SS-A52 antigen expression in thymic carcinoma accompanied with Sjögren syndrome

Abstract Rationale: The relationship between thymic tumors and Sjögren syndrome (SjS) is unknown, and surgical resection has not been optimized. Especially, thymic carcinoma with autoimmune disease is rare. Analysis of SS-A52, germinal centers, plasma cells, and Foxp3+ Treg in thymic carcinoma has never been reported, and their pathological roles in causing SjS have not been studied. Patient concerns: A 78-year-old man presented with sputum production and xerostomia while asleep. Chest computed tomography showed a homogeneous and hypodense mass in the anterosuperior mediastinum. Serum levels of the antinuclear antibody, antibody to SS-A, and antibody to SS-B were positive. Diagnoses: Thymic carcinoma (squamous cell carcinoma) and SjS. Interventions: Video-assisted thoracoscopic resection of the mediastinal tumor and postoperative radiation therapy was performed. Outcomes: The histological diagnosis was thymic squamous cell carcinoma. Histologically, the squamous carcinomatous cells were arranged in nests and cords in the fibrohyaline stroma with capsular invasion. In the stroma, dense lymphoid tissues containing large reactive germinal centers and many plasma cells were also noted. In the involuted thymus, CD20-positive mature lymphocytes infiltrated, and germinal centers were noted. Double immunohistochemical staining revealed that SS-A52 antigen was positive in both the carcinoma component and CD20-positive mature B cells. Postoperatively, the xerostomia persisted, and serum SS-A and SS-B remained positive. No evidence of carcinoma recurrence with chest computed tomography scan was observed at 18-months follow-up. Lessons: In the surgical treatment of thymic tumors with SjS, extended thymectomy might be worth considering to stop the progressive destruction of the targets of SjS-specific autoantibodies. However, the postoperative symptoms may not dramatically improve because the target organs might have changed irreversibly, and memory B cells might persist. This is the first report that demonstrated the SS-A52 antigen presentation in a thymic tumor to the best of our knowledge.


Introduction
Thymic tumors are sometimes accompanied by an autoimmune disease (AD). Myasthenia gravis (MG) remains the best-studied AD in terms of a coexisting thymic tumor. For example, approximately 10% to 15% of the patients with MG have a thymoma. [1] Type-A thymoma is rarely associated with MG (0%-33%); on the contrary, MG is more commonly associated with type B1-B3 (7%-71%) thymoma. [2] Morphologically, 58.8% of thymoma patients with MG (TMG) had thymic germinal centers (GC), whereas only 15.6% of the patients with thymoma but without MG showed GCs. [3] GCs are specialized microstructures found within secondary lymphoid tissues that produce long-lived antibody-secreting plasma cells and memory B cells during antigen presentation by follicular dendritic cells to T cells. [4,5] The number of GCs has been correlated with higher titers of antiacetylcholine receptor antibodies in MG patients. The patients showing chronicity of the GCs had persistence of the antigens, and the use of prednisolone therapy resulted in reduced GCs. [3,6] In addition to thymoma, thymic follicular hyperplasia with GCs is the most common morphology in early-onset MG and is also observed in remnant thymuses adjacent to thymomas in TMG (30%-50%). Thymic follicular hyperplasia is thought to be a source of autoantibodies. [2,6] This is a plausible explanation for the effectiveness of extended thymectomy (ET) as a treatment for MG. In ET, en bloc resection of the anterior mediastinal fat tissue, including the thymus, is performed. The resection borders were the diaphragm caudally, the thyroid grand orally, and the phrenic nerves laterally. [7] ET for MG showed significant improvements in postoperative clinical symptoms in both nonthymomatious [7,8] and thymomatous patients. [7] In addition, another immunoregulatory defect is observed in MG patients: thymic