Elgar Susanne Quabius

HPV DNA, E6*I-mRNA expression and p16INK4A immunohistochemistry in head and neck cancer – How valid is p16INK4A as surrogate marker?

It has been proposed that p16(INK4A) qualifies as a surrogate marker for viral oncogene activity in head and neck cancer (HNSCC). By analyzing 78 HNSCC we sought to validate the accuracy of p16(INK4A) as a reliable marker of active HPV infections in HNSCC. To this end we determined HPV DNA (HPVD) and E6*I mRNA (HPVR) expression status and correlated these results with p16(INK4A) staining. In tonsillar SCC 12/20 were HPVD+ and 12/12 of these showed active HPV infections whereas in non-tonsillar SCC 10/58 were HPVD+ and 5/10 showed active HPV infections. Thus, we prove about 8% of non-tonsillar SCC to be also correlated with HPV-associated carcinogenesis. Strikingly, 3/14 (21.4%) of tonsillar and non-tonsillar HPVD+/HPVR+ cases did not show p16(INK4A) overexpression and these cases would have been missed when applying initial p16(INK4A) staining only. However, in 13 cases negative for HPV, DNA p16(INK4A) was overexpressed. In conclusion, our data confirm tonsillar SCC to be predominantly but not only associated with active HPV infections. Furthermore, our data show that p16(INK4A) overexpression is not evident in a subgroup of HNSCC with active HPV infection. Definitive HPV data should therefore be utilized in diagnostics and treatment modalities of HPV positive and HPV negative HNSCC patients, resulting in a paradigm shift regarding these obviously different tumor entities.

Can peripheral blood γδ T cells predict osteonecrosis of the jaw? An immunological perspective on the adverse drug effects of aminobisphosphonate therapy†

Nitrogen‐bisphosphonates (n‐BP), often referred to as aminobisphosphonates, are the most commonly prescribed drugs for the treatment of disorders of bone fragility. However, long‐term continuous treatment predisposes certain individuals to serious rare side effects, such as bisphosphonate‐associated osteonecrosis of the jaw (BAONJ). n‐BP use is known to unintentionally activate a subset of innate T cells called Vγ9Vδ2 T cells, but the consequence of this chronic immune stimulation has remained unexplored. The primary objectives of this study were to 1) determine the fate of Vγ9Vδ2 T cells in osteoporotic patients on n‐BP therapy as a function of time and type of therapy; 2) evaluate the proportion of Vγ9Vδ2 T cells in patients who had recently experienced n‐BP–associated ONJ. We found there is a notable loss of Vγ9Vδ2 T cells over time in osteoporotic patients on n‐BP therapy, particularly those on intravenous (iv) therapy (Spearman r = −0.55, p < 0.0001 iv; r = −0.3, p < 0.03 oral) (n = 68); no difference was observed in total T cells, monocytes, or granulocytes. Importantly, the observed negative effect on Vγ9Vδ2 T cells coincides with the reported route of administration and timing of the rare occurrence of BAONJ. Patients (n = 6) who had experienced BAONJ were all found to be significantly deficient in Vγ9Vδ2 T cells (median = 0.07%) in comparison to age‐ and sex‐matched treatment‐naïve controls (N = 11; median = 2.40%), U = 0, p = 0.001; this was the only consistent difference in the leukocytes assessed. All BAONJ cases had an underlying condition that further contributed to impaired immunity. We propose Vγ9Vδ2 T cells show a strong potential to serve as harbingers of possible adverse immune effects of n‐BP therapy, particularly in those patients already having a compromised immune system as they may be most vulnerable to the development of conditions such as BAONJ.

Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood γδ T cells

Nitrogen-bisphosphonates (n-BP), such as zoledronate, are the main class of drugs used for the prevention of osteoporotic fractures and the management of cancer-associated bone disease. However, long-term or high-dose use has been associated with certain adverse drug effects, such as osteonecrosis of the jaw and the loss of peripheral of blood Vγ9Vδ2 T cells, which appear to be linked to drug-induced immune dysfunction. In this report we show that neutrophils present in human peripheral blood readily take up zoledronate, and this phenomenon is associated with the potent immune suppression of human peripheral blood Vγ9Vδ2 T cells. Furthermore, we found this zoledronate-mediated inhibition by neutrophils could be overcome to fully reconstitute Vγ9Vδ2 T cell proliferation by concomitantly targeting neutrophil-derived hydrogen peroxide, serine proteases, and arginase I activity. These findings will enable the development of targeted strategies to mitigate some of the adverse effects of n-BP treatment on immune homeostasis and to improve the success of immunotherapy trials based on harnessing the anticancer potential of peripheral blood γδ T cells in the context of n-BP treatment.

