Prognostic value of DNA flow cytometry in thymomas and thymic carcinomas.

Thymomas are the most common anterior mediastinal masses. Malignant potential and prognosis are unrelated to histologic appearance. Deoxyribonucleic acid (DNA) flow cytometry is of prognostic significance in a variety of tumors. We reviewed the records of 35 patients who on pathologic examination had a thymoma or thymic carcinoma. Flow cytometric studies, including DNA indices (ploidy) and S phase fraction, were done on paraffin block specimens from 31 patients. We believe this is the first report of DNA flow cytometric studies in thymic pathology. Mean survival was 63.5 +/- 13.3 months for patients with benign thymomas, 10.5 +/- 4.6 months for patients with malignant thymomas, and 19.3 +/- 4.1 months for patients with thymic carcinomas. Patients with benign thymomas lived significantly longer than those with malignant thymomas (P = .001) and thymic carcinomas (P = .03). DNA flow cytometry demonstrated four aneuploid tumors (two benign thymomas and two malignant thymomas). All thymic carcinomas were diploid. There was no statistically significant difference among the groups. The mean S phase fraction was 15.22% for benign thymomas, 11.15% for malignant thymomas, and 14.31% for thymic carcinomas. No statistically significant difference was found among the groups. We conclude that flow cytometry is not a useful guide to malignant potential or prognosis in thymomas and thymic carcinomas.

Polyoma-induced thymic epithelial tumors: analysis by 2D gel electrophoresis of tumors upon labeling the entire tumor bearing host.

Injection of neonatal C3H/Bi mice with polyoma virus (PTA-5) results after 4 to 20 weeks in the development of thymic epithelial tumors in 2/3 of the mice. These tumors appear to be the result of a neoplastic proliferation or transformation of thymic epithelial cells. The tumors contain immature lymphoid infiltrates characteristic of normal tissue and express the same function as normal tissue by providing the microenvironment for T cell maturation. To establish more comprehensively the relation of neoplastic to normal thymic epithelial cells, tumor-bearing mice and their normal counterparts were labeled in vivo by injection of S35-L-methionine. The stroma and lymphoid cells were separated and analyzed by two dimensional gel electrophoresis followed by radiofluorography. It was found that the epithelial portion of the tumor differed in several structural polypeptide components from normal tissue while the polypeptide composition of the thymocyte population remained unaltered. It was concluded that the neoplasia is confined to the epithelium compartment and the function of the neoplastic organ is maintained.

Low-grade B-cell lymphoma of mucosa-associated lymphoid tissue arising in the thymus. A thymic lymphoma mimicking myoepithelial sialadenitis.

We describe two cases of primary low-grade B-cell lymphoma of the thymus that showed histological features of low-grade B-cell lymphoma arising in mucosa-associated lymphoid tissue (MALT). The appearances most closely resembled MALT lymphoma arising in myoepithelial sialadenitis (MESA). In both cases, the tumor was excised. In one case, there has been no recurrence in 4 years of follow-up without further treatment; in the second case, the tumor has involved an axillary lymph node. Immunohistochemistry showed light-chain restriction in both cases, and the B-cell phenotype was similar to that previously described in MALT lymphomas. The occurrence of MALT lymphoma in the thymus is consistent with the presence of mucosal structures (Hassall's corpuscles) and with recent descriptions of a native B-cell population in this organ. The relationship of this previously undescribed thymic low-grade B-cell MALT lymphoma arising in the thymus has not yet been clarified.

Biochemical characterization of thymic hormones in thymoma tissues.

Thymic hormones induce T-cell markers and functions. These polypeptide hormones have also been shown by means of immunocytochemistry to localize in thymic epithelial cells. Employing biochemical isolation procedures, we have studied the concentration of two thymic hormones, prothymosin alpha and thymosin beta 4, in the thymus of three thymoma patients. After a brief boiling step, thymic tissue obtained from each patient was individually homogenized and centrifuged. The supernatant was then fractionated by gel filtration on Sephadex G-100 and further purified by reversed-phase high performance liquid chromatography (HPLC). Purified components were characterized by amino acid analysis and HPLC tryptic peptide mapping. Our results revealed that the extract from benign thymoma had both prothymosin alpha and thymosin beta 4, similar to normal human thymus. However, the thymus from a patient with invasive malignant thymoma contained only thymosin beta 4, but no prothymosin alpha. In the extract from an undifferentiated carcinoma, neither prothymosin alpha nor thymosin beta 4 could be detected. These results disclose the possible correlation of thymic hormones and the type and differentiation stage of thymomas. The inability of malignant thymic tumors to produce normal amounts of thymic hormones may contribute to their etiology. It is suggested that information on the thymic hormone content might add a new parameter to pathological diagnosis in thymic tumors.

Distribution of fibronectin and laminin in human thymoma.

