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MAGE antigen expression in monophasic and biphasic synovial sarcoma
MAGE Antigen Expression in Monophasic and Biphasic Synovial Sarcoma CRISTINA R. ANTONESCU, MD, KLAUS J. BUSAM, MD, KRISTIN IVERSEN, BA, DENISE KOLB, BS, KEREN COPLAN, MS, GIULIO C. SPAGNOLI, PHD, MARC LADANYI, MD, LLOYD J. OLD, MD, AND ACHIM A. JUNGBLUTH, MD Synovial sarcomas are high-grade malignant mesenchymal tumors carrying a pathognomonic cytogenetic alteration t(X;18) involving the SYT gene on chromosome 18 and either SSX1 or SSX2 on chromosome X. Morphologically, biphasic (BSS) and monophasic (MSS) variants can be distinguished. Cancer/testis (CT) antigens are expressed in a variety of malignant tumors, but not in normal tissues except in germ cells, primarily of the testis. Anti-MAGE monoclonal antibody (mAb) 57B previously showed a high incidence and homogenous reactivity pattern in a preliminary analysis of synovial sarcomas. This study was performed to analyze the expression of MAGE by immunohistochemistry with mAb 57B in 25 synovial sarcomas (12 monophasic, 13 biphasic), which were typed for the t(X;18)-derived
fusion transcript by reverse transcriptase polymerase chain reaction (19 SYT-SSX1, 6 SYT-SSX2). 57B immunoreactivity was present in 22 of 25 (88%) cases, and antigen expression was homogeneous in 14 of 22 57B-positive cases. Both morphological variants and both translocation types were immunoreactive; three SYT-SSX1 tumors (one MSS, two BSS) were 57B negative. Our study demonstrates that MAGE is frequently and homogeneously expressed in synovial sarcomas of both morphological variants and both translocation types, making these tumors an attractive target for MAGE antigen-based immunotherapy. HUM PATHOL 33:225-229. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: synovial sarcoma, MAGE antigen expression.
Genes encoding cancer-testis (CT) antigens are expressed in a variety of malignant neoplasms but not in normal tissues, except in germ cells, predominantly within the testis.1 The immune response to CT antigens is T cell mediated. Because testicular germ cells lack expression of HLA class I molecules, potential cytotoxic T cell– dependent immune reactions following vaccinations to CT antigens do not affect male germ cells.1 Due to the tumor-associated expression pattern, CT antigens are of major interest as targets for immunotherapy and may also serve as tools for diagnostic purposes. MAGE was the first CT antigen that was isolated,2 and an entire MAGE gene family has been recognized.3 Currently, more than 20 MAGE members can be distinguished, and several other CT antigens, including BAGE, GAGE, NY-ESO-1, and CT7, have been isolated.5,6 Current knowledge about the CT antigen expression is based mainly on reverse transcriptase-polymerase chain reaction (RT-PCR) studies, and only recently have data about the actual protein expression become available. Recent analyses using the anti-MAGE monoclonal antibody (mAb), 57B, demonstrated a mostly heterogeneous protein expression in a variety of epithelial malignant neoplasms,7,8 and most sarcoma types showed no MAGE antigen expression. Synovial sarco-
mas, however, revealed a high incidence of 57B-positive cases, with a homogeneous antigen expression in most of these cases.7 Synovial sarcomas are high-grade tumors9 that account for approximately 10% of all sarcomas and display two main morphologic subtypes: a monophasic purely spindle cell variant, and a biphasic type consisting of various amounts of spindle cells as well as a glandular epithelial component. Synovial sarcomas carry a pathognomonic chromosomal translocation, t(X;18)(p11;q11), leading to the fusion of the SYT gene on chromosome 18q11, to SSX1 or SSX2 (or rarely SSX4) on chromosome Xp11.10 Morphological and clinical parameters such as prognosis have been associated with SYT-SSX fusion types.11-13 The fusion transcript can be determined by RT-PCR analysis of either frozen or paraffin-embedded tissues.14,15 In our initial analysis, only a small number of synovial sarcomas were analyzed for their 57B immunoreactivity, and no correlation with the underlying SYT-SSX fusion transcript was performed.7 This study investigates the expression of MAGE antigen by immunohistochemistry as detected by mAb 57B in a larger number of monophasic and biphasic synovial sarcomas analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence and type of fusion transcript to elucidate possible associations of antigen expression with morphological subtype or the fusion transcript variant.
