(P 188) PRELIMINARY ASSESSMENT OF CIRCULATING INFLAMMATORY CYTOKINES IN EPITHELIOID HEMANGIOENDOTHELIOMA (EHE) PATIENTS AND IN AN EHE PATIENT-DERIVED XENOGRAFT BEFORE AND AFTER DOXORUBICIN AND SIROLIMUS

Objective: To report on the preliminary results of an Italian / British observational study supported by The EHE Rare Cancer Charity (UK) and The EHE Foundation (US), aimed at identifying prognostic and predictive factors in EHE, investigating the presence of inflammatory cytokines in the peripheral blood of patients (pts) affected by epithelioid hemangioendothelioma (EHE), and in a patient-derived model (PDX) of high-grade, WWTR1-CAMTA1-fused EHE, at baseline and after treatment with doxorubicin and sirolimus

Methods: Since May 2021, we collected the blood samples of adult pts affected by molecularly confirmed EHE who entered the study. Blood was taken at baseline and subsequently according to different study schedules, based on disease extent and treatment. Pts who entered the study were stratified in two groups (group A, high-risk; group B, low-risk) based on tumor and clinical characteristics. For comparison, we investigated a panel of inflammatory cytokines (Human XL Cytokine Array) in each blood sample and their blood concentration was evaluated overall, both in group A and B. The presence of the same cytokines was investigated in the blood of healthy donors.
In parallel, we engrafted 2 EHE surgical samples into SCID mice and successfully established a PDX from an adult pt affected by a high-grade EHE after three passages. This PDX model and the original clinical tumor were characterized and compared by histology, FISH, RNA sequencing. Following PDX disaggregation, we established 2D (monolayer) and 3D (spheroids) cell cultures. We comparatively assessed the anti-cancer activity of single-agent doxorubicin and sirolimus in the PDX model. The following dosages of sirolimus were tested: 5mg/Kg, 2.5mg/Kg and 1mg/Kg. We investigated the presence of the statistically significant inflammatory cytokines in the EHE PDX and in other sarcoma PDX models available at our institution for comparison, accounting also for their modulation following the treatment with sirolimus

Results: We have so far analyzed circulating cytokines in 23 pts with EHE (group A = 9; group B = 14). Circulating levels of IL-33R (ng/mL), GDF-15 (pg/mL) and IL-6 (pg/mL) were higher in plasma of EHE patients compared to healthy donors (P< 0.001) and in group A pts compared to group B (P=0.0079).

Histo-morphological and molecular assessment of EHE PDX, including the WWTR1-CAMTA1 fusion and overall transcriptomic profile, confirmed consistency with the originating clinical tumor. In the EHE PDX model, doxorubicin showed almost negligible activity while sirolimus induced 69-81% tumor volume inhibition. Tumours started to regrow after the end of treatment with sirolimus 1 mg/Kg and 2.5 mg/Kg while sirolimus 5 mg/Kg resulted in delayed tumor regrowth. Western blot analysis confirmed the downregulation of the m-TOR signaling after treatment with sirolimus. Consistently with in vivo results, sirolimus was more active than doxorubicin in terms of IC50 (0.1 vs 0.01 μM) on 2D cell line, where sirolimus resulted in slightly higher apoptosis induction compared to doxorubicin at the TUNEL assay.
We further analyzed the presence of GDF-15 in PDX models, as this was the cytokine with the most relevant difference between pts and healthy donors, as well as group A and B. Circulating levels of human GDF-15 were present in EHE PDX but not in healthy mice or mice with pleomorphic sarcoma xenotransplant. Also, GDF-15 was lower in EHE PDX treated with sirolimus (5 mg/Kg or 2.5 mg/Kg) compared to solvent. Finally, sirolimus reduced concentration of this cytokine in cell lines compared to control

Conclusion: These preliminary results suggest that the baseline presence of circulating inflammatory cytokines in EHE pts could predict a worse clinical behavior, with GDF-15 being the most interesting one. GDF-15 plasma level was found to be lowered in an EHE PDX model, to our knowledge the first EHE PDX generated so far, as an effect of treatment with sirolimus. A validation of the prognostic role of these cytokines and their value as a potential marker of response and progression is ongoing