and peripheral regulatory T cells (Treg) and effector T cells are functionally impaired. [9,10] Despite the accumulating knowledge, the relationship between thymic carcinoma and AD remains unclear. Studies that investigated the association of thymic carcinoma with AD are rare, and only a few cases have been reported, including MG, [11,12] autoimmune hepatitis, [13] stiff-man syndromes, [14] systemic lupus erythematosus (SLE) and hypertrophic pulmonary osteoarthropathy, [15] scleroderma, [16] and dermatomyositis. [17] According to another recent study, 1 out of 9 patients with micronodular thymic carcinoma with lymphoid B-cell hyperplasia, had MG, whereas 1 out of 9 had Sjögren syndrome (SjS). [18] In a previous report on thymic carcinoma with AD, GCs were reported. [19] Surgical resection of thymic carcinoma contributed to good control in some cases of AD [20,21] ; however, the relapse of AD was followed by postoperative metastasis of the tumor. [22] Furthermore, the relationship between thymic tumors and SjS remains unknown, and the surgical resection has not been optimized. The SS-A autoantigen is an intracellular RNA-protein complex that is the target of autoantibodies present in the sera of patients with SjS and SLE. [23,24] The SS-A antigenic system comprises 2 proteins: 52-kD polypeptide (SS-A52) and 60-kD polypeptide (SS-A60). [24] The mRNA expression of SS-A52 and SS-A60 was higher in SjS patients than that in controls. [25] SS-A52 is expressed in the cytoplasm of ductal cells of patients with SjS. [25] SS-A60 is found in the cytoplasm and nucleus of acinar cells and ductal cells of patients with SjS. [25] The role of these 2 polypeptides in SjS is unclear; however, antibodies, rather than autoantigens, seemed to injure the target organs. In 1 experiment, the IgG deposition and functional failure of the salivary glands following the surge of serum anti-SS-A52 antibodies after intravenous injection of SS-A52 into mice were reported. In the same report, the passive transfer of SS-A52 immune sera-induced salivary gland dysfunction. [26] In another study, anti-SS-A and anti-SS-B antibodies were detected in the lacrimal fluid, and their presence in the serum or tear fluid was associated with the severity of keratoconjunctivitis sicca. [27] However, the relationship between SS-A antigen and thymic tumors is still unclear. Additionally, the frequency of Foxp3+ Treg cells in salivary gland lesions in patients with SjS correlates with inflammation grade. [28] We herein report a case of thymic carcinoma with GCs complicated by SjS and examine the expression and distribution of SS-A52 antigen and Foxp3+ Treg cells in the resected specimen.

Case presentation
A 78-year-old man presented to our hospital with sputum production and xerostomia while asleep. Chest computed tomography showed a homogeneous and hypodense mass, 48 mm in size with smooth contours and without contrast enhancement in the anterosuperior mediastinum. Serum levels of anti-acetylcholine receptor antibody, soluble interleukin 2 receptor, carcinoembryonic antigen, a-fetoprotein, squamous cell carcinoma-related antigen, cytokeratin 19 fragment, and human chorionic gonadotropin b-subunit were within their normal ranges. The diagnosis of thymoma was suspected. However, serologic examination revealed the following: the antinuclear antibody, antibody to SS-A, and antibody to SS-B showed positive (1:640 with a speckled pattern, 1:>240, and 1:>320, respectively); rheumatoid factor, 113 IU/ml; anti-galactose-deficient IgG antibody level, 149.5 AU/ml; serum cryoglobulin, negative. He was diagnosed with SjS, fulfilling 2 of the 4 criteria items of the revised Japanese criteria for SjS. [29] Video-assisted thoracoscopic resection of the mediastinal tumor was performed. The thymic and adipose tissue around the tumor were also resected with the tumor to achieve complete resection. There was a lobulated and elastic mass in the thymus, measuring 45 Â 30 mm in size. We found no pleural dissemination of the tumor to the thoracic cavity.