From the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY; the Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY; and the Departments of Surgery and Research, University of Basel, Basel, Switzerland. Accepted for publication October 17, 2001. Address correspondence and reprint requests to Achim A. Jungbluth, MD, Ludwig Institute for Cancer Research at Memorial SloanKettering Cancer Center, 1275 York Ave, Box 32, New York, NY 10021. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3302-0014$35.00/0 doi:10.1053/hupa.2002.31295
MATERIALS AND METHODS Twenty-five cases of synovial sarcoma were retrieved from the archives of the Department of Pathology, Memorial SloanKettering Cancer Center. Representative blocks of formalinfixed paraffin-embedded tissues and snap-frozen corresponding tumor samples were chosen for further analyses. Most cases were used for previous analyses of synovial sarcoma.12,16
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TABLE 1. Immunohistochemical Typing of 25 Synovial Sarcomas with mAb 57B: Comparison of Antigen Expression, Morphology, and SYT-SSX Translocation Tumor Staining
⫹
Focal*
⫹⫹
⫹⫹⫹
Total Positive
⫹⫹⫹⫹
Total Negative
Histologic type
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
SYT-SSX1 SYT-SSX2 Total/Histologic type Total
1 1 2
0 0
0 0 0
5 5
0 0 0
1 1
1 1 2
1 1
3 4 7
4 4
5 6 11
11 11
1 0 1
2 2
2
5
1
3
11
22
3
*Focal: ⬍5%, ⫹: 5%–25%, ⫹⫹: ⬎25%–50%, ⫹⫹⫹: ⬎50%–75%, ⫹⫹⫹⫹: ⬎75%. Abbreviations: MSS, monophasic synovial sarcoma; BSS, biphasic synovial sarcoma.
mAb 57B, a murine IgG1, was described previously.17 Normal human testis was used as a positive control tissue. The 5-m sections were heated at 60°C overnight to ensure proper attachment. A heat-based antigen-retrieval method using citrate buffer (pH 6.0, 10 mM) and a steamer was performed before application of the primary antibody. The primary antibody was used at a concentration of 0.5 g/ml. The primary antibody was detected with a biotinylated horse anti-mouse secondary reagent (1:200; Vector Laboratories, Burlingame, CA) followed by an avidin-biotin-complex system (ABC-Elite; Vector Laboratories). DAB (Biogenix, San Ramon, CA) served as the chromogen. The extent of immunohistochemical staining was graded according to the immunopositive tumor area: focal: staining of single cells, not more than 5%, ⫹: ⬎5% to 25%, ⫹⫹: ⬎25% to 50%, ⫹⫹⫹: ⬎50% to 75%, ⫹⫹⫹⫹: ⬎75%. A negative control omitting the primary reagent was included in all assays. Tumor samples for molecular analysis were snap-frozen in liquid nitrogen and stored at ⫺70°C. Extraction of total RNA was based on the guanidinium isothiocyanate-phenol chloroform method. Analysis by RT-PCR for SYT-SSX transcripts was performed following standard protocols as described previously.12,16 RT-PCR products were separated by gel electrophoresis and visualized by ethidium bromide.
RESULTS The results of the morphological evaluation, the immunohistochemical staining pattern with mAb 57B, and the RT-PCR analysis of the fusion transcript are given in Table 1. A total of 25 synovial sarcomas were studied. Twelve cases were of the monophasic type whereas 13 cases showed a biphasic phenotype. None of our cases fulfilled the criteria of a poorly differentiated synovial sarcoma.18 RT-PCR revealed the SYT-SSX1 fusion transcript in 19 cases, comprising all 13 biphasic and 6 monophasic tumors, and the SYT-SSX2 fusion transcript in 6 monophasic lesions. Immunoreactivity with 57B was found in monophasic and biphasic synovial sarcomas (Fig 1) and in tumors of both translocation types. 57B was positive in 22 of 25 (88%) cases (11 biphasic, 11 monophasic). The 3 of 25 (12%) negative cases were 1 biphasic and 2 monophasic tumors, all carrying the SYT-SSX1 rearrangement. Of the 22 positive lesions, 14 showed homogeneous 57B reactivity (Fig 1A); that is, staining was present in ⬎50% of the tumors, 5 of which were biphasic (SYT-SSX1) and 9 of which were monophasic (4 SYT-SSX1, 5 SYT-SSX2).
Cytoplasmic staining was prevalent, but variable nuclear reactivity was also visible (Fig 1D). In the biphasic synovial sarcomas, staining could be seen in the spindle cell as well as in the epithelial glandular component. However, the staining of these components was variable even within the same tumor, and 57B negative glandular structures could be seen next to immunopositive glands (Fig 1B, C).
