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Ewing sarcoma is a bone and soft tissue tumor predominantly affecting adolescents and young adults. To characterize changes in anticancer drug activity and intratumor drug distribution during the evolution of Ewing sarcomas, we used immunodeficient mice to establish pairs of patient-derived xenografts (PDX) at early (initial diagnosis) and late (relapse or refractory progression) stages of the disease from three patients. Analysis of copy number alterations (CNA) in early passage PDX tissues showed that two tumor pairs established from patients which responded initially to therapy and relapsed more than one year later displayed similar CNAs at early and late stages. For these two patients, PDX established from late tumors were more resistant to chemotherapy (irinotecan) than early counterparts. In contrast, the tumor pair established at refractory progression showed highly dissimilar CNA profiles, and the pattern of response to chemotherapy was discordant with those of relapsed cases. In mice receiving irinotecan infusions, the level of SN-38 (active metabolite of irinotecan) in the intracellular tumor compartment was reduced in tumors at later stages compared to earlier tumors for those pairs bearing similar CNAs, suggesting that distribution of anticancer drug shifted toward the extracellular compartment during clonal tumor evolution. Overexpression of the drug transporter P-glycoprotein in late tumor was likely responsible for this shift in drug distribution in one of the cases., The work at Hospital Sant Joan de Deu was supported by associations of parents and families of children with cancer. The work of EdA was supported by the AECC Scientific Foundation (GCB13-1578), ISCIII-FEDER (PI14/01466 and PI17/00464), CIBERONC (CB16/12/00361), Asociación Pablo Ugarte and Fundación María García Estrada. The work of JM was supported by the AECC Scientific Foundation (GCB13-1578) and Asociación Pablo Ugarte. The work of AMC was supported by grants from the AECC Scientific Foundation, MINECO (SAF2011-22660), Fundacion BBVA (152/2011), the European Union Seventh Framework Programme (FP7/2007-2013) under Marie Curie International Reintegration Grant (PIRG-08-GA-2010-276998) and ISCIII-FEDER (CP13/00189 and CPII18/00009).

In addition to surgery, local tumor control in pediatric oncology requires new treatments as an alternative to radiotherapy. SN-38 is an anticancer drug with proved activity against several pediatric solid tumors including neuroblastoma, rhabdomyosarcoma and Ewing sarcoma. Taking advantage of the extremely low aqueous solubility of SN-38, we have developed a novel drug delivery system (DDS) consisting of matrices made of poly(lactic acid) electrospun polymer nanofibers loaded with SN-38 microcrystals for local release in difficult-to-treat pediatric solid tumors. To model the clinical scenario, we conducted extensive preclinical experiments to characterize the biodistribution of the released SN-38 using microdialysis sampling in vivo. We observed that the drug achieves high concentrations in the virtual space of the surgical bed and penetrates a maximum distance of 2 mm within the tumor bulk. Subsequently, we developed a model of subtotal tumor resection in clinically relevant pediatric patient-derived xenografts and used such models to provide evidence of the activity of the SN-38 DDS to inhibit tumor regrowth. We propose that this novel DDS could represent a potential future strategy to avoid harmful radiation therapy as a primary tumor control together with surgery In addition to surgery, local tumor control in pediatric oncology requires new treatments as an alternative to radiotherapy. SN-38 is an anticancer drug with proved activity against several pediatric solid tumors including neuroblastoma, rhabdomyosarcoma and Ewing sarcoma. Taking advantage of the extremely low aqueous solubility of SN-38, we have developed a novel drug delivery system (DDS) consisting of matrices made of poly(lactic acid) electrospun polymer nanofibers loaded with SN-38 microcrystals for local release in difficult-to-treat pediatric solid tumors. To model the clinical scenario, we conducted extensive preclinical experiments to characterize the biodistribution of the released SN-38 using microdialysis sampling in vivo. We observed that the drug achieves high concentrations in the virtual space of the surgical bed and penetrates a maximum distance of 2 mm within the tumor bulk. Subsequently, we developed a model of subtotal tumor resection in clinically relevant pediatric patient-derived xenografts and used such models to provide evidence of the activity of the SN-38 DDS to inhibit tumor regrowth. We propose that this novel DDS could represent a potential future strategy to avoid harmful radiation therapy as a primary tumor control together with surgery

