(P 311) QUANTIFICATION OF TUMOUR-INFILTRATING IMMUNE CELLS THROUGH DECONVOLUTION OF DNA METHYLATION DATA IN EWING SARCOMAS

Objective: Ewing Sarcomas (EWS) presents a major challenge in pediatric oncology due to its aggressive nature and poor prognosis, particularly in metastatic cases. Genetic fusions involving the EWSR gene and ETS family transcription factors are common in EWS, though other rarer fusions have also been identified. Current standard techniques like immunohistochemistry have failed to fully characterize the immune tumor microenvironment of EWS, hindering insights into tumor development and treatment strategies. Recent efforts apply gene expression datasets to quantify tumor-infiltrating immune cells in EWS. Similar deconvolution techniques can be also applied to DNA methylation (DNAm) arrays, which are much more stable compared to RNA-based methods. This study aims to characterize immune cell infiltration in EWS using DNAm array data.

Methods: We collected 30 EWS samples from 30 consecutive patients referred to Bambino Gesù Children’s Hospital. DNAm analysis was performed by EPIC arrays; data loading, normalization, deconvolution and survival analysis were then performed in R programming environment.

Results: The study cohort includes thirty samples of tumors with histological diagnosis of Ewing sarcoma from 32 consecutive patients aged less than 30 years old at the time of diagnosis. Patients’ characteristics are summarized in Table 1. The cohort included 17 females (53%) and 15 males (47%). The median age was 11 years old. Most samples (28/32) had the EWSR1::FLI1 translocation, while 4 had other translocations involving EWSR1 (2 EWSR1::ERG, 2 EWSR1::ETV4).
The immune cell deconvolution returned the abundance of 11 cell types, both relatively to the total amount of immune cells (relative abundance) and to the total tissue (absolute abundance).
Across the whole cohort, the most abundant cell types were T-memory lymphocytes (“Tmem”, mean percentage 37.49%), T-naïve lymphocytes (mean percentage 20.56%), neutrophils (mean percentage 14.10%), and natural killer (NK) cells (mean percentage 10.44%) (Figure 1A-B). We also checked these abundances among samples with EWSR1::FLI1 fusions and other rarer rearrangements. Samples with rarer translocations displayed a significantly lower mean content of dendritic cells (0% versus 0.59%) (Figure 2 A-B).
Follow-up information was available for 24/32 patients (Table 1). Samples with EWSR1::FLI1 translocation showed a significantly shorter overall survival than samples with rarer alterations. Moreover, Kaplan-Meier analysis revealed two variables significantly associated with overall survival. One was the absolute content of T-memory lymphocytes, that correlated with better outcomes (Bonferroni p-value=0.04) when it was higher than 2% (Figure 3 A), and the other was the absolute content of monocytes, that was associated with higher overall survival when lower than 0.82% (Figure 3B). Moreover, patients with tumor purity lower than 83.73% had a higher survival probability, although not significantly (Bonferroni p-value=0.11).

Conclusion: This study underscores the potential of DNAm arrays in providing robust insights into EWS immune profiles, offering a promising avenue for future research. Further investigations with larger cohorts are warranted to validate these findings and explore additional immune cell types influencing EWS outcomes.