Role of Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in relapsed acute promyelocytic leukaemia (APL) treated with oral arsenic trioxide

Dr Singh, Gill Harinder Harry   (Principal Investigator (PI))

Professor Kwong Yok Lam   (Co-Investigator)

Professor Leung Anskar Yu Hung   (Co-Investigator)

12

2015-02-12

2016-02-11

120000

Role of Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in relapsed acute promyelocytic leukaemia (APL) treated with oral arsenic trioxide

Acute promyelocytic leukaemia, Fms-like tyrosine kinase 3 internal tandem duplication, Oral arsenic trioxide, Relapse

Blood/Hematology,Cancer

201501159011

Seed Fund for Basic Research for New Staff

2014

Completed

Background. Acute promyelocytic leukaemia (APL) is a distinct subgroup of acute myeloid leukaemia (AML) characterized by translocation t(15;17) (q22;q12). Up to 20% of patients relapse after initial remission. Arsenic trioxide (As2O3) is highly effective for relapsed APL and is increasingly used as frontline treatment for newly diagnosed APL. It is anticipated that an increasing number of patients will be exposed to As2O3-based therapy and drug resistance to As2O3 is a consequent emerging clinical problem in the modern management of APL. We have developed an oral formulation of As2O3 and it is currently the standard of care for maintenance after first complete remission (CR1) and re-induction for relapsed APL in Hong Kong. Despite its efficacy as maintenance after CR1 and as re-induction in relapsed APL, subsequent relapse after oral As2O3-based treatment has been observed in up to 40% of patients and is associated with a dismal outcome. An association between the presence of fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) and relapse after exposure to oral As2O3 was observed. We hypothesize that FLT3-ITD+ APL clones persist and are selected during oral As2O3 therapy accounting for subsequent relapse. Objectives. To prove this hypothesis, we have designed a series of experiments that may enable us to understand the role of FLT3-ITD in relapsed APL following treatment with oral As2O3. The following issues will be addressed: 1. Do FLT3-ITD+ APL clones persist during and after oral As2O3 treatment? 2. Does FLT3-ITD in APL confer resistance to As2O3? 3. Does FLT3-ITD+ APL show differences in leukaemia-initiating activity compared with FLT3-WT APL? Methods and Plans. Objective 1. Do FLT3-ITD+ APL clones persist during and after oral As2O3 treatment? Retrospective patients with relapsed APL with FLT3-ITD will be studied. Serial archived peripheral blood (PB), bone marrow aspirate (BM) and cerebrospinal fluid (CSF) (in patients with central nervous system involvement) with relapsed APL during or after treatment with oral As2O3 will be evaluated for FLT3 mutations. Clonospecific primers will be designed based on the FLT3-ITD at relapse. Clonospecific polymerase chain reaction (PCR) of FLT3-ITD will be evaluated in the retrospective primary samples serially archived at initial diagnosis, remission and during previous oral As2O3 treatment. Objective 2. Does FLT3-ITD in APL confer resistance to As2O3? FLT3-ITD will be transfected to the human APL cell line NB4 to establish an in-vitro model of FLT3-ITD+ APL. Changes in cellular morphology, differentiation, apoptosis, proliferation, clonogenic activity associated with FLT3-ITD will be evaluated. Sensitivity of FLT3-ITD+ NB4 to arsenic trioxide with respect to apoptosis and differentiation and compared to FLT3 wild-type(WT) NB4. This will be repeated in other FLT3-ITD+ and FLT3-WT AML cell lines Objective 3. Does FLT3-ITD+ APL show differences in leukaemia-initiating activity compared with FLT3-WT APL? FLT3-WT and FLT3-ITD+ NB4 cell lines will be transplanted intravenously into 6-8 weeks old NSG-3GS mice and their engraftment will be enumerated. serial primary FLT3-WT and FLT3-ITD+ APL cells (mononuclear cells after density gradient centrifugation) that are oral As2O3 naïve and oral As2O3-resistant (those not responding to oral As2O3-based induction/re-induction or relapsing after exposure to oral As2O3-based maintenance) will be transplanted at the same cell doses for each patient. The absolute levels of engraftment, the lineage differentiation and engraftment kinetics as well as the presence of FLT3-ITD clones will be evaluated.