Angelica R. Putnam

Lipoblastoma (LPB): A Clinicopathologic and Immunohistochemical Analysis of 59 Cases

Lipoblastoma (LPB) is a benign neoplasm that occurs predominantly in early childhood. We investigated clinicopathologic features, associated conditions, immunohistochemistry, and outcome in 59 LPB identified from surgical pathology and consultation files. Pathology materials, cytogenetics reports, and medical records were reviewed. Immunohistochemistry for S100 protein, CD34, and Mib-1 was performed on formalin-fixed, paraffin-embedded tissue using standard techniques. Fifty-nine patients had 74 samples, including 14 patients with one or more with local recurrences among the 30 patients who had available follow-up information. There were 37 males and 22 females (ratio 1.7). Age at diagnosis ranged from 3 months to 16 years with 22% in the first year, 68% at 1 to 9 years, and 10% at 10 to 16 years. Sixty-four percent arose on the trunk, 27% on the extremities, and 8% in the head/neck. Forty-six percent had one or more recurrences. Tumor diameter ranged from 1.2 to 15.5 cm. The white to yellow cut surface showed variable lobulation and myxoid change. Histologically, nodules of adipose and myxoid tissue were demarcated by bands of fibrous tissue. The cells displayed a range of differentiation from multivacuolated lipoblasts to mature adipocytes. Mitoses were nonexistent to rare. Histologic variations included a subtle zonal architecture of fat maturation, abundant myxoid material, primitive mesenchymal cells, a focal plexiform vascular pattern, and multinucleated cells. All cases tested were immunoreactive for S100 and CD34; Mib-1 reactivity was absent to low. Cytogenetic aberrations included chromosome 8 abnormalities in 8 cases, nonspecific numerical abnormalities in 1 case, and a normal karyotype in 1 case. Ten patients had other medical conditions including macrocephaly, seizures, developmental delay, autism, congenital anomalies, Sturge-Weber syndrome, or a family history of multiple lipomas. In summary, this large series of LPB demonstrates its occurrence in older children and adolescents, documents a recurrence rate of 46% and confirms that the degree of adipocytic differentiation does not predict biologic behavior. An unexpected finding was the presence in 17% of patients of central nervous system disorders such as seizures, autism, and developmental delay, congenital anomalies, Sturge-Weber syndrome, or a family history of lipomas. These observations raise the question of whether predisposing genetic or other constitutional factors contribute to the development of LPB or whether LPB is indicative of a syndrome.

Molecular inversion probe analysis detects novel copy number alterations in Ewing sarcoma

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults, with dismal outcomes for metastatic and relapsed disease. To better understand the molecular pathogenesis of ES and to identify new prognostic markers, we used molecular inversion probes (MIPs) to evaluate copy number alterations (CNAs) and loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded (FFPE) samples, which included 40 ES primary tumors and 12 ES metastatic lesions. CNAs were correlated with clinical features and outcome, and validated by immunohistochemistry (IHC). We identified previously reported CNAs, in addition to SMARCB1 (INI1/SNF5) homozygous loss and copy neutral LOH. IHC confirmed SMARCB1 protein loss in 7-10% of clinically diagnosed ES tumors in three separate cohorts (University of Utah [N = 40], Childrens Oncology Group [N = 31], and University of Michigan [N = 55]). A multifactor copy number (MCN)-index was highly predictive of overall survival (39% vs. 100%, P < 0.001). We also identified RELN gene deletions unique to 25% of ES metastatic samples. In summary, we identified both known and novel CNAs using MIP technology for the first time in FFPE samples from patients with ES. CNAs detected by microarray correlate with outcome and may be useful for risk stratification in future clinical trials.

