Type B thymomas in patients with myasthenia gravis display a distinctive pattern of αß TCR and IL-7 receptor α expression on CD4+CD8+ thymocytes.

Thymoma is closely associated with myasthenia gravis (MG). However, due to the heterogeneity of thymoma and the intricate pathogenesis of MG, it remains unclear why some patients with thymoma develop MG and others do not. In this study, we conducted a comparative phenotype analysis of thymocytes in type B thymomas in patients with MG (MG (+) thymomas) and without MG (MG (-) thymomas) via fluorescence-activated cell sorting (FACS). Our results show that the developmental stages defined by the expression of CD3, CD4, and CD8 were largely maintained in both MG (+) and MG (-) thymomas, with CD4+CD8+ cells constituting the majority of thymocytes in type B thymoma, and no significant difference between this cell population was observed in MG (+) and MG (-) thymomas.We discovered that CD4+CD8+ thymocytes in MG (+) thymomas expressed low levels of αß TCR and high levels of IL-7 receptor α (IL-7Rα), whereas in MG (-) thymomas, CD4+CD8+ thymocytes exhibited the opposite pattern of αß TCR and IL-7Rα expression. These results suggest that the positive and negative selection processes of CD4+CD8+ thymocytes might differ between MG (+) thymomas and MG (-) thymomas. The expression of the Helios transcription factor is induced during negative selection and marks a group of T cells that have undergone negative selection and are likely to be deleted due to strong TCR binding with self-peptides/MHC ligands. We observed that the percentage of Helios-positive CD4SP T cells was greater in MG (-) than in MG (+) thymomas. Thus, the differentially regulated selection process of CD4+CD8+ thymocytes, which involves TCR and IL-7/IL-7Rα signaling, is associated with the presence of MG in type B thymomas.

Loss of YAP1 C-terminus expression as an ancillary marker for metaplastic thymoma: a potential pitfall in detecting YAP1::MAML2 gene rearrangement.

AIMS: Metaplastic thymoma is a rare thymic tumour characterized by Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) gene fusions resulting from an intrachromosomal inversion of chromosome 11. Immunohistochemistry with an antibody directed against the C-terminus of YAP1 has shown loss of expression in YAP1-rearranged vascular neoplasms, poromas, and porocarcinomas. This study aimed to validate an anti-YAP1 C-terminal antibody as an ancillary immunohistochemical marker for the diagnosis of metaplastic thymoma. MATERIALS AND METHODS: Ten metaplastic thymomas were selected for the current study. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed to detect YAP1::MAML2 fusions. We then performed immunohistochemistry to detect YAP1 C-terminus expression in 10 metaplastic thymomas, 50 conventional thymomas (10 each of type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, and type B3 thymoma) and seven thymic carcinomas. RESULTS: All 10 cases showed narrow split signals with a distance of nearly two signal diameters and sometimes had false-negative results in YAP1 and MAML2 break-apart FISH (BA-FISH). Abnormal colocalized signals of the YAP1::MAML2 fusion were observed in all 10 cases using fusion FISH (F-FISH) assays. Eight of 10 cases with adequate nucleic acids were successfully sequenced and all showed YAP1::MAML2 fusions; in two cases the fusions were detected by both DNA and RNA sequencing and in six cases by RNA sequencing only. YAP1::MAML2 fusion transcripts were identified in four cases by RT-PCR. Metaplastic thymoma showed loss of YAP1 C-terminus expression in all 10 (100%) cases. All other thymic neoplasms showed retained YAP1 C-terminus expression. CONCLUSION: YAP1 C-terminus immunohistochemistry is a highly sensitive and specific ancillary marker that distinguishes metaplastic thymoma from its mimics. BA-FISH assays could not effectively detect YAP1::MAML2 fusions due to the proximity of the two genes. Loss of YAP1 C-terminus expression is a reliable surrogate for the detection of YAP1::MAML2 fusions in metaplastic thymoma.

Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice.

