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Thymomas exhibit a unique genomic landscape, comprising the lowest on average total mutational burden among adult human cancers; a unique point mutation in the GTF2I gene in WHO type A and AB thymomas (and rarely others); almost unique KMT2A-MAML2 translocations in rare WHO type B2 and B3 thymomas; a unique YAP1-MAML2 translocation in almost all metaplastic thymomas; and unique miRNA profiles in relation to GTF2I mutational status and WHO histotypes. While most thymomas can be diagnosed solely on the basis of morphological features, mutational analyses can solve challenging differential diagnostic problems. No molecular biomarkers have been identified that predict the response of unresectable thymomas to chemotherapy or agents with known molecular targets. Despite the common and strong expression of PDL1 in thymomas, immune checkpoint inhibitors are rarely applicable due to the poor predictability of common, life-threatening autoimmune side effects that are related to the unrivaled propensity of thymomas towards autoimmunity.

A unique feature of thymomas is their unrivaled frequency of associated myasthenia gravis (MG). Previous studies reported that MG+ thymomas contain a larger number of mature "pre-emigrant" CD4+ T cells than MG- thymomas and that most thymomas do not contain AIRE expressing cells irrespective of MG status. These findings suggest that CD4+ T cells that mature inside the abnormal microenvironment of thymomas and egress to the blood are critical to the development of thymoma-associated MG (TAMG) irrespective of thymoma histotype. However, underlying mechanisms have remained enigmatic. To get hints to mechanisms underlying TAMG, we pursue three hypotheses: (i) Functional pathways with metabolic and immunological relevance might be differentially expressed in TAMG(+) compared to TAMG(-) thymomas; (ii) differentially enriched pathways might be more evident in immature lymphocyte-poor (i.e., tumor cell/stroma-rich) thymoma subgroups; and (iii) mechanisms leading to TAMG might be different among thymoma histological subtypes. To test these hypotheses, we compared the expression of functional pathways with potential immunological relevance ( N = 380) in relation to MG status separately in type AB and B2 thymomas and immature lymphocyte-rich and lymphocyte-poor subgroups of these thymoma types using the TCGA data set. We found that <10% of the investigated pathways were differentially upregulated or downregulated in MG+ compared to MG- thymomas with significant differences between AB and B2 thymomas. The differences were particularly evident, when epithelial cell/stroma-rich subsets of type AB and B2 thymomas were analyzed. Unexpectedly, some MG-associated pathways that were significantly upregulated in AB thymomas were significantly downregulated in B2 thymomas, as exemplified by the oxidative phosphorylation pathway. Conversely, the MG-associated pathway related to macrophage polarization was downregulated in MG+ AB thymoma and upregulated in MG+ B2 thymoma. We conclude that functional pathways are significantly associated with TAMG, and that some mechanisms leading to TAMG might be different among thymoma histological subtypes. Functions related to metabolisms, vascular and macrophage biology are promising new candidate mechanisms potentially involved in the pathogenesis of TAMG. More generally, the results imply that future studies addressing pathomechanisms of TAMG should take the histotype and abundance of tumor cells and non-neoplastic stromal components of thymomas into account., (Copyright © 2020 Yamada, Weis, Thelen, Sticht, Schalke, Ströbel and Marx.)

Background  Preoperative radiological assessment is important for clarification of surgical operability for advanced thymic tumors. Objective was to determine the feasibility of magnetic resonance imaging (MRI) with cine sequences for evaluation of cardiovascular tumor invasion. Patients and Methods  This prospective study included patients with advanced thymoma, who underwent surgical resection. All patients received preoperative computed tomography (CT) scan and cine MRI. Results  Tumor infiltration was surgically confirmed in the pericardium ( n  = 12), myocardium ( n  = 1), superior caval vein (SCV; n  = 3), and aorta ( n  = 2). A macroscopic complete resection was possible in 10 patients, whereas 2 patients with aortic or myocardial tumor invasion had R2 resection. The positive predictive value (PPV) was 50% for cine MRI compared with 0% for CT scan regarding myocardial tumor infiltration. The PPV for tumor infiltration of the aorta was 50%, with a higher sensitivity for the CT scan (100 vs. 50%). Infiltration of the SCV could be detected slightly better with cine MRI (PPV 75 vs. 66.7%). Conclusion  Cine MRI seems to improve the accuracy of preoperative staging of advanced thymoma regarding infiltration of cardiovascular structures and supports the surgical approach., (Georg Thieme Verlag KG Stuttgart · New York.)

Therapeutic options to cure advanced, recurrent, and unresectable thymomas are limited. The most important factor for long-term survival of thymoma patients is complete resection (R0) of the tumor. We therefore evaluated the response to and the induction of resectability of primarily or locally recurrent unresectable thymomas and thymic carcinomas by octreotide Long-Acting Release (LAR) plus prednisone therapy in patients with positive octreotide scans. In this open label, single-arm phase II study, 17 patients with thymomas considered unresectable or locally recurrent thymoma (n = 15) and thymic carcinoma (n = 2) at Masaoka stage III were enrolled. Octreotide LAR (30 mg once every 2 weeks) was administered in combination with prednisone (0.6 mg/kg per day) for a maximum of 24 weeks (study design according to Fleming´s one sample multiple testing procedure for phase II clinical trials). Tumor size was evaluated by volumetric CT measurements, and a decrease in tumor volume of at least 20% at week 12 compared to baseline was considered as a response. We found that octreotide LAR plus prednisone elicited response in 15 of 17 patients (88%). Median reduction of tumor volume after 12 weeks of treatment was 51% (range 20%-86%). Subsequently, complete surgical resection was achieved in five (29%) and four patients (23%) after 12 and 24 weeks, respectively. Octreotide LAR plus prednisone treatment was discontinued in two patients before week 12 due to unsatisfactory therapeutic effects or adverse events. The most frequent adverse events were gastrointestinal (71%), infectious (65%), and hematological (41%) complications. In conclusion, octreotide LAR plus prednisone is efficacious in patients with primary or recurrent unresectable thymoma with respect to tumor regression. Octreotide LAR plus prednisone was well tolerated and adverse events were in line with the known safety profile of both agents., Competing Interests: Dr. Kleylein-Sohn, Dr. May and Dr. Baierlein are employees of Novartis. Dr. Boy is an employee of Asclepios clinic. Prof. Schalke reports grants from Novartis within and outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors declare no conflicts of interest.

