Digital droplet PCR for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia by Immunoglobulin/T-cell receptor and BCR::ABL1 gene analysis. Preliminary comparative results.

Background. BCR::ABL1 transcript and immunoglobulin/T-cell receptor clonal gene rearrangements (IG/TR) can be used as biomarkers for minimal residual disease (MRD) monitoring in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). There is a growing interest in evaluating MRD using a “double-hit strategy”, especially in the adult setting, where data are lacking. Real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for MRD monitoring. It has, however, intrinsic issues that digital droplet PCR (ddPCR) may overcome. Aims. To carry out a parallel MRD monitoring at early time points (TPs) in adult Ph+ ALL patients by both molecular targets and, for BCR::ABL1, by both RQ-PCR and ddPCR to determine the concordance between the MRD values. Materials and methods. Samples were obtained from 28 adults with newly diagnosed Ph+ ALL, enrolled in the ongoing phase III GIMEMA ALL2820 clinical trial (NCT04722848). At diagnosis, all patients underwent IG/TR marker screening. MRD monitoring was performed by RQ-PCR and ddPCR for BCR::ABL1 quantification and by ddPCR for IG/TR. MRD results were considered discordant in case of a difference greater than one log10 or in case of a positivity/negativity. Results. The comparison between MRD values obtained by RQ-PCR and ddPCR using the BCR::ABL1 transcript showed an excellent degree of correlation (R2=0.996). Concerning IG/TR, a marker was identified in 20/28 patients, whereas 8/28 (29%) were no marker (NM). Moreover, in 4 patients (20%) it was not possible to design a sensitive allele-specific oligonucleotide (ASO) (NA). Sixteen samples were evaluated using both biomarkers by ddPCR. Overall, 7/16 (44%) samples were concordant by both targets, having either highly positive (2/7) or negative (5/7) MRD values, while 9/16 (56%) were discordant. Among these, 2/9 were MRD-positive by both targets but outside the concordance range, in 7/9 IG/TR MRD monitoring was negative, while BCR::ABL1 MRD monitoring was positive (range 10-3-10-1). In the 12 NM and NA patients, BCR::ABL1 ddPCR analysis revealed a quantifiable MRD in 8 cases (range 10-2-10-1), 1 proved positive not-quantifiable (PNQ), 2 were MRD-negative and 1 was not evaluable. Finally, in the NM subset, there were no differences in the type of fusion proteins (p190/p210) and enrichment of additional genetic lesions (Fig. 1), while the median WBC count at diagnosis was lower than in the other cases (6.27 x 109/l for NM vs 16.37 x 109/l for patients with IG/TR markers). Conclusions. BCR::ABL1 ddPCR is a sensitive tool for MRD monitoring, at least comparable to RQ-PCR. At variance, at least at early TPs, the degree of concordance between BCR::ABL1 transcript and IG/TR is suboptimal. Noteworthy, although IG/TR is the most widely used biomarker in children, the rate of NM in adult Ph+ ALL is high, suggesting that the BCR::ABL1 transcript remains the most sensitive target. Cohort expansion and clinical correlations are ongoing.