title: Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients.
abstract: FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2-2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Zuffa
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Franchini
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Papayannidis
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Baldazzi
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Simonetti
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Testoni
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Abbenante
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Paolini
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Sartor
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Sarah
authors.initials: S
authors.lastname: Parisi
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Marconi
authors.affiliation: Bone Marrow Transplant Unit, University of Brescia, Brescia, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Cattina
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Bochicchio
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Venturi
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Ottaviani
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Cavo
authors.affiliation: "Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Martinelli
mesh_terms: D000328:Adult; D000368:Aged; D005260:Female; D005500:Follow-Up Studies; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D009367:Neoplasm Staging; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020080:Tandem Repeat Sequences / Q000235:genetics*; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3
keywords: AML; FLT3; clonal evolution; minimal residual disease; ultra-deep sequencing