Establishment and Comprehensive Characterization of a Novel Preclinical Platform of Metastatic Retinoblastoma for Therapeutic Developments.

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.

EpiGe: A machine-learning strategy for rapid classification of medulloblastoma using PCR-based methyl-genotyping.

Molecular classification of medulloblastoma is critical for the treatment of this brain tumor. Array-based DNA methylation profiling has emerged as a powerful approach for brain tumor classification. However, this technology is currently not widely available. We present a machine-learning decision support system (DSS) that enables the classification of the principal molecular groups-WNT, SHH, and non-WNT/non-SHH-directly from quantitative PCR (qPCR) data. We propose a framework where the developed DSS appears as a user-friendly web-application-EpiGe-App-that enables automated interpretation of qPCR methylation data and subsequent molecular group prediction. The basis of our classification strategy is a previously validated six-cytosine signature with subgroup-specific methylation profiles. This reduced set of markers enabled us to develop a methyl-genotyping assay capable of determining the methylation status of cytosines using qPCR instruments. This study provides a comprehensive approach for rapid classification of clinically relevant medulloblastoma groups, using readily accessible equipment and an easy-to-use web-application.t.

The spectrum of morphological findings in pediatric central nervous system MN1-fusion-positive neuroepithelial tumors.

PURPOSE: Central nervous system high-grade neuroepithelial tumor with MN1 alteration (CNS-HGNET-MN1) is a rare entity defined by its DNA methylation pattern and pathologically considered to be high-grade with mixed patterns, stromal hyalinization, and with astrocytic differentiation. Our aim was to present six pediatric cases to contribute to the characterization of this group of tumors. MATERIAL AND METHODS: Six female patients aged 4 to 12 years with CNS tumors with MN1 alteration identified using genome-wide methylation arrays and/or RT-PCR were included. Clinicopathological, morphological, immunohistochemical, and molecular findings were analyzed. RESULTS: Tumor location was the parietal lobe in four and the intramedullary spinal cord in two. Two were morphologically diagnosed as ependymomas, one as gliofibroma, one as a HGNET-MN1 altered and the other two were difficult to classify. All were well-defined tumors, with a cystic component in three. Only two tumors had extensive stromal hyalinization, three had pseudopapillary formations, and four had other patterns. Multinucleated, clear, and rhabdoid cells were present. Necrosis and histiocyte clusters were also observed. Proliferative index was >10 in four. GFAP, EMA, CK, and SYN were variable, while Olig2 staining was mostly positive. Four of six patients with supratentorial tumors and complete resections were alive and tumor free after 2 to 10 years of follow-up. The two cases with medullary involvement and incomplete resections were alive and undergoing treatment 2 years after surgery. CONCLUSION: Neuroepithelial-MN1 tumors are challenging and suspicion requires molecular confirmation. Our pediatric data contribute to the knowledge for accurate diagnosis. Although further studies with a larger number of cases should be conducted in order to draw more robust conclusions regarding clinico-pathological features, here we present valuable pediatric data to increase the knowledge that may lead to the accurate management of this group of tumors.

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Medulloblastoma and Down Syndrome: An Extremely Rare Association.

Medulloblastoma has a reduced incidence in Down syndrome (DS). This protective characteristic has not been clarified yet. Here, we report the second case of SHH medulloblastoma and DS documented in the literature. A complete surgery was performed followed by reduced craniospinal irradiation dose and adjuvant chemotherapy. No evidence of tumor recurrence was observed. The overall survival was 9.1 years. No family history or physical stigma of other hereditary predisposition syndrome was found. In the elucidation of this extremely rare association, future case reports play an important role in defining the spectrum of brain tumors and their peculiar features in DS.

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.

[Targeted therapies with BRAF inhibitors for pediatric low- and high-grade gliomas with BRAFv600e mutation. Prof. Dr. Juan P. Garrahan Hospital experience]. / Terapias dirigidas con inhibidores del BRAF en gliomas pediátricos de alto y bajo grado con la mutación BRAFV600E. Experiencia del Hospital Prof. Dr. Juan P. Garrahan.

