Prognostic implications of p16 and HPV discordance in oropharyngeal cancer (HNCIG-EPIC-OPC): a multicentre, multinational, individual patient data analysis.

BACKGROUND: p16INK4a (p16) immunohistochemistry is the most widely used biomarker assay for inferring HPV causation in oropharyngeal cancer in clinical and trial settings. However, discordance exists between p16 and HPV DNA or RNA status in some patients with oropharyngeal cancer. We aimed to clearly quantify the extent of discordance, and its prognostic implications. METHODS: In this multicentre, multinational individual patient data analysis, we did a literature search in PubMed and Cochrane database for systematic reviews and original studies published in English between Jan 1, 1970, and Sept 30, 2022. We included retrospective series and prospective cohorts of consecutively recruited patients previously analysed in individual studies with minimum cohort size of 100 patients with primary squamous cell carcinoma of the oropharynx. Patient inclusion criteria were diagnosis with a primary squamous cell carcinoma of oropharyngeal cancer; data on p16 immunohistochemistry and on HPV testing; information on age, sex, tobacco, and alcohol use; staging by TNM 7th edition; information on treatments received; and data on clinical outcomes and follow-up (date of last follow-up if alive, date of recurrence or metastasis, and date and cause of death). There were no limits on age or performance status. The primary outcomes were the proportion of patients of the overall cohort who showed the different p16 and HPV result combinations, as well as 5-year overall survival and 5-year disease-free survival. Patients with recurrent or metastatic disease or who were treated palliatively were excluded from overall survival and disease-free survival analyses. Multivariable analysis models were used to calculate adjusted hazard ratios (aHR) for different p16 and HPV testing methods for overall survival, adjusted for prespecified confounding factors. FINDINGS: Our search returned 13 eligible studies that provided individual data for 13 cohorts of patients with oropharyngeal cancer from the UK, Canada, Denmark, Sweden, France, Germany, the Netherlands, Switzerland, and Spain. 7895 patients with oropharyngeal cancer were assessed for eligibility. 241 were excluded before analysis, and 7654 were eligible for p16 and HPV analysis. 5714 (74·7%) of 7654 patients were male and 1940 (25·3%) were female. Ethnicity data were not reported. 3805 patients were p16-positive, 415 (10·9%) of whom were HPV-negative. This proportion differed significantly by geographical region and was highest in the areas with lowest HPV-attributable fractions (r=-0·744, p=0·0035). The proportion of patients with p16+/HPV- oropharyngeal cancer was highest in subsites outside the tonsil and base of tongue (29·7% vs 9·0%, p<0·0001). 5-year overall survival was 81·1% (95% CI 79·5-82·7) for p16+/HPV+, 40·4% (38·6-42·4) for p16-/HPV-, 53·2% (46·6-60·8) for p16-/HPV+, and 54·7% (49·2-60·9) for p16+/HPV-. 5-year disease-free survival was 84·3% (95% CI 82·9-85·7) for p16+/HPV+, 60·8% (58·8-62·9) for p16-/HPV-; 71·1% (64·7-78·2) for p16-/HPV+, and 67·9% (62·5-73·7) for p16+/HPV-. Results were similar across all European sub-regions, but there were insufficient numbers of discordant patients from North America to draw conclusions in this cohort. INTERPRETATION: Patients with discordant oropharyngeal cancer (p16-/HPV+ or p16+/HPV-) had a significantly worse prognosis than patients with p16+/HPV+ oropharyngeal cancer, and a significantly better prognosis than patients with p16-/HPV- oropharyngeal cancer. Along with routine p16 immunohistochemistry, HPV testing should be mandated for clinical trials for all patients (or at least following a positive p16 test), and is recommended where HPV status might influence patient care, especially in areas with low HPV-attributable fractions. FUNDING: European Regional Development Fund, Generalitat de Catalunya, National Institute for Health Research (NIHR) UK, Cancer Research UK, Medical Research Council UK, and The Swedish Cancer Foundation and the Stockholm Cancer Society.

Tonsillar swabs and sputum predict SLPI- and AnxA2 expression in tonsils: A prospective study on smoking dependent SLPI- and AnxA2-expression, and tonsillar HPV infection.

