The lack of EphB3 receptor prevents bone loss in mouse models of osteoporosis.

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment.

Osteoporosis affects more than 200 million people, mostly women. Our work shows that the EphB3 receptor restricts bone formation, and its absence prevents bone loss in osteoporotic mice. The bone protection observed in EphB3-deficient mice is due to the presence of more bone-forming cells and fewer bone-degrading cells. Molecularly, we found that when there's no EphB3 in mesenchymal stem cells, some bone-promoting genes are increased while many inhibitors are reduced. Therefore, this receptor could become a key target for new therapies that would help to improve the quality of life for those suffering from bone diseases. We're really excited to share our findings with a broad audience, including patients, healthcare professionals, researchers, and the life sciences industry.

Fermenting Acerola (Malpighia emarginata D.C.) and Guava (Psidium guayaba L.) Fruit Processing Co-Products with Probiotic Lactobacilli to Produce Novel Potentially Synbiotic Circular Ingredients.

This study evaluated the effects of acerola and guava fruit processing co-products fermented with probiotic Lactobacillus acidophilus LA-05 and Lacticaseibacillus paracasei L-10 on the abundance of different intestinal bacterial groups and microbial metabolic activity during 48 h of in vitro fecal fermentation. Digested fermented fruit co-products increased the relative abundance of beneficial bacterial groups while overall decreasing or maintaining the relative abundance of non-beneficial bacterial groups, suggesting selective stimulatory effects on beneficial bacterial intestinal populations. The fermented co-products stimulated microbial metabolic activity due to decreased pH, sugar consumption, short-chain fatty acid production, phenolic compound and metabolic profile alteration, and high antioxidant capacity during fecal fermentation. Acerola and guava co-products have high nutritional value and bioactive compounds whose fermentation with probiotics improves their potential functionalities. The results show that fermented fruit co-products could induce beneficial changes in the relative abundance of several bacterial groups as well as in the metabolic activity of the human intestinal microbiota. These results highlight their potential as novel and circular candidates for use as synbiotic ingredients.

Prebiotic Activity of Pequi (Caryocar brasiliense Camb.) Shell on Lactobacillus and Bifidobacterium Strains: A Medicinal Food Ingredient.

Pequi is a native and popular fruit in Cerrado biome. The internal yellow-orange mesocarp is the edible fraction of the fruit, but its shell (peel and external mesocarp), which comprises 80% of the fruit, is not used by the agro-industry during fruit processing. There is a growing interest in the reduction of food loss and waste because of environmental, economic, and social impacts. So this study evaluated the chemical composition, antioxidant capacity, and in vitro prebiotic activity of pequi shell flour. Pequi shell flour was obtained from the lyophilization and milling of pequi shell. The content of dietary fibers, oligosaccharides, sugars, organic acids, total phenolics and tannins, polyphenol profile, and antioxidant capacity was determined in pequi shell flour. In addition, its prebiotic activity was evaluated on growth and metabolism of probiotics Lactobacillus and Bifidobacterium strains. Pequi shell flour has a high content of dietary fibers (47.92 g/100 g), soluble fibers (18.65 g/100 g), raffinose (2.39 g/100 g), and phenolic compounds (14,062.40 mg gallic acid equivalents/100 g). For the first time, the polyphenols epigallocatechin gallate, epicatechin, and procyanidin B2 were identified in this by-product. Pequi shell flour promoted greater growth of Lacticaseibacillus casei L-26 (at 24-48 h) and Bifidobacterium animalis subsp. lactis BB-12, as well as higher prebiotic activity scores than fructooligosaccharides (standard prebiotic). Pequi shell flour is rich in prebiotic compounds and has a high antioxidant and prebiotic potential. The promising results encourage its use as an ingredient with antioxidant and potential prebiotic properties to elaborate new functional foods and nutraceuticals.

Partial range of motion training elicits favorable improvements in muscular adaptations when carried out at long muscle lengths.

