David N. Shapiro

Activation of an imprinted allele of the insulin-like growth factor II gene implicated in rhabdomyosarcoma.

The insulin-like growth factor II (IGF2) gene is exclusively silent at the maternal allele in the mouse as well as in normal human tissues and is expressed at a high level in rhabdomyosarcoma (RMS). We report here that the normally imprinted allele of the IGF2 gene is activated in RMS tumors as well as in one RMS cell line. Since overexpression of IGF2 has been shown to be important in the pathogenesis of RMS, our data suggest that loss of imprinting (LOI) may lead to overexpression of IGF2 and play an important role in the onset of RMS. Furthermore, embryonal RMS usually has loss of heterozygosity (LOH) with paternal disomy of the IGF2 locus. One informative embryonal RMS tumor evaluated in this study was heterozygous at the IGF2 allele and had LOI, raising the possibility that LOI may be the functional equivalent of LOH in this tumor with both events leading to overexpression of IGF2.

Antisense-mediated reduction in insulin-like growth factor-I receptor expression suppresses the malignant phenotype of a human alveolar rhabdomyosarcoma.

The expression of the insulin-like growth factors (IGFs) and their receptors has been linked to cellular proliferation and tumorigenicity in a number of model systems. Since rhabdomyosarcoma cells express IGF-I receptors, an autocrine or paracrine loop involving this receptor and its ligands could be responsible in part for the growth characteristics of this tumor. To assess directly the role of the IGF-I receptor in rhabdomyosarcoma cell growth and tumorigenicity, a human alveolar rhabdomyosarcoma cell line with high IGF-I receptor expression was transfected with an amplifiable IGF-I receptor antisense expression vector. Four unique, transfected clones were analyzed and found to have reduced IGF-I receptor expression relative to the parental line. Integration of the antisense sequence was demonstrated by Southern blot analysis, and expression of antisense message in these clones was shown by S1 nuclease protection assay. Reduced IGF-I receptor surface expression in the transfectants was shown by decreased immunofluorescence with an IGF-I receptor monoclonal antibody and by decreased IGF-I binding as measured by Scatchard analysis. These clones had markedly reduced growth rates in vitro, impaired colony formation in soft agar, and failed to form tumors in immunodeficient mice when compared with vector-transfected clones. These results demonstrate that reduction of IGF-I receptor expression can inhibit both the in vitro and in vivo growth of a human rhabdomyosarcoma cell line and suggest a role for the IGF-I receptor in mediating neoplastic growth in this mesenchymally derived tumor.

MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature.

A typical case of alveolar soft part sarcoma was found to express in a strong and widespread fashion the marker MyoD1 protein. This nuclear phosphoprotein is the product of a regulatory gene that controls the commitment of a cell to myogenic lineage; it therefore provides strong and perhaps definitive evidence in support of the skeletal muscle nature of this enigmatic neoplasm.

Chromosomal assignment and genomic structure of Il15

Interleukin-15 (IL-15) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukin-2 (IL-2), presumably because IL-15 utilizes the beta and gamma chains of the IL-2 receptor. Murine IL-15 cDNA and genomic clones were isolated and characterized. The murine Il15 gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization.

Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35.

Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.

Solid alveolar rhabdomyosarcomas with the t(2;13). Report of two cases with diagnostic implications

Two patients having rhabdomyosarcomas with the recently described “solid variant” pattern of alveolar rhabdomyosarcoma are reported. Both tumors had aggressive cytologic features with monotonous sheets of round nuclei, but without fibrous septa, and both had been initially classified as embryonal rhabdomyosarcomas. Cytogenetic analyses of tumor explants from both cases revealed the t(2;13) chromosomal aberration typical of alveolar rhabdomyosarcoma. Both patients died of metastases, despite aggressive surgical and adjuvant therapy. This report represents the first karyotypic analyses of solid alveolar rhabdomyosarcomas and supports the inclusion of these tumors in the alveolar category despite the lack of fibrous septa.

Management and prognosis of head and neck sarcomas

Between 1962 and 1988, a total of 104 patients with head and neck rhabdomyosarcoma (RMS) and 17 patients with nonrhabdomyosarcoma (NRMS) were evaluated and treated at St. Jude Childrens Research Hospital. All parameningeal sites (middle ear, orbit, or nasopharynx) were excluded from further analysis; thus, 50 patients represent the cohort of head and neck sarcomas for this review. Survival was good in this group of patients, 28 of 50 being alive and disease-free at last follow-up. Twenty of the 38 patients with RMS were alive and disease-free. Similarly, 8 of the 12 patients with NRMS were disease-free at a median follow-up of 5 years. However, the site and size of the primary tumor impacted on the extent of the initial resection and further treatment in addition to surgery. Although the treatment policy evolved over time to a stage-specific strategy for treatment modalities, the data suggest that surgery alone may be sufficient initial therapy for a subset of patients. For patients in whom complete resection is not achieved, the addition of radiotherapy and chemotherapy may result in improved survival.

