Real-Time Detection of PML-RARA and RARA-PML Fusion Transcripts in High Risk Acute Promyelocytic Leukemia (APL) Treated with Arsenic Trioxide (ATO): Implications for Realization of Pre-Emptive Therapy for Molecular Relapse.
Authors: Grimwade David , Jovanovic J.V. , Diverio D. , Nugent E. , Ho A.Y. , Mufti G.J. , Hills R.K. , Solomon E. , Clark R.E. , Lo Coco F. , Burnett A.K. 
Published: April 2020

Abstract
A key challenge in the management of APL is to improve upon survival rates now achieved with ATRA and anthracycline-based chemotherapy. One approach involves minimal residual disease (MRD) monitoring to identify patients otherwise destined to relapse, whose outcome may be improved by pre-emptive therapy. There is also scope for improvement through reduction in induction deaths and treatment-related mortality, with recent studies suggesting that molecularly targeted therapy involving ATO can achieve long term remissions with limited toxicity. However, such strategies are critically dependent upon optimized schedules for MRD detection to rapidly identify those patients needing additional treatment. Yet, there are limited data concerning assay sensitivity, optimal sample type or kinetics of relapse, that are fundamental to delivery of MRD-directed therapy. We determined expression levels of PML-RARA and reciprocal RARA-PML transcripts in diagnostic BM samples from 102 patients using real-time quantitative PCR (RQ-PCR). Normalized PML-RARA transcript levels varied by 3-logs, equating with marked inter-patient variability in maximal achievable sensitivities for MRD detection, ranging between 1 in 102 and 1 in 105 (med. 1 in 103.8). RARA-PML fusion transcripts were expressed in 68/102 (67%). Parallel detection of both transcripts increased sensitivity for MRD detection in 35/102 (34%); yet, the detection limit was still &lt;1 in 104 in 49% due to the low level of fusion transcript expression. Serial MRD monitoring was undertaken in patients (n=32) treated with ATO for relapsed APL in UK and Eire since Nov 02: 22 in hematologic relapse (HR) and 10 treated pre-emptively in 1st molecular relapse (MR). RQ-PCR assessment prior to MR/HR showed a median increase in PML-RARA transcripts of 0.8 log/month (range 0.2–1.9). Analysis of paired BM and blood (PB) samples revealed that molecular conversion in BM invariably preceded that in PB; including a patient testing negative in PB despite a significant level of disease in BM (6% by FISH). Parallel use of the RARA-PML assay in evaluable patients increased rates of MRD detection, in some instances providing earlier warning of impending relapse. ATO was given using the conventional schedule (0.15mg/kg/d - Regimen A) or a regimen involving loading followed by intermittent dosing (0.3mg/kg/d x 5d, then 0.25mg/kg 2x/wk - Regimen B). No difference in kinetics of molecular response was observed; however amongst patients treated in HR, hyperleucocytosis was less common with Regimen B (peak WBC 5 vs 126 x 109/l, p=0.007), indicating that this schedule may favor apoptosis over differentiation and merits evaluation in newly diagnosed APL. OS from time of relapse was 60% at 3yrs; of 10 patients treated pre-emptively, 7 are in 2nd molecular CR at median of 23mo (7–40mo), 2 died in relapse, treatment is on-going in 1. This study highlights sample type, assay sensitivity (which demonstrates inter-patient and -sample variability) and kinetics of relapse as critical factors that need to be taken into account for the successful delivery of MRD-directed therapy, which is becoming increasingly pertinent to the management of other subsets of acute leukemia.