Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients

// Elisa Zuffa 1 , Eugenia Franchini 1 , Cristina Papayannidis 1 , Carmen Baldazzi 1 , Giorgia Simonetti 1 , Nicoletta Testoni 1 , Maria Chiara Abbenante 1 , Stefania Paolini 1 , Chiara Sartor 1 , Sarah Parisi 1 , Giovanni Marconi 1 , Federica Cattina 2 , Maria Teresa Bochicchio 1 , Claudia Venturi 1 , Emanuela Ottaviani 1 , Michele Cavo 1 , Giovanni Martinelli 1 1 “Seragnoli” Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy 2 Bone Marrow Transplant Unit, University of Brescia, Brescia, Italy Correspondence to: Cristina Papayannidis, e-mail: cristina.papayannidis@unibo.it Keywords: AML, FLT3, ultra-deep sequencing, clonal evolution, minimal residual disease Received: April 21, 2015 Accepted: August 24, 2015 Published: September 05, 2015 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.