| Abstract|| |
Background and Objectives: Synovial sarcomas (SS) are enigmatic soft tissue tumors, which are yet to have a defined cell of origin. SS have a variety of differential diagnosis depending upon the age of the patient and the site of presentation. This makes diagnosis cumbersome unless the specific fusion SS18:SSX is identified by reverse transcription-polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH). Immunohistochemistry is a useful tool in resource-poor settings in helping to narrow the differentials and help diagnose this tumor. This study set about assessing possible candidate immunohistochemical markers in their utility to recognize SS. Methods: Forty cases of SS, proven by FISH were included. A tissue microarray (TMA) was constructed, and immunohistochemistry was done using antibodies – TLE1 (OTI1F5), β-catenin (14), INI1 (MRQ-27), CK7 (OV-TL), CK19 (polyclonal), SS18 (polyclonal), calponin (CALP), and claudin1 (Polyclonal). The expression was analyzed to arrive at sensitivity and specificity. Results: TLE1 had a sensitivity of 92.5% and a specificity of 100%; β-Catenin had a sensitivity of 17.5% and specificity of 100%; Calponin had a sensitivity of 97.5% and a specificity of 81.25%; SS18 had a sensitivity of 95% and specificity of 100%; INI1 had a sensitivity of 95% and specificity of 100%; CK7 had a sensitivity of 90% and specificity of 87.5%; CK19 had a sensitivity of 90% and a specificity of 59.38%; and Claudin had a sensitivity of 85% and a specificity of 78.12%. Interpretation and Conclusions: The study showed both TLE1 and SS18 are robust diagnostic markers of synovial sarcoma with a sensitivity of 92% and 95%, respectively. INI1 can be used to discriminate SS from nonepithelioid and nonrhabdoid differentials. Calponin expression is helpful to differentiate poorly differentiated SS from its mimics. CK7 is a better marker than CK19 and can be used as a replacement for EMA in the initial screening panel. The use of claudin1 was restricted to delineating the epithelial component. β-Catenin had poor sensitivity, restricting its utility in SS.
Keywords: Beta-catenin, calponin, CK7, CK19, Claudin1, immunohistochemistry, INI1, SS18, synovial sarcoma, TLE1
|How to cite this article:|
Madakshira MG, Radotra BD, Kaman L, Saikia UN. Expression of TLE1, INI1, β-catenin, Claudin1, CK7, CK19, SS18 and calponin in synovial sarcoma. Indian J Pathol Microbiol 2021;64:707-16
|How to cite this URL:|
Madakshira MG, Radotra BD, Kaman L, Saikia UN. Expression of TLE1, INI1, β-catenin, Claudin1, CK7, CK19, SS18 and calponin in synovial sarcoma. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Dec 1];64:707-16. Available from: https://www.ijpmonline.org/text.asp?2021/64/4/707/328519
| Introduction|| |
Synovial Sarcoma (SS) is a malignant soft tissue neoplasm of uncertain histogenesis. It comprises 6% to 15%soft tissue sarcomas and is classified by the World Health Organisation (WHO) under a broad group of bone and soft tissue tumors. SS is unique, being a clinical, morphological, and genetically defined entity, with a wide age of occurrence and has been reported in almost any part of the body, including solid organs. SS is usually seen around the knee joint, which owes its misinterpreted “synovial” origins. The gross appearance of the tumor depends on the rate of growth and location. Histopathology subtypes include the classical biphasic, monophasic – epithelial and spindle cell types, and poorly differentiated types. The challenge for the histopathologist remains to establish the diagnosis of SS after differentiating it from the various morphological mimics, considering the varied clinicopathological heterogeneity. The importance of making a definitive diagnosis of SS for the patient is the better response shown by SS to chemotherapy, in comparison to other sarcomas. The current gold standard remains analysis of pathognomonic translocation SS18;SSX. This consistent reciprocal translocation, t (X; 18)(p11;q11) is found in all SS and involves the fusion of SS18 gene on chromosome 18 with either SSX1, SSX2, or rarely SSX4, all of which are on the X chromosome. This unique translocation can be detected by fluorescent in-situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). However, in resource-poor settings, the histopathologist may have to resort to immunohistochemistry for a definite diagnosis.
The currently available immunohistochemical markers for SS includeepithelial membrane antigen (EMA), cytokeratin (CK) cocktails, and mesenchymal marker vimentin to delineate the biphasic nature of the tumor. Literature is replete with a spectrum of additional immunohistochemical markers such as cluster differentiation 99 (CD99), B-cell lymphoma 2 (BCL2), CD56, and e-cadherin which aid in arriving at a diagnosis of SS.,,, However, these markers are also expressed by mimics of SS, leading to ambiguity in diagnosis. This dilemma has spurred the search for more specific markers which aid in the definitive diagnosis of SS without resorting to the identification of the translocation by FISH or RT-PCR. In recent times, antibodies against immunohistochemical markers such as calponin, β-catenin, claudin1, INI1 (SMARCB1), transducin-like enhancer protein 1 (TLE1), and SS18 have shown promising results in being superior differentiators of SS, for diagnosis.,,,,, These studies have singularly demonstrated the utility of the above-mentioned immunohistochemical markers in establishing the diagnosis of SS. Though, none have collectively evaluated them. The present study is an attempt to evaluate the expression of immunohistochemical markers calponin, β-catenin, claudin1, INI1, TLE1, SS18, CK7, and CK19 in diagnosed cases of SS to arrive at the most preferred set of the immunohistochemical panel to aid in the diagnosis of SS.
