A novel thymoma-associated autoimmune disease: Anti-PIT-1 antibody syndrome

Anti-PIT-1 antibody syndrome has recently been reported and characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with autoimmunity to a pituitary specific transcription factor PIT-1, which plays an essential role in GH-, PRL-, and TSH-producing cells. Although circulating anti-PIT-1 antibody and PIT-1-reactive cytotoxic T cells (CTLs) were detected in the patients, the pathophysiology and precise mechanisms for the autoimmunity remain unclarified. During the follow up, thymoma was diagnosed in all 3 cases with anti-PIT-1 antibody syndrome. Immunohistochemical analysis revealed that PIT-1 was strongly expressed in neoplastic cortical thymic epithelial cells. Importantly, after thymectomy, the titer of anti-PIT-1 antibody decreased and reactivity of CTLs toward PIT-1 diminished. These data strongly suggest that the aberrant expression of PIT-1 in the thymoma plays a causal role in the development of this syndrome. Thus, we define that this syndrome is a novel thymoma-associated autoimmune disease.

cells and their MHC antigen expression dictate positive selection, while medullary thymic epithelial cells that express autoantigens contribute to the negative selection 15 . It is well known that thymoma is closely associated with several autoimmune diseases such as myasthenia gravis (MG). Although the precise mechanisms remain unknown, it has been suggested that the aberrant expression of the antigen, acetylcholine receptor (AChR) in the tumor cells and a defect in the negative selection in thymoma may play a role in the development of autoimmunity [16][17][18] .
In this report, we demonstrate that patients with anti-PIT-1 antibody syndrome present with a thymoma and show substantial evidences that the thymoma plays a crucial role in the development of this disease.
The clinical characteristics of the 3 patients with anti-PIT-1 antibody syndrome were previously described in detail 8 and described briefly as follows: Patient 1. A 44-year-old man without growth and developmental delay who presented with facial, finger, and arm edema as a result of central hypothyroidism. Endocrinological provocative test revealed that the secretion of GH and PRL were completely blunted, and that of TSH was severely impaired. Patient 2. A 75-year-old man with a history of slowly progressive insulin-dependent diabetes mellitus showed central hypothyroidism. The anterior pituitary function was in level to what was observed in patient 1. Autopsy and histological analysis was performed. Patient 3. A 78-year-old man showed acquired central hypothyroidism. The anterior pituitary function was similar in level to what was observed in both patients 1 and 2. Circulating anti-PIT-1 antibody was detected in all these patients.

Results
Thymomas were diagnosed in all patients with anti-PIT-1 antibody syndrome. During the follow up, a mediastinal tumor was detected by checkup chest X-ray and subsequent computed tomography (CT) imaging confirmed the diagnosis in patient 1 ( Fig. 1a and b). The tumor was resected and histological analysis revealed a diagnosis of type B2 thymoma ( Fig. 1c and d). The patient did not undergo any immunotherapy including steroids during the clinical course. In patient 2, we examined the autopsy specimen and found a type AB thymoma (Fig. 1e). It is characterized by a dense infiltration of lymphocytes in these tumors. In addition, chest CT revealed an anterior mediastinal tumor with a suspicion of thymoma in patient 3 ( Fig. 1f and g). Because of the advanced age, the tumor has been carefully observed in this patient. These data indicate that although one case has not been histologically proven, all the patients with anti-PIT-1 antibody syndrome were associated with thymomas, suggesting that thymoma plays a role in the pathogenesis of this syndrome.

