Human papillomavirus and p53 polymorphism in codon 72 in head and neck squamous cell carcinoma.

The impact of a polymorphism of the wild-type human tumour suppressor gene p53(wt) on carcinogenesis is subject of controversy ever since a higher susceptibility of p53 to HPV-E6 mediated degradation when encoding for Arginine at codon 72 (p53Arg) was first reported. The issue remained unclear because various studies investigating this question for different tumour entities and different geographical regions demonstrated diverging results. In the present study, the HPV status and p53 genotype frequency of 42 head and neck cancers was analysed and compared to results reported in the recent literature. Applying PCR and cycle sequencing techniques, HPV DNA was demonstrated in 12/42 (29%) of the cases and the overall distribution of the p53 allele was: 64, 31 and 5% for p53Arg, p53Arg/Pro and p53Pro, respectively. There was no statistically significant association between HPV status and p53 genotype distribution. The results of our study and of the reviewed literature do not support a relevant role of the p53 polymorphism in head and neck carcinogenesis, either taken alone or in association with the HPV status.

Immunohistochemical staining of Ki-67 using the monoclonal antibody Ki-s11 is a prognostic indicator for laryngeal squamous cell carcinoma.

BACKGROUND: Proliferative activity has been shown to be of prognostic significance for several malignancies. Ki-67, a cell cycle associated antigen, is regarded as a promising proliferation marker. Very few results on the proliferative activity of head and neck cancer and their potential prognostic value are available. MATERIALS AND METHODS: The proliferative activity of 104 squamous cell carcinomas of the larynx (SCCL) was analyzed retrospectively with the monoclonal antibody Ki-S11 which specifically detects the Ki-67 antigen. Median follow-up time was 47 months. RESULTS: There was a statistically significant correlation (p<0.05) between histopathological grading, N-status and proliferative activity. There was also a significant difference for the 5-year survival between low and highly proliferating tumours. The patient group with low proliferating laryngeal cancer had a statistically (p<0.05) longer absolute and recurrence-free 5-year-survival time than patients with a highly proliferating cancer. CONCLUSION: These results show that Ki-67 staining of SCCL with Ki-S11 is a helpful prognostic indicator for squamous cell carcinoma of the larynx with a potential clinical application.

Expansion of circulating NKG2D+ effector memory T-cells and expression of NKG2D-ligand MIC in granulomaous lesions in Wegener's granulomatosis.

Expansion of circulating CD28- T-cells reminiscent of effector memory T-cells (T(EM)) has been reported in Wegener's granulomatosis (WG) recently. To investigate the role of T(EM) in WG, we analyzed the expression of the activating NK-receptor NKG2D and its ligand MIC on circulating T(EM) and in granulomatous lesions, respectively. NKG2D was anomalously expressed and preferentially detected on circulating CD4+CD28- T(EM) in WG. Compared to healthy controls, T(EM) display a more activated phenotype potentially favoring unbalanced proinflammatory responses in WG. Cluster-like formations of "Wegener's autoantigen" PR3 were surrounded by NKG2D+ and NKG2D-ligand MIC+ cells in WG-granulomata, but not in disease controls. Further, IL-15 - known to drive T(EM) differentiation and proliferation--was also expressed in WG-granulomata. Thus, through acquisition of NK-like "innate" properties, IL-15 stimulated NKG2D+ T(EM) could interact with MIC+ cells within WG-granulomata, thereby sustaining inflammation and autoimmunity and promoting self-perpetuating pathology in WG.

Vaccination strategy to target lysyl oxidase-like 4 in dendritic cell based immunotherapy for head and neck cancer.

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-1beta, TNF-alpha, 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-gamma 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-gamma 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.

Phytomedicines in Acute Rhinosinusitis: A Prospective, Non-interventional Parallel-Group Trial.

