Heterogeneous Expression of the SSX Cancer/Testis Antigens in Human Melanoma Lesions and Cell Lines1

[CANCER RESEARCH 60, 1654–1662, March 15, 2000] Heterogeneous Expression of the SSX Cancer/Testis Antigens in Human Melanoma Lesions and Cell Lines1

Nuno R. dos Santos,2 Ruurd Torensma,3 Teunis J. de Vries,3,4 Marco W. J. Schreurs, Diederik R. H. de Bruijn, Ellen Kater-Baats, Dirk J. Ruiter, Gosse J. Adema, Goos N. P. van Muijen, and Ad Geurts van Kessel Departments of Human Genetics [N. R. d. S., D. R. H. d. B., E. K-B., A. G. v. K.], Tumor Immunology [R. T., M. W. J. S., G. J. A.], and Pathology [T. J. d. V., D. J. R., G. N. P. v. M.], University Hospital Nijmegen, 6500 HB Nijmegen, the Netherlands

ABSTRACT Recently, when searching for melanoma tumor antigens, Tu¨reci et al. (9) found that the SSX2 gene was expressed in melanomas and, in The SSX genes, located on the X chromosome, encode a family of highly addition, several other malignancies. Further research disclosed that homologous nuclear proteins. The SSX1 and SSX2 genes were initially also SSX1 and two newly discovered SSX genes, SSX4 and SSX5, may identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as be expressed ectopically in a variety of cancers (10, 11). In contrast, the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in no SSX3 expression was observed in any of these malignancies. In different types of cancers, including melanomas. Because normal SSX addition, it was found that at least the SSX2 protein may act as a expression has been detected only in the testis and, at very low levels, the tumor-associated antigen (also designated as HOM-MEL-40), elicit- thyroid, these proteins are considered as new members of the still growing ing humoral immune responses in a subset of melanoma patients (9, family of cancer/testis antigens. These antigens are presently considered as 12). Due to the absence of expression in most normal tissues except targets for the development of cancer immunotherapy protocols. In the testis and the ectopic expression in a variety of malignancies, the present study, we developed a monoclonal antibody found to recognize SSX proteins are considered as new members of the CT5 family of SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and antigens (10). paraffin-embedded tissues. This antibody was used to investigate SSX The SSX genes exhibit nucleotide homologies ranging from 88 to expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the 95% and encode proteins of 188 amino acids that exhibit homologies testis was found to be restricted to spermatogenic cells, mainly spermato- ranging from 77 to 91%. Recent studies have shown that the SSX gonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and proteins are localized in the nucleus, exhibiting both diffuse and protein expression, although heterogeneously and at variable levels. punctated distribution patterns (13–15). In addition, it was found that -Treatment of an SSX-negative cell line with 5-aza-2؅-deoxycytidine, a the conserved COOH-terminus of SSX is able to repress the transcrip demethylating agent, led to SSX RNA and protein expression, indicating tion of a reporter gene (16). This domain is also responsible for a role for methylation in transcription regulation. Thirty-four of 101 nuclear localization, chromatin association, and colocalization with primary and metastatic melanoma cases and 2 of 24 common nevocellular Polycomb-group nuclear bodies (17). and atypical nevus cases showed SSX nuclear staining. Again, SSX ex- In the present study, we detected variable levels of SSX RNA pression was heterogeneous, ranging from widespread to scarce. Our expression in several human melanoma and other cancer cell lines. In findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic addition, we developed an anti-SSX monoclonal antibody suitable for trials. SSX protein detection in paraffin-embedded tissues. Using this antibody, we detected heterogeneous SSX expression in 9 of 18 melanoma cell lines, 34 of 101 primary and metastatic melanoma cases, INTRODUCTION and 2 of 24 common nevocellular and atypical nevus cases. Normal SSX expression in the testis was found to be confined to spermato- The SSX genes were initially identified as fusion partners of the SYT gonia. The implications of our findings for the use of SSX antigens as gene in human synovial sarcomas carrying a recurrent t(X;18)(p11.2; potential targets for immunotherapy are discussed. q11.2) translocation (reviewed in Ref. 1). Typically, these tumors express chimeric transcripts in which 3Ј sequences of either SSX1 or SSX2, both localized on the X chromosome, are fused to 5Ј sequences MATERIALS AND METHODS of SYT, which is localized on chromosome 18 (2–5). Expression studies in normal tissues showed that SSX transcripts are only found Cell Lines and Tissues. Eighteen human melanoma cell lines were used in this study (listed in Table 1), the origin of which was reported previously (18, in the testis and, at residual levels, the thyroid (4, 6). Preliminary 19). Eight nonmelanoma cell lines were also analyzed: UMSCC (head and evidence for the existence of additional SSX family members was neck squamous cell carcinoma), HeLa (cervical carcinoma), A431 (cervical provided by the detection of genomic fragments from Xp11 that epidermoid carcinoma), NT2D1 (testicular germ cell cancer), CaCo2 (colon hybridized to an SSX1 probe (7). The screening of a human testis carcinoma), A2243 (synovial sarcoma), U-4SS (sarcoma), and K562 (erythro- cDNA library led to the isolation of SSX3, which is localized in the leukemia). All cell lines were cultured and harvested as described previously region Xp11.1–11.2 (8). (18). To induce genome-wide demethylation, cultured cells were grown in DMEM medium containing 1 ␮M 5-aza-2Ј-deoxycytidine (Sigma). After 72 h Received 10/15/99; accepted 1/20/00. of incubation at 37°C, the cells were processed for RNA extraction or immu- The costs of publication of this article were defrayed in part by the payment of page nofluorescence microscopy. One hundred and twenty-five paraffin-embedded charges. This article must therefore be hereby marked advertisement in accordance with melanocytic lesions were examined by immunohistochemistry (listed in Table 18 U.S.C. Section 1734 solely to indicate this fact. 2). The lesions were surgically resected from patients treated at hospital centers 1 Supported by Grants NUKC95–912 and NUKC96–1351 from the Dutch Cancer Society (Koningin Wilhelmina Fonds) and Grant PRAXIS XXI/BD/3232/94 from the in Nijmegen, the Netherlands (88 lesions), Copenhagen, Denmark (39 lesions), Fundac¸a˜o para a Cieˆncia e Tecnologia (Lisbon, Portugal; to N. R. d. S.). Wu¨rzburg, Germany (8 lesions), and Brussels, Belgium (6 lesions). Special 2 To whom requests for reprints should be addressed, at University Hospital Nijmegen, care was taken that all specimens that were included in this multicenter P. O. Box 9101, 6500 HB Nijmegen, the Netherlands. Phone: 31-24-3618847; Fax: 31-24-3540488. 3 These authors contributed equally to this work. 5 The abbreviations used are: CT, cancer/testis; BT, biotinylated tyramine; DAC, 4 Present address: Department of Cell Biology and Histology, Academical Medical 5-aza-2Ј-deoxycytidine; GST, glutathione-S-transferase; RT, reverse transcription; mAb, Center, P. O. Box 22700, 1100 DE Amsterdam, the Netherlands. monoclonal antibody. 1654

