Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Refining measurable residual disease monitoring in Philadelphia-positive acute lymphoblastic leukemia adult patients: new insights from methodological and molecular standpoints

Measurable residual disease (MRD) negativity represents the primary endpoint in the management of adult Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. MRD assays must be highly sensitive and specific and the markers must be reliable and representative of the disease. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) can overcome some limitations of standard methodologies. In order to evaluate the best strategy for MRD monitoring, in this study were performed: i) BCR::ABL1-based MRD monitoring between RT-qPCR and ddPCR; ii) MRD evaluation by BCR::ABL1 fusion transcript and IG/TR clonal gene rearrangements ; iii) a comparison of the MRD concordance rate between the two markers; iii) a correlation with biologic features and clinical outcome. Samples derived from 111 adults enrolled in the ongoing phase III GIMEMA ALL2820 clinical trial. At diagnosis, patients underwent a screening for the identification of the predominant IG/TR rearrangement and for the IKZF1plus signature. Firstly, a BCR::ABL1-based comparative study was conducted on 156 MRD samples between the gold standard methodology, i.e. RT-qPCR, and ddPCR showing an optimal correlation degree (R2 = 0.93) and a concordance of 59.6%. Ninety-seven/111 (87.4%) cases were evaluable for IG/TR MRD monitoring. At day +70 (end of induction phase), 97 patients were studied and the concordance rate between BCR::ABL1 and IG/TR MRD monitoring was 46.4%; at day +133 (during consolidation phase), 70 patients were studied and the MRD concordance rate was 41.4%. Five patients experienced a hematologic relapse and a retrospective backtracking was carried out at a previous time-point: 1 was concordantly BCR::ABL1pos and IG/TRpos, 1 was BCR::ABL1pos and IG/TRneg, while the other 2 cases were both BCR::ABL1neg but IG/TRpos at 4.0E-05 and 2.0E-04, respectively, suggesting the presence at the onset of non Ph+ subclone. Overall, the concordance rate between BCR::ABL1 and IG/TR is limited, in line with the literature. Nevertheless, a double-hit strategy may be informative for MRD monitoring and possibly for the distinction between typical/lymphoid Ph+ ALL vs CML-like/multilineage Ph+ ALL and, more important, in some cases for predicting hematological relapse. Finally, a case report of an IKZF1plus patient with an extremely aggressive disease was extensively analyzed.