| Methods|| |
A retrospective and prospective study (2 years) was carried out. Formalin-fixed Pparaffin-embedded blocks were retrieved and utilized for the study material. A total of 75 cases of SS were screened for the study. The cases were reviewed by two experienced histopathologists to verify and confirm the diagnosis of SS. In addition, IHC panel including at least focal positivity for EMA (E29, Monoclonal, Dako Agilent, 1:300), and/or PanCK (AE1/AE3, Monoclonal, Dako Agilent, 1:300), Diffuse positivity for BCL2 (124, Monoclonal, Dako Agilent, 1:50) and CD99 (12E7, Monoclonal, Dako Agilent, 1:50) with the absence of CD34 (QBEnd10, Monoclonal, Dako Agilent, 1:50), were taken into account to arrive at a diagnosis. Areas of glandular differentiation and/or well-defined epithelial cell clusters were required to classify the type as BSS. A diagnosis of SPMSS was made with tumors having spindle cells with occasional cells showing epithelioid morphology. A tumor was classified as PDSS if it showed high-grade sarcomatous features with necrosis and brisk mitosis or a round cell morphology akin to Ewing sarcoma or cells which appeared to be intermediate between epithelioid and spindle cells including those with rhabdoid features.
All cases confirmed as SS following FISH using Vysis LSI SS18 dual color break-apart probe (Abbott Laboratories, USA). The probe was labeled at one end by green spectrum (3' end of SS18 gene) and the other end by orange spectrum (5' end of SS18). A probe was viewed as a split signal if separated green and orange signals were at a distance more than the size of two hybridization signals [Figure 1].
|Figure 1: Shows the typical histology of biphasic synovial sarcoma, with a low power image of a FISH preparation and, the expected negative (two fused red and green signals) and positive (one fused red/green signal and another break-a-part red and green signal)|
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Of the 75 cases screened, only 40 cases satisfied the inclusion criteria and conclusive results on break-a-part FISH. At least 50 nonoverlapping nuclei with distinct unequivocal signals were enumerated for each case. A case was designated as positive if it showed at least 15% of cells displayed a break apart signal. A nucleus was considered as harboring a split when there was one fused signal (yellow) and separately lying green and red signals placed at a distance of more than two hybridization signals.
The hematoxylin and eosin–stained slides of 40 selected cases were reviewed to mark the site for making a tissue microarray (TMA). A TMA was constructed with four cores in each case to account for tumor heterogeneity seen frequently in sarcomas. In addition, the array included representative cores of tumors with differential diagnosis of SS: fibrosarcoma, leiomyosarcoma, solitary fibrous tumor, carcinosarcoma, spindle cell melanoma, spindle cell carcinoma, neuroblastoma, Ewing tumor, dermatofibrosarcoma protruberans, biphasic mesothelioma, pleomorphic sarcoma, malignant peripheral nerve sheath tumor, spindle cell rhabdomyosarcoma, and gastrointestinal stromal tumor. Before TMA construction, a sector map was designed. The sector map specified a location within the TMA for each core sample, and it was used as a guide in both assembly and subsequent analysis. For the physical construction of the TMA, an automated UNITMA UATM 272B machine (South Korea) was used. Based on the planned sector map fed into the computer as an excel sheet, the machine removed representative 2-mm cores highlighted by white ink from the donor block and inserted them into the assigned hole in the recipient block (Quick Ray 6 × 10 matrix block of 2 mm cores, UNITMA, Korea). A 4-micron thick section of TMA-embedded paraffin block was used to carry out routine H and E staining and immunohistochemistry.
Immunohistochemistry was carried out on a Ventana Benchmark ST autostainer followed heat retrieval using antibodies against TLE1 (OTI1F5, Monoclonal, Abcam, 1:100), β-Catenin (14, Monoclonal, Cell Marque, 1:100), INI1 (MRQ-27, Monoclonal, Cell Marque, 1:100), CK7 (OV-TL, Monoclonal, Dako Agilent, 1:100), CK19 (Polyclonal, Abcam, 1:100), SS18 (Anti-SS18, Polyclonal, Abcam, 1:50), calponin (CALP, Monoclonal, Dako Agilent, 1:50), and claudin1 (Polyclonal, Abcam, 1:500). The chromogen used in all the reactions was diaminobenzidine. Both negative and positive controls were incorporated in each staining session.
The scoring of each of the antibodies are as follows:- TLE1: Positivity was taken as a nuclear expression in more than 1% tumor cells; Increasing grades of intensity were scored as 1, 2, and 3 similar to a study by Terry et al. Complete absence of nuclear expression (0) was taken as negative β-catenin: Adapting the IHC interpretation pattern used by Hasegawa et al. positivity was taken as membranous (1), nucleocytoplasmic (2), and nuclear (3) in more than 1% tumor cells. Complete absence of staining (0) was taken as negative INI1. As per the findings of Kohashi et al., positivity was taken as patchy loss (1) of nuclear expression and complete loss (2) of nuclear expression. Preserved intense nuclear expression in all tumor cells (0) was taken as negative. CK7 positivity was taken as a cytoplasmic expression of any intensity in more than 1% tumor cells (1). Lack of cytoplasmic positivity in any tumor cell was taken as negative (0). CK19 positivity was taken as a cytoplasmic expression of any intensity in more than 1% of tumor cells (1). SS18: Taking a cue from the study by He et al., positivity was taken as a nuclear expression in more than 1% of tumor cells; Increasing grades of intensity scored as 1, 2, and 3. Complete absence of nuclear expression (0) was taken as negative. Calponin: Positivity was taken as a cytoplasmic expression of any intensity in more than 1% of tumor cells (1). Lack of cytoplasmic positivity in any tumor cell was taken as negative (0). Claudin1: Positivity was taken as membranous expression of any intensity in more than 1% tumor cells (1). Lack of cytoplasmic positivity in any tumor cell was taken as negative (0).
