Virchows Archiv

Dockhorn-Dworniczak, B.; Schäfer, K.-L.; Dantcheva, R.; Blasius, S.; Böcker, W.; Burdach, S.; Strehl, S.; Winkelmann, W.; Valen, F.; Jürgens, H.

doi: 10.1007/BF00230345pmid: 7524975

One consistent feature of the Ewing's tumour family is the presence of a balanced translocation involving band q12 and band q24 of chromosome 22 and chromosome 11. Recent cloning of the chromosome breakpoint regions of t(11;22)(q24;q12) Ewing's sarcoma translocation has revealed that the breakpoints were localized within the Ewing's sarcoma gene (EWS gene) on chromosome 22 and the Fli-1 gene on chromosome 11. Molecular genetic techniques can thus be applied to the detection of the t(11;22) translocation in Ewing's tumours. By reverse transcription and polymerase chain reaction technique (RT-PCR) 11 Ewing's tumour derived cell lines, 12 primary Ewing's tumours, and 11 tumours after treatment were analysed for the occurence of the t(11;22) translocation. Furthermore, blood and bone marrow samples from 5 patients were available for RT-PCR. In 78% of the cell lines and 91% of the primary Ewing's tumours the t(11;22) translocation was detectable by RT-PCR. In bone marrow samples from a Ewing's sarcoma patient presenting in relapse tumour cells were detected by molecular genetic analysis. Our results indicate that molecular genetic detection of the t(11;22) translocation is valuable in the differential diagnosis of small round cell tumours and will provide important information for the staging and prognosis of Ewing's tumour.

Peringa, J.; Molenaar, W.; Timens, W.

doi: 10.1007/BF00230346pmid: 7952495

The Wilms' tumour (WT) is composed of blastema, epithelium and mesenchyme; the epithelium and possibly also the mesenchyme develop from the blastema, parallel to embryonal development. Since interactions between cell adhesion receptors and extracellular matrix (ECM) proteins play an important role in tissue maturation, we examined the expression of the integrin subunits α1–α6, β1 and β4, and of the ECM proteins fibronectin, laminin and collagen I and IV, in 20 frozen WT samples and in 5 fetal and 2 adult kidneys. The integrin and ECM protein distribution in tumour epithelium and mesenchyme showed strong similarities to that in their fetal counterparts, whereas the tumour blastema differed strongly from the fetal blastema. In the WT blastema different components were recognized. Undifferentiated blastema, characterized by expression of α3 and α6 and the virtual absence of ECM proteins. Blastema with epithelial commitment, showing increased expression of α3 and α6 and the appearance of α2 and, as a very early phenomenon, production of laminin. Blastema with mesenchymal commitment, with loss of α3 and α6 and expression of α1, α4 and α5 and presence of ECM proteins. It is speculated that the inability of the (undifferentiated) blastema to produce ECM proteins is related to its relatively high metastatic potential when compared with epithelium and mesenchyme.

Luzi, P.; Leoncini, L.; Funtò, I.; Bruni, A.; Lazzi, S.; Pacenti, L.; Tosi, P.

doi: 10.1007/BF00230347pmid: 7952496

Sinonasal non-Hodgkin's lymphomas (SNHLs) of B- or T-cell immunophenotype have been associated with Epstein-Barr virus (EBV) infection of neoplastic lymphoid tissue. Nine SNHLs were investigated using immunohistochemistry, the polymerase chain reaction (PCR) for EBV genome and in situ hybridization (ISH) for EBV encoded RNAs (EBER), immunoglobulin (CI-gHR) and clonal T-cell receptor (CTCβR) gene rearrangements. Eight cases were diagnosed as peripheral pleomorphic T-cell lymphomas (pPTCL). PCR showed the presence of EBV genome in eight cases; ISH for EBER led to the detection of positive cells in five cases. Late membrane protein (LMP) immunostaining was observed in three cases. No EBV positivity has been detected in control cases. The frequent association with EBV infection in the cases illustrated confirms the previous suggestions that EBV may have a role in the genesis of lymphomas of the sinonasal region.

