Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Characterization of adult Philadelphia negative B-cell Acute Lymphoblastic Leukemia (B-ALL) in cases enrolled in GIMEMA LAL1913 and GIMEMA LAL2317

The number of genetic lesions in adult patients with newly diagnosed B-lineage acute lymphoblastic leukemia (B-ALL) has largely increased with the use of genome-wide approaches. The detection of these aberrations can refine diagnosis, risk stratification and therapy. To assess the genomic landscape of cases negative for the major fusion genes (B-NEG-ALL), we screened patients enrolled in the GIMEMA LAL1913 and LAL2317 front-line trials. The aim of this study was to identify different genetic aberrations for extending the use of genetic-driven therapeutic approaches to all B-lineage ALL. We extensively evaluated about 200 adult patients using various techniques: BCR/ABL1-like predictor", targeted RNA sequencing to detect rearrangements, MLPA for copy number alteration and targeted DNA sequencing for mutations. Overall, 57 Ph-like cases were identified: 24 (44%) carried lesions involving CRLF2 or a tyrosine kinase (TK); furthermore, mutations in JAK/STAT pathway, overexpression of CRLF2, IKZF1 and IKZF1-plus deletion were significantly higher in these cases. Survival analyses of these cases showed an inferior EFS (event-free survival) and DFS (Disease-free survival) compared to that of non-Ph-like patients. Within non-Ph-like cases, the most common rearrangement involved ZNF384 gene: 17 cases (9%) with ZNF384-r were identified. The most frequent partner gene was EP300 (n=7), followed by TAF15 (n=4), TCF3 (n=2) and ARID1B (n=1). We also identified a novel ZNF384-r - ZNF384-SPI1 - in 1 case. ZNF384-r cases expressed high FLT3 levels and displayed an immunophenotype with a heterogeneous or negative expression of CD10, and an aberrant expression of myeloid antigens, CD13 and/or CD33. CNA analysis revealed additional aberration in only few cases and RAS pathway mutations (PTPN11, FLT3, NRAS and KRAS) were the most common. MRD analysis showed that 41.7%, 72.7%, 80% and 100% of ZNF384-r cases were negative or positive non-quantifiable at TP1, TP2, TP3 and TP4 respectively. In line with this finding, survival analyses showed that ZNF384-r patients had a significantly better (DFS) than other patients. We identified six patients (3%) carrying MEF2D rearrangement: MEF2D was rearranged to BCL9, HNRNPUL1 and FOXJ2, with the most common being BCL9 (n=4): common features were CDKN2A/B deletions and RAS pathway mutations. Outcome of these patients was very poor outcome, with 5 relapsing within 1 year. Finally, PAX5-r and IKZF2-r were observed in 2.1% and 1.6% of our cohort; given their rarity, no correlations were performed. Lastly, in 47% of non-Ph-like patients negative at targeted RNA sequencing, the most common lesions were IKZF1 deletions and RAS pathway mutations. Accurate and comprehensive identification of a full range of genetic alterations in ALL is important for diagnosis, risk stratification, implementation of targeted therapy, and sensitive monitoring of treatment response. The use of high throughput genome-wide technologies to identify genetic alterations and assess the total transcriptome of leukemic cells in B-ALL have provided tremendously informative insight into its pathogenesis. The data obtained by these technologies are important to refine patients’ stratification and pave the way for a more extended prognostic classification at the onset of disease, and to identify key deregulated pathways required for cellular transformation, which could be targeted by inhibitors, either at diagnosis or at relapse, particularly for high risk ALL subtypes.