Multikinase inhibitors sorafenib and sunitinib as radiosensitizers in head and neck cancer cell lines.

BACKGROUND: Radioresistance is a common feature of head and neck squamous cell carcinoma (HNSCC). We previously showed that the irradiation- activated vascular endothelial growth factor (VEGF)-extracellular signal-regulated kinase (ERK)-axis is fundamental for the survival of resistant tumors. In this study, we examined if treatment with potent multikinase (MK) inhibitors, sorafenib and sunitinib, could radiosensitize tumor cells. METHODS: Cultured HNSCC cell lines were treated with inhibitors and subsequently irradiated. Radiosensitizing effects were functionally assessed by annexin-V apoptosis and clonogenic assays and confirmed by Western blot. Additionally, we surveyed human HNSCC tissue microarrays (TMAs) for activated ERK expression. RESULTS: Based on combination indexes, we found that combining irradiation with both inhibitors exerted strong and supra-additive antitumor effects on clonogenic survival. Kinase inhibition enhanced irradiation-induced apoptotic rates and inhibited postradiogenic phospho-ERK-expression. Patients with recurrent HNSCC displayed significantly lower extracellular signal-regulated kinase phosphorylation (pERK) levels than relapse-free patients. CONCLUSION: We propose further evaluation of sorafenib and sunitinib as potential radiosensitizing agents in HNSCC treatment. © 2017 Wiley Periodicals, Inc. Head Neck 39: 623-632, 2017.

Olfaction in chemotherapy for head and neck malignancies.

OBJECTIVE: Systemic chemotherapy for different malignancies occurs alongside various side effects, including reduced sensory function. To date, little is known about the effect of chemotherapeutic agents on olfaction. The aim of this study was to provide new data about changes in sense of smell during chemotherapy among patients with advanced squamous cell carcinomas of the head and neck region. METHODS: In a prospective, controlled cohort study of patients undergoing up to three courses of chemotherapy (cis- or carboplatin, 5-fluorouracil and docetaxel), olfaction was evaluated prior to and directly following completing a cycle, as well as 3 weeks later with the beginning of the next cycle. For evaluation of sense of smell, the established Sniffin' Sticks test with a determination of threshold, discrimination and identification (TDI) was used. Thirty-three patients (44-85 years old, 25 men and 8 women) were included in the study. Most malignancies were located in the oropharynx. RESULTS: Among the 28 patients who scored normosmic or hyposmic at the beginning of the study, the mean decrease in TDI-score was 0.72 points (24.0-23.2) in the first cycle, 2.1 points (24.5-22.4) in the second cycle and 0.77 points (24.2-23.4) in the third cycle. The decrease during the second cycle was significant. Age (>55 years) had a significant (negative) influence in the first and the second cycles. Smoking only showed a tendency to decreased TDI-scores in chemotherapy. In-between consecutive cycles an increase in TDI-score was obvious (+1.0 points after the first and +1.5 points after the second cycle). CONCLUSION: Chemotherapy with cisplatin, 5-fluorouracil and docetaxel significantly affected sense of smell to a small extent. This effect was more pronounced in elderly patients and smokers. This fact must be taken into account as a possible additional negative effect in usually prevailing malnutrition in these patients. Furthermore, no cumulative effect of the administered therapeutic agents on olfaction could be proven during this study and recovery occurred within a 3-week period.

