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(American Journal of Pathology. 2003;162:781-792.)
© 2003 American Society for Investigative Pathology

Studies on the Molecular Pathogenesis of Extraskeletal Myxoid Chondrosarcoma—Cytogenetic, Molecular Genetic, and cDNA Microarray Analyses

Helene Sjögren*, Jeanne M. Meis-Kindblom*, Charlotte Örndal*, Peter Bergh{dagger}, Konrad Ptaszynski*, Pierre Åman*, Lars-Gunnar Kindblom* and Göran Stenman*

From the Department of Pathology,* Lundberg Laboratory for Cancer Research, and the Department of Orthopedic Surgery,{dagger} Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Extraskeletal myxoid chondrosarcomas (EMCs) are characterized by recurrent chromosome translocations resulting in fusions of the nuclear receptor TEC to various NH2-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 detectedin 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, includingt(9;17)(q22;q11–12), t(7;9;17)(q32;q22;q11), andt(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.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare and unique soft tissue malignancy that is clinically characterized by involvement of the proximal extremities and limb girdles of adults and has a frequently prolonged course despite a high incidence of local recurrences and metastases.1,2 Light microscopically, ultrastructurally, and histochemically, EMC shows some resemblance to embryonal cartilage.1-3 The morphological spectrum of EMC is wide, thus the differential diagnosis based on histology alone is at times problematic.2 The origin of EMC remains unclear and recent reports of neural-neuroendocrine differentiation in a subset of EMC has posed further questions regarding its histogenesis.4,5

Recent cytogenetic and molecular genetic studies of EMC have revealed three pathogenetically relevant chromosome translocations. The t(9;22)(q22;q12) translocation, found in ~75% of the cases, results in a fusion of the 5' part of the EWS gene to the TEC gene.6-9 The deduced chimeric protein consists of the NH2-terminal transactivation domain of EWS linked to the entire TEC protein. EWS encodes a putative RNA-binding protein and TEC (also called NR4A3, NOR1, MINOR, and CHN) encodes a novel orphan nuclear receptor belonging to the steroid/thyroid receptor gene super family.8-11 Similarly, the recurrent, less frequent (15% of cases), t(9;17)(q22;q11) results in a fusion of the NH2-terminal part of the EWS-related gene TAF2N to TEC.12-14 We recently identified yet a third variant translocation t(9;15)(q22;q21) that generates a fusion of the NH2-terminal part of the basic helix-loop-helix transcription factor TCF12 to TEC.15

The NH2-terminal parts of EWS and the related protein, TLS (FUS), are also regular fusion partners of different transcription factors in several other sarcomas, including Ewing’s sarcoma, clear cell sarcoma of tendons and aponeuroses, desmoplastic small round cell tumor, and myxoid liposarcoma.16 Presumably the TET family of fusion proteins (TLS, EWS, and TAF2N) exert their oncogenic effect by functioning as abnormal transcription factors.17 At least some fusion proteins, eg, EWS-FLI1 and EWS-TEC, may also function as dominant-negative inhibitors of splicing that affect splice site selection.18,19 Recently, it was shown that the AF2 (activation function-2) domain of TEC is essential for the transcriptional activity of the EWS-TEC fusion protein and that EWS-TEC, at least in part, interacts with the same transcriptional co-activators as the normal TEC protein.20

To gain further insights into the molecular pathogenesis of EMC we have extended and supplemented our previous investigations7,8,12,15 with cytogenetic, fluorescence in situ hybridization (FISH), spectral karyotype (SKY), and molecular analyses of five new tumors as well as with FISH and SKY analyses of previously reported tumors. In addition, we have used cDNA microarray to study the gene expression pattern in EMCs with differing fusion genes and morphologies.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tumor Material