In this review article, state-of-the-art diagnostic tools and innovative treatments of thymoma and thymic carcinoma (TC) are described with special respect to advanced tumour stages. Complete surgical resection (R0) remains the standard therapeutic approach for almost all a priori resectable mediastinal tumours as defined by preoperative standard computed tomography (CT). If lymphoma or germ-cell tumours are differential diagnostic considerations, biopsy may be indicated. Resection status is the most important prognostic factor in thymoma and TC, followed by tumour stage. Advanced (Masaoka-Koga stage III and IVa) tumours require interdisciplinary therapy decisions based on distinctive findings of preoperative CT scan and ancillary investigations [magnetic resonance imaging (MRI)] to select cases for primary surgery or neoadjuvant strategies with optional secondary resection. In neoadjuvant settings, octreotide scans and histological evaluation of pretherapeutic needle biopsies may help to choose between somatostatin agonist/prednisolone regimens and neoadjuvant chemotherapy as first-line treatment. Finally, a multimodality treatment regime is recommended for advanced and unresectable thymic tumours. In conclusion, advanced stage thymoma and TC should preferably be treated in experienced centres in order to provide all modern diagnostic tools (imaging, histology) and innovative therapy techniques. Systemic and local (hyperthermic intrathoracic chemotherapy) medical treatments together with extended surgical resections have increased the therapeutic options in patients with advanced or recurrent thymoma and TC., (© The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Background: Radical surgical resection of advanced thymic tumors invading either the heart or great vessels facing towards the heart is uncommonly performed because of the potential morbidity and mortality. To achieve a complete tumor resection, the use of cardiolpulmonary bypass (CPB) support might be necessary., Methods: Retrospective analysis of the results in six patients, who underwent radical tumor resection with CBP support., Results: Mean age was 46 years (27 to 66 years) and five patients were male. Tumor infiltration of the heart or the great vessels was evident in all patients. Five patients underwent induction therapy. Two patients were operated in complete cardioplegic arrest (antegrade cerebral perfusion: n = 1). Arterial cannulation of the ascending aorta (n = 5) or the femoral artery (n = 1) and venous cannulation of the right atrium (n = 4) or the femoral vein (n = 2) were performed. Resection of the left brachiocephalic vein (n = 6), resection of the superior caval vein (n = 2), the ascending aorta (n = 1) and the complete aortic arch with outgoing branches (n = 1) were performed. A macroscopic complete resection (R0/R1) was achieved in five patients, whereas one patient was resected incompletely (R2). In-hospital mortality was 0 %. Three (50 %) patients needed operative revision (hematothorax: n = 2, chylothorax: n = 1). All patients had a complicated postoperative course and developed respiratory insufficiency., Conclusions: Locally advanced thymoma/thymic carcinoma invading the heart or great vessels can be treated with radical surgical resection alongside with increased perioperative morbidity. The usage of CBP improves the chance of complete tumor resection in selected patients and might lead to a prolonged survival.

Thymoma-associated Myasthenia gravis (TAMG) is one of the anti-acetylcholine receptor MG (AChR-MG) subtypes. The clinico-pathological features of TAMG and its pathogenesis are described here in comparison with pathogenetic models suggested for the more common non-thymoma AChR-MG subtypes, early onset MG and late onset MG. Emphasis is put on the role of abnormal intratumorous T cell selection and activation, lack of intratumorous myoid cells and regulatory T cells as well as deficient expression of the autoimmune regulator (AIRE) by neoplastic thymic epithelial cells. We review spontaneous and genetically engineered thymoma models in a spectrum of animals and the extensive clinical and immunological overlap between canine, feline and human TAMG. Finally, limitations and perspectives of the transplantation of human and murine thymoma tissue into nude mice, as potential models for TAMG, are addressed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG - its associations with the CTLA4(high/gain-of-function) +49A/A genotype and with increased thymic export of naïve T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4(low) +49G(+) genotypes were more frequent (p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naïve T cells from the non-neoplastic thymuses of 36 LOMG patients was lower (p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of 'true LOMG' and an LOMG/early-onset MG overlapping group of patients between 40 and 60., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Extended thymoma resections including adjacent structures and pleurectomy/decortication (P/D) with hyperthermic intrathoracic chemotherapy (HITHOC) perfusion were performed in a multidisciplinary treatment regime., Patients and Methods: Between July 2000 and February 2012, 22 patients with Masaoka stage III (n = 9; 41%) and Masaoka stage IVa (n = 13; 59%) thymic tumors were included., Results: Mean age was 55 years (25-84 years) and 50% (11 out of 22) of patients were female. World Health Organization histological classification was as follows: B2 (n = 15), A (n = 1), B1 (n = 1), B3 (n = 2), and thymic carcinoma (C; n = 3). Radical thymectomy and partial resection of the mediastinal pleura and pericardium were performed. Of the 13, 9 patients with pleural involvement (stage IVa) received radical P/D followed by HITHOC (cisplatin). Macroscopic complete resection (R0/R1) was achieved in 19 (86%) patients. All patients received multimodality treatment depending on tumor stage, histology, and completeness of resection. Thirty-day mortality was 0% and three (13.6%) patients needed operative revision. Recurrence of thymoma was documented in five (22.7%) patients (stage III, n = 1; stage IVa, n = 4). Mean disease-free interval of patients with complete resection (n = 14 out of 22) was 30.2 months. After a mean follow-up of 29 months, 18 out of the 22 (82%) patients are alive. After P/D and HITHOC, 89% (8 out of 9 patients) are alive (current median survival is 25 months) without recurrence., Conclusions: Extended surgical resection of advanced thymic tumors infiltrating adjacent structures (stage III) or with pleural metastases (stage IVa) is safe and feasible. It provides a low recurrence rate and an acceptable survival. Additional HITHOC in patients with pleural thymoma spread seems to offer a better local tumor control., (Georg Thieme Verlag KG Stuttgart · New York.)