MORTALITY AND PATHOLOGICAL FINDINGS IN C (OPITZ TRIGONOCEPHALY) SYNDROME

Even as a rare multiple congenital anomalies/mental retardation syndrome, the C-syndrome (CS, or Opitz C-trigonoecephaly syndrome) is, at long last, beginning to attract attention because of its developmental and causal complexity. Also, the possibility that the apparently balanced translocation recently described in an affected Japanese boy may soon provide a molecular/causal insight into this disorder. The manifestations recorded in the previously published patients, those autopsied within recent years, and the unpublished instances in our files suggest that the CS is a heterogeneous genetic disorder, predominantly sporadic but with sufficient familial cases (at times with consanguinity) to allow postulation of an entity due to autosomal dominant mutations with a high rate of germinal mosaicism, or due to both autosomal dominant mutations and an autosomal recessive genocopy. In any event, elucidation of cause and pathogenesis of CS will, in due time, shed light on its developmental pleiotropy, rarity in liveborn infants, prevalence in stillborn fetuses, recurrence risk in humans, and occurrence in other animals (e.g., mice) to further understanding of pathogenesis.

Recurrence of Achondrogenesis Type 2 in Sibs: Additional Evidence for Germline Mosaicism

Achondrogenesis Type II (ACG2) is a lethal skeletal disorder caused by a dominant mutation in the type II collagen gene (COL2A1). Familial cases have been reported, suggesting both germline and somatic mosaicism. We report on two pregnancies from the same couple with gross, radiologic, and microscopic findings of ACG2. Molecular analysis of the second infant demonstrated heterozygosity for a c.2303G > A transition (p.Gly768Asp) in exon 33 of the COL2A1 gene. Although this mutation could not be proven by molecular studies in the first infant, identical findings in two affected pregnancies support germline mosaicism as the cause of ACG2 in this family.

