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Although external auditory canal cholesteatoma (EACC) was first described in 1850, its cause remains surprisingly unclear. Angiogenesis, the formation of new blood vessels, is essential to normal development and wound healing in adults. Abnormal regulation of angiogenesis has been implicated in the pathogenesis of several disorders. The aim of this study was to analyse angiogenesis regulator expression in EACC.Cryostat sections of 13 investigated EACC tissue samples and normal control tissue were immunostained for angiogenic hepatocyte growth factor (HGF)/scatter factor (SF), its c-Met receptor and vascular endothelial growth factor (VEGF) using a standard streptavidin-biotin complex procedure. Staining against von Willebrand factor (vWF) served as an endothelial marker. Statistical analysis was performed semiquantitatively.The assayed angiogenic factors were all present in the EACC tissue, and partly overexpressed. vWF was detected in the apical layers of the matrix epithelium. Positive immunoreactivity for c-Met and VEGF was detectable in all layers of the EACC epithelium; however, adjacent tissue did not express c-Met and VEGE. HGF/SF was predominantly expressed in the adjacent perimatrix tissue and fibroblasts in particular were stained positive.The presence of vWF in the apical part of the matrix depicted the attempt at angiogenesis in this part of the EACC. The detection of VEGF and c-Met in the epithelial part of the EACC implied that their origin may be epithelial, while HGF/SF may be secreted or stored in the adjacent mesenchymal EACC tissue. The angiogenic factors investigated seem to play an important role in establishing that EACC occurs by modulation of angiogenesis.

Tumors of the nasal cavity are located at a complex anatomic site and show a huge histological diversity. Although dealing with a rare malignancy, the last decade has brought – besides new histological and clinical classifications – a variety of new insights into etiological agents, tumor biology and therapeutic concepts as well as valuable overviews of rare histological subtypes. This review tries to disentangle the different medical and scientific aspects of the most frequently encountered histological types of tumors in the nasal cavity and the paranasal sinuses. We concentrate on epidemiology, classification, etiology, cytogenetics and molecular genetics, outcome and prognosis as well as treatment modalities, as far as the past few years have brought considerable new insights. Our principal aim is to provide the clinician with important data from publications of the last decade.

The aim of this study was to assess the value of topically applied estrogens in patients with hereditary hemorrhagic telangiectasia. Twenty-six patients with this disorder were treated with argon plasma coagulation and randomized into 2 groups: group A, which had postoperative application of estriol ointment (n = 14), and group B, which had postoperative application of dexpanthenol ointment (n = 12). Over a period of 12 months, the frequency and intensity of bleeding, the patient's satisfaction, and the success of the treatment were evaluated with a questionnaire. Before the operation, more than 90% of the patients in both groups complained of daily episodes of epistaxis. Twelve months after treatment, the frequency and intensity of bleeding had significantly decreased in group A as compared to group B. Of the patients in group A, 93% were satisfied with the treatment. Of the patients in group B, only 42% were satisfied with the treatment. In both groups, more than 90% of the patients were willing to undergo the same treatment again. The combined treatment approach with argon plasma coagulation and topical estriol enables us to significantly prolong the hemorrhage-free interval.

Specific immune therapy targeting residual areas of cancer cells may emerge as a powerful treatment strategy for head and neck squamous cell carcinoma (HNSCC). In order to define possible targets for immune therapy, we evaluated the frequency of two groups of tumor antigens-the RAGE and GAGE families-by means of reverse transcriptase polymerase chain reaction using primary HNSCCs (n = 28), mucosa specimens as normal controls (n = 10) and HNSCC cell lines (n = 6). By means of specific primer selection we could differentiate between RAGE-1, -2, -3 and -4, as well as between two groups of GAGE genes (GAGE-1,2,7 vs GAGE-3,4,5,6,8). While all mucosa tissues (from smokers and non-smokers) were negative for both antigen families, 24/28 investigated tumors were positive for up to 5 tumor antigens. Among the RAGE genes, RAGE-1-positive tumors were the most abundant (8/28), followed by RAGE-2 (7/28) and RAGE-4 (6/28). Differences in the locations of HNSCCs were reflected by different RAGE family members being expressed most frequently: larynx, RAGE-1; oropharynx, RAGE-2; and hypopharynx, RAGE-4. Primers against GAGE-1,2,7 and GAGE-3,4,5,6,8 revealed 6/27 and 16/27 positive tumors, respectively. This report suggests that RAGE genes and GAGE-3,4,5,6,8 may be promising candidates for specific immune therapy in HNSCC.