Aims: Morphological complexity hampers the histological classification of thymomas. Our aim was to determine whether the use of novel differentiation and maturation markers of cortical and medullary thymic epithelial cells (cTECs and mTECs) might provide an approach to understanding the underlying biology of these tumours., Methods and Results: Fifty-seven thymomas were studied by immunohistochemistry. The cortical markers used were B5T, PRSS16, and cathepsin V. The medullary markers used were CD40, claudin-4, AIRE, and desmin. Involucrin and cytokeratin 10 were used to study terminal mTEC maturation. Irrespective of histological subtype, most thymomas contained distinct areas with cortical and medullary differentiation. Type B1, type B2 and type AB thymomas showed marked bi-lineage differentiation, with lack of terminal mTEC maturation in type AB. Type AB thymomas were unique in showing areas where cells with either cortical or medullary differentiation were intimately 'mixed' at the single-cell level. Type B3 and type A thymomas showed only abortive lineage differentiation and maturation., Conclusions: Thymomas show highly characteristic patterns of bi-lineage TEC differentiation that reflect the histological subtypes recognized by the WHO classification. We hypothesize that thymomas arise from thymic precursor cells with different cortical and/or medullary maturation defects., (© 2013 John Wiley & Sons Ltd.)

Introduction: Patients with pleural thymoma spread (Masaoka stage IV a) should be treated within a multimodal treatment regime. However, the extent of local surgical resection to achieve optimal tumour control remains controversial., Patients and Methods: Prospective analysis between September 2008 and April 2013 of all patients with a Masaoka stage IV a thymoma, who underwent radical pleurectomy/decortication (P/D) followed by hyperthermic intrathoracic chemotherapy (HITHOC)., Results: A total of 11 patients (male n = 7; mean age 46.5 ± 11.4 years) with a primary stage IV a thymoma (n = 3) or thymoma with pleural relapse (n = 8) were included after successful transsternal thymoma resection. WHO histological classification was: B1 n = 1, B2 n = 6, B3 n = 3 and C n = 1. A radical P/D (5/11; 45 %) was extended with resection of the pericardium and diaphragm in 6/11 (55 %) patients. After surgical resection (91 % complete macroscopic R0/R1-resection) the HITHOC with cisplatin (100 mg/m2 body surface area (BSA) n = 7; 150 mg/m2 BSA n = 4) was performed for one hour at 42 °C. Operative revision was necessary in two patients (chylo- and hematothorax) with one patient also requiring temporary renal replacement therapy due acute renal failure (cisplatin 150 mg/m2 BSA). 30-day mortality was 0 %. Local recurrence (pulmonary n = 1, paravertebral n = 2) was documented in 3/10 (30 %) patients after R0/R1 resection. After a mean follow-up of 23 months the overall median survival was 27 months and 82 % (9/11) patients are still alive at the end of the study period., Conclusions: Masaoka stage IV a thymoma could be safely treated with lung-sparing radical P/D and HITHOC with cisplatin in a multimodality treatment regime. Early results with respect to recurrence and survival are encouraging, but further studies are warranted and we have to await long-term results., (Georg Thieme Verlag KG Stuttgart · New York.)

Autoimmune associations in myasthenia gravis (MG)-patients and their relatives have not been re-assessed since their separation into early- or late-onset MG (EOMG, LOMG), or thymoma-associated MG. Here, we analysed 226 EOMG-, 97 LOMG-, and 150 thymoma-patients for autoimmune disorders in themselves and their relatives. From 283 of them sera were tested for different organ- and non-organ-specific autoantibodies (autoAbs) by immunofluorescence test (IFT) and ELISA; genotyping was performed in 213 patients. Relatives with autoimmune disorders were reported by more patients with EOMG (40% of 210) than LOMG (20% of 89; p < 0.01) than thymomas (8% of 150; p < 0.001). In 150 genotyped EOMG-females, the known risk allele of the immuno-regulatory PTPN2 2 (R620W) appeared commoner in those with second autoimmune diseases (p ∼ 0.06), or with autoimmune relatives (p ∼ 0.03), than in those without. Organ-specific autoAbs were found in ∼ 30% of all MG-patients, autoAbs to striated muscle only in patients with thymoma-MG (62%) or LOMG (61%). Titers against adrenal cortex were lower in LOMG-patients. Disease-associated autoAbs against systemic targets or 'natural autoAbs' - except of autoAbs to nuclei - were uncommon in all groups (< 13%). Thus-with rare exceptions in EOMG and LOMG-we found minimal support for the notion that autoimmune patients have wide-ranging autoreactivity that causes disease only if it targets such Achilles' heels as the muscle acetylcholine receptor; even in thymoma-patients the autoAbs are sharply focused on a restricted range of muscle, cytokine and endocrine targets., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. All rights reserved.)