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3+/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3+/- and Phlda3-/- littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Aberrant Dendritic Cell Subsets in Patients with Myasthenia Gravis and Related Clinical Features.

INTRODUCTION: Dendritic cells (DCs) play critical roles in the pathogenesis of myasthenia gravis (MG), and a series of DC-based experimental strategies for MG have recently been developed. However, the definite roles of different DC subsets in the mechanism of MG have scarcely been covered by previous studies. The present study aimed to investigate the levels of three main DC subsets, plasmacytoid DCs (pDCs) (CD303 positive) and two distinct subsets of conventional DCs (cDCs), namely CD1c+ cDCs and CD141+ cDCs, in MG patients and analyze related clinical features. METHODS: From January 2016 to December 2020, 160 newly diagnosed MG patients and matched healthy controls (n = 160) were included in the study, and their clinical data were collected. The blood samples from MG patients before treatment and controls were collected for flow cytometry analysis. A total of 14 MG thymoma, 24 control thymoma, and 3 thymic cysts were used to immunostain the DC subsets. RESULTS: The flow cytometry analysis showed a significantly higher frequency of circulating pDCs, CD1c+ cDCs, and CD141+ cDCs in MG patients than in healthy controls (p < 0.001 for all). Patients with early-onset MG (<50 years old) had a lower frequency of circulating pDCs but a higher frequency of circulating CD1c+ cDCs than those with late-onset MG (≥50 years old) (p = 0.014 and p = 0.025, respectively). The frequency of circulating pDCs was positively associated with the clinical severity of late-onset MG patients (r = 0.613, p < 0.001). 64.3% (9/14) of MG thymoma is of type B2 under the World Health Organization classification, which is higher than that in control thymoma (33.3%, 8/24) (p = 0.019). For type B2 thymoma, there were significantly more pDCs but fewer CD1c+ cDCs in MG thymoma than in the controls. CONCLUSION: The distribution of aberrant pDCs, CD1c+ cDCs, and CD141+ cDCs in MG patients displayed age- and thymoma-related differences, which may contribute to the impaired immune tolerance and lead to the onset of MG.

HNRNPA2B1 as a potential therapeutic target for thymic epithelial tumor recurrence: An integrative network analysis.

Thymic epithelial tumors (TETs) are rare malignant tumors, and the molecular mechanisms of both primary and recurrent TETs are poorly understood. Here we established comprehensive proteomic signatures of 15 tumors (5 recurrent and 10 non-recurrent) and 15 pair wised tumor adjacent normal tissues. We then proposed an integrative network approach for studying the proteomics data by constructing protein-protein interaction networks based on differentially expressed proteins and a machine learning-based score, followed by network modular analysis, functional enrichment annotation and shortest path inference analysis. Network modular analysis revealed that primary and recurrent TETs shared certain common molecular mechanisms, including a spliceosome module consisting of RNA splicing and RNA processing, but the recurrent TET was specifically related to the ribosome pathway. Applying the shortest path inference to the collected seed gene module identified that the ribonucleoprotein hnRNPA2B1 probably serves as a potential target for recurrent TET therapy. The drug repositioning combined molecular dynamics simulations suggested that the compound ergotamine could potentially act as a repurposing drug to treat recurrent TETs by targeting hnRNPA2B1. Our study demonstrates the value of integrative network analysis to understand proteotype robustness and its relationships with genotype, and provides hits for further research on cancer therapeutics.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

Characterization of IL-10-producing regulatory B cells in thymoma.

