Hans U. Kasper

Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV‐DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV‐DNA and expression of p16 and EGFR were analyzed. The 5‐year disease‐free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty‐eight percent of the cases contained oncogenic HPV‐DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV‐DNA (p < 0.001). Univariate analysis of the 5‐year DFS revealed a significantly better outcome for patients with p16‐positive tumors (84% vs. 49%, p = 0.009). EGFR‐negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5‐year DFS of 93% for p16+/EGFR− tumors vs. 39% for p16−/EGFR+ tumors (p = 0.003) and to a 5‐year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5‐fold increased risk for relapse in patients with p16‐negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV‐positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

The Human Papillomavirus Type 8 E2 Protein Induces Skin Tumors in Transgenic Mice

Transgenic mice expressing early genes of the cutaneous human papillomavirus 8 (HPV8) spontaneously develop skin papillomas, epidermal dysplasia, and squamous cell carcinoma (6%). As the HPV8 protein E2 revealed transforming capacity in vitro, we generated three epidermal specific HPV8-E2-transgenic FVB/N mouse lines to dissect its role in tumor development. The rate of tumor formation in the three lines correlated with the different E2-mRNA levels. More than 60% of heterozygous line 2 mice, but none of the HPV8-negative littermates, spontaneously developed ulcerous lesions of the skin over an observation period of up to 144 weeks, beginning on average 74+/-22 weeks after birth. Most lesions presented infundibular hyperplasia and acanthosis combined with low-grade dysplasia. Severe dysplasia of the epidermis occurred in 6%. Two carcinomas revealed a sharply demarcated spindle-cell component. Only 3 weeks after a single UV irradiation, 87% of heterozygous line 2 and 36% of line 35 mice developed skin tumors. A rapidly growing invasive tumor composed of spindle cells arose 10 weeks after irradiation of a line-35 animal. The histology of skin cancers in HPV8-E2 mice is reminiscent of a subset of highly aggressive squamous cell carcinoma in immunosuppressed transplant recipients with a massive spindle-cell component.

Spontaneous tumour development in human papillomavirus type 8 E6 transgenic mice and rapid induction by UV-light exposure and wounding

Cutaneous human papillomavirus type 8 (HPV8) is carcinogenic in patients with epidermodysplasia verruciformis. Transgenic mice with the complete early region (CER) of HPV8 spontaneously developed papillomas, dysplasia and squamous cell carcinomas of the skin. To characterize the role of individual early genes in carcinogenesis, the E6 and E6/E7 genes were expressed separately in transgenic mice. Nearly all HPV8-E6-positive mice spontaneously developed multifocal tumours, characterized by papillomatosis, hyperkeratosis and varying degrees of epidermal dysplasia. In 6 % of the cases, the tumours became malignant, comparable with HPV8-CER mice. Thus, in the murine epidermis, E6 is the major oncogene necessary and sufficient to induce spontaneous tumour development up to the level of squamous cell carcinoma. To evaluate the synergistic effects of UV light and wound healing, the skin of HPV8 mice was irradiated with UVA/UVB light or wounded with punch biopsies. These treatments induced papillomatosis in HPV8-CER and -E6 mice within 3 weeks. Irradiation with UVA alone did not induce papillomatosis and UVB alone had a weaker effect than UVA/UVB, indicating a synergistic role of UVA in UVB-induced papillomatosis. An HPV8 infection persisting over decades in interaction with sun burns and wound healing processes may be a relevant cause of skin cancer in humans.

Portal Vein Arterialization Increases Hepatocellular Apoptosis and Inhibits Liver Regeneration

BACKGROUND Portal vein arterialization is performed in particular situations to guarantee sufficient blood flow in the portal vein. In addition, some authors have postulated a proliferation-promoting influence of portal vein arterialization on the liver tissue. However, portal vein arterialization is an unphysiological procedure: It increases portal blood flow and blood pressure as well as oxygenation of the liver tissue. On the other hand, it reduces the influx of hepatotrophic factors from the portal venous blood. The aim of these experiments was to investigate apoptosis and proliferation of hepatocytes during various conditions of the portal perfusion. MATERIALS AND METHODS After 70% liver resection in Lewis rats, the following four experimental groups were formed differing in portal perfusion: (I) hyperperfused, nonarterialized; (II) flow-regulated, nonarterialized; (III) hyperperfused, arterialized; (IV) flow-regulated, arterialized. A warm ischemia of 30 min was kept in all groups. RESULTS Portal vein arterialization of 70% reduced rat livers significantly reduced liver regeneration as shown by a significant reduction in liver weight, body weight, and liver function after 6 wk, in contrast to the group with 70% liver mass reduction and portal venous inflow of the portal vein. Furthermore, we found a significantly elevated number of apoptotic hepatocytes after portal vein arterialization. These results were independent from blood flow regulation of the arterialized portal vein, which caused no improvement of the results. CONCLUSIONS Portal vein arterialization should be performed only temporarily and is clinically not recommended as a permanent option, because of the increased hepatocellular apoptosis and the very distinctive, negative long-term effects on liver weight.