Background: Assessing the pharmacokinetics of intrapleurally administered cisplatin during hyperthermic intrathoracic chemotherapy perfusion (HITHOC) following pleurectomy/decortication in patients with malignant pleural mesothelioma or advanced thymoma with pleural spread., Methods: Pharmacokinetic analysis (ICP-MS) of intrapleural cisplatin with a dosage of 100 mg/m(2) (n = 5) or 150 mg/m(2) (n = 5) at 42°C perfusate temperature. Simultaneous pleural perfusion fluid and serum samples were collected at the beginning and every 15 min. Serum samples were collected at the end of the operation, 6, 12, and 24 hr postoperative., Results: Mean cisplatin levels in the perfusate slightly decreased during the HITHOC. The mean area under the curve ratios (AUC perfusate :AUC serum ) of cisplatin were nearly similar. The mean AUCs of cisplatin in the perfusate were approximately 58 and 55 times greater than detected in the serum. The mean peak of cisplatin in the serum was reached after 1 hr of HITHOC. The AUC of cisplatin in the serum did not significantly differ (P = 0.18) between both groups up to 24 hr after perfusion., Conclusions: HITHOC with cisplatin provides a pharmacological advantage of high local intrapleural cisplatin concentrations. Elevation of the cisplatin dosage to 150 mg/m(2) did not lead to a significant increase of the systemic cisplatin concentration., (Copyright © 2013 Wiley Periodicals, Inc.)

Objectives: A combination of cytoreductive surgery and hyperthermic intrathoracic chemotherapy perfusion (HITHOC) was performed for the treatment of primary and secondary pleural malignancies. We describe the perioperative management and our clinical experience., Methods: Between September 2008 and August 2011, eight patients with pleural manifestation of thymoma (Masaoka stage IVa) and eight patients with malignant pleural mesothelioma (MPM) were prospectively enrolled. Postoperative morbidity, recurrence and survival rates were analysed., Results: All the patients received multimodality therapy, including chemotherapy, radiation and surgical resection (pleurectomy/decortication) followed by the HITHOC procedure. Chemotherapy perfusion was performed with cisplatin (100-150 mg/m(2)) at 42°C for 1 h. Severe chemotherapy-related complications were not observed. Reoperation was necessary in two patients. There was no 30-day mortality. The median stay on the intensive care unit was 1 day, and the median duration of hospitalization was 15 days. Pleural recurrence of thymoma was evident in one thymoma patient 6 months after HITHOC. At mean follow-up of 22 months, seven thymoma patients (7/8; 88%) are alive without recurrence. Tumour progression was present in six mesothelioma patients (6/8; 75%). Four patients (50%) with MPM are alive, including two with no evidence of mesothelioma, and the median survival is 18 months., Conclusions: Cytoreductive surgery in combination with HITHOC can be performed with acceptable morbidity and mortality rates in selected patients. Patients should be evaluated by an interdisciplinary team to determine their eligibility for this therapeutic alternative. Early clinical results may encourage the use of this surgical option to provide better local tumour control in a multimodality treatment setting.

Objective: The aim of this study was to analyze the clinicopathological factors, treatment strategies and survival rates after surgical resection of thymoma., Methods: Between 12/1997 and 5/2010, 42 patients underwent surgical resection of the thymus. The presence of a thymoma was determined by histological examination in 23 patients, while patients with hyperplasia of the thymus (n = 19) were excluded from further analysis., Results: Myasthenia gravis coexisted in 9/23 (39.1%) patients. Thymomas were classified according to the Masaoka staging system (I: n = 6 [26.1%], IIa: n = 7 [30.4%], IIb: n = 2 [8.7%], III: n = 1 [4.4%], IVa: n = 7 [30.4%]) and the WHO histological classification (A: n = 4 [17.4%], AB: n = 5 [21.7%], B1: n = 1 [4.4%], B2: n = 8 [34.8%], B3: n = 3 [13%], C: n = 2 [8.7%]). Recurrence of thymoma was documented in three (13%) patients. After a mean follow-up of 58.4 months, 21 (91.3%) patients are alive. The overall survival rate was 95% and 87.8%, at 2 and 5 years, respectively. The disease-free interval at 5 years was 85% for the 17 (73.9%) patients with complete resection., Conclusions: Surgical resection of thymoma is the preferred treatment, because it is safe and effective with a low rate of recurrence and a good long-term survival. Advanced and invasive thymomas require a multimodal approach for better local tumor control and further improvement of prognosis., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Thymomas are tumors of thymic epithelial cells. They associate more often than any other human tumors with various autoimmune diseases; myasthenia gravis is the commonest, occurring in 10-50% of thymoma patients, depending on the World Health Organization-defined histologic subtype. Most thymomas generate many polyclonal maturing T lymphocytes but in disorganized microenvironments Failure to induce self-tolerance may be a key factor leading to the export of potentially autoreactive CD4 progeny, thus predisposing to autoimmune diseases. Normally, the master Autoimmune Regulator promotes expression of peripheral tissue-restricted antigens such as insulin by medullary thymic epithelial cells and induction of tolerance to them. The failure of approximately 95% of thymomas to express autoimmune regulator is another feature potentially contributing to autoimmunity.

Paraneoplastic autoimmune diseases associate occasionally with small cell lung cancers and gynecologic tumors. However, myasthenia gravis (MG) occurs in at least 30% of all patients with thymomas (usually present at MG diagnosis). These epithelial neoplasms almost always have numerous admixed maturing polyclonal T cells (thymocytes). This thymopoiesis-and export of mature CD4(+)T cells-particularly associates with MG, though there are rare/puzzling exceptions in apparently pure epithelial WHO type A thymomas. Other features potentially leading to inefficient self-tolerance induction include defective epithelial expression of the autoimmune regulator (AIRE) gene and/or of major histocompatibility complex class II molecules in thymomas, absence of myoid cells, failure to generate FOXP3(+) regulatory T cells, and genetic polymorphisms affecting T-cell signaling. However, the strong focus on MG/neuromuscular targets remains unexplained and suggests some biased autoantigen expression, T-cell selection, or autoimmunization within thymomas. There must be further clues in the intriguing serological and cellular parallels in some patients with late-onset MG but without thymomas-and in others with AIRE mutations-and in the contrasts with early-onset MG, as discussed here.

