PRO-C3, a Serological Marker of Fibrosis, During Childhood and Correlations With Fibrosis in Pediatric NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease in children and may lead to cirrhosis requiring liver transplant. Thus, prompt diagnosis of advanced fibrosis is essential. Our objectives were to examine PRO-C3 (a neo-epitope pro-peptide of type III collagen formation) levels across childhood/adolescence and associations with advanced fibrosis in pediatric NAFLD. This cross-sectional study included 88 children and adolescents with biopsy-proven NAFLD (mean age: 13.9 ± 2.9 years, 71% male) and 65 healthy participants (11.8 ± 4.5 years, 38% male). PRO-C3, and the bone remodeling biomarkers C-terminal telopeptide of type I collagen (CTX-I; bone resorption) and osteocalcin (N-MID; bone formation), were measured in serum by enzyme-linked immunosorbent assay. Fibrosis was assessed by liver biopsy in participants with NAFLD, who were categorized as having advanced (Ishak score ≥ 3) or none/mild fibrosis (Ishak score ≤ 2). Overall, PRO-C3 was similar in participants with NAFLD (median [interquartile range]: 20.6 [15.8, 25.9] ng/mL) versus healthy participants (19.0 [13.8, 26.0] ng/mL), but was significantly lower in older adolescents ≥ 15 years old (16.4 [13.0, 21.2] ng/mL) compared with children ≤ 10 years old (22.9 [18.1, 28.4] ng/mL; P < 0.001) or 11-14 years old (22.4 [18.3, 31.2] ng/mL; P < 0.001). PRO-C3 was also directly correlated with levels of CTX-I and N-MID (r = 0.64 and r = 0.62, respectively; both P < 0.001). Among participants with NAFLD, PRO-C3 was higher in those with advanced fibrosis (median [IQR]: 28.5 [21.6, 37.6]) compared with none/mild fibrosis (20.3 [18.2, 22.8]; P = 0.020) in models adjusted for age, sex, and body mass index z-score. However, associations were attenuated after additionally adjusting for bone-remodeling CTX-I (P = 0.09) or N-MID (P = 0.08). Conclusion: Collectively, these findings show that PRO-C3 levels are higher in children with advanced fibrosis in NAFLD, but are also influenced by age and pubertal growth spurt, assessed by bone remodeling biomarkers, and therefore may not be a reliable biomarker for liver fibrosis in pediatric NAFLD until late adolescence.

Development of a Plasma Screening Panel for Pediatric Nonalcoholic Fatty Liver Disease Using Metabolomics.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children, but diagnosis is challenging due to limited availability of noninvasive biomarkers. Machine learning applied to high-resolution metabolomics and clinical phenotype data offers a novel framework for developing a NAFLD screening panel in youth. Here, untargeted metabolomics by liquid chromatography-mass spectrometry was performed on plasma samples from a combined cross-sectional sample of children and adolescents ages 2-25 years old with NAFLD (n = 222) and without NAFLD (n = 337), confirmed by liver biopsy or magnetic resonance imaging. Anthropometrics, blood lipids, liver enzymes, and glucose and insulin metabolism were also assessed. A machine learning approach was applied to the metabolomics and clinical phenotype data sets, which were split into training and test sets, and included dimension reduction, feature selection, and classification model development. The selected metabolite features were the amino acids serine, leucine/isoleucine, and tryptophan; three putatively annotated compounds (dihydrothymine and two phospholipids); and two unknowns. The selected clinical phenotype variables were waist circumference, whole-body insulin sensitivity index (WBISI) based on the oral glucose tolerance test, and blood triglycerides. The highest performing classification model was random forest, which had an area under the receiver operating characteristic curve (AUROC) of 0.94, sensitivity of 73%, and specificity of 97% for detecting NAFLD cases. A second classification model was developed using the homeostasis model assessment of insulin resistance substituted for the WBISI. Similarly, the highest performing classification model was random forest, which had an AUROC of 0.92, sensitivity of 73%, and specificity of 94%. Conclusion: The identified screening panel consisting of both metabolomics and clinical features has promising potential for screening for NAFLD in youth. Further development of this panel and independent validation testing in other cohorts are warranted.

Natural History of NAFLD Diagnosed in Childhood: A Single-Center Study.

Little is known regarding the subsequent course of non-alcoholic fatty liver disease (NAFLD) diagnosed in childhood. The objectives of this single-center study were to gather data on long-term health outcomes and to assess the feasibility of contacting former pediatric patients. In a large pediatric medical center, electronic records were searched to initially identify 162 former patients who had a liver biopsy between 2000 and 2010. Of these, 44 subjects met the criteria for age at follow-up (≥18 year) and biopsy-proven NAFLD, and were recruited via postal and electronic mail. Participants were invited to complete a brief telephone survey on current health status. Supplemental data was also obtained from pediatric medical charts of all subjects. At NAFLD diagnosis, 18% of subjects had diabetes, 91% were obese, 61% had NASH, and 56% had fibrosis on biopsy. At follow-up, 10 subjects (23%) responded to the survey. Based on the survey and chart review, after a mean follow-up of 4.5 years, 5 additional subjects developed diabetes for a period prevalence of 30%, and most subjects (78%) remained obese at last follow-up. Additional prospective studies are needed to fully describe the longitudinal risks associated with pediatric NAFLD, and will require multi-dimensional strategies to successfully recruit former patients.

Congenital mesoblastic nephroma: a study of 19 cases using immunohistochemistry and ETV6-NTRK3 fusion gene rearrangement.