Sarcomas of the flexor fossae in children: Is amputation necessary?

Successful management of sarcomas of the extremities in children implies not only achievement of local control but also satisfactory function and maintenance of the growth potential. The popliteal and antecubital fossae, because of their complex neurovascular anatomy, all of which is essential, make resection with satisfactory margins difficult. We reviewed our experience with 14 patients (3 to 20 years old; median, 13 years) with soft tissue sarcomas arising in the popliteal (11 patients) or antecubital (3 patients) fossae. There were four rhabdomyosarcomas (3 alveolar, 1 embryonal) and 10 other sarcomas, the most frequent being synovial sarcoma (5 patients). Chemotherapy was given to all patients with rhabdomyosarcomas. The one patient presenting with metastatic disease was treated, after biopsy of the primary, by chemotherapy and radiation and survived 21 months. In three patients, the primary management was an above-the-knee amputation and two of three survived (3 and 43 months). In 10 patients a wide local excision of the primary tumor was performed. Radiation therapy was administered to five, either as external beam (3 patients) or as brachytherapy (2 patients). In this group, there were no local recurrences. Four patients remain free of disease (4 months to 18 years) and one developed pulmonary metastasis. Among the five non-irradiated patients, three developed local recurrences, requiring above-the-knee amputation for disease. The fourth patient relapsed in the lung and only one of the five is free of disease at 36 months. Of the 8 patients not treated with amputation, one acquired a leg length discrepancy, which required correction, and one has a minimal extension deficit of the knee.(ABSTRACT TRUNCATED AT 250 WORDS)

A phase II trial evaluation selective use of altered radiation dose and fractionation in patients with unresectable rhabdomyosarcoma

PURPOSE Between 1987 and 1991, 25 children with advanced rhabdomyosarcoma (20 with IRS Group 3 disease and 5 with Group 4 disease) were entered on a prospective study evaluating selective use of hyperfractionated irradiation (HFI) and reduced-dose conventionally fractionated irradiation (CFI), based on disease status following induction chemotherapy (ifosfamide or melphalan, followed by vincristine, adriamycin, and cyclophosphamide combination) with or without delayed surgery. METHODS AND MATERIALS Patients with gross disease following induction chemotherapy with or without delayed surgery, and whose primary tumor sites did not involve the central nervous system, received HFI (n = 12) at 1.1 Gy twice-a-day to 59.4-63.8 Gy total. Patients with parameningeal primaries and intracranial disease extension received HFI with initiation of therapy (n = 2). Those with microscopic disease following induction chemotherapy with or without delayed surgery (n = 11) received reduced-dose CFI to 40 Gy. Active follow-up ranges from 28-75 months (median = 43 months) with no patient lost to follow-up. RESULTS Eighteen patients (72%) are alive and without evidence of disease, including 8 of the children with gross residual disease postinduction therapy. The absolute 2-year continuous local tumor control rate is 86% for all patients. Among the 14 who received HFI, the absolute 2-year continuous local tumor control rate is 75% at 33 to 67 months (median = 38 months) postirradiation. Hyperfractionated irradiation was associated with expected enhancement of acute reactions, which all resolved with conservative medical management. Grade 4 or 5 acute toxicities were not seen. Significant late radiation morbidity has, thus far, been minimal and limited to Grade 1 and 2 events. Among the 11 who received reduced-dose CFI, the absolute 2-year continuous local tumor control rate is 100% at 25 to 70 months (median = 40 months) postirradiation. CONCLUSION This limited experience suggests that HFI to a dose level of 60 Gy can be used selectively in children with advanced rhabdomyosarcoma left with gross disease following induction chemotherapy, with or without delayed surgery, with an apparent improvement in local control, and minimization of potential late radiation toxicity. Concurrently, those left with microscopic disease following induction therapy can be selectively treated with reduced-dose CFI with excellent local control.

Rhabdomyosarcoma: From the Laboratory to the Clinic

The prospect of identifying and developing new agents for treatment of rhabdomyosarcomas is discussed in the light of current prognosis for children with advanced stage disease. Preliminary attempts to identify tumor-specific agents using in vitro cell culture show potential promise, but as yet remain unproven. The more complex system of identifying therapeutically active agents using human tumor xenografts has demonstrated usefulness. The potential problems associated with this system are discussed.