Introduction
The risk of relapse in retinoblastoma is currently determined by the presence of high‐risk histopathologic factors in the enucleated eye. However, the probability of developing ...metastatic disease is heterogeneous among these patients. Evaluating a biological marker to identify high‐risk patients could be useful in clinical setting. This study aims to evaluate whether the expression of TFF1, a surrogate for subtype 2 retinoblastoma, is a prognostic marker for relapse and death.
Methods
This multicenter cohort study included 273 patients, 48 of whom had extraocular disease. Immunohistochemical staining were performed for CRX, ARR3, TFF1, and Ki67. Tumors were classified as histological subtype 1 (HS1) if they had low or no expression of TFF1 (quick score (QS) ≤ 50) and as histological subtype 2 (HS2) if they expressed TFF1 diffusely (QS > 50). We studied the association between HS classification and outcome.
Results
Of 273 patients, 35.9% were classified as HS1, 59.3% as HS2 and 4.8% were not evaluable. In multivariate analysis, patients with HS2 tumors had a higher probability of relapse and death than those with HS1 (p < .0001 and p = .00020, respectively). We identified a higher‐risk subgroup among HS2 tumors, presenting non‐mutually exclusive expression of ARR3 and TFF1 and had an increased risk of relapse and death compared with tumors that displayed mutually exclusive expression (p = .012 and p = .027, respectively).
Conclusions
Expression of TFF1, especially when it is not‐mutually exclusive with ARR3, is an independent significant marker of poor outcome in retinoblastoma.
Abstract 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.
Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 ...protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.
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.
Tissue‐specific toxicity of SN38‐loaded nanofiber matrices—a new delivery system for localized treatment of cancer—is studied in a rat model resembling the clinical condition of pediatric cancer patients undergoing subtotal resection surgery. The product is safe and biocompatible in vital structures including blood vessels, nerves, and viscera, opening new clinical opportunities for this patient population.
Fundoscopy is the standard method for diagnosis and follow‐up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this ...work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell‐free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow‐up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus‐encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT‐ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non‐cancer patients). According to clinical criteria, we grouped patients as having progression‐free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus‐derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression‐free patients and control non‐cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut‐off point for discriminating between progressive and progression‐free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut‐off point in those patients responding to chemotherapy. In contrast, one non‐responder patient remained with values above the cut‐off during follow‐up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow‐up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.
Background The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive ...behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets. Methods Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O.sub.2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo. Results We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model. Conclusions Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB. Keywords: Neuroblastoma, Bone marrow, MIF, CXCR4, Hypoxia, 4-IPP
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.
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Current treatment of retinoblastoma involves using the maximum dose of chemotherapy that induces tumor control and is tolerated by patients. The impact of dose and schedule on the cytotoxicity of ...chemotherapy has not been studied. Our aim was to gain insight into the cytotoxic and antiangiogenic effect of the treatment scheme of chemotherapy used in retinoblastoma by means of different in vitro models and to evaluate potential effects on multi-drug resistance proteins. Two commercial and two patient-derived retinoblastoma cell types and two human vascular endothelial cell types were exposed to increasing concentrations of melphalan or topotecan in a conventional (single exposure) or metronomic (7-day continuous exposure) treatment scheme. The concentration of chemotherapy causing a 50% decrease in cell proliferation (IC50) was determined by MTT and induction of apoptosis was evaluated by flow cytometry. Expression of ABCB1, ABCG2 and ABCC1 after conventional or metronomic treatments was assessed by RT-qPCR. We also evaluated the in vivo response to conventional (0.6 mg/kg once a week for 2 weeks) and metronomic (5 days a week for 2 weeks) topotecan in a retinoblastoma xenograft model. Melphalan and topotecan were cytotoxic to both retinoblastoma and endothelial cells after conventional and metronomic treatments. A significant decrease in the IC50 (median, 13-fold; range: 3-23) was observed following metronomic chemotherapy treatment in retinoblastoma and endothelial cell types compared to conventional treatment (p<0.05). Metronomic topotecan or melphalan significantly inhibited in vitro tube formation in HUVEC and EPC compared to vehicle-treated cells (p<0.05). Both treatment schemes induced apoptosis and/or necrosis in all cell models. No significant difference was observed in the expression of ABCB1, ABCC1 or ABCG2 when comparing cells treated with melphalan or topotecan between treatment schedules at the IC50 or with control cells (p>0.05). In mice, continuous topotecan lead to significantly lower tumor volumes compared to conventional treatment after 14 days of treatment (p<0.05). Continuous exposure to melphalan or topotecan increased the chemosensitivity of retinoblastoma and endothelial cells to both chemotherapy agents with lower IC50 values compared to short-term treatment. These findings were validated in an in vivo model. None of the dosing modalities induced multidrug resistance mechanisms while apoptosis was the mechanism of cell death after both treatment schedules. Metronomic chemotherapy may be a valid option for retinoblastoma treatment allowing reductions of the daily dose.
BackgroundAntibody–drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote ...additional endogenous antitumor immune responses. Here, we characterized the immunomodulatory properties of D3-GPC2-PBD, a pyrrolobenzodiazepine (PBD) dimer-bearing ADC that targets glypican 2 (GPC2), a cell surface oncoprotein highly differentially expressed in neuroblastoma.MethodsADC-mediated induction of ICD was studied in GPC2-expressing murine neuroblastomas in vitro and in vivo. ADC reprogramming of the neuroblastoma tumor microenvironment was profiled by RNA sequencing, cytokine arrays, cytometry by time of flight and flow cytometry. ADC efficacy was tested in combination with macrophage-driven immunoregulators in neuroblastoma syngeneic allografts and human patient-derived xenografts.ResultsThe D3-GPC2-PBD ADC induced biomarkers of ICD, including neuroblastoma cell membrane translocation of calreticulin and heat shock proteins (HSP70/90) and release of high-mobility group box 1 and ATP. Vaccination of immunocompetent mice with ADC-treated murine neuroblastoma cells promoted T cell-mediated immune responses that protected animals against tumor rechallenge. ADC treatment also reprogrammed the tumor immune microenvironment to a proinflammatory state in these syngeneic neuroblastoma models, with increased tumor trafficking of activated macrophages and T cells. In turn, macrophage or T-cell inhibition impaired ADC efficacy in vivo, which was alternatively enhanced by both CD40 agonist and CD47 antagonist antibodies. In human neuroblastomas, the D3-GPC2-PBD ADC also induced ICD and promoted tumor phagocytosis by macrophages, which was further enhanced when blocking CD47 signaling in vitro and in vivo.ConclusionsWe elucidated the immunoregulatory properties of a GPC2-targeted ADC and showed robust efficacy of combination immunotherapies in diverse neuroblastoma preclinical models.
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