Usefulness of SS18-SSX antibody as a diagnostic marker for pulmonary metastatic synovial sarcoma

We retrieved the details of patients with SS, who underwent surgical resection between 2001 and 2019, from the Pathology Department of Shinshu University Hospital. The whole slides of the tumor of five patients were available for 10 pulmonary metastatic SS and the corresponding five primary sites (four in the limbs and one mediastinum), and of 93 clinical and histologic mimics, including 49 pulmonary metastatic bone and soft tissue sarcomas other than SS (osteosarcoma, chondrosarcoma, liposarcoma, malignant fibrous histiocytoma, Ewing’s sarcoma, leiomyosarcoma, fibrosarcoma, and undifferentiated pleomorphic sarcoma), 39 primary lung cancers (adenocarcinoma, squamous cell carcinoma, small cell lung cancer, large cell lung cancer, pleomorphic cell carcinoma, and carcinoid), and 5 intrathoracic solitary fibrotic tumors. All patients with SS underwent surgical resection of the primary origin and were diagnosed with SS by SS18 break-apart FISH to detect the SS18-SSX fusion gene. In metastatic SS specimens, two metastatic sites were selected from each patient, and a total of 10 specimens were stained. Chemotherapy had been initiated for all patients before the resection of the metastatic specimens. SS and other bone and soft tissue sarcomas were consecutive cases, including repeated pulmonary metastasectomies. Patients with SFT and primary lung cancer were randomly selected from our database. This study was approved by the Shinshu University Research Ethics Committee (No. 4870).

IHC staining was performed by manual methods. Specimens of whole tumors (SS, other bone soft tissue sarcoma, primary lung cancer, and solitary fibrotic tumor [SFT]) were paraffin-embedded and cut into 4-μm-thick sections. They were deparaffinized with ethanol and xylene, and endogenous peroxidase activity was blocked using methanol and 30% H₂O₂ solution for 30 min at room temperature. Protein blocking was performed using 1% bovine serum albumin [BSA]/phosphate-buffered saline [PBS] for 1 h at room temperature. The sections were incubated overnight at 4 °C with primary antibody against human SS18-SSX (clone E9X9V, 1:1000, Cell Signaling Technology, Danvers, MA, USA). The sections were washed in PBS three times and probed with an anti-rabbit IgG labeled with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan) for 1 h at room temperature. They were washed three times in PBS, and the immune complex was visualized using Histofine Simple Stain 3,3′-diaminobenzidine (Nichirei, Tokyo, Japan). After washing in water, the sections were counterstained with hematoxylin.

Immunostaining of intraoperative sealed cytology was performed similarly. The tumor was directly smeared onto the slide grass and then fixed with 99.5% ethanol. The subsequent immunostaining steps were the same.

Immunoactivity was reviewed by one pathologist (UT) and one thoracic surgeon (MK).

The characteristics of patients with SS are presented in Table 1. All SS of primary origin and pulmonary metastasis had already been diagnosed using SS18 break-apart FISH. The age at the identification of the primary tumor as SS was considered for each patient. In all five patients with SS, at least two metachronous metastasectomies were performed. The duration between the resection of primary sites and pulmonary metastasectomies ranged from 28 to 108 months. Cases 1, 3, 4, and 5 were monophasic spindle cell types, and Case 2 was biphasic type.

In Case 1, a 63-year-old woman with primary SS in the left ankle underwent pulmonary metastasectomy three times. IHC of SS18-SSX for pulmonary metastases was obtained from the first and second metastasectomy specimens. Figure 1A shows the computed tomographic findings of solitary pulmonary metastasis in the left lower lobe before the initial left wedge resection in this case.

In Case 2, a 28-year-old man had primary SS in the mediastinum. The large mass (60 × 60 × 40 mm) was protruding from the left side chest cavity (Fig. 2A, B), which was resected with combined pericardial resection by median sternotomy. In this case, the definitive diagnosis of SS was not reached until the initial metastasectomy, which was 108 months after the primary surgery when SS was finally diagnosed through comprehensive pathology workups including SS18 break-apart FISH for primary and metastatic sites. Figure 2C shows FISH for SS18 rearrangement in the primary tissue, demonstrating a disconnected SS18 gene. No evidence of recurrence was observed 17 months after the last pulmonary metastasectomy.

In Case 3, a 33-year-old man with primary SS in the left forearm underwent pulmonary metastasectomy four times and subsequent radiation therapy for right-sided pulmonary metastasis. Figure 1B shows the computed tomographic findings of the solitary pulmonary metastasis in the left lower lobe before left basal segmentectomy. IHC of SS18-SSX for pulmonary metastases was obtained from the first and second metastasectomy specimens.

In Case 4, a 51-year-old man had the primary origin as the left knee. He underwent pulmonary metastasectomy five times. The patient was followed up without any treatment. IHC of SS18-SSX for pulmonary metastases was obtained from the third and fourth metastasectomy specimens.

In Case 5, a 30-year-old man had the primary origin as the right forearm. He underwent pulmonary metastasectomies four times. IHC of SS18-SSX for pulmonary metastases was obtained from the second and third metastasectomy specimens. He had recurrence and was undergoing chemotherapy.

Figure 3 shows the histologic findings of SS18-SSX IHC and hematoxylin and eosin of 10 pulmonary metastatic SS and 5 corresponding primary sites from 5 patients with SS. The SS18-SSX fusion-specific antibody was positive with diffusely strong staining in all 10 metastatic SS samples. All five primary SS tumors were stained similarly to the corresponding metastatic SS. However, no staining of SS18-SSX was observed in the 93 clinical and histologic mimics (49 other bone and soft tissue sarcomas, 39 primary lung cancers, and 5 SFTs). Figure 4 shows the histologic findings of SS18-SSX IHC of 15 representative cases from 93 clinical and histological mimics. A summary of the IHC results is presented in Table 2.

Figure 5A shows the histologic finding of SS18-SSX IHC of a percutaneous needle biopsy specimen from the primary right forearm tumor in Case 5, demonstrating diffuse strong staining of SS18-SSX antibody similar to the surgically resected specimen (Fig. 3T). Figure 5B shows the cytological findings of SS18-SSX IHC of intraoperative touch imprint cytology specimen from the pulmonary metastatic site, demonstrating strong staining of the SS18-SSX antibody.