Statistical analysis of the data was done using Microsoft Excel and Medcalc software for descriptive analysis and to calculate the sensitivity, specificity, positive predictive value, and negative predictive value of the antibodies used in terms of diagnostic utility. An informed written consent was taken at the time of diagnosis from all prospective cases included in the study. No additional therapeutic intervention was carried out. The study has been approved by the institutional ethics committee. Ethical approval letter No. INT/IEC/2018/000922 dt 25 Jun 2018.
| Results|| |
Of the total of 75 cases of SS screened, 40 cases were selected for the study which satisfied the inclusion criteria. Amongst the 40 cases, there was a male preponderance with a male to female ratio of 1.6: 1. The cases included in this study spanned a wide age range with the youngest being 11 year old and the eldest being 73 year old. However, most of the cases fell between the age groups of 11 to 40 years comprising 72.5% of cases. The mean age in the study was 33 years. The most common anatomical site of involvement was around the knee joint constituting 47.5% (19 cases) of all cases. The extremities combined together formed three-quarters of the cases with a total of 30 out of 40 cases (75%). Three (7.5%) SS cases were arising in the head and neck region, with the origin being in the maxillary antrum, scalp, and intradural extramedullary site at the C6-C7 level, respectively. Two (5%) cases were had their origin from the anterior abdominal wall (trunk). Cases involving the internal organs (4 cases, 10%) were also part of the study, with 2 cases each arising from the lung and kidney, respectively. A single case (2.5%) with retroperitoneal origin was also part of the study. Of the 40 cases, four (10%) cases were recurrent SS. In all, the SPMSS was the dominant subtype with a total of 25 (62.5%) cases followed by BSS with 13 (32.5%) cases and 2 (5%) cases of PDSS. No case of monophasic epithelial SS was seen in the present series.
TLE1 immunohistochemistry [Figure 2]a, [Figure 2]b, [Figure 2]c stained 37 cases (92.5%) of SS, of which 70% (26 cases) had intense (2+) nuclear positivity and 27.5% (11 cases had less intense (1+) nuclear positivity. None of the other cases were considered as differential diagnosis stained for TLE1. Thus, TLE1 had a specificity and positive predictive value of 100% for SS. TLE1 showed a sensitivity of 92.5% with an overall accuracy of 95.83%, according to our observations [Table 1].
|Figure 2: (a-c) Case of monophasic SS: (a) H and E stain, 40×; (b) TLE1-IHC, 40×; (c) TLE1-IHC, 200× showing nuclear staining. (d-f) Case of biphasic SS; (d) H and E stain, 40×; (e) β-Catenin-IHC, 40×; (f) β-Catenin-IHC, 200× showing wild type membranous expression. (g-i) Case of monophasic SS: (g) H and E stain, 40×; (h) Calponin-IHC, 40×; (i) Calponin-IHC, 200× showing cytoplasmic expression. (j-l) Case of monophasic SS: (j) H and E stain, 40×; (k) SS18-IHC, 40×; (l) SS18-IHC, 200 × showing nuclear expression|
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Nuclear or nucleo-cytoplasmic staining for β-catenin was taken as positive [Figure 2]d, [Figure 2]e, [Figure 2]f. Only 7 (17.5%) SS cases showed a nucleo-cytoplasmic staining pattern, though none of the other differential diagnoses showed nuclear or nucleo-cytoplasmic expression for β-catenin. This showed a low sensitivity of 17.50%, but a specificity of 100% to β-catenin IHC [Table 1].
Cytoplasmic staining for calponin was seen in 97.5% (39 cases) of SS cases [Figure 2]g, [Figure 2]h, [Figure 2]i. Similar staining patterns are also seen in cases of leiomyosarcomas, gastrointestinal stromal tumor, mesothelioma, carcinosarcoma, malignant peripheral nerve sheath tumor, and pleomorphic sarcoma. Hence, we observed the overall sensitivity of calponin for SS was 97.5% with a negative predictive value of 95.93% [Table 1].
SS18 immunohistochemistry stained 38 cases (92.5%) of SS, with 20% (8 cases) showing an intense (2+) nuclear positivity [Figure 2]j, [Figure 2]k, [Figure 2]l. None of the differentials stained for SS18. This showed that SS18 had a specificity and positive predictive value of 100% for SS. SS18 showed a sensitivity of 92.5% with an overall accuracy of 95.83% [Table 1].