Controlled trial for long-term low-dose erythromycin after sinus surgery for chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: The efficacy of macrolides in chronic rhinosinusitis (CRS) is still under controversy. To date, only two double-blind, placebo-controlled studies have been published with differing results. None of these studies investigated the possible benefit of macrolides in the postoperative period. We conducted an investigator-initiated clinical trial using 250-mg erythromycin once a day over a period of 3 months, beginning the administration of either erythromycin or placebo 2 weeks after a surgical intervention for CRS. STUDY DESIGN: Randomized double-blind, placebo-controlled trial. METHODS: The concentrations of eosinophilic cationic protein (ECP) and myeloperoxidase in nasal secretion were chosen as primary outcome measures. Additionally, as a secondary outcome measure, changes in the Sino-Nasal Outcome Test-20 score, olfaction, saccharin transit time, nasal endoscopy score, and self-rating of nasal health using a visual analogue scale were evaluated. RESULTS: Sixty-seven patients after surgery for CRS with or without nasal polyps were screened, and 58 patients were randomized to the study groups. For the primary outcomes, the concentrations of ECP changed from 176.4 µl/l ± 79.0 to 226.1 µl/l ± 200.6 in the erythromycin group and from 186.9 µl/l ± 36.0 to 192.9 µl/l ± 189.2 in the placebo group; no statistical differences were found. Of the secondary outcomes, only the nasal endoscopy score showed a statistically significant improvement in the erythromycin group (from 2.6 ± 1.4 to 1.9 ± 1.5 points) compared to the placebo group (from 2.5 ± 1.3 to 2.6 ± 1.5 points). The subgroup of patients without nasal polyps in the erythromycin group showed a tendency to improvement in some secondary outcome criteria. CONCLUSIONS: A general recommendation for long-term, low-dose erythromycin treatment after surgery for CRS cannot be given. In patients with CRS without nasal polyps, a tendency to improved parameters was detected. LEVEL OF EVIDENCE: Ib.

Adenoids of patients with mucopolysaccharidoses demonstrate typical alterations.

OBJECTIVE: Tonsillar hypertrophy caused by the progressive accumulation of partially degraded glycosaminoglycans (GAGs) within the cells is a typical symptom in patients with mucopolysaccharidoses (MPS). We studied the tissue of adenoids and tonsils of patients suffering from MPS with special regard to characteristic morphological features serving as possible markers for diagnosis. METHODS: Adenoids of 87 patients and tonsils of 4 patients with MPS (2 patients with MPS I, 7 MPS II, 5 MPS IV and 10 MPS VI and 63 controls) and controls were examined. Examinations were repeated in a blinded manner by two pathologists. RESULTS: The key feature observed was a subepithelial "clearing" on scanning magnification, induced by perivascular accumulation of histiocytoid cell forms. Similar agglomerates could sometimes be found at the base of lymphoid follicles. In the blinded assessment a specificity of 92% (100% for adenoids) and a sensitivity of 100% were achieved. The inter-observer-consistency was 92% (100% for adenoids). In tonsillectomy specimens marked subepithelilal fibrosis can lead to a false-negative evaluation. CONCLUSIONS: Qualified histological examination could be an option for early diagnosis of MPS.

Phenotypic variability and risk of malignancy in SDHC-linked paragangliomas: lessons from three unrelated cases with an identical germline mutation (p.Arg133*).

CONTEXT: Mutations in the four subunits of succinate dehydrogenase (SDH) are the cause for the hereditary paraganglioma (PGL) syndrome types 1-4 and are associated with multiple and recurrent pheochromocytomas and PGLs. SDHC mutations most frequently result in benign, nonfunctional head-and neck PGLs (HNPGLs). The malignant potential of SDHC mutations remains unclear to date. OBJECTIVES: We report a patient with malignant PGL carrying a SDHC mutation and compare her case with two others of the same genotype but presenting with classic benign HNPGLs. Loss of heterozygosity (LOH) was demonstrated in the malignant PGL tissue. DESIGN: In three unrelated patients referred for routine genetic testing, SDHB, SDHC, and SDHD genes were sequenced, and gross deletions were excluded by multiplex ligation-dependent probe amplification (MLPA). LOH was determined by pyrosequencing-based allele quantification and SDHB immunohistochemistry. RESULTS: In a patient with a nonfunctioning thoracic PGL metastatic to the bone, the lungs, and mediastinal lymph nodes, we detected the SDHC mutation c.397C>T predicting a truncated protein due to a premature stop codon (p.Arg133*). We demonstrated LOH and loss of SDHB protein expression in the malignant tumor tissue. The two other patients also carried c.397C>T, p.Arg133*; they differed from each other with respect to their tumor characteristics, but both showed benign HNPGLs. CONCLUSIONS: We describe the first case of a malignant PGL with distant metastases caused by a SDHC germline mutation. The present case shows that SDHC germline mutations can have highly variable phenotypes and may cause malignant PGL, although malignancy is probably rare.

Reactive oxygen species activation of MAPK pathway results in VEGF upregulation as an undesired irradiation response.

BACKGROUND: Radioresistance limits the effectiveness of radiotherapy in head and neck squamous cell carcinoma. We previously demonstrated post-radiogenic mitogen-activated protein kinase (MAPK) pathway activation and vascular endothelial growth factor (VEGF) release resulting in reduced tumor cell response. Here, we examined the association of this mechanism with the induction of reactive oxygen species (ROS) under irradiation (IR). METHODS: Intracellular ROS after IR were measured. We modeled radiation-induced ROS by exposure of two SCC lines to H2 O2 and evaluated the impact of irradiation and ROS on ERK phosphorylation by Western blot, immunohistochemistry, and ELISA. RESULTS: We found elevated pERK levels after treatment with IR and H2 O2 , which could be distinctly suppressed by U0126. Immunohistochemistry and ELISA revealed increased intracellular VEGF levels after H2 O2 application. CONCLUSIONS: Our data show that irradiation-induced ROS activate the MAPK pathway and release of VEGF. As VEGF is known to be released after cellular distress resulting in cytoprotection, the described mechanism is potentially of importance for therapy success.

Malignant and benign sinonasal paragangliomas.

OBJECTIVES/HYPOTHESIS: To report on the clinical course and management of sinonasal paragangliomas (PGLs). STUDY DESIGN AND METHODS: Retrospective chart review of six patients with PGLs of the nasal cavity and paranasal sinuses. RESULTS: Three patients had tumors with malignant clinical behavior with cerebral metastases or infiltration of brain and local recurrence, despite surgery and/or radiotherapy, while three patients demonstrated a benign course. CONCLUSION: Sinonasal paragangliomas are frequently malignant. If malignant, they are very aggressive, with rapid local spread as well as high metastatic potential despite surgical resection; and they have a poor prognosis. Malignancy cannot be diagnosed on histology, but only on the basis of clinical behavior. Intracranial metastasis is commonly expected. Long-term follow-up, with particular emphasis put on the intracranial structures, is mandatory as recurrences or metastasis may occur even after a long time interval.

High incidence of extraadrenal paraganglioma in families with SDHx syndromes detected by functional imaging with [18F]fluorodihydroxyphenylalanine PET.

PURPOSE: Knowledge of the genetic backgrounds of hereditary syndromes, which are increasingly being characterized, enables genetic screening of family members of affected patients. Upon detection of a mutation, genetic counselling and clinical screening including imaging modalities and biochemical analyses are commonly performed. METHODS: Unaffected, mutation-positive relatives of index patients with hereditary paraganglioma syndromes were offered PET imaging with [(18)F]fluorodihydroxyphenylalanine and the incidence of pathological findings was retrospectively analysed in relation to mutations of the succinate dehydrogenase enzyme complex. PET only or PET/CT was performed in 21 individuals from eight families with SDHD, one family with SDHC and two families with SDHB mutations. Screening was offered every 2 to 5 years. RESULTS: Of the 21 individuals, 14 showed paraganglioma during screening. In particular, in only 2 of 15 patients with a SDHD mutation were the findings completely unremarkable on PET screening. However, false-negative lesions for abdominal manifestations in two SDHD-positive patients were detected. CONCLUSION: FDOPA PET is a sensitive imaging modality which should be offered to patients with a detected SDHx (SDHD) mutation, preferably using a hybrid technique.

Increased radioresistance via G12S K-Ras by compensatory upregulation of MAPK and PI3K pathways in epithelial cancer.

BACKGROUND: Irradiation-induced signaling via the 2 pathways, Raf-MEK-ERK and PI3K-Akt, is known to be closely associated with a limited response to radiotherapy. In the present study we analyzed the relevance of constitutively active K-Ras for postradiogenic pathway stimulation and the option of coordinated inhibition to overcome these rescue mechanisms. METHODS: We used 2 epithelial tumor cell lines as a model system, one of them harboring a G12S K-Ras mutation. Cells were irradiated and the effect of combined treatment with ionizing radiation and inhibitors on the expression of pERK and pAkt was determined by Western blotting. Additionally, clonogenic assays were performed to functionally analyze survival of the cell lines. RESULTS: Compared with the nonmutated cells we observed the G12S cell line showing a clearly reduced response to inhibitor treatment under irradiation. In the case of pharmacologic inhibition of 1 of the pathways a compensatory upregulation of the second cascade leading to increased clonogenic survival seems feasible. However, there was a good functional response of this cell line to double inhibition with both compounds represented by minimized colony forming ability. The activation of ERK and Akt after irradiation was confirmed in xenotransplants showing elevated postradiogenic protein levels. CONCLUSION: With our data we confirmed our hypothesis of postradiogenic constitutive activation of the 2 pathways both required for Ras-mediated radioresistance in epithelial cells. If this effect should prove itself as a general mechanism in Ras-mutated tumors, application of specific inhibitors to block both cascades in parallel could contribute to enhance radiosensitivity in these types of cancer.

Short-term hearing results after primary stapedotomy with nitinol and teflon-platinum prostheses for otosclerosis.

The aim of this study is to determine differences in postoperative air-bone gap (ABG) after placement of teflon-platinum or nitinol middle ear prostheses in primary stapedotomy patients with otosclerosis. Thirty otosclerosis patients (24 female, 6 male; age 10-61 years) with primary stapedotomy were studied prospectively. Before and after surgery, the mean and standard deviations of the ABG were measured at eight frequencies (0.25-4 kHz). Patients were randomized into one of two groups receiving either teflon-platinum or nitinol prostheses. Hearing results were assessed 1 year after surgery. To assess the joint influence of treatment and frequency on ABG reduction, a linear mixed model was used (significance level was p = 5%). The Tukey-Kramer method was used to adjust for multiple comparisons. Significant differences were found between treatment groups (p < 0.0001) and between frequencies within the same treatment group (p < 0.0001) but no interaction (p = 0.7963), i.e. the reduction of the conductive components over frequencies was nearly parallel in both groups. Overall, patients in the Teflon group had a larger reduction of conductive components, on average 8.0 dB more reduction, than patients in the nitinol group. However, after adjusting for multiple comparisons, we could not identify a single frequency with a significant difference in reduction of conductive components. Use of the teflon-platinum prosthesis results in statistically non-significant better ABG closure at 0.25-4 kHz 1 year postoperatively than the use of the nitinol prosthesis.

Cancer-associated fibroblasts do not respond to combined irradiation and kinase inhibitor treatment.

The emergence of radioresistance is a significant issue in the treatment of squamous cell carcinoma. We recently demonstrated that post-radiogenic extracellular signal-regulated kinase (ERK) signaling might decrease radiosensitivity in this cancer type. To further elucidate how tumor-organizing cell types respond to irradiation and ERK pathway inhibition, we analyzed one oral squamous cell carcinoma and one lung cancer cell line (HNSCCUM-02T, A549), fibroblasts (NIH3T3), primary normal and cancer-associated fibroblasts (CAFs) in vitro. Irradiated cells treated with mitogen-activated protein kinase (MAPK) inhibitor U0126 were screened for pERK levels. Post-radiogenic cellular responses were functionally analyzed by proliferation and colony assays. We found analogous pERK expression, proliferation and survival of tumor and normal fibroblast cells. CAFs did not show any response to treatment. We hypothesized that radiation and MAPK inhibition have no dose-limiting effect on tumor-surrounding normal tissue. As CAFs are considered to influence the radioresponse of the entire tumor, but are not affected by treatment themselves, potential CAF-mediated tumor protection should be considered in further studies.

Dysregulated survivin expression in nasal polyps of individuals with aspirin exacerbated respiratory disease.

BACKGROUND: A derailed balance of cell proliferation and apoptosis is presumed to result in cell hyperplasia as a typical feature of nasal polyps. Survivin, a protein of the inhibitors of the apoptosis family is proposed to promote polyp formation. However, studies concerning survivin expression in chronic rhinosinusitis (CRS) with nasal polyps are rare and the specificity of the survivin expression in nasal polyps from individuals with aspirin-exacerbated respiratory disease (AERD) has not been investigated. METHODS: Immunohistochemical survivin expression analysis was performed. Samples were taken from the ethmoid sinus of individuals with CRS with nasal polyps with and without AERD during sinus surgery and control specimens of the inferior turbinate from individuals without CRS. Cell cultures were stimulated with recombinant vascular endothelial growth factor (VEGF(165)) and the resulting survivin expression was analyzed by Western blot. RESULTS: The survivin expression of 61 specimens was analyzed by quantitative immunohistochemistry and a potential VEGF-dependant stimulation of survivin in cell cultures was investigated. The survivin expression in nasal polyps from individuals with AERD was increased compared with the controls (median, 1194 versus 927 arbitrary units [A.U.]; p = 0.054). Western blot analysis revealed in vitro a VEGF-dependant regulation of survivin in nasal polyps from individuals without AERD, but not in those with AERD (p = 0.05). CONCLUSION: Enhanced survivin expression might result in decreased apoptosis and cellular hyperplasia as a part of the largely unknown pathophysiology of nasal polyp formation. Furthermore, we hypothesize a pathological, VEGF-independent constitutive survivin expression in nasal polyps of individuals with AERD.

Low SPINK5 expression in chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: Chronic rhinosinusitis (CRS) is a multifactorial disease that probably arises as a result of genetic diversity and environmental factors. SPINK5 is a serine protease inhibitor, which is supposed to be an important regulator of epithelial barrier maintenance. The role of SPINK5 polymorphisms and expression in CRS, especially in individuals with aspirin intolerance, is unclear. STUDY DESIGN: SPINK5 single-nucleotide polymorphisms (SNPs) and SPINK5 expression levels were correlated with CRS without (CRSsNP) and with nasal polyps (CRSwNP), aspirin intolerance, asthma, and allergies. METHODS: One hundred four nasal tissue samples, 15 from patients with CRSsNP, 59 from patients with CRSwNP, and 30 from healthy controls of the inferior turbinate, were analyzed for their SPINK5 status. Genotypes of four SPINK5 single nucleotide polymorphism (SNPs; G1258A, G2475T, A2915G, and A1103G), as well as SPINK5 mRNA expression levels, were determined by polymerase chain reaction. RESULTS: No correlation between any SPINK5 SNP and CRSsNP, CRSwNP, or allergies and asthma was observed. The heterozygous SNPs G1258A and A1103G were observed more frequently in aspirin-intolerant patients; the homozygous (A/A) genotype of SNP 1258 and the homozygous (G/G) genotype SNP 1103 were less frequent. There was no correlation between the analyzed SNPs and the level of SPINK5 expression. It was noted that in individuals with CRSwNP, aspirin intolerance, and allergies, SPINK5 expression was lowered. CONCLUSIONS: G1258A and A1103G polymorphisms are distinctive for the aspirin intolerance syndrome. Lowered SPINK5 expression might be a contributing factor leading to CRS, and appears to be characteristic for patients suffering from aspirin intolerance and from allergies.

Vascular endothelial growth factor expression in nasal polyps of aspirin-intolerant patients.

OBJECTIVE: To study differences between aspirin-tolerant patients and aspirin-intolerant patients concerning vascular endothelial growth factor (VEGF) expression. Recent publications strongly suggest the involvement of VEGF and its receptors in the pathophysiologic process of nasal polyps. DESIGN: We subjected 43 polyp specimens to semiquantitative immunohistochemical analysis. We quantified VEGF and its receptors (Flk, Flt, and neuropilin) in all samples. To gain insight into potential VEGF-mediated cellular responses, we determined proliferative (Ki67) and apoptotic (caspase 3) indices. PATIENTS: Polyp samples were obtained from 22 aspirin-intolerant patients and from 21 aspirin-tolerant patients, and control specimens were obtained from 24 subjects with healthy nasal respiratory mucosa. SETTING: Laboratory; Department of Otorhinolaryngology-Head and Neck Surgery, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany. MAIN OUTCOME MEASURES: Expression levels of VEGF, VEGF receptors. and proliferative and apoptotic indices. RESULTS: We found higher expressed levels of VEGF and neuropilin and stronger proliferation in nasal polyps from aspirin-tolerant and aspirin-intolerant patients compared with controls. In polyps from aspirin-intolerant patients, VEGF was expressed at considerably higher levels compared with those from aspirin-tolerant subjects. Apoptotic activity remained unchanged in all 3 groups. CONCLUSIONS: Nasal polyps from aspirin-tolerant and aspirin-intolerant patients are characterized by strong proliferation and high levels of VEGF and neuropilin expression. Nasal polyps from aspirin-intolerant patients show distinctly increased VEGF levels. The relevance of these findings for future therapeutic approaches is yet to be determined.

Increased basic fibroblast growth factor release and proliferation in xenotransplanted squamous cell carcinoma after combined irradiation/anti-vascular endothelial growth factor treatment.

Novel strategies of cancer therapy combine irradiation and anti-angiogenic active compounds. However, little is known concerning the undesired cellular and molecular effects caused by this novel treatment concept. We used a mouse squamous cell carcinoma (SCC) xenotransplantation model to evaluate the potential undesired effects which compromise the success of this therapeutic combination. SCCs were subcutanously implanted in nude mice. Animals were treated with a fractionated irradiation scheme (5x4 Gy) alone or in combination with daily injections of anti-vascular endothelial growth factor (VEGF) antibodies. Controls remained untreated. Before and after treatment, resonance imaging (MRI), ultrasound and near-infrared spectrometry were used to evaluate tumor vessel integrity. Finally, tumors were explanted and VEGF, basic fibroblast growth factor (bFGF), vessel density, proliferation and apoptotic activity were analyzed by immunohistochemistry. Irradiation caused VEGF release and we found evidence for VEGF-mediated vessel protection. In the tumors derived from the combined treatment, blood volume was decreased, and apoptotic indices were increased. Remarkably, bFGF levels and proliferative indices were also increased. Combined irradiation/anti-VEGF treatment resulted in the desired VEGF depletion and increased tumor cell apoptosis. Nonetheless, bFGF and proliferation also increased, possibly suggesting a compensatory response. The application of additional targeted drugs may help develop more effective SCC treatments.

Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells.

Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.

Head and neck paragangliomas: Report of 175 patients (1989-2010).

BACKGROUND: Attention of the otorhinolaryngologist needs to be drawn to the versatile aspects of head and neck paragangliomas (PGLs). METHODS: This study is a retrospective, nonrandomized clinical study of all 175 individuals with PGLs treated in our department between 1989 and 2010. A genetic analysis was performed on 86 patients. RESULTS: The 175 patients presented 224 head and neck PGLs as well as 2 thyroid papillary carcinomas. Genetic analysis resulted in 1 patient positive for a von Hippel-Lindau (VHL) gene mutation and 34 for succinate dehydrogenase (SDH) gene mutations (22 SDHD, 7 SDHC, and 5 SDHB), 12 of the latter carrying a novel mutation. Thirty-three patients (18.9%) had multiple PGLs and 11 patients (6.3%) had a malignant paraganglioma. SDH-mutation carriers had multiple tumors in 64.7% and malignant paragangliomas in 20.6%. CONCLUSIONS: Multifocal occurrence, potential malignancy, genetic aspects, possible coincidence of thyroid carcinoma, and hormone production have to be considered in patients with head and neck PGLs.

Impact of ozone exposure on prostaglandin release in nasal polyps.

A dysregulation of the cyclooxygenases and a leukotriene/prostaglandin imbalance are assumed to be part of the pathogenesis of the aspirin (ASA) intolerance syndrome. Ozone is an air pollutant with known proinflammatory effects on exposed epithelia, however, its impact on the expression of the cyclooxygenases 1 and 2 (cox1/2) and prostaglandin E(2) (PGE(2)) in the nasal mucosa is not well known. Therefore, we analyzed cox expression and PGE(2) levels after ozone exposure in nasal mucosa and in nasal polyps considering ASA intolerance. Isolated epithelial nasal cells from control subjects without chronic rhinosinusitis (CRS), and those from patients with nasal polyps with and without ASA intolerance were cultured and exposed in vitro to ozone. Cox1/2 expression levels were analyzed by immunohistochemistry and PGE(2) release by ELISA. After ozone exposure cox1/2 expression remained unchanged in all the three groups. PGE(2) release was lowered in cell cultures from controls and from polyps of ASA tolerant but not in those of ASA intolerant patients after ozone exposure. In the latter, PGE(2) expression remained unchanged. Our in vitro data suggest that aspirin tolerant patients with polyps might be more affected by ozone compared to aspirin intolerant ones. The potential clinical impact of impaired PGE(2) expression caused by ozone on the functions of respiratory epithelia remains to be clarified.

Pharmacological genome demethylation increases radiosensitivity of head and neck squamous carcinoma cells.

Aberrant inactivation of tumor suppressor genes by promoter hypermethylation has been recognized as a crucial step of tumor development and is related to aggressiveness and therapy resistance. To identify potential novel treatment strategies, we evaluated pharmacological genome demethylation for the increase of irradiation treatment effectiveness in head and neck squamous cell carcinoma (HNSCC) in this in vitro study. HNSCC cells were cultured with 2 different concentrations of 5-azacytidine (5-Aza) for 72 h, followed by a single fraction irradiation with 4 or 50 Gy, respectively. To show successful genome demethylation, the methylation status of the tumor suppressor gene hic1 (hypermethylated in cancer) promoter was analyzed by methylation specific PCR (MSP) as well as hic1 transcription by quantitative RT-PCR. Survival, apoptosis, viability, and migration of the tumor cells were analyzed as functional parameters of combined treatment response. After 5-Aza treatment the hic1 promoter was demethylated and gene transcription restored demonstrating genome demethylation. 5-Aza treated cells tended to be less viable and showed decreased survival indicated by lower colony numbers. Apoptosis and migration were not affected. The combined application of irradiation and 5-Aza significantly reduced survival compared to the single treatments. Accordingly, apoptosis was strongly increased after combined 4 Gy/5-Aza treatment. Viability was not additionally affected by combined treatment. Migration was affected weakly by combined high dosage irradiation/5­Aza treatment. Our data show that the combined application of 5-Aza and irradiation is effective in vitro. A demethylating concept prior to irradiation should be further evaluated for its potential to reduce irradiation resistance.

Activation of mitogen-activated protein kinase extracellular signal-related kinase in head and neck squamous cell carcinomas after irradiation as part of a rescue mechanism.

BACKGROUND: Irradiation plays a pivotal role in head and neck squamous cell carcinoma (HNSCC) treatment. However, especially recurrent tumors frequently show increased radioresistance. We analyzed irradiation-stimulated mitogen-activated protein kinase (MAPK) signaling pathways to define cellular rescue mechanisms. METHODS: Irradiated HNSCC cells were screened for MAPK activation and results were confirmed and refined by functional analyses. Extracellular signal-regulated kinase (ERK) inhibitor U0126 application enabled us to specify postradiogenic cellular responses. Vascular endothelial growth factor (VEGF) levels were analyzed additionally. RESULTS: We observed a pronounced and time-dependent ERK stimulation. Pathway inhibition resulted in decreased radioresistance. Likewise, we found a decrease of VEGF release after inhibitor treatment. ERK activation was confirmed in xenotransplants showing elevated postradiogenic phospho-ERK (pERK) and VEGF levels. CONCLUSIONS: Our data give evidence for induction of ERK and successive VEGF release in HNSCC during radiotherapy, which might be partially explained by autoregulated cytoprotection maintained by pERK and potentially VEGF. In conclusion, targeting the ERK-VEGF axis might enhance the efficiency of radiotherapy.