High-resolution array CGH and gene expression profiling of alveolar soft part sarcoma

Abstract

Purpose: Alveolar soft part sarcoma (ASPS) is a soft tissue sarcoma with poor prognosis, and little molecular evidence exists for its origin, initiation, and progression. The aim of this study was to elucidate candidate molecular pathways involved in tumor pathogenesis.

Experimental design: We employed high-throughput array comparative genomic hybridization (aCGH) and cDNA-Mediated Annealing, Selection, Ligation, and Extension Assay to profile the genomic and expression signatures of primary and metastatic ASPS from 17 tumors derived from 11 patients. We used an integrative bioinformatics approach to elucidate the molecular pathways associated with ASPS progression. FISH was performed to validate the presence of the t(X;17)(p11.2;q25) ASPL-TFE3 fusion and, hence, confirm the aCGH observations.

Results: FISH analysis identified the ASPL-TFE3 fusion in all cases. aCGH revealed a higher number of numerical aberrations in metastatic tumors relative to primaries, but failed to identify consistent alterations in either group. Gene expression analysis highlighted 1,063 genes that were differentially expressed between the two groups. Gene set enrichment analysis identified 16 enriched gene sets (P < 0.1) associated with differentially expressed genes. Notable among these were several stem cell gene expression signatures and pathways related to differentiation. In particular, the paired box transcription factor PAX6 was upregulated in the primary tumors, along with several genes whose mouse orthologs have previously been implicated in Pax6 DNA binding during neural stem cell differentiation.

Conclusion: In addition to suggesting a tentative neural line of differentiation for ASPS, these results implicate transcriptional deregulation from fusion genes in the pathogenesis of ASPS.

Figure 1. Case of ASPS

A. Haematoxylin and Eosin (x10) of tumor highlighting the alveolar to solid pattern B. Positive nuclear TFE3 immunohistochemistry (x40). C–D. Interphase FISH with BAC-based probes harboring TFE3 and ASPL on a male patient with ASPS.

Figure 2. Top 323 differentially expressed (DE) genes

The heatmap depicts the expression of genes with t-test score greater than 3. Each row represents a gene. The samples (in columns) are grouped as 5 primary (green labels) and 8 metastatic (gold labels) sarcomas. Red and blue entries in the heatmap indicate high and low gene expressions respectively. The black horizontal line separates the genes up-regulated in primary sarcomas (above the line) from those up-regulated in metastases.

Figure 3. Gene Set Enrichment Anaylsis for all top differentially expressed genes

Each column represents a geneset that is either a key biological process/ pathway/ gene signature from the literature that is enriched among the top 323 DE genes. Each horizontal orange bar represents one of the top DE genes depicted in the sorted order t test score. The black horizontal line separates the genes up-regulated in primary sarcomas (above the line) from those up-regulated in metastases. The occurrence of Pax6, as a stem cell transcription factor, is marked with a green arrow and the distribution of its targets among the DE genes according to mouse ChIP studies [Sansom et al.] is shown with green marks beside the plot.

Figure 4. Differential Expression of Pax-6 Targets

The heatmap depicts the top DE genes (in rows) whose mouse orthologs show Pax-6 binding with DNA in ChIP studies [ Sansom et al.] in the process of neural stem cell differentiation in the developing mouse cerebral cortex. The samples (in columns) are grouped as primary (green labels) and metastatic (gold labels) sarcomas. Red and blue rectangles indicate high and low gene expressions respectively. The expression of the paired box transcription factor Pax6 is shown in the topmost row.

Figure 5. Composite analysis of the break at Xp11.23 and subsequent imbalance in 3 primary and 4 metastatic tumors

Blue horizontal line represents the location of the break at TFE3. Aberrations above the ADM-1 threshold of 10.0 are represented by the colored regions deviating to the left (relative loss to control) or right (relative loss to control). Imbalances in the primary and metastatic tumors are color coded as yellow and blue, respectively.