title: Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.
abstract: Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.
authors.affiliation: Children's Cancer Research Institute (CCRI), Zimmermannplatz 10, A-1090 Vienna, Austria. sandra.preuner@ccri.at.
authors.forename: Sandra
authors.initials: S
authors.lastname: Preuner
authors.affiliation: Red Cross Transfusion Service for Upper Austria, A-4017 Linz, Austria. agnes.barna@o.roteskreuz.at.
authors.forename: Agnes
authors.initials: A
authors.lastname: Barna
authors.affiliation: Department for Medical Statistics, Medical University of Vienna, A-1090 Vienna, Austria. florian.frommlet@meduniwien.ac.at.
authors.forename: Florian
authors.initials: F
authors.lastname: Frommlet
authors.affiliation: Children's Cancer Research Institute (CCRI), Zimmermannplatz 10, A-1090 Vienna, Austria. stefan.czurda@ccri.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Czurda
authors.affiliation: Children's Cancer Research Institute (CCRI), Zimmermannplatz 10, A-1090 Vienna, Austria. konstantin.byrgazov@ccri.at.
authors.forename: Byrgazov
authors.initials: B
authors.lastname: Konstantin
authors.affiliation: Imperial Molecular Pathology Laboratory, Hammersmith Hospital, Imperial College Healthcare National Health Service (NHS) Trust, London W12 0HS, UK. m.alikian@imperial.ac.uk.
authors.forename: Mary
authors.initials: M
authors.lastname: Alikian
authors.affiliation: Institute of Hematology and Blood Transfusion, 128 20 Prague, Czech Republic. katerina.machova@lf1.cuni.cz.
authors.forename: Katerina
authors.initials: K
authors.lastname: Machova Polakova
authors.affiliation: Hematology Department, Jagiellonian University, 31-501 Krakow, Poland. sachatom@gmail.com.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Sacha
authors.affiliation: Section for Hematology, Department of Medicine, University Hospital of Lund, 221 00 Lund, Sweden. johan.richter@med.lu.se.
authors.forename: Johan
authors.initials: J
authors.lastname: Richter
authors.affiliation: Children's Cancer Research Institute (CCRI), Zimmermannplatz 10, A-1090 Vienna, Austria. thomas.lion@ccri.at.|Department of Pediatrics, Medical University Vienna, A-1090 Vienna, Austria. thomas.lion@ccri.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lion
authors.affiliation: Red Cross Transfusion Service for Upper Austria, A-4017 Linz, Austria. christian.gabriel@o.roteskreuz.at.
authors.forename: Christian
authors.initials: C
authors.lastname: Gabriel
mesh_terms: D055028:Comparative Genomic Hybridization; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D004247:DNA; D016044:Fusion Proteins, bcr-abl
keywords: BCR-ABL1; CML; LD-PCR; NGS; pyrosequencing; quantitative analysis of mutant subclones