The study compared changes in strength and regional muscle hypertrophy between different ranges of motion (ROM) in the knee extension exercise. Forty-five untrained women were randomized to either a control group or to perform the exercise in one of the following 4 groups (0°=extended knee): Full ROM (FULLROM: 100°-30° of knee flexion); Initial Partial ROM (INITIALROM: 100°-65°); Final Partial ROM (FINALROM: 65°-30°); Varied ROM (VARROM: daily alternation between the ROM of INITIALROM and FINALROM). Pre- and post-training assessments included one repetition maximum (1RM) testing in the ROM corresponding to the initial, final and full ROM, and measurement of cross-sectional areas of the rectus femoris and vastus lateralis muscles at 40%, 50%, 60% and 70% of femur length in regard to regional muscle hypertrophy. Results showed that the INITIALROM group presented a greater relative increase than all groups at 70%, and at 50% and 60% the increases were greater than FINALROM, FULLROM, and non-training control (CON) groups. Moreover, FINALROM group presented similar changes compared to the CON group at 60% and 70%. In regard to 1RM, FINALROM and INITIALROM groups presented greater relative increases at the ROM trained, and no group showed greater increases than VARROM or INITIALROM, regardless the ROM tested. In conclusion, partial ROM training in the initial phase of the knee extension exercise promoted greater relative hypertrophy in certain muscle regions than training in other ROM configurations, and no group promoted a greater 1RM increase than VARROM group, which showed similar 1RM increases in the different ROMs tested.

The use of OCT in good visual acuity MOGAD and AQP4-NMOSD patients; with and without optic neuritis.

Myelin oligodendrocyte-antibody-associated disease (MOGAD) often presents with severe optic neuritis (ON) but tends to recover better than in aquaporin-4 antibody neuromyelitis optica spectrum disorder (AQP4-NMOSD). We measured OCT and VEP in MOGAD and AQP4-NMOSD eyes with good visual function, with or without previous ON episodes. Surprisingly, OCT and/or VEPs were abnormal in 84% MOGAD-ON versus 38% AQP4-NMOSD-ON eyes (p = 0.009) with good vision, compared with 18% and 17% respectively of eyes with no previous ON. A sub-group with macular OCT performed as part of a research study confirmed both retinal and macular defects in visually-recovered MOGAD eyes. These findings have implications for investigation and management of MOGAD patients.

Identifying hotspots of invasive alien terrestrial vertebrates in Europe to assist transboundary prevention and control.

This study aims to identify environmentally suitable areas for 15 of the most harmful invasive alien terrestrial vertebrates (IATV) in Europe in a transparent and replicable way. We used species distribution models and publicly-available data from GBIF to predict environmental suitability and to identify hotspots of IATV accounting for knowledge gaps in their distributions. To deal with the ecological particularities of invasive species, we followed a hierarchical approach to estimate the global climatic suitability for each species and incorporated this information into refined environmental suitability models within Europe. Combined predictions on environmental suitability identified potential areas of IATV concentrations or hotspots. Uncertainty of predictions identified regions requiring further survey efforts for species detection. Around 14% of Europe comprised potential hotspots of IATV richness, mainly located in northern France, UK, Belgium and the Netherlands. IATV coldspots covered ~ 9% of Europe, including southern Sweden and Finland, and northern Germany. Most of Europe (~ 77% area) comprised uncertain suitability predictions, likely caused by a lack of data. Priorities on prevention and control should focus on potential hotspots where harmful impacts might concentrate. Promoting the collection of presence data within data-deficient areas is encouraged as a core strategy against IATVs.

Eph/ephrin Signaling and Biology of Mesenchymal Stromal/Stem Cells.

