title: In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.
abstract: Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.
  
  
  
  a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS.
  
  
  
  DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes.
  
  
  
  DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy. simona.soverini@unibo.it.|Institute of Hematology "L. e A. Seràgnoli", Via Massarenti 9, 40138, Bologna, Italy. simona.soverini@unibo.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Soverini
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: De Benedittis
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Fausto
authors.initials: F
authors.lastname: Castagnetti
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gugliotta
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Mancini
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Luana
authors.initials: L
authors.lastname: Bavaro
authors.affiliation: Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
authors.forename: Katerina
authors.initials: K
authors.lastname: Machova Polakova
authors.affiliation: Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Linhartova
authors.affiliation: Division of Haematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Iurlo
authors.affiliation: Unit of Blood Disease and Stem Cell Transplantation, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Russo
authors.affiliation: Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Pane
authors.affiliation: Department of Clinical and Biological Sciences "S. Luigi Gonzaga" Hospital, University of Turin, Orbassano, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Saglio
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Gianantonio
authors.initials: G
authors.lastname: Rosti
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Cavo
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Baccarani
authors.affiliation: Hematology "L. e A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Martinelli
mesh_terms: D019008:Drug Resistance, Neoplasm; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics; D000071184:Pharmacogenomic Variants; D020641:Polymorphism, Single Nucleotide; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C581634:BCR-ABL1 fusion protein, human; D047428:Protein Kinase Inhibitors; D016044:Fusion Proteins, bcr-abl
keywords: BCR-ABL1; Chronic myeloid leukemia; Deep sequencing; Tyrosine kinase inhibitors; Warning