DISCUSSION Because of their almost exclusive presence in tumor tissue, CT antigens appear to be ideal targets for immunotherapeutic approaches. Because of the lack of appropriate serological typing reagents, until recently, data regarding the expression of most CT antigens were based mainly on mRNA studies, and actual antigen expression has been addressed only in some newer studies. This analysis used anti-MAGE mAb 57B, one of the few available reagents to CT antigens. 57B was generated to the MAGE-3 recombinant protein17 but was later found to be reactive to more than one MAGE gene product when tested on cells transfected with different MAGE genes.19 Recent analyses, however, indicate that mAb 57B reactivity with human tumors appears to cotype with MAGE-4 mRNA expression.20 The problem in determining 57B’s fine specificity arises because of the high homology of the MAGE genes in question.4 Although the definite specificity of 57B might need further analysis. 57B is one of few available MAGE-specific reagents, and several studies have used this antibody for assessment of the MAGE expression pattern.7,8,21,22 Serological reagents to other CT antigens have been developed, including mAb R5 to MAGE-4,23 mAb MA454 to MAGE-1,24 and mAb ES121 to NY-ESO-1.25 The picture that has evolved as a result of the immunohistochemical typing with these reagents is the predominantly heterogeneous expression pattern for CT antigens in most neoplasms tested. An exception is synovial sarcoma, which showed a high incidence and a homogeneous staining with mAb 57B in a limited number of cases analyzed.7 Synovial sarcomas are highgrade tumors that appear in two histomorphologic variants, the monophasic purely spindle cell type and the
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FIGURE 1. Immunohistochemical staining of synovial sarcomas with mAb 57B. (A) Monophasic synovial sarcoma with homogeneous immunoreactivity. (B) Biphasic synovial sarcoma, negative glandular component. (C) Biphasic synovial sarcoma, positive glandular component (*). (D) Biphasic synovial sarcoma, cytoplasmic and nuclear staining of spindle cell component. Original magnification 40⫻ (A), 200⫻ (B,C), 400⫻ (D).
biphasic variant consisting of an additional epithelial glandular component. Several recent reports and reviews have addressed these morphologic features as well as the clinical aspects of synovial sarcomas.18,26 This study confirms our previous findings, showing MAGE-antigen expression in almost 90% of our tested cases. Even more interestingly, half of the positive cases showed 57B immunoreactivity in more than 50% of the tumor areas. No predilection for either morphological variant is evident from our analysis. Hence both morphological variants of synovial sarcomas represent lesions with the best CT expression reported thus far. In our analysis, we also assessed the possibility of an association between the SYT-SSX fusion transcript structure and the 57B staining pattern. The karyotypic hallmark of synovial sarcomas is the recurrent chromosomal translocation (X;18), in which the SYT gene on chromosome 18q11 is fused to one of the SSX genes on chromosome Xp11.10,27 A family of five SSX genes, SSX1–SSX5, has been isolated,28 SSX1 and SSX2 are
most commonly involved in the translocation of synovial sarcomas, but rare tumors with SYT-SSX4 have also been reported.29 Tumors of either fusion transcript type were positive for mAb 57B, and in the biphasic variant, both the epithelial and spindle cell components were immunoreactive. This concurs with previous analyses, confirming that t(X;18) is present in both the spindle cell and the epithelial components.30 Although all three 57B-negative cases were carrying the SSX1 translocation (one monophasic, two biphasic), the more limited number of tumors carrying the SYT-SSX2 translocation in this study does not allow a definite statement as to whether they always express the MAGE antigen, and more SSX2 tumors need to be tested. SYT and SSX have been implicated in transcriptional processes exhibiting activating31 and repressing functions,32 respectively, and both gene products as well as products of the chimeric fusion were localized to the nucleus.33,34 Little is known about the function of MAGE. However, the immunoreactivity of 57B in our