Table 1 SSX expression in 18 melanoma and 8 nonmelanoma cell lines analysis results was achieved by sequencing of the cloned PCR products using SSX expression a vector-specific primer, the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer), and the ABI 373A automated DNA a b c d,e RT-PCR Expressed genes Immunoblotting IFA sequencer (Applied Biosystems). Melanoma cell lines Generation of the E3AS Anti-SSX mAb. Mice were immunized with 10 ϪϪϪ BLM ␮g of GST-SSX2 protein, produced, and isolated from Escherichia coli,as IF6 ϩϩϩ SSX1, SSX2 ϩϩϩ ϩϩϩϩϩ IF6m ϩϩϩ SSX2 ϩϩϩ ϩϩϩϩϩ previously reported (13). During the following weeks, collected serum was 530 ϩϩϩ SSX1, SSX2, SSX4 ϪϪ/ϩ tested for reactivity on formaldehyde-fixed and paraffin-embedded 1F6 Mel57 ϪϪϪ(SSX2-positive) and BLM (SSX2-negative) cells. A seropositive mouse was ϪϪϪ MV1 boosted i.v. for 2 subsequent days with 10 ␮g of GST-SSX2 protein. Two days MV3 Ϫ A375P ϩ SSX1, SSX2, SSX5 ϩϪ/ϩ after the last booster injection, the spleen was collected, and the spleen cells A375M ϩ SSX1, SSX2, SSX4 ϩϩwere isolated. These cells were fused with SP2/0 myeloma cells, using Omel2 ϩ SSX1 ϪϪ/ϩ PEG4000, in a ratio of 10 spleen cells:1 myeloma cell. The hybridomas were Ϫ Bowes screened by assessing the reactivity of the various supernatants toward 1F6 and MD3A Ϫ M24met Ϫ BLM cells by immunofluorescence analyses, immunohistochemistry, and im- E10 Ϫ munoblotting. Hybridomas secreting anti-SSX antibodies were subcloned sev- f 518A2 ϩϩϩ SSX2 ϩϩϩ ϩϩϩϩϩ eral times until achieving monoclonality. Finally, the obtained mAbs were ϩ f ϩϩϩ SK-mel-28 SSX2 assessed for staining of formaldehyde-fixed and paraffin-embedded 1F6 and M14 ϪϪϪ MZ2-MEL-3.0 ϩϩϩ SSX1 ϪϪ/ϩ BLM cells by immunohistochemistry. Nonmelanoma cell lines Immunoblotting Analysis. Cultured cells were lyzed in RIPA buffer [50 Ϫ UMSCC2 mM Tris (pH 8.0), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% HeLa ϪϪϪ SDS, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 4.8 A431 Ϫ NT2D1 Ϫ ␮g/ml aprotinin] following standard methods (20). Subsequently, equal K562 ϩϩϩ ND amounts of each cell extract were electrophoresed in SDS-polyacrylamide gels CaCo2 Ϫ and immunoblotted using the E3AS mAb (1:1000 dilution) and B39 polyclonal ϩ U-4SS SSX1 antibody (1:3000 dilution), essentially as previously described (13). As sec- A2243 Ϫ ondary antibodies, horseradish peroxidase-coupled goat antimouse (1:1000 a RT-PCR was done using primers that recognize all five SSX genes. Results: Ϫ, negative; ϩ, weak expression; ϩϩϩ, strong expression. dilution) and horseradish peroxidase-coupled goat antirabbit antibodies b The SSX genes were detected by cloning of RT-PCR products and restriction and (DAKO) were used. Detection was performed by incubating the blotted nitro- sequencing analysis of several clones. c Immunoblotting was performed using both the B39 and the E3AS antibodies. Results: Ϫ, negative; ϩ, weak expression; ϩϩϩ, strong expression. d Immunofluorescence analysis was performed using the E3AS antibody. The percent- Table 2 SSX expression in melanoma and nevus lesions age of positive cells was estimated by counting at least 150 cells and categorized as followed: ϩϩϩϩϩ, Ͼ80%; ϩϩϩϩ, 60–80%; ϩϩϩ, 40–60%; ϩϩ, 20–40%; ϩ, Tumorsa 1–20%; Ϫ/ϩ, Ͻ1%; Ϫ, negative. Percentage of positive cells Frequencyb e IFA, immunofluorescence analysis; ND, not determined. Primary tumors (n ϭ 35) f In addition to normal SSX2 cDNAs, a splice variant containing an additional 148-bp 0 20 (57) sequence was cloned. 1–5 7 (20) 5–25 2 (6) 25–50 3 (9) European study were fixed in 4% formaldehyde in PBS. Two testicular 50–75 0 biopsies (9-year-old and 41-year-old donors) and two normal thyroid paraffin- 75–100 2 (6) Total 14/35 (40) embedded specimens were also examined by immunohistochemistry. Locoregional metastases (n ϭ 17) RNA Isolation and RT-PCR. RNA was isolated using either the RNeasy 0 13 (76) Mini (Qiagen) or RNAzol B (Campro Scientific) kits. One to 5 ␮g of total 1–5 1 (6) RNA were reverse-transcribed using a random hexamer and SuperScript 5–25 0 Ϫ 25–50 1 (6) RNase H Reverse Transcriptase (Life Technologies) according to the man- 50–75 2 (12) ufacturer’s instructions. One to 2 ␮l of RT product were PCR-amplified using 75–100 0 Taq DNA polymerase (Life Technologies) following standard procedures. The Total 4/17 (24) ϭ sense primers SSX-Bcl (5Ј-TCTGATCATGCCCAAGAAGCCAGCAGAG- Lymph node metastases (n 38) Ј 0 26 (68) 3 ), which carries an artificial BclI restriction site (underlined), and SSX-start 1–5 7 (18) (5Ј-ACGGATCCCGTGCCATGAACGGAGACGAC-3Ј), which carries an ar- 5–25 2 (5) tificial BamHI site (underlined), were both used in combination with the 25–50 2 (5) antisense SSXL-rev primer (5Ј-TTGTCGACAGCCATGCCCATGTTC-G- 50–75 1 (3) Ј 75–100 0 TGA-3 ), which carries an artificial SalI restriction site (underlined), to am- Total 12/38 (32) plify 324-bp and 663-bp SSX fragments, respectively. PCR amplification of Distant-site metastasesc (n ϭ 11) PBGD, coding for the enzyme porphobilinogen deaminase, was included as a 0 7 (64) positive control for the RT products using the primers 5Ј-GCAGATGGCTC- 1–5 3 (27) Ј Ј Ј 5–25 1 (9) CGATGGTGA-3 (sense) and 5 -CTGGTAACGGCAATGCGGCT-3 (anti- 25–50 0 sense) giving rise to a 336-bp product. In some experiments, ␤-actin RT-PCR 50–75 0 was performed as another positive control using primers 5Ј-GCTACGAGCT- 75–100 0 GCCTGACGG-3Ј and 5Ј-GAGGCCAGGATGGAGCC-3Ј. SSX-Bcl/SSXL- Total 4/11 (36) Atypical nevi (n ϭ 12) rev PCR amplifications were performed for all cell lines, whereas SSX-start/ Total 2/12 (17)d SSXL-rev PCR was carried out in only nine cell lines (A375P, A375 M, BLM, Nevocellular nevi (n ϭ 12) 1F6, 1F6m, MV1, M14, Mel57, and 530). All PCR amplifications were Total 0/12 (0) performed using a GeneAmp PCR System 2400 (Perkin-Elmer). a n refers to the number of patients having one type of tumor lesion. In cases where a patient has more than one type of lesion, scoring refers to the lesion with highest value. Cloning, Restriction Analysis, and Sequencing. SSX PCR amplification b products were purified from agarose gels using the Easy-Pure kit (Biozym) and Number of lesions exhibiting the respective percentage of SSX-positive cells. The relative percentage to the total number of lesions is shown between parentheses. cloned into pGEM-T vectors using the pGEM-T Vector Systems kit (Promega) c Distant-site metastases included metastases to the lung, brain, liver, and skin. for further restriction analysis and sequencing. Confirmation of restriction d The two positive cases were scored at 5–25% of positive cells. 1655