Mesoblastic nephroma (MN) is the most common renal tumour in the first 3 months of life and accounts for 3-5% of all paediatric renal neoplasms. To further understand the morphological variants of MN, we identified 19 cases of MN (five classic, eight cellular and six mixed) and examined each case for markers known to be important in urogenital embryological development (PAX8, WT1 and RCC), stem cell associated markers (Oct 4, CD34 and c-kit), muscle/myofibroblastic markers (muscle specific actin, calponin and h-caldesmon), aberrant transcription factors, cell cycle regulation and other oncogenic proteins (p16, cyclin D1 and beta-catenin). Fluorescence in situ hybridisation (FISH) testing for ETV6-NTRK3 gene fusion/rearrangement revealed further differentiation between the subtypes with ETV6-NTRK3 gene fusion detected in 0/5 of the classic MN, 8/8 of the cellular MN and 5/6 of the mixed MN cohorts, respectively. Our results conclude that cyclin D1 and beta-catenin may be useful markers for differentiating between cellular MN and classic MN when the histology is not conclusive. The absence of expression of stem cell markers and markers involved in urogenital development suggests that MN is not a nephroma and most likely represents a soft tissue tumour, with congenital infantile fibrosarcoma representing cellular MN with a predilection to arise in the kidney. In addition, the immunophenotype and genetic fingerprint of mixed MN most likely represents a heterogenous group of tumours that are mostly cellular type, with areas that are phenotypically less cellular.

Rare presentation of four primary pediatric cardiac tumors.

Pediatric cardiac tumors are extremely rare and usually benign. We selected four unique cases of pediatric cardiac tumors from a 15-year period at our institution. The four chosen cases represent unique, rare primary tumors of the heart. Our selection includes a case of Rosai Dorfman disease without systemic involvement, which is, to our knowledge, the second case of isolated cardiac Rosai Dorfman disease in a child. We present a case of subtotal replacement of myocardium by granulocytic sarcoma with minimal bone marrow involvement, representing the first reported case in a child manifested as hypertrophic cardiomyopathy, as well as a case of a primary synovial sarcoma arising from the atrioventricular (AV) node, representing the fourth reported pediatric case of a cardiac synovial sarcoma, and it is the first to arise from the AV node. Finally, we present a primary congenital infantile fibrosarcoma of the heart, which is, to our knowledge, the first confirmed cardiac congenital infantile fibrosarcoma. These four cases represent the need for continued inclusion of rare cardiac conditions in a clinician's differential diagnosis. Furthermore, they present the need for more in-depth molecular and genomic analysis of pediatric cardiac tumors in order to identify their etiopathogenesis.

Atypical Marginal Zone Hyperplasia Is a Mimic for Lymphoma in Pediatric Transplant Recipients: Report of Two Patients.

Atypical marginal zone hyperplasia (AMZH) of mucosa-associated lymphoid tissue (MALT) closely resembles lymphoma in that it shows expansion of the marginal zones with prominent intraepithelial B lymphocytes, is immunoglobulin light-chain restricted, and may show aberrant CD43 expression. However, unlike lymphoma, it does not show rearrangement of the immunoglobulin heavy chain gene (immunoglobulin H [IgH]) by polymerase chain reaction (PCR), and it behaves in a benign fashion. We identified AMZH in 2 pediatric solid organ transplant recipients who presented with adenotonsillar hypertrophy. To date, the patients have experienced a self-limited course in the absence of treatment or reduction of immunosuppression. Atypical marginal zone hyperplasia is a pitfall for posttransplant lymphoproliferative disorder and MALT lymphoma in the pediatric solid organ transplant population. In transplant patients with a lambda-restricted B-cell clone and marginal zone hyperplasia in native MALT sites, PCR for IgH and IgK gene rearrangement is essential to prevent misdiagnosis.

Immunohistochemical Investigation of BRAF p.V600E mutations in thyroid carcinoma using 2 separate BRAF antibodies.

BACKGROUND: Approximately 45% of papillary thyroid carcinomas harbor BRAF p.V600E mutations and current practice algorithms endorse molecular testing for BRAF p.V600E. We assessed the utility of immunohistochemistry to detect BRAF p.V600E mutations in thyroid carcinomas using 2 separate BRAF monoclonal antibodies: one that detects both mutant and wild-type protein (pan-BRAF) and another that detects only the mutant protein (mut-BRAF). METHODS: We selected 41 formalin-fixed paraffin-embedded thyroid carcinomas (29 papillary, 1 follicular, 7 medullary, and 4 anaplastic) from 37 thyroidectomies and 4 fine-needle aspirations. Immunohistochemistry was performed using a pan-BRAF (clone EP152Y) or a mut-BRAF (clone VE1) monoclonal antibody. Tumors were considered positive if >10% of neoplastic cells showed moderate (2+) or strong (3+) cytoplasmic staining. BRAF p.V600E mutations were confirmed by molecular pyrosequencing, the gold standard for statistical analysis. RESULTS: pan-BRAF reactivity was observed in 80.5% (n=33) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 46.3% (n=19) were wild type. mut-BRAF reactivity was observed in 46.3% (n=19) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 12.2% (n=5) were wild type. The pan-BRAF antibody detected 14 more false positives (specificity: 29.6%, PPV: 42.4%) compared with the mut-BRAF antibody (specificity: 61.5%, PPV: 73.7%), but both antibodies detected the same 5 false positives. No false negatives were detected with either antibody (sensitivity and NPV 100.0% for both). CONCLUSIONS: The suboptimal specificity and PPV limits the diagnostic utility of both antibodies to reliably detect BRAF p.V600E mutations in thyroid carcinoma. However, both antibodies provide excellent sensitivity and NPV and either could be used to exclude BRAF wild-type thyroid carcinomas before molecular testing.

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition.

BACKGROUND: Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition. RESULTS: We isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment. CONCLUSION: These data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS.