¹Department of Clinical Genetics, Lund University Hospital, S-221 85 Lund, Sweden

²Department of Pathology, The Norwegian Radium Hospital, N-0310 Oslo, Norway

³Department of Oncology, The Norwegian Radium Hospital, N-0310 Oslo, Norway

⁴Department of Genetics, The Norwegian Radium Hospital and Institute for Cancer Research, N-0310 Oslo, Norway

Correspondence to: Ioannis Panagopoulos, Department of Clinical Genetics, Lund University Hospital, S-221 85 Lund, Sweden

Although most extraskeletal myxoid chondrosarcomas (EMC) are cytogenetically characterized by the translocation t(9;22)(q22;q12), another subset has recently been identified carrying a t(9;17)(q22;q11). Whereas the t(9;22) is known to result in fusion of the CHN (TEC) gene from 9q22 with the EWS gene from 22q12, creating a chimeric EWS/CHN, the genes involved in the t(9;17) of EMC are unknown. We examined two EMC with t(9;17)(q22;q11) and found that the CHN gene was recombined with the RBP56 gene from 17q11 to generate a chimeric RBP56/CHN. RBP56 has not previously been shown to be involved in tumorigenesis but it encodes a putative RNA-binding protein similar to the EWS and FUS (TLS) proteins known to play a pathogenetic role in several sarcomas. The presence of the RBP56/CHN chimeric gene in EMC with t(9;17)(q22;q11) shows that the N-terminal parts of EWS and RBP56 have similar oncogenic potential making them pathogenetically equivalent in oncoproteins arising from fusions with certain transcription factors.

chondrosarcoma; gene fusion; translocation genetics; karyotyping

Introduction

Extraskeletal myxoid chondrosarcoma (EMC) is a malignant soft tissue tumour found mostly in the extremities, but also occasionally in the tongue, chin, epiglottis, brachial plexus, chest wall, pleura, abdomen, buttock, inguinal region, testis and synovia (Liu-Shindo et al., 1989; Steffen et al., 1992; Elizalde et al., 1993). The disease is more common in middle-aged and elderly men but also occurs in women and children (Klijanienko et al., 1990). Long-term follow-up studies have shown that metastases can develop, especially in the lungs.

A translocation t(9;22)(q22;q12) has been found in several EMC but never in other tumours and, hence, seems to be pathognomonic for this disease (Mitelman, 1998). Recently, however, another translocation not previously described, t(9;17)(q22;q11) (Figure 1), was reported in two EMC, thus identifying a second cytogenetic subgroup of this tumour type (Bjerkehagen et al., 1999). The 9;22-translocation fuses the EWS gene from chromosome 22 with the CHN gene (also named TEC, NOR-1 or MINOR), which encodes an orphan nuclear receptor, from 9q22 (Labelle et al., 1995; Clark et al., 1996). The genes involved in t(9;17)(q22;q11) are unknown, but obviously CHN would be expected to be one of them.

The three genes EWS, FUS (or TLS) and RBP56 (also called hTAFii68 or TAF2N) are related in-as-much as they all encode similar RNA-binding proteins containing an RNP box in the central part and a degenerated repeat with the consensus sequence Ser-Tyr-Gly-Gln-Gln-Ser in the N-terminal region (Delattre et al., 1992; Crozat et al., 1993; Rabbitts et al., 1993; Bertolotti et al., 1996; Morohoshi et al., 1996). Whereas FUS (in 16p11) and EWS (in 22q12) are known to be arranged in several types of neoplasia, the tumorigenic involvement of RBP56 (in 17q11-12) has hitherto not been demonstrated.

FUS is rearranged in the t(12;16)(q13;p11) that characterizes myxoid liposarcoma (MLS) and in a subset of acute myeloid leukaemia (AML) with t(16;21)(p11;q22) (Crozat et al., 1993; Rabbitts et al., 1993; Ichikawa et al., 1994; Panagopoulos et al., 1994). In MLS, where the translocation results in the hybrid gene FUS/CHOP, the central and C-terminal parts of FUS are replaced by the full-length CHOP protein, a member of the bZIP family containing a leucine zipper structural motif (Crozat et al., 1993; Rabbitts et al., 1993). In AML with t(16;21), the N-terminal part of FUS is fused to the ETS DNA-binding domain and C-terminal part of ERG, a member of the ETS gene family, resulting in the formation of two hybrid genes, FUS/ERG and ERG/FUS (Ichikawa et al., 1994; Panagopoulos et al., 1994).