Thirty-eight SS cases showed partial (0.75%) or complete (87.5%) loss of nuclear staining for INI1 immunohistochemistry [Figure 3]a, [Figure 3]b, [Figure 3]c. All the differentials showed retained nuclear staining for INI1. Hence, the loss of nuclear expression of INI1 in SS had 95% sensitivity with an accuracy of 97.22% in the index study [Table 1].
|Figure 3: (a-c) Case of monophasic SS: (a) H and E stain, 40×; (b) INI1-IHC, 40×; (c) INI-1-IHC, 200× showing loss of nuclear staining with endothelial cells showing retained expression. (d-f) Case of biphasic SS; (d) H and E stain, 40×; (e) CK7-IHC, 40×; (f) Ck7-IHC, 200 × showing cytoplasmic expression. (g-i) Case of biphasic SS: (g) H and E stain, 40×; (h) CK19-IHC, 40×; (i) CK19-IHC, 200× showing cytoplasmic expression. (j-l) Case of biphasic SS: (j) H and E stain, 40×; (k) Claudin1-IHC, 40×; (l) Claudin1-IHC, 200× showing membranous expression|
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Cytoplasmic staining for CK7 was seen in 36 (90%) cases of SS [Figure 3]d, [Figure 3]e, [Figure 3]f. Amongst the differentials, mesothelioma and carcinosarcoma showed cytoplasmic staining for CK7. The overall sensitivity of CK7 for SS was 90% with a positive predictive value of 90.00% as per our observations [Table 1].
Cytoplasmic staining for CK19 was identified in 36 (90%) cases of SS [Figure 3]g, [Figure 3]h, [Figure 3]i. However, many of 13 differentials also expressed cytoplasmic CK19, including carcinosarcoma, mesothelioma, pleomorphic sarcoma, and gastrointestinal stromal tumor. This observation showed a specificity of CK19 for SS to be 59.38% with a negative predictive value of 76.39% [Table 1].
Membranous staining of claudin1 was seen in 34 (85%) cases of SS [Figure 3]j, [Figure 3]k, [Figure 3]l. Amongst the differentials, membranous claudin1 staining was identified in carcinosarcoma, Ewing sarcoma, and mesotheliomas. We found the sensitivity of claudin1 for SS was 85% with a positive predictive value of 82.93% [Table 1].
| Discussion|| |
SS is relatively rare soft tissue malignant tumor comprising 5% to 10% of all soft tissue sarcomas. We studied 40 cases of SS, with male gender preponderance having a male to female ratio of 1.6:1. Similar male preponderance has been seen in large epidemiological series by Sultan et al. (1268 cases) and Ferrari et al. (271 cases) with a male to female ratio of 1.13:1, respectively. SSs in their biphasic form shows evidence of partial epithelial differentiation. Due to its ambiguous histogenesis and cell of origin, SS has been classified under tumors of uncertain differentiation in 2013 WHO Classification of Tumors of Soft Tissue and Bone. The advent of cytogenetics led to the discovery of many characteristic mutations, in various tumors including soft tissue tumors. SS is now characterized by the presence of a reciprocal balanced translocation involving SS18 (SS18) gene on Chromosome 18 and SSX gene on the X chromosome leading to t (X; 18)(p11.2;q11.2). There are nine SSX genes described numbered as SSX1 to SSX9. However, only three possible fusion partners have been reported namely – SSX1, SSX2, and SSX4. SS18-SSX1 is the most common fusion product seen in 66% of SS. One-third of SS show SS18-SSX2 fusion product with SS18-SSX4 being reported in anecdotal cases. No other sarcoma has been shown to have the SS18-SSX translocation making it specific for SS. As the break-apart FISH was used in this study, it confirmed the translocation of the SS18 gene but did not identify the fusion product. The fusion gene can be analyzed by using specifically designed Fusion FISH probes or by RT-PCR.
Amongst 40 SS cases analyzed, there was wide age of presentation ranging from as young as 11 years to as old as 74 years. The mean age at presentation was 33 years, and the most commonly occurring age range was 11 to 40 years. Sultan et al. reported a median age at presentation of 34 years with 58.5% of cases being reported in the age range of 10 to 39 years. Similarly, Ferrari et al. reported a median age at presentation of 32 years with a wide age range of 5 to 87 years. This study suggests that though SS is a tumor of young adults, it can present with a wide age range.,,
The most common anatomical site of involvement was around the knee joint, forming 47.5% (19/40 cases) of all cases in our study, and the extremities together formed 75% of cases. Four cases had the involvement of internal organs (lung and kidney), which were also part of the study. Similarly, in series by Sultan et al., extremities formed the most common anatomical site of involvement with 70% of cases, whereas Ferrari et al. reported 85.6% cases involving the extremities. Head and neck involvement by SS was seen in 7% of cases in series by Sultan et al. Isolated cases and short case series involving uncommon sites and internal organs such as lungs and kidneys have also been rarely documented in the literature.,, This suggests that almost any site may be the primary site for SS. Nevertheless, extremities remain the most common site of involvement for SS, though intra-articular tumors are only anecdotal.,,
The most common subtype of SS in the present series was SPMSS (62.5%, 25/40 cases), followed by BSS (32.5%, 13/40 cases) and PDSS (5%, 2/40 cases). On the contrary, BSS was the most common subtype in series by Ferrari et al. comprising 42.8% cases, and subtyping was not reported by Sultan et al. in 51% of cases. However, in general, SPMSS has been reported to be more frequent. In general, similar to the present study, EPMSS is more hypothetical with most of the reported cases being an epithelial predominant biphasic SS having foci of spindle cells.,,
TLE1 gene was discovered to be consistently overexpressed in genome-wide analysis of SS by multiple studies., TLE1 protein is an integral part of WNT pathway and functions as transcription repressors. TLE1 has been shown to bind to the histones leading to the regulation of gene expression which is earmarked for transcriptional activators. Over the past few years, TLE1 has shown to be a robust discriminator of SS from its mimics.,,,,, The sensitivity ranges from 75% to 100% with specificity in the range of 92% to 96% [Table 2]. The antibody staining has the added advantage of being nuclear localization with minimal background staining. When compared with its morphological mimics, Kosemehmetoglu et al. showed that TLE1 was also expressed by peripheral nerve sheath tumors such as neurofibroma, schwannoma, and malignant nerve sheath tumor. Of these, malignant nerve sheath tumor is the closest differential in which frequency of TLE1 expression is about 30%, and SS can be differentiated using a panel of antibodies including keratins and CD99. The present study was in sync with the previous studies with an overall sensitivity of 92.5% as nuclear staining was seen across all three subtypes of SS.