Ten EMCs from nine patients were analyzed (Table 1) . The tumors from five of the patients (cases 1 to 5) have not been previously reported. The remaining five tumors from four patients (cases 6-I and 6-II as well as cases 7 to 9) have been previously reported regarding the expression of EMC-specific fusion transcripts, and all but two of these (cases 5 and 6-I) have also been cytogenetically analyzed.7,12,15 For SKY analysis, frozen, cultured tumor cells from three of the previously reported tumors were thawed, explanted, and harvested after 10 to 14 days in vitro.7,12 In addition, total RNA and/or cDNA were available for analyses in six EMCs. As a reference tumor for the cDNA microarray analysis, fresh frozen tumor tissue from a myxoid liposarcoma was used. Archival material from four additional myxoid liposarcomas was used for validation of the microarray results. All five myxoid liposarcomas expressed TLS-CHOP fusion transcripts.21


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  		Table 1. Clinical-Pathological Data on Nine EMC Cases

 
Light and Electron Microscopy and Immunohistochemistry

The 10 tumors were histologically reviewed and classified as either classical or cellular-solid variants of EMC as previously described.2,15 Tumors from all nine patients were obtained fresh, fixed in glutaraldehyde, and routinely processed for electron microscopy. Using standardized antigen retrieval techniques, all cases were analyzed immunohistochemically with regard to cytokeratins (AE1/AE3, KL1, and CAM 5.2), S100 protein, neuron-specific enolase, chromogranin A, synaptophysin, glial fibrillary acidic protein (GFAP), and Ki-67 (MIB1), as previously described.2 For validation of the cDNA microarray results the following antibodies and dilutions were used: RELB (1:25), COL3A1 (1:10), XRCC3 (1:50) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and GSTM-4 (1:500; Novocastra, Newcastle, UK). Antigen retrieval techniques, using citrate buffer (pH 6.0) or Tris-ethylenediaminetetraacetic acid (pH 9.0) and microwave heating (17 minutes) were applied. For detection, the Techmate (ChemMate; DAKO, Glostrup, Denmark) detection kit systems were used.

Cytogenetic and FISH Analyses

Primary cultures were established from fresh, unfixed specimens of four of the new EMC cases as previously described.15 Chromosome preparations were made from exponentially growing primary cultures or early passage cells (tumor cells from the three previously cultured EMCs), and these were subsequently G-banded and analyzed using standard procedures. Cytogenetic analysis of a second metastasis from a previously harvested EMC was also completed (case 6-I).12

FISH analysis was performed on metaphase chromosomes using wcp probes specific for the Y chromosome and chromosomes 9 and 17 (Vysis, Inc., Downers Grove, IL), and {alpha}-satellite probes specific for chromosomes 1, 2 (Vysis), and 14/22 (Appligene Oncor; Qbiogene, Carlsbad, CA). Chromosomes were counterstained with 4',6'-diamidino-2'-phenylindole dihydrochloride (DAPI). Interphase FISH analysis of formalin-fixed, paraffin-embedded tissue sections was performed using a paraffin pretreatment kit (Vysis) as recommended by the manufacturer. Five-µm sections were independently hybridized with {alpha}-satellite probes for chromosomes 7, 8, and 12, and telomeric probes for 1q and 19q (Vysis). The sections were counterstained with DAPI, and evaluated using a Zeiss Axioplan 2 Imaging fluorescence microscope (Carl Zeiss GmbH, Göttingen, Germany). At least 500 nuclei were scored with the exception of four sections hybridized with the 1q probe in which only 200 to 250 nuclei per section could be scored. All analyses were performed without knowing the case identity or cytogenetic findings. Ten sections were randomly selected, analyzed, and re-analyzed by two independent reviewers; the results obtained fell within a 5% range for each section studied. Five-µm sections of normal tonsil used as controls indicated that the basal background level of three signals per nuclei was in the range of 1 to 5% for all five probes tested. A specimen was therefore considered to be trisomic for a given chromosome if three separate signals for each respective probe could be detected in more than 10% of the nuclei analyzed.