INTRODUCTION: The objective of this prospective, multicenter, parallel-group, non-interventional clinical trial (NIT) was to characterize the effectiveness of a treatment with the phytomedicines ELOM-080 and BNO 1016 in patients with acute rhinosinusitis (ARS). METHODS: A total of 228 patients suffering from ARS took part in this NIT and were treated for a maximum of 14 days with either BNO 1016 or ELOM-080. Focus was on improvement of rhinosinusitis-associated pain/discomfort and nasal congestion in real-life conditions of primary care setting, as assessed by numeric and verbal rating scale, and five-point Likert scale. RESULTS: The course of the key ARS symptom facial pain demonstrated a faster recovery in patients with ELOM-080, when compared to BNO 1016. ELOM-080 tended to be superior for several ancillary criteria and induced significantly higher patient satisfaction with regard to the improvement of feeling of general illness. Physicians assessed both products to be very effective and well tolerated. Adverse drug reactions classified as gastrointestinal disorders occurred in both groups to a comparable extent. CONCLUSION: This trial demonstrated comparable effectiveness of a therapy of ARS with the phytomedicines ELOM-080 and BNO 1016, although the treatment with ELOM-080 resulted in a more rapid and more complete recovery in ARS key symptoms and tended to be superior for several ancillary criteria. Both treatments were well tolerated. TRIAL REGISTRATION NUMBER: NIS-6471. FUNDING: G. Pohl-Boskamp GmbH & Co. KG.

Human proteinase 3 (PR3) and its binding molecules: implications for inflammatory and PR3-related autoimmune responses.

Human proteinase 3 (PR3) is a multifunctional serine protease, mainly located in the azurophilic granules and on the cell surface of polymorphonuclear leukocytes (PMN). Cumulated data indicate that PR3, which is the main target autoantigen of antineutrophilic cytoplasmic antibodies (ANCA), interacts with several surface receptors and participates in the local inflammatory response. Herein, we summarize the efforts made to elucidate ANCA-binding epitopes of PR3, extended by data derived from the use of a random peptide library. The inserts for 107 peptides were obtained by panning of a random peptide library with PR3-ANCA(+) immunoglobulins. Analysis of the amino acid sequences of the inserted peptides derived from isolated positive clones suggested that they do not belong to linear epitopes of PR03 and possess a high proportion of positively charged amino acids. Furthermore, this article focuses on immune functions of PR3 with respect to PR3 modulation of cell activation via cleavage of protease-activated receptor-2 (PAR-2) and as binding protein for the proinflammatory cytokine IL-32alpha. Altogether, there are a number of (auto)molecules that bind to PR3, some of them even competitive and each binding interaction seems to have specific implications.

P53 codon 72 polymorphism in squamous cell carcinoma of the head and neck region.

BACKGROUND: The impact of codon 72 polymorphism of the human tumour suppressor gene p53 on the risk of developing squamous cell carcinomas of the head and neck (HNSCC) remains unclear because of contradictory results found by several studies. PATIENTS AND METHODS: We genotyped a group of 77 patients with advanced HNSCC by using a direct sequencing method. RESULTS: There were no significant differences in the age of the patients at the time of the first diagnosis nor in the 5-year survival rates. There was no additive effect between different risk factors (alcohol, nicotine) and codon 72 polymorphism. Compared to the frequency of homozygosity encoding for Arg/Arg in the Eurasian population given in literature, the present study has shown a significantly higher frequency of homozygosity for Arg/Arg at codon 72 than commonly detected. CONCLUSION: These findings may indicate codon 72 polymorphism as a risk factor for HNSCC or point to a high variability of codon 72 polymorphism among ethnic groups.

Detection of human papillomavirus DNA in benign and malignant sinonasal neoplasms.

Infections with human papillomaviruses are divided basically into three different infection types: those producing specific clinically visible lesions, those remaining subclinical, and those being latent. The assumed infection type thought to be present in tissue specimens has influence on the conclusions that can be made from an analysis, i.e. whether or not the HPV infection has a causal relationship with other epidemiological or molecular investigation observations. To determine whether HPV DNA detection in different entities of the upper aerodigestive tract represents a coincidental, persistent/latent or specific infection, 20 clinically intact mucosa specimens of the upper aerodigestive tract, 20 sinonasal polyps, 26 inverted papillomas, and 20 squamous cell carcinomas of the paranasal sinuses were investigated. HPV DNA was not detectable in specimens derived from clinically intact mucosa or in nasal polyps. Yet, three out of 26 inverted papillomas were HPV-positive, each showing double infection with HPV6 and 11. Four out of 20 squamous cell carcinomas were HPV16 positive. To our knowledge, we are presenting the first study contemporaneously analyzing benign as well as malignant non-proliferative and proliferative mucosal entities whilst applying identical methodical standards. The data corroborate the hypothesis that HPV DNA demonstration in tissue specimens represents a specific infection of the mucosa of the upper aerodigestive tract. It can thus be assumed that there is a causative involvement of HPV infections in the alteration of cell proliferation and in the case of infection with high risk HPV types even on progression to malignant transformation.

