SS18-SSX Antibody: A Useful Tool to Save Time and Reduce Costs in Synovial Sarcoma Diagnosis. Proposal of a Novel Diagnostic Algorithm

Abstract

Synovial sarcoma is a rare malignant mesenchymal neoplasm mostly affecting young adults, characterized by a specific translocation which results in the fusion of the SS18 gene on chromosome 18 with one of the three highly homologous SSX genes on chromosome X. Its morphological diagnosis, especially in monophasic or poorly differentiated variants, can be challenging because histological features often overlap with other malignant mesenchymal tumors. Until recently, the differential diagnosis mostly relied on the use of cytogenetic or molecular analyses to detect the specific t(X;18)(p11;q11) translocation, thus virtually restricting its correct identification to referral centers with a high histological and molecular pathology workflow. The recently commercialized highly sensitive and fusion-specific SS18-SSX antibody has significantly improved the approach to these tumors, representing a relatively cheap and easy to access tool for synovial sarcoma diagnosis. Through a retrospective analysis of 79 synovial sarcomas and histological mimickers, this study confirms the usefulness of the SS18-SSX antibody in the diagnosis of synovial sarcoma, particularly focusing on its application in the pathological response evaluation after neoadjuvant treatment as well as its time- and cost-saving advantages. Finally, we here propose a new diagnostic algorithm to apply into the routine practice.

Introduction

Synovial sarcoma (SS), a malignant mesenchymal neoplasm with a variable epithelial differentiation, accounts for 5–10% of all soft tissue sarcomas and affects predominantly adolescents and young adults with a predilection for the extremities.^1–4 The hallmark of SS is the presence of a pathognomonic translocation, t(X;18)(p11;q11), resulting in a fusion of the SS18 gene on chromosome 18 (named SYT) with one of the three highly homologous SSX genes on the X chromosome (SSX1, SSX2, and, rarely, SSX4).^1,5–7 This gene fusion is detected in more than 95% of cases. ⁸ Histologically, SS has a heterogeneous appearance and three histological variants have been described: monophasic, biphasic, and poorly differentiated.^9–11 Until a few years ago, a specific immunohistochemical (IHC) panel was not available and the diagnosis of SS relied on the detection of the t(X;18)(p11;q11) by molecular genetic or cytogenetic tests. ¹² Both fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR) analyses have been largely validated in the t(X;18)(p11;q11) detection, although cytogenetic evaluation is most widely used. ¹³ FISH analysis typically reveals a classical SS18 break-apart pattern, with only a small percentage of cases showing an atypical one that requires a second molecular test to confirm the translocation. ¹⁴ The FISH assay is not widely available in most laboratories, as it is considered a labor-intensive and expensive molecular analysis ¹⁵ and requires specific equipment as well as trained and dedicated staff. ⁸ Therefore, this molecular analysis is generally restricted to referral centers having a high workflow. ¹⁶ Recently, a fusion-specific SS18-SSX antibody has been commercialized and found to be highly sensitive and specific for SS diagnosis.^13,17–19 On a retrospective series of 400 sarcomas, including 100 SSs, Baranov and colleagues showed a 95% sensitivity and 100% specificity staining for SS, surpassing the specificity of previously used IHC markers. ¹⁹

Through a retrospective analysis of a series of SSs and histological mimickers, this study aims to confirm the usefulness of SS18-SSX IHC in the diagnosis of this tumor in terms of both sensitivity and specificity, focusing on the time- and cost-saving advantage of introducing the SS18-SSX IHC approach into routine diagnostics.

Materials and Methods

Case Series

A retrospective and consecutive series of 79 SSs and histological mimickers (including mostly undifferentiated sarcomas and carcinosarcomas) was collected from the pathology files of the Città della Salute e della Scienza hospital of Turin, between January 2009 and December 2022. Clinical data were retrospectively obtained from medical records, including sex, age at diagnosis, tumor size and location, date of histological diagnosis as well as type of treatment. In addition, for all cases, the number of IHC assays was recorded. Histological slides were independently reviewed by two different pathologists with an experience in soft tissue tumors (A.L. and M.P.), all blinded to the patient’s outcome. Tumor formalin-fixed paraffin-embedded blocks were available for all patients.