The distribution of fibronectin and laminin, two extracellular matrix and basement membrane components, was studied in 55 cases of thymoma using immunohistochemistry. The results were compared according to histologic type or clinical stage of the tumor. In addition, electron microscopic observation was done to clarify the extracellular structure of thymoma. Two staining patterns were seen. First, a diffusely or partially intricate network of fibers that contained fibronectin and laminin surrounded tumor cells in 21 out of 55 cases. Most of these cases were spindle cell thymomas and showed low invasive tumors. Second, fibers that contained fibronectin and laminin were restricted only to the septa, blood vessels, and perivascular spaces that did not show a network in the remaining 34 cases. Polygonal cell thymomas showed the latter staining pattern and these were more invasive tumors. We conclude that this network is a characteristic structure of spindle cell thymomas and is related to the invasiveness of the tumor.

Human epithelial thymic tumours: heterogeneity in immunostaining of epithelial cell markers and thymic hormones.

Different hormones (thymulin, thymosin alpha 1, vasopressin), antigenic markers of cortical and subcapsular/medullary thymic areas and tumour associated antigens were studied on paraffin or frozen section and cultures of human epithelial thymic tumours ('thymomas'). Thymulin, thymosin alpha 1 and for the first time vasopressin are found in most tumours. The epithelial cells of five 'thymomas' had markers of both cortical (TE3) and subcapsular/medullary thymic regions (A2B5 and/or TE4 and/or anti-p19). Leu-7, a marker of subcapsular epithelial cells was positive only in two tumours. The histological classification into cortical and medullary tumours does not correspond to our immunofluorescence results. The presence of these markers does not support the theory of different embryologic origin of the cortical and subcapsular/medullary epithelial cells. Transferrin receptors were detected on only some epithelial cells of thymic 'carcinomas'. Adenocarcinoma related antigen and carcino embryonic antigen only stained a few epithelial cells of all the tumours. There is no expected correlation between the presence of epidermal growth factor receptors on cell membranes and the number of proliferative cells stained by the anti-Ki67 antibodies. Immunostainings were heterogeneous according to the epithelial thymic tumours, independent of histological classification and not yet useful for prognosis.

Thymic carcinoma. A clinicopathologic study of 13 cases.

Thymic carcinoma (TCA) is a thymic epithelial neoplasm with obvious cytologic atypia. We studied 13 cases of TCA by light microscopy, immunohistochemistry, and electron microscopy and correlated the findings with clinical features. The patients' mean age was 54.2 years (range 30-74); the male/female ratio was 7/6. Twelve of the 13 patients presented with signs and symptoms caused by compression of mediastinal organs; the other patient was asymptomatic. Paraneoplastic syndromes were never seen. At thoracotomy, 11 tumors invaded or adhered to surrounding structures; the other two were encapsulated. The histologic types include squamous carcinoma including the lymphoepithelioma-like subtype (seven cases), small cell carcinoma (four cases), clear cell carcinoma (one case), and adenosquamous carcinoma (one case). Positive immunoperoxidase studies were as follows: keratin (13 cases), epithelial membrane antigen (EMA) (13 cases), leukocyte common antigen (none), carcinoembryonic antigen (CEA) (five cases), B72.3 (seven cases), Leu 7 (two cases), human placental alkaline phosphatase (none), vimentin (none), and chromogranin (one case). This profile is similar to those of normal thymus and thymoma except for the absence of CEA, B72.3, EMA in normal thymus, and the absence of CEA and B72.3 in thymoma. Electron-microscopic studies performed on eight cases showed glandular and squamous differentiation in one adenosquamous carcinoma, squamous differentiation in five squamous carcinomas, and neuroendocrine differentiation in one small-cell carcinoma. Nine patients died (three due to postoperative complications and six due to recurrences or metastasis at 3-36 months). Four patients (all with squamous carcinoma) were alive without disease at 2-60 months. The clinical and pathologic features were comparable with those of approximately 62 other cases of TCA previously reported. There are a number of well-defined histologic types of TCA that allow the pathologist to make a differential diagnosis of TCA from tumors extending or metastatic to thymus or other primary mediastinal tumors. Although neither asymptomatic presentation nor encapsulation improves the poor prognosis of TCA, the squamous carcinoma subtype is associated with a better outcome than the other subtypes. Based on the electronmicroscopic and immunohistochemical findings, the presence of normal thymic tissue at the periphery of several tumors, and the observation that several TCA arose from preexisting thymomas or thymic cysts, we conclude that TCA is derived from thymic epithelium.

Histochemical and immunohistochemical evidence of glandular differentiation in thymic carcinoma.

