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Legends to Supplementary Figures from EWS-FLI1 Fusion Protein Up-regulates Critical Genes in Neural Crest Development and Is Responsible for the Observed Phenotype of Ewing's Family of Tumors

Supplementary Figures 1-3 from EWS-FLI1 Fusion Protein Up-regulates Critical Genes in Neural Crest Development and Is Responsible for the Observed Phenotype of Ewing's Family of Tumors

Tumor-specific translocations are common in tumors of mesenchymal origin. Whether the translocation determines the phenotype, or vice versa, is debatable. Ewing's family tumors (EFT) are consistently associated with an EWS-FLI1 translocation and a primitive neural phenotype. Histogenesis and classification are therefore uncertain. To test whether EWS-FLI1 fusion gene expression is responsible for the primitive neuroectodermal phenotype of EFT, we established a tetracycline-inducible EWS-FLI1 expression system in a rhabdomyosarcoma cell line RD. Cell morphology changed after EWS-FLI1 expression, resembling cultured EFT cells. Xenografts showed typical EFT features, distinct from tumors formed by parental RD. Neuron-specific microtubule gene MAPT, parasympathetic marker cholecystokinin, and epithelial marker keratin 18 were up-regulated. Conversely, myogenesis was diminished. Comparison of the up-regulated genes in RD-EF with the Ewing's signature genes identified important EWS-FLI1 downstream genes, many involved in neural crest differentiation. These results were validated by real-time reverse transcription-PCR analysis and RNA interference technology using small interfering RNA against EWS-FLI1 breakpoint. The present study shows that the neural phenotype of Ewing's tumors is attributable to the EWS-FLI1 expression and the resultant phenotype resembles developing neural crest. Such tumors have a limited neural phenotype regardless of tissue of origin. These findings challenge traditional views of histogenesis and tumor origin.

3 Supplementary Figures from EWS-FLI1 Fusion Protein Up-regulates Critical Genes in Neural Crest Development and Is Responsible for the Observed Phenotype of Ewing's Family of Tumors

Supplementary Figure Legends from EWS-FLI1 Fusion Protein Up-regulates Critical Genes in Neural Crest Development and Is Responsible for the Observed Phenotype of Ewing's Family of Tumors

Background Dysembryoplastic neuroepithelial tumors (DNETs) are uncommon neural tumors presenting most often in children and young adults and associated with intractable seizures. Rare midline neoplasms with similar histological features to those found in DNETs have been described near the septum pellucidum and termed "DNET-like neoplasms of the septum pellucidum." Due to their rarity, these tumors have been described in just a few reports and their genetic alterations sought only in small series. Methods We collected 20 of these tumors for a comprehensive study of their clinical, radiological, and pathological features. RNA sequencing or targeted DNA sequencing was undertaken on 18 tumors, and genome-wide DNA methylation profiling was possible with 11 tumors. Published cases (n = 22) were also reviewed for comparative purposes. Results The commonest presenting symptoms and signs were related to raised intracranial pressure; 40% of cases required cerebrospinal fluid diversion. Epilepsy was seen in approximately one third of cases. All patients had an indolent disease course, despite metastasis within the neuraxis in a few cases. Radiologically, the septum verum/septal nuclei were involved in all cases and are the proposed site of origin for septal DNET (sDNET). Septal DNET showed a high frequency (~80%) of mutations of platelet derived growth factor receptor A (PDGFRA), and alterations in fibroblast growth factor receptor 1 (FGFR1) and neurofibromatosis type 1 (NF1) were also identified. In a genomic DNA methylation analysis alongside other neural tumors, sDNETs formed a separate molecular group. Conclusions Genetic alterations that are different from those of cerebral DNETs and a distinct methylome profile support the proposal that sDNET is a distinct disease entity.

The authors reviewed clinical features, surgical extent of resection, histologic parameters, and DNA content in 55 children with medulloblastomas and found that complete or near total resection, absence of tumor dissemination, tumor DNA aneuploidy, and low proliferative index correlated with a favorable clinical outcome. A scoring system was developed based upon these features to identify patients who, in the future, may benefit from more aggressive or novel therapeutic regimens. Patient age and sex and adjuvant chemotherapy did not significantly correlate with long-term survival. The data also suggest that tumors that have been designated as cerebellar neuroblastomas may be a distinct group of posterior fossa tumors, which may have a better prognosis.

Pulmonary infiltrates with eosinophilia (PIE) are a group of heterogeneous disorders having the common findings of lung disease and eosinophilia in the peripheral blood, bronchoalveolar lavage fluid, or pulmonary interstitium. Eleven cases of PIE syndromes were identified through a retrospective and prospective chart review: drug-induced (2), acute eosinophilic pneumonia (3), infant pulmonary eosinophilia (2), parasite-induced (2), Churg-Strauss syndrome (1), and atypical chronic PIE (1). Patient demographics, clinical presentation, and disease severity varied considerably among groups. Therapeutic interventions included bronchodilators (10), oxygen (7), corticosteroids (9), and mechanical ventilation (3). A single patient with acute eosinophilic pneumonia died. Our experience suggests that PIE syndromes are rare in childhood and that clinical presentation can vary widely. Because of the potential for significant morbidity and mortality, aggressive diagnostic evaluations are warranted, particularly in children with respiratory failure of unknown etiology.