Schmid, K.; Brink, M.; Freytag, G.; Böcker, W.; Kirchmair, R.; Fischer-Colbrie, R.; Heitz, P.; Klöppel, G.

doi: 10.1007/BF00230348pmid: 7952497

In the endocrine pancreas, chromogranins A and B as well as secretoneurin (a biologically active peptide processed endoproteolytically from secretogranin II) are most intensely expressed in alpha (glucagon) cells. We examined whether the functional status of neoplastic and nonneoplastic human alpha cells is reflected in the expression patterns of chromogranins/secretogranins. Neoplastic alpha cells were analysed immunocytochemically in six functioning glucagonomas and 37 nonfunctioning neuroendocrine tumours (29 with alpha cells) for their immunoreactivity to chromogranin A and B, as well as secretoneurin. There was no difference in the staining intensity for either peptide between glucagonomas and nonfunctioning, alpha cell containing tumours. Nonneoplastic alpha cells from patients with a functioning glucagonoma showed a decreased glucagon immunoreactivity, whereas the expression of chromogranin A (but not chromogranin B and secretoneurin) was as intense as in alpha cells not associated with glucagonoma syndrome. These results suggest that the expression of chromogranins/secretogranins in neoplastic alpha cells of the pancreas may be independently regulated from the cells' functional status. In nonneoplastic alpha cells there seems to be an association between glucagon production and chromogranin B and secretoneurin expression.

Tanaka, Y.; Tsugu, A.; Takamiya, Y.; Sato, O.; Abe, Y.; Yamazaki, H.; Ueyama, Y.; Tamaoki, N.; Nakamura, M.; Akatsuka, A.; Tsuruo, T.

doi: 10.1007/BF00230349pmid: 7952498

The P-glycoprotein (P-Gp) encoded by the human multidrug-resistance gene MDR1 has been suggested to play certain roles in the blood-brain barrier (BBB). However, the detailed mechanism of the activity of P-Gp in multidrug-resistance (MDR) remains unclear in human glioma. We examined the localization of P-Gp in human glioma by immunohistochemical (IHC) and immunoelectron microscopic (IEM) methods with anti P-Gp monoclonal antibodies (C219, MRK16). We also examined MDR1 expression in primary glioma and xenografts by reverse transcription-polymerase chain reaction (RT-PCR) with human MDR1-specific primers. The IHC study showed no P-Gp expression on tumour cells but it was present on capillary endothelial cells and IEM analysis showed definitive localization on their luminal surface. MDR1 gene expression was detected in eight primary glioma and three normal brain specimens by RT-PCR, but not in glioma xenografts. The lack of MDR1 expression in these cells appears to be a consequence of the replacement of the original human stroma, including blood vessels, by murine stroma in glioma xenografts. The unique distribution of P-Gp on the capillary blood vessels was confirmed in human glioma by the results of immunohistochemical and molecular biological studies.

Tannapfel, A.; Wittekind, C.; Yasui, W.; Yokozaki, H.; Tahara, E.

doi: 10.1007/BF00230350pmid: 7952499

Hepatocyte growth factor (HGF), identical to scatter factor, (SF) is a secretory glycoprotein from fibroblasts which dissociates and increases the motility of various types of epithelial cells. After treatment of three gastric carcinoma cell lines (MKN-28, MKN-45 and TMK-1) with HGF (10 ng/ml), TMK-1 cells lost their tight cell to cell contact and showed marked scattering, while the two other cell lines remained unaffected. To learn about the underlying mechanism of the HGF induced scattering, we examined the expression of adhesion molecules and growth factor/receptor systems at the mRNA and protein level. The observed scattering of treated TMK-1 cells was associated with a reduction in the expression of E- and P-cadherin protein. The respective mRNA levels remained unchanged after HGF/SF treatment. In the two other cell lines, which showed no scattering, there were no changes in the expression of E-and P-cadherin. All other growth factors and their receptors examined (TGF-α, EGFR, c-met and c-erbB2) remained constant and were not affected by HGF treatment. The results suggest that HGF/SF may regulate cell adhesion in gastric carcinomas via E-and P-cadherin expression at the protein level.

Smedts, F.; Ramaekers, F.; Link, M.; Vooijs, G.; Lauerova, L.; Troyanovsky, S.; Schijf, C.

doi: 10.1007/BF00230351pmid: 7524976

We investigated the expression of keratin subtypes 7, 8, 10, 13, 14, 17, 18 and 19 in the normal cervix, in cervical intraepithelial neoplasia (CIN) lesions and in cervical carcinomas, using a selected panel of monoclonal keratin antibodies, reactive with routinely processed, formalin fixed paraffin embedded tissue fragments. The reaction patterns derived for each keratin antibody were compared with known expression patterns of the various epithelia, previously examined in frozen tissues. Although the reactivity of the antibodies was generally acceptable, considerable modifications to the manufacturers' staining instructions were often necessary. For some antibodies, which were previously thought to be reactive with fresh frozen tissue only, we developed staining protocols rendering them reactive with routinely processed material. As with previous findings in frozen sections we observed increasing expression of keratins 7, 8, 17, 18 and 19 with increasing grade of CIN. In cervical carcinomas the differences in keratin detectability between the main categories were more pronounced than in frozen sections, probably due to fixation and processing. For routine pathology, keratin phenotyping of cervical lesions may be of value in classification. The fact that keratin 7 was detected for the first time in reserve cells, and that this keratin was also found to be expressed in a considerable number of CIN lesions and cervical carcinomas supports the suggestion that reserve cells are a common progenitor cell for these lesions.