|Table 2: Comparison with previous studies on TLE1 staining in SS, Number of SS cases shown in ( )|
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β-catenin protein is a part of a multifunctional complex that is present downstream the WNT pathway. Any mutation or abnormality in the sequence leads to stabilization of β-catenin protein leading to its accumulation in the cytoplasm and later its translocation into the nucleus. Once within the nucleus, β-catenin binds to an enhancer and stimulates the expression of genes involved in cell proliferation. Nuclear or cytoplasmic localization of β-catenin has been shown to correlate with poor prognosis in SS. β-catenin has shown a wide range of sensitivity (13% to 59%) in its nuclear and/or nucleocytoplasmic staining pattern amongst SS [Table 3]. This variation in expression may reflect selection bias, as β-catenin is shown to be translocated to the nucleus or accumulated in the cytoplasm in aggressive SS only. However, in our study, the morphological mimics neither showed cytoplasmic or nuclear staining pattern, indicating β-catenin to be a more specific than a sensitive marker for SS.
|Table 3: Comparison with previous studies on β-catenin staining in SS, Number of SS cases shown in ( )|
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Calponin protein binds to tropomyosin, calmodulin, and actin. The calponin gene is said to be one of the earliest markers of smooth muscle differentiation. However, the expression of calponin is not restricted to smooth muscle cells only. Other cells including myoepithelial cells and myofibroblasts also show cytoplasmic positivity, thereby doubting its specificity. Ono et al. demonstrated expression of calponin within both cultured cell lines and tumor samples of SS. Fisher et al.  extended their study to compare the expression of calponin in SS and its morphological mimics achieving a sensitivity of 98% [Table 4]. Fisher et al., suggested that though calponin is expressed by spindle cell mimics of SS such as leiomyosarcoma, dermatofibrosarcoma, and few fibrosarcomas. Thus, the utility of calponin remains in excluding round blue cell tumors such as Ewing sarcoma and neuroblastoma from the PDSS. The present study also demonstrated calponin to have a sensitivity of 97.5% across all three subtypes with negative staining in Ewing sarcoma and neuroblastoma.
|Table 4: Comparison with previous studies on Calponin staining in SS, Number of SS cases shown in ( )|
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The SS18 protein also known as SS18 protein is an integral part of the BAF (mSWI/SNF) complex, which is responsible for orchestrating the unwinding of chromatin and expose the DNA structure to either activate or inactivate transcription. The fusion protein of SS18: SSX in SS reversibly disrupts the BAF complex causing lysis of BAF47 protein and promotes proliferation by activation of SOX2. It was shown in an experiment by Hashimoto et al. that in-house antibodies produced against SS18:SSX fusion-derived protein stains the nuclei of SS. It was followed by a study by He et al. who showed a consistent and intense nuclear staining pattern for polyclonal anti-SS18 antibody (Santa Cruz) in SS with a sensitivity of 87%. The latest entrant is a monoclonal antibody against the fusion product SS18-SSX, Clone E9X9V, which has shown a sensitivity of 95%–100% and a specificity of 95%–100% [Table 5]., We used the polyclonal anti-SS18 antibody (Abcam) with a sensitivity of 95% in SS, indicating it to be another useful discriminator in confirming a diagnosis of SS.
|Table 5: Comparison with the previous study on SS18 staining in SS, Number of SS cases shown in ( )|
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The INI1 also known as SMARCB1, hSNF5, and BAF47 is part of BAF (SWI/SNF) complex and is ubiquitously expressed by all nucleated cells. INI1 is an important tumor suppressor gene and has been shown to be oncogenic in epithelioid sarcomas and malignant rhabdoid tumors, which show loss of nuclear expression of INI1 protein. In SS, the SS18:SSX fusion protein competes with INI1 (BAF47) in the BAF complex leading to the dissolution of dislodged INI1 protein. This should correspond to a decrease in INI1 protein within the nuclei which is reflected by the lack of nuclear expression of INI1 on immunohistochemistry. Over the past few years, partial (mosaic) to complete loss of nuclear expression of INI1 has been shown by multiple studies in SS.,,,, The sensitivity of INI1 loss in SS ranges from 69% to 92% with specificity reaching up to 100% [Table 6]. Only Mularz et al. showed a 21.2% sensitivity, who considered only complete loss of nuclear expression of INI1 staining for assessment. The present study showed a consistent reduced or mosaic-like INI1 nuclear expression amongst SS with a sensitivity of 95% and retained expression in the morphological mimics giving a specificity of 100%.