SKY Analysis

SKY analysis was performed in 7 of 10 EMCs (there was no material available for SKY analysis in cases 5 and 9, case 8 had been previously analyzed).15 The conditions for pretreatment, hybridization, posthybridization washes, and detection were essentially as recommended by the manufacturer (ASI-Applied Spectral Imaging Ltd., Migdal Ha'Emek, Israel). Image acquisition was achieved with the SpectraCube system (ASI) mounted on a Zeiss Axioplan 2 Imaging microscope equipped with a custom-designed optical filter cube (SKY-1; Chroma Technology, Brattleboro, VT) and a DAPI filter.22 Analysis of spectral images was performed using the SkyView software (ASI).

Isolation of RNA, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and Nucleotide Sequence Analyses

Total RNA was extracted from frozen tumor tissue using the Trizol (Invitrogen Life Technologies, Carlsbad, CA) method. For cDNA synthesis, 5 µg of total RNA were reverse-transcribed using the SuperScript Preamplification System according to the manufacturer’s manual (Invitrogen). An aliquot of 0.25 µg of the resulting first-strand cDNA was amplified using the appropriate primer sets. Thirty-six cycles of PCR (30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C) were performed with 1 µl of cDNA in 50-µl reaction volumes. The AmpliTaq Gold (Applied Biosystems, Foster City, CA) DNA polymerase was used for the amplification reactions. PCR primers used for amplification of normal TEC and CHI3L1 transcripts, TEC-containing fusion transcripts, and nucleotide sequence analysis are shown in Table 2 .8,12,15,23,24 As a control for intact RNA and cDNA, a RT-PCR reaction for expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on all cDNAs used (Table 2) . PCR products were purified and sequenced using an ABI Prism 310 Genetic Analyzer (Applied Biosystems) and the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The resulting sequences were analyzed using basic local alignment search tool (BLAST) searches (National Center for Biotechnology Information).


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  		Table 2. Primers Used for RT-PCR and Nucleotide Sequence Analyses

 
cDNA Microarray Analysis

Total RNAs from case 7 (classical EMC) and case 8 (cellular-solid variant of EMC) were selected for gene expression profiling using the Human Named Genes GeneFilter (GF211) Microarray Release 1 (Research Genetics, Huntsville, AL) containing ~4000 known genes and expressed sequence tags. As a reference tumor, total RNA from a myxoid liposarcoma with a TLS-CHOP fusion was selected. Three µg of total RNA was used as template in a cDNA synthesis reaction using the superscript first-strand synthesis system (Invitrogen) according to the manufacturer’s recommendations but with the following modifications: the dCTP was replaced with 10 µl of 33P-dCTP (NEN, Applied Biosystems), the mixture was subsequently incubated for 1 hour at 37°C and for 1 hour at 42°C. The cDNA probes were separated from free nucleotides and small molecules using a SpinX 200 spin column (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s recommendations. The cDNA probe mixture was subsequently denatured for 3 minutes at 96°C and added to the GF211 filters in 5 ml of hybridization solution (Research Genetics). Prehybridization and hybridization conditions were according to the manufacturer’s recommendations. After overnight incubation at 42°C, the filters were washed in 20 ml of washing solution (2x standard saline citrate, 1% sodium dodecyl sulfate) at 50°C for 2 x 20 minutes, followed by a high stringency wash (0.5x standard saline citrate, 1% sodium dodecyl sulfate) at 55°C for 2 x 15 minutes. The filters were wrapped in plastic film and the pattern of bound reactivity was scanned using a Molecular Imager FX Pro instrument (BioRad, Stockholm, Sweden).

