Assessment of SS18-SSX Fusion Specific Antibody in Carcinoma and Melanoma

Introduction

Synovial sarcoma is a soft tissue tumor that accounts for 7%–10% of all soft tissue sarcomas.^1,2 The tumor cells of synovial sarcoma do not originate from intra-articular synovium, but most likely from primitive mesenchymal cells.^3–5 Synovial sarcoma can be diagnostically challenging due to its nature to present as one of three distinct morphologies: monophasic, biphasic, and poorly differentiated. The poorly differentiated pattern can present with oval, round cell, or epithelioid morphology. In contrast, the monophasic pattern is predominantly composed of spindle cells with overlapping, hyperchromatic, ovoid to elongated nuclei. Lastly, the biphasic pattern contains spindle cells with variable amount of admixed epithelial component consisting of glands, cords, and nests. The varied histological patterns of synovial sarcoma can mimic a wide range of diagnoses from soft tissue sarcomas to poorly differentiated carcinomas. In this study, we aim to focus on one immunohistochemical stain (SS18::SSX, clone E9X9V) which has become the most useful stain for synovial sarcoma, and see if it has cross-reactivity with potential mimics.

To help aid in diagnosis, several immunohistochemical stains can be helpful for confirming the diagnosis of synovial sarcoma. Historically, keratin stains (EMA, KRT7, KRT19) have been used, especially to highlight the epithelial component. The stain CD99 is positive in over half of synovial sarcomas but it is not specific and can stain other lesions. ⁵ TLE1 has been demonstrated to show expression in over 90% of synovial sarcomas, but has also been seen expression in similar lesions such as malignant peripheral nerve sheath tumor and solitary fibrous tumor.^3,5,6 The aforementioned stains are no longer as widely used with the addition of this new immunohistochemical stain (SS18::SSX, clone E9X9V).

Synovial sarcoma is characterized by the presence of the t(X;18)(p11:q11) translocation, involving the SS18 (formerly SYT) and one of the SSX genes (mainly SSX1, SSX2, and rarely SSX4).^4,6–8 The SS18::SSX fusion genes are seen in more than 95% of synovial sarcoma tumors and the molecular detection of this gene alteration has been used as a gold standard for diagnostic confirmation.^6,7 SSX1, SSX2, and SSX4 gene expression has been seen in normal tissue including testis, thyroid, various carcinomas, and in melanomas.^6,9,10

SS18::SSX antibody, which targets the chimeric proteins created by SS18 gene fusion to SSX1, SSX2, and SSX4 alterations, has been created. This novel antibody has been shown to be extremely useful for confirming the diagnosis of synovial sarcoma. A study conducted by Jason Hornick and Christopher Fletcher demonstrated a sensitivity of 95% and specificity of 100% using antibody clone (E9X9V) when performed on 100 synovial sarcomas and over 300 non-synovial sarcomas. ⁶ They demonstrated no positive staining in any of the tested sarcoma. ⁶ A recent study demonstrated cross-reactivity of SS18::SSX with three of seven melanomas. ¹¹ This stain appears to have the highest specificity and sensitivity to date, and to our knowledge, no study has investigated SS18::SSX antibody staining in carcinomas.

Material and Methods

To perform this study, multiple tissue microarrays consisting of 100 carcinomas were retrieved from the pathology archives of SUNY Upstate Medical University from 2000 to 2007, and were used in a prior study. ⁵ The tumors selected included: 41 adenocarcinomas, 14 squamous cell carcinomas, seven breast carcinomas, five thyroid carcinomas, five renal cell carcinomas, five hepatocellular carcinomas, four mucoepidermoid carcinomas, four urothelial cell carcinomas, three adrenocortical carcinomas, two basal cell carcinomas, two neuroendocrine carcinomas, two small cell carcinomas, one each for seminoma, adenosquamous carcinoma, intraductal papillary mucinous neoplasm, adenoid cystic carcinoma, and ovarian serous. In addition, we used archival paraffin embedded blocks of five invasive melanoma specimens (one superficially spreading melanoma, three invasive nodular, one metastatic melanoma with spindle cell variant).

Four microarrays and five archival paraffin-embedded tissue blocks were sectioned at 3 microns and mounted to charged glass slides. IHC was performed with the Ventana Benchmark Ultra automated immunostainer (Ventana Medical System, Tucson, AZ), performing all steps from deparaffinization through counterstaining. Epitope retrieval was performed on the immunostainer using CC1 for 64 min at 95 C. The rabbit monoclonal anti-SS18::SSX antibody (Cell Signaling, Danvers, MA, USA) clone E9X9V was used at 1:400 dilution. Slides were incubated with the above primary antibody for 32 min at 37 C followed by detection with the UltraView DAB Universal Detection Kit. Post staining consisted of dehydration through graded alcohols, clearing in xylene and coverslipped with a Cytoseal 60 mounting media. The stain was counted as positive if any focal staining was identified within the tumor cells. Appropriate positive and negative tissue controls were used.

Results

Of the 100 various carcinomas and five melanomas, all of them stained negative for SS18::SSX fusion antibody. None of the tumors demonstrated any weak, moderate, or strong staining (Figure 1). All controls demonstrated appropriate staining.

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Figure 1.

H&E sections of different tumors (right) and corresponding SS18::SSX immunostian (left). Images are composed of squamous cell carcinoma (A), adenocarcinoma of the stomach (B), seminoma of testis (C), small cell carcinoma (D), urothelial carcinoma (E), and mucoepidermoid carcinoma (F).

Discussion

SS18 (SYT) gene rearrangement by FISH has been the gold standard for confirming the diagnosis of synovial sarcoma. TLE1 was a very specific immunohistochemistry marker; however, TLE1 has had many pitfalls showing cross-reactivity with different carcinomas. ⁵ More recently, the SS18::SSX fusion specific antibody has been shown to be very specific for synovial sarcoma. In this study, we examined multiple types of carcinomas and melanomas with the SS18::SSX antibody and demonstrated no cross reactivity.

Currently SS18::SSX fusion specific antibody has been shown to highly sensitivity and specific for synovial sarcoma when tested on various sarcomas. ⁶ Our study further supports the high sensitivity of this antibody for synovial sarcoma. The staining pitfalls that could pose a diagnostic dilemma with TLE1 are not seen with the fusion specific antibody, specifically poorly differentiated carcinoma. ⁵

An additional antibody alongside SS18::SSX has been tested for synovial sarcoma, which only detects the SSX portion (clone E5A2C) of the fusion. Studies have shown that SSX antibody is less specific (SS18::SSX 100% vs SSX 96%) for synovial sarcoma but more sensitive (SSX 100% vs SS18::SSX 95%) than its SS18::SSX counterpart. ⁵ SSX demonstrates cross reactive staining with other sarcomas that are FISH negative for SS18::SSX fusion including: sarcomatoid carcinoma, dedifferentiated liposarcoma, spindle cell sarcoma, malignant peripheral nerve sheath tumor, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, desmoplastic small round cell tumor, biphenotypic sinonasal sarcoma, and biphasic mesothelioma.^5,11

There have been a few studies showing cross-reactivity of the SS18::SSX fusion antibody with melanomas.^12,13 What was demonstrated in these studies was weak focal positivity for SS18::SSX antibody (E9X9V clone) within metastatic melanoma and dedifferentiated melanoma. ¹² This could pose a potential pitfall for synovial sarcoma diagnosis, but we were unable to get similar results with the stain using the E9X9V clone, the number tested was low, and this may be an area for future investigation.

In conclusion, currently the SS18::SSX antibody has a very high sensitivity and specificity for synovial sarcoma and may obviate the need to confirm the diagnosis of synovial sarcoma by molecular detection (i.e., FISH, RT-PCR, or next generation sequencing). While the current study did not demonstrate any staining in various carcinomas or melanoma, other studies have demonstrated rare staining in melanoma. Further studies in other tumor types may be warranted to assess the specificity of the SS18::SSX fusion specific antibody.