Hartwig Kosmehl

Evaluation of microvessel density by computerised image analysis in human renal cell carcinoma: Correlation to pT category, nuclear grade, proliferative activity and occurrence of metastasis

Abstract A high microvessel density is suspected to favour tumour progression and the occurrence of metastasis. To elucidate the significance of abundant vessels for the behaviour of human renal carcinomas, the microvessel density of 110 renal cell carcinomas was correlated to pT category, nuclear grade, proliferative activity, occurrence of metastasis and relapse-free survival interval. The microvessels were quantified using CD31 immunostaining of endothelial cells and computer-aided image analysis. The rules for reproducible microvessel counting, as defined by Weidner, were strictly observed. A statistically significant relationship between the microvessel density and nuclear grade, proliferative activity, occurrence of metastasis and relapse-free survival was found; only for tumour size could no such relation be seen. Perplexingly, there is a diminution of microvessel density in association with increasing nuclear grade, proliferative activity, relapse-free survival interval and frequency of metastasis. This finding is contradictory to the hypothesis that an increasing microvessel density indicates a worsening prognosis.

Evidence of ED-B+ fibronectin synthesis in human tissues by non-radioactive RNA in situ hybridization. Investigations on carcinoma (oral squamous cell and breast carcinoma), chronic inflammation (rheumatoid synovitis) and fibromatosis (Morbus Dupuytren)

Abstract The splicing variant of fibronectin containing the ED-B domain (oncofoetal fibronectin) occurs in foetal tissues, reparative processes, organ fibrosis and in tumour tissues. Consequently, a supportive effect of ED-B+ fibronectin for tissue remodelling and tumour progression is assumed. A non-radioactive RNA-RNA in situ hybridization protocol for the investigation of ED-B+ fibronectin synthesis applicable in human tissues is introduced. The ED-B+ fibronectin synthesis was investigated in human disease processes, for which the occurrence of ED-B+ fibronectin is well demonstrated by immunohistochemistry (rheumatoid arthritis, oral squamous cell carcinoma, invasive ductal carcinoma of the breast and nodular palmar fibromatosis). The ED-B+ fibronectin synthesis could be shown in lining cells and in endothelial cells of synovial villi in rheumatoid arthritis, in stromal cells of oral squamous cell carcinoma and invasive ductal carcinoma and in fibro-/myofibroblasts in the proliferative and early involutional phase of nodular palmar fibromatosis. By means of double labelling (α-smooth muscle actin immunostaining - ED-B+ fibronectin in situ hybridization), the ED-B+ fibronectin synthesis could be shown to be a typical feature of myofibroblasts. In contrast to the often diffuse ED-B+ fibronectin immunostaining, only a few synthetically active stromal cells were observed focally accentuated within the tumour, which were interpreted as hot spots of tumour-stroma interaction.

Fibrillary co-deposition of laminin-5 and large unspliced tenascin-C in the invasive front of oral squamous cell carcinoma in vivo and in vitro

Purpose: Invasion of oral squamous cell carcinoma (OSCC) is associated with laminin-5 (Ln-5) synthesis, focal Ln-5 loss from the basement membrane (BM), and Ln-5 depositions in the stroma beneath invading carcinoma cell complexes. Methods: The study is focused on the laminin-5 matrix reorganisation within the stroma of the OSCC invasive front outside the basement membrane region as well as in OSCC-fibroblast co-culture in relation to unspliced tenascin-C (Tn-CL) and ED-B+ fibronectin (ED-B+ fn) using confocal laser scanning microscopy. Results: In vivo, Ln-5 was demonstrated as fibrillary deposition in the invasive front. It was co-localised to Tn-CL. In pure OSCC cultures, Ln-5 was synthesised and deposited as a spot-like matrix. Fibrillary structures were not found. In contrast, in the OSCC-fibroblast co-culture, a fibrillary Ln-5 matrix organisation was revealed within the interface of OSCC cell-fibroblast complexes exclusively in co-distribution with Tn-CL and ED-B+ fn. Conclusion: At least in vitro, a carcinoma cell-stroma fibroblast interaction is indispensable for fibrillary Ln-5/Tn-CL matrix organisation. Behind the parallels to the initial basement membrane formation in organotypic cultures, the fibrillary multiprotein complexes at the OSCC cell-fibroblast interface are suggested as provisional basement membrane fragments with a possible supportive role for invasive tumour behaviour.