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2000 American Association for Cancer Research. SSX EXPRESSION IN HUMAN MELANOMA cellulose filters with enhanced chemiluminescence substrate (Amersham) and subsequent autoradiography. Cell Transfections and Indirect Immunofluorescence Assays. HeLa cells were grown in DMEM medium containing 10% FCS. Different SSX cDNAs (SSX1 to SSX4 and SSX2 deletion mutants) cloned in eukaryotic expression vectors containing epitope tags (pCATCH and pSG8-VSV) were transiently transfected into HeLa cells using Dosper liposomal reagent according to the manufacturer’s instructions (Boeringher Mannheim). Indirect immunofluorescence assays were performed as described previously (13). As primary and secondary antibodies, we used the E3AS mAb (1:10 dilution) and FITC-conjugated swine antimouse IgG (DAKO; 1:100 dilution). SSX Antigen Retrieval and Immunohistochemical Detection. As a test- ing system for immunohistochemical staining of paraffin-embedded pathology specimens, we embedded SSX-positive 1F6 and SSX-negative BLM cells in gelatin. These gelatin devices were fixed in 4% formaldehyde/PBS and embedded in paraffin. Four-␮m sections were deparaffinized and treated by microwave heating for 20 min at 650 W in 0.1 mM sodium citrate buffer (pH 6.0). The slides were adjusted to room temperature for 30 min and preincubated for 10 min in 20% normal horse serum in PBS. This solution was decanted, and the E3AS mAb was applied as hybridoma culture supernatant for 60 min at room temperature. We used an avidin-biotin complex-peroxidase method for antibody detection as previously described (21). The signal was enhanced with catalyzed reporter deposition, an amplification method developed in our laboratory based on the deposition of BT (22). The BT precipitate was then visualized with peroxidase-labeled avidin and 3-amino-9-ethylcarba- zole as a substrate. The normal testis and thyroid specimens were analyzed by immunohistochemistry using a similar protocol but without the use of BT. In addition, these samples were stained with E3AS mAb preincubated with GST-SSX2 or GST-SYT peptide to assess the antibody specificity. Scoring. For each section, the percentage of positive melanocytic cells was estimated. Each section was assigned to one of the following percentage categories: 0%, 1–5%, 5–25%, 25–50%, 50–75%, and 75–100%. Positive melanocytic staining was scored when at least 1% of melanocytic cells were stained. The scoring was performed by two observers (N. R. d. S. and T. J. d. V.), but doubtful cases were analyzed and judged by a panel of four observers (N. R. d. S., T. J. d. V., G. N. P. v. M., and D. J. R.).