Mesenchymal stromal/stem cells (MSCs) have emerged as important therapeutic agents, owing to their easy isolation and culture, and their remarkable immunomodulatory and anti-inflammatory properties. However, MSCs constitute a heterogeneous cell population which does not express specific cell markers and has important problems for in vivo homing, and factors regulating their survival, proliferation, and differentiation are largely unknown. Accordingly, in the present article, we review the current evidence on the relationships between Eph kinase receptors, their ephrin ligands, and MSCs. These molecules are involved in the adult homeostasis of numerous tissues, and we and other authors have demonstrated their expression in human and murine MSCs derived from both bone marrow and adipose tissue, as well as their involvement in the MSC biology. We extend these studies providing new results on the effects of Eph/ephrins in the differentiation and immunomodulatory properties of MSCs.

Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma.

Gliomas are the most common intracranial primary tumors, for which very few therapeutic options are available. The most malignant subtype is the glioblastoma, a disease associated with a 5-year survival rate lower than 5%. Given that research in glycobiology continues highlighting the role of glycans in tumor cell biology, it offers an interesting niche for the search of new therapeutic targets. In this study, we characterized aberrant glycosylation and its impact on cell biology over a broad panel of high- and low-grade glioma cell lines. Results show high expression of terminal Lewis glycans, mainly SLex, and overexpression of sialyl- and fucosyltransferases involved in their biosynthesis in high-grade glioma cell lines. Moreover, we report an association of complex multi-antennary N-glycans presenting ß1,6-GlcNAc branches with the high-grade glioma cells, which also overexpressed the gene responsible for these assemblies, MGAT5. In addition, downmodulation of N-glycosylation by treatment with the inhibitors Tunicamycin/Swainsonine or MGAT5 silencing decreased SLex expression, adhesion and migration in high-grade glioma cells. In contrast, no significant changes in these cell capacities were observed in low-grade glioma after treatment with the N-glycosylation inhibitors. Furthermore, inhibition of histone deacetylases by Trichostatin A provoked an increase in the expression of SLex and its biosynthetic related glycosyltransferases in low-grade glioma cells. Our results describe that aggressive glioma cells show high expression of Lewis glycans anchored to complex multi-antennary N-glycans. This glycophenotype plays a key role in malignant cell behavior and is regulated by histone acetylation dependent mechanisms.

Comparison of two surgical approaches for Laparoscopic Ovum Pick Up in Ewes / Comparação de duas abordagens cirúrgicas para aspiração folicular laparoscópica em ovelhas

The present study had as an aim to evaluate a right lateral access as an alternative method to laparoscopic ovum pick-up (LOPU) in sheep. Twenty-four Santa Ines ewes were randomly assigned in two groups with twelve animals each: RLD - positioned in right lateral decubitus, with 10º head-down tilt; and DD - positioned in dorsal decubitus with 35º head-down tilt. The following parameters were evaluated every 10 minutes during the procedure: total surgical time (ST), visualized follicles (VF), aspirated follicles (AF), recovered oocytes (RO), mean arterial pressure (MAP), heart rate (HR), respiratory rate (fR) and end tidal CO2 pressure (EtCO2). Pre and postoperative arterial hemogasometry parameters (PaO2, PaCO2, pH, CHCO3 and BE) were also evaluated; and serum fibrinogen levels (SFL) on postoperative period. The values of VF, AF, RO, fR, PaO2, pH, CHCO3, BE and SFL were similar between groups, although ST, HR, MAP, EtCO2 and PaCO2 were higher in LG. Regarding operative periods, PaO2 and pH were lower after surgery (PaO2: 79.1±16.4; 79.2±11.7mmHg; pH: 7.30±0.09; 7.32±0.08) in both groups when compared to preoperative (PaO2: 80.1±14.3; 83.4±10.5 mmHg; pH: 7.38±0.05; 7.39±0.05) while PaCO2 (43.6±4.6; 41.9±5.4mmHg) and CHCO3 (22.8±1.5; 22.7±3.0mmol/L) increased (PaCO2: 54.3±10.9; 46.9±6.3mmHg; CHCO3: 24.8±3.4; 24.4±2.7mmol/L) postoperative. This alternative decubitus presented is a viable procedure and did not differ in oocyte recovery rates in ewes. However, entails cardiorespiratory major alterations compared to conventional procedure, making its practical applicability limited.(AU)

