EMMPRIN (CD147) expression and differentiation of papillary thyroid carcinoma: implications for immunocytochemistry in FNA cytology.

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tumour progression, invasion and metastasis. EMMPRIN expression has been demonstrated in several tumours, but its expression profile in thyroid cancer remains unclear. METHODS: We evaluated the expression profile of EMMPRIN at various stages of differentiation of thyroid carcinoma, including 20 cases of well-differentiated papillary carcinoma (WDPC), 15 cases of papillary carcinoma with a poorly differentiated carcinoma component (PC/PDC) and four cases with an undifferentiated carcinoma (UDC) component, using paraffin-embedded sections for immunohistochemical stains. Also, we used 32 fine needle aspiration cytology and imprint smears from the same cases for immunocytochemical stains. The staining results were evaluated with a scoring system. RESULTS: Immunohistochemical staining showed that EMMPRIN expression was absent or weak in almost all WDPC specimens, whereas it was moderate or strong in PDC and UDC components. In tumours that showed a gradual morphological transformation from WDPC to PDC components, the expression of EMMPRIN was progressively stronger from the areas of WDPC to those of PDC. WDPC, PC/PDC and UDC had expression scores of 4.9, 45.0 and 245.7, respectively. Results of immunocytochemical staining showed almost the same staining profile as those of immunohistochemical staining. The cytological atypia of EMMPRIN-positive cells was greater than that of negative cells. CONCLUSION: These results indicated that EMMPRIN expression correlates significantly with the degree of dedifferentiation of thyroid carcinoma. This study demonstrates the feasibility of expression of EMMPRIN using fine needle aspiration samples. Therefore, immunocytochemical analysis of EMMPRIN may be a novel aid to evaluate the differentiation of thyroid carcinoma.

Dipeptidyl peptidase IV (DPP IV/CD26) staining predicts distant metastasis of 'benign' thyroid tumor.

Because follicular thyroid carcinoma is extremely difficult to diagnose, several cases were encountered which have been rediagnosed as carcinoma due to distant metastasis. In the search for a method of correctly diagnosing 'benign' thyroid tumor, dipeptidyl peptidase (DPP) IV immunostaining was applied to 10 cases whose diagnoses had been corrected to follicular thyroid carcinoma because of distant metastases. The positive rate of immunostaining using paraffin sections in the rediagnosed follicular thyroid carcinoma group (7/10) was much higher than that of the control group (1/29), which consisted of 15 cases of follicular thyroid adenoma and 14 cases of nodular hyperplasia. These results suggested that pre- or postoperative DPP IV staining is useful for predicting distant metastasis of 'benign' thyroid tumor.

Characterization of gastric Na+/I- symporter of the rat.

Characterization of gastric Na+/I- symporter (NIS) of the rat was carried out. Sequencing of the open reading frame of gastric NIS mRNA showed only three nucleotide changes when compared with FRTL-5 NIS cDNA, and two of these changes led to amino acid changes. The results of Northern blot analysis showed that abundant NIS mRNA was expressed in the stomach when compared with other organs. Western blot analysis using gastric mucosa and FRTL-5 lysates detected the difference in molecular weight between FRTL-5 and gastric mucosa lysates, suggesting abnormal posttranslational modification of gastric NIS protein. Immunohistochemically, gastric NIS protein was located in the cornification layer of the stratified squamous epithelium of the pars proventricularis and in parietal cells and on the apical border of surface epithelial cells of the pars glandularis. Gastric NIS protein was present in tubulovesicular structures and lysosomes in parietal cells by immunoelectron microscopy. Gastric NIS protein exists to trap I- from the gastric lumen, except in parietal cells. Results indicated that a very large amount of gastric NIS mRNA is expressed to be translated, whereas only a small amount of immature gastric NIS protein is detected. This may indicate that immature gastric NIS protein rapidly degrades to peptides.

[Apoptosis in Hashimoto's thyroiditis].