Pairwise comparative analyses of the resulting images were performed using the software Pathways 4.0 (Research Genetics). Normalization was performed using the default data point protocol (Pathways 4.0). All sample data point intensities were divided by the mean sample intensity of all clones. All selected hybridization spots were inspected visually and data points that were difficult to interpret were omitted from further analysis. The control data points containing total genomic DNA showed up to four times difference between the samples. We therefore considered genes with at least eight times differential signal strength and at least two times above background to be differentially expressed. Expression ratios were calculated by dividing the normalized values of the selected genes from the two EMC tumors by the normalized value of the same genes from the reference tumor.

To verify the cDNA microarray results we studied the expression of four differentially overexpressed genes in EMC (CHI3L1, RELB, GSTM4, and XRCC3) and one gene that was differentially overexpressed in the myxoid liposarcoma reference tumor (COL3A1). CHI3L1 was assessed by RT-PCR, and RELB, GSTM4, XRCC3, and COL3A1 by immunohistochemistry. The expression of the five genes was studied in all 10 EMCs as well as in the five myxoid liposarcomas (including the reference tumor).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Clinicopathological Characteristics

Seven men and two women between 42 and 71 years of age were included in this series. Six primary tumors were intramuscular and located in the lower extremity (five, thigh; one, lower leg), one was intramuscular in the upper arm, one was intragluteal, and the remaining tumor involved the soft tissues around the symphysis pubis and inguinal regions. Tumor sizes ranged from 4 to 22 cm in greatest dimension.

Seven of nine primary tumors were classified as classical EMC (Figure 1A and Table 1 ). One of these cases had hypocellular foci, one had cells with rhabdoid features, and another had in addition to classical areas (case 4, Figure 1B ) cellular areas with rare pleomorphic foci (Figure 1C) associated with increased mitoses and focal necrosis. The remaining two cases, cases 5 and 8 (Figure 1D) , were classified as cellular-solid variants of EMC. Local recurrences and metastases were morphologically similar to the primary tumors.



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  		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).

 
Tumors from all nine patients were analyzed ultrastructurally. In addition to demonstrating the characteristic features of EMC,2 eight tumors revealed abundant microtubules; these were packed in intracisternal aggregates in five cases and freely distributed in the cytoplasm in three cases. Six of nine tumors had dense core neurosecretory granules (Figure 1E and Table 1 ).

Chromogranin A and synaptophysin were detected immunohistochemically in two tumors (Figure 1, F and G) , S100 protein was found in three tumors, and all but one tumor were positive for neuron-specific enolase. Cytokeratins and GFAP were not detected in any case. Ki67 immunolabeling with MIB1 ranged from <1 to 25%. Clinical follow-up data for all patients is tabulated in Table 1 .

Cytogenetic, SKY, and FISH Analyses

Cytogenetic analysis was performed in five previously not analyzed EMCs (cases 1 to 4 and 6-I). Diploid or near-diploid karyotypes predominated in all tumors. The karyotypic findings based on G-banding in combination with FISH and/or SKY are summarized in Table 3 . Clonal structural aberrations were detected in four tumors; a fifth tumor (case 6-I) showed trisomy 7 as the sole clonal change. Two tumors had a t(9;22)(q22;q12) and a third tumor had a del(22)(q12–13) (Figure 2; A to C) . SKY analysis did not reveal any hidden rearrangements of chromosomes 9 or 22 in the latter case. In addition, both tumors with t(9;22) had several other structural and numerical abnormalities. Notably, one case had a jumping translocation in which the segment 1q12-qter was translocated onto dissimilar chromosome segments, ie, Yq, 3p, 15p, and 22q (Figure 2A) . Because all four clones also contained two intact chromosomes 1, the net result of these rearrangements is partial trisomy for 1q. This case had also a single cell with a t(1;17)(p36;q12) as the sole anomaly. All of the remaining cells analyzed had the t(9;22). Three different areas sampled from case 4 showed a complex t(7;9;17)(q23;q22;q11) and a del(10)(q21) in all areas (Figure 2, D and E) ; areas A and B also contained several other more or less complex rearrangements (Figure 2E) . SKY analysis of case 7 confirmed the dup(1) and t(9;22) originally identified on the basis of G-banding (Figure 2F) .7


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  		Table 3. Summary of Cytogenetics, SKY, FISH, and RT-PCR Analyses in 10 EMCs from Nine Patients

 


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  		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.

