Gunnar Wichmann

Interleukin-17-producing T helper cells in autoimmunity

With all the incredible progress in scientific research over the past two decades, the trigger of the majority of autoimmune disorders remains largely elusive. Research on the biology of T helper type 17 (T(H)17) cells over the last decade not only clarified previous observations of immune regulations and disease manifestations, but also provided considerable information on the signaling pathways mediating the effects of this lineage and its seemingly dual role in fighting the invading pathogens on one hand, and in frightening the host by inducing chronic inflammation and autoimmunity on the other hand. In this context, recent reports have implicated T(H)17 cells in mediating host defense as well as a growing list of autoimmune diseases in genetically-susceptible individuals. Herein, we summarize the current knowledge on T(H)17 in autoimmunity with emphasis on its differentiation factors and some mechanisms involved in initiating pathological events of autoimmunity.

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas

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.

Current aspects of targeted therapy in head and neck tumors

This review focuses on the current and upcoming options of targeted therapy (biologicals) in head and neck squamous cell carcinoma (HNSCC) with special regard to conceptual integration in future strategies. Epidermal growth factor receptor (EGFR) is the most prominent candidate for therapeutic targeting because of its more than 90% expression rate in HNSCC and influence on the regulation of proliferation, apoptosis, metastasis, angiogenesis and cell differentiation. The point of view of head and neck surgeons is mainly adjusted to reach a balance between targeted, minimal ablative surgery and the evidence-based demand of oncologic accurate surgery with clear margins and, if needed, adjuvant or primary systemic chemoradiation. Therefore, the long-term effects of chemoradiation regimens, such as dysphagia, aspiration and laryngeal immobility caused by fibrosis, are just beginning to be studied and are becoming one of the major problems in the ongoing treatment of HNSCC. In this context, molecular targeting biologicals with a different toxicity profile and hopefully less late damage to functionally important tissues may open new strategies in primary and adjuvant treatment of HNSCC. Besides cetuximab and other EGFR targeting mAbs, this review focuses on receptor and non-receptor tyrosine kinase inhibitors, which further might play a role in the future treatment of HNSCC. To complete the current picture, the problem of multi drug resistance in cancer progenitor cells, targeting members of several relevant pathways and novel agents like pemetrexed and enzastaurin, are discussed in a broader sense of targeted therapy.

The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer

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 (DNA+ RNA‐) are similar to HPV‐negative (DNA‐) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response‐related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA‐ tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53‐mutation status for patient stratification and identify associations of an immune response‐related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC.

Assay-based response evaluation in head and neck oncology: requirements for better decision making

This article gives an overview on different current strategies of assay-based response evaluation in head and neck squamous cell carcinomas (HNSCC) and critically summarizes their role and needs for future clinical evaluation. Due to a growing amount of data of phase III clinical trials of multimodality treatment options for HNSCC, treatment planning in regard to optimal outcome is becoming an interdisciplinary challenge. New concepts such as induction chemotherapy with bi- or ternary combinations of chemotherapeutics, integration of targeted therapies, concurrent and sequential chemoradiation concepts, and multimodality-based organ preservation strategies strongly compete with traditional definitive surgical procedures. Moreover, the outcome is difficult to predict due to heterogeneity of a tumor’s response, impaired late functional outcome, and increased late toxicity if simultaneously applied to radiation. Retrospectively looking at non-responders with tumors classified as resectable, primary surgery is very likely to have achieved better results, since chemoradiation causes a high degree of early and late toxicities leading to extremely complicated terms and conditions in surgery following current multimodal therapeutic strategies. Unfortunately, predictive information on response characteristics of a given tumor before starting the therapy is not available in daily routine, although heterogeneity in response of a given tumor entity to treatments has been known for decades. Therefore, current therapy strategies for HNSCC still have to ignore this fact, creating an urgent need for the development of proper predictive assays. There are interesting clinical observations showing that response on induction chemotherapy may predict the outcome after radiotherapy. Some trials use this empiric phenomenon to pre-select non-responders for primary surgical treatment avoiding severe salvage complications after failure of complete chemoradiation treatment. Moving one step further, recent literature and our own investigations implicate that response evaluation of the individual patient’s HNSCC in a suitable ex vivo assay just before starting the treatment is mature for clinical research. To this end, essential needs and hints are addressed and discussed.

