A Comparison of Droplet Digital PCR and RT-qPCR for BCR-ABL1 Monitoring in Chronic Myeloid Leukemia
Authors: Fava Carmen , Berchialla Paola , Petiti Jessica , Bochicchio Maria Teresa , Izzo Barbara , Giugliano Emilia , Ottaviani Emanuela , Errichiello Santa , Rege Cambrin Giovanna , Venturi Claudia , Luciano Luigiana , Daraio Filomena , Calistri Daniele , Martino Alessandro , Saglio Giuseppe , Martinelli Giovanni , Pane Fabrizio , Cilloni Daniela , Gottardi Enrico Marco 
Published: November 2019

Background: Monitoring of BCR-ABL1 molecular levels is essential for the management of Chronic Myeloid Leukemia (CML) patients treated with Tyrosine Kinases Inhibitors (TKIs). Real Time Quantitative PCR (RT-qPCR) is currently the standard method for assessing molecular remission (MR) in patients with CML. Recently, droplet digital PCR (ddPCR) has emerged to provide a more accurate detection of minimal residual disease (MRD). In order to hypothesize the use of this new technology in the clinical practice, in the era of TKI discontinuation, we designed a multi-centric study to evaluate the potential value of ddPCR in diagnostic routine.
Aims of the study were:1) the evaluation of the agreement between the measures obtained by ddPCR and RT-qPCR and 2) the assessment of the repeatability of the two methods.
Methods: Total RNA was extracted from 37 CML patients using Maxwell 16 LEV simplyRNA Blood kit (Promega), following the manufacturer's instructions. Samples were divided in 5 groups based on molecular response (MR) as follow: group 1, MR<3, n=5; group 2, MR 3, n=5; group 3, MR 4, n=9; group 4, MR 4.5, n=9 and group 5, MR 5, n=9. BCR-ABL1 p210 was quantified by RT-qPCR and ddPCR in 3 different Italian laboratories namely Lab A, Lab B and Lab C. Lab B and C performed 1 amplification session each, while Lab A performed 3 independent sessions. All ddPCR experiments were performed using Kit QXDxTM BCR-ABL%IS (Bio-Rad) on the QX200 system (Bio-Rad), according to the manufacturer's instructions. For RT-qPCR experiments, BCR-ABL1 p210 was quantified with three different methods: by using the One-Step BCR-ABL P210 ELITe MGB Kit (ELITech Group), according to manufacturer's protocol (Lab A); by using the One-Step Philadelphia SensiQuant Kit (Bioclarma), according to the manufacturer's instructions (Lab B) and as described in Gabert et al (Lab C). The results obtained were corrected for the laboratory-specific conversion factor, as recommended by Standard Operating Procedures of Labnet CML network (GIMEMA group). The target quantification for each sample was expressed as BCR-ABL1/ABL1 %IS for both RT-qPCR and ddPCR.
Statistical analysis: Bland-Altman analysis was performed. For the measurement of the agreement we reported the bias, which is the mean of the difference between the methods, the 95% limits of agreement and the coefficient of variation. Residual variance and coefficients of repeatability (i.e. the upper limits of a prediction interval for the absolute difference between two measurements by the same method on the same item) were computed to achieve the second endpoint. An analysis of sensitivity on the labs was also carried out. All analyses were stratified by the level of disease.
Results: A total of 370 measures were included in the analysis, 185 for ddPCR and 185 for RT-qPCR divided as follow: 50 for group 1 and for group 2, 90 for group 3, 4 and 5. In Table 1 we reported the median and interquartile range (IQR) for all levels of disease. Results of the Bland-Altman analysis are shown in Table 2. The coefficients of variation, which expresses the standard deviation as a percentage of the mean, were 2.35, 2.31, 1.10, 1.34, 39.12 in group 1, 2, 3, 4 and 5 respectively. The repeatability coefficients of ddPCR were smaller than qRT-PCR across all the levels of disease, showing a slightly better precision of ddPCR (Table 2).
Conclusions: Higher coefficients of variation in group 1 and 2 were probably due to a greater heterogeneity of the patients. In fact, BCR-ABL1/ABL1 levels by RT-qPCR ranged between 1.43 and 6.94 and between 0.10 and 0.25 in group 1 and in group 2 respectively (Table 1). Sensitivity analysis showed that the high coefficient of variation in group 5 can be explained almost all by the variability observed in Lab B. Coefficient of repeatability of ddPCR was always smaller than RT-qPCR for all level of disease showing a slightly better precision. Our results showed that ddPCR has a good agreement with RT-qPCR and it is more precise to quantify BCR-ABL1 transcript levels, particularly for MR 4 and MR 4.5. Thus, ddPCR may be valuable in diagnostic routine.

Disclosures
Fava: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Martino:Bio-Rad: Employment. Saglio:Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy. Martinelli:Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Cilloni:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.