Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation

Abstract

Synovial sarcoma is an aggressive spindle cell sarcoma with two major histological subtypes, biphasic and monophasic, defined respectively by the presence or absence of areas of glandular epithelial differentiation. It is characterized by a specific chromosomal translocation, t(X;18)(p11.2;q11.2), which juxtaposes the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene on chromosome X. The chimeric SYT-SSX products are thought to function as transcriptional proteins that deregulate gene expression, thereby providing a putative oncogenic stimulus. We investigated the pattern of gene expression in synovial sarcoma using cDNA microarrays containing 6548 sequence-verified human cDNAs. A tissue microarray containing 37 synovial sarcoma samples verified to bear the SYT-SSX fusion was constructed for complementary analyses. Gene expression analyses were performed on individual tumor samples; 14 synovial sarcomas, 4 malignant fibrous histiocytomas, and 1 fibrosarcoma. Statistical analysis showed a distinct expression profile for the group of synovial sarcomas as compared to the other soft tissue sarcomas, which included variably high expression of ERBB2, IGFBP2, and IGF2 in the synovial sarcomas. Immunohistochemical analysis of protein expression in tissue microarrays of 37 synovial sarcomas demonstrated strong expression of ERBB2 and IGFBP2 in the glandular epithelial component of biphasic tumors and in solid epithelioid areas of some monophasic tumors. Fluorescence in situ hybridization analysis indicated that the ERBB2 overexpression was not because of gene amplification. Differentially expressed genes were also found in a comparison of the expression profiles of the biphasic and monophasic histological subgroups of synovial sarcoma, notably several keratin genes, and ELF3, an epithelial-specific transcription factor gene. Finally, we also noted differential overexpression of several neural- or neuroectodermal-associated genes in synovial sarcomas relative to the comparison sarcoma group, including OLFM1, TLE2, CNTNAP1, and DRPLA. Our high-throughput studies of gene expression patterns, complemented by tissue microarray studies, confirm the distinctive expression profile of synovial sarcoma, provide leads for the study of glandular morphogenesis in this tumor, and identify a new potential therapeutic target, ERBB2, in a subset of cases.

Figure 1.

a: Hierarchical clustering dendrogram separating synovial sarcomas (orange) and MFH/fibrosarcoma (blue). The dendrogram shown on top was generated using a list of 50 genes with the most power to separate the groups based on weight value and a significant expression level in synovial sarcoma, [average ln(RRI) for the synovial sarcomas ≥1.5]. The weight value is listed to the right. A pseudocolored representation of gene expression ratios is shown according to the scale below. b: Multidimensional scaling plot for all 19 tumor samples studied. The similarity of gene expression profiles between any pair of tumor samples was assessed by Pearson correlation coefficients based on expression levels for the 50 genes presented in a. Orange dots denote synovial sarcoma and blue dots denote MFH or fibrosarcoma. c: Genes significantly expressed in biphasic synovial sarcoma as compared to monophasic synovial sarcoma. The four columns to the left represent the expression levels for the biphasic tumors, and the 10 columns to the right represent expression levels for the monophasic tumors. For inclusion, each gene met the following criteria: average ln(RRI) ≥2 for the biphasic and ≤1 for the monophasic tumors, red intensity >500, and ratio ≥1.2 for all biphasic tumors. The color scale is as in b.

Figure 2.

Composite figure showing patterns of ERBB2 and IGFBP2 immunoreactivity in synovial sarcomas from a TMA. Biphasic synovial sarcoma SS-51 shows 3+ ERBB2 immunoreactivity in the glandular epithelial component. Monophasic synovial sarcoma SS-67 shows 2+ ERBB2 immunoreactivity in a solid epithelioid area. Biphasic synovial sarcoma SS-93 shows intense immunoreactivity for IGFBP2 in its epithelial component (lumen formation seen in other fields). Biphasic synovial sarcoma SS-52 shows 2+ ERBB2 immunoreactivity and moderate-to-strong immunoreactivity for IGFBP2 in the glandular epithelial component. In contrast, BCL2 immunostaining, a known marker of the spindle cell component in synovial sarcomas, outlines the nests of primarily BCL2-negative epithelial cells in SS-52.