benedittis

Poláková

et al. 2013

Blood

149

162

View full text Add to dashboard Cite

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A Differential Fluorescent Receptor for Nucleic Acid Analysis

Bengtson

Kolpashchikov

2013

ChemBioChem

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Differential receptors use an array of sensors to recognize analytes. Each sensor in the array can recognize not one, but several analytes with different rates, so a single analyte triggers a response of several sensors in the array. The receptor thus produces a pattern of signals that is unique for each analyte, thereby enabling identification of a specific analyte by producing a “fingerprint” pattern. We applied this approach for the analysis of DNA sequences of Mycobacterium tuberculosis strains that differ by single nucleotide substitutions in the 81‐bp hot‐spot region that imparts rifampin resistance. The technology takes advantage of the new multicomponent, selfassembling sensor, which produces a fluorescent signal in the presence of specific DNA sequences. A differential fluorescent receptor (DFR) contained an array of three such sensors and differentiated at least eight DNA sequences. The approach requires only one molecular‐beacon‐like fluorescent reporter, which can be used by all three sensors. The DFR developed in this study represents a cost‐efficient alternative to molecular diagnostic technologies that use fluorescent hybridization probes.

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Detection of BCR-ABL1 mutations that confer tyrosine kinase inhibitor resistance using massively parallel, next generation sequencing

2015

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Detection of BCR-ABL1 mutations that confer resistance to tyrosine kinase inhibitors is important for management of patients with t(9;22);BCR-ABL1-positive (Ph+) leukemias. Testing is often performed using Sanger sequencing (SS) which has relatively poor sensitivity. Given the widespread adoption of next generation sequencing (NGS), we sought to reevaluate the testing in the context of NGS methods. We developed an NGS-based BCR-ABL1 mutation test on the Ion Torrent Personal Genome Machine (PGM) to test for resistance mutations, primarily in the kinase domain in BCR-ABL1. We analyzed 508 clinical samples from patients with Ph+ leukemias. In a subset of these samples (n = 97), we conducted a comparison of the NGS results to a classical SS-based test. NGS facilitated detection of low-level mutations (<20 % allele frequency) that were not detectable by SS. In a subset of cases with multiple mutations, NGS was also able to determine if two mutations were on the same molecule (compound) or on separate molecules (polyclonal) but this was limited by the distance between mutated positions and by the effects of apparent distance-dependent PCR recombination. We found 22 compound mutations that centered on one or two key residues including two novel compound mutants: Q252H/Y253H and F311Y/F359I. The advantages of NGS make it a superior method for inventorying BCR-ABL1 resistance mutations. However, data analysis may be complicated by short read lengths and the effects of PCR recombination.

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A pyrosequencing-based test for detection and relative quantification of theBCR-ABL1T315I point mutation

Abstract: The described test is useful for detection and relative quantification of the T315I point mutation in chronic myelogenous leukaemia in a sensitive, specific and reproducible manner.

Cited by 6 publications

References 26 publications

Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

A Differential Fluorescent Receptor for Nucleic Acid Analysis

Detection of BCR-ABL1 mutations that confer tyrosine kinase inhibitor resistance using massively parallel, next generation sequencing

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