Expression of TLE-1 and CD99 in Carcinoma: Pitfalls in Diagnosis of Synovial Sarcoma
Daniel J. Zaccarini, MD, Xiaobing Deng, PhD, Jamie Tull, BS, Charlene Maciak, BS, Alfredo L. Valente, MD, and Shengle Zhang, MD
Abstract: The characteristic immunoprofile for the diagnosis of synovial sarcoma, a neoplasm of unclear tissue origin, is ex- pression of transducer-like enhancer of split 1 (TLE-1), CD99, partial expression of cytokeratin, and epithelial membrane an- tigen by immunohistochemistry (IHC). Diagnostic dilemma or misdiagnosis can occur due to overlap in IHC and morphology with carcinomas, and particularly poorly differentiated and metastatic tumors. The frequency of TLE-1 and CD99 ex- pression in carcinomas by IHC has not been previously assessed. We evaluated TLE-1 and CD99 expression in various carcino- mas and evaluated the expression of the SS18 (SYT) gene re- arrangement (a characteristic biomarker for synovial sarcoma) in tumors with TLE-1 and/or CD99 expression. Immunostains of TLE-1 and CD99 were performed in 100 various carcinomas. Seven of the 98 cases (7%) of carcinomas showed TLE-1 ex- pression, including 1 each of prostate adenocarcinoma (ADCA), esophageal ADCA, basal cell carcinoma, adrenocortical carci- noma, endometrial ADCA, ovarian serous carcinoma, and small cell carcinoma. Twenty-one of the 100 cases (21%) of carcinomas demonstrated CD99 expression, including 6 prostate ADCA, 3 esophageal ADCA, 5 squamous cell carcinomas, 2 hepatocellular carcinomas, 1 each for endometrial ADCA, renal cell carcinoma, urothelial cell carcinoma, neuroendocrine car- cinoma, and mucoepidermoid carcinoma. An esophageal AD- CA was positive for both TLE-1 and CD99. None of the carcinomas with positive TLE-1 (n = 7) or CD99 (n = 21) by IHC showed SS18 gene rearrangement by fluorescent in situ hybridization. TLE-1 and CD99 expression were identified in 7% and 21% of carcinomas, respectively. This is a potential pitfall in the IHC interpretation for diagnosis of synovial sar- coma. SS18 gene rearrangement by fluorescent in situ hybrid- ization is helpful for the diagnostically challenging cases, either for confirmation or exclusion of synovial sarcoma.
Key Words: synovial sarcoma, TLE-1, poorly differentiated, carcinoma, CD99, pitfalls
(Appl Immunohistochem Mol Morphol 2016;00:000-000)
From the State University of New York Upstate Medical University, Syracuse, NY.
Reprints: Daniel J. Zaccarini, MD, Department of Pathology, SUNY Upstate Medical University, 750 East Adams Street Syracuse, NY 13210 (e-mail: zaccarid@upstate.edu).
S ynovial sarcoma accounts for approximately 10% of all soft tissue malignancies, and usually occurs in the extremities. Contrary to its name, it rarely occurs in the synovial cavity, instead it can occur anywhere in the body, mainly para-articular regions.1 The monophasic fibrous pattern is the most common histologic subtype; however, biphasic patterns and poorly differentiated patterns occur. The poorly differentiated pattern typically shows oval, round cell and/or epithelioid morphology, which may mimic poorly differentiated carcinoma. In contrast, poorly differentiated and/or metastatic carci- noma may show spindle cell or sarcomatoid pattern, overlapping with synovial sarcoma in histology.
The immunohistochemical approach helps in con- firming the diagnosis of synovial sarcomas. Most histo- logic subtypes express cytokeratin (CK) markers, especially the epithelial component, particularly EMA, CK7, and CK19.1-3 More than half of synovial sarcomas express CD99 and BCL-2.2 Transducer-like enhancer of split 1 (TLE-1) is a more specific biomarker for differ- entiating synovial sarcoma from other soft tissue tu- mors.4 6 However, TLE-1 expression can be seen in some other soft tissue neoplasms, such as peripheral nerve sheath tumor and fibrous solitary tumor, as well as mes- othelioma.4-7 In practice, we have seen rare cases of car- cinoma with intense staining for TLE-1, which has caused confusion and difficulty in diagnosing synovial sarcoma.
Synovial sarcoma is characterized by the t(X;18)(p11.2;q11.2) translocation, which fuses the SS8 gene on chromosome 18 to either SSX1, SSX2, or SSX4 on chromosome X. Fluorescent in situ hybridization (FISH) with SS18 (SYT) gene break-apart probe can detect these translocations indirectly, and is very sensitive and specific for diagnosing synovial sarcoma. In this study, we tested expression of TLE-1 and CD99, the 2 most useful biomarkers by immunohistochemistry (IHC) for the diagnosis of synovial sarcoma, in a variety of carcinomas. We study their frequency and specificity, as compared with SS18 gene break-apart by FISH, a gold standard for the diagnosis of synovial sarcoma.