RESULTS Several SSX Genes Are Ectopically Expressed in Melanoma Cell Lines. The discovery of SSX2 ectopic expression in several human cancers, including melanoma (9), prompted us to evaluate its expression in a panel of established human melanoma cell lines (Table 1). Using primers that recognize and amplify the 3Ј ends of the five known SSX genes (SSX1 to SSX5), we observed a specific band of 324 bp in 9 of 18 melanoma cell lines tested (Fig. 1A; Table 1). Eight nonmelanoma cell lines were also analyzed, and only the U-4SS and K562 cell lines were found to be SSX-positive (Table 1). In all cases, RT-PCR amplification of PBGD mRNA, a housekeeping gene, was included as a control to ascertain the appropriate quality and quantity Fig. 1. Detection of SSX gene expression in human melanoma cell lines by RT-PCR. of the different RNA samples (Fig. 1B). In nine of the melanoma cell A, SSX RT-PCR with primers SSX-Bcl and SSXL-rev yielded an expected band of 324 bp in cell lines 530, 1F6m, A375P (weak), and 518A2 and in a positive control (plasmid lines, an additional SSX RT-PCR reaction was performed using a containing SSX2 cDNA), whereas cell line MD3A and a blank control were negative. primer set that amplifies full-length SSX mRNAs (Fig. 1C). Accord- Besides, an additional band of ϳ450 bp was detected in cell line 518A2 (arrowhead). B, ingly, the cell lines that were previously found to be SSX-positive upper panel, SSX RT-PCR with primers SSX-start and SSXL-rev yielded an expected band of 663 bp in cell lines A375P, A375 M (both weak), 1F6, and 1F6m and in a positive gave rise to a specific band of 663 bp, whereas negative cell lines control (plasmid containing SSX2 cDNA), whereas cell lines BLM, MV1, M14, and remained so, as expected (Fig. 1B). Mel57 and a blank control were negative. Lower panel, RT-PCR amplification of PBGD, Restriction digestion of the short (324 bp) PCR products using a housekeeping gene, yielding a 336-bp product. As a size marker, a 100-bp DNA ladder (Life Technologies) is shown in both panels. C, diagram depicting relevant restriction sites different enzymes known to discriminate between SSX1, SSX2, and present in the five known SSX cDNAs (B, BglII; E, EcoRV; L, LspI; H, HpaI; P, PvuII; SSX3 (8; Fig. 1C) indicated that more than one SSX gene was ex- S, SmaI). Also depicted are the locations of the primers used to amplify the SSX cDNA sequences (bottom arrows). The primers recognize sequences located in distinct exons, pressed in four of nine positive melanoma cell lines. To identify the thereby preventing the amplification of putative contaminating DNA. corresponding cDNAs, the PCR fragments were cloned into plasmid vectors and propagated in E. coli. Several of these clones were isolated and subjected to restriction and sequencing analyses (Fig. fragments with higher molecular weight (ϳ450 bp) were also ampli- 1C). By doing so, we found in three cell lines only SSX1 (Omel2, fied from cell lines 518A2 and SK-mel-28 (Fig. 1A, arrowhead). MZ2-MEL-3.0, and U-4SS) and in three other cell lines only SSX2 These fragments were found to correspond to an alternatively spliced (1F6m, 518A2, and SK-mel-28) PCR products (Table 1). Besides, SSX2 variant containing an additional intronic sequence of 148 bp 1656