All cases were deidentified and coded by a pathology staff member not involved in the study, and all data were accessed anonymously. The study was conducted in accordance with the principles set out in the Declaration of Helsinki. Considering the retrospective nature of this research protocol and that it had no impact on patients’ care, no specific written informed consent was required.

SS18 FISH

All samples were analyzed by means of FISH analysis. Based on H&E staining, the appropriate tumor area was selected. Sections for FISH were cut at 4 µm. DNA FISH was performed using the ZytoLight SPEC SS18 Dual Color Break Apart Probe (Zytovision; Bremerhaven, Germany). At first, sections were deparaffinized and then were pretreated using Aquarius Tissue Pretreatment Kit (Cytocell Ltd.; Oxford Gene Technology, 418 Cambridge Science Park, Milton Road, Cambridge, CB4 0PZ, UK). Subsequently, digestion of tissue and hybridization overnight using SPEC SS18 Dual Color Break Apart Probe in a HyBrite (Abbott Molecular Inc.; IL) was carried out for each sample. Following hybridization, the slides were washed with 2× saline sodium citrate NP40 (Abbott Molecular Inc.). After air-drying, slides were counterstained with 4′,6-diamidino-2-phenylindole (1,4-phenylenediamine in phosphate-buffered saline and glycerol) (Abbott molecular Inc.). Slides were analyzed using a Zeiss Axio Imager Z1 fluorescent microscope (Carl Zeiss S.p.A.; Via Varesina, 162, 20156 Milano MI). For analysis, 10 invasive areas on each slide were selected and automatically acquired at 40× magnification with the motorized Zeiss Axio Imager Z1 automatic scanning system. Metafer 4 software was used to analyze the results.

FISH data were classified according to Papp et al. criteria. ¹⁴ Specifically, cases were considered positive when (1) a typical/classical SS18 break-apart FISH pattern with one fusion (1F) signal, one green (1G) signal, and one red (1R) signal was observed in 15% or more of the neoplastic cells, and (2) an atypical SS18 break-apart FISH pattern with a 1F signal followed by a 1R signal or a 1G signal was reported in a sufficient and representative percentage (at least 20%) of tumor cells. Cases without translocation signals or with a percentage of translocation less than 15% were considered negative. ¹⁷

SS18-SSX Immunohistochemistry

SS18-SSX immunohistochemistry was performed in all cases. Specifically, SS18-SSX IHC was performed on the same tissue block in which SS18-FISH analysis was performed. Three-µM-thick serial paraffin sections were processed by immunohistochemistry using an automated platform (Ventana BenchMark AutoStainer; Ventana Medical Systems, Tucson, AZ) with antibody against SS18-SSX (clone E9X9V #72364; Cell Signaling Technology, Danvers, MA) at 1:500 dilution and 1-hr incubation. Appropriate positive controls were included for each slide. IHC reactions were reviewed by two pathologists (R.S. and F.S.) blinded to the FISH data. IHC was scored according to both the percentage of positive neoplastic cells and their staining intensity, as reported by Baranov et al. ¹⁹

FISH and IHC Cost Analysis

Cost analysis was investigated for both methods considering consumable materials, only. Reagent costs were calculated as a result of the volumes and amounts of reagent needed to process one sample.

