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Abstract: Comparative genomic hybridisation (CGH) is a technique which identifies gains and losses of DNA sequence copy number in tumours. We used CGH to search for genetic changes in one of the most aggressive malignancies – anaplastic thyroid carcinoma (ATC). For this purpose, we analysed tumour specimens of nine ATCs and DNA of two ATC cell lines. CGH detected aberrations in 10 of 11 samples, with a mean number of gains or losses per carcinoma of 4.8 (range 0–13). Total or partial changes of chromosome 8 (n=6), including gains or losses of 8p (n=6) or 8q (n=5) were those detected most frequently. Chromosome 5p was amplified in five cases. Gains in two of three samples were found for 3q, 7p, 11q and 20q. Gains in a fewer number were seen for 1p (1 case), 1q (1), 7q (2), 9q (2), 11p (2), 12q (1), 14 (1), 15 (1), 17q (2), 18p (2), 18q (1), 20p (1), 21 (2), Xp (2) and Xq (2). Losses were less frequent than gains and observed for 1p (2 cases), 1q (1), 2p (1), 2q (2), 3p (2), 3q (1), 4q (2), 6q (1), 9p (2), 9q (1), 18p (1), 18q (1) and Y (2). Examples of analysis of tumour sections and cell lines performed by fluorescence in situ hybridisation (FISH) confirmed the gains and losses found by CGH and detected additional signals for 8q21 in tumour cells in a sample with no gains or losses normally in CGH. The results suggest that aberrations of 5p, 8p and 8q, which are rarely found in differentiated thyroid carcinoma, may play an important role in the development of ATC. Therefore, these chromosomes could harbour gene loci potentially involved in the aggressiveness of neoplastic tumours, as shown in tumours such as in this study for ATC.

Although eosinophilic granulocytes are frequently observed in lymphatic tissue of Hodgkin's patients, no substantial data reveal the prognostic role, if any, of tissue eosinophilia. Thus, eosinophilia was analyzed histologically in 1511 diagnostic biopsy specimens of patients treated under protocol therapy of the German Hodgkin's Lymphoma Study Group between 1988 and 1994. Prominent eosinophilia was seen in 38% of cases, which differed among the histologic types of Hodgkin's disease (HD): none in lymphocyte predominant, 14% in lymphocyte rich classical, 40% in nodular sclerosis grade 1 (NS-1), 55% in nodular sclerosis grade 2, 43% in mixed cellularity (MC), and 54% in lymphocyte depleted. In a multivariate analysis, tissue eosinophilia proved to be the strongest prognostic factor for freedom from treatment failure (P < .001) and overall survival (P < .001) in a stage-stratified model. Among NS-1 patients, the effect was highly significant. In MC, no significant effect of eosinophilia on survival could be demonstrated. Eosinophils secrete CD30 ligand that is capable of binding to CD30 positive HD cells. The activation of TRAF2, followed by NF-kappaB, which occurs on CD30L/CD30 binding, may explain the neoplastic proliferation and apoptosis protection of HD cells. TRAF2 is also activated by EBV-LMP expression, which is detectable in the majority of MC but not NS cases. In addition to the possibility that eosinophils are only passive indicators for other unknown prognostic determinants, it may be concluded that the positive clinical outcome of eosinophilia-negative NS cases could be due to lower NF-kappaB activity.

Although eosinophilic granulocytes are frequently observed in lymphatic tissue of Hodgkin's patients, no substantial data reveal the prognostic role, if any, of tissue eosinophilia. Thus, eosinophilia was analyzed histologically in 1511 diagnostic biopsy specimens of patients treated under protocol therapy of the German Hodgkin's Lymphoma Study Group between 1988 and 1994. Prominent eosinophilia was seen in 38% of cases, which differed among the histologic types of Hodgkin's disease (HD): none in lymphocyte predominant, 14% in lymphocyte rich classical, 40% in nodular sclerosis grade 1 (NS-1), 55% in nodular sclerosis grade 2, 43% in mixed cellularity (MC), and 54% in lymphocyte depleted. In a multivariate analysis, tissue eosinophilia proved to be the strongest prognostic factor for freedom from treatment failure (P< .001) and overall survival (P< .001) in a stage-stratified model. Among NS-1 patients, the effect was highly significant. In MC, no significant effect of eosinophilia on survival could be demonstrated. Eosinophils secrete CD30 ligand that is capable of binding to CD30 positive HD cells. The activation of TRAF2, followed by NF-kappaB, which occurs on CD30L/CD30 binding, may explain the neoplastic proliferation and apoptosis protection of HD cells. TRAF2 is also activated by EBV-LMP expression, which is detectable in the majority of MC but not NS cases. In addition to the possibility that eosinophils are only passive indicators for other unknown prognostic determinants, it may be concluded that the positive clinical outcome of eosinophilia-negative NS cases could be due to lower NF-kappaB activity.

