PS937 RANDOMIZED PHASE III HOVON-100 STUDY OF CLOFARABINE COMBINED WITH STANDARD TREATMENT IN ADULT PATIENTS WITH NEWLY DIAGNOSED ALL (2019)

Background: In Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), minimal residual disease (MRD) is the strongest independent prognostic factor. Q-RT-PCR represents the gold-standard approach, but it is limited by the lack of standardization across laboratories. Moreover, a fraction of patients with very low MRD levels are classified as positive not-quantifiable (PNQ), which represent a clinical gray zone. Droplet digital PCR (ddPCR) is an alternative approach which is based on an absolute quantification without the need of a standard curve, has the potential to overcome some limitations of RQ-PCR and has shown to be advantageous in other lymphoproliferative disorders. In Ph+ ALL, few data have been reported and there are no guidelines for ddPCR MRD analysis and interpretation. Aims: 1) To develop a ddPCR assay, by establishing the reaction parameters (i.e. cDNA input and PCR conditions) for the quantification of BCR/ABL1 p190 and p210 transcripts; 2) to assess the limit of detection (LOD), specificity and reproducibility of the assay; 3) to compare ddPCR and Q-RT-PCR in samples from patients enrolled in the GIMEMA LAL2116 clinical trial. Methods: To perform ddPCR experiments we followed Biorad recommendations and used the same primers and probes of Q-RT-PCR (BIOMED1). After PCR, the plate was loaded into the QX200 Droplets Reader and data analyzed by the QuantaSoft analysis Software v1.7.4. Only replicates with a number of droplets ≥9000 were deemed acceptable. For results interpretation, we followed the FIL MRD NETWORK recommendations. Three volumes of cDNA (i.e. 1 μL, 2.5 μL and 5 μL) were tested to define the optimal input. To evaluate the specificity of the assay, we tested 4 pools of mononuclear cells from 4 healthy donors and not template controls. To assess the reproducibility, we tested 3 diagnostic Ph+ ALL samples and their 10–1, 10–2, 10–3 dilutions in 2 independent experiments. To define the LOD, we tested serial dilutions of the lowest point (10 copies) of a plasmid standard curve corresponding to 1x10–4; 5x10–5; 1x10–5; 5x10–6; 1x10–6, in a logarithmic scale. Finally, we compared Q-RT-PCR and ddPCR in 10 samples at diagnosis and 35 collected during MRD monitoring. Results: The set-up experiments revealed that: 1) 1 μL of undiluted cDNA for diagnostic samples and 5 μL for follow-up samples are required; 2) the maximum sensitivity achieved by the method is 1x10–5 and reproducible sensitivity is 5x10–5. We documented a high intra- and inter-run reproducibility. The comparison of Q-RT-PCR and ddPCR on diagnostic samples highlighted a high concordance between the two methods (Pearson correlation coefficient = 0.72). The analysis of PNQ cases by Q-RT-PCR revealed that 55.5% (15/27) proved quantifiable by ddPCR, 25.9% (7/27) were confirmed as PNQ and 18.5% (5/27) were negative by ddPCR. The analysis of negative cases by Q-RT-PCR confirmed their negativity in 75% (6/8) also by ddPCR, while 25% (2/8) were classified as PNQ. Summary/Conclusion: ddPCR is as sensitive as Q-RT-PCR and can provide a more accurate quantification in PNQ cases by Q-RT-PCR, allowing to recover their quantification in 55% of cases. At present, clinical correlates analyses cannot be performed given the short follow-up period of patients. Finally, the application of ddPCR in clinical protocols with well-defined international guidelines is needed to define whether it can result in a refined management of Ph+ ALL outcome.