Analysis of On-Target and Off-Target Resistance Factors Associated with Gilteritinib Efficacy in Relapsed or Refractory AML Patients with FLT3 Mutations
Authors: Iwata Satoshi , Ishikawa Yuichi , Ushijima Yoko , Miyao Kotaro , Harada Yasuhiko , Nishiyama Takahiro , Hosono Naoko , Watamoto Koichi , Sakaida Emiko , Niimi Keiko , Kobayashi Miki , Ozeki Kazutaka , Nishida Tetsuya , Kurahashi Shingo , Ozawa Yukiyasu , Kim Jeong Hui , Naruse Seara , Nakashima Marie , Kawashima Naomi , Kuwatsuka Yachiyo , Kiyoi Hitoshi 
Published: December 2024

Background: The clinical application of FLT3 inhibitors has improved the prognosis of AML with FLT3 mutation, but the short duration of response to FLT3 inhibitors and the emergence of resistant clones remain challenges. The mechanisms of FLT3 inhibitor resistance can be divided into two categories: cellular intrinsic factors such as acquisition of resistance gene mutations, and extracellular factors, such as FLT3 ligand (FL) stimulation. Particularly, the attenuated inhibitory effect of FLT3 inhibitors through the activation of wild-type FLT3 which is co-expressed in the most of AML cells by FL stimulation needs to be clarified in clinical practice. In this study, we aimed to elucidate the factors affecting gilteritinib efficacy and the mechanisms of resistance using patient samples.
Methods: This study included patients 16 years of age or older with relapsed or refractory AML with FLT3 mutation, and who were scheduled to receive gilteritinib. Clinical information, patient plasma, and AML cells from bone marrow or peripheral blood were collected over time. FL concentrations were measured at the points defined in the study plan and plasma inhibitory activity assay (PIA) assay was performed using 32D cells co-expressing wild-type FLT3 and ITD-FLT3. Targeted sequencing was performed on samples collected at study enrollment and at relapse or refractory stage. Primary endpoint was the association between PIA assay and best clinical response. Secondary endpoints were the relation between the clinical response to gilteritinib and the gene mutation profiles.
Results: 30 patients (15 males and 15 females) were enrolled from April 14, 2020, to December 20, 2022. Median patient age was 68 years. (range 34-84). Median observation period was 815 days (range 193-1173). 26 of the 30 patients were positive for FLT3-ITD mutation and five for TKD mutation with one patient carrying both mutations. Of the 30 patients, 18 had received no chemotherapy within four weeks prior to gilteritinib except for hydroxyurea. Eight received intensive chemotherapy within 15 days, and four were treated with other therapies prior to gilteritinib. The composite complete remission (CRc) rate for all patients was 50%, median overall survival was 250 days and median progression-free survival was 170 days. Coexisting genetic mutations frequently identified were NPM1 (47%), DNMT3A (40%), RUNX1 (17%), TET2 (17%), IDH2 (13%), CEBPA (10%), U2AF1 (10%), and WT1 (10%) mutations, however, there was no correlation with prognosis. The median plasma FL concentration at the initiation of treatment was 0 pg/ml (range 0-199), and patients who reached CRc in the patients with no prior intensive chemotherapy within 4 weeks showed a gradual increase in FL concentration, which was significantly higher at day 29 compared to patients who did not achieve CRc (median 240 pg/ml vs 45 pg/ml, P=0.017).The inhibitory level of FLT3 phosphorylation in PIA assay using plasma on Day 29 of gilteritinib treatment did not correlate with best response in patients with or without CRc achievement. However, patients in prior intensive chemotherapy group showed a significant rapid increase in plasma FL levels on days 4 to 29 after initiation of gilteritinib compared to those in the no prior treatment group, and a decrease in FLT3 phosphorylation inhibitory activity from Day 4 to Day 15 by PIA assay. Of 18 patients with refractory or relapsed disease after gilteritinib treatment, 6 patients were found to be FLT3 mutation negative, and 6 patients had acquired mutations involved in activation of RAS/MAPK pathway.
Conclusion: No correlation was found between the best clinical response and inhibition level of FLT3 phosphorylation in PIA assay at day 29 of gilteritinib monotherapy. In this study, there was no relation between treatment response and on- or off-target resistance mechanisms, suggesting the involvement of multiple factors such as activation of pathways implicated in other drug susceptibilities. In the group of patients who received prior potent chemotherapy within two weeks, there was a rapid increase in plasma FL concentration and a decrease in FLT3 inhibitory activity in PIA assay. In combination of chemotherapy and FLT3 inhibitor, the increase in FL concentration due to chemotherapy and the possibility of decreased inhibitory activity of FLT3 inhibitors during the period of increased FL concentration in AML cells co-expressing wild-type FLT3 should be considered.