Prenatal death in Smith–Lemli–Opitz/RSH syndrome

Smith–Lemli–Opitz (RSH) syndrome is an autosomal recessive disorder involving virtually all organ systems. Severity varies widely, but all degrees of severity are due to the disturbance of the same genetic/biochemical system involving a deficiency of cholesterol [Irons et al., 1993]. The enzyme 7dehydrocholesterol reductase, which is abnormal in RSH syndrome, catalyzes the last step of endogenous cholesterol synthesis [Moebius et al., 1998; Wassif et al., 1998; Waterham et al., 1998]. The gene, DHCR7, which encodes this enzyme, is located on chromosome region 11q13. Some 100 different mutations in the DHCR7 gene have been identified in more than 200 patients with the syndrome [Opitz, 2001]. Cholesterol is a precursor of steroid hormones and bile acids. It is an important component of myelin, mitochondrial and cell membranes [Opitz, 2001; Opitz et al., 2002]. Decreased 7DHCR activity leads to cholesterol deficiency and subsequent accumulation of the precursor molecules 7and 8-dehydrocholesterol. Prenatally, there is a direct correlation between the severity of the phenotype and 7DHC levels in the amniotic fluid. The clinical condition can range from prenatal death with malformations of multiple organ systems to minimal physical effects and only mild behavior problems and mental impairment in children and adults [Nowaczyk et al., 1999, 2001]. Malformations observed in the RSH syndrome include Yshaped 2–3 toe syndactyly, polydactyly, unilobate lungs, renal dysplasia, or agenesis, Hirschsprung disease, complex cardiac malformations, cataracts, central nervous malformations such as microcephaly and agenesis of corpus callosum, oropharyngeal malformations (cleft palate), genital ambiguity in genetic males, and facial abnormalities such as anteverted nostrils, micrognathia, and apparently low-set ears [Smith et al., 1964; Cunniff et al., 1997; Kelley, 2000]. Opitz et al. [2002] described three cases studied pathologically. Case 1 involved a spontaneous miscarriage of an approximately 19-week gestational age female fetus. Ultrasonography had demonstrated an enlarged third ventricle, thickening of the myocardium and oligohydramnios. Autopsy documented a large secundum atrial septal defect, muscular ventricular septal defect, hypertrophy of the left ventricle and enlargement of the right ventricle. The severely hypoplastic lungs were unilobate with an incipient fissure on the right. Hepatomegaly, bilateral renal agenesis with rudimentary ureters, and a bifid uterus were identified. The umbilical cord had three vessels. Case 2 was confirmed by gas chromatography mass-spectrometry of amniotic fluid. The cholesterol content was 6.2 mg/ml; 7and 8-dehydrocholesterol levels were 2.2 and 0.9 mg/ml, respectively. The stillborn male fetus was delivered at 22 weeks of gestation. Ultrasonography at 21 weeks had shown short limbs, micropenis, and possible hydrocephalus. Autopsy findings included fused eyelids, a nose with a single midline nasal pit and choanal atresia, high arched palate with broad alveolar ridges, postminimus of the left hand, postaxial polydactyly of the feet, syndactyly of the 2nd and 3rd, and 5th and 6th toes bilaterally, and nuchal edema. The external genitalia were female. The left lung was unilobate and the right lung was bilobate. The brain was a small solid mass (OFC 17 cm, more compatible with atelen/aprosencephaly than cebocephaly). The umbilical cord had two vessels. Case 3 was a term, live born infant. A short umbilical cord, cleft palate and abnormal oropharynx were noted at delivery. The infant had upward slanting palpebral fissures, small pupils, cataracts, a flattened nose, and a small tongue, mouth and mandible. The ears were angulated posteriorly. The infant had bilateral 2/3 toe syndactyly and polydactyly of both second toes, 3rd degree hypospadias, and unilateral cryptorchidism. The diagnosis was confirmed on the basis of hypocholesterolemia (19 mg/dl) and hyperdehydrocholesterolemia (180 mg/ ml). At 61 days of life, cholesterol treatment had increased the cholesterol levels to 55 mg/dl, but the infant had no psychomotor development. He developed hypertension, reflux, delayed gastric emptying, and suffered numerous upper and lower respiratory infections which ultimately lead to his death at age 7.5 months. Angle et al. [1998] reported an atypical case of RSH syndrome with hydrops, uncharacteristic facial appearance, and absence of 2/3 toe syndactyly in a 46,XY fetus who died at birth. Antenatal ultrasonography was abnormal and maternal uE3 levels were low. Cultured skin fibroblasts showed elevated 7DHC levels and abnormalities of cholesterol synthesis. In the atypical, but biochemically confirmed case of Seller et al. [1995] there was oligohydramnios with severe Pottersequence changes, webbing of axillae and crura (fetal akinesia), small right thumb and severe hypoplasia of right 5th finger, absence of left 5th finger, partial cutaneous syndactyly of toes 2/3 bilaterally, female external genitalia with testes and ducts, agenesis of right kidney and ureter and severe hypoplasia of the left, unilobate left lung, bilobed right lung, one pair of cervical ribs, and non-ossification or absence of vertebrae S4 and S5. Thus, postaxial oligodactyly may be the developmental equivalent of postaxial polydactyly. In the biochemically confirmed infant of Ness et al. [1997] there was, in addition to typical malformations, biventricular myocardial hypertrophy, PDA, aortic coarctation, bicuspid aortic valve, hepatomegaly with severe cholestasis, early septal fibrosis, marked bile staining of meninges with strikingly abnormal gyral pattern, mild hydrocephalus with porencephaly, absence of corpus callosum, and a hypoplastic cerebellum. Histologically there was an abnormality of cortical migration with four instead of six layers, and a severe lack of myelination. Also noted were depletion of thymocytes, enlarged hyperchromatic nuclei of the pancreatic islet cells *Correspondence to: Dr. Juliana G. Szakacs, Department of Pathology, University Utah School of Medicine, 50 N., Medical Drive, Salt Lake City, UT 84132. E-mail: Juliana.Szakacs@path.utah.edu

The effect of surgical margins on outcomes for low grade MPNSTs and atypical neurofibroma

While convention defines atypical neurofibroma as benign and low‐grade malignant peripheral nerve sheath tumors (MPNSTs) as malignant, sparse outcomes data exist for these tumors. This study reviews clinical outcomes of surgically resected low‐grade MPNST and atypical neurofibroma, focusing on the effect of surgical margins on outcome.

Prenatal death in Fraser syndrome.