Statistical Analyses

All analyses were performed using Stata 17.0 Statistical Software (StataCorp; College Station, TX). Differences in the distribution of the variables assessed according to clinicopathological parameters were analyzed using parametric and non-parametric tests (Student’s t-test, Pearson’s chi-square test, and Wilcoxon’s rank test). For the evaluation of concordance between the diagnosis of SS made by FISH and by IHC, the overall concordance was calculated through the sum of true positives and true negatives compared with the total number of cases by calculating Cohen’s κ index to eliminate the random component. The interpretation of the κ values was performed according to the following guidelines: κ 0.01–0.20 = none to slight agreement; κ 0.21–0.40 = fair agreement; κ 0.41–0.60 = moderate agreement; κ 0.61–0.80 = substantial agreement; κ 0.81–1.00 = almost perfect agreement.

Results

Clinical Characteristics of the Entire Cohort of 79 Patients

Our retrospective cohort included 79 patients, 44 males (56%) and 35 females (44%). The patients’ age ranged from 7 to 80 years, with a median age of 43 years. For each case, the number of IHC assays performed for diagnostic purposes ranged from 3 to 19, with a median of 8 IHC reactions. Among the 79 cases of SS and histological mimickers, 40 cases were originally diagnosed as SS, 32 cases were diagnosed as other soft tissue sarcomas (including undifferentiated sarcomas and isolated cases of malignant peripheral nerve sheath tumors, malignant solitary fibrous tumor, and Ewing’s sarcoma), and 7 cases were diagnosed as carcinosarcoma.

Table 1 summarizes the main features of the entire cohort.

Table 1.

Summary of Clinicopathological Parameters of the Entire Cohort (SSs and Histological Mimics).

	Total (N = 79)	Non-SSs	SSs	p Value
Sex
M	44	25	19	0.137
F	35	14	21
Age
Median (range)	43 (7–80)	56 (11–80)	36 (7–76)	0.002
Histological diagnosis
SS	40	-	40	-
Carcinosarcoma	7	7	-
Other sarcoma	32	32	-
FISH (sec. Papp et al.)
Positive, classical pattern	36	-	36	-
Positive, atypical pattern	4	-	4
Negative	39	39	-
Number of IHC for diagnosis
Median (range)	8 (3–19)	8 (4–18)	7 (3–19)	0.304
SS18-SSX IHC
Negative	41	39	2	<0.001
Positive	38	0	38

Abbreviations: FISH, fluorescence in situ hybridization; IHC, immunohistochemical; SS, synovial sarcoma.

Patient’s Characteristics, FISH, and IHC Analyses on 40 Cases Originally Diagnosed as SS

In 40 patients, FISH analysis yielded a positive result, thus confirming the original morphological diagnosis of SS. In this subgroup, 19 cases (47.5%) were males and 21 cases (52.5%) were females. The patients’ age ranged from 7 to 76 years, with a median age of 36 years; specifically, 20 cases (50%) were aged <36 years. In this subgroup, in 25 patients (62.5%) the tumor was located in the lower extremities; in 7 patients (17.5%) in the upper extremities; in 3 patients (7.5%) in the trunk; and in 2 patients (5%) in the head and neck region; a visceral localization was observed in 3 of the 40 patients (7.5%). The tumor size ranged from 0.6 to 15 cm (median: 6.9 cm). Histologically, the most represented subtype was monophasic SS in 19 of the 40 cases (47.5%); the remaining cases were diagnosed as either biphasic (17 cases; 42.5%) or poorly differentiated SS (4 cases; 10%). Neoadjuvant treatment was administrated to 23 of the 40 patients (57.5%).

For each case, an IHC panel, comprising a median of seven biomarkers, was originally performed for diagnostic purposes (range: 3–19). IHC approach was not standardized and differed among cases. The most frequently assessed biomarkers included CD99, BCL-2, epithelial membrane antigen (EMA), vimentin, cytokeratin, and desmin. TLE1 was never performed because it was not available at our institution.

A typical/classical SS18 break-apart FISH pattern was observed in 36 of the 40 cases (90%), whereas an atypical cytogenetic pattern was observed in 4 of the 40 cases (10%) characterized by 1F + 1R signals.