Summary A monoclonal antibody 7A9 was raised against the tumour-associated glycoprotein TAG-12 purified from T47-D breast carcinoma cells. In immunoblots from cytosol of T47-D cells and from sera of breast cancer patients, antibody 7A9 detects the high molecular weight mucin-like TAG-12 antigen. A series of paraffin sections of normal, benign and malignant mammary tissues have been studied with monoclonal antibody 7A9 and the immunoalkaline phosphatase method. In resting gland, proliferating gland and fibroadenoma ducts, reactivity of 7A9 was mainly restricted to luminal membranes of epithelial cells and secretions. 77/79 primary breast carcinomas including ductal, lobular and various other carcinoma types showed cytoplasmic and/or membrane-associated staining with 7A9 in most tumour cells. Metastases (31/31) from different sites were also positive. Strong immunoreactivity with single tumour cells was noted in cytological preparations from freshly resected breast cancer tissue. Thus, monoclonal antibody 7A9 seems to be very useful for the targeting of breast carcinoma cells.

Fluorescence in situ hybridization (FISH) is a new technique that allows demonstrating of the bcr/ablgene fusion in bone marrow cells of patients with Philadelphia translocation (Ph)-positive chronic myeloid leukemia (CML). In this study, bone marrow samples of 150 patients were investigated routinely by interphase FISH, cytogenetics, and bone marrow histopathology. In 20 patients with reactive hyperplasia of the granulopoiesis and normal karyotypes, FISH revealed nonspecific bcr/ablfusion signals at a mean frequency of 2.7 of the cells examined. The cutoff level for specific fusion signals was set at three times the standard deviation (9.0). None of the 29 cytogenetically Ph-negative patients with myeloproliferative disease other than CML had fusion signals exceeding 9. The mean frequency of specific fusion signals in nontreated patients with CML (n = 59) was 92.7, and 49.3 in patients with CML who received therapy (n = 42). For diagnosing Ph-positive CML, interphase FISH has been faster, more reliable, and more sensitive than cytogenetics, which was successful in 54 of 59 patients investigated at first diagnosis but only in 27 of 42 patients receiving therapy, and it failed to detect Ph-positive cells in three patients with CML. However, small percentages of less than 9.0 of cells with bcr/ablfusion signals were below the threshold of interphase FISH, thereby limiting its use for detecting minimal residual disease.

RNA in situ hybridization technique (RNA-ISH) is hampered by formalin fixation of tissues. This necessitates a pretreatment step of enzymatic digestion. However, to achieve the best results, digestion times and concentrations of enzymes have to be adapted for every specimen in a cumbersome procedure.

Abstract: The influence of postmortem damage of tissues on the immunohistochemical diagnosis of wound age has not as yet been clarified. We utilized antibodies against the proteinase inhibitors α-1-antichymotrypsin and α-2-macroglobulin, fibronectin and lysozyme to study samples of skin which had been intact intravitally, but were damaged postmortem either by autolysis or compression with a surgical clamp at the time of dissection. Even in the absence of autolysis, antibodies against the proteinase inhibitors and fibronectin exhibited staining of tissue margins. Autolysis caused an increase in false positive results. In contrast, antibodies against lysozyme did not give false positive staining. There were no antigens sensitive to postmortem clamping and false positive results were not observed. Antibodies against proteinase inhibitors are not useful for the diagnosis of wound age because of a high number of false positive reactions in marginal areas. Fibronectin also showed false positive band-shaped staining patterns at the tissue margin. In addition, autolytic processes increase the number of false positives. The antibody against lysozyme is much less sensitive to autolysis and no false positive reactions were observed in our series of tests.

Involvement of the CNS by a granulosa cell tumor of the ovary is rare. We describe a patient with leptomeningeal and cerebral cortex infiltration by this tumor. The diagnosis was confirmed by CSF cytology and immunocytochemistry using monoclonal antibodies against human inhibin.