MATERIALS AND METHODS
Cases
Tissue microarrays (TMA) with 100 various carci- nomas from the pathology archives of SUNY Upstate
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| TABLE 1. Summary of TLE-1 and CD99 Expression by IHC and SS18 Gene Break-Apart in a Variety of Carcinomas | ||||||
|---|---|---|---|---|---|---|
| Subtype of Carcinoma | TLE-1 Positive | TLE-1, Negative | CD99 Positive | CD99 Negative | SS18 Positive by FISH | SS18 Negative by FISH |
| Squamous cell carcinoma | 0 | 13 | 4 | 8 | 0 | 4 |
| Basaloid SqCC | 0 | 1 | 1 | 1 | 0 | 1 |
| Basal cell carcinoma | 1 | 0 | 0 | 1 | 0 | 1 |
| Papillary thyroid carcinoma | 0 | 3 | 0 | 4 | 0 | 0 |
| Medullary thyroid carcinoma | 0 | 1 | 0 | 1 | 0 | 0 |
| Ductal carcinoma | 0 | 4 | 0 | 4 | 0 | 0 |
| Lobular carcinoma | 0 | 1 | 0 | 1 | 0 | 0 |
| Invasive mammary carcinoma | 0 | 1 | 0 | 1 | 0 | 0 |
| Tubular carcinoma | 0 | 1 | 0 | 1 | 0 | 0 |
| RCC | 0 | 5 | 1 | 4 | 0 | 1 |
| UCC | 0 | 4 | 1 | 2 | 0 | 1 |
| HCC | 0 | 5 | 2 | 2 | 0 | 2 |
| Neuroendocrine carcinoma | 0 | 1 | 1 | 1 | 0 | 1 |
| Seminoma | 0 | 1 | 0 | 1 | 0 | 0 |
| Mucoepidermoid carcinoma | 0 | 4 | 1 | 3 | 0 | 1 |
| Adenosquamous | 0 | 1 | 0 | 1 | 0 | 0 |
| Adrenocortical adenoma | 1 | 2 | 0 | 3 | 0 | 1 |
| IPMN | 0 | 1 | 0 | 1 | 0 | 0 |
| Adenoid cystic carcinoma | 0 | 1 | 0 | 1 | 0 | 0 |
| Small cell carcinoma | 1 | 1 | 0 | 1 | 0 | 1 |
| Serous carcinoma (ovary) | 1 | 0 | 0 | 1 | 0 | 1 |
| Malignant mixed germ cell tumor | 0 | 1 | 0 | 1 | 0 | 0 |
| Prostate adenocarcinoma | 0 | 19 | 6 | 10 | 0 | 7 |
| Esophageal adenocarcinoma | 0 | 3 | 3 | 1 | 0 | 4 |
| Gastric adenocarcinoma | 0 | 4 | 0 | 4 | 0 | 0 |
| Colon adenocarcinoma | 0 | 5 | 0 | 5 | 0 | 0 |
| Lung adenocarcinoma | 0 | 2 | 0 | 5 | 0 | 0 |
| Endometrial adenocarcinoma | 1 | 5 | 1 | 5 | 0 | 2 |
| Pancreatic adenocarcinoma | 0 | 1 | 0 | 5 | 0 | 0 |
| Total | 7 | 91 | 21 | 79 | 0 | 28 |
| HCC indicates hepatocellular carcinoma; IPMN, intraductal papillary mucinous neoplasm; RCC, renal cell carcinoma; SqCC, squamous cell carcinoma; UCC, urothelial cell carcinoma. | ||||||
Copyright @ 2016 Wolters Kluwer Health, Inc. All rights reserved.
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Medical University from 2000 to 2007 were used for the study, which include: 41 adenocarcinomas (ADCA), 14 squamous cell carcinomas (SqCC), 7 breast carcinomas, 5 thyroid carcinomas, 5 renal cell carcinomas (RCC), 5 hepatocellular carcinomas (HCC), 4 mucoepidermoid carcinomas (MEC), 4 urothelial cell carcinomas (UCC), 3 adrenocortical carcinomas (ACC), 2 basal cell carcinomas (BCC), 2 neuroendocrine carcinomas (NEC), 2 small cell carcinomas (SCC), 1 each for seminoma, adenosquamous carcinoma, intraductal papillary mucinous neoplasm, adenoid cystic carcinoma, ovarian serous carcinoma, and malignant mixed germ cell tumor.
IHC
IHC was performed on the TMA sections (3 to 5 um) with Ventana Benchmark Ultra automated im- munostainers (Ventana Medical System, Tucson, AZ) with appropriate positive and negative controls. TLE-1 antibody (Clone 1F5, prediluted) was purchased from Sigma-Aldrich (St Louis, MO). CD99 (Clone EPR3097Y, prediluted) was also purchased from Sigma-Aldrich. TLE-1 expression in ≥ 10% nuclei with ≥ moderate in- tensity was defined as positive, ≥ 10% nuclei with weak staining as equivocal, and <10% as negative. Mem- branous CD99 expression was evaluated with the same cut-off values.