Eighteen cases of primary thymic carcinoma were reviewed from the viewpoint of glandular differentiation. Squamous differentiation was evident in 14 cases (83%). Immunohistochemical study revealed secretory component (SC)-positive carcinoma cells in 12 cases (67%), most of which were also associated with squamous differentiation. Three of these 12 cases contained areas with a definite glandular or microcystic structure with occasional epithelial mucin, and were diagnosed as adenosquamous carcinoma. Review of patients' medical records revealed that thymic carcinomas with a glandular element were more often resectable at surgery, and had a much better prognosis than those without a glandular element. However, further study on larger number of cases is necessary to confirm this relationship. Because SC-positive epithelial cells do exist in the non-neoplastic thymus, the presence of a glandular component suggests another direction of morphological and/or functional differentiation of thymic carcinoma cells in addition to the well-known squamous differentiation.

[Immunohistochemical demonstration of neuropeptide Y in the normal human thymus and in thymoma]. / Dimostrazione immunoistochimica del neuropeptide Y nel timo umano normale e nel timoma.

The distribution of neuropeptide Y-like immunoreactivity in normal thymic parenchyma and in thymoma was evaluated in order to characterize the typical aspects of the thymic peptidergic pattern of innervation in normal and pathological conditions. The possible role of neuropeptide Y in neuromodulation of the immunological response is discussed.

Proteins with epitopes of the acetylcholine receptor in epithelial cell cultures of thymomas in 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.

Growth-promoting effect of oestriol in a lymphoma lacking oestrogen receptors.

Various doses (1 microgram to 10 mg) of oestriol (E3) were intraperitoneally injected into mice immediately after subcutaneous inoculation of an oestrogen receptor-negative lymphoma cell line (KE-5) established from a spontaneously developed AKR thymic lymphoma. The growth of KE-5 cells was markedly promoted by E3 at the early stage of tumour growth. At this stage, 1 microgram E3 enhanced tumour growth significantly and the maximum effect was obtained with 1 mg E3. Normal female mice showed a higher incidence and shorter latency than males. However, once tumours became palpable, the tumour growth rate appeared to be unaffected. Histological observations using Alcian blue and colloidal iron revealed a marked increase of hyaluronic acid in the subcutaneous connective tissue of the tumour-injection site within 3-5 days after intraperitoneal administration of 1 mg E3. Biochemical analyses showed a rapid and marked increase in skin hyaluronic acid content to over 3 times the control levels (0.25 +/- 0.10 mg g-1 skin) within 3 days of E3 administration. Subcutaneous inoculation of KE-5 cells together with hyaluronic acid (0.2 mg) resulted in markedly enhanced tumour growth, particularly at the early stage. These results suggest that an increase in stromal hyaluronic acid content is the most likely mechanism responsible for the promoting effect of E3 on KE-5 cells.

Primary cultures of human thymic epithelial tumors. Morphological and immunocytochemical characterization.

Primary cultures are introduced as a method for an immunocytochemical and functional characterization of epithelial cells (ECs) from human thymic epithelial tumors. Neoplastic ECs were obtained after enzymatic digestion of the tumor tissue with dispase. The ECs were kept in culture for up to 1.5 months. Over this period a progressive decline in their proliferation rate was observed. In all five cases studied, ECs showed a co-expression of keratin and vimentin intermediate filaments in vitro as well as strong expression of major histocompatibility complex (MHC)-class I antigens and a progressive loss of MHC-class II antigens. Acetylcholine receptor (AchR)-epitopes were detected immunohistochemically by using monoclonal antibodies (mAbs) to the cytoplasmic site of the alpha-chain if the AchR. Epitopes were found in three of five thymomas in vivo and to a varying degree in all five cases in vitro.

Characterization of human corticotrophin-releasing hormone and pro-opiomelanocortin-related peptides in a thymic carcinoid tumour responsible for Cushing's syndrome.

Severe Cushing's syndrome developed in a man of 35 years. Plasma ACTH and lipotrophin hormone levels were supranormal, and dexamethasone failed to stop their production. An ACTH-producing thymic carcinoid tumour was found to be responsible for the Cushing's syndrome. The tumour tissue contained pro-opiomelanocortin (POMC)-mRNA and POMC-related peptides. In addition, human corticotrophin-releasing hormone (h-CRH) (0.25 ng/mg wet tissue) was identified in the tumour extract. Among a series of extracts from two normal and three tumoral (Nelson's syndrome) pituitary glands, six non-pituitary POMC-producing tumours and five normal thymuses examined, only the extract from the thymic tumour of our patient contained h-CRH. The molecule isolated had the same properties as synthetic h-CRH (dilution, Sephadex G 50 chromatography). Circulating h-CRH levels, however, were normal. The possible involvement of such ectopic CRH production in the aetiology of Cushing's syndrome remains uncertain.

Degree of malignancy of thymic epithelial tumors in terms of nuclear DNA content and nuclear area. An analysis of 39 cases.