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A-producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis.

Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) inhibits T-cell activation and interleukin-2 (IL-2) production. The PTPN22(gain-of-function)+1858T(+) genotypes predispose to multiple autoimmune diseases, including early-onset (non-thymomatous) myasthenia gravis (MG). The disease association and the requirement of IL-2/IL-2 receptor signaling for intrathymic, negative T-cell selection have suggested that these genotypes may weaken T-cell receptor (TCR) signaling and impair the deletion of autoreactive T cells. Evidence for this hypothesis is missing. Thymoma-associated MG, which depends on intratumorous generation and export of mature autoreactive CD4(+) T cells, is a model of autoimmunity because of central tolerance failure. Here, we analyzed the PTPN22 +1858C/T single nucleotide polymorphism in 426 German Caucasian individuals, including 125 thymoma patients (79 with MG), and investigated intratumorous IL-2 expression levels. Unlike two previous studies on French and Swedish patients, we found strong association of PTPN22 +1858T(+) genotypes not only with early-onset MG (P=0.00034) but also with thymoma-associated MG (P=0.0028). IL-2 expression in thymomas with PTPN22 +1858T(+) genotypes (P=0.028) was lower, implying weaker TCR signaling. We conclude that the PTPN22(gain-of-function) variant biases towards MG in a subgroup of thymoma patients possibly by impeding central tolerance induction.

Generation of autoreactive CD4(+) effector T cells and defective production of regulatory CD4(+) T cells inside thymomas contribute to the development of myasthenia gravis (MG) in >90% of MG(+) thymomas. The molecular basis of these abnormalities is unknown. We report here that a) expression levels of class II major histocompatibility complex (MHCII) genes are variably decreased in thymomas, most prominently in histological WHO types A and AB; b) epithelial cells of type A and AB thymomas exhibit signal transducer and activator of transcription (STAT-1)-related defects of interferon-gamma (IFN-gamma) signaling and human leukocyte antigen (HLA)-DR expression in vitro; c) the promoter III (pIII)- and pIV-driven splice variants of the MHCII transactivator (CIITA) play a key role in MHCII gene expression in thymus and thymomas; and d) the pIV CIITA promoter is heavily methylated in thymomas. Recently, we also found that expression of the autoimmune regulator (AIRE) gene is absent from approximately 95% of thymomas. Among all theses abnormalities, only better preserved expression levels of MHCII (P < 0.001) in thymomas were significantly associated with the presence of MG. Taking the association of a gain-of-function polymorphism of the CTLA-4 and PTPN22 gene with MG in thymomas into account, we conclude that these acquired cellular abnormalities of the thymoma microenvironment in concert with inherited genetic high-risk polymorphisms of immunoregulatory genes have an impact on intratumorous thymopoiesis and appear to tip the balance toward central tolerance failure and development of MG. The findings imply that IFN-gamma and STAT-1 signaling play a role in MHCII expression in the human thymus and in the pathogenesis of paraneoplastic MG.

Thymomas are thymic epithelial neoplasms, associated with a variety of autoimmune disorders (especially myasthenia gravis), that apparently result from aberrant intra-tumourous thymopoiesis and export of inefficiently tolerized T-cells to the periphery. The autoimmune regulator (AIRE) drives the expression of self-antigens in the thymic medulla and plays an essential role in 'central' tolerance in both humans and mice. However, while inactivating AIRE mutations result in the 'autoimmune polyendocrinopathy syndrome type 1' (APS-1), its major features are not well reproduced in AIRE-knock-out mice. Therefore, alternative human disease scenarios with concomitant AIRE deficiency may be valuable tools to test conclusions drawn from mouse models. Here we show, in a large series, that approximately 95% of thymoma patients are 'chimeric'; expression of AIRE and major AIRE-related autoantigens (eg insulin) were undetectable in their tumours but maintained in their remnant thymic tissue and lymph nodes. Notably, despite the AIRE-deficient thymopoiesis in thymomas, disorders and autoantibodies typical of APS-1 were distinctly uncommon in these patients. The one striking similarity was in the recently observed neutralizing anti-type I interferon (IFN) antibodies, which are found at diagnosis in 100% of patients with APS-1 and in approximately 60% of patients with thymomas, as we show here. We conclude that APS-1 type autoantigens must be protected from autoimmunity by mechanisms that do not extend to the muscle autoantigens so frequently targeted in thymoma patients but so rarely recognized in APS-1. Thus our findings argue strongly for a tolerogenic function of AIRE beyond its role in negative T-cell selection in human thymopoiesis, and/or for specific autoimmunization against muscle in thymomas., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Myasthenia gravis (MG) in thymoma patients depends critically on intratumorous generation and export of mature autoreactive CD4+ T cells. Why non-MG thymomas fail to produce CD4+ T cells is unknown. We studied three single-nucleotide polymorphisms of the cytotoxic T-lymphocyte-associated antigen 4(CTLA4) gene in thymoma patients, nonthymoma early-onset MG patients, and control subjects. Surprisingly, the CTLA4high genotype +49A/A, which is protective against several autoimmune diseases, exerted a prominent predisposing effect to paraneoplastic MG in thymoma patients. The unusual disease association with a CTLA4high genotype implies a unique pathogenesis of paraneoplastic MG, with high CTLA4 levels possibly supporting the nontolerogenic selection of CD4+ T cells in MG-associated thymomas.

Myasthenia gravis (MG) is the prime autoimmune manifestation of thymomas. We investigated the generation of T cells with a regulatory phenotype (T(R)) in thymomas with and without associated MG. In patients with MG(+) thymomas, maturation and export of T(R) cells but not of other T-cell subsets was significantly reduced. We conclude that imbalance between effector and regulatory T cells in thymomas may be involved in modulation of onset and/or severity of MG.