Significance In HER2-amplified breast cancers, HER2 inhibitors have been very successful as adjuvant therapy but not as monotherapy. Here, we demonstrate that coamplification of a HER2 intronic miRNA causes intrinsic resistance to HER2 inhibitors by indirectly down-regulating the pro-apoptotic NOXA. Importantly, coinhibition with MCL-1 inhibitors overcomes this resistance., HER2 (ERBB2) amplification is a driving oncogenic event in breast cancer. Clinical trials have consistently shown the benefit of HER2 inhibitors (HER2i) in treating patients with both local and advanced HER2+ breast cancer. Despite this benefit, their efficacy as single agents is limited, unlike the robust responses to other receptor tyrosine kinase inhibitors like EGFR inhibitors in EGFR-mutant lung cancer. Interestingly, the lack of HER2i efficacy occurs despite sufficient intracellular signaling shutdown following HER2i treatment. Exploring possible intrinsic causes for this lack of response, we uncovered remarkably depressed levels of NOXA, an endogenous inhibitor of the antiapoptotic MCL-1, in HER2-amplified breast cancer. Upon investigation of the mechanism leading to low NOXA, we identified a micro-RNA encoded in an intron of HER2, termed miR-4728, that targets the mRNA of the Estrogen Receptor α (ESR1). Reduced ESR1 expression in turn prevents ERα-mediated transcription of NOXA, mitigating apoptosis following treatment with the HER2i lapatinib. Importantly, resistance can be overcome with pharmacological inhibition of MCL-1. More generally, while many cancers like EGFR-mutant lung cancer are driven by activated kinases that when drugged lead to robust monotherapeutic responses, we demonstrate that the efficacy of targeted therapies directed against oncogenes active through focal amplification may be mitigated by coamplified genes.

In HER2 -amplified breast cancers, HER2 inhibitors have been very successful as adjuvant therapy but not as monotherapy. Here, we demonstrate that coamplification of a HER2 intronic miRNA causes intrinsic resistance to HER2 inhibitors by indirectly down-regulating the pro-apoptotic NOXA. Importantly, coinhibition with MCL-1 inhibitors overcomes this resistance. HER2 (ERBB2) amplification is a driving oncogenic event in breast cancer. Clinical trials have consistently shown the benefit of HER2 inhibitors (HER2i) in treating patients with both local and advanced HER2+ breast cancer. Despite this benefit, their efficacy as single agents is limited, unlike the robust responses to other receptor tyrosine kinase inhibitors like EGFR inhibitors in EGFR -mutant lung cancer. Interestingly, the lack of HER2i efficacy occurs despite sufficient intracellular signaling shutdown following HER2i treatment. Exploring possible intrinsic causes for this lack of response, we uncovered remarkably depressed levels of NOXA, an endogenous inhibitor of the antiapoptotic MCL-1, in HER2 -amplified breast cancer. Upon investigation of the mechanism leading to low NOXA, we identified a micro-RNA encoded in an intron of HER2, termed miR-4728, that targets the mRNA of the Estrogen Receptor α (ESR1). Reduced ESR1 expression in turn prevents ERα-mediated transcription of NOXA, mitigating apoptosis following treatment with the HER2i lapatinib. Importantly, resistance can be overcome with pharmacological inhibition of MCL-1. More generally, while many cancers like EGFR -mutant lung cancer are driven by activated kinases that when drugged lead to robust monotherapeutic responses, we demonstrate that the efficacy of targeted therapies directed against oncogenes active through focal amplification may be mitigated by coamplified genes.