Follicular dendritic cells (FDC) constitute an essential component of B cell follicles, normally serving as antigen-presenting cells and as cells regulating the germinal center reaction. FDC sarcoma is a very rare tumor. We report the case of a 55-year-old male patient who showed persistent cervical lymph node swelling. On biopsy the tumor consisted of spindle cells with nuclear atypia. Immunophenotypic investigation of the tumor tissue revealed a FDC sarcoma of a cervical lymph node. The tumor cells were characterized immunohistochemically especially by the expression of CD21 and focal expression of CD68. In conclusion, FDC sarcoma has to be considered in cases of tumors of uncertain origin in cervical lymph nodes, tonsils, soft palate, or nasopharynx. For clinical practice, it is important to note that sarcomas in cervical lymph nodes are not necessarily metastases but can be primary tumors.

Surgical reduction of the inferior turbinate remains a widely used therapy for nasal obstruction caused by hyperplastic turbinates. Current methods are costly and time-consuming, require nasal packing, and cause extended postoperative swelling. Argon plasma coagulation (APC) is a new, innovative technique in otorhinolaryngological surgery that is based on high-frequency electrical current. The current is transmitted through ionized argon gas (plasma) contact-free to the tissue surface and creates a coagulation and devitalization zone with limited penetration. Forty-five patients with nasal obstruction were treated under local anesthesia for reduction of the inferior turbinates with APC. Seventy-six percent of the patients reported an improvement of postoperative swelling within the first week. After 3 months, 86% reported having better nasal airflow than they had before the operation. Macroscopically, after 4 weeks the turbinate was covered by normal mucosa in 51% of the patients. Crust formation was minimal, and no bleeding occurred. Turbinate reduction with APC is an alternative technique with many advantages, and the results are comparable to those of standard techniques. Ninety-five percent of the patients were satisfied with the operation.

Juvenile-onset recurrent respiratory papillomatosis is a relatively uncommon disease that presents clinically with symptoms ranging from hoarseness to severe dyspnea. Human papilloma viruses type 6 and 11 are important in the etiology of the papillomata and are most probably transmitted from mother to child during childbirth. Although spontaneous remission is frequent, a rare fatal course because of pulmonary spread or malignant transformation has occurred. CO2 laser evaporation of papillomas and adjuvant drug therapy using lymphoblastoid alpha-interferon are the most common treatment modalities at present. However, several other treatment modalities have been tried with varying success. Recent advances in basic research and different therapeutic approaches are reviewed.

Angiogenesis is crucial for tumor growth and metastasis. In several tumors, microvascular density has been shown to correlate with metastasis and aggressiveness. Basic fibroblast growth factor (bFGF) has potent angiogenic activity and has been identified in a wide variety of malignancies including head and neck squamous cell carcinomas (HNSCC). Material and Methods Frozen sections of 50 HNSCC were immunostained for von Willebrand factor and bFGF. Microvessels were counted by light microscopy; bFGF expression was studied at the light and electron microscopic level. Laryngeal cancer cell line HlaC79 was incubated with interferon (IFN) alpha and beta. bFGF quantification was performed by ELISA, and antiproliferative effects were determined by BrdU assay.The mean number of blood vessels (77.5 +/- 23.7) is significantly increased in HNSCC compared with controls (17.1 +/- 5.9). bFGF protein expression was detected in all HNSCC but not in control tissue. An correlation between bFGF expression and mean number of microvessels was found (p.001). However, no correlation between bFGF expression and the main clinicopathologic features was shown. The long-term exposure (144 hr) of HNSCC cells to noncytostatic concentrations of IFN alpha and beta (10 U/mL) down-regulated the protein production of bFGF.bFGF expression and angiogenesis are enhanced in HNSCC. The higher microvessel density in HNSCC with strong bFGF expression supports the importance of bFGF for tumor angiogenesis. IFN alpha and beta treatment leads to a down-regulation of bFGF expression independent of their antiproliferative effects, suggesting that IFN treatment might result in a reduction of angiogenesis in HNSCC.