EWS is rearranged in Ewing sarcoma (ES) and related primitive neuroectodermal tumours (PNET) by means of a t(11;22)(q24;q12) or one of four variant translocations, t(21;22)(q22;q12), t(7;22) (p22;q12), t(17;22)(q12;q12) and t(2;22)(q33;q12) (Delattre et al., 1992; Zucman et al., 1993b; Sorensen et al., 1994; Jeon et al., 1995; Kaneko et al., 1996; Urano et al., 1996; Peter et al., 1997). In all five translocations, the same N-terminal part of EWS is fused to the ETS DNA-binding domains of either FLI1 from 11q24, ERG from 21q22, ETV1 from 7p22, ETV4/E1AF from 17q12 or FEV from 2q33, all of which are members of the ETS gene family (Delattre et al., 1992; Zucman et al., 1993b; Giovannini et al., 1994; Sorensen et al., 1994; Jeon et al., 1995; Kaneko et al., 1996; Urano et al., 1996; Peter et al., 1997). In addition, the N-terminal part of EWS is fused with the DNA-binding domain of ATF1, a member of the bZIP family, in clear cell sarcomas of tendons and aponeuroses with t(12;22)(q13;q12), with the DNA-binding domain of WT1 in the intra-abdominal desmoplastic small round cell tumour with t(11;22)(p13;q12), and with the CHOP gene in myxoid liposarcomas with translocations between 12q13 and 22q12 (Zucman et al., 1993a; Ladanyi and Gerald, 1994; Panagopoulos et al., 1996).

In the examples given above, the N-terminal part of FUS or EWS was fused to the DNA-binding domain of the genes CHOP, ATF1, FLI1, ERG, ETV1, ETV4/E1AF, FEV or WT1 in tumorigenic, specific translocations affecting the respective gene loci. Since RBP56 maps to the proximal region of 17q which harbours one of the breakpoints of the t(9;17), we decided to find out if this gene was fused with CHN in EMC carrying a t(9;17)(q22;q11).

Results

PCR with a TAF308F and CHN823R primer combination amplified a 422 bp fragment from the cDNA from both tumours. Nested PCR with a TAF348F and CHN789R primer combination amplified a 382 bp fragment and PCR with a TAF444F and CHN789R primer combination amplified a 253 bp cDNA fragment (Figure 2). To verify the presence of an RBP56/CHN chimeric transcript, the 382 bp and 253 bp PCR amplified fragments were analysed by direct sequencing. This showed that, in both tumours, exon 6 of the RBP56 gene was fused in frame to position -2 of the CHN cDNA immediately before the initiating methionine (Figure 3).

Discussion

The present study demonstrates the formation of an RBP56/CHN fusion gene in two EMS carrying the translocation t(9;17)(q22;q11) (Figure 1). It appears, therefore, that the translocations t(9;22)(q22;q12), giving rise to an EWS/CHN fusion gene, and t(9;17)(q22;q11), giving rise to an RBP56/CHN fusion gene, are pathogenetically equivalent in EMS and that both are pathognomonic for this tumour type. Cytogenetically (Mitelman, 1998; Bjerkehagen et al., 1999) as well as at the molecular level, the former rearrangement seems to be the more common: Labelle et al. (1995) detected the EWS/CHN gene in five out of seven EMC, Brody et al. (1997) found it in six of eight EMC, and Antonescu et al. (1998) described it in seven of the nine EMC cases examined by them. It is tempting to speculate that some, if not all, of the EMC cases that were negative for the EWS/CHN fusion gene in these series had the alternative RBP56/CHN chimeric gene.

Two main EWS/CHN transcripts have been reported for the standard t(9;22)(q22;q12) in EMC. In type 1, EWS exon 12 is fused to position -2 of the CHN cDNA. In type 2, exon 7 of the EWS gene is fused to position -176 of the CHN cDNA resulting in a novel open reading frame with the addition of 59 amino acids between the fusion point and the initiating methionine of CHN (Labelle et al., 1995; Clark et al., 1996). In the RBP56/CHN transcripts detected in the two cases of EMC with t(9;17), exon 6 of RBP56, which corresponds to exon 7 of EWS, is fused to position -2 of the CHN cDNA. Thus, the putative RBP56/CHN chimeric protein contains the N-terminal part of RBP56, a region rich in Ser, Gly, Gln and Tyr, and the full-length normal CHN protein. In analogy with what is presumed to be the case for the EWS/CHN fusion protein, RBP56/CHN is likely to be a potent transcriptional activator wielding its tumorigenic influence by action on as yet unknown target genes involved in cell proliferation. It seems, therefore, that the N-terminal parts of the RBP56 and EWS proteins are functionally equivalent when fused with the transcription factor CHN, leading in both instances to the same malignant phenotype (EMC). This implies a situation parallel to that in myxoid liposarcomas, in which the N-terminal parts of the FUS and EWS proteins are functionally equivalent when fused to the transcription factor CHOP (Zinszner et al., 1994; Panagopoulos et al., 1996). The observed role of RBP56, EWS and FUS in the oncogenic activation of transcription factors CHOP (FUS or EWS in MLS) and CHN (RBP56 or EWS in EMC) (Figure 4) raises the question of whether they may also be interchangeable in other tumour types.