Overall, 38 of the 40 cases (95%) were SS18-SSX positive at IHC analysis. Among the positive cases, 37 of the 38 cases (97.4%) showed a moderate/strong and homogeneous nuclear staining pattern in more than 50% of neoplastic cells (score 4/5+ according to Baranov et al.) (Figure 1). ¹⁹ In the remaining positive case, the percentage and intensity of staining were lower, probably due to suboptimal tissue handling in the preanalytical phase (score 3+ with a weak positivity).

Figure 1.

(A–C) Monophasic SS (A: H&E, 200×, selected area 400×) with a diffuse and homogeneous strong nuclear staining for SS18-SSX antibody (B: SS18-SSX IHC, 200×) as well as a positive FISH analysis showing a typical SS18 break-apart pattern (C: FISH 1F + 1G + 1R pattern). (D–F) Monophasic SS (D: H&E, 200×, selected area 400×) with a diffuse and strong nuclear staining for SS18-SSX antibody (E: SS18-SSX IHC, 200×) but with an atypical SS18 break-apart FISH pattern (F: FISH 1F + 1R, white arrow). (G–I) Biphasic SS (G: H&E, 200×, selected area 400×) with a negative staining for SS18-SSX antibody (H: SS18-SSX IHC, 200×) and a classical SS18 break-apart FISH pattern (I: FISH 1F + 1G + 1R). Scale bar 200 µm. Abbreviations: FISH, fluorescence in situ hybridization; SS, synovial sarcoma; IHC, immunohistochemical; 1F, one fusion; 1G, one green; 1R, one red.

In all four cases harboring an atypical SS18 break-apart FISH pattern, IHC resulted diffusely positive with a percentage of tumor cells as 4/5+ with strong nuclear staining (Figure 1).

In the subgroup of patients undergoing neoadjuvant treatment, immunohistochemistry was assessed both in pre-treatment and post-treatment samples. The SS18-SSX antibody maintained its expression in post-treatment specimens, easily highlighting the residual viable tumor areas in the tissue sections (Figure 2).

Figure 2.

Monophasic SS in a 7-year-old patient. (A–C) Pretreatment biopsy: a monomorphic spindle cell proliferation (A: H&E, 200×) with a diffuse and strong nuclear staining for SS18-SSX antibody (B: SS18-SSX IHC, 200×) and an atypical SS18 break-apart FISH pattern (C: FISH 1F + 1R, white arrow). (D and E) Post-treatment sample: a single and marginal focus of residual disease in a background of diffuse hyaline fibrosis is highlighted by SS18-SSX antibody (D: H&E, 200×; E: SS18-SSX IHC, 200×). Scale bar 200 µm. Abbreviations: FISH, fluorescence in situ hybridization; IHC, immunohistochemical; SS, synovial sarcoma; 1F, one fusion; 1R, one red.

Tables 1 and 2 summarize the main characteristics of the SS subgroups.

Table 2.

Summary of Patient’s Clinicopathological Parameters of SS Cases.

	Total (N = 40)	Sex
		M	F
Age
Median (range)	36 (7–76)	41 (15–76)	33 (7–76)
Tumor size (cm)
Median (range)	6.9 (0.6–15)	6.9 (2–15)	6.5 (0.6–13)
Tumor site
Lower extremities	25	9	16
Upper extremities	7	5	2
Trunk	3	2	1
Head-neck	2	1	1
Visceral	3	2	1
% of SS18-SSX-positive nuclei
Median (range)	95 (0–99)	92 (70–99)	95 (0–99) a
SS subtype
Biphasic	17	7	10
Monophasic	19	10	9
Poorly differentiated	4	2	2
FISH
Positive, classical pattern	36	17	19
Positive, atypical pattern	4	2	2
Neoadjuvant treatment
No	10	6	4
Yes	23	9	14

Abbreviations: FISH, fluorescence in situ hybridization; SS, synovial sarcoma.

^aTwo cases were negative.