Vascular endothelial growth factor (VEGF) has potent angiogenic activity and has been identified in a wide variety of malignancies, including head and neck squamous cell carcinoma (HNSCC). The tumour-suppressor gene p53 has been thought to regulate VEGF. Cryostat sections of 33 head and neck squamous cell carcinomas (HNSCC) were immunostained for VEGF using a standard streptavidin-biotin complex procedure. To evaluate angiogenesis, microvascular density was counted by staining endothelial cells immunohistochemically using anti-vWF monoclonal antibody. The p53 gene status was analysed using a PCR-SSCP analysis and direct sequencing. VEGF positive staining was detected in 18 (55%) out of 33 tumours. VEGF immunoreactivity did not correlate with the main clinicopathological characteristics of the patients (localization, T-stage, N-status, histological grading). Statistical analysis gave a clear correlation between the tumour vascularity and the VEGF protein expression (p = 0.0036). VEGF negative tumours showed a lower mean number of microvessels per microscopic field (60.3 +/- 15.5) than VEGF positive tumours (79.6 +/- 22.9). P53 mutations were identified in 12 (36.4%) of 33 tumours. The association of p53 mutations and VEGF expression level was significant (0.027). The higher microvessel density in VEGF positive tumours supports the importance of VEGF for tumour angiogenesis in HNSCC. Our results support the hypothesis of a p53 regulation on the angiogenic process through a VEGF up-regulation.

Bei der hereditaren hamorrhagischen Teleangiektasie (Morbus Osler-Rendu-Weber) stellt die rezidivierende Epistaxis fur den Patienten eine grose Belastung und ein oft nur schwer therapeutisch angehbares Problem dar. Bisherige beschriebene Therapieformen beinhalten die Dermoplastik, die Elektro- und Laserkoagulation, die Brachytherapie mit Iridium sowie die systemische Gabe von Ostrogenen. Ein Therapieansatz mit der Argon-Plasma-Koagulation (APC) erscheint aufgrund theoretischer Uberlegungen in hohem Mase erfolgversprechend. Bei diesem in der HNO bislang noch nicht angewendeten Koagulationsverfahren ist die Tiefenwirkung durch den Desik-kationseffekt auf die Schleimhaut auf ca. 1–2 mm beschrankt, wodurch das Risiko einer Schadigung benachbarter Gewebe minimal ist. Die APC enfaltet ihre koagulierende Wirkung bevorzugt am Ort der hochsten elektrischen Leitfahigkeit, demzufolge insbesondere an frischen Blutungsherden sowie an Blutgefasen. Vier multipel vorbehandelte Patienten mit rezidivierender Hb-relevanter Epistaxis bei Teleangiektasien in beiden Nasenhaupthohlen wurden mit der APC therapiert und der pra- und postoperative Befund makroskopisch und histologisch dargestellt. Die Patienten waren postoperativ mit dem Ergebnis sehr zufrieden. Die Frequenz und Intensitat der Blutungen hatte deutlich abgenommen. Die ersten klinischen Erfahrungen bestatigen die theoretischen Erwagungen, welche die APC als eine sinnvolle Alternative zu bisherigen Behandlungsformen bei der hamorrhagischen Teleangiektasie erscheinen lassen.

The growth of tumor cells can be regulated by a variety of cytokines. To investigate the pathogenesis of head and neck cancer and explore a new therapeutic approach for the carcinoma, the role of interleukin-6 (IL-6) in the growth of a human head and neck squamous cell carcinoma (HNSCC) cell line was examined. Whether or not IL-6 is increased in HNSCC and whether or not IL-6 antisense oligonucleotide treatment could decrease proliferation and angiogenic activity of HNSCC cell lines, was determined. Established human HNSCC cell lines were screened for IL-6 expression at both mRNA and protein levels. By using a 15-mer antisense phosphorothioate oligonucleotide targeting a sequence in the second exon of the IL-6 gene, modulation of IL-6 and vascular endothelial growth factor (VEGF) expression was examined in UMSCC IIA in cell supernatants by capture enzyme-linked immunosorbent assay (ELISA), and in cell lysates by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, cell growth was determined by cell count. Endothelial cell migration was measured using a modified Boyden chamber. IL-6 was identified in the supernatant of the cell culture medium, indicating that these cells secreted IL-6, and the mRNAs of IL-6 were shown to be present in the cell lysates. IL-6 antisense oligonucleotide treatment resulted in a significant reduction of IL-6 protein expression compared to the sense control. The antisense oligonucleotides targeting IL-6 mRNA, also, inhibited cell growth and IL-6 production as well as VEGF expression. The addition of conditioned medium from IL-6 antisense-treated tumor cells resulted in decreased endothelial cell migration and tubule formation. Taken together, these findings indicate that endogenous IL-6 plays an important role in the growth of HNSCC and exerts its action by an autocrine growth mechanism, and that therapeutic trials with antisense oligonucleotides targeted to IL-6 mRNA may have some value for the treatment of HNSCC due to a decrease of neovascularization.