We report for the first time on a nano-drug delivery system based on glucosylated polymeric nanomicelles to actively target the second-generation tyrosine kinase inhibitor dasatinib to glucose-avid pediatric sarcomas by the intravenous route. After a comprehensive physicochemical characterization that confirmed the substantially lower critical micellar concentration and the higher encapsulation capacity of the glucosylated amphiphilic nanocarrier with respect to the pristine counterpart, we showed a 9-fold decrease of the half maximal inhibitory concentration of dasatinib in a rhabdomyosarcoma cell line, Rh30, in vitro. In immunodeficient mice bearing the glucose-avid Rh30 xenograft, we revealed that the glucosylated polymeric nanomicelles increased the delivery of dasatinib in the tumor parenchyma. Conversely, the exposure of off-target tissues and organs to the drug was substantially reduced. Upon experimental confirmation that most patient-derived xenograft (PDX) models of pediatric sarcomas overexpress glucose transporter 1 (GLUT-1), we demonstrated the selective accumulation of dasatinib in a patient-derived rhabdomyosarcoma model in vivo. Conversely, the reference dose administered by the oral route was not tumor-selective. Finally, the improved nanocarrier pharmacokinetics led to prolonged median survival of mice bearing a clinically relevant PDX model of alveolar rhabdomyosarcoma from 19 days for the untreated controls to 27 days for the targeted therapy.

Delivery of chemotherapy in the surgical bed has shown preclinical activity to control cancer progression upon subtotal resection of pediatric solid tumors, but whether this new treatment is safe for tumor-adjacent healthy tissues remains unknown. Here, Wistar rats are used to study the anatomic and functional impact of electrospun nanofiber matrices eluting SN-38-a potent chemotherapeutic agent-on several body sites where pediatric tumors such as neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma arise. Blank and SN-38-loaded matrices embracing the femoral neurovascular bundle or in direct contact with abdominal viscera (liver, kidney, urinary bladder, intestine, and uterus) are placed. Foreign body tissue reaction to the implants is observed though no histologic damage in any tissue/organ. Skin healing is normal. Tissue reaction is similar for SN-38-loaded and blank matrices, with the exception of the hepatic capsule that is thicker for the former although within the limits consistent with mild foreign body reaction. Tissue and organ function is completely conserved after local treatments, as assessed by the rotarod test (forelimb function), hematologic tests (liver and renal function), and control of clinical signs. Overall, these findings support the clinical translation of SN-38-loaded nanofiber matrices to improve local control strategies of surgically resected tumors.

Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo. Fil: Monterrubio, Carles. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Paco, Sonia. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Olaciregui, Nagore G.. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Pascual Pasto, Guillem. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Vila Ubach, Monica. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Cuadrado Vilanova, Maria. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Ferrandiz, M. Mar. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Castillo Ecija, Helena. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Kuplennik, Nataliya. Technion-Israel Institute of Technology; Israel Fil: Jungbluth, Achim. Memorial Sloan-Kettering Cancer Center; Estados Unidos Fil: de Torres, Carmen. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Lavarino, Cinzia. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Cheung, N. K. V.. Memorial Sloan-Kettering Cancer Center; Estados Unidos Fil: Mora, Jaume. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Sosnik, Alejandro Dario. Technion-Israel Institute of Technology; Israel Fil: Montero Carcaboso, Angel. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España

INTRODUCTION: Inadequate drug delivery due to an intact blood-brain barrier (BBB) could explain part of the resistance to chemotherapeutic agents in diffuse intrinsic pontine glioma (DIPG). Convection-enhanced delivery (CED) is an alternative mode of local delivery and irinotecan, and its active compound SN‐38, are potentially effective agents. In this study we aim to perform prolonged CED in the pontine area of rats using irinotecan and newly designed SN‐38 loaded polymer nanoparticles (NPs). METHODS: Efficacy of irinotecan and SN‐38 against patient-derived HSJD-DIPG-007 cells was determined in vitro. Stability and release of SN‐38 from the NPs was tested with a dialysis chamber. Prolonged CED (200 μL in 24 hours using an Alzet Osmotic pump) was performed in naïve rats, and maximum tolerated dose (MTD) was determined by neurological follow-up using Rota-rod testing. Orthotopic HSJD-DIPG-007 xenografts were treated with the MTD of SN-38NPs, irinotecan, or vehicle (NaCl 0.9%). RESULTS: IC50 of SN‐38 against HSJD‐DIPG-007 cells was in the nanomolar range. IC50 of irinotecan was 1000 fold higher. Rota-rod scores in rats with HSJD-DIPG-007 tumors declined significantly before weight loss could be objectified. Stability of SN‐38 and release from NPs was adequate for the aimed experiment. Prolonged CED with SN-38 and irinotecan in the pontine area was tolerated at 1.2 μg (~8 μg/kg) and 0.08 mg (~5 mg/kg), respectively. These dosages did not show efficacy against HSJD-DIPG-007 tumors. DISCUSSION: This study shows that prolonged CED in the rat pons using potent chemotherapeutic agents is feasible and safe; it validates stability of newly‐designed NPs containing SN38 in the used vehicle, and it shows Rota-rod testing to be an effective tool for studying transitory functional changes related to toxicity in the pons and cerebellum of rats. The lack of efficacy in treating HSJD-DIPG-007 tumors identifies important new starting points for further research.

