In vivo and in vitro growth of alveolar soft part sarcoma (ASPS)

269 Alveolar soft part sarcoma (ASPS), a rare malignant neoplasm found predominantly in adolescents and young children, is characterized by a non-reciprocal chromosomal translocation in which the C-terminal region of the transcription factor TFE3 (X chromosome) is fused to the N-terminal region of the gene ASPL (Chromosome 17). Clinically, ASPS is characterized by periods of latency, extremely slow growth and a predilection for metastasis to the lung and brain. The fragility and slow growing nature of ASPS tumor cells in a milieu of rapidly proliferating stromal cells has made it extremely difficult to establish in-vivo and in-vitro models of the disease. We report here the development of an ASPS culture system, permitting the first in-vitro experiments intended to explain the phenotypic effects of the ASPS translocation. In-vivo growth of ASPS from several patients was achieved as a subcutaneous xenograft in sex-matched NOD.SCID\NCr mice. To confirm tumor origin, a representative xenograft was harvested 200 days folllowing implantation. Expression of ASPL-TFE3 fusion transcript was confirmed using RT-PCR and histological analysis revealed PAS-Diastase resistant crystals and intense nuclear staining with antibodies to both TFE3 and to a 25aa peptide homologous to the ASPL-TFE3 junction. Multiple lung metastases were present in this mouse and the tumor exhibited characteristic histopathology and immunohistochemistry. Non-enzymatic disaggregation of the tumor enabled in-vitro culture of cells growing with an estimated doubling time of 180 days, similar to that observed in-vivo. A cytokine/growth factor secretory profile of these in-vitro cultured xenograft cells was performed to better understand their signaling pathways. The results demonstrated the secretion of numerous cytokines/growth factors whose genes were localized to chromosome 17. This suggests that the chromosomal translocation t(X;17)(p11;q25) results in disregulation of multiple genes on chromosome 17. This observation was supported and extended by microarray analysis of 12 additional ASPS clinical samples of ASPS, constituting both primary tumors and metastases. Relative levels of the fusion transcript in each tumor were determined by quantitative RT-PCR, and these values were used to standardize microarray data and illuminate ASPS specific gene expression in a background of contaminating stromal cells. This novel technique revealed that a statistically significant (p