Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members.

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.

Incidence, morphology, and ultrastructure of spontaneous thymoma--the most common neoplasm in W/Nhg rats.

Spontaneous thymoma was observed with an incidence of 97 and 36% in female and male rats, respectively, from an inbred Wistar/Neuherberg strain (W/Nhg). The thymomas often caused dyspnea and were occasionally the direct cause of death. The neoplasms resembled human thymomas and showed a variable cell composition, ranging from mainly lymphocytic to mainly epithelial. The detailed ultrastructural findings are described and compared with those in other rat thymomas and in human thymomas. A characteristic feature of all dividing lymphocytes was the presence of often multilayered, confronting cisternae. As in more than 50% of human thymomas, W/Nhg rat thymomas were not associated with myopathies or any other possibly autoimmune diseases. They may thus offer a useful model for the study of thymoma without associated parathymic syndromes.

[Clinicopathologic study of metaplastic thymoma].

OBJECTIVE: To study the clinicopathologic characteristics of metaplastic thymoma. METHODS: Resection specimens of 3 cases of metaplastic thymoma were studied by light microscopy, immunohistochemistry and electron microscopy. RESULTS: All the 3 patients were females and aged 33, 58 and 45 years respectively. Histologically, a biphasic growth pattern, consisting of intimate admixture of epithelial cells and spindle cells, was noted. The epithelial cells showed mild cytologic atypia, sometimes nuclear grooves and pseudonuclear cytoplasmic inclusions. These cells were arranged in nests and anastomosing cords. Mitotic figures were rarely seen. On the other hand, the spindle cells were bland-looking, mitotically inactive and arranged in fascicles. Immunohistochemical study showed that the epithelial cells strongly expressed cytokeratin (AE1/AE3) but not vimentin or CD5. The proliferation index, as demonstrated by Ki-67 immunostaining, was about 3% to 5%. In contrast, the spindle cells were diffusely positive for vimentin and epithelial membrane antigen. Staining for CD5 and CD20 was negative. The background lymphocytes were positive for CD3, but not for TdT and CD99. Ultrastructurally, well-formed desmosomes or hemidesmosomes were identified in the epithelial element. They were not detected within the spindle cells. CONCLUSION: Metaplastic thymoma is a rarely encountered indolent or low-grade thymic tumor and may represent a distinct clinicopathologic entity.

Absence of protein kinase C in nuclei of EL4 mouse thymoma cells.

Since evidence indicates that phorbol ester-induced production of interleukin 2 requires transcription, we investigated the possibility that the phorbol ester receptor acts directly in the nuclei of EL4 thymoma cells. Using a procedure that minimized plasma membrane contamination (as measured by 5'-nucleotidase activity) and maintained the integrity of the double nuclear membrane, we were unable to detect specific binding of [3H]phorbol 12,13-dibutyrate in nuclei of unstimulated cells. Treatment of cells with phorbol 12,13-dibutyrate (100 nM, 37 degrees C) for up to 6 h did not cause appearance of phorbol ester binding capacity in nuclei (4 +/- 8% of homogenate value; 5'-nucleotidase activity = 10 +/- 3%) despite translocation of 40% of the cytosolic binding capacity to the plasma membrane fraction. The failure to detect nuclear binding capacity in treated cells was not due to occupation of nuclear sites with unlabeled ligand; effective exchange binding was demonstrated by recovery of total homogenate binding capacity in treated cells of 82 +/- 13% of that in untreated cells. Treatment of isolated nuclei with DNase to liberate DNA binding proteins also failed to reveal any nuclear phorbol ester binding capacity. Assay of nuclei for protein kinase C enzymatic activity gave similar negative results. These data argue strongly against a direct action of the intact phorbol ester receptor (or the phorbol ester binding fragment) in the transcriptional activation of interleukin 2 in EL4 cells but cannot rule out the possibility of a role for the catalytic fragment.

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse.

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl2 and 5 mM MgCl2, and centrifuged at 52 000 X g for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of (Na+ + K+)-ATPase, Mg2+-ATPase and 5'-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40 degrees C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.

A simple and rapid method for the preparation of plasma membranes.