In head and neck squamous cell carcinoma (HNSCC), metastatic dissemination to regional lymph nodes serves as a major prognostic indicator for incipient disease progression and constitutes the guideline for subsequent therapeutic strategies. In this study, whether intratumoral (IT) and peritumoral (PT) lymphatic vessel density (LVD) might be a predictive indicator to the risk of lymph node metastasis was investigated.Tumour lymph vessels in fresh frozen sections of 105 head and neck cancer were quantified by immunostaining for the lymphatic endothelial marker LYVE-1. These results underwent correlation with the nodal status of the patient.There was a significant relationship between a high IT LVD and nodal metastasis (N+) (p=0.049, Mann-Whitney test). Analysed separately by anatomic regions, a significant correlation was only shown in oral carcinoma (p=0.032, Mann-Whitney test). Intratumoral LVD was lower compared to peritumoral LVD. Logistic regression, however, showed that the only predictive parameter for the nodal status was the localisation of the primary tumour but not LVD.This study confirmed that IT LVD is low in HNSCC. In this group of tumours there was a significant correlation between IT LVD and nodal involvement.

Tonsillectomy is one of the more common surgical procedures in childhood, yet the proper determination of its indications requires extensive clinical experience and is often complicated by difficulties in interpreting the child’s complaints, combined with parental expectations and the recommendations of the physicians who have already been taking care of the child. Moreover, physicians from the various medical specialties dealing with such patients tend to base their recommendations about surgery on highly divergent clinical experiences and points of view. Tonsillar surgery, particularly for children, has evolved to some extent in recent years. Time-tested or innovative techniques of partial tonsillar resection are now being used more frequently (again). Not only scientific journals, but also the popular press, continue to report case after case of fatal complications after tonsillectomy, usually involving postoperative hemorrhage. Operative mortality in childhood, though rare, is of particular concern. This prospect creates a special challenge not just for surgeons and for the hospitals where these operations are performed, but also for the physician initially determining the indication for surgery. In view of these facts, this review article will contain a discussion of the current state of knowledge regarding the indications, preoperative risk assessment, operative techniques, and postoperative complications of tonsillectomy in children, with particular attention to the current national and international guidelines and consensus statements. Prevalence Tonsillectomy is one of the more commonly performed operations in childhood. After reading this article, the reader should be able to understand the indications for tonsillectomy in childhood in an interdisciplinary context, know what preoperative tests are needed to assess surgical risks according to the current recommendations, and implement this knowledge in practice, understand the basics of surgical technique and postoperative management in order to initiate the necessary steps in case of postoperative hemorrhage.

To date, the underlying genomic changes in benign and malignant tumors of salivary-gland and paranasal-sinus origin are poorly understood. This is due in part to the low incidence of these tumors and the enormous histological variety of tumors within this head and neck region. We examined 58 of these tumors (14 adenoid cystic carcinomas, 9 adenocarcinomas, 5 cylindrical carcinomas, 11 pleomorphic adenomas, and 19 inverted papillomas) by dual fluorescence in situ hybridization (FISH) with centromere-specific probes on six chromosomes (3, 7, 9, 11, 17, and 18) for numerical changes. In adenoid cystic carcinomas, monosomy of chromosome 17 and polysomy of chromosomes 3, 9 and 11 were most frequently encountered. In adenocarcinomas, monosomy of chromosome 17 and polysomy of chromosomes 7 and 11 were most frequent. In cylindrical cell carcinomas, polysomy of chromosomes 7, 9, 11 and 17 was present in the majority of tumors. Disomy is rare, even in benign tumors. Polysomy is more frequent in malignant tumors than in benign. Tetrasomy is found almost only in malignant tumors. In summary, the occurrence of polysomy might reflect a step towards malignancy in tumors of the salivary glands and paranasal mucosa. Polysomy of chromosome 11 could be defined as typical for all investigated histological types of malignant tumor in this region of the head and neck.

Interleukin (IL)-6 plays a central role as a differentiation and growth factor of tumor cells. IL-6 has been identified in a wide variety of malignancies, including head and neck squamous cell carcinomas (HNSCC). The aim of this study was to investigate the association between the serum levels of IL-6 in HNSCC patients and the biological characteristics of the tumor as well as the clinicopathological status of the patients. The circulating level of IL-6 in sera from patients with various HNSCC (n = 90) as well as from healthy normal controls (n = 39) was investigated. Serum IL-6 concentrations were determined as serum immunoreactivity using a quantitative sandwich enzyme immunoassay technique. For statistical analysis, the Kruskal-Wallis test was performed. The majority of the patients with HNSCC were found to have high serum IL-6 concentrations. The IL-6 levels in the sera of patients with cancer ranged from below the detection limit to 312.8 pg/ml (mean, 19.5 pg/ml). In contrast, the IL-6 serum levels in 39 healthy individuals ranged from below the detection limit to 52.2 pg/ml (mean, 6.0 pg/ml), with the concentration being significantly higher in HNSCC patients (p0.001). Furthermore, the correlation of the IL-6 serum concentration with tumor stage was significant (p = 0.04). Accordingly, there was a significant difference of IL-6 serum concentration of tumors with positive and negative lymph nodes (p = 0.045), with concentration being significantly higher in lymph node-positive tumors. Our data on elevated IL-6 serum levels in the majority of HNSCC cancer patients and its correlation with tumor stage and lymph node status suggest that serum IL-6 reflects the proliferative activity of the tumor in patients with head and neck cancer. IL-6 serum determinations might serve as a biological marker and help to identify advanced head and neck tumors.