|Table 6: Comparison with the previous study on INI1 staining in SS, Number of SS cases shown in ( )|
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The BSS is characterized by epithelial differentiation, which is demonstrated by the staining pattern of pan-cytokeratin cocktails and EMA. The use of cytokeratin subsets as a tool for discrimination of SS from its morphological mimics has been studied by Miettinen et al., who showed more frequent focal expression of simple keratins CK7 and CK19 amongst SS. Smith et al. in their study demonstrated the utility of CK7 and CK19 staining in SPMSS to differentiate it from malignant peripheral nerve sheath tumors. In a study by Machen et al., the sensitivity of CK7 and CK19 amongst PDSS was 52% and 43%, respectively, the absence of CK7 and occasional positivity of CK19 amongst the Ewing/PNET tumors makes CK7 a better discriminator of poorly differentiated SS from its morphological mimics [Table 7] and [Table 8]. We observed both CK7 and CK19 to have a similar sensitivity of 90% with CK7 having a superior specificity of 87.5% in our cases. This indicates that CK7 can be used as keratin of choice in the initial panel of IH.C
|Table 7: Comparison with the previous study on CK7 staining in SS, Number of SS cases shown in ( )|
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|Table 8: Comparison with the previous study on CK19 staining in SS, Number of SS cases shown in ( )|
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The epithelial element of SS such as glands and tubules have been known to possess tight intercellular junctions. The protein components of tight junctions include occludins, ZO1, and claudin1. There is only one study on claudin1 expression amongst SS and its co-relation with ultrastructure. Billings et al. showed that claudin1 showed consistent expression along the membrane in the epithelial component of BSS with only focal aberrant expression in a few SPMSS. Similarly, the current study highlighted the epithelial elements of BSS with only occasional SPMSS showing focal interrupted positivity along the cell membrane. Thus, the overall sensitivity of claudin-1 for SS was 85% [Table 9]. However, the utility of claudin1 shall remain in demonstrating the epithelial elements in cases of spindle cell predominant SS.
|Table 9: Comparison with the previous study on Claudin1 staining in SS, Number of SS cases shown in ( )|
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To conclude, SS remains an enigmatic tumor with a wide age range and subtypes. It has a multitude of morphological patterns which can occur at any age and at any anatomical site. This unpredictability makes the diagnosis challenging for the histopathologist. Judicious use of an initial panel of immunohistochemistry including CK7, CD34, TLE1, or SS18 is recommended for arriving at a diagnosis. INI1 is useful to distinguish SS from a tumor having a nonepithelioid and nonrhabdoid morphology. In a similar light, calponin can be useful to exclude a possibility of poorly differentiated SS from its mimics such as Ewing/PNET tumor. The cost of immunohistochemistry for a panel of sixantibodies would be cheaper, being less than half the price of FISH analysis. However, in cases of equivocal IHC results, confirmation by FISH or RT-PCR needs to be resorted to, which is the gold standard diagnostic tool for a definite diagnosis of SS of all subtypes.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Goldblum JR, Weiss SW, Folpe AL. Enzinger and Weiss's Soft Tissue Tumors E-Book. Elsevier Health Sciences; 2013. 1052-70 p.
Suurmerijer AJH, Ladanyi M, Nielson TO. Synovial Sarcoma. In: Lokuhetty D, White VA, Cree IA. WHO Classification of Tumors - Soft tissue and Bone tumors. 5th Edition. Lyon: International Agency for Research on Cancer;2020.p290-3.
Haagensen CD, Stout AP. Synovial sarcoma. Ann Surg 1944;120:826-42.
Cadman NL, Soule EH, Kelly PJ. Synovial sarcoma. An analysis of 134 tumors. Cancer 1965;18:613-27.
Ferrari A, Gronchi A, Casanova M, Meazza C, Gandola L, Collini P, et al
. Synovial sarcoma: A retrospective analysis of 271 patients of all ages treated at a single institution. Cancer 2004;101:627-34.
Clark J, Rocques PJ, Crew AJ, Gill S, Shipley J, Chan AM-L, et al
. Identification of novel genes, SS18 and SSX, involved in the t (X; 18)(p11. 2; q11. 2) translocation found in human synovial sarcoma. Nat Genet 1994;7:502-8.
Amary MF, Berisha F, Bernardi FD, Herbert A, James M, Reis-Filho JS, et al
. Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: Analysis of conventional RT-PCR, qRT-PCR and dual color FISH as diagnostic tools for synovial sarcoma. Mod Pathol 2007;20:482-96.
Dei Tos A, Wadden C, Calonje E, Sciot R, Pauwels P, Knight J, et al
. Immunohistochemical demonstration of glycoprotein p30/32mic2 (CD99) in synovial sarcoma. A potential cause of diagnostic confusion. Appl Immunohistochem Mol Morphol 1995;3:168-73.
Hirakawa N, Naka T, Yamamoto I, Fukuda T, Tsuneyoshi M. Overexpression of bcl-2 protein in synovial sarcoma: A comparative study of other soft tissue spindle cell sarcomas and an additional analysis by fluorescence in situ
hybridization. Hum Pathol 1996;27:1060-5.
Olsen SH, Thomas DG, Lucas DR. Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol 2006;19:659-68.
