Abstract
Alveolar rhabdomyosarcoma (ARMS) is characterized by one of three translocation states: t(2;13) (q35;q14) producing PAX3-FOXO1, t(1;13) (p36;q14) producing PAX7-FOXO1, or translocation-negative. Tumors with t(2;13) are associated with greater disease severity and mortality than t(1;13) positive or translocation negative patients. Consistent with this fact, previous work concluded that a molecular analysis of RMS translocation status is essential for the accurate determination of prognosis and diagnosis. However, despite this knowledge, most diagnoses rely on histology and in some cases utilize fluorescence in situ hybridization (FISH) probes unable to differentiate between translocation products. Along these same lines, diagnostic RT-PCR analysis, which can differentiate translocation status, is unable to determine intratumoral translocation heterogeneity, making it difficult to determine if heterogeneity exists and whether correlations exist between this heterogeneity and patient outcomes. Using newly developed FISH probes, we demonstrate that intratumoral heterogeneity exists in ARMS tumors with respect to the presence or absence of the translocation product. We found between 3 and 98% of cells within individual tumor samples contained a translocation event with a significant inverse correlation (R2 = 0.66, p = 0.001) between the extent of intratumoral translocation heterogeneity and failure-free survival of patients. Taken together, these results provide additional support for the inclusion of the molecular analysis of these tumors and expand on this idea to support determining the extent of intratumoral translocation heterogeneity in the diagnosis of ARMS to improve diagnostic and prognostic indicators for patients with these tumors.
Highlights
Rhabdomyosarcoma (RMS) is the one of the most common pediatric soft tissue sarcomas in the United States
While adequate to determine the presence of a FOXO1-containing genetic alteration, the use of this probe requires subsequent RT-PCR analysis to identify the exact translocation event, with RT-PCR being unable to provide more detail about the potential intratumoral heterogeneity of the translocation
We performed fluorescence in situ hybridization (FISH) analysis on all samples using our probes for PAX3, PAX7, and FOXO1 analyzing 100 individual cells for each sample
Summary
Rhabdomyosarcoma (RMS) is the one of the most common pediatric soft tissue sarcomas in the United States. In addition to histological subtype and translocation status, additional factors have been reported to affect prognosis, including age at diagnosis, stage of tumor progression, clinical grade of the tumor (based on the extent of residual disease after surgery), location of the primary tumor, and the presence of metastasis (Lawrence et al, 1987; Crist et al, 1995, 2001; Raney et al, 2001) Despite this information, standard practice in the diagnosis of ARMS in the United States includes the determination of histological phenotype, immunohistochemistry to determine expression levels of muscle specific genes, and occasionally fluorescence in situ hybridization (FISH) to determine translocation status (Morotti et al, 2006). While adequate to determine the presence of a FOXO1-containing genetic alteration, the use of this probe requires subsequent RT-PCR analysis to identify the exact translocation event, with RT-PCR being unable to provide more detail about the potential intratumoral heterogeneity of the translocation
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