CIP/KIP family proteins entitled p21(WAF1/CIP1) and p27(KIP1) have key positions in cell cycle regulation leading to an arrest of cell proliferation. They are supposed to enable a repair process of DNA damage. In several human tumors, a loss of these proteins is associated with poor clinical outcome. The role of these cell cycle regulators in tumors of salivary gland and paranasal sinus origin is still unclear. In this study it was intended to demonstrate and compare the expression of p21, p27, and p53 in benign and malignant tumors of salivary glands and paranasal sinuses. Protein expression was detected by conventional immunohistochemistry (IHC). Additionally, we performed tyramide signal amplified immunohistochemistry (TSA-IHC) for p21 and p53 levels. Nine adenoid cystic carcinomas, 5 adenocarcinomas, 4 cylindrical cell carcinomas, as well as 30 pleomorphic adenomas and 26 inverted papillomas, were studied. In 78% of all adenoid cystic carcinomas a complete loss of p27 expression could be identified, whereas 60% of the adenocarcinomas overexpressed the protein. The majority of cylindrical cell carcinomas showed distinct cytoplasmic accumulation of p27. All malignant tumors turned out to be positive for p21 after performing TSA-IHC, although 72% of those samples had shown weak to negative protein levels in conventional immunostaining. Immunohistochemical results of CIP/KIP proteins were compared to p53 expression as well as to main clinical parameters. The study sheds new light upon the role of CIP/KIP protein family in tumors of salivary glands and paranasal sinuses. Furthermore, it is the first description of p21 and p53 TSA-IHC in these tumor types.

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is an autosomal dominant disorder of the fibrovascular tissue. It is characterized by the classic triad of (muco-)cutaneous telangiectases, arteriovenous malformations with recurrent epistaxis and hemorrhages, and inheritance. A wide variety of clinical manifestations in HHT have been described. In more than 90% of the patients, nosebleeds are the first predominant symptom, therefore ENT physicians often play a key role as far as diagnosis and management of the disease are concerned. In spite of recent diagnostic and therapeutic progress, a cure for this often burdening and handicapping disease is still not available. Apart from affecting the nose, arteriovenous malformations (AVMs) may also affect the skin, lungs, brain, liver and gastrointestinal tract. The two known genes that are implicated in HHT are endoglin (ENG) located on chromosome 9q33-q34 and activin-receptor-like kinase (ALK1) located on chromosome 12q13. Mutations of ENG are observed in HHT type 1 with an incidence up to 40% for pulmonary AVMs, whereas mutations of ALK1 are observed in HHT type 2 with an incidence of only 14% for pulmonary AVMs, which clinically distinguishes these two types of mutation. The emphasis of this paper is mainly on the clinical manifestation, molecular genetics and diagnosis of HHT, taking account of current literature on HHT in order to better understand the complexity of the disease. Recent therapeutic options in the treatment of HHT have been omitted from this paper as they are subject of a following paper. HHT is more common than previously thought and shows a broad range of different clinical organ manifestations that can be sources of substantial morbidity and mortality, making HHT a continuing challenge for many sub-specialties where interdisciplinary diagnostic screening is mandatory in the management of the disease.

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. We determined whether VEGF antisense oligonucleotide treatment can decrease the angiogenic activity of HNSCC cell lines in vitro and of HNSCC xenografts in vivo. Established human HNSCC cell lines were screened for VEGF expression at both mRNA and protein levels. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined the modulation of VEGF expression in cell line supernatants by capture ELISA and in cell lysates by Western blotting. Human endothelial cells were grown in conditioned medium produced from the treated tumor cells. Endothelial cell proliferation was determined by cell count, and endothelial cell migration was measured using a modified Boyden chamber. Mice with HNSCC xenografts were treated with PBS, VEGF antisense or sense oligonucleotides (10 mg/kg i.p. injection, 3 times/week), respectively, and tumor volumes were measured for 5 weeks. VEGF antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to treatment with the sense control. Although the growth rate of the tumor cell lines was not affected, the addition of conditioned medium from VEGF antisense-treated tumor cells resulted in decreased endothelial cell proliferation and migration. VEGF antisense oligonucleotide treatment of HNSCC xenografts resulted in a significant tumor growth suppression. These results suggest that downmodulation of VEGF using antisense oligonucleotides may be a potential therapy for the inhibition of angiogenesis in HNSCC.

