Epilepsy with faint capillary malformation or reticulated telangiectasia associated with mosaic AKT3 pathogenic variants.

Capillary malformations (CMs) are the most common type of vascular anomalies, affecting around 0.3% of newborns. They are usually caused by somatic pathogenic variants in GNAQ or GNA11. PIK3CA and PIK3R1, part of the phosphoinositide 3-kinase-protein kinase B-mammalian target of rapamycin pathway, are mutated in fainter CMs such as diffuse CM with overgrowth and megalencephaly CM. In this study, we present two young patients with a CM-like phenotype associated with cerebral anomalies and severe epilepsy. Pathogenic variants in PIK3CA and PIK3R1, as well as GNAQ and GNA11, were absent in affected cutaneous tissue biopsies. Instead, we identified two somatic pathogenic variants in the AKT3 gene. Subsequent analysis of the DNA obtained from surgically resected brain tissue of one of the two patients confirmed the presence of the AKT3 variant. Focal cortical dysplasia was also detected in this patient. Genetic analysis thus facilitated workup to reach a precise diagnosis for these patients, associating the vascular anomaly with the neurological symptoms. This study underscores the importance of searching for additional signs and symptoms to guide the diagnostic workup, especially in cases with atypical vascular malformations. In addition, it strongly emphasizes the significance of genotype-phenotype correlation studies in guiding clinicians' informed decision-making regarding patient care.

Identification of immunosuppressive factors in retinoblastoma cell secretomes and aqueous humor from patients.

The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor-associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163+ protumoral M2-like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2-like polarization. M2-like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8+ tumor-infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD-L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3+ Tregs infiltrating tumors. We confirmed the pathology results using single-cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single-cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2-like polarization upon exposure to retinoblastoma-conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.

Treatment-driven selection of chemoresistant Ewing sarcoma tumors with limited drug distribution.

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.

Therapeutic targeting of the RB1 pathway in retinoblastoma with the oncolytic adenovirus VCN-01.

Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressor RB1 VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.

Corrigendum: Immune Response Generated With the Administration of Autologous Dendritic Cells Pulsed With an Allogenic Tumoral Cell-Lines Lysate in Patients With Newly Diagnosed Diffuse Intrinsic Pontine Glioma.

[This corrects the article on p. 127 in vol. 8, PMID: 29755954.].

Immune Response Generated With the Administration of Autologous Dendritic Cells Pulsed With an Allogenic Tumoral Cell-Lines Lysate in Patients With Newly Diagnosed Diffuse Intrinsic Pontine Glioma.

BACKGROUND AND OBJECTIVE: Diffuse intrinsic pontine glioma (DIPG) is a lethal brainstem tumor in children. Dendritic cells (DCs) have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT) clinical trial with the use of autologous dendritic cells (ADCs) pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy). METHODS: Nine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV) were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase. RESULTS: Vaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients) and specific (8/9 patients) antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC). Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF) of two patients. Vaccine administration resulted safe in all patients treated with this schema. CONCLUSION: These preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

Increased delivery of chemotherapy to the vitreous by inhibition of the blood-retinal barrier.

Treatment of retinoblastoma -a pediatric cancer of the developing retina- might benefit from strategies to inhibit the blood-retinal barrier (BRB). The potent anticancer agent topotecan is a substrate of efflux transporters BCRP and P-gp, which are expressed at the BRB to restrict vitreous and retinal distribution of xenobiotics. In this work we have studied vitreous and retinal distribution, tumor accumulation and antitumor activity of topotecan, using pantoprazole as inhibitor of BCRP and P-gp. We used rabbit and mouse eyes as BRB models and patient-derived xenografts as retinoblastoma models. To validate the rabbit BRB model we stained BCRP and P-gp in the retinal vessels. Using intravitreous microdialysis we showed that the penetration of the rabbit vitreous by lactone topotecan increased significantly upon concomitant administration of pantoprazole (P=0.0285). Pantoprazole also increased topotecan penetration of the mouse vitreous, measured as the vitreous-to-plasma topotecan concentration ratio at the steady state (P=0.0246). Pantoprazole increased topotecan antitumor efficacy and intracellular penetration in retinoblastoma in vitro, but did not enhance intratumor drug distribution and survival in mice bearing the intraocular human tumor HSJD-RBT-2. Anatomical differences with the clinical setting likely limited our in vivo study, since xenografts were poorly vascularized masses that loaded most of the vitreous compartment. We conclude that pharmacological modulation of the BRB is feasible, enhances anticancer drug distribution into the vitreous and might have clinical implications in retinoblastoma. CHEMICAL COMPOUNDS INCLUDED IN THIS MANUSCRIPT: Topotecan (PubChem CID: 60700) Pantoprazole sodium (PubChem CID: 15008962).

Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8.

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.

Preclinical platform of retinoblastoma xenografts recapitulating human disease and molecular markers of dissemination.

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.

Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes.

BACKGROUND: cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. RESULTS: Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. CONCLUSIONS: These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks.

Combined Microdialysis-Tumor Homogenate Method for the Study of the Steady State Compartmental Distribution of a Hydrophobic Anticancer Drug in Patient-Derived Xenografts.

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.

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies.

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.

Content and traffic of taurine in hippocampal reactive astrocytes.

Taurine is one of the most abundant free amino acids in the mammalian central nervous system, where it is crucial to proper development. Moreover, taurine acts as a neuroprotectant in various diseases; in epilepsy, for example, it has the capacity to reduce or abolish seizures. In the present study, taurine levels has been determine in mice treated with Kainic Acid (KA) and results showed an increase of this amino acid in hippocampus but not in whole brain after 3 and 7 days of KA treatment. This increase occurs when gliosis was observed. Moreover, taurine transporter (TAUT) was found in astrocytes 3 and 7 days after KA treatment, together with an increase in cysteine sulfinic acid decarboxylase (csd) mRNA, that codifies for the rate-limiting enzyme of taurine synthesis, in the hippocampus at the same times after KA treatment. Glial cultures enriched in astrocytes were developed to demonstrate that these cells are responsible for changes in taurine levels after an injury to the brain. The cultures were treated with proinflammatory cytokines to reproduce gliosis. In this experimental model, an increase in the immunoreactivity of GFAP was observed, together with an increase in CSD and taurine levels. Moreover, an alteration in the taurine uptake-release kinetics was detected in glial cells treated with cytokine. All data obtained indicate that astrocytes could play a key role in taurine level changes induced by neuronal damage. More studies are, therefore, needed to clarify the role taurine has in relation to neuronal death and repair.

Regulation of exocytotic protein expression and Ca2+-dependent peptide secretion in astrocytes.

Vesicular transmitter release from astrocytes influences neuronal development, function and plasticity. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we show that a variety of SNARE and Munc18 isoforms are expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca(2+)-dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. Moreover, 8Br-cAMP treatment greatly increased the cellular content of the peptidic vesicle marker secretogranin-2. Release assays performed on cAMP-treated astrocytes showed that basal and stimulated secretogranin-2 secretion are dependent on [Ca(2+)](i). As shown release of the chimeric hormone ANP.emd from transfected cells, cAMP-induced differentiation in astrocytes enhances Ca(2+)-regulated peptide secretion. We conclude that astroglial cells display distinctive molecular components for exocytosis. Moreover, the regulation of both exocytotic protein expression and Ca(2+)-dependent peptide secretion in astrocytes by differentiating and activating agents suggest that glial secretory pathways are adjusted in different physiological states.