Saito T, Oda Y, Sakamoto A, Tamiya S, Kinukawa N, Hayashi K, et al
. Prognostic value of the preserved expression of the E-cadherin and catenin families of adhesion molecules and of β-catenin mutations in synovial sarcoma. J Pathol 2000;192:342-50.
Fisher C, Montgomery E, Healy V. Calponin and h-caldesmon expression in synovial sarcoma; the use of calponin in diagnosis. Histopathology 2003;42:588-93.
Horvai AE, Kramer MJ, O'Donnell R. β-Catenin nuclear expression correlates with cyclin D1 expression in primary and metastatic synovial sarcoma: A tissue microarray study. Arch Pathol Lab Med 2006;130:792-8.
Billings SD, Walsh SV, Fisher C, Nusrat A, Weiss SW, Folpe AL. Aberrant expression of tight junction-related proteins ZO-1, claudin-1 and occludin in synovial sarcoma: An immunohistochemical study with ultrastructural correlation. Mod Pathol 2004;17:141-9.
Rekhi B, Jambhekar NA. Immunohistochemical validation of INI1/SMARCB1 in a spectrum of musculoskeletal tumors: An experience at a Tertiary Cancer Referral Centre. Pathol Res Pract 2013;209:758-66.
Seo SW, Lee H, Lee HI, Kim HS. The role of TLE1 in synovial sarcoma. J Orthop Res 2011;29:1131-6.
He R, Patel RM, Alkan S, Hammadeh R, Weiss SW, Goldblum JR, et al
. Immunostaining for SS18 protein discriminates synovial sarcoma from other soft tissue tumors: Analysis of 146 cases. Mod Pathol 2007;20:522-8.
Terry J, Saito T, Subramanian S, Ruttan C, Antonescu CR, Goldblum JR, et al
. TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies. Am J Surg Pathol 2007;31:240-6.
Hasegawa T, Yokoyama R, Matsuno Y, Shimoda T, Hirohashi S. Prognostic significance of histologic grade and nuclear expression of β-catenin in synovial sarcoma. Hum Pathol 2001;32:257-63.
Kohashi K, Oda Y, Yamamoto H, Tamiya S, Matono H, Iwamoto Y, et al
. Reduced expression of SMARCB1/INI1 protein in synovial sarcoma. Mod Pathol 2010;23:981-90.
Sultan I, Rodriguez-Galindo C, Saab R, Yasir S, Casanova M, Ferrari A. Comparing children and adults with synovial sarcoma in the surveillance, epidemiology, and end results program, 1983 to 2005: An analysis of 1268 patients. Cancer 2009;115:3537-47.
Thway K, Fisher C. Synovial sarcoma: Defining features and diagnostic evolution. Ann Diagn Pathol 2014;18:369-80.
Becher R, Wake N, Gibas Z, Ochi H, Sandberg AA. Chromosome changes in soft tissue sarcomas. J Natl Cancer Inst 1984;72:823-31.
Przybyl J, Sciot R, Rutkowski P, Siedlecki JA, Vanspauwen V, Samson I, et al
. Recurrent and novel SS18-SSX fusion transcripts in synovial sarcoma: Description of three new cases. Tumour Biol 2012;33:2245-53.
Skytting B, Nilsson G, Brodin B, Xie Y, Lundeberg J, Uhlén M, et al
. A novel fusion gene, SS18-SSX4, in synovial sarcoma. J Natl Cancer Inst 1999;91:974-5.
Shipley J, Crew J, Birdsall S, Gill S, Clark J, Fisher C, et al
. Interphase fluorescence in situ
hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma. Am J Pathol 1996;148:559-67.
Fisher C. Synovial sarcoma. Ann Diagn Pathol 1998;2:401-21.
Essary LR, Vargas SO, Fletcher CD. Primary pleuropulmonary synovial sarcoma: Reappraisal of a recently described anatomic subset. Cancer 2002;94:459-69.
Divetia M, Karpate A, Basak R, Desai SB. Synovial sarcoma of the kidney. Ann Diagn Pathol 2008;12:333-9.
Puffer RC, Daniels DJ, Giannini C, Pichelmann MA, Rose PS, Clarke MJ. Synovial sarcoma of the spine: A report of three cases and review of the literature. Surg Neurol Int 2011;2:18.
] [Full text]
Ishida T, Iijima T, Moriyama S, Nakamura C, Kitagawa T, Machinami R. Intra-articular calcifying synovial sarcoma mimicking synovial chondromatosis. Skeletal Radiol 1996;25:766-9.
Baird K, Davis S, Antonescu CR, Harper UL, Walker RL, Chen Y, et al
. Gene expression profiling of human sarcomas: Insights into sarcoma biology. Cancer Res 2005;65:9226-35.
Nagayama S, Katagiri T, Tsunoda T, Hosaka T, Nakashima Y, Araki N, et al
. Genome-wide analysis of gene expression in synovial sarcomas using a cDNA microarray. Cancer Res 2002;62:5859-66.
Da Yuan XY, Yuan Z, Zhao Y, Guo J. TLE1 function and therapeutic potential in cancer. Oncotarget 2017;8:15971-6.
Foo WC, Cruise MW, Wick MR, Hornick JL. Immunohistochemical staining for TLE1 distinguishes synovial sarcoma from histologic mimics. Am J Clin Pathol 2011;135:839-44.