Human papillomavirus infection in head and neck cancer: The role of the secretory leukocyte protease inhibitor

We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non-malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases.

Synthetic mRNAs for manipulating cellular phenotypes: an overview.

Availability of high quality synthetic mRNAs (syn-mRNAs) has enabled progress in their applications. Important structural features and quality requirements are discussed. Developments in the application of mRNA-mediated manipulation of cells are presented (i) mRNA-directed expression of antigens in dendritic cells for vaccination projects in oncogenesis, infectious disease and allergy prevention; (ii) reprogramming of human fibroblasts to induced pluripotent stem cells with their subsequent differentiation to the desired cell type; (iii) applications in gene therapy.

Geographical and anatomical influences on human papillomavirus prevalence diversity in head and neck squamous cell carcinoma in Germany

The increased knowledge regarding HPV-infections in head and neck squamous cell carcinoma (HNSCC) has unexpectedly contributed to several uncertainties related to i) prevalence diversities depending on tumour site and geographical origin of the patients, ii) proportion of HPV-driven tumours among HPV-DNA-positive cases, and iii) identification of patients with HPV-attributed survival benefit. To investigate this heterogeneity, we analysed 307 HNSCC cases (tonsillar, n=135; non-tonsillar, n=172) from eight health care centers mostly from Northern Germany and determined HPV-DNA/mRNA and p16INK4A-status and combined results with the patient outcome. Overall HPV-DNA prevalence rate was 23.5% (72/307); attributed to: 43.7% (59/135) and 7.6% (13/172) tonsillar and non-tonsillar cases, respectively. Among these, 96.6% tonsillar and 38.5% non-tonsillar SCC were HPV-mRNA-positive. Although the study cohort was composed of patients from regions of rather close proximity, prevalence rates showed diversities of up to 40% in HNSCC subsite analysis with the lowest prevalence for tonsillar SCC in metropolitan areas (22.2%) vs. 50.9% in rural areas. Survival analysis identified p16INK4A alone as strongest predictor, followed by HPV-DNA-status alone or in combination with p16INK4A. This survival benefit was shown for tonsillar and non-tonsillar cases. Smoking significantly correlated with HPV-status, however, it does not influence survival when stratified for HPV. In conclusion, the data emphasize the urge for further data on HPV-infection in HNSCC to, e.g. clarify to what extent survival benefits of p16INK4A-positive patients are truly attributed to HPV-infections.

Autosomal-dominant striatal degeneration is caused by a mutation in the phosphodiesterase 8B gene.

Autosomal-dominant striatal degeneration (ADSD) is an autosomal-dominant movement disorder affecting the striatal part of the basal ganglia. ADSD is characterized by bradykinesia, dysarthria, and muscle rigidity. These symptoms resemble idiopathic Parkinson disease, but tremor is not present. Using genetic linkage analysis, we have mapped the causative genetic defect to a 3.25 megabase candidate region on chromosome 5q13.3-q14.1. A maximum LOD score of 4.1 (Theta = 0) was obtained at marker D5S1962. Here we show that ADSD is caused by a complex frameshift mutation (c.94G>C+c.95delT) in the phosphodiesterase 8B (PDE8B) gene, which results in a loss of enzymatic phosphodiesterase activity. We found that PDE8B is highly expressed in the brain, especially in the putamen, which is affected by ADSD. PDE8B degrades cyclic AMP, a second messenger implied in dopamine signaling. Dopamine is one of the main neurotransmitters involved in movement control and is deficient in Parkinson disease. We believe that the functional analysis of PDE8B will help to further elucidate the pathomechanism of ADSD as well as contribute to a better understanding of movement disorders.