 
FISH analysis showing the in vivo frequencies of trisomy 1q, 7, 8, 12, and 19 in tissue sections revealed the presence of one or more trisomies in seven of nine EMCs (Table 3) . Using conventional cytogenetics, trisomies were detected in five of nine tumors. Thus, the combination of these two methods detected trisomies in all but one tumor. The most common gains were +7 (five tumors), +1q (four tumors), and +12 (four tumors). Five tumors had multiple trisomies and four cases had single trisomies.

Detection of EMC-Specific Fusion Transcripts

To search for possible EWS-TEC, TAF2N-TEC, and TCF12-TEC fusion transcripts in cases 1 to 5, we performed RT-PCR experiments using the appropriate primer combinations. Amplification with the primer set EWS ex. 7 fwd and TEC RevC resulted in a product of 745 bp in cases 1 and 2, corresponding to a type 1 fusion in which EWS exon 12 is fused in frame to TEC exon 3 (Figure 3A) . In cases 3 and 5 amplification with the same primer set resulted in shorter fragments of 415 bp and 567 bp, respectively (Figure 3A) . The 415-bp fragment in case 3 corresponds to a type 2 fusion in which EWS exon 7 is fused to TEC exon 2. The 567-bp fragment in case 5 represents a novel fusion in which EWS exon 10 is fused to a 72-bp sequence derived from intron 2 of the TEC gene (EMC type 5 fusion, GenBank accession no. AF524261) (Figure 3B) . This sequence corresponds to a previously unrecognized TEC exon, which we have designated exon 2b. Analysis of the genomic sequence surrounding this exon revealed the presence of the highly conserved GT/AG sequences at the donor and acceptor splice sites, respectively. The fact that a sequence identical to exon 2b previously has been recognized in mitogen-induced TEC transcripts in various cell types,25 indicates that it represents an alternative TEC exon. In case 4 amplification with the primers TAF2N 314U23 and TEC RevA generated a 300-bp fragment (Figure 3A) consistent with a fusion of TAF2N exon 6 to TEC exon 3. The identities of the putative fusion transcripts were confirmed by nucleotide sequence analysis (data not shown). RT-PCR analysis revealed that the normal TEC transcript (amplified as a 517-bp fragment using the primer set TEC fwdF and TEC RevC) encoded by the unrearranged TEC allele was expressed in all 10 cases of EMC (data not shown).



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  		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.

 
Gene Expression Profiles of EMCs with Different Morphologies and Fusion Transcripts

The gene expression profiles of two EMCs with differing histologies (classical myxoid versus cellular-solid variant) and fusion transcripts (EWS-TEC versus TCF12-TEC) were compared with the profile of the reference tumor, a myxoid liposarcoma with a known TLS-CHOP fusion transcript. We chose myxoid liposarcoma as a reference tumor because this tumor may simulate EMC and expresses related fusion transcripts (TLS-CHOP or EWS-CHOP). Analysis of scatter plots revealed that the expression profiles of the two EMCs were remarkably similar despite differing histologies and expression of different fusion transcripts (Figure 4A) . None of the analyzed genes in the two tumors showed a difference in expression ratio that was >3. In contrast, the expression profiles of the two EMCs were very different from that of the myxoid liposarcoma (Figure 4B) . Among the genes with an expression ratio >8, there were 66 genes common to both EMCs, two genes overexpressed in case 7, and 22 genes overexpressed in case 8. A list of the 35 genes with the highest expression ratios in both EMCs is shown in Table 4 (a complete list of the expression data are available on request). The two genes with the highest expression ratios in both tumors were CHI3L1 and METTL1. As previously mentioned, EMC may show ultrastructural and immunohistochemical evidence of neural-neuroendocrine differentiation (Table 1) . Among the genes with an expression ratio >=8, there were at least four genes encoding neural-neuroendocrine markers, including SCG2 (chromogranin C), NEF3 (neurofilament 3), GFAP, and GAD2 (glutamate decarboxylase 2). The latter gene was only overexpressed in case 8.