This tumor was diagnosed as squamous cell carcinoma of the thymus (Masaoka stage II) based on the World Health Organization classification. [30] Histologically, the neoplasms were composed of atypical polygonal epithelial cells arranged in a sheet-like pattern, and the tumor nodules were separated by abundant reactive lymphoid stroma (Fig. 1A). The epithelial cells were round to oval in shape, with large nuclei containing dense chromatin and prominent nucleoli. The mitotic count was 5 to 8/ 10 HPF, and keratinization was observed. The surrounding lymphoid stroma was composed of densely packed proliferating small lymphocytes, many plasma cells, and large lymphoid follicles with GCs Immunostainings for CD3, CD5, CD20, CD21, CD138, widespectrum antipancytokeratin (AE1AE3), SS-A52, and Foxp3 were performed in controls (thymic squamous cell carcinoma without the autoimmune disease [Sq/AD -]) and, in our case (Sq +SjS), according to the manufacturer's protocols. Tumor specimens of Sq/ADwere obtained from 3 patients with thymic squamous cell carcinoma without any autoimmune disease who underwent surgery around the same time in our hospital. The primary antibodies used were antihuman 52-kD Ro/SSA mouse monoclonal antibody (clone D-12; dilution rate, 1:100; Santa Cruz Biotechnology, Dallas, TX) and antihuman Foxp3 mouse monoclonal antibody (clone 236A/E7; dilution rate, 1:100; eBioscience Inc., San Diego, CA). To confirm the expression of SS-A52, a dual-fluorescent staining for SS-A52/CD3, SS-A52/ CD20, and SS-A52/AE1AE3 was also performed manually in our case using the Opal 4-color manual IHC kit (Akoya Biosciences, San Francisco, CA), according to the manufacturer's protocols. For nuclear staining, 4',6-diamidino-2-phenylindole (DAPI) was also stained and shown in blue. The reaction product was fluorescently labeled with Opal 520 and Opal 690 fluorophore working solution (Opal 4-color manual IHC kit; Akoya Biosciences) and was observed under a fluorescent microscope BZ-X810 (Keyence Corporation, Osaka, Japan). Immunohistochemical studies showed that the carcinoma component was positive for AE1AE3 and CD5 (Fig. 1A-C). The surrounding lymphoid stroma contained CD3-positive small lymphocytes (Fig. 1D). CD20-positive B lymphocytes were seen in the center of GCs and surrounding lymphoid stroma in reactive pattern (Fig. 1E). SS-A52 was diffusely positive in the cytoplasm of the carcinoma cells (Fig. 1F). In the center of the GCs, there were small foci of CD21-positive dendritic cells (Fig. 1G). CD3positive T lymphocytes and numerous CD138-positive plasma cells were observed in the interfollicular area (Fig. 1H). In the dual-fluorescent staining for SS-A52/AE1AE3, SS-A52 was positive in tumor cells that were also positive for AE1AE3 ( Fig. 2A-C). In the dual-fluorescent staining for SS-A52/CD3 and SS-A52/CD20, the co-expression of SS-A52 and CD20 was partially observed, but that was not the case with SS-A52 and CD3 ( Fig. 2D-I). In the involuted thymus surrounding the carcinoma, prominent GCs were noted, and the aggregated lymphocytes were mainly CD20-positive B cells that were weakly positive for SS-A52 (Fig. 3). In contrast, GC formation and SS-A52 positivity were not observed in Sq/AD -(data not shown). Foxp3-positive lymphocytes infiltrated the squamous cell carcinoma and lymphoid stroma (Fig. 4A). In Sq/AD -, few lymphocytes infiltrated the carcinoma component and fibrous stroma, but they were negative for Foxp3 (Fig. 4B).
The postoperative course was uneventful, and the patient underwent adjuvant radiation therapy with a total dose of 50 Gy divided into 30 fractions. After radiation therapy, cevimeline hydrochloride hydrate (30 mg/day) was started, and xerostomia improved slightly. At the 18-month follow-up, no evidence of tumor recurrence was detected. However, serum levels of the antinuclear antibody, SS-A antibody, SS-B antibody, and rheumatoid factor remained unchanged. Both the Saxon test and Schirmer test remained positive.