Expression of the transmembrane protein tyrosine phosphatase RPTPα in human oral squamous cell carcinoma

Abstract Little is known about the role of protein-tyrosine phosphatases (PTPs), the cellular counterparts of protein-tyrosine kinases, both for normal growth regulation and for its dysregulation in cancer. The receptor-like PTPα (RPTPα) may play a positive role in growth regulation and has been shown to be overexpressed in colon carcinoma. An RNA/RNA in situ hybridisation protocol for RPTPα as well as RPTPα immunohistochemistry was developed to evaluate RPTPα expression in oral squamous cell carcinomas (OSCCs) of different histological grade and to reveal the synthetically active cells and their tissue distribution. In well-differentiated OSCC (G1), RPTPα mRNA could be detected by in situ hybridisation exclusively in stroma cells (fibro/myofibroblasts and inflammatory cells). A higher histological grade (G2/G3) was associated with an increased number of RPTPα-synthesising carcinoma cells haphazardly distributed within invading tumour areas. Consistent results were obtained by immunocytochemistry. Thus, both carcinoma dedifferentiation and stroma recruitment and activation seem to be associated with an upregulation of RPTPα expression in OSCC. The results speak in favour of the important role of activation of stroma fibro/myofibroblasts influencing the biological behaviour of epithelial tumours and also suggest that elevated RPTPα expression may be a more general marker for proliferating or dedifferentiated cells.

Fibronectin Splice Variants – Prognostic Markers for the Stage of Renal Interstitial Fibrosis in the Rat

Background/Aims: Renal interstitial fibrosis (RIF) is the main cause for progressive renal failure, but its pathogenic factors are not well known. In animal models of renal fibrogenesisdone thus far an increase of total fibronectin (FN) mRNA has been proved. Recent studies have pointed to a key role of the splice variant EIIIA+-FN and EIIIB+-FN for the development of organ fibrosis. However, a broader knowledge of the distribution of these different FN mRNA isoforms is still lacking. Our aim was to study the particular expression of the EIIIA+-FN and EIIIB+-FN during the process of fibrogenesis in two rat models and to evaluate the FN isoforms as diagnostic/prognostic marker for the stage of interstitial damage in rat kidneys. Methods: Kidneys of unilateral ureteral obstruction (UUO) and control rats were removed in intervals of 5, 14 or 21 days after surgery. For the investigation of kidney damage due to uranyl nitrate (UN), rats obtained a single i.p. dose of 5 mg/kg body weight UN and were killed 2, 10 and 20 weeks thereafter. The quantitative RT-PCR method was used to estimate the total FN, EIIIA+-FN and EIIIB+-FN transcription rate. Results: In the UUO model, a significant augmentation of both isoforms was obtained in the kidneys in the first 5-day interval, which was more pronounced at the 21-day interval. In the UN-treated kidneys there appeared only a continuous increase of EIIIA+-FN and the splice variant EIIIB+-FN failed to show a shift in these animals as compared to the controls. Conclusion: Both animal models generated fibrogenic damages of the tubulointerstitium, whereas only the UUO resulted in progressive fibrosis. Absence of EIIIB+-FN seems to enhance the progression of fibrogenesis.

