Ewing sarcoma (EwS) is the second most common bone and soft tissue tumor, affecting primarily adolescents and young adults. Patients with secondary EwS are excluded from risk stratification in several studies and therefore do not benefit from new therapies. More knowledge about patients with EwS as secondary malignant neoplasms (SMN) is needed to identify at-risk patients and adapt follow-up strategies. Epidemiology, clinical characteristics, and survival analyses of EwS as SMN were analyzed in 3844 patients treated in the last three consecutive international EwS trials, EICESS 92, Euro-E.W.I.N.G. 99, and EWING 2008. Forty-two cases of EwS as SMN (approximately 1.1% of all patients) were reported, preceded by a heterogeneous group of malignancies, mainly acute lymphoblastic leukemias (n = 7) and lymphomas (n = 7). Three cases of EwS as SMN occurred in the presumed radiation field of the primary tumor. The median age at diagnosis of EwS as SMN was 19.4 years (range, 5.9–72) compared with 10.8 years (range, 0.9–51.2) for primary EwS. The median interval between first malignancy and EwS diagnosis was 7.4 years. The 3-year overall survival (OS)/event-free survival (EFS) was 0.69 (SE = 0.09)/0.53 (SE = 0.10) for localized patients and 0.36 (SE = 0.13)/0.29 (SE = 0.12) for metastatic patients (OS: p = 0.02; EFS: p = 0.03). Survival in patients with EwS as SMN did not differ between hematologic or solid primary malignancies. EwS as SMN is rare; however, survival is similar to that of primary EwS, and its risk-adjusted treatment should be curative, especially in localized patients.

Background: Multimodality treatment of Ewing sarcoma (EwS), a highly malignant bone and soft tissue tumor, provides a cure in most patients, but in the metastatic stage the prognosis remains bleak; new systemically effective therapeutic options are urgently needed. The multikinase inhibitor lestaurtinib (CEP701) is an orphan drug that has been clinically studied in various carcinomas, refractory neuroblastoma, and acute myeloid leukemia, but has not yet established itself as a standard therapy. Methods: We performed an in vitro screening of 142 compounds with different mechanisms of action at two different concentrations and measured cell viability 120 hours after the first of two days of treatment. Lestaurtinib was further tested as a lead compound in various cell biology assays in a group of EwS cell lines genetically representing clinically predominant tumor characteristics. Results: Lestaurtinib was among the 21 of 142 (14.8%) compounds tested that were sensitive, i.e., reduced cell viability to below 20% at 1 µM and/or 10 µM in at least four of five EwS cell lines tested. In an expanded group of six other EwS cell lines, 72 hours of treatment with lestaurtinib resulted in significantly reduced cell growth compared to human mesenchymal stem cells. In several colony formation assays, a significant reduction in the number or size of colonies was observed, both when treatment occurred prior to colony growth and when colonies that had already formed were treated. Lestaurtinib induced apoptosis with a significant increase in PI+/FTIC+-driven cells, significantly promoted anoikis, and prevented cell migration by FACS, low adherence flow cytometry analysis, and scratch assays, respectively. Cell cycle analyses by FACS showed significant G2-M cell cycle arrest after 24 hours of treatment. Lestaurtinib also displayed a synergistic cytotoxic effect with doxorubicin, an established agent for the treatment of EwS, and exhibited a favorable dose-reduction index in all EwS cell lines tested. Notably, treatment with lestaurtinib significantly reduced doxorubicin dose at high efficacy levels (Fa 0.97) and low combination index values. Conclusion: Our results highlight the in vitro efficacy of lestaurtinib in EwS and warrant further testing in an in vivomodel as a single agent and in combination with doxorubicin. Citation Format: Samet Dayi, Christiane Schaefer, Anne Albers, Kiruthiga Balusamy, Marc Hotfilder, Maximilian Kerkhoff, Leonhard Koch, Hatice Önder, Claudia Rössig, Uta Dirksen, Stefan K. Zöllner. Preclinical testing of lestaurtinib (CEP701) as a single and combination agent for the treatment of Ewing sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6730.

Resection margin adequacy plays a critical role in the local control of sarcomas. Fluorescence-guided surgery has increased complete resection rates and local recurrence-free survival in several oncological disciplines. The purpose of this study was to determine whether sarcomas exhibit sufficient tumor fluorescence (photodynamic diagnosis (PDD)) after administration of 5-aminolevulinic acid (5-ALA) and whether photodynamic therapy (PDT) has an impact on tumor vitality in vivo. Sixteen primary cell cultures were derived from patient samples of 12 different sarcoma subtypes and transplanted onto the chorio-allantoic membrane (CAM) of chick embryos to generate 3-dimensional cell-derived xenografts (CDXs). After treatment with 5-ALA, the CDXs were incubated for another 4 h. Subsequently accumulated protoporphyrin IX (PPIX) was excited by blue light and the intensity of tumor fluorescence was analyzed. A subset of CDXs was exposed to red light and morphological changes of both CAMs and tumors were documented. Twenty-four hours after PDT, the tumors were excised and examined histologically. High rates of cell-derived engraftments on the CAM were achieved in all sarcoma subtypes and an intense PPIX fluorescence was observed. PDT of CDXs resulted in a disruption of tumor-feeding vessels and 52.4% of CDXs presented as regressive after PDT treatment, whereas control CDXs remained vital in all cases. Therefore, 5-ALA mediated PDD and PDT appear to be promising tools in defining sarcoma resection margins (PDD) and adjuvant treatment of the tumor bed (PDT).