Discussion
In this report, a specific autoantigen was found in the tumor tissues of a patient with thymic squamous cell carcinoma with SjS. We believe that the autoantigen-expressing tumor tissues may be the culprit for SjS. Moreover, the non-resected involuted thymus may still be responsible for the postoperative SjS because B-cell infiltration was found. Therefore, ET may be useful for thymic tumors with SjS to normalize the postoperative serum anti-SS-A antibody levels. This is the first report to reveal positive SS-A52 expression in thymic carcinoma via immunohistochemistry staining to the best of our knowledge. In our patient, the distribution patterns of CD3 and CD20 were relatively compartmented, and CD21 showed a network pattern of GC. They are considered similar structures of active GCs in the lymph nodes. [31] However, Foxp3-positive lymphocytes were found in the tumor cells and among infiltrated lymphocytes of Sq +SjS. Although the Treg cells may be activated to repress the abnormal immune responses, they might not prevent SjS. Based on these findings, SS-A52 might be expressed by the tumors, and the autoantibodies might be produced by the B cells in extranodal GCs, which might be enough to destroy the local exocrine glands or skin and cause the SjS.
Interestingly, our case showed similar findings to those of TMG on immunohistochemistry staining: peripheral lymphocytes infiltration and GCs. Abnormal T-cell functionality, probably caused by the inflammation of the tumor and B-cell infiltration to organize GCs, has been observed in the thymus with early-onset MG. [32] Recent research showed that the titers of anti-acetylcholine receptor antibody were significantly higher in TMG patients with numerous thymic GCs than in TMG patients with fewer GCs, supporting the hypothesis that thymic GCs were associated with MG in thymoma patients. [3] A similar pathogenesis might exist in the thymic tumors with SjS. We reviewed past reports on thymic tumors accompanied with SjS in patients who underwent surgery (n = 16, Table 1). Lymphoid follicles were observed in 7 out of the 16 cases (no descriptions and no images ET for thymic tumors with SjS may normalize the postoperative serum autoantibodies and stop the progression of SjS, but it does not always improve the symptoms. First, ET may reduce the incidence of postoperative autoantibodies. In our case, the postoperative serum level of anti-SS-A antibody was positive. The non-resected thymus may be the culprit because GCs, B-cell infiltration, and SS-A52 expression were found in the surrounding involuted thymus. If the hypothesis that autoantibody injures the target organs is true, then ET may reduce the number of B cells, normalize serum autoantibodies, and stop further progression of SjS. Second, another plausible explanation for the persisting symptoms could be the irreversible destruction of the exocrine glands. In a recent study (n = 66 vs n = 67), B-cell depletion by rituximab in patients with SjS showed no clinical improvement in dry mouth, salivary, and lacrimal flow rates, among others. [46] This result may be explained by the fact that the function of the exocrine glands was irreversibly destroyed in many cases. In previous case reports on thymic tumors, postoperative improvement of the symptoms of SjS was found in only 2 out of the 7 cases with no recurrence of tumor, as shown in Table 1. We speculate that the surgery might have been performed at a relatively early stage of SjS in those 2 cases and at a progressive stage close to irreversible in the remaining 5 cases without improvement. Therefore, ET of thymic tumors with SjS may normalize the serum autoantibodies and stop the progression of SjS yet not  www.md-journal.com Table 1 Case reports on surgeries performed on thymic tumors with SjS.

Postoperative course
Year and reference Country Sex

Corrections
The corresponding author email was originally misspelled as hshino@med.kindai.ac.jp and has been corrected to hshio no@med.kindai.ac.jp.
In the 1984 row of Table 1, the Chemotherapy, prednisolone or other medication column appeared incorrectly as plasma exchange and has been corrected to plasma exchange and prednisolone.