Dissecting Tumor Heterogeneity and Therapy Resistance Markers in Pediatric Burkitt Lymphoma

Burkitt lymphoma (BL) is the most common subtype of non-Hodgkin lymphoma (NHL) in pediatric age. Despite current chemotherapy regimens are extremely effective, the outcome for patients with primary refractory or relapsed disease still remains very poor, with a survival rate of less than 30%. With the aim of implementing new biomarkers for risk assessment, this thesis is structured around two main objectives. The first focuses on the characterization of mutations in TP53 gene to define their prognostic relevance; the second investigates the transcriptional heterogeneity of BL at single cell level, with the goal of identifying features associated with therapy resistance. To this end, we conducted a mutational study of the DNA binding domain (DBD) of TP53 in a cohort of 214 BL pediatric patients. In parallel, we performed single-cell transcriptomic analysis on a small cohort of tumor samples to explore tumor heterogeneity and identify molecular features distinguishing therapy responders from non-responders. In line with previous studies conducted by the UK and German groups, TP53 variants were detected in 40.7% of our cases. The presence of TP53 mutations was associated with a significantly inferior outcome (3-year PFS of 91%±3% for TP53 wild-type vs. 76%±5% for patients bearing TP53 mutations, p-value 0.005). The multivariate analysis, which also considered the main clinical parameters, showed the prognostic impact of TP53 mutations for the early identification of “high risk” BL patients. Interestingly, TP53 mutations at hotspot residue R248 were characterised by a different prognostic relevance depending on the specific amino acid substitution. Patients with the R248W variant had an increased risk of relapse, whereas those with the R248Q substitution had outcomes comparable to TP53 wild-type cases. Single-cell transcriptomic analysis revealed an unexpected and high inter- and intra-tumoral heterogeneity. Additionally, numerous transcripts were found to be differentially expressed among patients with different prognoses, including Tropomyosin 2 (TPM2), a member of the actin filament binding protein family, which was confirmed to be significantly higher in the majority of relapsed cases at both RNA and protein levels in an independent cohort of 99 BL cases (p < 0.05). We demonstrated also a significant correlation between high TPM2 transcript expression and MDD positivity, suggesting a potentially interesting role for TPM2 in cell motility. To support this hypothesis, functional studies have been performed, including migration and invasion assays. We also plan to generate a TPM2 knockout model using CRISPR-Cas9 technology to investigate the functional consequences of TPM2 loss on tumor cell morphology and migration. In conclusion, we confirmed the prognostic impact of TP53 mutational status, which will be included in the upcoming B-NHL 2025 treatment protocol, excluding the R248Q variant, for the early identification of pediatric patients with BL at higher risk of treatment failure. The aberrant expression of TPM2 in non-responder cases strongly suggested its contribution in therapy resistance of BL. This novel observation not only provides a new potential biomarker for early detection of patients at high risk of refractory disease, but also instructs on the need to further investigate the role of cytoskeleton features in pediatric BL pathogenesis.