Cryptophthalmos may be partial or complete, unilateral or bilateral, apparently nonsyndromal or syndromal. A recent study of 2 stillborn infants at the University of Utah prompted an analysis of the developmental aspects of the syndromal form (Fraser syndrome). We conclude that, per se, cryptophthalmos is a developmental field defect on the basis of heterogeneity (autosomal dominant and recessive forms) and phylogeneity (occurrence also in the pheasant, rabbit, pigeon, dog, and mouse). In humans this autosomal recessive disorder maps to 4q21, is homologous to the bleb (bl/bl) mouse, and is due to mutations in the FRAS1 gene that codes for a 4007 amino acid protein 85% identical to the Fras1 gene of the bleb mouse. Commonest anomalies in humans are cryptophthalmos, cutaneous syndactyly of digits, abnormal ears and genitalia, renal agenesis, and congenital heart defects. Almost half of affected infants are stillborn or die in infancy, and mental retardation is common. The pathogenesis evidently involves abnormal epithelial integrity during prenatal life. Older (mostly German) publications, some dating to the 19th century, provide a fascinating historical insight into the process of syndrome delineation.

C/EBPβ-1 promotes transformation and chemoresistance in Ewing sarcoma cells

CEBPB copy number gain in Ewing sarcoma was previously shown to be associated with worse clinical outcome compared to tumors with normal CEBPB copy number, although the mechanism was not characterized. We employed gene knockdown and rescue assays to explore the consequences of altered CEBPB gene expression in Ewing sarcoma cell lines. Knockdown of EWS-FLI1 expression led to a decrease in expression of all three C/EBPβ isoforms while re-expression of EWS-FLI1 rescued C/EBPβ expression. Overexpression of C/EBPβ-1, the largest of the three C/EBPβ isoforms, led to a significant increase in colony formation when cells were grown in soft agar compared to empty vector transduced cells. In addition, depletion of C/EBPβ decreased colony formation, and re-expression of either C/EBPβ-1 or C/EBPβ-2 rescued the phenotype. We identified the cancer stem cell marker ALDH1A1 as a target of C/EBPβ in Ewing sarcoma. Furthermore, increased expression of C/EBPβ led to resistance to chemotherapeutic agents. In summary, we have identified CEBPB as an oncogene in Ewing sarcoma. Overexpression of C/EBPβ-1 increases transformation, upregulates expression of the cancer stem cell marker ALDH1A1, and leads to chemoresistance.

Classic adamantinoma with osteofibrous dysplasia-like foci and secondary aneurysmal bone cyst.

Adamantinoma, a rare bone lesion of the tibia and fibula, has two distinct variants, classic adamantinoma and osteofibrous dysplasia-like adamantinoma. Composite lesions have not been described. Aneurysmal bone cyst is a benign cystic lesion which may also occur in the tibia and fibula. We report an unusual case of classic adamantinoma with osteofibrous dysplasia-like areas and foci of secondary aneurysmal bone cyst with prominent giant cells. A lesion was diagnosed in a 17-year-old girl with a 14-year history of a slowly enlarging left tibial mass and increasing deformity. Pathologically, the predominant pattern was classic adamantinoma, with minor foci of osteofibrous dysplasia-like adamantinoma and areas of secondary aneurysmal bone cyst with abundant multinucleated giant cells. We report the clinical, radiologic, and pathologic features of this case, and summarize lesions associated with secondary aneurysmal bone cyst. To our knowledge, the association of adamantinoma with secondary aneurysmal bone cyst has not been previously reported.

Unilateral Sclerocornea and Tracheal Stenosis: Unusual Findings in a Patient with Goldenhar Anomaly

The Goldenhar anomaly (GA) is a heterogeneous field defect of uncertain cause and wide variability of expression, characterized by facial phenotypes, usually asymmetric and unilateral, accompanied by various combinations and gradations of cardiac, skeletal, renal, and central nervous system defects. We report the pathologic findings in a 5-month-old boy with GA, tracheal stenosis, and left unilateral sclerocornea. To the best of our knowledge this is the first description of sclerocornea in a patient with GA.