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The goals of this work were to identify factors favoring patient-derived xenograft (PDX) engraftment and study the association between PDX engraftment and prognosis in pediatric patients with Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. We used immunodeficient mice to establish 30 subcutaneous PDX from patient tumor biopsies, with a successful engraftment rate of 44%. Age greater than 12 years and relapsed disease were patient factors associated with higher engraftment rate. Tumor type and biopsy location did not associate with engraftment. PDX models retained histology markers and most chromosomal aberrations of patient samples during successive passages in mice. Model treatment with irinotecan resulted in significant activity in 20 of the PDXs and replicated the response of rhabdomyosarcoma patients. Successive generations of PDXs responded similarly to irinotecan, demonstrating functional stability of these models. Importantly, out of 68 tumor samples from 51 patients with a median follow-up of 21.2 months, PDX engraftment from newly diagnosed patients was a prognostic factor significantly associated with poor outcome (p = 0.040). This association was not significant for relapsed patients. In the subgroup of patients with newly diagnosed Ewing sarcoma classified as standard risk, we found higher risk of relapse or refractory disease associated with those samples that produced stable PDX models (p = 0.0357). Overall, our study shows that PDX engraftment predicts worse outcome in newly diagnosed pediatric sarcoma patients.

Diffuse intrinsic pontine glioma (DIPG) is a chemo-resistant, incurable pediatric tumor of the central nervous system (CNS). The blood−brain barrier (BBB) remains intact in the course of the disease, preventing drugs from entering the brain and resulting in therapeutic failure. The topoisomerase I inhibitor SN-38 shows strong anticancer activity in a patient-derived DIPG cell line in vitro, though a low CNS bioavailability and anti-DIPG efficacy in vivo. In this work, we produced SN-38-loaded polymeric nanoparticles of an amphiphilic chitosan (CS)-g-poly(methyl methacrylate)-poly(acrylic acid) copolymer that were surface-modified with a peptide shuttle that improves transport across the BBB. Drug-loaded nanoparticles displayed a size of ∼200 nm (intensity distribution) and a ζ-potential of +16 mV. The cytocompatibility and endocytosis assayed in DIPG cells (both attached and in suspension) indicated that the nanoparticles are compatible and mainly internalized by clathrin-mediated endocytosis and that the anticancer activity of SN-38 is preserved after nanoencapsulation. In addition, a tandem permeability/anticancer activity study utilizing a coculture model of BBB endothelial cells and DIPG cell spheroids demonstrated that the modified nanoparticles cross a BBB endothelial cell monolayer to a higher extent than the unmodified counterparts and are taken up by DIPG cells. After 72 h of exposure, both SN-38-loaded nanoparticles killed ∼84-88% of the DIPG cells in suspension, indicating that they reach a concentration above the inhibitory concentration 50 of the drug. Finally, the brain accumulation of the drug-loaded nanoparticles upon intravenous injection to Hsd:ICR mice was preliminarily characterized by light sheet fluorescence microscopy. As opposed to unmodified SN-38-loaded nanoparticles, the modified counterparts bind the brain blood vessels and accumulate in the cerebral parenchyma to a large extent. These results confirm the potential of this nanotechnology platform to deliver anticancer agents to the brain in DIPG and other brain tumors with fully conserved BBB. KEYWORDS: diffuse intrinsic pontine glioma (DIPG), amphiphilic polymeric nanoparticles, peptide shuttles, targeting of the central nervous system (CNS)

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Background Successful engraftment of human cancer biopsies in immunodeficient mice correlates with the poor prognosis of patients. This was reported 30 years ago for children with neuroblastoma, but the standard of care treatment evolved significantly during the last 15 years, leading to improved survival of these patients. Here, we evaluated the association of patient‐derived xenograft (PDX) engraftment and prognosis in patients receiving up‐to‐date treatments for cancers classified as metastatic (stage M) high‐risk neuroblastoma (HR‐NB) by the International Neuroblastoma Risk Group Staging System (INRGSS). Methods We obtained biopsies from patients with stage M HR‐NB. We inoculated biopsy fragments subcutaneously in mice. We studied the association of PDX engraftment with event‐free survival (EFS) and overall survival (OS) of patients. Results Since 2009, we established 17 PDX from 97 samples of 66 patients with stage M HR‐NB, with a follow‐up of at least two years. Factors associated with higher probability of engraftment were the death as outcome (p = .0006) and the amplification of the gene MYCN in tumors (p = .0271). Patients whose biopsies established a PDX had significantly shorter EFS and OS (p = .0039 and .0002, respectively) than patients whose samples did not engraft. The association of PDX engraftment and OS was significant in patients without MYCN amplification (p = .0041), but not in patients with MYCN amplification (p = .2707). Conclusion Positive PDX engraftment is a factor related to poor prognosis and fatal outcome in patients with stage M HR‐NB treated with up‐to‐date therapies.

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma. Citation Format: Guillem Pascual-Pasto, Claudia Resa-Pares, Angel M. Carcaboso. Low Bcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in 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 6249.