A simple and rapid method for preparing plasma membranes from isolated cells or tissues is described. The membranes were characterised (a) biochemically by an analysis of specific marker enzymes, (b) by quantitation of cell surface receptors, and (c) immunologically by their ability to elicit specific allogeneic responses from cytotoxic T cells in secondary in vitro stimulations. Based on both biochemical and immunologic criteria, plasma membranes prepared by the method described here are of equal or greater 'purity' compared to those prepared by two other methods that are most widely used to date and the yields are several-fold higher.

Undifferentiated epithelial-rich invasive malignant thymoma: complete response to cisplatin, vinblastine, and bleomycin therapy.

Two cases of complete remission plus one almost complete and another partial response of undifferentiated, invasive epithelial malignant thymoma using the combination of cisplatin, vinblastine, and bleomycin (PVB), are reported in four patients treated with this combination. Radiotherapy was instituted after completing the fourth course of chemotherapy in three patients. One patient died from intercurrent infection after the fourth cycle of combination chemotherapy. Three patients remain free of disease at the end of the treatment program. PVB appears to be highly active in this disease and deserves more extensive evaluation in multicenter clinical trials.

Sex steroid hormone receptors in human thymoma.

In this study we examined the immunohistochemical localization of sex steroid receptors for estrogen alpha (ER alpha) and ER beta, progesterone-A (PR-A) and PR-B, and androgen (AR) in human thymoma (n = 132) and correlated these findings with various clinicopathological parameters. We used RT-PCR and real-time PCR to further study the expression of these receptors in 20 thymoma cases. Immunoreactivity for all sex steroid receptors was detected in the nuclei of thymoma epithelial cells. The percentage of immunopositive cases and the H-score values for each receptor (mean +/- SD) were: ER alpha, 66% and 85.8 +/- 80.2; ER beta, 7% and 7.2 +/- 8.7; PR-A, 4% and 2.7 +/- 4.9; PR-B, 49% and 55.8 +/- 68.3; and AR, 15% and 14.1 +/- 11.7, respectively. The results of real-time PCR were consistent with those of immunohistochemistry, especially results for ER alpha, PR-B, and AR. A significant positive correlation was detected between immunoreactivity for ER alpha and PR-B. ER alpha immunoreactivity was inversely correlated with tumor size, clinical stage, WHO classification, and Ki-67 labeling index. In addition, the status of ER alpha immunoreactivity was significantly associated with a better clinical outcome in thymoma patients. Results from our study suggest that estrogens may inhibit thymoma growth via ER alpha, and that ER alpha immunoreactivity may act as a prognostic factor in human thymoma.

Thymoma with pseudosarcomatous stroma: report of an unusual histologic variant of thymic epithelial neoplasm that may simulate carcinosarcoma.

Six cases are described of an unusual type of primary thymic epithelial neoplasm characterized by a biphasic epithelial/spindle cell morphology that closely resembled a carcinosarcoma. The patients were two women and four men 28-70 years of age. The tumors presented clinically as asymptomatic anterior mediastinal masses found incidentally on routine chest radiographs. All patients were treated by complete surgical excision. Grossly, the tumors consisted of well-circumscribed, encapsulated masses that measured 6-14 cm in greatest diameter and showed a gray-white, homogeneous, rubbery cut surface. Histologically, the lesions were composed of anastomosing islands and cords of oval to polygonal epithelial cells displaying large nuclei with occasional prominent nucleoli and rare mitotic figures, separated by areas containing a highly cellular spindle cell proliferation without nuclear atypia. Thymic remnants could be identified in the periphery of the lesions in four cases. Immunohistochemical stains showed diffuse strong positivity for keratin and focally for epithelial membrane antigen (EMA) in the epithelial cell component, and strong positivity for vimentin and focally for actin in the spindle cell stromal component. Stains for keratin, EMA, desmin, S-100 protein, and CD34 were negative in the spindle stromal cells in all cases except one, in which EMA positivity was present; CD5 stains were negative in the epithelial cells in all cases examined. Electron microscopic examination in one case showed well-formed desmosomes and tonofilaments in the epithelial elements, as well as features indicative of fibroblastic differentiation in the spindle stromal cells. Because of the unusually florid spindle cell stromal component and the focally atypical features of the epithelial cells, some of these tumors initially were misinterpreted as examples of carcinosarcoma. Clinical follow-up in five cases showed that the patients were alive and without evidence of disease over a period of 5-20 years (mean follow-up 10 years), suggesting a benign or very low grade malignant biologic behavior. The present cases appear to represent an unusual, previously undescribed morphologic variant of thymoma characterized by a prominent pseudosarcomatous stromal component. Because of the distinctive histologic appearance and indolent clinical behavior, these lesions should be distinguished from other more aggressive anterior mediastinal neoplasms displaying a biphasic morphology.

Ectopic hamartomatous thymoma. A distinctive benign lesion of lower neck.

Five cases of a distinctive tumor located in the soft tissues of the lower neck in adults are described. The lesion is characterized microscopically by the presence of four components: cellular areas made up of spindle elements; epithelial islands composed of solid nests, trabeculae and cysts; mature adipose tissue; and lymphocytes, sometimes arranged in a Hassall's corpuscle-like fashion. Electron microscopy and immunostaining for keratin showed that the spindle cell component was of epithelial nature. These tumors were supraclavicular or suprasternal in location, and no local recurrence has developed in any case following local resection. We interpret these lesions as benign tumors arising on the basis of a developmental defect. We think that they are derived from the third branchial arch and that they are composed of abnormal thymic tissue.

Thymomas express epitopes shared by the ryanodine receptor.

Myasthenia gravis (MG) patients with thymoma have antibodies against ryanodine receptor (RyR) of skeletal and heart muscle. In this study, thymomas were examined for reactivity with a panel of polyclonal rabbit antibodies against various short peptides of RyR. An antibody against peptide C2 in the transmembrane region of RyR stained thymoma epithelial cells in cryosections of 17/23 thymomas, and detected a 40-kDa peptide in Western blotting of a thymoma membrane fraction. The other RyR antibodies did not react with thymoma tissue. The anti-C2 RyR antibody did not react with normal thymus, tonsil or carcinoma of colon. The results strongly indicate that epithelial thymoma cells express an epitope shared by the transmembrane region of skeletal and cardiac muscle RyR.

Extracellular matrix of the human thymus: immunofluorescence studies on frozen sections and cultured epithelial cells.

The immunohistochemical detection of elements of the human thymic extracellular matrix in situ and in vitro is described. In the normal thymus, the intracapsular and intraseptal fibers were strongly labeled by anti-type I collagen antiserum. Basement membranes bordering the capsule, septae, and perivascular spaces were intensely stained by anti-type IV collagen, anti-fibronectin, and anti-laminin sera. In hyperplastic myasthenia gravis thymuses, the major changes consisted of discontinuities of the basement membrane adjacent to clusters of epithelial (keratin-containing) cells, among which an unusual connective framework (densely labeled by all the antisera) was observed. In vitro, most epithelial cells were strongly labeled by antifibronectin serum and to a lesser extent by the anti-type IV collagen and anti-laminin sera. In addition, fibronectin, laminin, and type IV collagen were detected in the intercellular spaces bordering the epithelial cells in culture. Results show that thymic epithelial cells participate in the synthesis of extracellular matrix elements, which as a result of their localization and influence on epithelial cell growth, should be regarded as constitutive components of the thymic microenvironment.

Kappa opioid receptors expressed on three related thymoma cell lines. Differences in receptor-effector coupling.

