Defining the Landscape of Resistance Mutations in the Context of Modern Treatment Protocols for Acute Promyelocytic Leukemia (APL).
Authors: Jovanovic Jelena V , Guidez Fabien , Ravandi Farhad , Duprez Estelle , Patel Mukunder , Mills Ken , Kornblau Steven , Aldouri Maadh , Auger Martin , Burnett Alan K , Solomon Ellen , Grimwade David 
Published: April 2020

Abstract
Despite the remarkable success of targeted therapy with all-transretinoic acid (ATRA), acquired resistance remains a potential problem in the treatment of APL. Early studies showed that a substantial proportion of patients with recurrent disease exhibited some degree of clinical resistance to ATRA and in approximately 30% this was associated with acquisition of mutations in the ligand-binding domain (LBD) of the PML-RARα oncoprotein. However, a feature of these early studies was the treatment of patients for prolonged periods with ATRA used as a single-agent for remission induction and/or maintenance. Treatment strategies for the management of APL have developed over the last two decades, with ATRA generally being given simultaneously with anthracycline-based chemotherapy and with 6-MP and methotrexate in maintenance. Despite improvements in outcome, 10–15% of patients still relapse following such schedules. Moreover, the acquisition of ATRA resistance remains potentially relevant, since this agent is still frequently used as a component of relapse therapy. Therefore, to establish rates of resistance due to PML-RARα LBD mutations we analyzed a cohort of 34 patients with molecular (n=12) or hematological relapse (n=22) following standard ATRA and anthracycline-based treatment protocols (i.e. MRC, PETHEMA or AIDA), including 9 entered in the MRC AML15 trial. In addition, we studied relapse material derived from 2 patients treated with a chemotherapy-free approach involving ATRA, arsenic trioxide (ATO) and gemtuzumab ozogamicin (GO) (Estey et al, Blood 2006). Screening for mutations in the region encoding the PML-RARα LBD was performed by long-range RT-PCR and sequence analysis. The assay was optimized using a resistant subclone of the NB4 cell line (NB4-LR2); interestingly we found two mutations in the PML-RARα LBD region, namely F310L and E401X, rather than the published Q411X mutation. Only 2 of 34 patients (~6%) relapsing following ATRA and anthracycline-based chemotherapy were found to have alterations in the PML-RARα LBD, which were novel missense mutations (L224H and Y208D), mapping to the extreme 5′ portion of the domain. Both had received PETHEMA/AIDA-based treatment schedules, with hematologic relapses occurring 33 and 12.5 months from diagnosis. In neither case was the resistance mutation detected in pre-treatment marrow samples. With no knowledge of the underlying resistance mutation, the patient with the L224H mutation was re-treated with single-agent ATRA, which failed to induce a second remission, at which stage they were commenced on ATO. Interestingly, pyrosequencing revealed that the L224H mutation was present in only a minor fraction of leukemic cells (~10%) at the time of relapse, suggesting that the mutant subclone was not a primary factor in disease recurrence and was unlikely to have arisen in the APL stem cell compartment. Although there was no net change in PML-RARA transcript levels following ATRA treatment for relapse, there were clear changes in the APL population dynamics with regression of the major wild-type PML-RARα clone and outgrowth of the L224H mutant subclone. This responded to ATO, inducing molecular remission which has been maintained for 14.5 months. ATO was also used to treat relapse in the patient with the Y208D mutation in PML-RARα; sensitivity of the mutant clone was suggested by the rapid development of hyperleukocytosis and differentiation syndrome. However, blood products could not be used for religious reasons and the patient died on re-induction. Neither of the patients relapsing following first-line therapy with the chemotherapy-free ATRA+ATO+GO schedule exhibited LBD mutations. Overall, this study suggests that the incidence of LBD mutations with conventional treatment schedules in which chemotherapy is given simultaneously with ATRA is lower than previously thought and that these do not necessarily arise at the APL stem cell level. Our data support the use of ATO as first-line therapy for relapse, which can induce molecular remissions in patients with ATRA-resistant disease due to LBD mutations. While neither of the cases studied in which relapse occurred after the chemotherapy-free schedule developed LBD mutations, it remains to be seen whether secondary ATRA resistance re-emerges as a more frequent occurrence with greater use of deintensified treatment schedules as these are evaluated in large scale clinical trials.