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  		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.

 

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  		Table 4. List of the 35 Genes with the Highest Expression Ratios in EMC Cases 7 and 8, Relative to the Myxoid Liposarcoma Reference Tumor

 
We validated the microarray data by studying the expression of four highly overexpressed genes in EMC and one gene that was overexpressed in the myxoid liposarcoma reference tumor. RT-PCR analysis of the expression of CHI3L1 revealed that it was expressed in cases 7 and 8 as well as in the other eight EMCs but not in the five myxoid liposarcomas analyzed (Figure 5) . The authenticity of the amplified product was confirmed by nucleotide sequence analysis. Similarly, high expression of RELB, GSTM4, and XRCC3 was confirmed by strong immunoreactivity in cases 7 and 8 as well as in the eight other EMCs. The RELB immunostaining was predominantly cytoplasmic (Figure 1H) (REL proteins may be found in either the cytoplasm or nucleus depending on the cell type and state of the cell),26 the GSTM4 reaction was cytoplasmic and nuclear (Figure 1J) , and the XRCC3 staining nuclear in six EMCs and predominantly cytoplasmic in three cases (data not shown). The reference tumor was only focally positive for RELB and GSTM4 (Figure 1, I and K) and negative for XRCC3. Of the other four myxoid liposarcomas two were focally positive andtwo negative for RELB; three were focally positive and one negative for GSTM4, and one was positive and three negative for XRCC3. COL3A1 was strongly expressed(cytoplasmic staining) in the reference tumor as well as in the other four myxoid liposarcomas. In contrast, the EMC cases 7 and 8 were only focally positive for COL3A1. Of the other eight EMCs, six were entirely negative and two focally positive for COL3A1 (data not shown).



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  		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).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Previous cytogenetic studies of EMC have been primarily confined to conventional chromosome analysis.6,7,27-33 Here we have used a combination of chromosome banding, FISH, and SKY to obtain detailed information about the karyotypic profiles of EMC. Detailed comparison of the G-banded karyotypes with the SKY and DAPI-band images allowed us to map the breakpoints in all but one of the marker chromosomes recorded. Including the 10 EMCs presented here, cytogenetic information regarding 26 tumors (25 patients) is available in the literature.6,27-33 Collectively, the most striking observation is the finding of recurrent rearrangements of chromosome 9 with consistent breakpoints at 9q22 in 22 tumors. Fifteen of the 26 tumors had a t(9;22)(q22;q12) or variants thereof, six a t(9;17)(q22;q11–12) or variants thereof, and one case a t(9;15)(q22;q21). Of the four remaining tumors, three had apparently cryptic rearrangements resulting in EWS-TEC or TAF2N-TEC fusions. Thus, all but 1 of 26 cases of EMC with known karyotypes have shown translocations with recurrent breakpoints at 9q22 or cryptic rearrangements resulting in TEC-containing fusion transcripts. The only case deviating from this pattern is a tumor described by Bridge and colleagues34 that had a t(2;13)(q32;p12) as the sole anomaly. Whether this case expressed any of the EMC-specific fusion transcripts is not known. Because the translocations t(9;22)(q22;q12), t(9;17)(q22;q11–12), t(9;15)(q22;q21), and variants of these have not been found in any other tumors (Mitelman Database of Chromosome Aberrations in Cancer. Edited by F Mitelman, B Johansson, F Mertens. http://cgap.nci.nih.gov/chromosomes/mitelman, 2001) they can be regarded as pathognomonic for EMC.