The mouse thymoma R1.1 cell line was shown previously to express a single high-affinity kappa 1 opioid receptor that is negatively coupled through a pertussis toxin-sensitive G-protein to adenylyl cyclase. This study compared opioid receptor binding and inhibition of adenylyl cyclase activity in three unique derivatives of the R1.1 cell line. Membranes from the R1.G1 and R1E/TL8x.1.G1.OUAr.1 (R1EGO) cell lines bound both [3H]U69,593 and [3H](-)-bremazocine with similar affinities compared with R1.1 membranes, whereas membranes from the R1E/TL8x.1 (R1E) cell line did not possess any opioid binding sites, detected by radioreceptor binding. The Bmax values for [3H]U69,593 and [3H]-(-)-bremazocine binding to R1.G1 and R1EGO cell membranes were, respectively, 3- and 6-fold greater than those obtained with the parent R1.1 cell line. GTP and its nonhydrolyzable analog, Gpp(NH)p, inhibited [3H]U69,593 binding to all three cell lines. Stimulation of low-Km GTPase activity by the kappa-selective agonist (-)U50,488 was greatest in R1.G1 membranes, followed by R1EGO and R1.1. The maximal inhibition of forskolin-stimulated adenylyl cyclase activity by (-)U50,488 was 66 +/- 2% in R1.G1 and 49 +/- 2% in R1EGO, compared with 37 +/- 1% in R1.1 membranes. Whereas maximal inhibition of adenylyl cyclase activity did not correlate with receptor number among cell lines, the inhibition of cyclic AMP production did correlate with stimulation of low-Km GTPase activity. The R1.1 cell line and its derivatives, R1.G1 and R1EGO, express a similar type of kappa opioid receptor, which exhibits differences in coupling to G-proteins and to adenylyl cyclase among cell lines. These cell lines provide an excellent model system for studying the regulation of opioid receptor-adenylyl cyclase coupling efficiency.

Thymic neoplasia in two male siblings.

Thymic tumors are uncommon, and malignant variants of such neoplasms are rare. Hence, the association of two siblings with aggressive tumors of the thymus is not likely to be a coincidence. We report on two brothers with thymic epithelial neoplasms, one being a thymic carcinoma and the other an invasive spindle cell thymoma with associated hypogammaglobulinemia.

Immunologic and ultrastructural characterization of the small cell population in malignant thymoma.

A case of malignant thymoma is presented. The electron microscopic (EM) findings of the lymphocyte-epithelial relationships were similar to these previously described. Utilizing specific monoclonal antibodies to differentiate thymocytes from T-lymphocytes, the small cell population with ultrastructural features of lymphoid cells consisted of true thymocytes rather than peripheral T-lymphocytes. The latter finding may prove to be of importance in differentiating thymomas from reactive or most neoplastic lymphocytic processes involving the thymus gland.

Primary thyroid thymoma: a distinct clinicopathologic entity.

A 51-year-old man presented with a paratracheal tumor. He had undergone resection of a thyroid tumor 15 years previously; at that time, the histologic diagnosis had been anaplastic carcinoma. When the tumor recurred, the presumptive clinical diagnosis was medullary thyroid carcinoma. Histologic examination revealed a poorly differentiated epithelial tumor with immunoreactivity for keratins, carcinoembryonic antigen, and, focally, S-100 protein. The tumor was negative for calcitonin and thyroglobulin. There were scattered lymphocytes and plasma cells. Ultrastructural examination showed elongated epithelial cells with prominent desmosomes and bundles of cytoplasmic tonofilaments but no secretory granules; amyloid was not present ultrastructurally or histochemically. The characteristic ultrastructural and immunocytochemical features and the clinical behavior of this tumor verify the existence of primary thyroid thymoma. This new primary thyroid neoplasm is of clinical importance, considering the more benign behavior of primary thyroid thymoma than of other tumors in the differential diagnosis of this lesion.

False aneuploidy in benign tumors with a high lymphocyte content: a study of Warthin's tumor and benign thymoma.

The quality of results of flow cytometric DNA content analysis of formalin-fixed, paraffin-embedded tissue may be affected by a number of preanalytical variables. We performed flow cytometric DNA content analysis on two types of benign tumors to investigate the effect of a prominent lymphocytic component: Warthin's tumor (N = 20) and benign thymoma (N = 8). Malignant tumors (N = 23) were included as DNA aneuploid controls. All tissues studied were archival material processed using Hedley's technique either without prolonged rehydration in water (day 0 samples) or with 24- or 48-hour rehydration (day 1 and day 2 samples, respectively). Image cytometric DNA ploidy analysis was also performed on most cases. Eight cases (40%) of Warthin's tumor and five cases (63%) of benign thymoma showed either hyperdiploid peaks or marked asymmetry on the day 0 DNA histograms; nine of the malignant tumors were aneuploid. The DNA histogram abnormalities of the benign tumors could be gated out by excluding the lymphocyte nuclei. None of the DNA indices of the benign tumors corresponded with expected deviations based on published chromosomal studies. All of the DNA histogram abnormalities of the benign tumors disappeared and/or fused with the main peaks on the day 1 or day 2 samples, except for one case of benign thymoma. All the DNA aneuploid peaks on the malignant tumors persisted with prolonged rehydration. Image cytometric DNA analysis showed a diploid pattern in all benign tumors. We conclude that a high lymphocyte content may be a cause of false aneuploidy in these benign tumors. Furthermore, the degree of rehydration appears to be an important factor in achieving optimum fluorochrome staining of DNA.

