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.

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.

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.

[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.

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.

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.

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.

A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes.

Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.

Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA.

Abelson murine leukemia virus induces oligoclonal pre-B lymphoma in mice. The expression of the v-abl oncogene in target cells does not appear to be sufficient for tumor induction in several mouse strains, and additional genetic events are thought to be required. We postulated that the helper Moloney murine leukemia virus might induce these events, and its potential role as an insertional mutagen was assessed by the search of a common helper provirus integration site in Abelson murine leukemia virus lymphomas. Molecular cloning of cellular sequences adjacent to Moloney proviruses enabled us to identify a cellular region, designated Ahi-1, which was found occupied by the helper proviruses in 16% of Abelson pre-B-cell lymphomas. All proviruses for which the precise integration site within Ahi-1 could be mapped were found to be in the same orientation. Ahi-1 has been mapped to mouse chromosome 10 and represents a new common proviral integration site. These data suggest that the helper virus contributes to the induction of secondary genetic events which may be important for the development of Abelson murine leukemia virus-induced pre-B-cell lymphoma.

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.

Subunit interactions within the T-cell antigen receptor: clues from the study of partial complexes.

The T-cell antigen receptor is a multisubunit complex composed of seven transmembrane chains (alpha beta gamma delta epsilon zeta 2). Subunit interactions within this complex were defined by analyzing the subunit composition of partial complexes. These partial complexes were observed in mutant and tumor T cells that fail to synthesize one or more of the receptor chains or in fibroblasts transfected with genes encoding T-cell antigen receptor chains. In addition, partial complexes were generated by immunoprecipitation with antibodies that cause selective dissociation of T-cell antigen receptor chains. The alpha and beta chains were found to form a disulfide-linked dimer in the absence of any of the other chains. The gamma, delta, and epsilon chains were also efficiently associated in the absence of a complete heterodimer. Complexes composed only of delta epsilon or gamma epsilon could be observed. Both these dimers, as well as the gamma delta epsilon trimer, could form stable complexes with alpha beta, even in the absence of zeta 2. The zeta 2 dimer could bind directly to alpha beta. In the absence of a complete clonotypic heterodimer, zeta 2 was not stably associated with gamma delta epsilon. These observations suggest a model in which alpha beta interacts directly with the gamma delta epsilon trimer and zeta 2, with less-direct interaction between the latter two.

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.

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.

[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.

Immunohistochemical studies in human thymomas. Localization of thymosin and various cell marker.

Forty five human thymomas were studied immunohistochemically using antibodies to thymosin x-1, thymosin beta-3, cortical epithelium of human thymus (UH-1), mouse thymic nurse cells (Th-3) and Leu-7. Most thymomas were found to contain thymosin x-1 (80%) and thymosin beta-3 (89%). Also used in the study were a new monoclonal antibody (UH-1), which reacts with the epithelial cells forming a meshwork in the cortex of the normal newborn thymus and Leu 7, which reacts with subcapsular epithelial cells in the outer thymic cortex. The combined use of UH-1 and Leu-7 was found to identify neoplastic epithelial cells of thymic cortical origin in thymomas. Approximately 80% (37/45) of the thymomas in the present study reacted with Leu-7, UH-1 or both antibodies, and were thus considered to be derived from cortical thymic epithelium. Of the eight thymomas which were negative with both Leu-7 and UH-1, four were histologically of mixed type characterized by the formation of epithelial cell islands. All four of these thymomas were positive with thymosin and were therefore considered to be of medullary origin. Ten of the thymoma were associated with myasthenia gravis; all were positive with UH-1 and were consider to be of cortical origin.

A novel form of GD1c ganglioside IV3 (NeuAc alpha 2-8 NeuGc)Gg4Cer from murine X-ray induced thymoma.

Four disialoganglioside fractions were isolated from X-ray induced thymoma in C57Bl/6 mice. On the basis of compositional and methylation analyses as well as degradation with specific exoglycosidases a novel form of GD1c ganglioside NeuAc alpha 2-8 NeuGc alpha 2-3 Gal beta 1-3 GalNAc beta 1-4 Gal beta 1-4 GlcCer was identified.

Gangliosides shed by tumor cells enhance tumor formation in mice.

The role of tumor cell membrane gangliosides in tumor formation was probed using a series of cloned murine AKR lymphoma cell lines. Tumor formation was directly related to high expression and shedding of membrane gangliosides. In vivo, as little as 1 pmol of purified total gangliosides of highly tumorigenic cells, injected intradermally with poorly tumorigenic cells (which lacked and did not shed gangliosides), markedly increased the tumorigenicity of these cells in syngeneic normal mice. Thus, gangliosides shed by tumor cells are a previously unrecognized, extremely potent enhancer of tumor formation in vivo.

Increase in S-100b protein content in thyroid carcinoma.

S-100b protein was detectable in the soluble fraction of thyroid tissue. The concentration of S-100b protein in thyroid carcinoma tissue was three to five times higher than in normal thyroid tissue and thyroid adenoma. It is, however, not higher in the thyroid tissue of Graves' disease. The increase of S-100b protein concentration was not remarkable in carcinomatous tissue of the stomach and other digestive organs. The calmodulin content in the thyroid carcinoma tissue increased but the increment was low compared to that of S-100b protein. These data suggest that S-100b protein may play a significant role in cell maturation or differentiation.

Structure of a new disialoganglioside GD1c from spontaneous murine thymoma.

A major mono- and a di-sialoganglioside were isolated and purified to homogeneity from a spontaneous thymoma that occurs in AKR mice. Compositional and methylation analyses and the use of exoglycosidases established the monosialoganglioside to be alpha Neu(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----4)Cer and the disialoganglioside to be alpha NeuAc(2----8)alpha NeuAc(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----1)Cer (GD1c). A possible pathway for the biosynthesis of this disialoganglioside is presented.