Ex vivo analysis of antineoplastic agents in precision-cut tissue slices of human origin: effects of cyclooxygenase-2 inhibition in hepatocellular carcinoma.

Abstract: Cultures of precision‐cut tissue slices allow the investigation of substance effects on human tissues under in vivo‐like conditions over a limited time span. We have adapted the model for direct analyses of antineoplastic substances on tumor tissues. We have recently demonstrated that selective cyclooxygenase‐2 (COX‐2) inhibitors strongly suppress growth of human hepatocellular carcinoma (HCC) cells in vitro and nude mouse HCC implants by inducing apoptosis and reducing proliferation. We have now analyzed the effects of COX‐2 inhibition on human tumor tissue. Three hundred micrometer slices of tumorous and non‐tumorous liver tissue from three surgically resected HCCs were cultured with increasing concentrations of the selective COX‐2 inhibitor Meloxicam (20–200 μM) for 6, 12, 24, and 48 h. The cultured tissue slices were analysed morphologically and by immunohistology for proliferation (Ki‐67), apoptosis (M30), and COX‐2 expression. COX‐2 was expressed in all HCCs and in the non‐tumorous liver tissue. Cytoplasmic COX‐2 immunoreactivity in HCCs increased during culturing time. In two of three cases, COX‐2 inhibition significantly increased tumor cell apoptosis in HCCs, whereas the low basal apoptosis rate in the non‐tumorous liver parenchyma did not change. Tumor cell proliferation was mildly reduced, but the changes did not reach statistical significance. These results demonstrate that the precision‐cut tissue slice culture model is a useful tool to analyze directly drug‐dependent antitumorous or unwanted organ‐specific effects. The analysis of COX‐2 inhibition lends further support to the antineoplastic effects previously demonstrated in vitro and in animal models.

Conspicuity of zones of ablation after radiofrequency ablation in porcine livers: Comparison of an extracellular and an SPIO contrast agent

To compare conspicuity of zones of ablation on nonenhanced, gadopentetate dimeglumine‐(Gd‐DTPA) and ferucarbotran‐(SPIO)‐enhanced magnetic resonance (MR) images.

Induction of early murine cytomegalovirus infection by different reporter gene-associated recombinant viruses.

Summary.  Murine cytomegalovirus (MCMV) has provided useful models for acute, chronic and latent CMV infection because of its similarities in structure and biology with human CMV. We report the induction of acute MCMV hepatitis with different bacterial artificial chromosome (BAC)‐cloned virus constructs [MCMV‐SEAP which includes the gene for secreted alkaline phosphatase (SEAP) under Rous sarcoma virus (RSV)‐promoter control, MCMV‐GFP which includes the gene for enhanced green fluorescent protein (eGFP) under HCMV‐ie promoter control, MCMV‐HBs includes the gene for hepatitis B surface antigen (HBsAg) under simian virus (SV)40‐promoter control and the DeltaMC95.21 virus in which the m152 gene was deleted and substituted by the reporter gene lacZ] in order to elucidate the histopathological changes together with different reporter‐gene products in the liver tissue and the effect of the deletion of a certain gene. All the virus constructs induced a similar mild acute hepatitis which had its climax from days 3 to 5 post‐infection in immunocompetent mice. In situ, the reporter‐gene products beta‐galactosidase and secreted alkaline phosphatase could be visualized in relation to the inflammatory changes. The composition of the invading cell populations did not change even in the absence of the m152 gene. Additionally discrete inflammatory changes were seen in kidney and serosa while the other organs were not involved. This model helps us understand the immunological and histopathological mechanisms of the CMV‐induced hepatitis, which plays an important role especially in the immunocompromised patient. The morphological changes can be analysed while the respective reporter gene product is expressed by the virus construct.