Purpose: Thymic epithelial tumors (TET) are rare epithelial neoplasms of the thymus with considerable histologic heterogeneity. This retrospective study focused on the correlation of WHO-defined TET histotypes with survival and tumor recurrence in a large cohort of patients receiving different modes of treatment., Patients and Methods: Two hundred twenty-eight patients were followed for up to 21 years (median, 60 months; range, 1 to 252 months) after primary surgery. Forty-two patients received adjuvant radiotherapy (mean dose, 53 Gy), and 33 patients received adjuvant chemotherapy., Results: Seventy-six (88%) of 86 patients with WHO type A, AB, and B1 thymomas were treated by surgery alone, with three tumor relapses after 3 to 10 years (median, 3.4 years). Twelve of 67 patients with WHO type B2 and B3 thymomas in Masaoka stages I and II were treated by adjuvant radiotherapy without evidence of tumor recurrence after 1 to 12 years (median, 4 years). Among 75 patients with B2 and B3 thymomas with incomplete resection or a tumor stage III or higher, the recurrence rate was 34% (n = 23) after 0.5 to 17 years (median, 5 years) in patients receiving adjuvant radiochemotherapy, compared to 78% (seven of nine patients) in patients without adjuvant radiochemotherapy. Incomplete tumor resection was associated with a high recurrence rate (65%) and a poor prognosis (P <.01)., Conclusion: The long-term outcome of TET patients is related to tumor stage, WHO histotype, completeness of surgical removal, and type of treatment. Prospective trials are warranted to formally address the efficacy of adjuvant therapy in the treatment of localized and advanced malignant TETs.

Thymomas are the only tumors that are proven to generate mature T cells from immature precursors. It is unknown, however, whether intratumorous thymopoiesis has an impact on the peripheral T-cell pool and might thus be related to the high frequency of thymoma-associated myasthenia gravis. This study shows, using fluorescence-activated cell sorting-based analyses and T-cell proliferation assays, that thymopoiesis and T-cell function in thymomas correspond with immunologic alterations in the blood. Specifically, the proportion of circulating CD45RA(+)CD8(+) T cells is significantly increased in patients with thymoma compared with normal controls, in accordance with intratumorous T-cell development that is abnormally skewed toward the CD8(+) phenotype. Moreover, it is primarily the proportion of circulating CD45RA(+)CD8(+) T cells that decreases after thymectomy. The results also demonstrate that T cells reactive toward recombinant autoantigens are distributed equally between thymomas and blood, whereas T-cell responses to foreign antigen (ie, tetanus toxoid) are seen only among circulating T cells and not among thymoma-derived T cells. These functional studies support the hypothesis that thymopoiesis occurring within thymomas alters the peripheral T-cell repertoire. Because many thymomas are enriched with autoantigen-specific T cells, a disturbance of circulating T-cell subset composition by export of intratumorous T cells may contribute to paraneoplastic autoimmune disease arising in patients with thymoma. (Blood. 2000;96:3872-3879)

Intratumorous expression of a 153-kd protein (p153), which contains an acetylcholine receptor-like epitope, is the only tumor marker described to date that significantly associates with thymoma in paraneoplastic myasthenia gravis (MG). Here, we report that p153 is identical to the midsize neurofilament, as verified by immunohistochemistry, immunofluorescence, and western blot analysis. Furthermore, the acetylcholine receptor-like epitope of the midsize neurofilament (NF-M) was identified by peptide epitope mapping. We also show, using T-cell proliferation assays, a significantly increased response of intratumorous T cells to a recombinant midsize neurofilament fragment in thymoma patients with MG compared with MG patients with thymic follicular hyperplasia or thymoma patients without MG. The T cells of thymic follicular hyperplasia and thymoma patients without MG seem to be unresponsive to NF-M. In contrast, we found increased T-cell responses to recombinant acetylcholine receptor fragments in MG patients in general compared with non-MG patients. Increased T-cell responses to NF-M in patients with paraneoplastic MG might be the result of an abnormal positive selection of immature T cells within thymomas, caused by the expression of NF-M in neoplastic thymic epithelial cells. Our results offer further evidence that NF-M expression in thymomas is an autoantigenic determinant in MG.

Objective: To investigate the association of MG with the transcription of muscular or neuronal acetylcholine receptor (AChR) subunit genes in thymomas., Background: Many steps in the pathogenesis of MG have been elucidated but, with rare exceptions, its etiology is unknown. In patients with MG with thymoma, the tumor probably elicits autoimmunity to AChR, but it is enigmatic why MG develops in some patients but not in others., Methods: Reverse transcriptase (RT)-PCR, immunohistochemistry, and immunofluorescence studies were carried out to investigate AChR expression in 35 patients with thymoma. Statistical analysis was used to specify significant differences between thymoma subtypes., Results: Considering all thymomas (n = 35), no correlation was found between MG status and AChR gene expression as detected by RT-PCR. However, when histologically defined thymoma subtypes were studied separately, transcription of the muscular AChR P3A- alpha-subunit gene was significantly associated (alpha < 0.01) with the occurrence of MG in mixed thymomas (n = 17), but not in thymomas of the cortical type. For the other muscular AChR subunits (P3A+ alpha isoform, beta, gamma, delta, and epsilon) and the alpha2 and beta4 neuronal AChR subunits, no such correlation was detected., Conclusions: Expression of the P3A AChR alpha-subunit gene might be important for the pathogenesis of MG in mixed thymomas, suggesting etiologic heterogeneity of paraneoplastic MG among patients with histologically different thymoma subtypes.