Materials and methods

Cases

The clinicopathological description and cytogenetic findings of the two cases have previously been described in detail (Bjerkehagen et al., 1999). Cases 1 and 2 in our report correspond to cases 3 and 4 in the series examined by Bjerkehagen et al. (1999). Both tumours were phenotypically EMC and both had a t(9;17)(q22;q11) (Figure 1) as their only chromosomal abnormality.

RT - PCR and sequencing analysis

The primers used for PCR amplification and sequence analysis are presented in Table 1. RBP56 primers were based on the full length RBP56 cDNA sequence (Bertolotti et al., 1996; Morohoshi et al., 1996) and CHN primers on the reported CHN cDNA sequence (Hedvat and Irving, 1995; Labelle et al., 1995; Clark et al., 1996; Ohkura et al., 1996).

RT - PCR was carried out for the detection of RBP56/CHN chimeric transcripts using total RNA as the starting material. Total RNA was extracted using the Trizol reagent according to the manufacturer's instructions (Gibco - BRL). Four and 2 g from cases 1 and 2, respectively, were reversely transcribed in 20 l reaction volume containing 50 mM Tris-HCl pH 8.3 (at 25°C), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 1 mM of each dNTP, 37 units RNA guard (Pharmacia AB), 10 pmol random hexamers, 1 g Oligo (dT)₁₇ and 200 units M-MLV Reverse Transcriptase (Gibco - BRL). The reaction was carried out at 37°C for 60 min, then heated for 5 min at 99°C and kept at 5°C.

In the first PCR amplification, the 50 l reaction volume contained 20 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂, 0.2 mM of each dNTP, 1 unit PlatinumTaq polymerase (Gibco - BRL), 0.5 M of each of the outer primers (Table 2), and 2 l of the cDNA. The first PCR products were diluted 1 : 100 and 2 l of this dilution were amplified in a second 50 l PCR with the same composition as in the first PCR, except that 1 mM MgCl₂ and the inner primers were used (Table 2). After an initial denaturation at 94°C for 5 min, 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C were run using a MJ Research PCT-200 DNA Engine, followed by an final extension for 10 min at 74°C.

Ten l of the PCR products were analysed by electrophoresis through 1.5% agarose gels, stained with ethidium bromide, and photographed.

For sequence analysis, the RT - PCR amplified RBP56/CHN cDNA fragments were run on 1.5% agarose gels, purified using Qiagen gel extraction kit and directly sequenced using the dideoxy procedure with Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) on the Applied Biosystems Model 373A DNA sequencing system.

Acknowledgements

We thank Margareth Isaksson for excellent technical assistance. This study was supported by grants from the Swedish and Norwegian Cancer Societies and from the Medical Faculty of Lund University. Accession No. AJ243810.

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Figure 1 Partial karyotype of homologue pairs 9 and 17 from the EMC of case 2 showing the t(9;17)(q22;q11). The rearranged chromosome 9 is to the right and the rearranged chromosome 17 to the left. Arrowheads indicate breakpoints

Figure 2 RT - PCR analysis of the two EMC with t(9;17)(q22;q11). Total RNA was reversely transcribed and cDNA was used as template in a first PCR amplification using TAF308F and TEC823R primer combination. The first PCR products were diluted 1 : 100 and 2 l of this dilution were subsequently nested PCR amplified. In both samples, PCR with TAF348F and CHN789R primer combination amplified a 382 bp fragment (lanes 1 and 2) and PCR with TAF444F and CHN789R primer combination amplified a 253 bp cDNA fragment (lanes 3 and 4). B1, blank for the TAF348F and TEC789R primer combination. B2, blank for the TAF444F and TEC789R primer combination. M, 100 bp DNA ladder

Figure 3 (a) Schematic representation of the chimeric cDNA fragment of RBP56/CHN detected using RT - PCR in both EMC with t(9;17)(q22;q11). Numbers refer to exons of the RBP56 and CHN genes. The position and direction of the primers are indicated. (b) Partial sequence chromatogram showing the junction (arrow) of the RBP56 and CHN genes. (c) Nucleotide sequence of the amplified RBP56/CHN chimeric cDNA fragment determined after direct sequencing of the fragment using the TAF478F and CHN444R primers. Double underlining shows the junction of the RBP56 and CHN genes. In both cases, the exon 6 of the RBP56/hTAFii68 gene is fused, in frame, to position -2 of the CHN cDNA immediately before the initiating methionine. The primers TAF478F and CHN744R used for sequence are underlined

Figure 4 Diagram showing the currently known involvement of EWS, FUS and RBP56 with different transcription factor genes in various types of neoplasia

Table 1 Primers used for PCR and sequencing