The BRAFV600E point mutation plays a key role in the tumorigenesis of many gliomas. Inhibiting its product is part of the innovative therapies emerging in recent years. Knowing the role of these treatments is essential. The aim of this experience was to describe the clinical-radiological response of pediatric BRAFV600E mutated gliomas treated with BRAF inhibitors. To this end, a descriptive and retrospective study was performed in patients under 16 years of age with BRAFV600E gliomas, who received vemurafenib or dabrafenib at Hospital Garrahan. Thirteen patients treated in the last 7 years were included: 9 were low-grade and 4 high-grade gliomas. The median age at diagnosis was 8.6 years (0.89-14.04) and at start of targeted therapy was 11.62 years (3.64-15.42). All patients had previously a surgical procedure, and 12/13 had received another therapy prior BRAF inhibition: 11 chemotherapy (in one case, up to 4 different protocols) and 4 radiotherapy. Under targeted therapy, tumour response was obtained in 10 patients (size reduction equal to or greater than 25%), and best response was observed in the first 6 months of treatment in 7 children. Four patients progressed under treatment (all high-grade gliomas) and 2 progressed shortly after stopping the inhibitor (both low-grade gliomas). Five patients had grade 3-4 toxicity, with subsequent full recovery. A good and sustained clinical-radiological response, with acceptable tolerance, is described in patients with BRAFV600E mutated low-grade gliomas treated with BRAFV600E inhibitors. In contrast, the response in patients with high-grade gliomas was intermediate and of short duration, with early tumour progression.

La mutación puntual V600E del gen BRAF juega un papel fundamental en la tumorigénesis de muchos gliomas. La inhibición de su producto forma parte de terapias innovadoras emergentes en los últimos años. Conocer el rol de estos tratamientos resulta imprescindible. El objetivo del trabajo fue describir la respuesta clínico-radiológica en niños con gliomas BRAFV600E mutado tratados con inhibidores BRAF. Para ello se realizó un estudio descriptivo y retrospectivo en pacientes menores de 16 años con gliomas BRAFV600E mutado que recibieron vemurafenib o dabrafenib en el Hospital Garrahan. Trece pacientes tratados en los últimos 7 años fueron incluidos: 9 gliomas de bajo grado y 4 de alto grado. La mediana de edad al diagnóstico fue 8.6 años (0.89-14.04) y del comienzo del inhibidor 11.62 años (3.64-15.42). Inicialmente, todos habían realizado tratamiento quirúrgico, y 12/13 recibieron previamente otra terapia: 11 quimioterapia (eventualmente hasta 4 líneas distintas) y 4 radioterapia. Con la terapia dirigida, 10 pacientes tuvieron una disminución tumoral mayor o igual al 25%, quedando evidenciada en 7 niños la mejor respuesta dentro de los 6 meses del inicio. Hubo 4 progresados intratratamiento (todos alto grado), y 2 progresados prontamente luego de suspender el inhibidor (ambos bajo grado). Cinco presentaron efectos adversos grado 3-4, con recuperación ad-integrum. Se describe una buena y sostenida respuesta clínico-radiológica, con tolerancia aceptable, en pacientes con gliomas de bajo grado BRAFV600E mutado tratados con inhibidores BRAFV600E. En contraste, la respuesta en pacientes con gliomas de alto grado fue intermedia y de poca duración, con progresión tumoral precoz.

Craniospinal irradiation as part of re-irradiation for children with recurrent medulloblastoma.

BACKGROUND: Many studies have demonstrated in the last years that once medulloblastoma has recurred, the probability of regaining tumor control is poor despite salvage therapy. Although re-irradiation has an emerging role in other relapsed brain tumors, there is a lack of strong data on re-irradiation for medulloblastoma. METHODS: This is a retrospective cohort study of patients aged 18 years or under, treated at least by a second course of external beam for recurrence medulloblastoma at Garrahan Hospital between 2009 and 2020. Twenty-four patients met eligibility criteria for inclusion. All patients received upfront radiotherapy as part of the curative-intent first radiotherapy, either craniospinal irradiation (CSI) followed by posterior fossa boost in 20 patients or focal posterior fossa radiation in 4 infants. The second course of radiation consisted of CSI in 15 and focal in 9. The 3-year post first failure OS (50% vs. 0%; p = 0.0010) was significantly better for children who received re-CSI compared to children who received focal re-irradiation. Similarly, the 3-year post-re-RT PFS (31% vs. 0%; p = 0.0005) and OS (25% vs. 0%; p = 0.0003) was significantly improved for patients who received re-CSI compared to patients who received focal re-irradiation. No symptomatic intratumoral haemorrhagic events or symptomatic radionecrosis were observed. Survivors fell within mild to moderate intellectual disability range, with a median IQ at last assessment of 58 (range 43-69). CONCLUSIONS: Re-irradiation with CSI is a safe and effective treatment for children with relapsed medulloblastoma; improves disease control and survival compared with focal re-irradiation. However this approach carries a high neurocognitive cost.

A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression.

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.

Paediatric Langerhans Cell Histiocytosis Disease: Long-Term Sequelae in the Hypothalamic Endocrine System.