Dreidimensionales In-vitro-Invasionsmodell für orale Plattenepithelkarzinome

Da die Invasion von Tumorzellen ein räumlich strukturierter Prozeß unter Beteiligung der Tumorzelle und des benachbarten Stromas ist, erfordern In-vitro-Invasionsmodelle das Vorhandensein von Äquivalenten beider Kompartimente in räumlich definierter Anordnung. Die Evaluation von Differenzierung, invasivem Potential und Basalmembranformierung neuer Zellinien humaner oraler Plattenepithelkarzinome (OSCC) wurde in vitro unter dem Einfluß humaner Fibromatosefibroblasten im 3-D-Kokultursystem (Kollagen-Typ-I-Gel/ Fibroblasten – Matrix-in-vitro-Invasionsmodell) untersucht. Die OSCC-Zellinien wurden auf Kollagen-I-Matrix mit und ohne eingeschlossene Fibro-/Myofibroblasten (kultiviert aus nodulärer Palmarfibromatose) kultiviert. Die Bewertung der Invasion erfolgte in der konventionellen HE-Färbung nach Paraffineinbettung. Immunohistochemische Untersuchungen auf Cytokeratin (MNF116), Involukrin (Sy5), Vimentin (V9), Kollagen IV (CIV22), Laminin (α1-Kette, 4C7) und Ki-67 (MIB1) sowie Transmissionselektronenmikroskopie wurden zur Beurteilung eingesetzt. Es wurden folgende neue OSCC-Zellinien etabliert: PE/CA-PJ15, PE/CA-PJ34, PE/CA-JP41. Die Invasion erfolgte ausschließlich in Fibroblasten-haltiges Kollagen-I-Gel mit Herausbildung eines mehrschichtigen, epithelähnlich gegliederten Tumorzellverbandes. Eine polarisierte Expression von Cytokeratin und Involukrin wurde ausschließlich in den superfiziellen Zellen gefunden. Es erfolgte kein immunhistochemischer/elektronenmikroskopischer Nachweis einer strukturellen Basalmembran trotz der Expression von Kollagen IV und Laminin durch Tumorzellen in der zweidimensionalen Zellkultur. Es ergaben sich keine Unterschiede in der Proliferation bei Kultivierung mit und ohne Fibroblasten. Eine Invasion der Karzinomzellen verlangt somit die Anwesenheit von Fibroblasten im Gel; das Fehlen einer Basalmembran bedeutet nicht per se invasives Wachstum. Dieses Ergebnis spricht für eine wesentliche Beteiligung des Tumorstromafibroblasten an der Karzinominvasion. Die Stratifizierung des Epithelverbands und die terminale Differenzierung der epithelialen Tumorzellen sind nicht an die Ausbildung einer strukturellen Basalmembran geknüpft. Because tumour cell invasion is a three-dimensional process involving tumour cells as well as stromal cells, tumour invasion models should include equivalents of both compartments in a spatially defined organisation. Aims: Evaluation of differentiation and invasive potential in relation to basement membrane (BM) formation of new human oral squamous cell carcinoma (OSCC) cell lines under the influence of fibromatosis fibroblasts in a 3D co-culture system (collagen type I/fibroblast-containing matrix). Material and methods: OSCC cell lines were established and cultured on collagen type I matrix with and without the inclusion of fibro-/myofibroblasts derived from nodular palmar fibromatosis). Evaluation of invasion was done by conventional H & E staining after paraffin embedding; immunohistochemistry was performed for cytokeratins (MNF 116), involucrin (Sy5), vimentin (V9), collagen type IV (CIV22), laminin (α1-chain, 4C7), and Ki-67 (MIB1); and electron microscopy followed. Results: The OSCC cell lines PE/CA-PJ15, PE/CA-PJ34, PE/CA-PJ41 were invasive exclusively on gels containing fibroblasts with stratification of the multilayerd OSCC cell coat and polarised expression of cytokeratin and involucrin. There was no evidence for BM, either by immunohistochemistry or by electron microscopy, although OSCC are immunohistochemically positive for collagen type IV and laminin in the 2D cell culture. There were no differences in proliferative activity of the gels with and without fibroblasts. Conclusions: Carcinoma cell invasion depends on the presence of fibroblasts in the gel indicating the important role of tumor stromal fibro-/myofibroblasts during carcinomic invasion. The absence of a BM does not, per se, imply an invasive tumour growth. Moreover, stratification and terminanl differentiation of epithelial tumour cells occur independently of a structural BM.