O presente estudo teve como objetivo avaliar o acesso lateral direito como um método alternativo para a recuperação de oócitos por laparoscopia (LOPU) em ovelha. Vinte e quatro ovelhas Santa Inês foram distribuídas aleatoriamente em dois grupos com 12 animais: grupo RLD - posicionado em decúbito lateral direito, cefalodeclive com 10º de inclinação; grupo DD - posicionado em decúbito dorsal em cefalodeclive, inclinação de 35º. Foram avaliados, a cada 10 minutos, durante o procedimento cirúrgico: tempo total da cirurgia (ST), folículos visualizados (VF), folículos aspirados (AF), oócitos recuperados (RO), pressão arterial média (MAP), frequência cardíaca (FC), frequência respiratória (fR) e pressão final de CO2 (EtCO2). Também foram avaliados os parâmetros de hemogasometria arterial pré-operatória e pós-operatória (PaO2, PaCO2, pH, CHCO3 e BE), bem como os níveis séricos de fibrinogênio (SFL) no período pós-operatório. Os valores de VF, AF, RO, fR, PaO2, pH, CHCO3, BE e SFL foram semelhantes entre os grupos, embora ST, FC, MAP, EtCO2 e PaCO2 tenham sido maiores em RLD. Os parâmetros PaO2 e pH foram menores após a cirurgia (PaO2: 79,1±16,4; 79,2±11,7mmHg; pH: 7,30±0,09; 7,32±0,08) em ambos os grupos em relação ao momento pré-cirúrgico (PaO2: 80,1±14,3; 83,4±10,5mmHg; pH: 7,38±0,05; 7,39±0,05), enquanto PaCO2 (43,6±4.6; 41,9±5,4mmHg) e CHCO3 (22,8±1,5; 22,7±3.0mmol/L) aumentaram (PaCO2: 54,3±10,9; 46,9±6,3mmHg; CHCO3: 24,8±3,4; 24,4±2,7mmol/L) após a cirurgia. O decúbito lateral é uma alternativa viável para LOPU e não apresenta diferença para a taxa de recuperação oocitária em ovelhas. No entanto, promove alterações cardiorrespiratórias em comparação com o decúbito dorsal, tornando a sua aplicabilidade prática limitada.(AU)

Aberrant O-glycosylation modulates aggressiveness in neuroblastoma.

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.

Data to model risks for recolonizing wolves in Scandinavia through the integration of territory presence and human-driven mortalities.

This dataset article describes the data and sources used to model risks for the recolonizing wolf (Canis lupus) in Sweden and Norway in the article "Integrated spatially-explicit models predict pervasive risks to recolonizing wolves in Scandinavia from human-driven mortality" (Recio et al., 2018). Presences on wolf territories were used to model the potential distribution of the species. Presences of human-driven mortalities including traffic collisions, culling (protective/defensive, and licensed hunting), and illegal killing (i.e. poaching) were used to model predictions on the distribution of these mortalities. Sources for the independent variables used for the models are also described.

Antibody-dependent cell-mediated cytotoxicity induced by active immunotherapy based on racotumomab in non-small cell lung cancer patients.

Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients' treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients.

Synergistic potentiation of the anti-metastatic effect of anti EGFR mAb by its combination with immunotherapies targeting the ganglioside NGcGM3.