Aberrant expression of PIT-1 protein in the neoplastic cortical thymic epithelial cells. It is well
known that thymoma is causally associated with several autoimmune diseases such as MG. Because acquired immune intolerance to PIT-1 plays a pivotal role in the development of anti-PIT-1 antibody syndrome 10,13,19 , we hypothesized that PIT-1-reactive T cells may be positively selected in the thymoma tissues. Since it has been reported that the aberrant expression of AChR is associated with autoimmunity to AChR in MG 20 , we analyzed the expression of PIT-1 protein in the thymoma tissue. First, we verified that the serum of patients with thymoma without hypopituitarism did not have anti-PIT-1 antibody ( Table 1, Fig. 2a). It indicated that anti-PIT-1 antibody is specific for the patients of anti-PIT-1 antibody syndrome. Intriguingly, immunoblotting analysis demonstrated a substantial expression of PIT-1 protein in the thymoma of patient 1 (Fig. 2b, lane 6). In contrast, PIT-1 protein was not detected in the thymoma tissues from patients with MG or without MG (Fig. 2b,  Thymectomy drastically improved the immune response to PIT-1. We examined the titer of anti-PIT-1 antibody by serial dilution using preserved sera before and after thymectomy in patient 1 ( Fig. 3a and Supplementary Figure). Intriguingly, before thymectomy, the titer remained unchanged; however, after thymectomy the titer decreased by one eighth during the following 2 years. We further evaluated the peripheral CTLs reacted with PIT-1 protein before and after thymectomy. Convincingly, CTLs reacted with PIT-1 protein, as detected by ELISpot assay, diminished after thymectomy ( Fig. 3b and c).

Discussion
In this report, we have demonstrated that thymoma was associated with all patients with anti-PIT-1 antibody syndrome. PIT-1 was highly expressed in the neoplastic cortical thymic epithelial cells in the two cases examined. Importantly, after thymectomy, the titer of anti-PIT-1 antibody substantially decreased and specific CTLs for PIT-1 diminished in one patient who underwent surgery. These data strongly suggest that thymoma that aberrantly expressed PIT-1 plays an essential role in the development of this disease.
It is well known that thymoma is closely associated with the pathogenesis of MG. Thymomas are histologically classified into types A, AB, B1, B2, and B3 (WHO classification) 21 . It has been reported that autoimmune diseases are associated particularly with types AB and B 22 , as observed with our cases. Approximately 10% of patients with autoimmune MG have a thymoma, which plays a role in disease initiation through multiple mechanisms including the expression of self-antigens such as AChR and impaired negative selection of autoreactive T cells 23 . In anti-PIT-1 antibody syndrome, it is speculated that the aberrant expression of PIT-1 evoked positive selection for PIT-1-reactive T cells and the defect in negative selection in thymoma tissue induced breakdown in immune tolerance for PIT-1.
One of the key questions is the reason for the aberrant expression of PIT-1 in the thymoma. Recently, it has been reported that mutations of epigenetic regulatory genes are common in thymic neoplasms 24 , suggesting that epigenetic changes such as histone modification, chromatin remodeling, and DNA methylation pathways may occur and cause a dysregulated expression of silencing gene in thymoma. It is possible that such epigenetic changes cause the aberrant expression of PIT-1 in the thymoma tissue of anti-PIT-1 antibody syndrome. It has also been reported that PIT-1 was aberrantly expressed in some tumor tissues, such as breast cancer and  acute myeloid leukemia cells, and plays an important role in the carcinogenesis, promotion of tumor growth and metastasis 25,26 . Although the mechanisms, in which PIT-1 was aberrantly expressed in the breast cancer have not been elucidated, it has been reported that deregulation of several embryonic transcription factors including POU homeobox genes were expressed at high levels in human breast cancer [27][28][29] . Similar mechanisms might be present in the thymoma tissue of anti-PIT-1 antibody syndrome.
In MG, the anti-AChR antibody is directly responsible for the pathogenesis 23,30 . Anti-AChR antibodies are the most common type of the pathogenesis of MG. Anti-AChR antibody activates the classical complement pathway, formation of the membrane attack complex initiated by activated C3, causes severe structural injury of endplates and lyses the postsynaptic membrane 31 . We examined the pathogenic role of anti-PIT-1 antibody in detail but we could not find any effects 13 , suggesting that this antibody is only a marker of this syndrome. On the other hand, PIT-1-reactive CTLs plays an essential role in the development of the disease 13 . In MG, it has been speculated that the first alteration in intratumorous T cell thymopoiesis may involve the generation of muscle-specific CTLs, and the resulting muscle damage could lead to various autoimmune responses including a production of autoantibodies against AChR and intracellular antigens 32 . It is speculated that anti-PIT-1 antibody may be produced after the injury of PIT-1 expressing cells by CTL and the resulting exposure of the nuclear protein PIT-1.
Interestingly, not only did the titer of anti-PIT-1 antibody decrease but peripheral PIT-1-reactive CTLs also diminished after thymectomy without any immunosuppressive therapy, suggesting that the thymoma played an essential role in the breakdown of immune tolerance for PIT-1. In MG, disease remission or improvement is expected following thymectomy 33 . In the patients with anti-PIT-1 antibody syndrome who underwent thymectomy, although the titer of antibody and PIT-1-reactive CTLs decreased, we could not observe an improvement of anterior pituitary function at two years after thymectomy ( Table 2). As shown in the autopsy case, GH-, PRL-, TSH-, and PIT-1-positive cells were absent, with inflammation and marked fibrosis observed in the anterior pituitary 10 . This suggests that when hormone secretion was impaired, target cells might already be irreversibly injured by CTLs. However, because an increase in remission rates over 7 to 10 years has been reported in MG 34 , it is necessary to follow anterior pituitary function in the patients over the long term. It has been reported that some thymoma patients may have impaired anterior pituitary function; however, detailed examination has not been performed 35 . Although the prevalence of anti-PIT-1 antibody syndrome in patients with thymoma is unknown, it is warranted to screen pituitary function in patients with thymoma especially presenting with symptoms related to hypothyroidism.
It is also known that thymoma is associated with paraneoplastic syndrome, especially paraneoplastic neurological syndrome such as paraneoplastic encephalomyelitis and paraneoplastic limbic encephalitis [36][37][38] . Most of the patients reveal autoantibodies against the common antigens between the tumor and central nervous system. Some of the antibodies play a pathogenic role in the development of disease and the resection of tumor is efficacious. In these aspects, it is considerable that anti-PIT-1 antibody syndrome is a paraneoplastic syndrome. It is speculated not only thymoma but also other tumors with an aberrant expression of PIT-1 protein may cause this syndrome.
In conclusion, we define anti-PIT-1 antibody syndrome as a thymoma-associated autoimmune disease, which exhibits an acquired and specific GH, PRL, and TSH deficiency.