FISH and IHC: Comparison of Methods

A significant positive correlation was observed between FISH and IHC analyses regarding SS18-SSX translocation detection (p<0.001) (Table 3). A concordance of 97.5% between the two methods was observed (Cohen’s κ: 0.94). Diagnostic IHC method for SS diagnosis demonstrated a sensitivity of 95% and a specificity of 100%.

Table 3.

Concordance Between SS18-SSX FISH and IHC in SS Diagnosis.

	Total	SS18-SSX IHC		p Value
		Negative	Positive
FISH-negative	39	39	0	<0.001
FISH-positive	40	2	38

Abbreviations: FISH, fluorescence in situ hybridization; IHC, immunohistochemical; SS, synovial sarcoma.

Costs and Turnaround Time Analysis: FISH Versus IHC

In our high workflow laboratory, FISH method represents a routine investigation with a cost of about 57€ estimated per case (personnel cost excluded). Regarding SS18-SSX IHC, a total cost of 12€ (comprising reagents and the antibody, only) was assessed. The raw savings between the two methods results therefore in 45€.

The time required for a FISH analysis is two working days in our laboratory, excluding the interpretation time that is experience dependent and provided by dedicated personnel. In contrast, for IHC, the run-time is estimated to be 1 day, and interpretation is done directly by the pathologist in charge of the case.

Discussion

Histological diagnosis of SS is based on the detection of its pathognomonic translocation t(X:18) which is reported in more than 95% of cases.^8,20 Until a few years ago, its evaluation could only be carried out by molecular investigations, especially by FISH, which appears to be the most widely used method.^12,13,21 Recently, a fusion-specific antibody able to identify the t(X:18) translocation by immunohistochemistry has been developed and commercialized, paving the way for a new diagnostic approach to SS. ¹⁹

The first goal we aimed at in this study was to confirm the high sensitivity and specificity of the SS18-SSX antibody in the diagnosis of SS in a retrospective cohort of SSs and its histological mimickers. Our results report a sensitivity of 95% and a specificity of 100% and are thus in perfect agreement with recently published data^{13,17,19,22–27}. In the largest reported series (400 tumors including 100 SSs and 300 mimics), Baranov et al. who suggested for the first time that the IHC approach could replace molecular investigations to support SS morphological diagnosis ¹⁹ have obtained values perfectly overlapping with those presently reported. Moreover, in a series of 36 SSs, Tay et al. documented an SS18-SSX antibody sensitivity of at least 91% and a specificity of 100%. ¹⁷ A 100% specificity for SS18-SSX antibody allows to identify, among histologically suspected cases, only those that are truly SSs, thus avoiding misclassification in non-SS neoplasms. As a result, the introduction of the SS18-SSX antibody in the daily diagnostic routine could represent a significant change in the diagnostic approach, bypassing FISH analysis in most cases. Conversely, a sensitivity of 95% does not rule out that some true cases of SS may go undetected, but we want to underline that they represent a minor subgroup. In particular, this subclass could consist of SS cases with uncommon gene fusions and therefore requiring a second molecular diagnostic step. ¹⁹ Moreover, because a diffuse and homogeneous strong nuclear reaction is observed in almost all cases, the easy interpretation of the IHC reaction is of further value.

The second goal we focused on was the correlation between SS18-SSX IHC and SS18 FISH pattern. In our series, we identified four cases with an atypical SS18 break-apart FISH pattern, and specifically 1F + 1R in more than 20% of nuclei. Interestingly, all these cases resulted diffusely positive at IHC analysis with a percentage of positive cells ranging from 70% to 99%. Previously, only Tay et al. focused on this issue. ¹⁷ In their series, they collected 10 cases of morphologically suspected SS presenting an atypical FISH pattern; among those, only 50% were positive at IHC analysis with the novel SS18-SSX antibody. Through subsequent next-generation sequencing (NGS) analysis, only these latter cases were confirmed to be SS. These observations emphasize not only that SS18-SSX IHC is more specific than FISH in the diagnosis of SS but also that immunohistochemistry has an excellent correlation with NGS data. ¹⁷