FISH
FISH with SS18 (SYT) break-apart probes (Abbott Molecular Inc., Des Plaines, IL) were performed on TMA sections (3 to 5 pm). Deparaffinization was accomplished with xylene (3 times, 5 min each), ethanol (2 times, 1 min each), and then dried. The slides were then pretreated using Paraffin Pretreatment Kit IV; washing in NaSCN for 15 minutes subsequently by protease digestion for another 20 minutes. Probes were added, and then the slides were placed on a HYBrite at 37℃ for 5 minutes, followed by 37℃ overnight with coverslips. After wash- ing, the slides were counterstained with DAPI I and were examined using a fluorescence microscope with suitable
filters. FISH signals were counted on no less than 40 morphologically intact, nonoverlapping nuclei. When a 2 fusion signal (yellow) pattern was present it was in- terpreted as normal. One fusion (red and green over- lapping), 1 green, and 1 orange signals was interpreted as positive for break-apart rearrangement. According to our earlier validation, a ratio > 0.10 of break-apart signals to intact signals was considered positive (Table 1).
RESULTS
Seven of the 98 cases (7%) of carcinomas showed TLE-1 expression, including 1 each of prostate ADCA, esophageal ADCA, BCC, ACC, endometrial ADCA, ovarian serous carcinoma, and SCC (Figs. 1-3). Twenty- one of the 100 cases (21%) of carcinomas showed CD99 expression, including 6 prostate ADCA, 3 esophageal ADCA, 5 SqCC, 2 HCC, 1 each for endometrial ADCA, RCC, UCC, NEC, and MEC (Fig. 4). An esophageal ADCA was positive for both TLE-1 and CD99. None of the carcinomas with positive TLE-1(n = 7) or CD99 (n = 21) by IHC showed SS18 gene rearrangement by FISH (Fig. 5 and Table 1).
DISCUSSION
In this study TLE-1 and CD99 expression were identified in 7 out of 98 (7%) and 21 out of 98 (21%) cases of carcinomas, respectively. Excluding soft tissue neo- plasms, important differential diagnoses for synovial sarcoma also include sarcomatoid and poorly differ- entiated carcinomas as tumor location, histology, and IHC between the 2 entities may overlap and therefore create diagnostic dilemmas. For instance, sarcomatoid carcinomas can have a spindle cell appearance, whereas poorly differentiated carcinomas can have a round or ovoid cell morphology, which mimics typical or poorly differentiated synovial sarcomas. This study demonstrates that a fair number of carcinomas can express TLE-1 and CD99, and caution must be taken when using these im- munostains for diagnosing synovial sarcoma.
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Most studies related to TLE-1 staining in synovial sarcomas have compared it to other mesenchymal tu- mors. Multiple studies have shown that several other mesenchymal tumors can show TLE-1 positivity, includ- ing neurofibromas, schwannomas, malignant peripheral nerve sheath tumors, and solitary fibrous tumors. 4,6,8 Although staining can occur in other soft tissue tumors, TLE-1 is generally specific for synovial sarcoma, espe- cially when there is diffuse moderate to intense ex- pression.6,9 A study examining high-grade sarcomas demonstrated that TLE-1 was a specific biomarker for synovial sarcoma among these tumors.1º TLE-1 ex- pression has also been seen in malignant mesothelioma; however, the SYT-SSX translocation has not been iden- tified yet.7,11,12 Expression of TLE-1 was also seen in non- neoplastic tissue, including basal keratinocytes, perineural
cells, endothelial cells, mesothelial cells, and adipocytes.4 To the best of our knowledge, this study of TLE-1 ex- pression in carcinomas is novel in the field.
CD99 expression was seen in 21% of carcinomas in this study. CD99 is a product of the MIC2 gene, and is expressed in a wide variety of normal tissue and tumors.13-16 Therefore, it is not surprising to see nonspecific staining with just CD99. In combination with other specific markers, such as TLE-1, EMA, CK19, and BCL-2, it would be helpful for diagnostic purposes. BCL-2 expression was not included in our study as it had shown positivity in other tumor types, including BCC, adrenal cortical carcinoma, spindle cell epi- theliomas of the vagina, and SqCC.17-21
In conclusion, there are pitfalls in interpreting TLE- 1 and CD99 expression for diagnosing synovial sarcoma as expression is present in a fair number of carcinomas as
well. In daily practice, one should be aware that carci- nomas can have overlapping features with synovial sar- coma in terms of location, histology, and IHC. SS18 (SYT) gene rearrangement by FISH or other molecular methods, such as RT-PCR and cytogenetics, should be considered for diagnostically challenging cases, either for conformation or exclusion of synovial sarcoma. A defin- itive diagnosis is important as synovial sarcomas have been shown to be more sensitive to alkylating agents.22
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