Methods
Patients. This study was approved by the ethics committee of Kobe University Graduate School of Medicine and all methods were performed in accordance with the guidelines. The patients provided a written informed consent for the analysis. Titration analysis of anti-PIT-1 antibody, ELISpot assay, and western-blotting analysis were performed using samples from patient 1. Immunohistochemical analysis of thymoma tissues were performed in both patients 1 and 2. In patient 3, the tumor has not been resected and has carefully been observed.
The clinical characteristics of control patients with thymoma without anti-PIT-1 antibody syndrome were shown in Table 1. Enzyme-Linked Immunospot (ELISpot) assay. ELISpot assays were performed according to the manufacture's instruction as previously described 11 . Briefly, aliquots of 2.0 × 10 5 isolated lymphocytes from peripheral blood per well were incubated with recombinant PIT-1 protein (10 μ g/mL, Santa Cruz Biotechnology) in 96-well microtiter plates pre-coated with anti-human IFN-γ antibody for 48 hours (ELISpot PRO for Human IFN-γ ; Mabtech, Stockholm, Sweden). After washing, biotin-conjugated anti-cytokine antibody (7-B6-1) was added and incubated followed by streptavidin conjugated with alkaline phosphatase. Finally, 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate solution was added and incubated for 48 hours. Stimulation by Ab-mCD3-2 was used as a positive control. All assays were performed with quadruplicate for each antigen. The spots were counted viewing through a microscope by 2 independent investigators in a blinded way. Homogeneously stained spots were evaluated as positive.