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study speaks for a nuclear localization of the MAGE gene product in at least some synovial sarcomas. MAGE (as well as other CT antigens) and SSX are both located on chromosome X; MAGE genes are located on the long arm (Xq28),4 and SSX maps to the short arm (Xp11).27 An upregulation of MAGE genes by direct involvement during the translocation process appears unlikely because of the huge genetic distance between these genes. Whether the genome-wide demethylation that has been implicated in the activation of MAGE35 is associated with the SYT-SSX fusion remains to be investigated. Although the underlying mechanism is poorly understood, our study shows that monophasic as well as biphasic synovial sarcomas have a high incidence and a striking homogeneity of MAGE antigen expression, regardless of the associated SYT-SSX fusion transcript type. Several vaccination trials using MAGE antigens in melanoma patients have produced promising results, generating tumor responses in several patients and reporting no immunological reactions involving testicular tissue.36,37 Based on our study, synovial sarcomas must be considered potential tumors for vaccinationbased cancer therapy. Further studies analyzing the association between MAGE gene expression and the genetic events occurring in synovial sarcomas will provide a better understanding of the biology of both CT antigens and synovial sarcomas. REFERENCES 1. Boon T, Old LJ: Cancer tumor antigens. Curr Opin Immunol 9:681-683, 1997 2. Coulie PG, Weynants P, Lehmann F, et al: Genes coding for tumor antigens recognized by human cytolytic T lymphocytes. J Immunother 14:104-109, 1993 3. Lucas S, De Plaen E, Boon T: MAGE-B5, MAGE-B6, MAGEC2, and MAGE-C3: Four new members of the MAGE family with tumor-specific expression. Int J Cancer 87:55-60, 2000 4. De Plaen E, Arden K, Traversari C, et al: Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 40:360-369, 1994 5. Boel P, Wildmann C, Sensi ML, et al: BAGE: A new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity 2:167-175, 1995 6. Chen YT, Gure AO, Tsang S, et al: Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library. Proc Natl Acad Sci USA 95:6919-6923, 1998 7. Jungbluth AA, Busam KJ, Kolb D, et al: Expression of MAGEantigens in normal tissues and cancer. Int J Cancer 85:460-465, 2000 8. Busam KJ, Iversen K, Berwick M, et al: Immunoreactivity with the anti-MAGE antibody 57B in malignant melanoma: Frequency of expression and correlation with prognostic parameters. Mod Pathol 13:459-465, 2000 9. Skytting B: Synovial sarcoma. A Scandinavian Sarcoma Group project. Acta Orthop Scand Suppl 291:1-28, 2000 10. Limon J, Mrozek K, Mandahl N, et al: Cytogenetics of synovial sarcoma: Presentation of ten new cases and review of the literature. Genes Chromosomes Cancer 3:338-345, 1991 11. Renwick PJ, Reeves BR, Dal Cin P, et al: Two categories of synovial sarcoma defined by divergent chromosome translocation breakpoints in Xp11.2, with implications for the histologic sub-classification of synovial sarcoma. Cytogenet Cell Genet 70:58-63, 1995 12. Antonescu CR, Kawai A, Leung DH, et al: Strong association of SYT-SSX fusion type and morphologic epithelial differentiation in synovial sarcoma. Diagn Mol Pathol 9:1-8, 2000
13. Kawai A, Woodruff J, Healey JH, et al: SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma. N Engl J Med 338:153-160, 1998 14. Argani P, Zakowski MF, Klimstra DS, et al: Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases. Mod Pathol 11:65-71, 1998 15. Lasota J, Jasinski M, Debiec-Rychter M, et al: Detection of the SYT-SSX fusion transcripts in formaldehyde-fixed, paraffin-embedded tissue: A reverse transcription polymerase chain reaction amplification assay useful in the diagnosis of synovial sarcoma. Mod Pathol 11:626-633, 1998 16. Antonescu CR, Leung DH, Dudas M, et al: Alterations of cell cycle regulators in localized synovial sarcoma: A multifactorial study with prognostic implications. Am J Pathol 156:977-983, 2000 