Kühler-Obbarius, C.; Milde-Langosch, K.; Löning, T.; Helling-Giese, G.; Salfelder, A.; Peimann, Claus

doi: 10.1007/BF00230352pmid: 7952500

Seven hundred and twelve patients from cancer screening, pregnancy care, outpatient clinics for patients at risk for cervical dysplasia and human immunodeficiency virus (HIV) infection were tested simultaneously for cytological aberrations and human papillomavirus (HPV). Classification of these cases, and of all cytology records throughout 1991 and 1992 was performed according to the “Münchner Nomenklatur” and the Bethesda classification. HPV-directed polymerase chain reaction analysis was carried out with general primers, patients at risk for cervical dysplasia were tested by subsequent hybridization with HPV 16 and 18 probes. Patients from cancer screening and pregnancy care showed similar HPV prevalences ranging between 19.4%–24.6%. In contrast, patients from dysplasia and HIV units were infected in 56.2%–62.3% and 75.0%–76.9% respectively in centre of disease control stage III–IV. HPV detection rates in patients from dysplasia and HIV units increased gradually from 40.1%–52.9% in non-suspicious smears to 80.8%–100% in atypical smears. High risk HPV 16 and 18 infections were detected in 64% of smears with cytological evidence of HPV infection (koilocytosis) to 84.2% in severe dysplasia. Following the Bethesda guidelines, 2.9%–14.7% of all smears initially reported as Pap 2 K (suggestive of HPV infection) would be qualified as risk lesions (low grade squamous intraepithelial lesions), although they tested HPV negative in more than a third of cases. Thus, when using the Bethesda system, HPV analysis is needed to prevent overclassification and overtreatment. The “Münchner Nomenklatur” avoids this dilemma by not mixing morphological statements on infection, atypia and cancer risk.

Carbonelli, D.; Durán, H.; Schwint, A.; Molinari de Rey, B.

doi: 10.1007/BF00230353pmid: 7952501

Nucleolar organizer regions stained selectively with a silver colloid technique (AgNOR) were evaluated during the process of tumour promotion in the skin of mice. Tumour promotion and control skin samples were processed for identification of AgNOR by light microscopy and submitted to a morphometric study of the following AgNOR-related variables: nuclear area (V.NUC); AgNOR number per nucleus (N.NOR); single AgNOR area (V.NOR); total AgNOR area per nucleus (TV.NOR) and proportion of nucleus occupied by AgNOR (TV.NOR/V.NUC). N.NOR exhibited significant differences between control and tumour tissue, but in the promotion period, N.NOR did not exhibit a significant rise until week 24. V.NOR and TV.NOR rose significantly as early as 2 weeks after the onset of promotion when the cells fail to exhibit unusual microscopic features. The significant increase in AgNOR material at the beginning of the promotion period reveals the potential value of the variables assessed in the early quantitative evaluation of cellular alterations which could be linked to the probability of tumour development. Rise in AgNOR material would indicate transcriptional activation leading to an increase in protein synthesis and, ultimately, to the expression of an altered phenotype.

Rinaldi, N.; Barth, T.; Henne, C.; Mechtersheimer, G.; Möller, P.

doi: 10.1007/BF00230354pmid: 7524977

The expression of the β1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were α6β1-positive but lacked α1 through α5. In mild inflammation type A synoviocytes neo-expressed α1, α3, and α5 chains. In severe inflammation both type A and B synoviocytes expressed α3, α4, α5, and α6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the β1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The α1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), and interferon-γ (INF-γ). This effect was enhanced by combining IL-1β and TNF-α. Expression of the α3 chain was up-regulated by IL-1β and, more intensely, by IFN-γ. Transforming growth factor β (TGF-β) inhibited the up-regulating effect of IL-1β and antagonized the effect of IFN-γ on α3 chain expression. Expression of the α5 chain was up-regulated significantly by co-stimulation through IL-1β together with TGF-β or TNF-α. Thus, the β1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1β, TNF-α, IFN-γ, and TGF-β are likely to be among the effectors regulating β1 integrin expression in synoviocytes in vivo.