Jagdis A, Rubin BP, Tubbs RR, Pacheco M, Nielsen TO. Prospective evaluation of TLE1 as a diagnostic immunohistochemical marker in synovial sarcoma. Am J Surg Pathol 2009;33:1743-51.
Knösel T, Heretsch S, Altendorf-Hofmann A, Richter P, Katenkamp K, Katenkamp D, et al
. TLE1 is a robust diagnostic biomarker for synovial sarcomas and correlates with t (X; 18): Analysis of 319 cases. Eur J Cancer 2010;46:1170-6.
Kosemehmetoglu K, Vrana JA, Folpe AL. TLE1 expression is not specific for synovial sarcoma: A whole section study of 163 soft tissue and bone neoplasms. Mod Pathol 2009;22:872-8.
Rekhi B, Basak R, Desai SB, Jambhekar NA. Immunohistochemical validation of TLE1, a novel marker, for synovial sarcomas. Indian J Med Res 2012;136:766-75.
] [Full text]
Ono H, Yoshikawa H, Ueda T, Yamamura H, Kudawara I, Manou M, et al
. Expression of smooth muscle calponin in synovial sarcoma. Sarcoma 1999;3:107-13.
Miettinen MM, Sarlomo-Rikala M, Kovatich AJ, Lasota J. Calponin and h-caldesmon in soft tissue tumors: Consistent h-caldesmon immunoreactivity in gastrointestinal stromal tumors indicates traits of smooth muscle differentiation. Mod Pathol 1999;12:756-62.
Kosho T, Miyake N, Carey JC. Coffin–Siris syndrome and related disorders involving components of the BAF (mSWI/SNF) complex: Historical review and recent advances using next generation sequencing. Am J Med Genet C Semin Med Genet 2014;166C: 241-51.
Kadoch C, Crabtree GR. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. Cell 2013;153:71-85.
Hashimoto N, Araki N, Yoshikawa H, Myoui A, Matsumine A, Kaneko M, et al
. SS18-SSX fusion proteins in synovial sarcomas: Detection and characterization with new antibodies. Cancer Lett 2000;149:31-6.
Baranov E, McBride MJ, Bellizzi AM, Ligon AH, Fletcher CD, Kadoch C, et al
. A novel SS18-SSX fusion-specific antibody for the diagnosis of synovial sarcoma. Am J Surg Pathol 2020;44:922-33.
Zaborowski M, Vargas AC, Pulvers J, Clarkson A, de Guzman D, Sioson L, et al
. When used together SS18-SSX fusion-specific and SSX C-terminus immunohistochemistry are highly specific and sensitive for the diagnosis of synovial sarcoma and can replace FISH or molecular testing in most cases. Histopathology 2020;77:588-600.
Arnold MA, Arnold CA, Li G, Chae U, El-Etriby R, Lee C-CR, et al
. A unique pattern of INI1 immunohistochemistry distinguishes synovial sarcoma from its histologic mimics. Hum Pathol 2013;44:881-7.
Hollmann TJ, Hornick JL. INI1-deficient tumors: Diagnostic features and molecular genetics. Am J Surg Pathol 2011;35:e47-63.
Ito J, Asano N, Kawai A, Yoshida A. The diagnostic utility of reduced immunohistochemical expression of SMARCB1 in synovial sarcomas: A validation study. Hum Pathol 2016;47:32-7.
Mularz K, Harazin-Lechowska A, Ambicka A, Kruczak A, Rozmus-Piętoń M, Marchińska-Osika U, et al
. Specificity and sensitivity of INI-1 labeling in epithelioid sarcoma. Loss of INI1 expression as a frequent immunohistochemical event in synovial sarcoma Loss of INI1 expression as a frequent immunohistochemical event in synovial sarcoma. Pol J Pathol 2012;63:179-83.
Rekhi B, Vogel U. Utility of characteristic 'W eak to A bsent'INI 1/SMARCB 1/BAF 47 expression in diagnosis of synovial sarcomas. APMIS 2015;123:618-28.
Miettinen M, Limon J, Niezabitowski A, Lasota J. Patterns of keratin polypeptides in 110 biphasic, monophasic, and poorly differentiated synovial sarcomas. Virchows Arch 2000;437:275-83.
Smith TA, Machen SK, Fisher C, Goldblum JR. Usefulness of cytokeratin subsets for distinguishing monophasic synovial sarcoma from malignant peripheral nerve sheath tumor. Am J Clin Pathol 1999;112:641-8.
Machen S, Fisher C, Gautam R, Tubbs R, Goldblum J. Utility of cytokeratin subsets for distinguishing poorly differentiated synovial sarcoma from peripheral primitive neuroectodermal tumour. Histopathology 1998;33:501-7.
Fisher C. Synovial sarcoma: Ultrastructural and immunohistochemical features of epithelial differentiation in monophasic and biphasic tumors. Hum Pathol 1986;17:996-1008.
Nusrat A, Parkos C, Verkade P, Foley C, Liang T, Innis-Whitehouse W, et al
. Tight junctions are membrane microdomains. J Cell Sci 2000;113:1771-81.
Saito T, Oda Y, Yamamoto H, Kawaguchi K-i, Tanaka K, Matsuda S, et al
. Nuclear β-catenin correlates with cyclin D1 expression in spindle and pleomorphic sarcomas but not in synovial sarcoma. Hum Pathol 2006;37:689-97.
Uma Nahar Saikia
Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]