For determination of the degree of malignancy among thymic epithelial tumors, the DNA content and area of nuclei in 13 cases each of noninvasive thymoma, invasive thymoma, and thymic carcinoma were investigated by cytofluorometry and morphometry. The nuclear DNA content was determined in terms of the mean nuclear DNA content, DNA histogram pattern, and the occurrence of the aneuploid stem cell line. The mean nuclear DNA content of the thymic carcinoma was significantly higher than that of both subgroups of thymoma (P less than 0.01), but there was no significant difference between noninvasive and invasive thymomas. The aneuploid stem cell line appeared in 92.3% of thymic carcinomas, one case (7.7%) of invasive thymomas, and none of noninvasive thymomas. Abnormal DNA histogram patterns were seen in 53.8% of thymic carcinomas and none of the thymomas. The mean nuclear area increased significantly in the increasing order of noninvasive thymoma, invasive thymoma, and thymic carcinoma (P less than 0.01). The cytofluorometric and morphometric results demonstrated a significant difference in degree of malignancy between thymic carcinoma and thymoma; however, there was a trend toward an increasing degree of malignancy from noninvasive to invasive thymomas, yet there was a sizeable overlap in results between the two groups. Therefore, these two methods are not satisfactory for predicting the behavior of an individual case of noninvasive or invasive thymoma.

Immunohistochemical studies on a human thymic epithelial cell subset defined by the anti-cytokeratin 18 monoclonal antibody.

Two monoclonal antibodies respectively recognizing cytokeratins (CK) 18 and 19 were applied to the human thymic epithelium (in vivo and in vitro) in normal and pathological conditions, including 12 thymomas. We observed that in both normal and hyperplastic thymuses (from patients with myasthenia gravis) virtually the entire epithelial network was CK19-positive as were the majority of cells growing in culture. In four thymomas, however, the expression of cytokeratin 19 was not detected by immunofluorescence. On the other hand, CK18 was expressed by a discrete subset of medullary thymic epithelial cells in normal and in hyperplastic thymuses. Among the thymomas a large majority was either negative or contained few isolated CK18-positive cells scattered within the tumour. Conversely, in the two undifferentiated epithelial thymomas, virtually all the tumoral network was strongly labeled with the anti-CK18 monoclonal antibody. The present investigation thus not only defines the human thymic epithelial cell subset on the basis of differential cytokeratin expression but also indicates that anti-CK antibodies with single cytokeratin specificities can be regarded as useful tools to study the heterogeneity of thymomas.

Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein.

Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and "ligand blotted" with 125I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed.

[Analysis of nuclear DNA histograms in thymomas].

The nuclear DNA content of normal thymuses and thymomas were measured by microphotometry. The DNA histograms were analyzed for 1) peak values, 2) cell with DNA contents of 4C or more (tetraploidy), and 3) average histograms. The DNA histograms of normal thymuses had peak values around 2C. However, the peak value on thymomal histograms moved to the 4C region (tetraploidy). Advances in stages were followed by an increase in the number of cells with high DNA contents. The number of cells with DNA contents of 8C or more (octaploidy) were significant only in stage III. Thymomal histograms were different from those of normal thymuses. The DNA histograms of thymomas changed with advances in stage, which may indicate that the epithelial cells of thymomas become neoplastic.

T cell receptor beta-chain genes in BW5147 and other AKR tumors. Deletion order of murine V beta gene segments and possible 5' regulatory regions.

The AKR thymoma BW5147 has rearranged both of its TCR beta-chain loci, using the same J beta region (J beta 2.5) in each, but with different V beta gene segments. Although the two rearrangements are expressed approximately equally in cytoplasmic RNA, the principle of allelic exclusion is maintained because only one rearrangement is in-frame and capable of encoding a functional protein. In hybridomas made with BW5147 as the fusion partner, this protein may combine with the alpha-chain protein derived from the normal cell to form new Ag/MHC specificities. An analysis of the sequences upstream from the BW5147 rearrangements and additional V regions suggests that two conserved sequences, 10 and nine nucleotides in length and located adjacent to each other 70 to 100 nucleotides 5' of the initiation codon, may be important in the expression of TCR beta-chain genes. Although B and T cells derive from common stem cells, no sequences are observed in T cells that are homologous to the octamer located 5' of all Ig genes. This implies that at least some of the sequences that regulate transcription are not shared in the two major types of lymphocytes. A survey of BW5147 and six other AKR thymomas using probes for 10 of the 18 known V region families indicates a distribution of V beta rearrangements in the tumors consistent with that found in thymocytes. Four of these tumors have apparent VDJ rearrangements on both chromosomes, with the deletion of other V beta gene segments. These data suggest that the primary mechanism of VDJ beta rearrangement is by looping out and excision of the intervening DNA and that most of the V regions are located 5' to the C region. These data were also used to develop a deletion order of the V beta gene segments in the TCR beta-chain locus.