BACKGROUND: Regulatory B cells (Bregs) are a subset of B cells that secrete interleukin 10 (IL-10) and play a vital role in suppressing the immune response. The aim of this study was to evaluate the proportion of Bregs in patients with thymoma. METHODS: The proportions of subgroups of Bregs in 23 patients with thymoma and 15 healthy controls were detected by flow cytometry. The serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels of the subjects were measured using a cytometric bead array (CBA). RESULTS: The proportions of circulating IL-10+ B cells, IL-10+CD24hiCD38hi Bregs, and IL-10+CD24hiCD27+ Bregs and the serum IL-10 level were significantly higher in patients with thymoma than in the control group and were negatively correlated with the Karnofsky Performance Scale (KPS) score. The serum levels of cytokines IL-2, IL-6, IFN-γ, and TNF-α were higher and serum IL-17A level was lower in patients with thymoma. Patients with advanced-stage thymoma exhibited significantly higher proportions of IL-10-producing Bregs and a higher serum IL-10 level. After tumour resection, the frequency of circulating IL-10+CD24hiCD38hi Bregs and the serum IL-10 level were significantly decreased in patients with thymoma. The serum IL-10 levels exhibited the best accuracy in assessing the risk of thymoma occurrence in this study. CONCLUSIONS: The expression of IL-10 produced by Bregs is increased in patients with thymoma, particularly those with advanced-stage disease, which may suggest that Bregs are involved in the pathogenesis and progression of thymoma.

Oncogenic Runx1-Myc axis in p53-deficient thymic lymphoma.

p53 deficiency and Myc dysregulation are frequently associated with cancer. However, the molecular mechanisms linking these two major oncogenic events are poorly understood. Using an osteosarcoma model caused by p53 loss, we have recently shown that Runx3 aberrantly upregulates Myc via mR1, a Runx consensus site in the Myc promoter. Here, we focus on thymic lymphoma, a major tumour type caused by germline p53 deletion in mice, and examine whether the oncogenic Runx-Myc axis plays a notable role in the development of p53-deficient lymphoma. Mice lacking p53 specifically in thymocytes (LP mice) mostly succumbed to thymic lymphoma. Runx1 and Myc were upregulated in LP mouse lymphoma compared with the normal thymus. Depletion of Runx1 or Myc prolonged the lifespan of LP mice and suppressed lymphoma development. In lymphoma cells isolated from LP mice, knockdown of Runx1 led to Myc suppression, weakening their tumour forming ability in immunocompromised mice. The mR1 locus was enriched by both Runx1 and H3K27ac, an active chromatin marker. LP mice with mutated mR1 had a longer lifespan and a lower incidence of lymphoma. Treatment with AI-10-104, a Runx inhibitor, improved the survival of LP mice. These results suggest that Myc upregulation by Runx1 is a key event in p53-deficient thymic lymphoma development and provide a clinical rationale for targeting the Runx family in p53-deficient malignancies.

Arginase Pathway Markers of Immune-Microenvironment in Thymic Epithelial Tumors and Small Cell Lung Cancer.

BACKGROUND: Key regulators of antitumor immunity such as arginase-1 and the adenosine pathway may have an important role in modulating the effect of immunotherapy. Here, we investigated the expression profile of these immune-related biomarkers in thymic epithelial tumors (TETs) and small cell lung cancer (SCLC), 2 solid tumors where immune checkpoint inhibitors have activity. MATERIALS AND METHODS: Immunohistochemical staining was performed using tissue microarrays of 123 TET (110 thymoma and 13 thymic carcinoma) and 125 SCLC cases. The expression profile of the following immune-related biomarkers was assessed: arginase-1, CD39, CD73, A2AR, PD-L2, and CD15. The expression profile was also correlated with clinical data. RESULTS: No sample was positive for arginase-1. In the adenosine pathway, the prevalence of positive staining for CD39, CD73, and A2AR was 4.9%, 2.5%, and 69.2%, in TETs and 0%, 1.7%, and 50.8%, in SCLC. The multivariate analysis showed that CD39 expression was significantly associated with worse disease related survival (hazard ratio [HR], 10.36; 95% confidence interval [CI]: 2.01-53.47; P= .005) and a shorter time-to progression (HR, 11.35; 95% CI, 2.11-61.23; P = .005) in TETs. Other biomarkers were not associated with disease related survival or time to progression in TETs. No biomarker was associated with survival in SCLC. CONCLUSION: Arginase-1 was not detectable in TETs and SCLC. Expression of markers in the adenosine pathway were present in both TETs and SCLC. CD39 expression in tumor cells may identify subsets of patients with TETs with an unfavorable prognosis.

