Brain tumors are the leading cause of cancer‐related death in children. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of pediatric brain cancers are limited and hard to establish. We present a protocol that enables efficient generation, expansion, and biobanking of pediatric brain cancer organoids. Utilizing our protocol, we have established patient‐derived organoids (PDOs) from ependymomas, medulloblastomas, low‐grade glial tumors, and patient‐derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the tumors from which they were derived. We also showed that PDOs can be xenografted. Most interestingly, when subjected to the same routinely applied therapeutic regimens, PDOs respond similarly to the patients. Taken together, our study highlights the potential of PDOs and PDXOs for research and translational applications for personalized medicine.

Familial intrahepatic cholestases (FICs) are a heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. Three distinct forms are described: FIC1 and FIC2, associated with low/normal GGT level in serum, which are caused by impaired bile salt secretion due to defects in ATP8B1 encoding the FIC1 protein and defects in ABCB11 encoding bile salt export pump protein, respectively; FIC3, linked to high GGT level, involves impaired biliary phospholipid secretion due to defects in ABCB4, encoding multidrug resistance 3 protein. Different mutations in these genes may cause either a progressive familial intrahepatic cholestasis (PFIC) or a benign recurrent intrahepatic cholestasis (BRIC). For the purposes of the present study we genotyped 27 children with intrahepatic cholestasis, diagnosed on either a clinical or histological basis. Two BRIC, 23 PFIC and 2 BRIC/PFIC were identified. Thirty-four different mutations were found of which 11 were novel. One was a 2Mb deletion (5’UTR- exon 18) in ATP8B1. In another case microsatellite analysis of chromosome 2, including ABCB11, showed uniparental disomy. Two cases were compound heterozygous for BRIC/PFIC2 mutations. Our results highlight the importance of the pathogenic role of novel mutations in the three genes and unusual modes of their transmission.

Focal adhesion kinase (FAK) is over-expressed and is correlated with aggressiveness in adult hepatocellular carcinoma (HCC). Inhibition of FAK decreases HCC invasiveness by down-regulating Enhancer of Zeste homolog 2 (EZH2), an epigenetic controller, that acts in transcriptional repression of a large number of genes via histone 3 methylation of lysine 27 (H3K27me3). Here, we investigated the hepatic expression of total FAK, EZH2, H3K27me3, and proliferating cell nuclear antigen (PCNA) in 17 pediatric HCCs and 8 healthy livers (CTRL). Quantitative imaging analysis showed that FAK gene/protein expression is up-regulated in HCCs compared to CTRL and, among tumor samples the levels of this gene/protein are significantly higher in cirrhotic HCCs than in a healthy milieu. Accordingly, the protein levels of EZH2 were also significantly increased in HCCs from a cirrhotic background. Intriguingly, the protein expression of FAK, EZH2, and PCNA significantly inversely correlated with tumor size. Finally, in HCC samples, mainly in cirrhotic background, the up-regulation of FAK gene positively correlated with that observed in β-Catenin gene. Conclusion: FAK gene/protein is over-expressed in pediatric HCCs concomitantly to EZH2 protein and β-Catenin gene, with a more significant up-regulation in a cirrhotic background. This triad of interactors deserves further investigations for translational application.

P.R375W (Fibrinogen Aguadilla) is one out of seven identified mutations (Brescia, Aguadilla, Angers, Al du Pont, Pisa, Beograd, and Ankara) causing hepatic storage of the mutant fibrinogen γ. The Aguadilla mutation has been reported in children from the Caribbean, Europe, Japan, Saudi Arabia, Turkey, and China. All reported children presented with a variable degree of histologically proven chronic liver disease and low plasma fibrinogen levels. In addition, one Japanese and one Turkish child had concomitant hypo-APOB-lipoproteinemia of unknown origin. We report here on an additional child from Turkey with hypofibrinogenemia due to the Aguadilla mutation, massive hepatic storage of the mutant protein, and severe hypo-APOB-lipoproteinemia. The liver biopsy of the patient was studied by light microscopy, electron microscopy (EM), and immunohistochemistry. The investigation included the DNA sequencing of the three fibrinogen and APOB–lipoprotein regulatory genes and the analysis of the encoded protein structures. Six additional Fibrinogen Storage Disease (FSD) patients with either the Aguadilla, Ankara, or Brescia mutations were investigated with the same methodology. A molecular analysis revealed the fibrinogen gamma p.R375W mutation (Aguadilla) but no changes in the APOB and MTTP genes. APOB and MTTP genes showed no abnormalities in the other study cases. Light microscopy and EM studies of liver tissue samples from the child led to the demonstration of the simultaneous accumulation of both fibrinogen and APOB in the same inclusions. Interestingly enough, APOB-containing lipid droplets were entrapped within the fibrinogen inclusions in the hepatocytic Endoplasmic Reticulum (ER). Similar histological, immunohistochemical, EM, and molecular genetics findings were found in the other six FSD cases associated with the Aguadilla, as well as with the Ankara and Brescia mutations. The simultaneous retention of fibrinogen and APOB-lipoproteins in FSD can be detected in routinely stained histological sections. The analysis of protein structures unraveled the pathomorphogenesis of this unexpected phenomenon. Fibrinogen gamma chain mutations provoke conformational changes in the region of the globular domain involved in the “end-to-end” interaction, thus impairing the D-dimer formation. Each monomeric fibrinogen gamma chain is left with an abnormal exposure of hydrophobic patches that become available for interactions with APOB and lipids, causing their intracellular retention and impairment of export as a secondary unavoidable phenomenon.