Cardiomyocyte behavior on biodegradable polyurethane/gold nanocomposite scaffolds under electrical stimulation

Following a myocardial infarction (MI), cardiomyocytes are replaced by scar tissue, which decreases ventricular contractile function. Tissue engineering is a promising approach to regenerate such damaged cardiomyocyte tissue. Engineered cardiac patches can be fabricated by seeding a high density of cardiac cells onto a synthetic or natural porous polymer. In this study, nanocomposite scaffolds made of gold nanotubes/nanowires incorporated into biodegradable castor oil-based polyurethane were employed to make micro-porous scaffolds. H9C2 cardiomyocyte cells were cultured on the scaffolds for one day, and electrical stimulation was applied to improve cell communication and interaction in neighboring pores. Cells on scaffolds were examined by fluorescence microscopy and scanning electron microscopy, revealing that the combination of scaffold design and electrical stimulation significantly increased cell confluency of H9C2 cells on the scaffolds. Furthermore, we showed that the gene expression levels of Nkx2.5, atrial natriuretic peptide (ANF) and natriuretic peptide precursor B (NPPB), which are functional genes of the myocardium, were up-regulated by the incorporation of gold nanotubes/nanowires into the polyurethane scaffolds, in particular after electrical stimulation.

Head and neck cancer cells and xenografts are very sensitive to palytoxin: decrease of c-jun n-terminale kinase-3 expression enhances palytoxin toxicity

ObjectivesPalytoxin (PTX), a marine toxin isolated from the Cnidaria (zooanthid) Palythoa caribaeorum is one of the most potent non-protein substances known. It is a very complex molecule that presents both lipophilic and hydrophilic areas. The effect of PTX was investigated in a series of experiments conducted in head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts.Materials and methodsCell viability, and gene expression of the sodium/potassium-transporting ATPase subumit alpha1 (ATP1AL1) and GAPDH were analyzed in HNSCC cells and normal epithelial cells after treatment with PTX using cytotoxicity-, clonogenic-, and enzyme inhibitor assays as well as RT-PCR and Northern Blotting. For xenograft experiments severe combined immunodeficient (SCID) mice were used to analyze tumor regression. The data were statistically analyzed using One-Way Annova (SPSS vs20).ResultsSignificant toxic effects were observed in tumor cells treated with PTX (LD50 of 1.5 to 3.5 ng/ml) in contrast to normal cells. In tumor cells PTX affected both the release of LDH and the expression of the sodium/potassium-transporting ATPase subunit alpha1 gene suggesting loss of cellular integrity, primarily of the plasma membrane. Furthermore, strong repression of the c-Jun N-terminal kinase 3 (JNK3) mRNA expression was found in carcinoma cells which correlated with enhanced toxicity of PTX suggesting an essential role of the mitogen activated protein kinase (MAPK)/JNK signalling cascades pathway in the mechanisms of HNSCC cell resistance to PTX. In mice inoculated with carcinoma cells, injections of PTX into the xenografted tumors resulted within 24 days in extensive tumor destruction in 75% of the treated animals (LD50 of 68 ng/kg to 83 ng/kg) while no tumor regression occurred in control animals.ConclusionsThese results clearly provide evidence that PTX possesses preferential toxicity for head and neck carcinoma cells and therefore it is worth further studying its impact which may extend our knowledge of the biology of head and neck cancer.

The antileukoprotease secretory leukocyte protease inhibitor (SLPI) and its role in the prevention of HPV-infections in head and neck squamous cell carcinoma

Recently, we demonstrated a significant inverse correlation between HPV-infection and SLPI-expression suggesting that SLPI protects against HPV-infection of HNSCC. Here we analyzed in a single lab setting 307 formalin-fixed paraffin-embedded HNSCC cases (tonsillar n = 135; non-tonsillar: n = 172) from eight health care centers. Samples were analyzed for SLPI gene- and protein-expression. Annexin A2, its heterotetramer A2t, putatively facilitating HPV- and SLPI-cell entry, was measured to study the correlation between SLPI and annexin A2. Data were correlated with tobacco consumption and HPV-status. Overall, HPV-DNA prevalence was 23.5% (72/307); attributed to: 43.7% (59/135) tonsillar and 7.6% (13/172) non-tonsillar cases. Smoking resulted in 6.44-fold increased and HPV-infection in 3.46-fold decreased SLPI-gene expression in all HNSCC with similar significant results obtained in tonsillar and non-tonsillar SCC separately. Correlating annexin A2- and SLPI-gene expression showed a significant surplus of annexin A2 in HPV-positive tumors (4.21× more annexin A2) and 6.72× more annexin A2 than SLPI in nonsmokers in all HNSCCs and similar significant results for both tumor entities separately. The surplus of annexin A2 in non-smokers and HPV-positive patients supports our hypothesis that decreased SLPI levels facilitate HPV-infection i.e., increased SLPI-expression may protect against HPV-infection of tonsillar and non-tonsillar SCC.