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Humanized immunodeficient mice serve as critical models for investigating the functional interplay between transplanted human cells and a pre-reconstituted human immune system. These models facilitate the study of molecular and cellular pathogenic mechanisms and enable the evaluation of the efficacy and toxicity of immunotherapies, thereby accelerating their preclinical and clinical development. Current strategies rely on inefficient, long-term/delayed hematopoietic reconstitution by CD34+ hematopoietic progenitors or short-term reconstitution with peripheral blood mononuclear cells (PB-MNCs) associated with high rates of graft-versus-host disease (GvHD) and an inefficient representation of immune cell populations. Here, we hypothesized that immunologically naïve cord blood mononuclear cells (CB-MNCs) could serve as a superior alternative, providing long-lasting and functionally effective immune reconstitution. We conducted a comprehensive comparison between the non-obese diabetic (NOD).Cg-Prkdc∧ˆscid-IL2rg∧ˆtm1Wjl/SzJ (NSG) and NSG-Tg(CMV-IL3,CSF2,KITLG)∧ˆ1Eav/MloySzJ (NSGS) immunodeficient mouse models following humanization with either PB-MNCs or CB-MNCs. We assessed the engraftment dynamics of various human immune cells over time and monitored the development of GvHD in both models. For the most promising model, we extensively evaluated immune cell functionality in vitro and in vivo using sarcoma and leukemia xenografts. Humanizing NSGS mice with CB-MNCs results in a rapid, robust, and sustained representation of a diverse range of functional human lymphoid and myeloid cell populations while minimizing GvHD incidence. In this model, human immune cell populations significantly impair the growth and engraftment of sarcoma and B-cell acute lymphoblastic leukemia cells, with a significant inverse correlation between immune cell levels and tumor growth. This study establishes a fast, efficient, and reliable in vivo platform for various applications in cancer immunotherapy, particularly for exploring the complex interactions between cancer cells, immune cells, and the tumor microenvironment in vivo , prior to clinical development., Competing Interests: Competing interests: PM is founder of the spin-off OneChain Immunotherapeutics, which has no connection with the present research. The remaining authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Basic and translational research in pediatric cancer are essential to improve patient care. To critically assess the developments achieved in these areas in Latin America, we systematically reviewed information published between 2013 and 2023., Methods: Studies of basic and translational research performed by investigators in Latin America evaluating pediatric malignant solid and central nervous system tumors were retrieved from PubMed. Original articles published in English between 2013 and 2023 were included. Collaborations among Latin American authors or among Latin American authors working with researchers from other continents were also included. Studies were excluded if they focused only on adults or on basic research in tumor biology not specifically related to the tumor types analyzed in this review., Results: A total of 550 articles were retrieved, but after removal of duplicates, 514 articles were included in the analysis, the majority of which were authored by researchers affiliated with institutions in Argentina, Brazil and Mexico. These countries also had the highest number of collaborations on original articles published with authors from Europe and North America. Argentina had the highest number of collaborations on original publications, with coauthors from Brazil and Uruguay. The median impact factor of the 244 journals in which articles were published was 3.5. The most commonly studied tumors were osteosarcomas, neuroblastomas and medulloblastomas; the most commonly studied areas were molecular analysis, tumor cell biology and biomarkers., Conclusions: In Latin America, research in pediatric oncology is on the agenda, despite a notable disparity in publication rates and frequency of collaboration between countries. There is a need to strengthen scientific collaboration within Latin America and with countries from other continents to promote research and to develop novel treatment strategies that reflect the local needs of children in Latin America who have solid tumors and brain cancer., Competing Interests: Conflicts of interest. None declared.

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., (© 2023 Wiley Periodicals LLC.)