It has been suggested that apoptosis plays a pivotal role in the pathogenesis of autoimmune diseases. In Hashimoto's thyroiditis which is a typical organ-specific autoimmune disease, Fas-FasL-mediated apoptosis has been demonstrated as the mechanism of follicular epithelial cell death in which Fas is expressed by IL-1 beta stimulation of FasL is constitutively expressed on follicular epithelial cells. The processes involved in this finding and some questions concerning epithelial cell death are presented. The thyroid tissue of Hashimoto's thyroiditis was examined for DNA fragmentation of follicular epithelial cells by the TUNEL method. DNA fragmentation was observed more frequently on thyroid follicles in the area adjacent to lymphoid cell follicles than on those in the central area. Electron microscopic study supported the results of TUNEL study. Immunohistochemical study on Fas and FasL expression on follicular epithelial cells of various thyroid diseases showed that Fas and FasL were strongly expressed on follicular epithelial cells in Hashimoto's thyroiditis and thyroid cancer. Epithelial cells of patients with Graves' disease and adenomatous goiter, however, were scarcely stained. Fas and FasL expression on follicular epithelial cells were well correlated. In vitro study on follicular epithelial cells clarified that FasL was constitutively expressed on epithelial cells not only in Hashimoto's thyroiditis but also in nontoxic goiter. Fas expression was induced by IL-1 beta stimulation. IL-1 beta stimulation also brought about apoptosis of epithelial cells and epithelial cells killed Fas-positive target cells. Therefore, it was concluded that FasL expressed constitutively on follicular epithelial cells interacts with Fas on epithelial cells expressed by IL-1 beta stimulation to induce apoptosis of epithelial cells.

Expression of CD26/dipeptidyl peptidase IV in adult T cell leukemia/lymphoma (ATLL).

The association of CD26/dipeptidyl peptidase IV (DPPIV) and human T lymphotropic virus type I (HTLV-I) was studied by two approaches. First, we examined the expression of CD26 in peripheral blood mononuclear cells (PBMC) from the patients with adult T cell leukemia/lymphoma (ATLL), an HTLV-I-related malignancy. The expression of CD26 on the surface of PBMC was decreased in all 20 patients with ATLL compared with those from normal individuals (P < 0.01) and the expression of the CD26 gene transcript was not detectable in seven out of eight patients with ATLL. Then we compared the quantity of viral DNA in CD26-negative (CD26-) and CD26-positive (CD26+) cells obtained from 17 HTLV-I healthy carries by using a polymerase chain reaction method. The CD26-cells had a higher copy number of viral DNA than CD26+ cells. These findings indicate that HTLV-I has in vivo tropism to CD26- cells, suggesting that some phenotypes of ATLL cells reflect the in vivo cellular tropism of HTLV-I.

Differential expression of dipeptidyl peptidase IV (CD26) and thyroid peroxidase in neoplastic thyroid tissues.

This paper reports the results of research to examine the possibility of using the differential expression of two enzymes, dipeptidyl peptidase IV (DPPIV/CD26, EC: 3.4.14.5) and thyroid peroxidase (TPO, EC: 1.11.1.7), as histochemical markers histopathologically to diagnose thyroid carcinomas. The research is based on previous reports that DPPIV/CD26 is overexpressed in differentiated thyroid carcinoma tissues, and that TPO activity is very low in thyroid carcinoma tissues. Differential expression of the two enzymes in 32 thyroid tissues of various thyroid diseases was studied by Northern blot analysis and histochemical analysis. On Northern blot analyses, all 14 differentiated thyroid carcinomas (11 papillary carcinomas and 3 follicular carcinomas) overexpressed DPPIV/CD26 mRNA, whereas all 17 benign thyroid tissues (4 normal thyroid tissues, 4 Graves' diseases, 2 adenomatous goiters and 7 follicular adenomas) showed faint mRNA expression of DPPIV/CD26. All 17 benign thyroid tissues expressed high levels of TPO mRNA, whereas all 11 papillary carcinomas strongly underexpressed TPO mRNA. Histochemically, all 17 benign tissues were DPPIV/CD26 negative and strongly TPO positive, while all 11 papillary carcinomas were strongly DPPIV/CD26 positive and TPO negative. Two of 3 follicular carcinomas were histochemically positive for the two enzymes. A medullary carcinoma did not show any mRNA expression of either enzyme. These results suggest that the differential expression of these two enzymes can be applied to study the thyroid tumorigenesis.

[CD26/dipeptidyl peptidase IV and thyroid peroxidase as molecular markers for differentiated thyroid carcinoma].

