Deborah E. Schofield

Molecular Classification of Rhabdomyosarcoma—Genotypic and Phenotypic Determinants of Diagnosis: A Report from the Children's Oncology Group

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.

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. Ewings 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 Ewings 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 Ewings 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.

Fibrosarcoma in infants and children: application of new techniques

Fibrosarcomas occurring in infants and young children are intriguing tumors. Although biologically more benign than their counterparts occurring in old patients, they are histologically similar. Microscopically, they fit in one end of a spectrum of fibrous proliferations occurring in the pediatric population. Prompted by recent karyotypic reports of nonrandom gains of chromosomes 8, 11, 17, and 20 in infantile fibrosarcomas, we retrospectively analyzed 12 infantile fibrosarcomas utilizing fluorescence in-situ hybridization (FISH) with chromosome 8-, 11-, 17-, and 20-specific probes. Parallel studies were performed on fibrosarcomas occurring in older children and young adults and also on cellular fibromatoses, myofibroma-toses, and fibromatoses. Gains of chromosomes 8, 11, 17, and 20 (in various combinations) were observed in 11 of 12 fibrosarcomas occurring in infants <2 years of age. Extra copies of chromosomes 17 and 20 were observed in a fibrosarcoma occurring in a 5-year-old child but no abnormalities were detected by FISH in four additional fibrosarcomas occurring in patients aged 6–17 years. One of three cellular fibromatoses was characterized by extra copies of chromosome 8, 11, 17, and 20. Similar findings were not observed in any of the noncellular fibromatoses or in myofibromatoses.

Intestinal Neuronal Dysplasia

Summary 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 Childrens Hospital of Pittsburgh. Cases of proven Hirschsprungs disease were excluded. In 38 biopsies from 38 patients, we found a mild to moderate increase in mucosal acetyl-cholinesterase 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 Hirschsprungs 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.

Selective Usage of D-Type Cyclins by Ewing’s Tumors and Rhabdomyosarcomas

The genetic mechanisms that control proliferation of childhood musculoskeletal malignancies, notably Ewing’s tumor (ET) and rhabdomyosarcoma (RMS), remain largely unknown. Most human cancers appear to overexpress at least one of the G1 cyclins (cyclins D1, D2, D3, E1, and E2) to bypass normal regulation of cell cycle G1 progression. We compared the gene expression profiles of 7 ET and 13 RMS primary tumor samples and found overexpression of cyclin D1 in all 7 ET samples. In contrast, RMS samples expressed higher levels of cyclin D2, cyclin D3, and cyclin E1. This was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot. The relative roles of RAS-extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3′-kinase (PI3K)-AKT pathways in the regulation of D-type cyclin expression in these tumors were then assessed. Inhibition of either pathway reduced expression of cyclins D1, D2, and D3 in RMS lines, whereas only PI3K inhibitors blocked cyclin D1, D2, and D3 expression in ET lines. Furthermore, PI3K-AKT appeared to regulate D-type cyclin transcription in RMS lines through FKHR and FKHRL1. Finally, the role of the ET-associated EWS-FLI1 fusion gene in regulating D cyclin expression was studied. Inhibition of EWS-FLI1 expression in the TC71 ET line decreased cyclin D1 levels but increased cyclin D3 levels. In contrast, induction of EWS-FLI1 expression in the RD RMS cell line increased cyclin D1 expression but decreased cyclin D3 expression. Our results demonstrate distinct regulation of D-type cyclins in ET and RMS and indicate that EWS-FLI1 can modulate the expression of D-type cyclins independent of cellular backgrounds.

Acetylcholinesterase-stained suction rectal biopsies in the diagnosis of Hirschsprung's disease

Summary: Conflicting reports in the literature regarding the sensitivity and specificity of the acetylcholinesterase (AChE) stain in establishing or excluding the diagnosis of Hirschsprungs 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.

cdna microarray analysis of global gene expression in sarcomas

Sequencing of the human genome and rapidly evolving microarray technology have combined to provide investigators with the ability to analyze individual tumors and groups of tumors for global patterns of gene expression. Few of these types of studies have been performed on rhabdomyosarcomas and osteogenic sarcomas, including cell lines and animal models. Groups of expressed genes that may characterize rhabdomyosarcomas and their subgroups and separate them from other types of tumors have been identified. More specifically, genes involved in myogenesis or the inhibition of myogenesis have been identified, as have genes that may play a role in metastatic activity in osteogenic sarcomas. Also, a study documenting the consistent and specific gene expression profile of gastrointestinal stromal tumors has been published. While the data regarding gene expression patterns in sarcomas is accruing, numerous investigators are working on developing and enhancing bioinformatic skills and tools such that the vast amount of data can be converted into knowledge regarding biology, therapeutic responsiveness or resistance, and prognosis.

An unusual round cell tumor of the tibia with granular cells

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 Ewings sarcoma, and small-cell osteosarcoma. We favor the former interpretation, although we raise the possibility that it may be a unique lesion.

Expression Profiling of Bone Tumors

A variety of neoplasms arise in bone. Many of these, such as osteogenic sarcoma, are relatively unique to bone, while others, such as hemangiomas and lymphomas, are more ubiquitous in their origins. This chapter will focus on the malignant tumors whose primary site of origin is bone and, in particular, those tumors whose cell of origin is mesenchymal rather than hematopoietic. The most common mesenchymal tumors of bone are osteosarcomas, chondrosarcomas, and Ewing’s sarcomas. As Ewing’s sarcomas are discussed in more detail in Chapter 16, they will be addressed here in a comparative fashion with osteosarcoma and other common sarcomas of childhood.