Basaloides Plattenepithelkarzinom (basaloid-squamöses Karzinom) des Mundbodens

The basaloid squamous cell carcinoma (BSCC) is a variant of squamous cell carcinoma, having histologically distinctive features and appearing in the oral cavity, upper respiratory tract and esophagus. Histological hallmarks are the presence of a basaloid component in intimate association with squamous cell carcinoma. The basaloid component is characterized by tightly packed nests of cells with scant cytoplasm and hyperchromic nuclei without visible nucleoli and an increased mitosis rate. Basaloid squamous cell carcinoma is said to have a higher malignant potential than common oral squamous cell carcinoma with an increased incidence of regional lymph-node metastases and distant metastases. Our finding of a Ki-67 index of 30% and the immunohistochemical demonstration of p53 protein speaks well for enhanced aggressive biological behavior. The differential diagnosis includes the adenoid cystic, mucoepidermoid, neuroendocrine, adenosquamous and conventional oral squamous cell carcinoma. Because of early dissemination, radical surgical treatment and additional radiation therapy are considered necessary. Our findings indicate that partial clinical and histological tumor regression occurs after systemic neoadjuvant chemotherapy.Das basaloide Plattenepithelkazinom (basaloid-squamöses Karzinon; basaloid squamous cell carcinoma) ist eine Plattenepithelkarzinomvariante, die durch ihr histologisches Erscheinungsbild definiert ist und im Bereich der Mundhöhle, der oberen Atemwege und im Ösophagus auftritt. Histologische Merkmale sind die Verbindung eines Plattenepithelkarzinomanteils mit einer basaloiden Karzinomkomponente, die durch dicht gepackte Zellnester mit spärlichem Zytoplasma, hyperchromatische Zellkerne ohne abgrenzbare Nukleoli und eine hohe Mitoserate gekennzeichnet ist. Den basaloiden Plattenepithelkarzinomen wird gegenüber den gewöhnlichen oraden Platten-epithelkarzinomen ein höheres malignes Potential mit gesteigerter lymphatischer und hämatogener Metastasierung zugeschrieben, welches in der eigenen Fallbeobachtung durch einen Ki 67-Index von 30% und durch die massive immunhistochemische p53-Expression bestätigt wird. Die Differential-diagnose zum adenoidzystischen, mukoepidermoiden, neuroendokrinen, adenosquamösen Karzinom sowie zum konventionellen oralen Plattenepithelkarzinom wird dargestellt. Aufgrund einer frühen Disseminierung wird neben der radikalen Chirurgie eine Strahlentherapie als notwendig erachtet und eine systemische Chemotherapie erwogen. In der eigenen Fallbeobachtung konnte infolge einer neoadjuvanten Chemotherapie eine partielle klinische und histologische Tumorregression beobachtet werden, so daß eine präoperative Chemotherapie beim basaloiden Plattenepithelkarzinom als sinnvoll angesehen werden kann. The basaloid squamous cell carcinoma (BSCC) is a variant of squamous cell carcinoma, having histologically distinctive features and appearing in the oral cavity, upper respiratory tract and esophagus. Histological hallmarks are the presence of a basaloid component in intimate association with squamous cell carcinoma. The basaloid component is characterized by tightly packed nests of cells with scant cytoplasm and hyperchromic nuclei without visible nucleoli and an increased mitosis rate. Basaloid squamous cell carcinoma is said to have a higher malignant potential than common oral squamous cell carcinoma with an increased incidence of regional lymph-node metastases and distant metastases. Our finding of a Ki-67 index of 30% and the immunhisto-chemical demonstration of p53 protein speaks well for enhanced aggressive biological behavior. The differential diagnosis includes the adenoid cystic, mucoepidermoid, neuroendocrine, adenosquamous and conventional oral squamous cell carcinoma. Because of early dissemination, radical surgical treatment and additional radiation therapy are considered necessary. Our findings indicate that partial clinical and histological tumor regression occurs after systemic neoadjuvant chemotherapy.