Langerhans cell histiocytosis (LCH) is a disorder of the mononuclear phagocyte system that can affect almost any organ and system. The most common central nervous system (CNS) manifestation in LCH is the infiltration of the hypothalamic-pituitary region leading to destruction and neurodegeneration of CNS tissue. The latter causes the most frequent endocrinological manifestation, that is, central diabetes insipidus (CDI), and less often anterior pituitary hormone deficiency (APD). The reported incidence of CDI is estimated between 11.5 and 24% and is considered a risk factor for neurodegenerative disease and APD. Three risk factors for development of CDI are recognized in the majority of the studies: (1) multisystem disease, (2) the occurrence of reactivations or active disease for a prolonged period, and (3) the presence of craniofacial bone lesions. Since CDI may occur as the first manifestation of LCH, differential diagnosis of malignant diseases like germ cell tumours must be made. APD is almost always associated with CDI and can appear several years after the diagnosis of CDI. Growth hormone is the most commonly affected anterior pituitary hormone. Despite significant advances in the knowledge of LCH in recent years, little progress has been made in preventing long-term sequelae such as those affecting the hypothalamic-pituitary system.

Clinical and molecular characterization of mitochondrial DNA disorders in a group of Argentinian pediatric patients.

OBJECTIVE: To describe the clinical and molecular features of a group of Argentinian pediatric patients with mitochondrial DNA (mtDNA) disorders, and to evaluate the results of the implementation of a classical approach for the molecular diagnosis of mitochondrial diseases. METHODS: Clinical data from 27 patients with confirmed mtDNA pathogenic variants were obtained from a database of 89 patients with suspected mitochondrial disease, registered from 2014 to 2020. Clinical data, biochemical analysis, neuroimaging findings, muscle biopsy and molecular studies were analyzed. RESULTS: Patients were 18 females and 9 males, with ages at onset ranging from 1 week to 14 years (median = 4 years). The clinical phenotypes were: mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome (n = 11), Leigh syndrome (n = 5), Kearns-Sayre syndrome (n = 3), Chronic Progressive External Ophthalmoplegia (n = 2), Leber hereditary optic neuropathy (n = 2), myoclonic epilepsy associated with ragged-red fibers (n = 1) and reversible infantile myopathy with cytochrome-C oxidase deficiency (n = 3). Most of the patients harbored pathogenic single nucleotide variants, mainly involving mt-tRNA genes, such as MT-TL1, MT-TE and MT-TK. Other point variants were found in complex I subunits, like MT-ND6, MT-ND4, MT-ND5; or in MT-ATP6. The m.13513G > A variant in MT-ND5 and the m.9185 T > C variant in MT-ATP6 were apparently de novo. The rest of the patients presented large scale-rearrangements, either the "common" deletion or a larger deletion. CONCLUSIONS: This study highlights the clinical and genetic heterogeneity of pediatric mtDNA disorders. All the cases presented with classical phenotypes, being MELAS the most frequent. Applying classical molecular methods, it was possible to achieve a genetic diagnosis in 30% of the cases, suggesting that this is an effective first approach, especially for those centers from low-middle income countries, leaving NGS studies for those patients with inconclusive results.

Intracranial germ cells tumour: a single institution experience in Argentina.

BACKGROUND: Intracranial germ cell tumor (iGCT) represents a rare and heterogeneous group, with variable incidence and diverse treatment strategies. Although multiagent chemotherapy with reduced radiotherapy strategy has been applied by several cooperative groups in North America and Western Europe, there is a paucity of data to understand if this combined regimen is suitable in low-middle income countries (LMIC). METHODS: We evaluate the outcome in a cohort of iGCT treated by SIOP-CNS-GCT-96 strategy at hospital J.P Garrahan in Argentina over the last 20 years. Radiation field and dose included focal radiotherapy (FRT) before 2009 or focal radiotherapy plus whole ventricular radiotherapy (WVRT) after 2009 for localized germinoma and FRT or FRT plus WVRT or CSI for non germinomatous germ cell tumors (NGGCT) RESULTS: Sixty iGCT were identified; 39 germinoma and 21 NGGCT. Median follow-up was 6.57 years (range 0.13-20.5). 5-year PFS and OS were 83.5% (95% CI [165.53-223.2]) and 88.7% (95% CI [169.84-223.2]) for the germinoma group, while for the NGGCT group were 75% (95% CI [133.27-219.96]) and 64.2% (95% CI [107.38-201.81]) respectively. The localized germinoma group showed poor results between 2000 and 2009 with 5-year PFS and OS of 69 and 75% respectively, and an excellent outcome between 2010 and 2019 with a 5-years PFS and OS of 92.8 and 100%. A univariable analysis identified this difference in survival as related to the field of radiotherapy, specifically whole ventricular radiotherapy. FRT increased the risk of recurrence in localized germinoma, involving not only ventricular relapses; but spinal cord and disseminated disease as well. There were no relapses of localized NGGCT after FRT and FRT plus WVRT. CONCLUSION: Herein we demonstrate that intensive chemotherapy followed by FRT plus WVRT for germinoma is a feasible and effective strategy, warranting further study in the developing world.