Somatostatin receptor scintigraphy with 111In-[DTPA-D-Phe1]-octreotide has the potential for visualizing primary and recurrent thymomas in patients with myasthenia gravis, whereas thymic hyperplasias fail to accumulate somatostatin analog peptides. We demonstrate somatostatin receptor imaging findings in a patient with a mixed encapsulated thymoma which exhibited intense 111In-[DTPA-D-Phe1]-octreotide uptake in early and late scans. In another patient with a history of malignant thymoma 111In-[DTPA-D-Phe1]-octreotide accumulation was clearly seen in a mass suspected to be a recurrence. This paper describes the imaging protocol including Single Photon Emission Computed Tomography (SPECT) and discusses the clinical applications of this feasible functional imaging method in patients with thymomas.

To gain insight into the pathogenesis of thymoma-associated myasthenia gravis, thymocyte maturation was investigated in mixed and cortical thymomas by three-color flow cytometry. Although we detected cells at all recognizable stages, we noted an unusual increased percentage of early CD4+/CD3- thymocytes--especially in mixed thymoma--and a pronounced decreased percentage of mature CD4+/CD3+ cells in cortical thymomas as well. The percentage of CD3+/CD69+ cells that arose after positive selection was reduced in both thymoma subtypes compared with control thymuses, which suggests differences in the rate or efficiency of positive selection particularly in mixed thymomas. Mature T cells in 10 of 11 thymomas were not activated in situ as shown by the absence of CD25 expression. After stimulation with recombinant human acetylcholine receptor alpha-subunit fragments, thymocytes from 8 of 11 thymomas of both subtypes proliferated more strongly than those from controls, regardless of whether the donors were myasthenic. Responses of residual thymus cells to tetanus toxoid correlated well with those of autologous blood T cells, whereas those from the thymomas clearly did not--implying minimal colonization of thymomas by mature recirculating T cells. In conclusion, our results show that cortical and mixed thymomas exhibited differences in thymocyte maturation. Nevertheless, both thymoma subtypes seem to contribute to the pathogenesis of paraneoplastic myasthenia gravis by generating naive but potentially autoaggressive T cells; in some thymomas, these cells may then be actively immunized inside the tumor.

Autoantibodies against both striated muscle proteins, particularly titin, and the acetylcholine receptor are a hallmark of thymoma-associated myasthenia gravis. However, the stimulus for these responses remains enigmatic as whole titin is not detectable in these tumors. This study reports that in thymomas with cortical differentiation many of the neoplastic epithelial cells expressed low and medium molecular weight neurofilaments detected with several antibodies (on selections and blots) and at the RNA level (by reverse transcriptase polymerase chain reaction). Moreover, higher molecular weight forms sharing at least one epitope with titin were detectable slightly less frequently, as were the more strongly phosphorylated epitopes. In stark contrast, in medullary and mixed thymomas, and especially in the normal thymus, immunoreactivity with anti-neurofilament antibodies was rare. This aberrant overexpression of a titin epitope by epithelial cells with antigen-presenting phenotype in an inappropriate cortical microenvironment suggests that they might autosensitize maturing T cells there and so initiate anti-titin autoimmunity in these patients.

Based on a study of 26 cases, the well-differentiated thymic carcinoma is described as a distinct organotypical carcinoma of the thymus with low-grade malignancy. It is characterized by a predominance of epithelial cells with usually low mitotic rate, an epidermoid differentiation with slight to moderate cytological atypia, the constant presence of interepithelial immature cortical thymocytes, lobular growth, and formation of epithelial palisades around perivascular spaces. The tumor occurs at age 14 to 76 years in both sexes. An association with myasthenia gravis is found in 77% of the patients, and 83% of the tumors show invasion of adjacent organs or endothoracic metastasis at primary operation. This rate is higher than in cortical thymomas (47%) but lower than in other thymic carcinomas (92%). Two of 18 patients with follow-up died of tumor recurrence and pleural metastasis. Well-differentiated thymic carcinoma can be related to cortical thymoma by common morphological features and a similar immunophenotype of epithelial cells. It must be differentiated from the lymphocyte-depleted cortical thymomas after corticosteroid treatment and from the benign epithelial-rich medullary thymomas.

To investigate the relationship between anti-acetylcholine receptor (AchR) autoimmunity and the occurrence of thymoma in a particular group of myasthenia gravis (MG) patients we analyzed DNA and RNA from MG-associated thymomas and control tissue by Southern and Northern blotting, respectively, using the AchR alpha, beta, gamma, delta and epsilon-subunit cDNAs or oligonucleotides as probes. Restriction analysis of genomic DNA showed an organization of AchR subunit genes in thymomas identical with that of normal tissues. In particular, in thymomas, there was no deletion of exon 4 of the alpha-subunit which encodes the main immunogenic region of the AchR. Dot and Northern blot analysis did not reveal transcription of any AchR subunit gene in thymomas. Instead, an RNA nucleotide sequence was identified in MG-associated thymomas that hybridized to an AchR oligonucleotide probe coding for amino acids 371-378 of the AchR alpha-subunit. With this sequence as a probe, three DNA restriction fragments in addition to a restriction fragment of the AchR alpha-subunit gene could be identified in the human gene. The findings suggest that proteins with extensive homology to the AchR are not expressed in MG-associated thymomas. However, there are three genomic loci in thymoma genomes with a very restricted homology to the AchR alpha-subunit gene. One of these loci might code for the cross-reacting AchR epitope detected in almost all MG-associated thymomas in contrast to thymomas without MG.

Expression by neoplastic thymic epithelial cells of acetylcholine-receptor (AChR) epitopes is associated with the presence of AChR autoantibodies and the development of myasthenia gravis. We studied thymic tumours from patients with and without myasthenia gravis for the expression of neurofilament epitopes by immunohistochemistry with four monoclonal antibodies. There was very little antibody binding in control samples (healthy thymus, or thymitis) or in medullary and mixed thymomas, but neurofilament epitopes were strongly expressed in all cortical thymomas and thymic carcinomas. In addition, the frequency of serum autoantibodies against axons was significantly higher among myasthenic patients with thymic epithelial tumours than among age-matched controls (7/10 vs 3/50; p less than 0.01).