17. Kocher T, Schultz-Thater E, Gudat F, et al: Identification and intracellular location of MAGE-3 gene product. Cancer Res 55:2236-2239, 1995 18. van de Rijn M, Barr FG, Xiong QB, et al: Poorly differentiated synovial sarcoma: An analysis of clinical, pathologic, and molecular genetic features. Am J Surg Pathol 23:106-112, 1999 19. Rimoldi D, Salvi S, Schultz-Thater E, et al: Anti-MAGE-3 antibody 57B and anti-MAGE-1 antibody 6C1 can be used to study different proteins of the MAGE-A family [letter]. Int J Cancer 86:749751, 2000 20. Landry C, Brasseur F, Spagnoli GC, et al: Monoclonal antibody 57B stains tumor tissues that express gene MAGE-A4. Int J Cancer 86:835-841, 2000 21. Cheville JC, Roche PC: MAGE-1 and MAGE-3 tumor rejection antigens in human germ cell tumors. Mod Pathol 12:974-978, 1999 22. Chambost H, Van Baren N, Brasseur F, et al: Expression of gene MAGE-A4 in Reed–Sternberg cells. Blood 95:3530-3533, 2000 23. Takahashi K, Shichijo S, Noguchi M, et al: Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis. Cancer Res 55:3478-3482, 1995 24. Jungbluth AA, Stockert E, Chen YT, et al: Monoclonal antibody MA454 reveals a heterogeneous expression pattern of MAGE-1 antigen in formalin-fixed paraffin embedded lung tumors. Br J Cancer 83:493-497, 2000 25. Jungbluth AA, Chen YT, Stockert E, et al: Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues. Int J Cancer 92:856-860, 2001 26. Skytting B, Meis-Kindblom JM, Larsson O, et al: Synovial sarcoma—Identification of favorable and unfavorable histologic types: A Scandinavian sarcoma group study of 104 cases. Acta Orthop Scand 70:543-554, 1999 27. Clark J, Rocques PJ, Crew AJ, et al: Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat Genet 7:502-508, 1994 28. Gure AO, Tureci O, Sahin U, et al: SSX: A multigene family with several members transcribed in normal testis and human cancer. Int J Cancer 72:965-971, 1997 29. Skytting B, Nilsson G, Brodin B, et al: A novel fusion gene, SYT-SSX4, in synovial sarcoma [letter]. J Natl Cancer Inst 91:974-975, 1999 30. Kasai T, Shimajiri S, Hashimoto H: Detection of SYT-SSX fusion transcripts in both epithelial and spindle cell areas of biphasic synovial sarcoma using laser capture microdissection. Mol Pathol 53:107-110, 2000 31. Brett D, Whitehouse S, Antonson P, et al: The SYT protein involved in the t(X;18) synovial sarcoma translocation is a transcriptional activator localised in nuclear bodies. Hum Mol Genet 6:15591564, 1997 32. Lim FL, Soulez M, Koczan D, et al: A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas. Oncogene 17:20132018, 1998 33. Soulez M, Saurin AJ, Freemont PS, et al: SSX and the synovial-sarcoma-specific chimaeric protein SYT-SSX co-localize with the human Polycomb group complex. Oncogene 18:27392746, 1999 34. dos Santos NR, de Bruijn DR, Balemans M, et al: Nuclear
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MAGE IN SYNOVIAL SARCOMAS (Antonescu et al) localization of SYT, SSX and the synovial sarcoma-associated SYT-SSX fusion proteins. Hum Mol Genet 6:1549-1558, 1997 35. de Smet C, De Backer O, Faraoni I, et al: The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc Natl Acad Sci USA 93:7149-7153, 1996 36. Marchand M, Van Baren N, Weynants P, et al: Tumor re-
gressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 80:219-230, 1999 37. Marchand M, Weynants P, Rankin E, et al: Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3 [letter]. Int J Cancer 63:883-885, 1995
229
fusion transcript by reverse transcriptase polymerase chain reaction (19 SYT-SSX1, 6 SYT-SSX2). 57B immunoreactivity was present in 22 of 25 (88%) cases, and antigen expression was homogeneous in 14 of 22 57B-positive cases. Both morphological variants and both translocation types were immunoreactive; three SYT-SSX1 tumors (one MSS, two BSS) were 57B negative. Our study demonstrates that MAGE is frequently and homogeneously expressed in synovial sarcomas of both morphological variants and both translocation types, making these tumors an attractive target for MAGE antigen-based immunotherapy. HUM PATHOL 33:225-229. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: synovial sarcoma, MAGE antigen expression.