We present the clinical, radiographic and histopathologic findings of an unusual tumor that originated in the diaphysis of the tibia in a 10-year-old boy. Clinical symptoms had been present for approximately 2 years and radiographic abnormalities for > or = 8 months before biopsy and subsequent resection of the neoplasm. The child is doing well 4 1/2 years later. Microscopically, the tumor was not typical of any bone tumor with which we are familiar. It was a round-cell tumor with extensive fibrosis, prominent cytoplasmic granularity, and isolated immunoreactivity for vimentin, epithelial membrane antigen, and antichymotrypsin. Ultrastructural examination uncovered the presence of both well-formed desmosomes and cell-associated basement membrane material in addition to abundant phagolysosomes. Classification of this tumor is a challenge; the differential diagnosis includes atypical adamantinoma, atypical Ewing's sarcoma, and small-cell osteosarcoma. We favor the former interpretation, although we raise the possibility that it may be a unique lesion.

Nerve growth factor (NGF) has the potential to induce cellular differentiation in various neoplastic and nonneoplastic cell lines. In this study, our aim was to determine NGF receptor (NGFr) status in medulloblastoma specimens and cell lines and to investigate whether NGF could act as a potential differentiating agent for this common pediatric brain tumor. Paraffin-embedded tumor tissue from 10 patients with the diagnosis of medulloblastoma was retrospectively analyzed to determine the frequency of NGFr expression. Of the 10 tumor specimens evaluated, 4 were positive for NGFr; however, NGFr staining was confined to only 5 to 8% of the cells in a randomly scattered pattern. No colocalization was present with neuronal, glial, or vascular structures. In addition, two medulloblastoma cell lines established in our laboratory were also evaluated for NGFr. In this study, we also examined the effects of retinoic acid, 12-O-tetradecanoyl-phorbol-13-acetate, and NGF on medulloblastoma cell lines to evaluate their effect on morphological differentiation and NGFr expression. Although these agents failed to cause NGFr expression in our cell lines, morphological alteration was noticed in only one of the cell lines with retinoic acid. Therefore, because of the lack of de novo or induced NGFr expression, it is unlikely that NGF will be useful as a potential therapeutic differentiating agent for medulloblastomas.

The purpose of this article is to summarize some of the current issues in diagnostic histopathology and to provide an update of both molecular and cytogenetic studies of the most commonly encountered pediatric brain tumors. Most of the discussion focuses on astrocytomas, primitive neuroectodermal tumors and medulloblastomas, and ependymomas because they make up the majority of these neoplasms. A few comments on choroid plexus tumors are also included.

We encountered an infant with clinical and histopathologic features of Gaucher's disease (infantile, type 2) with normal glucocerebrosidase (d-glucosyl- N -acylsphingosine glucohydrolase, E.C.3.2.1.45) activity. Biochemical analysis was performed on leukocytes, cultured skin fibroblasts, and liver. Normal activity of glucocerebrosidase previously has been reported in an older child with juvenile onset (type 3) Gaucher's disease and attributed to a deficiency of a sphingolipid activator protein. These rare cases illustrate and expand our concept of Gaucher's disease and may have both diagnostic and therapeutic implications.

(1992). Case 4 Intestinal Neuronal Dysplasia in a Case of Sigmoid Stenosis. Pediatric Pathology: Vol. 12, No. 2, pp. 275-280.

Rhabdomyosarcoma (RMS) in children occurs as two major histological subtypes, embryonal (ERMS) and alveolar (ARMS). ERMS is associated with an 11p15.5 loss of heterozygosity (LOH) and may be confused with nonmyogenic, non-RMS soft tissue sarcomas. ARMS expresses the product of a genomic translocation that fuses FOXO1 (FKHR) with either PAX3 or PAX7 (P-F); however, at least 25% of cases lack these translocations. Here, we describe a genomic-based classification scheme that is derived from the combined gene expression profiling and LOH analysis of 160 cases of RMS and non-RMS soft tissue sarcomas that is at variance with conventional histopathological schemes. We found that gene expression profiles and patterns of LOH of ARMS cases lacking P-F translocations are indistinguishable from conventional ERMS cases. A subset of tumors that has been histologically classified as RMS lack myogenic gene expression. However, classification based on gene expression is possible using as few as five genes with an estimated error rate of less than 5%. Using immunohistochemistry, we characterized two markers, HMGA2 and TFAP2ss, which facilitate the differential diagnoses of ERMS and P-F RMS, respectively, using clinical material. These objectively derived molecular classes are based solely on genomic analysis at the time of diagnosis and are highly reproducible. Adoption of these molecular criteria may offer a more clinically relevant diagnostic scheme, thus potentially improving patient management and therapeutic RMS outcomes.