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-ß1 and focal adhesion kinase activation.

Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin ß1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin ß1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin ß1 and promotes neuroblastoma cell migration.

Recurrent Somatic Chromosomal Abnormalities in Relapsed Extraocular Retinoblastoma.

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.

Lung destruction secondary to intrapulmonary migration of a ventriculoperitoneal shunt catheter: report of an unusual case and literature review.

Ventriculoperitoneal shunt placement for the treatment of hydrocephalus is one of the most common pediatric neurosurgical procedures. Complications, including infections, catheter obstruction, shunt breakdown, and hemorrhage, have been described in the literature. Occasionally, however, uncommon and devastating complications occur. We report a case of a 10-year-old female patient who at birth underwent surgical closure of lumbar myelomeningocele and placement of a CSF shunt at another center. Her neurosurgical follow-up was poor. She presented at our institution with a history of recurrent pneumonia. Control chest X-rays showed a right pulmonary infiltrate with lung retraction and mediastinal shift. Chest and brain CT scans confirmed the intrapulmonary location of the distal catheter tip and ventricular dilation. Surgical shunt revision was performed with removal of the intrapulmonary catheter and placement of a new intraperitoneal catheter. Subsequently, right pneumonectomy was performed with good postoperative recovery of the patient. Intrathoracic migration of the distal catheter of the CSF shunt is an extremely rare complication that may produce severe morbidity. To our knowledge, there have been no previous reports on extensive lung destruction secondary to intrathoracic and intrapulmonary ventriculoperitoneal shunt migration. In patients with CSF shunts and pulmonary symptoms, intrapulmonary catheter migration should be considered.

Detailed Clinical and Histopathological Description of 8 Cases of Molecularly Defined CNS Neuroblastomas.

Central nervous system neuroblastoma with FOXR2 activation (CNS NB FOXR2) has recently been described as a class of brain tumors sharing common genetic events and a highly similar DNA methylation profile. Most of these tumors have previously been diagnosed as primitive neuroectodermal tumor (PNET). Whereas the entity of PNET has been removed from the WHO classification of brain tumors in its current edition, CNS neuroblastoma was kept as an entity, but still lacks any molecular detail. Here, we describe 8 cases of CNS NB FOXR2 focusing on histomorphological and immunohistochemical features and include magnetic resonance imaging (MRI) for 2 of these cases. MRI revealed large supratentorial masses in superficial location with prominent cysts and necrosis, but little edema. Diffusion and enhancement characteristics were variable. Histological analyses showed that most of the cases displayed neuronal differentiation with necrosis, endothelial proliferation, and high vascularity. Immunohistochemistry revealed strong expression of synaptophysin, MAP2, and OLIG2 as well as moderate proliferation. These findings suggest that tumors with the molecular diagnosis of CNS NB FOXR2 may fit well into the WHO entity of CNS neuroblastoma. Our findings may be helpful when establishing an integrated diagnosis and may be indispensable if molecular data are unavailable.

Pathogenic Variants in the Myosin Chaperone UNC-45B Cause Progressive Myopathy with Eccentric Cores.

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.

Treatment response of CNS high-grade neuroepithelial tumors with MN1 alteration.

Central nervous system high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1) is a rare recently described entity. Fourteen CNS HGNET-MN1 patients were identified using genome-wide methylation arrays/RT-PCR across seven institutions. All patients had surgery (gross total resection: 10; subtotal resection: four) as initial management followed by observation alone in three patients, followed by radiotherapy in eight patients (focal: five; craniospinal: two; CyberKnife: one) and systemic chemotherapy in three patients. Seven patients relapsed; five local and two metastatic, despite adjuvant radiotherapy, of which three died. Treatment of CNS HGNET-MN1 remains a major treatment challenge despite aggressive surgical resections and upfront radiotherapy, warranting new approaches to this rare malignancy.

Clinical, Genomic, and Pharmacological Study of MYCN-Amplified RB1 Wild-Type Metastatic Retinoblastoma.

An uncommon subgroup of unilateral retinoblastomas with highly aggressive histological features, lacking aberrations in RB1 gene with high-level amplification of MYCN (MCYNamplRB1+/+) 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 MCYNamplRB1+/+ 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 MCYNamplRB1+/+ that may be associated with chemotherapy resistance and in vitro/in vivo models that serve as basis for tailoring therapy in these cases.