Gene expression profiling of head and neck squamous cell carcinoma using cDNA microarrays.

The molecular mechanisms causing the development of squamous cell carcinomas in the head and neck region are mostly unknown. Identification of molecular changes which are involved in carcinogenesis might play a key role in improving the diagnosis, therapy and prognosis of patients with carcinomas in the head and neck. The purpose of the study was to identify transcriptional alterations of apoptosis associated genes between normal mucosa and tumor tissue. We measured the mRNA expression of 408 apoptosis associated genes by microarray-technique in normal upper aerodigestive tract mucosa (n=4), and in cancer tissue (n=8) of squamous cell carcinomas of the upper aerodigestive tract. RT-PCR was performed to confirm the microarray results. A hierarchial cluster analysis, based on 22 selected marker-genes showing a separation of the two tissue types supports the hypothesis of a specific expression pattern associated with tumor development. Additionally, we found 11 genes associated with anti-apoptotic processes to be upregulated while 12 genes associated with proapoptotic functions as well as 5 DNA-replication and chromosome cycle associated genes were found to be downregulated in the tumor tissue. Furthermore 6 of 8 genes which are known to be associated with lymphocyte activation were upregulated in tumor edge tissue. These results represent a first step for the diagnostic use of microarrays in squamous cell carcinomas of the head and neck region and might improve the understanding of the molecular mechanisms of carcinogenesis in the head and neck region.

HPV16 DNA in histologically confirmed tumour-free neck lymph nodes of head and neck cancers.

BACKGROUND: Human papillomavirus (HPV) has been demonstrated in lymph node neck metastases (NM) of HPV-positive squamous cell carcinomas of the head and neck (HNSCC), underscoring the possible role of HPV for HNSCC progression. Reports on HPV infections in histopathologically tumour-free lymph-nodes of the SCC of the uterine cervix developing higher rates of lymph-node metastases and recurrences later in the survey of the patients was the starting point of the present study. MATERIALS AND METHODS: The presence of HPV-DNA in primary tumours (PT, n=45), NM (n=45) and histologically confirmed tumour-free neck lymph-nodes (LN, n=102) of HNSCC from 60 patients was analysed by PCR and Southern blot hybridisation. RESULTS: A highly positive correlation of simultaneous HPV-DNA detection in PT and NM was demonstrated. In the case of HPV-positivity of PT and/or NM [24/60 cases (40%)], 11/24 (45.8%) LN contained HPV-DNA, as well. Accepting HPV demonstration as a marker for the presence of micro-metastasis, HPV analysis would result in an upstaging of the N category in 4 out of these 11 patients. CONCLUSION: Considering the high agreement of HPV-DNA detection in PT and simultaneous HPV-DNA demonstration in the draining NM corroborating the monoclonal character of the tumour cells, the HPV-DNA presence in LN seems to be indicative of micro-metastasis in these lymph nodes. Thus, HPV analysis might be another powerful tool for the definition of the N-status of HPV-positive HNSCC.

Human papillomaviruses in lymph node neck metastases of head and neck cancers.