Genes encoding cancer-testis (CT) antigens are expressed in a variety of malignant neoplasms but not in normal tissues, except in germ cells, predominantly within the testis.1 The immune response to CT antigens is T cell mediated. Because testicular germ cells lack expression of HLA class I molecules, potential cytotoxic T cell– dependent immune reactions following vaccinations to CT antigens do not affect male germ cells.1 Due to the tumor-associated expression pattern, CT antigens are of major interest as targets for immunotherapy and may also serve as tools for diagnostic purposes. MAGE was the first CT antigen that was isolated,2 and an entire MAGE gene family has been recognized.3 Currently, more than 20 MAGE members can be distinguished, and several other CT antigens, including BAGE, GAGE, NY-ESO-1, and CT7, have been isolated.5,6 Current knowledge about the CT antigen expression is based mainly on reverse transcriptase-polymerase chain reaction (RT-PCR) studies, and only recently have data about the actual protein expression become available. Recent analyses using the anti-MAGE monoclonal antibody (mAb), 57B, demonstrated a mostly heterogeneous protein expression in a variety of epithelial malignant neoplasms,7,8 and most sarcoma types showed no MAGE antigen expression. Synovial sarco-
mas, however, revealed a high incidence of 57B-positive cases, with a homogeneous antigen expression in most of these cases.7 Synovial sarcomas are high-grade tumors9 that account for approximately 10% of all sarcomas and display two main morphologic subtypes: a monophasic purely spindle cell variant, and a biphasic type consisting of various amounts of spindle cells as well as a glandular epithelial component. Synovial sarcomas carry a pathognomonic chromosomal translocation, t(X;18)(p11;q11), leading to the fusion of the SYT gene on chromosome 18q11, to SSX1 or SSX2 (or rarely SSX4) on chromosome Xp11.10 Morphological and clinical parameters such as prognosis have been associated with SYT-SSX fusion types.11-13 The fusion transcript can be determined by RT-PCR analysis of either frozen or paraffin-embedded tissues.14,15 In our initial analysis, only a small number of synovial sarcomas were analyzed for their 57B immunoreactivity, and no correlation with the underlying SYT-SSX fusion transcript was performed.7 This study investigates the expression of MAGE antigen by immunohistochemistry as detected by mAb 57B in a larger number of monophasic and biphasic synovial sarcomas analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence and type of fusion transcript to elucidate possible associations of antigen expression with morphological subtype or the fusion transcript variant.
From the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY; the Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY; and the Departments of Surgery and Research, University of Basel, Basel, Switzerland. Accepted for publication October 17, 2001. Address correspondence and reprint requests to Achim A. Jungbluth, MD, Ludwig Institute for Cancer Research at Memorial SloanKettering Cancer Center, 1275 York Ave, Box 32, New York, NY 10021. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3302-0014$35.00/0 doi:10.1053/hupa.2002.31295
MATERIALS AND METHODS Twenty-five cases of synovial sarcoma were retrieved from the archives of the Department of Pathology, Memorial SloanKettering Cancer Center. Representative blocks of formalinfixed paraffin-embedded tissues and snap-frozen corresponding tumor samples were chosen for further analyses. Most cases were used for previous analyses of synovial sarcoma.12,16
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TABLE 1. Immunohistochemical Typing of 25 Synovial Sarcomas with mAb 57B: Comparison of Antigen Expression, Morphology, and SYT-SSX Translocation Tumor Staining
⫹
Focal*
⫹⫹
⫹⫹⫹
Total Positive
⫹⫹⫹⫹
Total Negative
Histologic type
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
MSS
BSS
SYT-SSX1 SYT-SSX2 Total/Histologic type Total
1 1 2
0 0
0 0 0
5 5
0 0 0
1 1
1 1 2
1 1
3 4 7
4 4
5 6 11
11 11
1 0 1
2 2
2
5
1
3
11
22
3
*Focal: ⬍5%, ⫹: 5%–25%, ⫹⫹: ⬎25%–50%, ⫹⫹⫹: ⬎50%–75%, ⫹⫹⫹⫹: ⬎75%. Abbreviations: MSS, monophasic synovial sarcoma; BSS, biphasic synovial sarcoma.
mAb 57B, a murine IgG1, was described previously.17 Normal human testis was used as a positive control tissue. The 5-m sections were heated at 60°C overnight to ensure proper attachment. A heat-based antigen-retrieval method using citrate buffer (pH 6.0, 10 mM) and a steamer was performed before application of the primary antibody. The primary antibody was used at a concentration of 0.5 g/ml. The primary antibody was detected with a biotinylated horse anti-mouse secondary reagent (1:200; Vector Laboratories, Burlingame, CA) followed by an avidin-biotin-complex system (ABC-Elite; Vector Laboratories). DAB (Biogenix, San Ramon, CA) served as the chromogen. The extent of immunohistochemical staining was graded according to the immunopositive tumor area: focal: staining of single cells, not more than 5%, ⫹: ⬎5% to 25%, ⫹⫹: ⬎25% to 50%, ⫹⫹⫹: ⬎50% to 75%, ⫹⫹⫹⫹: ⬎75%. A negative control omitting the primary reagent was included in all assays. Tumor samples for molecular analysis were snap-frozen in liquid nitrogen and stored at ⫺70°C. Extraction of total RNA was based on the guanidinium isothiocyanate-phenol chloroform method. Analysis by RT-PCR for SYT-SSX transcripts was performed following standard protocols as described previously.12,16 RT-PCR products were separated by gel electrophoresis and visualized by ethidium bromide.
