Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: Refining the interplay between minimal residual disease and genomic features in Philadelphia-positive and Philadelphia-like acute lymphoblastic leukemia

First project The outcome of the patients with Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) has markedly improved with the introduction of the chemo-free strategy with tyrosine kinase inhibitors (TKIs), but some patients experience disease relapse due to the development of point mutations in the BCR::ABL1 kinase domain (KD), that impair the drug binding. The detection of ABL1 mutations before the occurrence of an overt hematologic relapse and the rapid switch to another TKIs is pivotal for an optimal clinical management of Ph+ ALL patients. Thus, the aim of this project was applied digital droplet PCR (ddPCR) for the detection of the T315I BCR::ABL1 KD mutation in adult Ph+ ALL and compared it with Sanger sequencing (SS) method, for the BCR::ABL1 KD mutational screening. We analyzed 16 adult patients enrolled in the Gruppo Italiano Malattie EMatologiche dell’Adulto (GIMEMA) LAL2116 clinical trial who experienced a minimal residual disease (MRD) increase during treatment: by SS, we found that 8 cases proved wild type (WT) and 8 developed mutations (7 with the T315I and 1 with the E255K). By analyzing the same time point (TP) samples by ddPCR, we indeed confirmed the presence of the T315I mutation in SS positive cases. To determine if ddPCR could anticipate the detection of the T315I mutation compared to SS we analyzed a previous TP when MRD levels were lower and we found that ddPCR proves a sensitive method to detect the T315I mutation also at very low levels of disease. Furthermore, ddPCR appears useful to predict molecular relapse before the increase in MRD at levels for which SS is not sufficiently sensitive. For the above mentioned reasons, the ddPCR has the potential to overcome the limitations of the SS. Second project Philadelphia (Ph)-like ALL is a subgroup of B-ALL with a transcriptional profile that resembles that of Ph+ ALL but lacks the BCR::ABL1 transcript and characterized by an unfavorable outcome and a higher relapse rate with both conventional and intensive treatment. The genetic landscape of Ph-like ALL is heterogeneous and several molecular pathways are involved. ALL is the first neoplasm where the assessment of early response to therapy by MRD monitoring is pivotal for guiding therapeutic choices. Currently, real-time quantitative PCR (RQ-PCR) by immunoglobulin/T cell receptor (IG/TR) gene rearrangement marker is the standard molecular method for MRD assessment. For the above mentioned reasons, the role of the Ph-like signature has been investigated in the context of the GIMEMA LAL2317 trial - designed for newly diagnosed adult Ph-negative (Ph-) B-ALL - where the patients received an intensive chemotherapy regimen and two cycles of bispecific monoclonal antibody blinatumomab. The primary goal of the study was MRD negativity after the first cycle of blinatumomab. Despite achievement MRD negativity after blinatumomab treatment Ph-like ALL patients tend to relapse. The main objective of this project was to redefine the outcome of these Ph-like patients by correlating their genomic characteristics and MRD status. Indeed, based on the gene fusions at presentation and on the MRD status, we could identify 3 subgroups of Ph-like ALL. These findings suggest that Ph-like ALL cases should be followed with different markers in addition to IG/TR gene rearrangements, particularly in cases with well-defined fusion genes, who are at a very high risk of relapse. In this sense, a refined and rapid genetic characterization at diagnosis of Ph-like ALL cases is warranted for a more personalized and targeted patient management.