Conflicting reports in the literature regarding the sensitivity and specificity of the acetylcholinesterase (AChE) stain in establishing or excluding the diagnosis of Hirschsprung's disease (HD) prompted this review of 497 rectal biopsies performed on 455 children. Using hematoxylin and eosin (H&E) to stain formalin-fixed, paraffin-embedded tissue sections is our preferred method of identifying ganglion cells. In this series, however, there were eight children with HD, and nine without HD in whom the AChE-stained portion of the sample provided invaluable diagnostic information not obtained from the concomitant, formalin-fixed, H&E-stained portion of the sample. The AChE stain also provided at least suggestive evidence of HD in some of the anal or anorectal biopsy specimens.

Congenital teratoma rarely occurs in the craniofacial region. We present an infant with a nasal tip teratoma that was documented by ultrasonographic examination. The tumor was excised at 1 day of life, and the splayed upper and lower lateral cartilages were apposed primarily. The remaining redundant skin was excised in a second stage. To our knowledge, this is the first reported case of a nasal tip teratoma.

Twin pregnancies with a complete hydatidiform mole (CHM) and a coexisting fetus have an aggressive postevacuation behavior; it is, therefore, important to differentiate these cases from partial hydatidiform moles that rarely require treatment for late sequelae. It has been presumed that twin pregnancies with a CHM and a coexistent fetus are dizygotic gestations, but this has not been confirmed in most cases. The authors investigated the sex chromosomal constitution of paraffin-embedded, formalin-fixed placental tissues in nine pregnancies histopathologically diagnosed as twin gestations with CHM and coexisting fetus, using fluorescent in situ hybridization (FISH) with X- and Y-chromosomal probes. Normal placental tissues showed an even sex distribution—four cases: X signal only, presumably female; four cases: X and Y signals, presumably male. In contrast, all molar tissues of these same pregnancies hybridized with the Xchromosomal probe only. Thus, in four of nine cases, gender differences (ie, different sex chromosome content) in molar villi (X chromosome only, cytogenetic female) versus normal villi (both sex chromosomes, cytogenetic male) confirmed the histopathological diagnosis of dizygotic twinning; a strict relationship between villous morphology (molar vs normal) and chromosomal gender was observed in each instance. This study illustrates that use of FISH on paraffin-embedded tissues can retrospectively establish dizygotic twinning in this unusual type of molar gestation.

A 3-year-old girl of Navajo heritage had intractable diarrhea beginning at 4 days of age and resulting in long-term hyperalimentation. Investigation before multivisceral transplantation included biopsies of the rectum, stomach, duodenum, and liver. The diagnosis of microvillus inclusion disease was established by documentation of microvillus inclusions in duodenal epithelial cells. A trial of somatostatin therapy was ineffective in controlling the diarrhea. Subsequently, a multivisceral organ transplant provided a unique opportunity to establish the gastrointestinal extent of involvement of this disease. Ultrastructural microvillus inclusions were identified in the duodenum, jejunum, ileum, and colon, but not in the gallbladder. A few inclusions also were documented in gastric antral epithelial cells. Alkaline phosphatase stains performed on paraffin-embedded material showed a few inclusions in the antrum of the stomach and many inclusions throughout the small intestine, primarily in surface epithelial cells but also in upper crypt cells.

Four hundred ninety-eight acetylcholinesterase-stained suction rectal biopsies from 456 children were examined to assess the occurrence of "isolated" or "primary" intestinal neuronal dysplasia at Children's Hospital of Pittsburgh. Cases of proven Hirschsprung's disease were excluded. In 38 biopsies from 38 patients, we found a mild to moderate increase in mucosal acetylcholinesterase staining and abundant submucosal ganglion cells (large, irregular ganglia or at least five ganglia per HPF). This group was clinically heterogeneous with a frequent history of prematurity and small left colon/meconium plug syndrome, protein/formula intolerance, or obstructive anatomic gastrointestinal abnormalities. We feel that isolated "intestinal neuronal dysplasia," as diagnosed by suction rectal biopsy and the above criteria, is a descriptive biopsy appearance. Rather than characterizing a unique disease entity, it is encountered in a variety of clinical situations. Based on our observations and review of the literature, we therefore conclude that "intestinal neuronal dysplasia" not associated with Hirschsprung's disease, neurofibromatosis, or multiple endocrine neoplasia syndrome has yet to be well defined in clinical and histopathologic terms. The histologic diagnosis, at this time, should be reserved for those rare, florid cases of parasympathetic hyperganglionosis that are documented by adequate tissue sampling.

We describe a congenitally malformed heart with usual atrial arrangement and concordant atrioventricular connexions in which a solitary arterial trunk left the base of the heart and supplied the coronary and systemic arteries. Pulmonary blood supply was derived from systemic to pulmonary collateral arteries but, in addition, hypoplastic intrapericardial pulmonary arteries were found in each lung hilum. The two sets of hilar arteries were discontinuous, but it seemed likely that this discontinuity had been artefactually produced by prior dissection. Irrespective of this matter, the left pulmonary arteries were connected by a thread-like atretic cord to the left-sided sinus of the solitary arterial trunk. This indicates that, initially, there had been a common trunk which now demonstrates pulmonary atresia. The significance of this lesion is discussed in the light of the nomenclature and description of hearts with common arterial trunk.