We detected a fusion transcript in all 10 EMCs; EWS-TEC, TAF2N-TEC, and TCF12-TEC fusions were found in tumors from five, three, and one patient, respectively.12,15 Tumors lacking typical EMC-specific translocations may also express fusion transcripts, as illustrated in cases 3 and 6. The former had a del(22)(q12) and two apparently normal chromosome 9 homologues and expressed an EWS-TEC transcript, whereas both tumors in the latter case had apparently normal chromosomes 9, 17, and 22 and expressed TAF2N-TEC transcripts. Cryptic fusions have recently been detected also in Ewing’s sarcoma.35 From a practical, diagnostic standpoint, these results further emphasize the specificity, sensitivity, and utility of demonstrating TEC-containing fusion transcripts in the diagnosis of EMC. This is especially apparent with cases lacking typical EMC-specific translocations and with cellular-solid variants of EMC, which are very difficult to recognize on a purely histological basis.

We also found expression of the unrearranged, normal TEC allele in all 10 EMCs with fusion transcripts. This is in contrast to Brody and colleagues24 who found that the normal TEC allele was not expressed in two EMCs with EWS-TEC fusions. Experiments using a semiquantitative RT-PCR approach have, however, indicated that the expression level of the normal TEC transcript is very low compared to that of the TEC fusion transcripts (unpublished observations), suggesting that the strong expression of the TEC fusion genes is likely to be driven by the promoter regions of the 5'-partner genes.

Secondary chromosome abnormalities seem to be relatively common in EMC. In the total material of 26 cytogenetically analyzed cases, 50% had recurrent, secondary abnormalities, notably partial trisomy for 1q and/or single or multiple trisomies. In this study, FISH analysis of tissue sections of nine EMCs revealed that the in vivo frequency of trisomies was more common than suggested by cytogenetics (Table 3) . In the 26 collective cases of EMC, the most frequent trisomies (based on cytogenetic and FISH analyses) are +1q (seven cases), +7 (seven cases), +12 (six cases), +19 (four cases), and +8 (three cases). Because information about clinical data, follow-up, and morphology are unfortunately lacking in most of the previously published EMCs, there is little if anything known about possible correlations between clinicopathological parameters and the genetics of these tumors. In our series, we found no apparent correlation between any of the trisomies, histological features, type of translocation/fusion gene, or prognosis. We also found that classical versus cellular-solid subtype of EMC and evidence of neural-neuroendocrine differentiation did not correlate with cytogenetic and molecular characteristics. Further studies in larger series with long-term follow-up are, however, necessary to evaluate the presence or absence of correlations among the above-mentioned factors.

In EMCs with partial trisomy for 1q, the breakpoints were at 1q12–21 (four cases) and 1q25 (one case). Notably, all EMCs with an extra copy of 1q also had a t(9;22). In one of the current cases an extra copy of 1q was involved in a jumping translocation. To the best of our knowledge this is the first example of a jumping translocation in EMC and the second example of this aberration in a solid tumor.36 Previous molecular studies of jumping translocation breakpoints in neoplasia have shown that they might involve oncogenes, resulting in amplification of the affected gene, eg, ABL and/or CD3-MLL.37 Whether the 1q21 breakpoint in the jumping translocations in our case affects an oncogene is not known. There are several candidate genes in this region of 1q, including PRUNE, JTB, and EAT/mcl-1.38-40 One of these, PRUNE, is of particular interest since it was recently shown to be amplified and overexpressed in sarcomas.39 PRUNE encodes a protein that negatively regulates nm23-H1, a known metastasis suppressor in, for example, colon and breast cancers.