A polymorphism at codon 72 of the human tumor suppressor p53 determines translation into either arginine or proline. Yet, the impact of this amino acid variability on the risk to develop malignant tumors, particularly carcinomas associated with human papilloma virus (HPV) infections, remains unresolved because of contradictory results. To address a potential correlation between the different genotypes and the manifestation of squamous cell carcinomas of the head and neck (SCCHN), we determined the p53 codon 72 in 193 healthy subjects and 122 unselected SCCHN with known HPV status. Furthermore, loss of allele-specific transcription was analyzed in p53 codon 72 heterozygous (Arg/Pro) SCCHN and correlated with HPV 16 and/or 18 E6 transcript expression. We found a moderately increased risk (odds ratio, 1.86; 95% confidence interval, 1.0-3.3) for individuals with germ line heterozygosity to develop SCC of the pharynx. On the other hand, p53 codon 72 polymorphic variants, most notably the Arg/Arg genotype, showed no association with the presence of HPV 16 and/or 18 E6 transcript. Moreover, there was no evidence for HPV-driven selection in SCCHN with allele-specific loss of transcription. Our data suggest that the p53 codon 72 polymorphism has a minor impact on the development of SCCHN.

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumour progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. Tumour treatment with anticancer agents might have an effect on the secretion of VEGF. Therefore, we determined whether certain chemotherapeutic agents stimulate VEGF secretion in HNSCC and whether VEGF antisense oligonucleotide treatment can modulate these effects in vitro.The effect of chemotherapeutic agents (Cisplatin, Carboplatin and 5-FU) on the production of VEGF was investigated on established human HNSCC cell lines at both mRNA and protein levels. By using a 21-mer VEGF antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA.The treatment of HNSCC cell lines with chemotherapeutic agents resulted in a significant induction of VEGF production. Carboplatin most prominently induced the release of VEGF from the tumour cells. VEGF antisense oligonucleotide treatment resulted in a significant reduction of chemotherapy-induced VEGF up-regulation compared to sense control.Induction of VEGF secretion might contribute to the frequently observed drug resistance of HNSCC to chemotherapeutic agents. This molecular effect might be reduced by the use of VEGF antisense oligonucleotides in head and neck cancer therapy.

Problem: The knowledge of the genomic changes in rare tumors of the salivary gland and the paranasal sinus is very limited. Methods: We examined 28 benign and malignant tumors of salivary gland and paranasal sinus origin, including 9 adenoid-cystic carcinomas, 5 adenocarcinomas, and 4 cylindrical cell carcinomas by fluorescence-in-situ-hybridization (FISH) of chromosomes 3, 7, 9, 11, 17, and 18. Results: In adenoid-cystic carcinomas, there was a high percentage of tumors with trisomy of chromosomes 9, 11, and 17 (6/9, 6/9, 5/9). In cylindrical cell carcinomas, three quarters of the tumors showed trisomy of all examined chromosomes, which is compatible with a triploidy in these tumors. Among all malignant tumors, aneusomy was most frequently encountered in adenocarcinomas. Conclusion: Altogether, the intratumoral heterogeneity is less pronounced in malignant tumors of salivary gland and paranasal sinus origin than in head and neck squamous cell carcinomas. Significance: Our results are a step toward a more profound understanding of the underlying genomic changes in these rare tumors. Support: None reported.

Interleukin-18 (IL-18), a recently described cytokine secreted mainly by macrophages, stimulates interferon-gamma (IFN-gamma) production by natural killer cells and T cells. The purpose of this study was to determine tissue expression and serum levels of IL-18 in head and neck squamous cell carcinoma (HNSCC) and to evaluate ethanol and endotoxin-driven cytokine secretion. In 24 patients with primary HNSCC and 28 healthy controls, PBMC were isolated and incubated with 50 mM ethanol, LPS (doses 25 ng/ml, 250 ng/ml, 2500 ng/ml) and both agents for 24 h. Levels of IL-18 in serum, and cell supernatants were analysed by capture ELISA, IL-18 tissue level by immunoblotting. Serum levels of IL-8, IL-10 and IL-12, IFN-gamma, and endotoxin plasma levels were also determined. Statistical analysis involved Welch t-test and Page's test for trend. The majority of patients with HNSCC had high concentrations of serum IL-18. The level of IL-18 in the sera of these patients had a mean level of 271.7 pg/ml, while the mean IL-18 serum level in healthy controls was 174,0 pg/ml (p