Exploiting mesothelin in thymic carcinoma as a drug delivery target for anetumab ravtansine.

BACKGROUND: Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. METHODS: We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody-drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma. RESULTS: Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth. CONCLUSIONS: These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo.

Thymic stroma and TFII-I: towards new targeted therapies.

Thymic epithelial tumors (TETs) have been characterized at the molecular level through bioptic sections and cell lines. Despite these advances, there is a need for a more thorough characterization of the thymic stroma in thymoma, particularly because of the diversity of cell types that populate the tumor and the absence of a healthy thymic counterpart. Recent work on healthy pediatric thymi - both in vitro and at the single-cell level - now sets the stage for new studies on their neoplastic counterparts. Furthermore, general transcription factor IIi (GTF2I), a thymoma-specific oncogene, as well as some of its SNPs, are increasingly associated with autoimmune disease, a significant feature of thymomas. We summarize recent discoveries in the field and discuss the development of new targeted therapies.

Osteoblast-specific inactivation of p53 results in locally increased bone formation.

Inactivation of the tumor suppressor p53 (encoded by the Trp53 gene) is relevant for development and growth of different cancers, including osteosarcoma, a primary bone tumor mostly affecting children and young adolescents. We have previously shown that deficiency of the ribosomal S6 kinase 2 (Rsk2) limits osteosarcoma growth in a transgenic mouse model overexpressing the proto-oncogene c-Fos. Our initial aim for the present study was to address the question, if Rsk2 deficiency would also influence osteosarcoma growth in another mouse model. For that purpose, we took advantage of Trp53fl/fl mice, which were crossed with Runx2Cre transgenic mice in order to inactivate p53 specifically in osteoblast lineage cells. However, since we unexpectedly identified Runx2Cre-mediated recombination also in the thymus, the majority of 6-month-old Trp53fl/fl;Runx2-Cre (thereafter termed Trp53Cre) animals displayed thymic lymphomas, similar to what has been described for Trp53-deficient mice. Since we did not detect osteosarcoma formation at that age, we could not follow our initial aim, but we studied the skeletal phenotype of Trp53Cre mice, with or without additional Rsk2 deficiency. Here we unexpectedly observed that Trp53Cre mice display a unique accumulation of trabecular bone in the midshaft region of the femur and the humerus, consistent with its previously established role as a negative regulator of osteoblastogenesis. Since this local bone mass increase in Trp53Cre mice was significantly reduced by Rsk2 deficiency, we isolated bone marrow cells from the different groups of mice and analyzed their behavior ex vivo. Here we observed a remarkable increase of colony formation, osteogenic differentiation and proliferation in Trp53Cre cultures, which was unaffected by Rsk2 deficiency. Our data thereby confirm a critical and tumorigenesis-independent function of p53 as a key regulator of mesenchymal cell differentiation.

Increased carbohydrate antigen 19-9 expression in a thymic neuroendocrine tumor.

Here, we report a case of carbohydrate antigen (CA) 19-9-producing mediastinal neuroendocrine tumor (NET) (atypical carcinoid). A 54-year-old woman with no specific relevant medical history was referred to our hospital because of increased CA19-9 (95.3 U/ml) detected on health screening. Chest computed tomography (CT) revealed an anterior mediastinal mass without localized lymphadenopathy. Thoracic surgery was performed and the histopathological diagnosis was thymic CA19-9-positive NET. The patient developed mediastinal lymph node metastasis at 1 year (CA19-9: 413 U/ml) and multiple bone metastases 4 years (CA19-9: 2303 U/ml) after surgery. Increased CA19-9 levels paralleled the clinical courses of relapse. To our knowledge, this is the first report of CA19-9-producing thymic NET.

PD-1 preferentially inhibits the activation of low-affinity T cells.

Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.

Survival and Functional Immune Reconstitution After Haploidentical Stem Cell Transplantation in Atm-Deficient Mice.