RESULTS The results of the morphological evaluation, the immunohistochemical staining pattern with mAb 57B, and the RT-PCR analysis of the fusion transcript are given in Table 1. A total of 25 synovial sarcomas were studied. Twelve cases were of the monophasic type whereas 13 cases showed a biphasic phenotype. None of our cases fulfilled the criteria of a poorly differentiated synovial sarcoma.18 RT-PCR revealed the SYT-SSX1 fusion transcript in 19 cases, comprising all 13 biphasic and 6 monophasic tumors, and the SYT-SSX2 fusion transcript in 6 monophasic lesions. Immunoreactivity with 57B was found in monophasic and biphasic synovial sarcomas (Fig 1) and in tumors of both translocation types. 57B was positive in 22 of 25 (88%) cases (11 biphasic, 11 monophasic). The 3 of 25 (12%) negative cases were 1 biphasic and 2 monophasic tumors, all carrying the SYT-SSX1 rearrangement. Of the 22 positive lesions, 14 showed homogeneous 57B reactivity (Fig 1A); that is, staining was present in ⬎50% of the tumors, 5 of which were biphasic (SYT-SSX1) and 9 of which were monophasic (4 SYT-SSX1, 5 SYT-SSX2).
Cytoplasmic staining was prevalent, but variable nuclear reactivity was also visible (Fig 1D). In the biphasic synovial sarcomas, staining could be seen in the spindle cell as well as in the epithelial glandular component. However, the staining of these components was variable even within the same tumor, and 57B negative glandular structures could be seen next to immunopositive glands (Fig 1B, C).
DISCUSSION Because of their almost exclusive presence in tumor tissue, CT antigens appear to be ideal targets for immunotherapeutic approaches. Because of the lack of appropriate serological typing reagents, until recently, data regarding the expression of most CT antigens were based mainly on mRNA studies, and actual antigen expression has been addressed only in some newer studies. This analysis used anti-MAGE mAb 57B, one of the few available reagents to CT antigens. 57B was generated to the MAGE-3 recombinant protein17 but was later found to be reactive to more than one MAGE gene product when tested on cells transfected with different MAGE genes.19 Recent analyses, however, indicate that mAb 57B reactivity with human tumors appears to cotype with MAGE-4 mRNA expression.20 The problem in determining 57B’s fine specificity arises because of the high homology of the MAGE genes in question.4 Although the definite specificity of 57B might need further analysis. 57B is one of few available MAGE-specific reagents, and several studies have used this antibody for assessment of the MAGE expression pattern.7,8,21,22 Serological reagents to other CT antigens have been developed, including mAb R5 to MAGE-4,23 mAb MA454 to MAGE-1,24 and mAb ES121 to NY-ESO-1.25 The picture that has evolved as a result of the immunohistochemical typing with these reagents is the predominantly heterogeneous expression pattern for CT antigens in most neoplasms tested. An exception is synovial sarcoma, which showed a high incidence and a homogeneous staining with mAb 57B in a limited number of cases analyzed.7 Synovial sarcomas are highgrade tumors that appear in two histomorphologic variants, the monophasic purely spindle cell type and the
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FIGURE 1. Immunohistochemical staining of synovial sarcomas with mAb 57B. (A) Monophasic synovial sarcoma with homogeneous immunoreactivity. (B) Biphasic synovial sarcoma, negative glandular component. (C) Biphasic synovial sarcoma, positive glandular component (*). (D) Biphasic synovial sarcoma, cytoplasmic and nuclear staining of spindle cell component. Original magnification 40⫻ (A), 200⫻ (B,C), 400⫻ (D).