OBJECTIVES Little is known about the extent of intratumoral genetic heterogeneity in head and neck squamous cell carcinoma (HNSCC). MATERIAL Therefore, we examined 79 stage III and IV primary HNSCCs and matched lymph node metastases for over- and underrepresentation of specific chromosome regions by comparative genomic hybridization. RESULTS The overall ratio of gains and losses was higher in metastases (M) than in primary (P) tumors (4/1 vs. 2.5/1). Gains of 3q (78.1% P vs. 87.5% M) and 11q (78.1% P vs. 62.5% M), and deletions of 3p (43.8% P vs. 34.4% M) and 9p (31.3% P vs. 15.6% M) were most frequently detected. The highest rate of intratumoral discordance was observed for primary tumors and corresponding metastases (32.8%) compared to matched pairs of 2 metastases (26.5%), and of 2 anatomically distinct sides of 1 primary tumor (24.3%). Furthermore, the discordance rate was dependent on the primary tumor site (oral cavity 49.2%, oropharynx 31%, hypopharynx 30.3% and larynx 27.3%). In some tumors, the extent of genomic discordance argues against a monoclonal origin. CONCLUSION We demonstrate a high individual variation of intratumoral genomic heterogeneity depending on the localization and selection of matched pairs. These findings are of specific importance in view of establishing prognostic markers.

Intratumoral genomic heterogeneity, which can be defined as both intersample and intrasample heterogeneity, is still a poorly understood phenomenon in head and neck squamous cell carcinoma (HNSCC) with presumed implications on tumor behavior and even prognosis. We analyzed 89 tumor specimen from 37 HNSCC patients by fluorescence in situ hybridization (dual-FISH) using specific DNA probes binding to centromeric sites of 6 chromosomes to investigate intratumoral heterogeneity. A derivation from disomy in at least 1/6 chromosomes was detected in 88/89 (99%) specimen. In 33% of these samples, a change in ploidy could be suspected. Intrasample heterogeneity was detected in 68/89 (76%). Intrasample heterogeneity was more pronounced in primary tumors than in metastatic tumors. Analysis of the intersample heterogeneity revealed notable differences between the 6 chromosomes with the highest discordance detected for chromosome 3 (46%) and the lowest for chromosome 11 (27%). Following our results, it seems important to us to underline that intratumoral heterogeneity exists as intra- and sample heterogeneity in HNSCC. Altogether, trisomic cells were significantly more frequent in primary tumors than in metastases (p=0.01) while, in turn, monosomic cells were significantly more frequent in metastases (p=0.029). In individual cases the extent of discordance between corresponding samples made a common clonal precursor unlikely. In these cases, the synchronous development of a primary tumor and a carcinoma of unknown primary ('CUP syndrome'), otherwise undetected, should be considered.

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro and of HNSCC xenografts in vivo. Established human HNSCC cell lines were screened for VEGF expression at both mRNA and protein levels. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell (EC) proliferation was determined by cell count and EC migration was measured using a modified Boyden chamber. Mice with HNSCC xenografts were treated with PBS, VEGF antisense or sense oligonucleotides (10 mg/kg; i.p. injection), respectively and tumor volumes were measured for 5 weeks. VEGF antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense control. Although the growth rate of the tumor cell lines was not affected, addition of conditioned medium from VEGF antisense-treated tumor cells resulted in decrease of endothelial cell proliferation and migration. VEGF antisense oligonucleotide treatment of HNSCC xenografts resulted in a significant tumor growth suppression. These results suggest that downmodulation of VEGF using antisense oligonucleotides may be a potential therapy for the inhibition of angiogenesis in HNSCC.

Little is known about the extent of intratumoral genetic heterogeneity in head and neck squamous cell carcinoma (HNSCC). We therefore examined 79 stage III and IV primary HNSCCs (P) and matched lymph node metastases (M) for over- and underrepresentation of specific chromosome regions by comparative genomic hybridization (CGH). The overall ratio of gains and losses was higher in metastases than in primary tumors (4/1 vs. 2.5/1). Gains of 3q (78.1% P vs. 87.5% M) and 11q (78.1% P vs. 62.5% M) and deletions of 3p (43.8% P vs. 34.4% M) and 9p (31.3% P vs. 15.6% M) were most frequently detected. The highest rate of intratumoral discordance was observed for primary tumors and corresponding metastases (32.8%) compared with matched pairs of two metastases (26.5%) and of two anatomically distinct sides of one primary tumor (24.3%). Furthermore, the discordance rate was dependent on the primary tumor site (oral cavity 49.2%, oropharynx 31%, hypopharynx 30.3%, and larynx 27.3%). In some tumors, the extent of genomic discordance argues against a monoclonal origin. In conclusion, we found a high individual variation of intratumoral genomic heterogeneity depending on the localization and selection of matched pairs. These findings are of specific importance in view of establishing prognostic markers.