ysis. Absence of the most NH2-terminal 110 amino acids (as in standard SYT-SSX fusions; FLAG-SYT-SSX2) led to loss of E3AS mAb staining. Conversely, COOH-terminal deletions up to amino acid 81 did not abrogate E3AS staining. Because absence of the 24

most NH2-terminal amino acids did not prevent E3AS recognition, the epitope of this mAb maps between amino acids 25 and 80 (Fig. 2). The E3AS mAb Specifically Detects SSX Protein in Human Testis. To test whether the E3AS mAb could also recognize normal SSX expression in paraffin-embedded tissues, we analyzed two testicular and two thyroid biopsies by immunohistochemistry. One bi- opsy was obtained from an adult male testis, whereas the other was obtained from a prepuberal male testis. SSX expression in mature Fig. 2. Diagram depicting different SSX2 deletion mutants fused to a peptide tag (FLAG, VSV) and expressed in transiently transfected HeLa cells. Each of these fusion testis was found to occur only in the nuclei of spermatogenic cells, proteins was checked for E3AS mAb recognition by indirect immunofluorescence anal- mainly in spermatogonia and occasionally in primary spermatocytes ysis, as indicated in the right column. The numbers indicate the amino acid positions. close to the basement membrane (Fig. 3). The expression was heterogeneous, and only a fraction of spermatogonia was stained. Like- (GenBank accession no. AF190791). By analyzing clones derived wise, in immature testis, SSX expression was found to occur in a from the remaining four positive cell lines (1F6, 530, A375P, and portion of spermatogonial cells, the predominant cell type present A375 M), we obtained evidence for the expression of the SSX4 and (data not shown). Neither interstitial cells nor Sertoli cells were SSX5 genes recently identified by Gu¨re et al. (10) in three of them. stained by the E3AS mAb. Attesting the specificity of the mAb E3AS, SSX4 expression was detected in the 530 and A375 M cell lines, we found that preincubation with the immunizing antigen (GST- whereas SSX5 expression was detected only in the A375P cell line SSX2) abolished nuclear staining of spermatogenic cells. In contrast, (Table 1). The SSX1 and SSX2 genes were both expressed in these four preincubation with an unrelated protein (GST-SYT) had no effect on cell lines. It should be noted that no SSX3 gene expression was found E3AS staining (data not shown). Surprisingly, none of the thyroid in any of the melanoma cell lines tested. tissue sections examined revealed SSX staining. Generation and Characterization of an Anti-SSX mAb. For SSX Protein Is Expressed in Several Melanoma Cell Lines. further analysis at the protein level, we set out to generate a mouse Thirteen melanoma cell lines were analyzed by immunoblotting (B39 mAb against SSX proteins. Several hybridomas were produced, and and E3AS antibodies) and immunofluorescence microscopy (E3AS because we were particularly interested in developing an antibody mAb; Table 1). Immunoblotting using the B39 antibody revealed able to detect protein from paraffin-embedded tissues, each hybri- specific bands of 29 kDa in 1F6, 1F6m, and 518A2 cell lysates, as doma was assessed for its ability to detect SSX expression in paraffin- well as in a positive control (COS-1ϩSSX2; Fig. 5A). Less intense embedded 1F6 cells. BLM cells, which were SSX-negative by RT- bands of similar weight could be discerned in the immunoblotted PCR, were used as a negative control. By doing so, we identified one SK-mel-28, A375P, and A375 M cell lysates. Identical results were hybridoma (E3AS) that was able to specifically detect SSX in 1F6 but obtained using the E3AS mAb (not shown). Immunofluorescence not in BLM cells (Fig. 4, a-c). analysis using the E3AS mAb disclosed nuclear stained cells in 9 of To determine the specificity of the E3AS mAb, HeLa cells were 13 analyzed cell lines. However, the percentage of positive cells transiently transfected with different epitope-tagged SSX expression varied markedly between cell lines. In three of them (1F6, 1F6m, and constructs (SSX1 to SSX4) and subjected to indirect immunofluores- 518A2), the majority of cells were positive (Ͼ80%), whereas five cence analysis. We found that the E3AS mAb was able to detect other cell lines (Omel2, 530, A375P, A375 M, and MZ2-MEL-3.0) SSX2, SSX3, and SSX4 but not SSX1 protein in the nucleus of HeLa showed very low numbers of positive cells (Ͻ2%). The SK-mel-28 cells (not shown). The SSX5 protein was not tested. The B39 poly- cell line showed an intermediate percentage of positive cells (ϳ25%; clonal antibody (13) was also found to recognize SSX2, SSX3, and Table 1). SSX4 but not SSX1 (not shown). Interestingly, SSX1 contains the SSX Proteins Are Heterogeneously Expressed in Melanoma most dissimilar amino acid sequence in the SSX family of proteins Lesions. Using the E3AS mAb, we observed SSX nuclear staining in (10), suggesting that the E3AS mAb recognizes a less conserved 36 of 125 (29%) melanocytic lesions. Apart from two atypical nevi epitope. To determine the location of the E3AS mAb epitope, we that displayed weakly positive cells (Fig. 4k), no SSX staining was transiently transfected HeLa cells with several SSX2 deletion mutants found in the remaining benign human melanocytic lesions (Fig. 4l). (Fig. 2) and tested them for reactivity by immunofluorescence anal- Within the malignant lesions, the frequency of E3AS positivity ranged

