The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin-dependent kinases p16 INK4a . In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type-specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence-labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT-PCR were performed, and for p16 INK4a detection, immunohistochemistry was performed. With 21/39 (53%) HPV-positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16 INK4a staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16 INK4a expression was of statistical significance (p 5 0.02). Kaplan-Meier estimations revealed better survival outcome for patients with HPV-positive tumors with detectable E6/E7 mRNA and p16 INK4a overexpression (p 5 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16 INK4a staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16 INK4a as a surrogate marker with the potential to impact the standard of care of HPV DNA-positive head and neck carcinomas.Malignant tumors of the upper aerodigestive tract account for $7% of all cancers worldwide, 1 with squamous cell carcinomas (SCCs) constituting the largest portion among the different tumor entities. 2 Even though a majority of SCCs of the head and neck (HNSCC) seems associated to etiologic factors as carcinogenetic substances in tobacco smoke and alcohol, 3 an increase of especially younger patients without reported misuse of these environmental factors in history has been recognized. 4 Therefore, an increase in knowledge of other factors such as the infection with human papillomaviruses (HPV) promoting carcinogenesis in especially the latter patient group gains in importance.Epidemiologic data demonstrate HPV DNA prevalences of about 20-30% concerning all anatomical tumor sites of the head and neck region, 4-6 with the SCC of the Waldeyer's tonsillar (squamous cell carcinogenesis of tonsillar cancers, TSCC) ring showing an HPV DNA-positive rate of up to 60% as particularly predestined for an infection with HPV. 4,5,7 Recent data from Sweden describe HPV DNA infections in tonsillar carcinomas in even up to 85% of the investigated patients. 8 HPV DNA-positive HNSCCs, again with accentuation of those derived from the Waldeyer's tonsillar ring, are considered as being ...

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Epithelial cellular fibronectin is frequently repressed after malignant transformation in a variety of cancers. This change has been associated with a loss of contact inhibition. To determine if these findings are unique to malignant processes and to identify mechanisms responsible for fibronectin suppression, we investigated fibronectin expression patterns in 46 head and neck carcinomas, 16 samples of adenoid tissue, and 10 benign mucosal biopsies. We report fibronectin suppression in 78% of the head and neck cancer samples, occurring most prominently within tumor cells, as opposed to the adjacent stroma which exhibited abundant fibronectin. Interestingly, fibronectin was also strongly repressed in chronically inflamed adenoid samples. We showed that fibronectin suppression is mediated by different mechanisms in both benign as well as malignant scenarios: In adenoids, macrophages and T-cells were visualized throughout epithelium that has lost its tight cellular array, allowing leukocyte passage. We have shown that tumor necrosis factor-• secreted by macrophages is capable of inducing epithelial derangement via activator protein-1 and nuclear factor-κB mediated fibronectin suppression. In head and neck carcinomas, we identified human papilloma virus early protein-2 as a fibronectin transcription inhibitor. We conclude that epithelial fibronectin suppression may not be a hallmark of malignancy, because it can concur with benign processes that involve leukocyte migration. Furthermore, our data suggest that the pattern of fibronectin suppression within the tumor structure largely depends on the cancer cell-stroma relation, which could explain previous conflicting reports on its repression or overexpression along with malignant transformation. In addition, our data support an involvement of human papilloma virus as a mechanism of carcinogenesis mediated via a loss of fibronectin gene expression.

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Abstract. Overexpression of lysyl oxidase (LOX) is associated with the invasive potential of metastatic breast and head and neck cancer (HNC) cells and reduced metastasis-free and overall survival. Recently, we have demonstrated up-regulation of a new member of the LOX family, lysyl oxidase-like 4 (LOXL4), in invasive HNC revealed a significant correlation between LOXL4 expression and local lymph node metastases and higher tumour stages. The objective of this study was to examine whether cellular LOXL4 may provide an effective target for cell-meditated immunotherapy in invasive tumours associated with LOXL4 overexpression. As a feasibility study we expressed LOXL4 mRNA in immature dendritic cells derived from human peripheral blood mononuclear cells (PBMC). LOXL4 protein expression was ascertained using Western blotting and immunocytochemistry with polyclonal rabbit anti-LOXL4 antibody. The successfully transfected immature dendritic cells (DCs) were induced to mature with GM-CSF, IL-4, IL-1ß, TNF-α, IL-6, and PGE2, and then used to stimulate T cell enriched non-adherent fraction of PBMC. LOXL4 specific T cell stimulation induced cytotoxic T lymphocyte (CTL) response was monitored using IFN-γ secretion from the non-adherent PBMC fraction exposed to mature, LOXL4 transfected DCs acting as the antigen presenting target cells. LOXL4-DC stimulated T cells produced higher IFN-γ secretion compared to unstimulated T cells and T cells stimulated with untransfected DCs, in the presence of the pan-DR-epitope (PADRE). These initial results demonstrated the potential for LOXL4-transfected DCs to serve as efficient tumour vaccine and support their suitability as a vaccination strategy applicable to cancer patients with tumour specific up-regulation of LOXL4.

Abstract. Lysyl oxidases are a family of five copperdependent amine oxidases including LOX, LOXL, LOXL2, LOXL3 and LOXL4. LOX and LOXL are essential for the assembly and maintenance of extracellular matrixes. LOXL2, LOXL3 and LOXL4, secreted and active enzymes, were also noted in association with diverse tumor types. We have recently reported overexpression of the LOXL4 mRNA and protein and a close relation of LOXL4 with the pathogenesis of head and neck squamous cell carcinomas (HNSCC). In this study, we analyzed the organization of the LOXL4 gene and addressed the regulatory mechanisms responsible for the overexpression. We demonstrated de novo transcription of the LOXL4 gene in HNSCC, but not in normal squamous epithelial cells. Analysis of the consecutive promoter region spanning positions -960 to -1 identified binding sites for several transcription factors. Promoter constructs containing selected specific promoter regions and consensus binding sites exhibited significantly increased reporter gene activity in HNSCC cells, but not in normal epithelial cells in transient coexpression experiments. The activity profiles of some of these constructs were similar in both cell types indicating that elements of the basic transcriptional regulatory mechanisms remained intact in HNSCC cells. DNA-binding experiments demonstrated that nuclear extracts from HNSCC cells have increased binding activity to the TATA (-25) and the SP1 (-181) sites compared to normal epithelial cells, suggesting that these transcription factors are involved in the upregulation of LOXL4 gene expression in HNSCC.