Differential expression of two enzymes, dipeptidyl peptidase IV (CD26/DPP IV) and thyroid peroxidase (TPO), in neoplastic thyroid tissues was studied by Northern blot analysis and histochemical analysis using 31 thyroid tissue specimens of various thyroid diseases. On Northern blot analysis, all 16 differentiated carcinomas (12 papillary and 4 follicular carcinomas) overexpressed CD26/DPP IV mRNA, whereas all 14 benign tissue specimens (4 normal thyroid, 4 Graves' disease, 2 adenomatous goiters and 4 follicular adenomas) showed faint expression of CD26/DPP IV mRNA. All 14 benign tissues expressed high levels of TPO mRNA, whereas all 12 papillary carcinomas strongly underexpressed TPO mRNA. A medullary carcinoma did not show any mRNA expression of either enzyme. TPO mRNA expression in differentiated carcinomas did not always correlate with mRNA expression of thyroglobulin, thyroid stimulating hormone receptor, and thyroid transcription factor-1. Northern blot analysis also revealed that CD26/DPP IV is a more specific marker of differentiated carcinoma than three proto-oncogenes previously reported to increase mRNA expression in thyroid carcinomas: c-met, c-erbB-2, and EGF-R. Histochemically, all 14 benign tissues were CD26/DPP IV negative and strongly TPO positive, while all 12 papillary carcinomas were strongly CD26/DPP IV positive and TPO negative. Three of 4 follicular carcinomas were histochemically positive for the two enzymes. These findings suggest that the differential expression of these two enzymes can be applied to study the thyroid tumorigenesis.

High expression of heat shock protein 60 in follicular cells of Hashimoto's thyroiditis.

Heat shock protein 60 expression in the thyrocytes of Hashimoto's thyroiditis and Graves' disease was studied immunohistochemically. Thyrocytes of Hashimoto's thyroiditis showed high expression of heat shock protein 60 not only in tall eosinophilic-cells but also in low and flattened cells, although the former was stained with moderate to strong staining intensity and the latter weakly to moderately. Follicular cells around lymphoid cell follicles were stained more intensely, whereas cells apart from lymphoid cell follicles were stained weakly to moderately. In Graves' disease, only follicular cells around lymphoid cell follicles were stained with varying intensities. Since the pattern in all positive staining was granular, it was thought that heat shock protein 60 overexpressed in thyrocytes located on mitochondria. To investigate the immunological role of overexpression of self heat shock protein 60 in the thyrocytes of Hashimoto's thyroiditis, gamma delta TCR+ T-cells in the tissue and the IgG class of anti-self heat shock protein 60 antibodies were studied. gamma delta TCR+ T-cells were detected among lymphoid cells scattered in interfollicular connective tissue. No difference in antibody level was seen among subjects with Hashimoto's thyroiditis, Graves' disease and normal subjects. Self heat shock protein 60 overexpression in the thyrocytes of Hashimoto's thyroiditis may play a disease-modifying role, although it does not influence the anti-self heat shock protein 60 antibody level.

Identification of biclonal (duplex) leukaemic cells expressing either CD4+/CD8- or CD4-/CD8+ from a patient with adult T-cell leukaemia/lymphoma.

A 24-year-old Japanese woman was admitted to our hospital in 1987 with a chief complaint of skin eruptions, and was diagnosed as having chronic ATLL. In 1993 the leucocyte count increased gradually to 126.0 x 10(9)/l with 91.5% abnormal lymphocytes expressing two different types of antigenicity, either CD+/CD8- or CD4-/CD8+. Monoclonal integration of human T-cell lymphotropic virus type-I proviral DNA was detected at different sites of the genomic DNA in each cell type. These studies clearly indicate that CD4+/CD8- and CD4-/CD8+ leukaemic cells originated from two independent clones.

Apoptosis in thyroid tissue from patients with Hashimoto's thyroiditis.

To clarify whether apoptosis of thyroid follicular epithelial cells occurs at the tissue level in autoimmune thyroiditis, 17 specimens of thyroid tissues with Hashimoto's thyroiditis were stained for fragmented DNA. Almost all nuclei of follicular epithelial cells forming atrophic thyroid follicles surrounded by mononuclear cell infiltration and fibrosis showed positive staining. With increasing distance from lymphoid cell follicles, the percentage of follicular epithelial cells with DNA fragmentation-positive nuclei decreased (30-80%). Electron microscopic study revealed the existence of epithelial cells with shrunk and condensed nuclei. The frequency of those cells in different areas was almost compatible with that of cells with fragmentation-positive nuclei. These findings suggest that apoptosis plays an important role in the thyroid tissue injury in autoimmune thyroiditis.