CONCLUSION: The results of this study corroborate earlier findings that human papillomavirus (HPV)16 is the most prevalent type of HPV in squamous cell carcinomas of the head and neck (SCCHNs) and reinforce a possible influence of HPV on SCCHN progression by showing that the majority of HPV-positive patients harbor HPV16 (or HPV33) both in their primary tumors and in lymph node neck metastases (LNNMs). OBJECTIVE: HPVs are causally associated with carcinomas of the uterine cervix and have also been linked to a subset of SCCHNs. In order to further investigate the predicted causative role of HPV in SCCHNs, we analyzed pairs of primary tumors and LNNMs or LNNMs alone for the presence of HPV DNA using polymerase chain reaction (PCR). MATERIAL AND METHODS: DNA was extracted from fresh frozen tissue samples of primary tumors and the corresponding LNNMs of 18 patients and from LNNMs alone in 17 patients. For the detection and typing of HPV, PCR was performed using both type-specific and consensus primer pairs, followed by Southern hybridization and, in selected cases, sequencing of the PCR products. RESULTS: Of the 35 patients investigated, 22 (63%) were found to have HPV DNA in their tumors: HPV16 DNA in 21 cases and HPV33 in 1. The highest HPV prevalence was detected in tumors of Waldeyer's tonsillar ring (8/9 patients; 89%). Of the 18 patients in whom primary tumors and LNNMs were analyzed, 7 (39%) were HPV-positive in both samples (HPV16, n = 6; HPV33, n = 1), in 3 (17%) the primary tumors were HPV-negative and the LNNMs HPV16-positive and in 1 (5.5%) the primary tumor contained HPV16 and the LNNM was negative. Interestingly, of the 7 patients in whom LNNMs had been detected only several months after diagnosis and treatment of the primary tumors, only 1 showed infection with HPV (HPV33).

Cyfra 21-1 in diagnosis of distant metastases of head and neck carcinoma.

Follow-up examinations of patients might detect local tumor recurrences at a curable stage, which is more difficult for distant metastases (DM). Recently, elevated Cyfra 21-1 serum levels (CySL) could be shown not only to correlate with HNSCC-tumor size but also with development of DM. We focussed on the CySL of 476 HNSCC patients as a first step. At first time diagnosis, besides regular staging procedures, these patients were screened for CySL higher than 3.3 ng/ml. Seventeen out of 476 (3.9%) patients showed DM. Seventeen out of 19 patients (89.5%) presented elevated CySL: A further 830 patients with HNSCC were tested for changes in CySL in the course of disease (cut-off value 3.3 ng/ml). Seventy-one out of 830 patients (8.6%) showed elevated CySL. Tumor growth was found in 50 out of 71 patients (70.4%). In 54% of these patients (27 out of 50) DM were detected. Routine screening for CySL can lead to timely detection of DM in HNSCC, despite its fairly low sensitivity.

Overexpression of a novel lysyl oxidase-like gene in human head and neck squamous cell carcinomas.

BACKGROUND: To compare the mRNA expression profile of head and neck squamous cell carcinoma (HNSCC) cells and normal epithelial cells. MATERIALS AND METHODS: Differential display was used to trace genes showing differential expression in HNSCC cells. Rapid amplification of cDNA ends (RACE) was carried out to identify 5'upstream sequences followed by Northern hybridisation for verification of the differential mRNA expression. RESULTS: An overexpression of a 59 bp gene fragment was detected in HNSCC cells in contrast to normal epithelial cells. After cloning and sequencing the gene fragment, high grade of homology was found with a human cDNA full insert sequence (fis). After amplification and sequencing of the 5'end of the fis clone, subsequent database searches showed an exact match with the coding sequence of the human lysyl oxidase-like gene 4 (LOXL4). The differential expression of this 4.8 kb LOXL4-mRNA was confirmed by Northern hybridisation. CONCLUSION: The data presented in this work might emphasize the involvement of LOXL4 in molecular processes of the genesis or progression of head and neck carcinomas.

p53 autoantibodies as tumor marker in head and neck squamous cell cancer.

p53 autoantibodies (AAB) are a new serological parameter with unknown potential in patients with malignancies. The reason why and the mechanism by which they develop is still unclear. So far, only in a limited number of studies has the usefulness of p53 AAB in the follow-up of cancer patients been shown. In this study 32 patients with head and neck cancer, seropositive for p53 AAB in their serum detected with an ELISA, were followed-up for at least 42 months. In 9 out of 32 p53 seropositive AAB head and neck cancer patients a correlation with the clinical course of the disease was seen. The remaining 23 of the p53 AAB-positive patients did not demonstrate any significant AAB titer changes during the follow-up and no significant correlation with the clinical course was observed. In conclusion, the clinical value of p53 AAB as a tumor marker for patients with head and neck cancer seems to be limited.

Prognostic relevance of the proliferation marker REPP86 for laryngeal cancer.

BACKGROUND: There is a clear correlation between proliferative activity and the biological behavior of cancer, which might have an impact on the patients' prognosis and consequences for the individual therapy concept. REPP86 (restrictedly expressed proliferation-associated protein 86) is a proliferation-associated protein expressed in S-, G(2)- and M-phases of the cell cycle, regarded as a promising proliferation marker and has not yet been examined in squamous cell carcinoma of the larynx (SCCL). MATERIALS AND METHODS: REPP86 was analyzed retrospectively in 104 SCCL using the monoclonal antibody Ki-S2. Proliferative activity was correlated with tumor stage, histopathological grading, patients' survival and the results we recently published on Ki-67 staining in SCCL. Median follow-up time was 47 months. RESULTS: A significant correlation (p<0.05) between histopathological grading, N-status and proliferation activity was observed. The patient group consisting of low proliferating laryngeal cancer showed a statistically longer absolute (p<0.05) and relapse-free (p=0.001) 5-year survival time than the group with a high proliferating tumor. Compared to the Ki-67 staining results, the REPP86 antibody better predicts the relapse-free 5-year-survival. CONCLUSION: Our results indicate that REPP86 staining of SCCL with Ki-S2 is a helpful prognostic indicator for SCCL and better predicts the relapse-free survival than Ki-67 staining in SCCL.

Metalloproteinases and their inhibitors: influence on tumor invasiveness and metastasis formation in head and neck squamous cell carcinomas.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in tumor invasiveness. This study investigates the expression status of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in head and neck squamous cell carcinomas (HNSCC). METHODS: Of 48 laryngeal squamous cell carcinoma (LSCC) biopsies and 10 HNSCC cell lines, mRNA was isolated, reversely transcribed, and subjected to polymerase chain reaction (PCR) amplifying MMP-1, MMP-2, MMP-9, MMP-10, TIMP-1, and TIMP-2. Silver nitrate-stained gel electrophoresis demonstrated MMP and TIMP expression status. Exemplary immunohistochemistry and zymography confirmed translation and enzyme activity. RESULTS: Densitometric analysis revealed MMP-2 expression and lymph node metastases to be positively and TIMP-1 and TIMP-2 to be negatively correlated with lymph node metastases. TIMP-2 expression and tumor size were negatively correlated. MMP-1, MMP-9, and MMP-10 expression were not correlated to metastasis formation or tumor size. CONCLUSIONS: Our results suggest that MMP-2 expression enhances, whereas TIMP-1 and TIMP-2 both suppress, cancer spread in LSCC.

Human papillomaviruses in head and neck cancer: 8 year-survival-analysis of 73 patients.

Depending on the primary tumour's anatomical location, squamous cell carcinoma of the head and neck (HNSCC) shows HPV prevalences between 20 and 30% for oro-, hypopharyngeal as well as laryngeal SCC and up to over 50% for SCC of the Waldeyer's tonsillar ring. There is persistent controversy on the role of HPV infection in HNSCC-progression, and on the influence of these infections on the final clinical outcome. To evaluate the possible relevance of HPV infection on survival and prognosis, 73 patients with HNSCC were investigated statistically with a median follow-up time of 28 (0.3-94) months. The statistical analysis revealed no differences in the overall survival of HPV-positive and HPV-negative cancer patients. A correlation between decreased survival and increased lymph node status was expected. Patients with carcinomas of the Waldeyer's tonsillar ring with a high HPV prevalence rate as compared to tumours of other anatomical locations revealed a better survival. Moreover, an association between HPV positivity and higher lymph node status at time of first diagnosis, and a better survival of HPV-positive patients compared to HPV-negative patients given the same initial nodal status (N0 vs. N1-N2b vs. N2c-N3) could be demonstrated. The influence of HPV on the patient's survival can only be observed statistically in combination with other prognostic factors, as the lymph nodal status of the patients. The better prognosis of survival of HPV-positive vs. the HPV-negative patients with lymph node neck metastasis is attributable to a better response of the HPV-positive group to therapy, especially radiotherapy.