Translational research in retinoblastoma – a pediatric tumor that originates during the development of the retina – would be improved by the creation of new patient-derived models. Using tumor samples from enucleated eyes we established a new battery of preclinical models that grow in vitro in serum-free medium and in vivo in immunodeficient mice. To examine whether the new xenografts recapitulate human disease and disseminate from the retina to the central nervous system, we evaluated their histology and the presence of molecular markers of dissemination that are used in the clinical setting to detect extraocular metastases. We evaluated GD2 synthase and CRX as such markers and generated a Taqman real-time quantitative PCR method to measure CRX mRNA for rapid, sensitive and specific quantification of local and metastatic tumor burden. This approach was able to detect 1 human retinoblastoma cell in 100.000 mouse brain cells. Our research adds novel preclinical tools for the discovery of new retinoblastoma treatments for clinical translation. Fil: Pascual Pasto, Guillem. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Olaciregui, Nagore G.. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Vila Ubach, Monica. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Paco, Sonia. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Monterrubio, Carles. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Rodriguez, Eva. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Winter, Ursula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Sant Joan de Deu Barcelona; España. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Batalla Vilacis, Mireia. Fundacio Sant Joan de Deu; España Fil: Catala, Jaume. Hospital Sant Joan de Deu Barcelona; España Fil: Salvador, Hector. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Parareda, Andreu. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Schaiquevich, Paula Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Suñol, Mariona. Hospital Sant Joan de Deu Barcelona; España Fil: Mora, Jaume. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Lavarino, Cinzia. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: de Torres, Carmen. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España Fil: Chantada, Guillermo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Carcaboso, Angel M.. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España

PURPOSE: To develop a reproducible microdialysis-tumor homogenate method for the study of the intratumor distribution of a highly hydrophobic anticancer drug (SN-38; 7-ethyl-10-hydroxycamptothecin) in neuroblastoma patient-derived xenografts. METHODS: We studied the nonspecific binding of SN-38 to the microdialysis tubing in the presence of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in the perfusate. We calibrated the microdialysis probes by the zero flow rate (ZFR) method and calculated the enhancement factor (f = extrapolated SN-38 concentration at the ZFR / SN-38 concentration in the dialysed solution) of HPBCD. We characterized the extravasation of HPBCD to tumors engrafted in mice. In vivo microdialysis and terminal homogenate data at the steady state (subcutaneous pump infusions) were used to calculate the volume of distribution of unbound SN-38 (Vu,tumor) in neuroblastoma. RESULTS: HPBCD (10% w/v) in the perfusate prevented the nonspecific binding of SN-38 to the microdialysis probe and enhanced SN-38 recovery (f = 1.86). The extravasation of HPBCD in the tumor during microdialysis was lower than 1%. Vu,tumor values were above 3 mL/g tumor for both neuroblastoma models and suggested efficient cellular penetration of SN-38. CONCLUSIONS: The method contributes to overcome the limitations of the microdialysis technique in hydrophobic drugs and provides a powerful tool to characterize compartmental anticancer drug distribution in xenografts. Fil: Monterrubio, Carles. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España Fil: Paco, Sonia. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España Fil: Vila Ubach, Mónica. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España Fil: Rodríguez, Eva. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España Fil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina Fil: Lavarino, Cinzia. Technion - Israel Institute of Technology; Israel Fil: Schaiquevich, Paula Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; Argentina Fil: Sosnik, Alejandro. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España Fil: Mora, Jaume. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España Fil: Carcaboso, Ángel M.. Hospital Sant Joan de Déu Barcelona; España. Fundació Sant Joan de Déu; España

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.