Purpose: Although there have been improvements in the management of metastatic retinoblastoma, most patients do not survive, and all patients suffer from multiple short- and long-term treatment toxicities. Reliable and informative models to assist clinicians are needed. Thus we developed and comprehensively characterized a novel preclinical platform of primary cell cultures and xenograft models of metastatic retinoblastoma to provide insights into the molecular biology underlying metastases and to perform drug screening for the identification of hit candidates with the highest potential for clinical translation., Methods: Orbital tumor, bone marrow, cerebrospinal fluid, and lymph node tumor infiltration specimens were obtained from seven patients with metastatic retinoblastoma at diagnosis, disease progression, or relapse. Tumor specimens were engrafted in immunodeficient animals, and primary cell lines were established. Genomic, immunohistochemical/immunocytochemical, and pharmacological analysis were performed., Results: We successfully established five primary cell lines: two derived from leptomeningeal, two from orbital, and one from lymph node tumor dissemination. After the intravitreal or intraventricular inoculation of these cells, we established cell-derived xenograft models. Both primary cell lines and xenografts accurately retained the histological and genomic features of the tumors from which they were derived and faithfully recapitulated the dissemination patterns and pharmacological sensitivity observed in the matched patients., Conclusions: Ours is an innovative and thoroughly characterized preclinical platform of metastatic retinoblastoma developed for the understanding of tumor biology of this highly aggressive tumor and has the potential to identify drug candidates to treat patients who currently lack effective treatment options.

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., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

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., (© 2022 The Pathological Society of Great Britain and Ireland.)

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas., (© 2021. The Author(s).)

Most reports about copy number alterations (CNA) in retinoblastoma relate to patients with intraocular disease and features of children with extraocular relapse remain unknown, so we aimed to describe the CNA in this population. We evaluated 23 patients and 27 specimens from 4 centers. Seventeen cases had extraocular relapse after initial enucleation and six cases after an initial preservation attempt. We performed an analysis of CNA and BCOR gene alteration by SNP array (Single Nucleotide Polymorfism array), whole-exome sequencing, IMPACT panel and CGH array (Array-based comparative genomic hybridization). All cases presented CNA at a higher prevalence than those reported in previously published studies for intraocular cases. CNA previously reported for intraocular retinoblastoma were found at a high frequency in our cohort: gains in 1q (69.5%), 2p (60.9%) and 6p (86.9%), and 16q loss (78.2%). Other, previously less-recognized, CNA were found including loss of 11q (34.8%), gain of 17q (56.5%), loss of 19q (30.4%) and BCOR alterations were present in 72.7% of our cases. A high number of CNA including 11q deletions, 17q gains, 19q loss, and BCOR alterations, are more common in extraocular retinoblastoma. Identification of these features may be correlated with a more aggressive tumor warranting consideration for patient management.

An uncommon subgroup of unilateral retinoblastomas with highly aggressive histological features, lacking aberrations in RB1 gene with high-level amplification of MYCN ( MCYN ampl RB1 +/+) has only been described as intra-ocular cases treated with initial enucleation. Here, we present a comprehensive clinical, genomic, and pharmacological analysis of two cases of MCYN ampl RB1 +/+ with orbital and cervical lymph node involvement, but no central nervous system spread, rapidly progressing to fatal disease due to chemoresistance. Both patients showed in common MYCN high amplification and chromosome 16q and 17p loss. A somatic mutation in TP53 , in homozygosis by LOH, and high chromosomal instability leading to aneuploidy was identified in the primary ocular tumor and sites of dissemination of one patient. High-throughput pharmacological screening was performed in a primary cell line derived from the lymph node dissemination of one case. This cell line showed resistance to broad spectrum chemotherapy consistent with the patient's poor response but sensitivity to the synergistic effects of panobinostat-bortezomib and carboplatin-panobinostat associations. From these cells we established a cell line derived xenograft model that closely recapitulated the tumor dissemination pattern of the patient and served to evaluate whether triple chemotherapy significantly prolonged survival of the animals. We report novel genomic alterations in two cases of metastatic MCYN ampl RB1 +/+ that may be associated with chemotherapy resistance and in vitro/in vivo models that serve as basis for tailoring therapy in these cases.