Expression of functional Fas antigen on adult T-cell leukemia.

The expression of Fas antigen was analyzed in the peripheral blood mononuclear cells of 12 patients with adult T-cell leukemia (ATL) by flow cytometry. The induction of apoptosis in these cells by adding an anti-Fas antibody and the expression of activated T-cell surface antigens, CD25 and CD26, were also studied. It appears that the cells in ATL expressed a significantly larger number of Fas antigens than those in normal subjects (p < 0.01) and their fluorescent intensity was also shown to be much stronger in ATL (p < 0.01). The large number of ATL cells showed apoptosis in a short-term culture in the presence of the anti-Fas antibody. There was no difference in the expression of Fas antigen among ATL cells with different phenotypes of CD4+/CD8-, CD4-/CD8- and CD4+/CD8+ as well as with clinical subtypes of ATL. Interestingly, the expression of Fas and CD26 antigens showed a negative correlation (p < 0.01, r = 0.78). The strong expression of functional Fas antigen in ATL leads to the impression that anti-Fas antibody could be one of the treatment modalities for ATL which is known to be a very difficult disease to cope with.

Expression of dipeptidyl aminopeptidase IV activity in human lung carcinoma.

Dipeptidyl aminopeptidase IV (DAP IV) staining was examined in various histological types of lung carcinomas to evaluate this enzyme activity. A total of 45 lung carcinomas were examined for their enzyme activity. Almost all (93.1%) cases of adenocarcinoma were positive for DAP IV activity, whereas all cases of squamous cell carcinoma, small cell carcinoma, large cell carcinoma and carcinoid were negative. DAP IV activity of microsomes in lung carcinomas was significantly higher in papillary adenocarcinomas than in squamous cell carcinomas. These data suggest that DAP IV may be a good marker to distinguish adenocarcinoma from other histological types of lung carcinoma.

Diagnostic usefulness of dipeptidyl aminopeptidase IV monoclonal antibody in paraffin-embedded thyroid follicular tumours.

Monoclonal antibodies to dipeptidyl aminopeptidase IV (DAP IV, EC 3.4.14.5) were raised and selectively applied to paraffin-embedded sections of thyroid carcinoma. Five monoclonal antibodies were found to stain paraffin sections of thyroid carcinomas. Using one of these antibodies (44-4), we studied retrospectively aberrant expression of DAP IV in thyroid carcinoma to determine whether immunohistochemical staining with DAP IV antibody is useful in pathological diagnosis. In almost all cases of thyroid follicular and papillary carcinoma, tumour cells were positive (99.0 per cent) with DAP IV, whereas the cases of follicular adenoma showed a low incidence (27.1 per cent) of positive staining. Follicular adenoma with incomplete capsular invasion had a higher positive incidence (50 per cent) than follicular adenoma without incomplete capsular invasion (9.6 per cent). In positive staining cases previously diagnosed as benign tumours, 11 benign cases reacting positively with DAP IV were rediagnosed as carcinoma after re-examination of more thyroid paraffin block sections or serial sections. These findings suggest that DAP IV monoclonal antibody is very useful in distinguishing thyroid follicular carcinoma from follicular adenoma.

Immunohistochemical localization of dipeptidyl aminopeptidase IV in thyroid papillary carcinoma.

The localization of dipeptidyl aminopeptidase IV expressed aberrantly in thyroid carcinoma was studied by immunoelectron microscopy using a monoclonal antibody to the enzyme with special reference to enzyme-histochemical staining of the enzyme. Five thyroid papillary carcinomas were investigated including two lymph-node metastases. All cases showed the dense immunoreaction product on the apical membrane and only traces of the product on lateral membranes, endoplasmic reticulum and nuclear membranes. In one case only, the dense product was observed on basal tubular structures. Analysis, using immunogold labelling on pre-embedded cryosections, revealed that dipeptidyl aminopeptidase IV was localized on the luminal surface of cancer cells. Two different distribution patterns of dipeptidyl aminopeptidase IV activity staining, diffuse and apical patterns, reported previously were thought to be due to different amounts of dipeptidyl aminopeptidase IV in the cytoplasm of cancer cells. This enzyme-histochemical staining method is useful for pathological diagnosis of thyroid tumours and can be applied to clinical materials. The enzyme localization is revealed by the staining pattern.

[Detection of immature granulocytes and atypical and/or abnormal lymphocytes using scatterplots of Coulter STKS].