The coincidence of autoantibodies against the acetylcholine receptor (AChR) and muscle striational antigens (SA) is a characteristic finding in thymoma-associated myasthenia gravis (MG), but their origins are still unresolved. Some common muscle antigens that were shown to be targets of anti-SA autoantibodies in thymoma-associated MG have also been detected in normal or neoplastic thymic epithelial cells, suggesting that the release of (eventually altered) antigens from the thymic tumors could elicit SA autoimmunity. In contrast to this model, we report here that titin, which is a recently reported target of SA autoimmunity, is not expressed in thymomas. In addition, we show that skeletal muscle type-II fibers exhibit a striational immunoreactivity with monoclonal antibody mAb155, which was previously identified to label a very immunogenic cytoplasmic epitope of the AChR and neoplastic epithelial cells of MG-associated thymomas. We conclude from these findings that titin autoimmunity in thymoma-associated MG is either due to a molecular mimicry mechanism involving tumor antigens (other than titin) or is a secondary phenomenon following release of titin from muscle. Based on the common immunoreactivity of the AChR, a striational antigen and thymoma, we suggest as the pathogenetic mechanism of thymoma-associated MGa "circulus vitiosus" in which SA autoimmunity could help maintain the AChR autoimmunity that is primarily elicited by the thymomas.

Thymic epithelial tumors from myasthenia gravis (MG) patients and non-neoplastic thymuses were investigated by immunohistochemistry for the expression of neurofilament (NF) epitopes. There was little immunoreactivity confined to the medulla in non-neoplastic thymuses and a faint staining only for a 200 kD NF epitope in medullary and mixed thymomas. In contrast, cortical thymomas and well-differentiated thymic carcinomas expressed epitopes of the 68 kD and 160 kD NF. Demonstrating anti-axonal and anti-NF autoantibodies in thymoma patients we conclude that "false-positive T cell selection" is a mechanism of autoimmunity in paraneoplastic MG.

Immunohistochemical studies have shown that almost all thymomas of myasthenia gravis patients contain at least one protein sharing an antigenic determinant with the nicotinic acetylcholine receptor (AchR) of human muscle. We describe the characterization of this protein (p153) which has a molecular weight of 153 and an isoelectric point of 5.0. By treatment of p153 with endoglycosidases, no significant glycosylation has been detected. Immunologically, p153 crossreacts with monoclonal antibodies against the amino acid sequence 371-378 of the alpha-chain of the AchR. No cross-reactivity to the main immunogenic region of the AchR nor an alpha-bungarotoxin binding site are found. By Western blotting, p153 was generally neither detectable in normal tissues nor extrathymic tumors with the exception of paraganglioma and neuroblastoma. In conclusion, the structure of p153 is apparently unrelated to the AchR from muscle or the alpha-bungarotoxin binding proteins from thymoma. Since there is no evidence for an AchR expression in thymoma, the antigenic homology of p153 with the nicotinic AchR might be relevant for triggering an intrathymomatous autosensitization of maturing T cells and could be responsible for the high association of thymomas with myasthenia gravis.

Thymomas from 12 patients with myasthenia gravis (MG) were investigated for the presence of epitopes of the alpha-subunit of the nicotinic acetylcholine receptor (AchR) using monoclonal antibodies (MAb) reacting against the AchR. In all but two of the tumors epitopes corresponding to antigenic determinants located on the cytoplasmic side of the AchR were identified. From eight thymomas cell lines were established that have been kept in culture for up to 6 months. The cultured cells expressed the same AchR-epitopes as did the primary tumors. During early passages the percentage of epithelial cells positive for the AchR epitopes approximately mirrored the percentage of positive cells in the original tumors. With passaging the relative number of positive cells usually declined but in some cultures an increase was observed. Three cell lines that showed extensive staining with an MAb against the AchR were radiolabeled to characterize the antigen. From protein extracts of these three cell lines proteins of 45 kd and 156 kd molecular weight (MW) were precipitated. These proteins are different from other proteins described in the context of both thymomas and MG. The negative reactivity with MAb against other epitopes of the alpha-subunit, especially against the main immunogenic region (MIR), speaks in favor of membrane-associated proteins of only limited crossreactivity to the AchR. A previous study found an almost exclusive occurrence of these AchR-epitopes in thymomas associated with MG, but not in other thymomas of similar histologic type. The expression of the proteins described here could therefore play a role in the triggering of the autoimmune process against the AchR of the motor, endplate in MG patients.

Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) inhibits T-cell activation and interleukin-2 (IL-2) production. The PTPN22gain-of-function+1858T(+) genotypes predispose to multiple autoimmune diseases, including early-onset (non-thymomatous) myasthenia gravis (MG). The disease association and the requirement of IL-2/IL-2 receptor signaling for intrathymic, negative T-cell selection have suggested that these genotypes may weaken T-cell receptor (TCR) signaling and impair the deletion of autoreactive T cells. Evidence for this hypothesis is missing. Thymoma-associated MG, which depends on intratumorous generation and export of mature autoreactive CD4(+) T cells, is a model of autoimmunity because of central tolerance failure. Here, we analyzed the PTPN22 +1858C/T single nucleotide polymorphism in 426 German Caucasian individuals, including 125 thymoma patients (79 with MG), and investigated intratumorous IL-2 expression levels. Unlike two previous studies on French and Swedish patients, we found strong association of PTPN22 +1858T(+) genotypes not only with early-onset MG (P=0.00034) but also with thymoma-associated MG (P=0.0028). IL-2 expression in thymomas with PTPN22 +1858T(+) genotypes (P=0.028) was lower, implying weaker TCR signaling. We conclude that the PTPN22gain-of-function variant biases towards MG in a subgroup of thymoma patients possibly by impeding central tolerance induction. [ABSTRACT FROM AUTHOR]