Studies on the molecular pathogenesis of extraskeletal myxoid chondrosarcoma-cytogenetic, molecular genetic, and cDNA microarray analyses

Abstract

Extraskeletal myxoid chondrosarcomas (EMCs) are characterized by recurrent chromosome translocations resulting in fusions of the nuclear receptor TEC to various NH(2)-terminal partners. Here we describe the phenotypic, cytogenetic, and molecular genetic characteristics of a series of 10 EMCs. Using spectral karyotyping and fluorescence in situ hybridization, clonal chromosome abnormalities were detected in all but one tumor. A t(9;22)(q22;q12) translocation was found in three cases; a del(22)(q12-13)in one case; and variant translocations, including t(9;17)(q22;q11-12), t(7;9;17)(q32;q22;q11), and t(9;15)(q22;q21), were detected in one case each. Recurrent, secondary abnormalities, including trisomy 1q, 7, 8, 12, and 19, were found in seven tumors. All tumors contained translocation-generated or cryptic gene fusions, including EWS-TEC (five cases, of which one was a novel fusion), TAF2N-TEC (four cases), and TCF12-TEC (one case). cDNA microarray analysis of the gene expression patterns of two EMCs and a myxoid liposarcoma reference tumor revealed a remarkably distinct and uniform expression profile in both EMCs despite the fact that they had different histologies and expressed different fusion transcripts. The most differentially expressed gene in both tumors was CHI3L1, which encodes a secreted glycoprotein (YKL-40) previously implicated in various pathological conditions of extracellular matrix degradation as well as in cancer. Our findings suggests that EMC exhibits a tumor-specific gene expression profile, including overexpression of several cancer-related genes as well as genes implicated in chondrogenesis and neural-neuroendocrine differentiation, thus distinguishing it from other soft tissue sarcomas.

Figure 1.

A: Classical EMC, case 2. B: Classical areas with uniform, bland spindle cells in a myxoid matrix, corresponding to area A in case 4. C: Same case, area C showing prominent pleomorphism and associated with an increased proliferative index. D: Unusual, entirely cellular and solid variant of EMC, case 8. E: Ultrastructural detection of scattered tumor cells with dense core granules of neurosecretory type, case 3. F and G: Distinct immunoreactivity for chromogranin (F) and synaptophysin (G), case 6. H and J: Strong immunoreactivity for RELB (predominantly cytoplasmic) (H) and GSTM4 (cytoplasmic and nuclear) (J) in case 7. I and K: The myxoid liposarcoma reference tumor is predominantly negative for RELB (I) and focally, weakly positive for GSTM4 (K).

Figure 2.

Partial G-banded and partial and complete SKY karyotypes of case 1 (A), case 2 (B), case 3 (C), case 4C (D), case 4B (E), and case7 (F). Translocated chromosome segments are indicated. Breakpoints are denoted by arrowheads.

Figure 3.

A: Detection of TEC-containing chimeric transcripts by RT-PCR using EWS-TEC-specific primers in case 1 (lane 1), case 3 (lane 2), case 2 (lane 3), case 5 (lane 6), and TAF2N-TEC-specific primers in case 4, areas A, B, and C (lanes 7, 8, and 9). Lanes 4, 5, and 10 are controls (PCR reactions with EWS-TEC- and TAF2N-TEC-specific primers but without cDNA template). The DNA Molecular Weight Marker VIII (Boehringer Mannheim) was used as a molecular weight marker (lane M). B: Nucleotide and deduced amino acid sequences of the EWS-TEC breakpoint junction in case 5. The fusion point is indicated by a vertical line and the ATG initiation codon in TEC is indicated by an asterisk. The previously unrecognized TEC exon 2b is underlined.

Figure 4.

Comparison of gene expression profiles by means of scatter plots of log intensities. Comparisons of the expression profiles of the two EMC cases 7 and 8 (A), case 7 (B) versus the myxoid liposarcoma reference tumor. Each point in a scatter plot represents 1 of the 4000 genes and expressed sequence tags analyzed.

Figure 5.

RT-PCR analysis of the expression of CHI3L1 in EMC cases 7 (lane 1), 8 (lane 2), 4B (lane 3), and 5 (lane 4) as well as in four myxoid liposarcomas (lanes 5 to 8). Lane 9 is a control (PCR reactions with CHI3L1- and GAPDH-specific primers but without cDNA template) and lane M is a molecular weight marker (DNA Molecular Weight Marker VIII, Boehringer Mannheim). A 550-bp GAPDH fragment was co-amplified with the 270-bp CHI3L1 fragment as an internal standard. Expression of CHI3L1 was only detected in the four EMC samples (lanes 1–4).