Hematopoietic stem cell transplantation (HSCT) has been proposed as a promising therapeutic opportunity to improve immunity and prevent hematologic malignancies in Ataxia-telangiectasia (A-T). However, experience in the transplantation strategy for A-T patients is still scarce. The aim of this study was to investigate whether different approaches of HSCT are feasible in regard to graft versus host response and sufficient concerning functional immune reconstitution. Atm-deficient mice were treated with a clinically relevant non-myeloablative host-conditioning regimen and transplanted with CD90.2-depleted, green fluorescent protein (GFP)-expressing, and ataxia telangiectasia mutated (ATM)-competent bone marrow donor cells in a syngeneic, haploidentical or allogeneic setting. Like syngeneic HSCT, haploidentical HSCT, but not allogeneic HSCT extended the lifespan of Atm-deficient mice through the reduction of thymic tumors and normalized T-cell numbers. Donor-derived splenocytes isolated from transplanted Atm-deficient mice filled the gap of cell loss in the naïve T-cell population and raised CD4 cell functionality up to wild-type level. Interestingly, HSCT using heterozygous donor cells let to a significantly improved survival of Atm-deficient mice and increased CD4 cell numbers as well as CD4 cell functionality equivalent to HSCT using with wild-type donor cells. Our data provided evidence that haploidentical HSCT could be a feasible strategy for A-T, possibly even if the donor is heterozygous for ATM. However, this basic research cannot substitute any research in humans.

Potential Prognostic Value of Preoperative Leukocyte Count, Lactate Dehydrogenase and C-Reactive Protein in Thymic Epithelial Tumors.

Thymic epithelial tumors are the most common mediastinal tumors. Surgery is the mainstay of treatment and complete resection provides the best survival rate. However, advanced tumors often require multimodality treatment and thus we analyzed the prognostic potential of routine circulating biomarkers that might help to risk-stratify patients beyond tumor stage and histology. Preoperative values for white blood cell count (WBC), C-reactive protein (CRP) and lactate dehydrogenase (LDH) were analyzed in 220 thymic epithelial tumor patients operated between 1999 and 2018. Increased CRP levels (>1 mg/dl) were significantly more often measured in thymic carcinoma and neuroendocrine tumors when compared to thymoma. LDH serum activity was higher in thymic neuroendocrine tumors when compared to thymoma or thymic carcinoma. The median disease specific survival was significantly longer in thymoma cases than in thymic carcinoma and neuroendocrine tumors. Increased preoperative LDH level (>240 U/L) associated with shorter survival in thymus carcinoma (HR 4.76, p = 0.0299). In summary, higher CRP associated with carcinoma and neuroendocrine tumors, while LDH increased primarily in neuroendocrine tumors suggesting that biomarker analysis should be performed in a histology specific manner. Importantly, preoperative serum LDH might be a prognosticator in thymic carcinoma and may help to risk stratify surgically treated patients in multimodal treatment regimens.

Identification and Characterization of Non-Coding RNAs in Thymoma.

BACKGROUND Thymoma is the most common tumor of the anterior mediastinum, and can be caused by infrequent malignancies arising from the epithelial cells of the thymus. Unfortunately, blood-based diagnostic markers are not currently available. High-throughput sequencing technologies, such as RNA-seq with next-generation sequencing, have facilitated the detection and characterization of both coding and non-coding RNAs (ncRNAs), which play significant roles in genomic regulation, transcriptional and post-transcriptional regulation, and imprinting and epigenetic modification. The knowledge about fusion genes and ncRNAs in thymomas is scarce. MATERIAL AND METHODS For this study, we gathered large-scale RNA-seq data belonging to samples from 25 thymomas and 25 healthy thymus specimens and analyzed them to identify fusion genes, lncRNAs, and miRNAs. RESULTS We found 21 fusion genes, including KMT2A-MAML2, HADHB-REEP1, COQ3-CGA, MCM4-SNTB1, and IFT140-ACTN4, as the most frequent and significant in thymomas. We also detected 65 differentially-expressed lncRNAs in thymomas, including AFAP1-AS1, LINC00324, ADAMTS9-AS1, VLDLR-AS1, LINC00968, and NEAT1, that have been validated with the TCGA database. Moreover, we identified 1695 miRNAs from small RNA-seq data that were overexpressed in thymomas. Our network analysis of the lncRNA-mRNA-miRNA regulation axes identified a cluster of miRNAs upregulated in thymomas, that can trigger the expression of target protein-coding genes, and lead to the disruption of several biological pathways, including the PI3K-Akt signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. CONCLUSIONS Our results show that overexpression of this miRNA cluster activates PI3K-Akt, FoxO, HIF-1, and Rap-1 signaling pathways, suggesting pathway inhibitors may be therapeutic candidates against thymoma.