Neuroendocrine differentiation in thymic epithelial tumors with special reference to thymic carcinoma and atypical thymoma.

To determine the neuroendocrine (NE) features of thymic epithelial tumor, immunohistochemistry and electron microscopy studies were performed on eight NE tumors (thymic carcinoids) and 26 non-NE tumors (nine thymic carcinomas, five atypical thymomas, and 12 thymomas other than lymphocytic thymoma). Immunohistochemical studies were performed with antibodies against general markers for NE cells (synaptophysin, alpha subunit of a guanine nucleotide-binding protein, Go, and small-cell lung carcinoma cluster 1 antigen), and a broad panel of antibodies for hormonal substances. Thymic carcinoid showed synchronous diffuse immunoreactivity for the three NE markers and contained cells that were positive for a variety of hormonal products: human chorionic gonadotropin (hCG) alpha-subunit (eight of eight), hCG beta-subunit (three of eight), adrenocorticotropic hormone (ACTH) (three of eight), calcitonin (two of eight), calcitonin gene-related peptide (two of eight), and serotonin (one of eight). Conversely, although positivity for NE markers was neither synchronous nor diffuse in non-NE tumors, seven of nine thymic carcinomas, three of five atypical thymomas (focal or dispersed distribution), and none of the five thymomas were positive for at least two of these NE markers. A small number of neoplastic cells were positive for hCGalpha-subunit or ACTH in three thymic carcinomas and one atypical thymoma. Ultrastructurally, dense core granules (DCG) were much more frequent in thymic carcinoid, but several DCG-like granules were identified in 12 of 13 non-NE tumors with or without immunoexpression of NE markers. The presence of focal or dispersed NE cells in thymic carcinoma and atypical thymoma may reflect multidirectional differentiation within the tumor, which, like cytological atypia, epithelial CD5 expression, and lack of immature T cell infiltration, may be another feature of this group at thymic tumors.

Ectopic hamartomatous thymoma: a case report with immunohistochemical and ultrastructural studies.

A case of ectopic hamartomatous thymoma (EHT) arising in the supraclavicular region of a 52-year-old male is presented. The well-defined tumor measuring 1.7x1.5x0.7 cm consisted of three components: spindle cell (70%), epithelial (25%), and adipose (5%). The spindle cell component was characterized by sheet-like, haphazard and short fascicular arrangements of bland spindle cells. Neither mitotic figures nor cellular pleomorphism were found. Admixed with, and adjacent to, the spindle cell areas was an obviously epithelial component of variable appearance, ranging from glandular spaces lined by mainly cuboidal clear cells, irregularly anastomosing cords, and strands of epithelial cells to irregular solid nests of squamous epithelium with dark and clear cytoplasm. Myoepithelial cells were also observed. Immunohistochemically, the spindle cells were strongly and diffusely positive for cytokeratins and some of them were positive for BRST2, alpha-smooth muscle actin, and CD10. The tumor was negative for S-100 protein, glial fibrillary acidic protein, and CD34. Ultrastructurally, tonofilaments and desmosomes were observed in the spindle cells. The findings indicate an epithelial origin. The patient was well without recurrence or metastasis 8 months after excision. Pathologists and clinicians should be aware of the existence of ectopic hamartomatous thymoma in the supraclavicular or suprasternal region and should differentiate it from a high-grade sarcoma, such as biphasic synovial sarcoma or glandular malignant peripheral nerve sheath tumor.

Hodgkin's disease of the thymus (granulomatous thymoma) and myasthenia gravis: a unique association.

A 19-year-old girl who had myasthenia gravis was found to have thymic Hodgkin's disease or "granulomatous thymoma." The myasthenia regressed completely following surgical removal of the thymic lesion. This association of Hodgkin's disease of the thymus and myasthenia appears unique. Possibly pathogenetic implications are discussed.