Several Anti-EGFR mAbs are register for the treatment of human cancer. However, their impact on patients overall survival has been limited by tumor resistance. N-Glycolyl variant of GM3 ganglioside (NGcGM3) is specifically expressed in some human tumors, and it has been associated with a poor prognosis. Several reports have documented that GM3 physically associates to EGFR inhibiting its ligand depend phosphorylation, but it also facilitates an alternative/compensatory signaling cascade mediated by Uroquinase Plasminogen Activator Receptor (uPAR) and integrin α5ß1 interaction. However, the difference between NGc and N-Acetylated (NAc) variants of GM3 regarding such interactions is unknown. We hypothesized that enrichment of NGcGM3 expression in tumors relates to advantages of this ganglioside, on ensuring both EGFR and uPAR pathways optimal function. We explored the impact of combining an anti-EGFR (7A7 mAb) with anti-NGcGM3 therapies: NGcGM3/VSSP vaccine or 14F7 mAb. Both combinations synergistically increase overall survival in two models of lung metastasis: 3LL-D122 and 4T1; but combination with NGcGM3/VSSP vaccine is significantly more effective. In 3LL-D122-metastasis, of mice treated with the best combination, both EGFR and uPAR/α5ß1 integrin pathways are turn off (I.e expression of uPAR/α5ß1; and phosphorylation of EGFR, Stat3, Src and FAK are reduced); and tumor angiogenesis is decreased. Interestingly, combination treatment increases tumor infiltrating CD4+T, CD8+T and NK+-cells. Furthermore, a positive clinical outcome is reported for a cancer patient treated with an anti-EGFR mAb and anti-NGcGM3 therapy. Overall, our results support the combination of anti EGFR antibodies with therapies targeting NGcGM3 to increase their efficacy in future clinical trials.

Eficacia de la Ecografia preoperatoria para orientar la cánula de colecistoscopia. Estudio en cerdos / Preoperative sonography eficacy for cholecystoscopy cannula orientaton, evaluaton on pigs

Antecedentes: la colecistoesclerosis colecistoscópica es un procedimiento de sesión única en etapa de perfeccionamiento, diseñado para lograr la eliminación definitiva de la vesícula biliar con anestesia locorregional. Comprende la remoción de litos y eliminación de epitelio vesicular vía colecistoscópica, para lo cual requiere la colocación de una cánula de colecistoscopia desde la pared abdominal hasta el fondo vesicular. Objetivo: evaluar si la Ecografia preoperatoria es eficaz para definir el lugar donde debe realizarse el ojal de pared, para que permita a la cánula llegar al fondo vesicular, de manera que el eje de la cánula y el eje de la vesícula coincidan, y que el acceso de instrumental a la luz vesicular sea fuido. Material y métodos: se planificó utlizar Ecografia preoperatoria en una serie de operaciones en cerdos, para definir la posición de la vesícula con respecto a las tres dimensiones del espacio, y prolongar en forma virtual el eje del órgano hacia la pared abdominal e identificar el lugar adecuado para confeccionar el ojal de pared. Resultados: se realizó una serie de 5 operaciones de acuerdo con lo planificado; se logró acceder rápidamente con la cánula de colecistoscopia al fondo vesicular y se constató que el eje de la cánula y el eje de la vesícula coincidieron en todos los casos. Conclusiones: la experiencia ofrece un frme indicio de que la Ecografia preoperatoria es muy eficaz para definir el lugar adecuado en la pared abdominal para colocar la cánula de colecistoscopia.

Background: the Cholecystoscopic Cholecystosclerosis is a single session procedure, under enhancing process, which pursues a definitive Gallbladder eliminaton by means of stone removal and mucous membrane ablaton through Operatory Cholecystoscopy, under local-regional anesthesia, and requires the inserton of a Cholecistoscopy Cannula from the abdominal wall toward the gallbladder fundus. Objective: to assess the efectiveness of the Preoperative Sonography when it comes to finding the right place for the Cholecystoscopy Cannula inserton. It is important to achieve the Cholecystoscopy Cannula being introduced in a way that its axis matiches the Gallbladder´s axis, in order to provide a suitable passage for instruments between the Cannula and the Gallbladder´s lumen. Material and Method: preoperative sonography was planned to be performed in a series of surgical interventons on pigs, in order to determine the positon of the gallbladder in its three spatal dimen-sions, allowing a virtual projecton of the organ´s axis toward the abdominal wall, thus pointing to the most suitable place for the Cholecystoscopy Cannula inserton. Results: fve procedures were performed as planned, the Cholecystoscopy Cannula rapidly reached the gallbladder fundus and coincidence between cannula axis and gallbladder axis was achieved in all the cases. Conclusions: preoperative sonography seems to be very eficient for choosing the right place for Cholecystoscopy Cannula inserton accordingly to this experience.