Fig. 3. Immunohistochemical staining of normal testicular tissue using the anti-SSX E3AS mAb. A, seminiferous tubule exhibiting nuclear stained early spermatogenic cells, mainly spermatogonia (red; arrows). Magnification, ϫ200. B, higher magnification (ϫ400) of another seminiferous tubule showing SSX nuclear staining in spermatogonial cells.

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Fig. 4. SSX immunohistochemical staining in melanoma cell lines and melanocytic lesions. a and b, nuclear staining of the positive control cell line 1F6. c, no staining is found in the SSX-negative BLM cell line. d-h, examples of SSX nuclear staining in primary melanoma lesions. Positive cells could appear as widespread (d), as foci (e), as scattered (f), and as isolated (g). h, in some cases, cytoplasmic staining of mitotic cells (arrows) was observed next to nuclear staining. i, membrane staining of tumor cells besides nuclear (arrowhead) staining in a lymph node metastasis. j, staining of fibroblast-like cells in a lymph node metastasis. k, weak staining of nuclei in an atypical nevus. l, no staining in a common nevocellular nevus. Magnifications: b, bar ϭ 6.7 ␮m; a, c-i, and k, bar ϭ 13.5 ␮m; j and l, bar ϭ 27 ␮m. 1658

Table 3 Levels of SSX immunostaining in different tumors from 32 patients with Table 3 Continued primary and metastatic lesions a Tumor SSX staininga Tumor SSX staining vVB FM58 Primary 1 Primary 5 Lymph node metastasis 0 Lymph node metastasis 1 BB FM62 Primary 1 Primary 1 Lymph node metastasis 0 Lymph node metastasis 1 LK FM66 Primary 0 Primary 0 Locoregional metastasis 0 Lymph node metastasis 0 FvW FM72 Primary 0 Primary 1 Lymph node metastasis 0 Lymph node metastasis 0 Lymph node metastasis 1 FM76 Skin metastasis 0 Primary 3 Skin metastasis 0 Locoregional metastasis 3 Skin metastasis 1 Lymph node metastasis 2 Skin metastasis 0 FM77 dK Primary 3 Primary 0 Lymph node metastasis 0 Locoregional metastasis 0 FM92 M Primary 0 Primary 0 Locoregional metastasis 0 Lymph node metastasis 0 FM93 Lung metastasis 0 Primary 0 Liver metastasis 1 Locoregional metastasis 0 Brain metastasis 0 FM94 V Primary 0 Primary 0 Locoregional metastasis 0 Lymph node metastasis 1 a Tumors were scored as: 0, negative; 1, 1–5%; 2, 5–25%; 3, 25–50%; 4, 50–75%; 5, vHS 75–100% positive cells. Primary 0 Locoregional metastasis 0 Ru Primary 0 from 24% in locoregional metastases to 40% in primary melanomas Locoregional metastasis 0 (Table 2). Among all lesions studied, SSX expression was very Locoregional metastasis 0 heterogeneous, with the majority of the cases exhibiting low numbers Lymph node metastasis 0 B1 of positive cells (Ͻ25%; Table 2). Large SSX-negative areas were Primary 0 observed in several tumors. Four main staining patterns could be Lymph node metastasis 0 B2 distinguished in both primary and metastatic melanomas: (a) wide- Primary 0 spread, with Ͼ75% of positive cells (Fig. 4d); (b) focal, with positive Lymph node metastasis 2 cells clustered in a restricted area from the tumor (Fig. 4e); (c) B3 Primary 0 scattered, with a low percentage of positive cells present in several Lymph node metastasis 0 areas of the tumor (Fig. 4f); and (d) isolated, with few positive cells K localized in largely negative areas (Fig. 4g). Apart from the typically Primary 0 Locoregional metastasis 0 observed nuclear staining, some cases showed aberrant patterns. In Locoregional metastasis 0 one primary tumor, cytoplasmic staining of mitotic cells was observed Locoregional metastasis 4 E next to nuclear staining in other cells (Fig. 4h). In seven melanoma Primary 2 lesions (two primary melanomas and five metastases), tumor cells Locoregional metastasis 0 showed cytoplasmic or membrane staining, either exclusively or in Ro Primary 0 conjunction with the typical nuclear staining (Fig. 4i). Also, one Locoregional metastasis 0 lymph node metastasis exhibited SSX staining in the cytoplasm of CT-P fibroblast-like cells adjacent to tumor cells (Fig. 4j). Primary 2 Lymph node metastasis 1 Because 32 patients had both primary and metastatic tumors, we FM2 investigated whether the status of SSX expression varied in the Primary 0 Lymph node metastasis 1 different lesions from each patient (Table 3). Fourteen patients devel- FM3 oped both primary and metastatic SSX-negative lesions, whereas in Primary 5 six patients, all lesions showed SSX nuclear positivity. Six patients Lymph node metastasis 3 FM9 had SSX-negative primary melanomas and at least one SSX-positive Primary 0 metastasis, whereas six other patients had SSX-positive primary tu- Lymph node metastasis 0 mors and negative metastases. Furthermore, no correlation was found FM28 Primary 1 between SSX expression and Clark staging, Breslow tumor thickness, Lymph node metastasis 0 or age of the patients. FM39 Primary 0 SSX Expression Is Induced by a Genome-wide Demethylating Lymph node metastasis 0 Agent. To assess whether SSX expression might be regulated by FM55 demethylation, we treated one SSX-negative (BLM) and one SSX- Primary 0 Locoregional metastasis 0 positive (K562) cell line with DAC. RNA was extracted from these FM57 cell cultures before and after DAC treatment, and SSX expression was Primary 1 evaluated by RT-PCR. Treatment with 1 ␮M of DAC for 72 h resulted Lymph node metastasis 1 in de novo induction of SSX expression in BLM cells and a clear 1659

