Digital droplet PCR at the time of TKI discontinuation in chronic‐phase chronic myeloid leukemia patients is predictive of treatment‐free remission outcome

Abstract

To the Editor The introduction of tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML) and close molecular monitoring of the disease has opened the way to the possibility of discontinuing therapy for those patients who witness long-lasting and stable responses. Several trials have explored the possibility of obtaining a treatment-free remission (TFR), both with imatinib and with nilotinib or dasatinib, with percentages of patients who maintained a major molecular response (MMR) without treatment ranging between 40% and 60%. The EUROSKI trial showed a molecular relapse-free survival at 36 months of 50%, discontinuing 750 patients in MR4. Predictive factors associated with a positive outcome after discontinuation were the duration of median imatinib treatment (more than 5.8 years) and the median duration of MR4 prior to discontinuation. With imatinib, about 34% of patients achieved a stable and confirmed deep molecular response (DMR) by RQ-PCR in a median of 8 years of treatment, whereas with dasatinib and nilotinib frontline more than 50% of patients reached this response after 5 years. No data have so far been reported by digital droplet PCR (ddPCR) in clinical practice in order to evaluate the relapse rate of patients who stop treatment. Aim of our study was to evaluate the predictive role of ddPCR in 50 CML patients who had reached a stable undetectable MR4.5 IS previously evaluated with RQ-PCR and then stopped treatment outside of clinical trial. After centrifugation of the sample, buffy coat was recovered and residual red cells were lysed using EL buffer (Qiagen), according to standard procedures. Subsequently, the cells were washed in PBS and counted, and the pellet was resuspended in 1 mL of Trizol reagent (ThermoFisher Scientific). RNA was extracted following standard procedures, quantified, and the quality assessed by running an aliquot on an agarose gel. RNA was maintained at −80 C until use. cDNA was obtained by retrotranscription of 1 μg of total RNA using VILO III RNA Master mix (ThermoFisher Scientific), according to the manufacturer s instructions. The ddPCR assay was performed using the Biorad-QX200 platform (Bio-Rad Laboratories, GmbH, Munich, Germany). cDNA samples were placed into a QX200 droplet generator to partition each samples into 20,000 nanoliter-sized droplets. To test the BCR-ABL fusion transcript and the ABL control gene, we used the same primers and probes according to the standardized protocol developed within the Europe Against Cancer (EAC) program. The ddPCR reaction was performed using 10 μL of ddPCR supermix for probes, 300nM of each primers, 200mM of probe, and 200 ng of cDNA in a total volume of 20 μL. Each reaction mixture was partitioned into approximately 20,000 droplets and then amplified at the following conditions: 95 C for 10 minutes, followed by 40 cycles of 94 C for 30 seconds, 60 C for 1 minute, and a final cycle of 98 C for 10 minutes. At the end, droplets were read using the QX200 droplet-reader. Analysis of the ddPCR data was performed with the QuantaSoftware (Bio-Rad). According to the manufacturer s application, only analyses that led to a number of droplets greater than or equal to 10 000 per replicate were considered acceptable; the threshold was manually set using the FAM channel. In order to define the LOD for the BCR/ABL assays, serial dilutions of a plasmid standard curve were tested. These dilutions were escalated into an increasing number of replicates for each dilution point. Samples were tested in triplicate and repeated in two independent experiments to confirm the results that were expressed as the absolute number of BCR-ABL copies. Informed consent was obtained from all patients included in the study. The characteristics of patients studied are shown in Table 1. The median duration of TKI treatment was 11.9 years (range 3.3-17.9), and the median duration of stable MR4.5 was 2.5 years (range 1.5-7.4). In 28 patients (56%), ddPCR confirmed the negativity of the samples, whereas in 22 (44%), positivity was detected with a median of 0.04 (0.011-0.1) BCR-ABL1 copies per microliter. Of the 28 negative patients, 24 (86%) have not relapsed following discontinuation after a median follow-up of 13 months (range 11-28), while four (14%) have relapsed after a median time of 6 months (range 4-27). In the setting of ddPCR-negative patients, comparison between ddPCR and RQ-PCR in prospective longitudinal monitoring after discontinuation showed that the results obtained by ddPCR anticipated the relapse by 3 months. Within the ddPCR-positive cohort, with the same median follow-up, 11 patients (50%) did not relapse, while 11 (50%) relapsed after a median of 3 months (range 1-5). According to the ddPCR status at the time of discontinuation, TFR outcome was statistically significant between the two groups (P = .026, Figure 1). We compared ddPCR-positive versus ddPCR-negative patients but did not identify differences in terms of median age (59.2 in the positive group vs 58.4 in negative patients, P = .922), Sokal risk stratification (P = .684), previous IFN treatment (31.8% in the positive group vs 25% in the negative group, P = .70), type of TKI received (P = .250), or type of transcript (b3a2 64% and b2a2 36% in ddPCR-negative group compared to 47% b3a2 and 53% b2a2 Received: 21 May 2019 Revised: 7 July 2019 Accepted: 11 July 2019