The importance of urban gardens in supporting children's biophilia.

Exposure to and connection with nature is increasingly recognized as providing significant well-being benefits for adults and children. Increasing numbers of children growing up in urban areas need access to nature to experience these benefits and develop a nature connection. Under the biophilia hypothesis, children should innately affiliate to nature. We investigated children's independent selection of spaces in their neighborhoods in relation to the biodiversity values of those spaces, in three New Zealand cities, using resource-selection analysis. Children did not preferentially use the more biodiverse areas in their neighborhoods. Private gardens and yards were the most preferred space, with the quality of these spaces the most important factor defining children's exposure to nature. Children's reliance on gardens and yards for nature experiences raises concerns for their development of a nature connection, given disparities in biodiversity values of private gardens in relation to socioeconomic status, and the decline in sizes of private gardens in newer urban developments.

Minimal Disseminated Disease in Nonmetastatic Retinoblastoma With High-Risk Pathologic Features and Association With Disease-Free Survival.

IMPORTANCE: Fatal metastatic relapse may occur in children with retinoblastoma and high-risk pathologic features (HRPFs). Minimal dissemination (MD) may be an additional tool for risk estimation. The use of cone-rod homeobox (CRX) transcription factor messenger RNA for MD evaluation in metastatic retinoblastoma was previously reported, but no data in nonmetastatic cases with HRPFs are available. OBJECTIVES: To evaluate whether MD is detectable in patients with nonmetastatic retinoblastoma and to assess its prognostic effect on disease-free survival (DFS). DESIGN, SETTING, AND PARTICIPANTS: This single-institution cohort study of patients with nonmetastatic retinoblastoma and HRPFs used prospectively defined inclusion criteria and a sampling strategy to procure bone marrow (BM) and cerebrospinal fluid (CSF) samples from May 1, 2007, through October 31, 2013. Median follow-up was 38 months (range, 8-89 months). Survival analysis was closed in December 2015, and no further updates were made after that point. INTERVENTIONS: The study evaluated CRX messenger RNA by quantitative polymerase chain reaction in BM and CSF at diagnosis and follow-up. In 14 patients, GD2 synthase was used instead of CRX for CSF evaluation. Patients were treated under uniform guidelines. MAIN OUTCOMES AND MEASURES: Metastatic relapse. RESULTS: The study included 96 children (median age at study inclusion, 26 months; range, 1-168 months; 46 male [47.9%]; 50 female [52.1%]) with nonmetastatic retinoblastoma and HRPFs (isolated massive choroidal invasion in 14, postlaminar optic nerve invasion in 51 [26 with concomitant massive choroidal and 13 with scleral invasion], 12 with scleral invasion without postlaminar optic nerve invasion, and 7 with tumor at the resection margin of the optic nerve) were evaluated at the time of primary or secondary enucleation. Minimal dissemination was detected in 9 patients (7 BM samples and 2 CSF samples) and was associated with extension beyond the resection margin of the optic nerve and scleral involvement, but only the former was independently associated (adjusted odds ratio, 57.0; 95% CI, 4.8-678.2; P = .001). In addition, MD occurred in 8 of the 43 International Intraocular Retinoblastoma Classification group E eyes with glaucoma (18.6%) and in 8 of 80 (10%) and 1 of 16 children (6.3%) who underwent primary or secondary enucleation, respectively. Children with MD had a 3-year DFS of 0.78 compared with 0.98 in those without MD (95% CI for the difference in DFS, 0.17-0.23; P = .004). CONCLUSIONS AND RELEVANCE: These findings identified a high-risk population of children with retinoblastoma and HRPFs with MD. Because the number of events was small, these results, which suggest that children with International Intraocular Retinoblastoma Classification group E retinoblastoma and glaucoma have a higher risk of MD at diagnosis, should not be considered definitive at this time.