The cytotoxic effects of the organophosphates chlorpyrifos and diazinon differ from their immunomodulating effects

Some organophosphate insecticides have immunomodulating capacities, but it is unknown whether different compounds within this class affect the immune system to the same extent. In this in vitro study, human immortalized T-lymphocytes or bronchial epithelial cells were treated with diazinon or chlorpyrifos in the absence or presence of cellular stress factors, thereby mimicking a stimulated immune system. Cytotoxicity was determined and cytokine release or cytokine-promoter studies were performed to study immunomodulatory effects of these chemicals, whereby the same concentrations of chlorpyrifos and diazinon were used. Results showed that chlor- pyrifos was cytotoxic at concentrations ≥ 250 μM, whereas diazinon was not toxic at concentrations up to 1 mM. The immunomodulatory effects of these two compounds were similar for most cytokine promoters tested and induction of cellular stress enhanced these effects. The results were compared to data obtained with blood mononuclear cells, which confirmed the results of stably transfected cell lines, but refer to a higher sensitivity of primary cells. In conclusion, these two pesticides act in a different manner on cell viability and on some immune parameters, but cell viability was not linked to immunomodulation. The results also imply that healthy and diseased individuals are differentially affected by these pollutants.

Therapeutic options for treatment of Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare malignant neuroendocrine carcinoma of the skin. Owing to the aggressiveness of this tumor and the bad overall survival, we reviewed the therapeutic strategies, including surgery, radiation, and chemotherapy to find out the potentially best treatment option for one patient treated at our hospital. In addition, we investigated MCC biopsies using the FLAVINO assay to find out if individual chemoresponse testing might be a useful supplement in decision-making for the optimal therapeutic option for our MCC patient. The different results achieved using cisplatin, docetaxel, and cetuximab led to the conclusion that an individual chemoresponse testing in a predictive short-time assay might potentially be a useful diagnostic tool in identifying potentially effective chemotherapy treatments.

Single Tissue Samples from Head and Neck Squamous Cell Carcinomas are Representative Regarding the Entire Tumor’s Chemosensitivity to Cisplatin and Docetaxel

Background: In multimodal therapy concepts for advanced head and neck squamous cell carcinoma (HNSCC), a valid predictive assay for the quick detection of efficient chemotherapeutic agents is desirable. Questionable so far was whether tissue samples of about 100 mg correctly reflect the chemoresponse of a whole HNSCC. This was proven using an ex-vivo colony-forming assay. Materials and Methods: Of 14 HNSCC, 3 biopsies each were taken from separate sites, minced, and collagenase digested. HNSCC digests were added to microtiter plates containing serial dilutions of chemotherapeutic agents or medium as control. After 72-h incubation, wells were washed and cultures methanol-fixed before Giemsa-staining. Epithelial colonies were counted. Results: 11/14 HNSCC (78.6%) showed sufficient colony formation allowing reliable cut-off detection. Cut-off concentrations (complete chemotherapeutically suppressed colony formation) between 3.3 μM and >50 μM cisplatin, and 0.55 μM and 17.6 μM docetaxel were detected. Inhibition of colony formation to 50% of colonies detected in controls (IC50) was found between 0.2 μM and 17.9 μM cisplatin or 1.5 μM and 13.7 μM docetaxel. Cut-off concentrations and IC50 of the HNSCC fragments showed a strong correlation (docetaxel: r > 0.80, p < 0.005; cisplatin: r > 0.67, p < 0.044), while being only insignificantly different in the t-test for paired samples (docetaxel: p > 0.163; cisplatin: p > 0.167). Conclusion: In most cases, tissue samples of about 100 mg allow a representative assessment of chemoresponse of HNSCC.

Key molecules in the differentiation and commitment program of T helper 17 (Th17) cells up-to-date

The mechanisms underlying autoimmunity and cancer remain elusive. However, perpendicular evidence has been evolved in the past decade that T helper (Th)17 cells and their related molecules are implicated in initiation and induction of various disease settings including both diseases. Meanwhile, extensive research on Th17 cells elucidated various molecules including cytokines and transcription factors as well as signaling pathways involved in the differentiation, maturation, survival and ultimate commitment of Th17 cells. In the current review, we revise the mechanistic underpinnings delivered by recent research on these molecules in the Th17 differentiation/commitment concert. We emphasize on those molecules proposed as targets for attaining potential therapies of various autoimmune disorders and cancer, aiming both at dampening the dark-side of Th17 repertoire and simultaneously potentiating its benefits in the roster of the antimicrobial response.

Sensitivity and specificity of antibodies against HPV16 E6 and other early proteins for the detection of HPV16-driven oropharyngeal squamous cell carcinoma

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%], Cohens 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.