Scatterplots of the Coulter STKS were studied in order to make better analyses of morphological abnormality of leukocytes. The analytical pattern of a sample failed to completely prevent blood coagulation, especially platelet aggregation, showed the poor separation between lymphocyte and neutrophil populations. On the scatterplot, a small population was occasionally observed in the lower area of normal lymphocyte population with elevation of room temperature, being thought an artificial population. When atypical and/or abnormal lymphocytes increased, the pattern was characterized by the distribution of large lymphoid cells spreading over monocyte population area. The characteristics became clear in proportion to the percentage of large lymphoid cells. To detect immature granulocytes, we introduced a criterion originated in our laboratory. Using the detection criterion, we could obtain the satisfactory results with sensitivity of 85%, specificity of 93%, efficiency of 91% and coefficient correlation (r) of 0.73. It is concluded that the pattern analysis and the detection criterion are useful in the experiments using routine laboratory samples and valuable in clinical implication.

Dipeptidyl aminopeptidase IV staining of cytologic preparations to distinguish benign from malignant thyroid diseases.

Staining for dipeptidyl aminopeptidase IV (DAP IV [EC: 3.4.14.5]) activity was applied to aspiration biopsy specimens or imprint preparations of surgical biopsy specimens from thyroid tumors. Material was obtained from 55 patients with histologically proven thyroid diseases: 9 with papillary carcinoma, 5 with follicular carcinoma, 11 with follicular adenoma, 13 with adenomatous goiter, 8 with Hashimoto's thyroiditis, and 9 with other benign conditions. Most tumor cells, follicular lumina in cell clusters, and intranuclear inclusions were strongly positive for DAP IV in all examples of papillary or follicular carcinoma. In contrast, only a few epithelial cells were labeled for DAP IV in follicular adenoma and adenomatous goiter. Some Hürthle cells in Hashimoto's thyroiditis also were positive for DAP IV. When a DAP IV scoring system based on the percentage of positive cells and staining intensity was used, all benign tissues except one (from a follicular adenoma) were found to have extremely low scores. These results indicate that staining for DAP IV activity is a simple but useful tool to aid in distinguishing benign from malignant thyroid neoplasms.

Expression of dipeptidyl aminopeptidase IV activity in thyroid carcinoma.

Dipeptidyl aminopeptidase IV activity staining was performed in various thyroid tissues to evaluate this enzyme activity as a thyroid tumor marker. A total of 195 thyroid tissues were tested for their enzyme activity expression. All papillary and follicular carcinomas, 40 cases and 3 cases, respectively, showed enzyme activity, although two other carcinomas, one medullary and one anaplastic, were not stained. Follicular adenoma expressed enzyme activity in 4 of 26 cases. Fifty-two cases with adenomatous goiter, 54 with Graves' disease and 13 with chronic thyroiditis were judged to be negative. Five normal thyroids expressed no activity except for occasional positive staining of capillary endothelia. These data suggest that dipeptidyl aminopeptidase IV activity staining is very useful for pathological diagnosis of thyroid tumors.

Application of the avidin-biotin-complex method for the light microscopic analysis of lymphocyte subsets with monoclonal antibodies on air-dried smears.

A study was undertaken of the application of the avidin-biotin-peroxidase complex (ABC) method to the monoclonal antibody MAbs staining of mononuclear cells in hematologic and cytodiagnostic materials. Satisfactory cell morphology and immunoreactivity of surface antigens were observed when the slides were fixed in 80% acetone in phosphate-buffered saline or in 60% acetone in 0.03 M citric acid buffer solution (pH 5.4). Unstained air-dried preparations could be preserved for two weeks at room temperature in a desiccator and for one year at -70 degrees C after fixation. An excellent immunoreaction, even with a weak surface antigen, was observed by inhibition of endogenous peroxidase after the secondary antibody reaction; reactions of weak antigens tended to be obscured when the inhibition was performed before the first antibody reaction. Use of the Giemsa stain as a counterstain made it possible to readily observe the cell morphology; therefore, white blood cell analysis could be performed simultaneously when peripheral blood smears were studied. The positive rate of immunoreaction by an immunofluorescent method was well correlated with that obtained by the ABC method. The ABC method proved to be an excellent immunocytochemical technique for detecting cell surface antigens with high sensitivity and specificity; furthermore, it is useful for cell morphology studies and yields permanent preparations.