For the first time, we here demonstrated the usefulness of using SS18-SSX IHC in the setting of neoadjuvant treatments. Preoperative radiotherapy or chemotherapy is recommended for large tumors in which a close margin is foreseen to preserve neurovascular structures, as well as in young patients with high-risk tumors. In cases that underwent neoadjuvant treatment, the histological report should include an indication of the amount of residual disease. Especially in cases of monophasic SS, the evaluation of foci of residual viable tumor cells is not an easy task, also due to the presence of a fibrotic, hyaline, and/or necrotic background. ² A few years ago, the European Organisation for Research and Treatment of Cancer—Soft Tissue and Bone Sarcoma Group proposed a new standardized approach for the pathological evaluation after neoadjuvant treatment. ²⁸ This five-tier response score, based on the assessment of the volume of the stainable residual tumor, is very difficult to apply, especially when the pathological response is nearly complete (response grade A–C).^28,29 In this regard, our study showed how the use of SS18-SSX antibody allowed us to easily identify persistent supposedly viable neoplastic foci, highlighting residual cells with a strong and specific nuclear staining, even in the context of dense and diffuse fibrous reaction.

Finally, the introduction of SS18-SSX IHC in daily routine practice may represent an advantage in terms of cost and run-time. The use of SS18-SSX antibody would initially allow peripheral centers to independently manage cases morphologically suspected to be SS, avoiding displacement to referral hospitals and saving time. In fact, immunohistochemistry is a low-cost and rapid method available in most laboratories, whereas FISH is concentrated mostly in referral centers and is considered a labor-intensive and expensive molecular analysis that requires specific equipment as well as trained and dedicated staff. ¹⁵ As there is not a standardized approach to SS in pathology, the diagnosis often comes after performing an extensive IHC panel to rule out other histological mimickers. This imposes an important economic investment to which a molecular approach must often be added to confirm diagnosis. Applied to real life, considering a median of seven IHC reactions and one FISH assay in SS diagnosis, we observe a total cost of 141€ per case (specifically, 7 × 12€ for IHC + 57€ for FISH). Supposing to use SS18-SSX antibody as a first IHC step for each morphologically suspected SS, and three additional IHC reactions (such as S100, EMA, and cytokeratin, according to our experience), the total incurred cost would be 48€. Therefore, in tumors with a positive SS18-SSX reaction, there would be relevant economic savings. Thus, the second molecular step (FISH, RT-PCR, or NGS) could be restricted to morphologically highly suspicious cases with negative IHC.

Based on the above, we propose a novel diagnostic algorithm to be applied in morphologically suspicious cases of SS, as described in Figure 3. Assuming a first IHC approach to screen patients, only a small percentage of cases suspected to be SS, but with negative IHC reaction, would be sent to a referral center for a second molecular step, thus saving time and significantly reducing the cost.

Figure 3.

Proposal of a new diagnostic algorithm for cases morphologically suspicious for SS. Abbreviations: SS, synovial sarcoma; RT-PCR, reverse transcription polymerase chain reaction; IHC, immunohistochemical; EMA, epithelial membrane antigen; FISH, fluorescence in situ hybridization; NGS, next-generation sequencing.

In conclusion, our study underlines the following:

1. SS18-SSX antibody is a valid and easy-to-interpret diagnostic tool in SS diagnosis as much as a concrete substitute for a molecular analysis, which is able to identify also SS cases with an atypical FISH pattern;

2. SS18-SSX antibody allows an easier identification and evaluation of the amount of residual disease in patients who underwent neoadjuvant treatment;

3. The introduction of SS18-SSX antibody in the daily diagnostic routine, as a first step in histologically suspected SS, represents an economic advantage, as well as a consistent saving in run-time;

4. The acquisition of SS18-SSX antibody in the IHC marker panel could allow an SS diagnosis in virtually all pathology laboratories.