Downregulation of DNMT3a expression by RNAi and its effect on NF-κBs expression of thymic epithelial cells.

OBJECTIVE: To understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire). METHODS: Tissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software. RESULTS: In 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased. CONCLUSIONS: Our study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment.

Expression patterns for Bcl-2, EMA, ß-catenin, E-cadherin, PAX8, and MIB1 in thymomas.

The expression of immunohistochemical markers has been extensively investigated in thymomas to assist in the differential diagnosis. We have studied six select markers to determine their utility in the evaluation of these tumors. A series of 126 thymomas including 33 type A, 27 type AB, 20 type B1, 22 type B2, and 24 type B3, were examined utilizing a tissue microarray (TMA) technique with antibodies to e-cadherin, ß-catenin, PAX8, bcl-2, EMA, and MIB-1. Keratin AE1/AE3 and p63 were used for quality control. A significant finding was strong and consistent positivity for bcl-2 in type A (90%) and type AB (88.8%) thymoma, while 100% of B1, B2, and B3 were negative. The distribution of e-cadherin and ß-catenin was not useful for differential diagnosis. E-cadherin and ß-catenin were expressed in a high proportion of all the tumors (92-100%), except for B2 thymoma which showed only 45% expression. A significant increase in the expression of the MIB-1 proliferation marker (mean: 12.8% nuclear positivity) was also observed in B3 thymoma compared with the other histologic types. Statistical significance was confirmed using Kruskal's non-parameterized test for distribution. EMA was generally negative except for spindle cells in the fibrous septa in types A and AB thymoma. PAX8 showed less consistent nuclear staining than p63 and was only widely expressed in 55.7% of cases. Bcl-2 may serve as a useful marker to separate spindle cell thymomas (Type A and AB) from the other types, and the MIB1 proliferation index may be of use to differentiate type B2 from type B3 thymoma.

Expression of cancer testis antigens in thymic epithelial tumors.

Cancer testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. However, to our knowledge, expression of the antigens in thymic epithelial tumors has not been examined yet. We examined the immunohistochemical expression of five CTAs (MAGE-A, NY-ESO-1, MAGE-C1, SAGE and GAGE7) in 192 cases of thymic epithelial tumor. The CTAs were variably expressed in the thymic epithelial tumors. Type B component of type AB thymomas, type B1/B2/B3 thymomas, and thymic carcinomas showed a generally positive correlation between the malignancy grades and positive expression rates in four CTAs other than MAGE-C1. In thymic squamous cell carcinomas (SqCCs), four antigens except for MAGE-C1 showed high expression rates ranging from 23.1% to 43.6%. In the prognostic analysis, a positive expression of SAGE (P = 0.0485) and GAGE7 (P = 0.0289) were associated with a shorter overall survival in type B2/B3 thymomas, respectively. In thymic SqCC, a positive MAGE-A expression was significantly associated with an increased level of programmed death ligand in tumor-infiltrating lymphocytes (P = 0.0181). We showed (i) a frequent CTA expression, (ii) a general correlation of CTA expression with tumor malignancy grades and (iii) a prognostic impact in some of the CTAs.