Feasibility of Implementing Minimal Residual Disease Monitoring in Acute Promyelocytic Leukemia Patients Treated in Developing Countries
Authors: Lange Ana Paula Alencar de Lima , Lima Ana Sílvia Gouvea , Jacomo Rafael , Melo Raul A , Bittencourt Rosane , Pasquini Ricardo , Pagnano Katia B. , Fagundes Evandro M. , Chauffaille Maria de Lourdes , Chiattone Carlos S , LoCoco Francesco , Grimwade David , Rego Eduardo M. 
Published: October 2019

Abstract
Minimal Residual Disease (MRD) monitoring is recognized as a clinically useful tool for the management of Acute Promyelocytic Leukemia (APL) and has been performed by reverse transcriptase Polymerase Chain Reaction (RT-PCR) and Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR). The vast majority of the published studies were conducted in developed countries within well established clinical trials, but the feasibility of MRD monitoring in developing countries is controversial. Here we describe the experience of Brazilian centers that participated in the International Consortium on Acute Promyelocytic Leukemia (IC-APL). In the present study, the participating centers were located at distances up to 2.500 km of central national laboratory and delivery sample time is less than two days. The aim of this study was to determine the feasibility and compare the effectiveness of RQ-PCR and RT-PCR in the MRD research in the context of IC-APL. We analyzed 398 bone marrow (BM) samples from 74 Brazilians patients with de novo APL; mean follow-up of 18 months. Samples were collected at diagnosis (n=74), at post-induction (n=48) after the third consolidation (n=41), and at maintenance (n=235). Standardized assays developed by Europe Against Cancer (EAC) program were used. Clinical characteristics were similar among the full cohort (patients from Brazil [n=122], Mexico [n=30], Chile [n=23], Uruguay [n=8]). Thirty-nine (52%) patients were classified as intermediate risk. PML breakpoint was bcr1 (n=45), bcr2 (n=1), and bcr3 (n=27). The median NCN of transcripts at diagnosis was 0.5151 (n=41) and 0.4690 (n=27) for the bcr1 and bcr3 subtypes, respectively. At the end of induction, there was a reduction of about 3 logs (0.0004 for bcr1 and 0.0005 for bcr3). In this stage, six discrepant cases were observed, all positive by RQ-PCR. There were no relapsed cases. 66/74 (89%) patients completed induction phase and achieved complete remission, and 8/74 (10%) died of hemorrhagic causes. The rate of molecular remission in our study was 37.5% (18/48) by RQ-PCR and 50% (24/48) by RT-PCR. Considering samples obtained at the end of consolidation phase, one discrepant result was detected as negative by RT; however it was not confirmed by RQ-PCR. Both cases did not relapse. The analysis for RQ-PCR was performed in 64% (41/64) of samples. Two patients have died of infectious diseases during the consolidation phase. All patients achieved molecular remission based on the results of RT-PCR. One patient was positive by RQ-PCR (NCN: 0.00006), but all subsequent samples were negative for both techniques, and the patient is alive, in remission. The median NCN of all samples after the third consolidation phase was 0.00001 PML-RARA/104 copies of ABL copies, regardless of the breakpoint. Within 53 patients who have completed the third cycle of consolidation, only 48 started the maintenance. 235 samples were evaluated during maintenance; 87.6% (206/235) were negative by RQ-PCR technique and 94.4% (222/235) by RT-PCR. The median NCN of all samples in the maintenance was 0.00001 PML-RARA/104 copies of ABL copies. The RQ-PCR technique proved to be predictive of relapse in three out of four cases of molecular relapse. All three cases showed positive results for RT-PCR during the early stages of the maintenance cycle and were confirmed by a second sample taken within 15 days. These results are confirmed in literature, observing that most patients who had negative PCR after consolidation relapsed after few months. In one case of molecular relapse, the RQ-PCR analysis provided much earlier warning of recurring disease, testing positive 5 and 6 months, respectively, before documentation of molecular relapse by conventional RT-PCR assay. In two cases, the negative result was performed by RT-PCR post induction, and was not confirmed by RQ-PCR, which remained positive on subsequent samples, and was confirmed once by RT-PCR after long time. According to transcripts numbers at maintenance, there was a decrease of approximately 5 logs when compared to samples after third consolidation. RQ-PCR technique was more sensitive than RT-PCR, providing earlier warning of relapse, thereby allowing greater opportunity for successful delivery of pre-emptive therapy. In sum, the implementation of the IC-APL allowed the improvement of laboratory standards in parallel to advances in clinical management.

Disclosures
No relevant conflicts of interest to declare.