Mutations and interaction with high-risk human papillomavirus (HPV) E6 oncoprotein are well-established mechanisms of p53 inactivation. In a series of 123 unselected squamous cell carcinomas of the head and neck (SCCHN), we performed sequence analysis of the entire coding region of p53 transcript and determined the presence of the E6 transcripts of HPV 16 and 18. Aberrant p53 transcripts were identified in 97 (79%) SCCHN. HPV 16 and/or 18 E6 transcripts were detected in 37 (30%) tumor specimens, including 20 (77%) of the 26 p53 wild-type tumors. The likely inactivation of p53 in 117 (95%) of the 123 SCCHN suggests that this event could be obligatory in the multistep process of carcinogenesis.

Overexpression of the epidermal growth factor receptor (EGFR) is thought to play a key role in the development of head and neck squamous cell carcinoma (HNSCC) primarily through its effect on promoting uncontrolled cell proliferation. Blocking EGFR ligand binding might also inhibit angiogenesis and down-regulate the production of angiogenic factors. Angiogenesis is increased in various human tumors, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. The vascular endothelial growth factor (VEGF) is thought to be the most important angiogenic factor. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human EGFR mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell migration was measured using a modified Boyden chamber. EGFR antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense oligonucleotide control. Addition of conditioned medium from EGFR antisense-treated tumor cells resulted in decreased endothelial cell migration. In conclusion, therapeutic strategies targeting EGFR signaling in head and neck cancer might have an antitumor effect mediated in part by inhibition of tumor angiogenesis.

Very little is known about possible intra-tumoural genetic heterogeneity between primary tumours and lymph node metastases in head and neck squamous cell carcinoma (HNSCC). To investigate this phenomenon, we analysed 96 micro-dissected tumour samples for allelic imbalance at four of the most frequently altered chromosomal locations in HNSCC (3p14.2; 9p21; 11q23.3; 17p13.1) using microsatellite markers. From 23 patients, matched pairs of primary tumour and lymph node metastasis were analysed. Discordance in the allelic distribution was identified in 8 cases (35%). With one exception, the metastasis contained a more balanced allelic status than the primary tumour. In contrast, in a group of 25 tumours with two anatomically different samples from the primary tumour site, discordance was identified in only 3 tumours (13%). These results are compatible with the dissemination of subclones from the primary tumour site with a more balanced allelotype in the metastasis. In our opinion, several scenarios could explain this phenomenon. From a clinical point of view, genetic discordance between the metastasis and the primary tumour must be taken into consideration when establishing molecular biologic markers for choice of therapy and prognosis in head and neck cancer.

The huge majority of head and neck squamous cell carcinoma (HNSCC) show alterations of p53 either on the genetic level or on the protein level. Allelic imbalance (AI)/loss of heterozygosity (LOH) on 17p at the p53 locus is frequent in HNSCC. However, the complex relationship between these phenomena is poorly understood in HNSCC. We investigated one group of 39 HNSCC for: a) allelic imbalance on 17p using 4 microsatellite markers located throughout this chromosomal arm; b) mutations of p53 in exons 5-9; and c) overexpression of p53 using two antibodies located on opposite ends of the protein. AI/LOH was detected in 44% at the locus TP53, rising to 69% when regarding all 4 markers on 17p. Therefore, our data are in line with the assumption of additional tumour suppressor genes on 17p in HNSCC. A nuclear accumulation of p53 (51%) was independent from the antibody and the recognised epitope. At the first glance there was no correlation between overall p53 mutation (36%) and overexpression. However, it appeared that, with very few exceptions, only nonsense mutations did not lead to p53 overexpression, while missense mutations did. As overexpression of p53 was 15% more frequent than p53 mutations and only 35% of the tumours with p53 overexpression carried a p53 mutation, our data support the hypothesis of additional mechanisms of p53 overexpression. AI/LOH at the p53 locus in 83% of all tumours with a p53 mutation is in line with Knudson's theory of inactivation of tumour suppressor genes.