biphasic variant consisting of an additional epithelial glandular component. Several recent reports and reviews have addressed these morphologic features as well as the clinical aspects of synovial sarcomas.18,26 This study confirms our previous findings, showing MAGE-antigen expression in almost 90% of our tested cases. Even more interestingly, half of the positive cases showed 57B immunoreactivity in more than 50% of the tumor areas. No predilection for either morphological variant is evident from our analysis. Hence both morphological variants of synovial sarcomas represent lesions with the best CT expression reported thus far. In our analysis, we also assessed the possibility of an association between the SYT-SSX fusion transcript structure and the 57B staining pattern. The karyotypic hallmark of synovial sarcomas is the recurrent chromosomal translocation (X;18), in which the SYT gene on chromosome 18q11 is fused to one of the SSX genes on chromosome Xp11.10,27 A family of five SSX genes, SSX1–SSX5, has been isolated,28 SSX1 and SSX2 are
most commonly involved in the translocation of synovial sarcomas, but rare tumors with SYT-SSX4 have also been reported.29 Tumors of either fusion transcript type were positive for mAb 57B, and in the biphasic variant, both the epithelial and spindle cell components were immunoreactive. This concurs with previous analyses, confirming that t(X;18) is present in both the spindle cell and the epithelial components.30 Although all three 57B-negative cases were carrying the SSX1 translocation (one monophasic, two biphasic), the more limited number of tumors carrying the SYT-SSX2 translocation in this study does not allow a definite statement as to whether they always express the MAGE antigen, and more SSX2 tumors need to be tested. SYT and SSX have been implicated in transcriptional processes exhibiting activating31 and repressing functions,32 respectively, and both gene products as well as products of the chimeric fusion were localized to the nucleus.33,34 Little is known about the function of MAGE. However, the immunoreactivity of 57B in our
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study speaks for a nuclear localization of the MAGE gene product in at least some synovial sarcomas. MAGE (as well as other CT antigens) and SSX are both located on chromosome X; MAGE genes are located on the long arm (Xq28),4 and SSX maps to the short arm (Xp11).27 An upregulation of MAGE genes by direct involvement during the translocation process appears unlikely because of the huge genetic distance between these genes. Whether the genome-wide demethylation that has been implicated in the activation of MAGE35 is associated with the SYT-SSX fusion remains to be investigated. Although the underlying mechanism is poorly understood, our study shows that monophasic as well as biphasic synovial sarcomas have a high incidence and a striking homogeneity of MAGE antigen expression, regardless of the associated SYT-SSX fusion transcript type. Several vaccination trials using MAGE antigens in melanoma patients have produced promising results, generating tumor responses in several patients and reporting no immunological reactions involving testicular tissue.36,37 Based on our study, synovial sarcomas must be considered potential tumors for vaccinationbased cancer therapy. Further studies analyzing the association between MAGE gene expression and the genetic events occurring in synovial sarcomas will provide a better understanding of the biology of both CT antigens and synovial sarcomas. REFERENCES 1. Boon T, Old LJ: Cancer tumor antigens. Curr Opin Immunol 9:681-683, 1997 2. Coulie PG, Weynants P, Lehmann F, et al: Genes coding for tumor antigens recognized by human cytolytic T lymphocytes. J Immunother 14:104-109, 1993 3. Lucas S, De Plaen E, Boon T: MAGE-B5, MAGE-B6, MAGEC2, and MAGE-C3: Four new members of the MAGE family with tumor-specific expression. Int J Cancer 87:55-60, 2000 4. De Plaen E, Arden K, Traversari C, et al: Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 40:360-369, 1994 5. Boel P, Wildmann C, Sensi ML, et al: BAGE: A new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity 2:167-175, 1995 6. Chen YT, Gure AO, Tsang S, et al: Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library. Proc Natl Acad Sci USA 95:6919-6923, 1998 7. Jungbluth AA, Busam KJ, Kolb D, et al: Expression of MAGEantigens in normal tissues and cancer. Int J Cancer 85:460-465, 2000 8. Busam KJ, Iversen K, Berwick M, et al: Immunoreactivity with the anti-MAGE antibody 57B in malignant melanoma: Frequency of expression and correlation with prognostic parameters. Mod Pathol 13:459-465, 2000 9. Skytting B: Synovial sarcoma. A Scandinavian Sarcoma Group project. Acta Orthop Scand Suppl 291:1-28, 2000 10. Limon J, Mrozek K, Mandahl N, et al: Cytogenetics of synovial sarcoma: Presentation of ten new cases and review of the literature. Genes Chromosomes Cancer 3:338-345, 1991 11. Renwick PJ, Reeves BR, Dal Cin P, et al: Two categories of synovial sarcoma defined by divergent chromosome translocation breakpoints in Xp11.2, with implications for the histologic sub-classification of synovial sarcoma. Cytogenet Cell Genet 70:58-63, 1995 12. Antonescu CR, Kawai A, Leung DH, et al: Strong association of SYT-SSX fusion type and morphologic epithelial differentiation in synovial sarcoma. Diagn Mol Pathol 9:1-8, 2000
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