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2000 American Association for Cancer Research. SSX EXPRESSION IN HUMAN MELANOMA increase of expression in the K562 cell line (Fig. 5B). The effect of DAC treatment on SSX expression was also assessed at the protein level for BLM cells. Immunofluorescence microscopy revealed that DAC treatment resulted in SSX nuclear staining in about 9–13% of the cultured cells (Fig. 5C). In contrast, no cells were stained in the absence of DAC. Furthermore, incubation with higher concentrations of DAC (10 ␮M) did not increase the percentage of SSX-positive BLM cells (ϳ7%; not shown).

DISCUSSION The SSX gene family is comprised of five members, including the SSX1 and SSX2 genes, that are fused with the SYT gene in t(X;18)- positive synovial sarcomas (4, 5). Recently, it was found that SSX1, SSX2, SSX4, and SSX5, but not SSX3, RNAs may be aberrantly expressed in various tumor types, in particular, human melanoma (9, 11). Besides, normal SSX gene expression, as detected at the RNA level, was found to be restricted to testicular and thyroid tissues (4, 6). In the present study, we developed an anti-SSX-specific mAb and evaluated the levels of SSX protein expression in normal testicular Fig. 5. A, immunoblot analysis of several human melanoma cell lines using the and thyroid tissues, a panel of cancer cell lines, common nevocellular anti-SSX B39 polyclonal antibody (13). Cell lines 1F6, 1F6m, 518A2, A375P, A375 M, and SK-mel-28 revealed an SSX-specific band of 29 kDa. B, induction of SSX expression and atypical nevus lesions, and primary and metastatic melanoma in cell lines treated with DAC. BLM and K562 cells were grown in the absence (Ϫ)or lesions. presence (ϩ)of1␮M DAC and subsequently subjected to RT-PCR using the primers Of 18 melanoma cell lines studied by RT-PCR, 9 were found to SSX-start/SSXL-rev (Fig. 1C). BLM cells were SSX-negative before and SSX-positive after DAC treatment. Untreated K562 cells expressed SSX at a relatively low level express at least one SSX gene, SSX1 and SSX2 being most frequently (compared with e.g., 518A2 cell line); this expression was increased after DAC treatment. expressed (Table 1). The levels of SSX expression were variable, as The RNA levels were identical in all isolates as revealed by RT-PCR amplification of ␤-actin. C, indirect immunofluorescence analysis of untreated and DAC-treated cultured also found by Gu¨re et al. (10) in their panel of melanoma cell lines. BLM cells using the anti-SSX E3AS mAb. SSX nuclear staining (green) was observed in In three pairs of melanoma cell lines derived from the same patient about 9–13% of the cells after incubation with 1 ␮M DAC. BLM cells grown in standard (A375P/A375 M, 1F6/1F6m, and MV1/MV3), the overall presence or medium did not show any positive cells. Respective 4Ј,6-diamidino-2-phenylindole nuclear counterstaining (blue) is shown in the lower panels. Original magnification, ϫ630. absence of SSX expression was concordant. However, the SSX family members were different in each pair, suggesting that the expression of certain SSX genes might have been switched on or off after cell line Thirty-two patients from our series developed both primary and establishment. Using a newly developed mAb (E3AS) that recognizes metastatic melanomas (Table 3), allowing the assessment of whether the NH2 terminus (amino acids 25–80) of SSX2, SSX3, and SSX4 SSX expression could be related to tumor progression. Because six (but not SSX1) proteins, we searched for expression in 13 of 18 patients had SSX-positive primary melanomas and SSX-negative me- melanoma cell lines by immunoblotting and immunofluorescence. In tastases, we conclude that expression of these proteins does not confer contrast to the former method, which revealed SSX bands in only six a clonal selective advantage to melanoma cells in vivo. This is in cell lines, immunofluorescence analysis allowed the detection of SSX contrast with the reported association between MAGE expression and protein in all cell lines that were found to be SSX-positive by RT- tumor progression and increased aggressiveness (19, 23). PCR. However, the percentage of SSX-positive cells varied consid- The SSX proteins are members of the still growing family of CT erably among these cell lines (Table 1). A low percentage of SSX- antigens. All CT antigens (e.g., MAGE, BAGE, GAGE, NY-ESO-1/ positive cells was observed in two cell lines (530 and MZ2-MEL-3.0) LAGE-1, SCP-1, CT7, and SSX) share common characteristics: (a) that showed strong SSX expression by RT-PCR. This discrepancy is normal expression in the testis only, (b) ectopic expression in malig- likely due to the fact that the E3AS mAb does not detect SSX1 nancies of various histological origins, (c) existence of several family expression. Altogether, these results indicate that the different levels members, and (d) localization on the X chromosome (10, 24, 25). of SSX mRNA detected by RT-PCR are not due to differential However, not all CT antigens fully comply with these rules. For intensities of transcriptional activity but rather to cell-to-cell hetero- example, several MAGE genes are also expressed in the placenta (26), geneity. SSX1 and SSX2 RNAs are detected in the thyroid (4, 6), and the SCP-1 The E3AS mAb was designed and found to be suitable for the gene is localized on chromosome region 1p12-p13 (27). SSX expres- detection of SSX protein in paraffin-embedded tissues. Using this sion at the protein level was detected in the testis but not in two antibody, we analyzed 125 cases of benign and malignant melanocytic normal thyroid specimens examined by immunohistochemistry (this lesions by immunohistochemistry. Heterogeneous SSX expression report). Additional RT-PCR analyses were not performed on these was found in 40% of primary melanomas analyzed. This percentage is samples due to lack of fresh and/or frozen material. However, our similar to what has been observed by other investigators using RT- results combined with those of others that did find SSX expression in PCR (43% [11]), but it may be underestimated because SSX1 expres- the thyroid by RT-PCR (4, 6) suggest that this expression may be too sion is not detected by this antibody. Yet, this underestimation may low to be detected at the protein level. Alternatively, the discrepancy not be of major significance because others found that only 1 of 37 may be explained by a bias in sample biopsies. (3%) melanomas tested expressed SSX1 alone (11). The heterogeneity Immunohistochemical detection of SSX proteins in the testes of of SSX expression in the melanocytic lesions was striking, ranging two donors revealed nuclear expression in spermatogonia, both from from widespread to scarce. This finding parallels our observations in mature and immature testes. Several studies have shown that sper- the melanoma cell lines and, therefore, indicates that SSX-expressing matogenic cells undergo profound changes in methylation patterns cells do not undergo positive or negative selection during cell line (28–30). In several cases, it was observed that hypomethylation of establishment. certain promoter sequences occurs at the early stages of spermatogen- 1660

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2000 American Association for Cancer Research. SSX EXPRESSION IN HUMAN MELANOMA esis (i.e., in spermatogonia), thereby being linked to the activation of ACKNOWLEDGMENTS transcription (29, 31, 32). SSX expression in spermatogonia suggests that also these genes may be activated after demethylation of their We thank Drs. S. A. Aaronson, J. Ponte´n, and C. van Roozendaal for kindly providing the A2243, U-4SS, and NT2D1 cell lines, respectively. We are also promoter sequences. Our finding that DAC incubation induces SSX grateful to Bert Janssen, Aı¨cha Fourkour, and Miriam Smeets for invaluable expression in otherwise non- or low-expressing cells further supports technical assistance. Members of the pathology subgroup of the European this hypothesis. A similar phenomenon has been observed for Organization for Research and Treatment of Cancer-melanoma cooperative MAGE-1, a gene that is also primarily expressed in spermatogonia group are acknowledged for providing primary and metastatic melanoma (33) and is activated by DAC treatment (19, 34). specimens. Klaus Hou-Jensen (Copenhagen, Denmark), Eva B. Bro¨cker (Wu¨r- The testis is an immune privileged organ because spermatogenic zburg, Germany), and Nathalie Renard (Brussels, Belgium) generously pro- cells do not express HLA class I and II antigens at the cell surface (35, vided paraffin-embedded specimens. 36). Concomitantly, the testis has a so-called blood-testis barrier in the seminiferous tubuli generated by the Sertoli cells. Because ectopic REFERENCES expression of CT antigens may thus lead to an autologous cellular 1. Geurts van Kessel, A., dos Santos, N. 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Cancer Res 2000;60:1654-1662.

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