Importance: Comprehensive understanding of the genomic and gene-expression differences between retinoblastoma tumors from patients with bilateral disease may help to characterize risk and optimize treatment according to individual tumor characteristics., Objective: To compare the genomic features between each eye and a specimen from an orbital relapse in patients with bilateral retinoblastoma., Design, Setting, and Participants: In this case, 2 patients with retinoblastoma underwent upfront bilateral enucleation. Tumor samples were subjected to genomic and gene-expression analysis. Primary cell cultures were established from both of the tumors of 1 patient and were used for gene-expression studies., Main Outcomes and Measures: Whole-exome sequencing was performed on an Illumina platform for fresh tumor samples and DNA arrays (CytoScan or OncoScan) were used for paraffin-embedded samples and cell lines. Gene-expression analysis was performed using Agilent microarrays. Germinal and somatic alterations, copy number alterations, and differential gene expression were assessed., Results: After initial bilateral enucleation, patient 1 showed massive choroidal and laminar optic nerve infiltration, while patient 2 showed choroidal and laminar optic nerve invasion. Patient 1 developed left-eye orbital recurrence and bone marrow metastasis less than 1 year after enucleation. Both ocular tumors showed gains on 1q and 6p but presented other distinct genomic alterations, including an additional gain in 2p harboring the N-myc proto-oncogene (MYCN) in the left tumor and orbital recurrence. Similar copy number alterations between the orbital recurrence and the left eye supported the origin of the relapse, with an additional 11q loss only detected in the orbital relapse. Specimens from patient 2 showed common copy number gains and losses, but further evolution rendered a 2p gain spanning MYCN in the left tumor. For this patient, microarray expression analysis showed differential expression of the MYCN and the forkhead box protein G1 (FOXG1) gene pathways between the left and right tumors., Conclusions and Relevance: Differential genomic and gene expression features were observed between tumors in 2 patients with bilateral disease, confirming intereye heterogeneity that might be considered if targeted therapies are used in such patients. Chromosomal alteration profile supported the origin of the orbital recurrence from the homolateral eye in 1 patient. Loss in chromosome 11q may have been associated with extraocular relapse in this patient.

In this retrospective study of patients with overt orbital retinoblastoma, we evaluated minimally disseminated disease (MDD) in bone marrow and cerebrospinal fluid (CSF) using CRX and/or GD2 synthase as markers. Ten patients were evaluated-five (50%) at diagnosis and five upon relapse. MDD was detected in four cases (one in the bone marrow, two in the CSF, and in one case in both sites). All patients received chemotherapy and four received orbital radiotherapy. Seven patients relapsed or progressed and all of them died. Three patients remain in complete remission. There was no apparent correlation between MDD and the outcome., (© 2019 Wiley Periodicals, Inc.)

Trilateral retinoblastoma (TRb) presents a management challenge, since intracranial tumours are seldom times resectable and quickly disseminate. However, there are no risk factors to predict the final outcome in each patient., Objective: To evaluate minimal disseminated disease (MDD) in the bone marrow (BM) and the cerebrospinal fluid (CSF) at diagnosis and during follow-up and reviewing its potential impact in the outcome of patients with TRb., Methods and Analysis: We evaluated MDD in five patients with TRb, detecting the mRNA of CRX and/or GD2 , in samples from BM and CSF, obtained at diagnosis, follow-up and relapse., Results: Treatment involved intensive systemic chemotherapy in four patients, one did not receive this treatment and died of progression of the disease. Two patients underwent stem cell rescue. Three patients had leptomeningeal relapse and died. One patient remains disease-free for 84 months. RB1 mutations were identified in the five patients, all of them were null mutations. At diagnosis, one patient had tumour cells in the CSF, and none had the BM involved. Only one case of four presented MDD during follow-up in the CSF, without concomitant detection in the BM. On leptomeningeal relapse, no case had MDD in the BM. In all these cases, cells in the CSF were positive for GD2 and/or CRX ., Conclusion: CSF dissemination always concluded in the death of the patient, without concomitant systemic dissemination denoting the importance of increasing treatment directed to the CSF compartment. The MDD presence could indicate a forthcoming relapse., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: CONFLICTS OF INTEREST All authors declare no conflict of interest. V.I. Segatori, M.R. Gabri, and D.F. Alonso are members of the National Council of Scientific and Technical Research (CONICET, Argentina). H.A. Cuello and M. Alberto are research fellows from CONICET, and R. Aschero from. ANPCyT. K. Madauss is a full-time employee from Glaxo Smith Kline.