Microarray analyses of the gene expression profiles of two EMCs revealed a remarkably distinct and uniform expression profile in both tumors despite the fact that they had different histological appearances (one classical and one cellular-solid type) and expressed different fusion transcripts (one EWS-TEC and one TCF12-TEC). In contrast, the expression profile of the reference tumor, a myxoid liposarcoma, was quite different, suggesting that EMC exhibits a tumor-specific gene expression profile that distinguishes it from myxoid liposarcoma and probably other soft tissue sarcomas. This conclusion is supported by recent studies of the pattern of global gene expression in several other types of soft tissue sarcomas, including synovial sarcoma, gastrointestinal stromal tumors, and subgroups of leiomyosarcoma, all of which have rather consistent and homogeneous expression profiles.41,42 Our findings suggest that differences in morphology and/or expression of fusion transcripts are not associated with any major differences in gene expression profiles in EMC.

Among the genes with the highest expression ratios in EMCs were several potentially significant genes such as CHI3L1, METTL1, and RELB. CHI3L1 encodes a chitinase-like protein (YKL-40, cartilage glycoprotein-39), which is a major secreted protein of human articular chondrocytes and synovial fibroblasts and a marker of late macrophage differentiation.23,43 Elevated serum levels of CHI3L1 have been observed in patients with rheumatoid arthritis and hepatic fibrosis and cirrhosis.44,45 It has been suggested that the protein may function in remodeling or degradation of extracellular matrix, and could therefore play a role also in tumor invasion.23,46 High serum levels of this protein have been associated with short survival in patients with colorectal and recurrent breast cancers.46,47 Recently, two microarray analyses also identified CHI3L1 as highly overexpressed in high-grade malignant gliomas48 and papillary thyroid carcinomas.49 Using RT-PCR, we confirmed that CHI3L1 was expressed in all 10 EMCs of our study but not in any of the five myxoid liposarcomas, indicating that overexpression of CHI3L1 is a consistent finding in EMC. Because CHI3L1 is a secreted protein, it could conceivably be useful as a serum marker monitoring disease progression in EMC patients.

METTL1 encodes a methyltransferase-like protein, which is also of potential interest in relation to cancer because abnormalities of, for example, DNA methylation are consistently found in human neoplasia.50,51 RELB, a member of the nuclear factor-{kappa}B family of transcription factors, has been implicated in the control of cell proliferation, differentiation, and apoptosis.52,53 Overexpression of RELB has previously been observed in chondrosarcoma cells stimulated with interleukin-1ß.54 Immunohistochemical analysis of RELB expression confirmed our microarray data by demonstrating intense staining of all 10 EMCs. Interestingly, the microarray also identified overexpression of MYB, a target gene of RELB,55 in both EMCs.

We also observed expression of at least two genes implicated in chondrogenesis (CHI3L1 and CHST1) and several genes encoding neural-neuroendocrine markers (SCG2, NEF3, GFAP, and GAD2). In addition, prominent immunoreactivity for chromogranin A and synaptophysin were found in 3 of 10 tumors and neurosecretory granules in six of nine tumors. These observations are in line with the recent detection of partial neural-neuroendocrine differentiation in a subset of EMC.4,5 The presence of abundant microtubules in eight of our nine cases further suggests neural-neuroendocrine differentiation, since microtubule-associated protein-2 (MAP-2) and class III ß-tubulin, major components of microtubules found in neurons and their derivatives, have been recently detected in EMC.56 Whether or not the TEC-containing fusion genes are responsible for the induction of a neural-neuroendocrine differentiation program or whether this is an inherent feature of the progenitor cell of EMC is at present unclear. Notably, EWS-ETS fusion genes are able to induce epithelial and neuroectodermal differentiation in NIH 3T3 cells.57


   Footnotes
 
Address reprint requests to Prof. Göran Stenman, Lundberg Laboratory for Cancer Research, Department of Pathology, Göteborg University, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. E-mail: goran.stenman{at}llcr.med.gu.se

Supported by grants from the Swedish Cancer Society, the IngaBritt and Arne Lundberg Research Foundation, the Johan Jansson Foundation for Cancer Research, and the Assar Gabrielsson Research Foundation.

Accepted for publication November 15, 2002.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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