Digital Droplet PCR for Minimal Residual Disease Assessment in Philadelphia-Positive Acute Lymphoblastic Leukemia Using IG/TR Genes and BCR/ABL1 as Markers. Preliminary Results of a Comparative Analysis

Abstract

Beldinanzi M, Della Starza I, contributed equally Background. The Philadelphia (Ph) chromosome is the most frequent genetic abnormality in adult acute lymphoblastic leukemia (ALL). BCR/ABL1 fusion transcript levels and clonal immunoglobulin/T-cell receptor (IG/TR) gene rearrangements are used as biomarkers for the study of minimal residual disease (MRD) in Ph+ ALL, with IG/TR used mainly in pediatric cohorts. There is evidence suggesting a poor concordance between these two molecular markers and there is a growing interest in evaluating MRD using a "double-hit strategy", especially in the adult setting where data are lacking. The real-time quantitative polymerase chain reaction (RQ-PCR) represents the current gold standard for MRD monitoring. It carries, however, some intrinsic limitations that digital droplet PCR (ddPCR), an innovative evolution of standard method, could overcome. Aim of this study. To carry out in adult Ph+ ALL patients a parallel MRD monitoring at early time points by both molecular targets - BCR/ABL1 gene fusion and clonal IG/TR gene rearrangements - by ddPCR. For BCR/ABL1 this was carried out by both RQ-PCR and ddPCR, in order to determine the concordance between the MRD values. Materials and Methods. The study comprised samples derived from 17 adults with newly diagnosed Ph+ ALL enrolled in the ongoing phase III GIMEMA LAL2820 clinical trial. Bone marrow (BM) samples were collected at the end of the induction phase (day +70) with ponatinib in the experimental arm or with a chemotherapy backbone plus imatinib in the control arm. At diagnosis, all patients underwent also a IG/TR gene rearrangements screening and MRD monitoring was performed by RQ-PCR and ddPCR for the quantification of BCR/ABL1 transcript levels and by ddPCR for IG/TR rearrangements, as described. MRD results were considered discordant in case of a difference greater than one log10 or in case of positivity/negativity. Results. The comparison between MRD values obtained in all samples by RQ-PCR and ddPCR using BCR/ABL1 mRNA level as biomarker showed an excellent correlation degree (R2=0.99). At variance, by IG/TR screening, it was possible to identify a clonal rearrangement only in 11/17 cases (65%), while 6/17 (35%) had no marker (NM) (3 p190, 3 p210; 2 IKZF1plus, 2 IKZF1 loss only and 2 IKZF1 WT). Moreover, of the 11 patients with a clonal rearrangement, in 3 (27%) (3 p190; 1 IKZF1plus, 2 IKZF1 WT) it was not possible to design a sensitive allele-specific oligonucleotide (ASO) for MRD monitoring. Overall, 8 samples were evaluated using both molecular markers by ddPCR, showing a poor correlation (R2=-0.21) and a low concordance rate. Indeed, 3/8 (37.5%) samples were concordant by both targets, either having highly positive (2/3) or negative (1/3) MRD values, while 5/8 (62.5%) were discordant; among the discordant cases, 1/5 was MRD-positive by both targets but outside the concordance range, while in 4/5 IG/TR MRD monitoring was negative, while BCR/ABL1 MRD monitoring was positive (range 10-3-10-1). Finally, in the 9 patients who failed IG/TR MRD monitoring, the BCR/ABL1 RQ-PCR analysis revealed a quantifiable MRD positivity in 6 cases (ranged 10-1-10-3), 2 proved "positive non-quantifiable" (PNQ) and 1 was MRD-negative (Figure 1). Conclusions. In adult Ph+ ALL, ddPCR with BCR/ABL1 as marker proved to be a sensitive and reliable tool for MRD monitoring at least comparable to RQ-PCR, showing a very high concordance rate. At variance, at least at early time points, the degree of concordance between BCR/ABL1 fusion transcript levels and IG/TR clonal rearrangements appeared suboptimal. Although IG/TR gene rearrangements are the most widely used biomarker for MRD monitoring in pediatric patients, the rate of failure in IG/TR marker identification in the adult Ph+ ALL setting is higher than in other subtypes. These findings suggest that the BCR/ABL1 fusion transcript remains the main sensitive target in adult Ph+ ALL and lead to hypothesize that in in this setting IG/TR clonal rearrangements represent a surrogate marker. Cohort expansion is warranted and clinical correlations are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal