Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA1 RNA-) are similar to HPV-negative (DNA-) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.

To determine the sensitivity and specificity of HPV16 serology as diagnostic marker for HPV16-driven oropharyngeal squamous cell carcinoma (OPSCC), 214 HNSCC patients from Germany and Italy with fresh-frozen tumor tissues and sera collected before treatment were included in this study. Hundred and twenty cancer cases were from the oropharynx and 94 were from head and neck cancer regions outside the oropharynx (45 oral cavity, 12 hypopharynx and 35 larynx). Serum antibodies to early (E1, E2, E6 and E7) and late (L1) HPV16 proteins were analyzed by multiplex serology and were compared to tumor HPV RNA status as the gold standard. A tumor was defined as HPV-driven in the presence of HPV16 DNA and HPV16 transformation-specific RNA transcript patterns (E6*I, E1 E4 and E1C). Of 120 OPSCC, 66 (55%) were HPV16-driven. HPV16 E6 seropositivity was the best predictor of HPV16-driven OPSCC (diagnostic accuracy 97% [95%CI 92-99%], Cohen's kappa 0.93 [95%CI 0.8-1.0]). Of the 66 HPV-driven OPSCC, 63 were HPV16 E6 seropositive, compared to only one (1.8%) among the 54 non-HPV-driven OPSCC, resulting in a sensitivity of 96% (95%CI 88-98) and a specificity of 98% (95%CI 90-100). Of 94 HNSCC outside the oropharynx, six (6%) were HPV16-driven. In these patients, HPV16 E6 seropositivity had lower sensitivity (50%, 95%CI 19-81), but was highly specific (100%, 95%CI 96-100). In conclusion, HPV16 E6 seropositivity appears to be a highly reliable diagnostic marker for HPV16-driven OPSCC with very high sensitivity and specificity, but might be less sensitive for HPV16-driven HNSCC outside the oropharynx.

Available literature indicated that sirolimus therapy might be effective for lymphatic malformations. However, further randomized controlled studies are required to analyze the efficacy and long-term adverse events and to clarify the potential role for sirolimus in the management of lymphatic malformations.

Background:Human head and neck squamous cell carcinoma (HNSCC) fundamentally vary in their susceptibility to different cytotoxic drugs and treatment modalities. There is at present no clinically accepted test system to predict the most effective therapy for an individual patient.Methods:Therefore, we established tumour-derived slice cultures which can be kept in vitro for at least 6 days. Upon treatment with cisplatin, docetaxel and cetuximab, slices were fixed and paraffin sections were cut for histopathological analysis.Results:Apoptotic fragmentation, activation of caspase 3, and cell loss were observed in treated tumour slices. Counts of nuclei per field in untreated compared with treated slices deriving from the same tumour allowed estimation of the anti-neoplastic activity of individual drugs on an individual tumour.Conclusion:HNSCC-derived slice cultures survive well in vitro and may serve not only to improve personalised therapies but also to detect mechanisms of tumour resistance by harvesting surviving tumour cells after treatment.

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Exposure to molds diminishes the numbers of T-helper type 1 (Th1) cells in the peripheral blood of children and is a risk factor for the development of allergic diseases (results of LARS: Leipzig Allergy Risk Children Study, Mueller et al. 2002). We hypothesized that mycotoxins are responsible for this effect and therefore investigated the influence of citrinin, gliotoxin, and patulin on human peripheral blood mononuclear cells (PBMC). CD3/CD28-stimulated PBMC of healthy donors were incubated for 24 h with the mycotoxins in serial dilutions and triplicates. Vitality and proliferation were tested using the MTT assay and T-cell function by the expression of cytokines (ELISA, intracellular cytokine staining, and real-time polymerase chain reaction (RT-PCR) for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). The cytokine secretion was inhibited at concentrations 2-130 times lower compared to vitality (ELISA versus MTT assay). The strongest inhibition of cytokine expression was found for IFN-gamma: 8.3 microg/mL citrinin, 34.2 ng/mL gliotoxin, and 64.8 ng/mL patulin caused a 50% inhibition of the IFN-gamma release (50% inhibitory dose, ID(50)). For IL-4 release the corresponding ID(50) values were 21.6 microg/mL citrinin, 82.8 ng/mL gliotoxin, and 243.2 ng/mL patulin. Furthermore, 3 ng/mL patulin caused a significant increase of IL-4 but a significant suppression of IFN-gamma. On the mRNA level, after 24 h an unaltered or enhanced IL-4 was observed compared to a reduced IFN-gamma expression. Using a method of intracellular cytokine staining, we were able to show that the described effects are caused by a reduction of the number of IFN-gamma-producing T lymphocytes rather than by a reduced functional capacity of the single cell. We suggest that mycotoxins primarily cause stronger inhibition of IFN-gamma-producing Th1 cells, which may lead to T-cell polarization toward the Th2 phenotype and may raise the risk for the development of allergies.

This study shows an elevated number, but pronounced functional impairment, of EPCs in patients on long-term HD therapy. The latter may result in limited endothelial repair, which, in turn, may contribute to endothelial dysfunction in this particular group of patients.