Previous retrospective studies have elucidated a correlation between secretory leucocyte protease inhibitor (SLPI) and Annexin A2 (AnxA2), patient smoking status and tonsillar human papilloma virus (HPV) status. The current study assessed these parameters prospectively and to the best of our knowledge, analyzed SLPI-/AnxA2-expression for the first time in tonsillar swabs and sputum. Samples were obtained from 52 patients with tonsillar squamous cell carcinoma and 163 patients with tonsillar hyperplasia (H; n=56) and chronic or recurrent tonsillitis (CRT; n=107). HPV-DNA, SLPI and AnxA2 gene expression was analyzed in sputum, tonsillar swabs and tissue by performing reverse transcription-quantitative PCR. Results were compared with smoking status, revealing that smoking resulted in significantly increased SLPI gene expression in all biomaterials of all cases. SLPI-gene expression was significantly decreased in all HPV-DNA-positive samples (tissue/swab/sputum), while AnxA2 was significantly increased in all HPV-DNA-positive samples. Results from swabs and sputum were able to predict SLPI- and AnxA2 gene expression of the corresponding tonsil. The current prospective study confirmed previous retrospective results underlining this hypothesis: Smoking enhances SLPI-expression, preventing HPV-binding to AnxA2. HPV-binding to AnxA2 appears essential for successful cell-entry. SLPI/AnxA2-gene expression in swabs and sputum reflect their expression in tonsillar tissue. Accordingly, a positive AnxA2/SLPI-ratio in sputum/swabs could possibly be used to reduce HPV-associated carcinogenesis, by performing tonsillectomy or HPV-vaccination in patients with positive AnxA2/SLPI-ratios.

Sealing Behavior of a New Material Combination in Two-Part Dental Implant Systems: An In Vitro Examination.

PURPOSE: To test a newly introduced implant-abutment material combination against bacterial endotoxin leakage in a human whole blood assay. MATERIALS AND METHODS: Two dental implant systems with internal connections and the following material combinations at the implant-abutment interface (IAI) were used (implant material/abutment material): yttrium-stabilized tetragonal zirconium dioxide (Y-TZP)/polyetherketoneketone (PEKK), and titanium (Ti/Ti). Test implants were inoculated with lipopolysaccharide (LPS) and sealed and submerged in human whole blood. Untreated implants served as the control groups. Changes in gene expression levels of inflammatory markers indicating LPS leakage were assessed after 1, 8, and 24 hours using quantitative real-time polymerase chain reaction. RESULTS: In the Y-TZP/PEKK test group, a significant influence of the implant system (P < .001) on increases in gene expression indicating leakage were detected after 8 hours for TLR-4 and after 24 hours for interleukin 1-ß and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), indicating microleakage of LPS at the IAI. In the Ti/Ti test group, differences in gene expression were found only for NF-κB after 8 hours. CONCLUSION: The internal hexalobe IAI of two-piece dental implants fabricated from Y-TZP and PEKK do not prevent LPS molecular microleakage.

Erroneous expression of NKG2D on granulocytes detected by phycoerythrin-conjugated clone 149810 antibody.

BACKGROUND: The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, γδ T cells and small subsets of CD4 T lymphocytes. During the course of an extensive flow cytometry phenotyping of immune cells in the peripheral blood of patients with glioblastoma multiforme (GBM) we noticed an unexpected expression of NKG2D receptor on granulocytes using the phycoerythrin (PE)-conjugated clone 149810 antibody. METHODS: Peripheral blood samples from 35 patients with GBM and 22 age-matched healthy control (HC) donors were analyzed using flow cytometry, imaging cytometry and real-time quantitative reverse transcription PCR to validate the observed expression of NKG2D receptor on myeloid cells. RESULTS: Reactivity with PE-149810 was mostly observed on granulocytes from GBM patients on dexamethasone treatment where it correlated with inferior survival rates. Surprisingly, such NKG2D expression on granulocytes was not observed using the allophycocyanin (APC)-conjugate of the same clone 149810 antibody or an indirect staining procedure with unconjugated clone 149810 antibody. Moreover, the PE-conjugate of a different anti-NKG2D clone (1D11) also did not stain granulocytes. Imaging cytometry indicated cell surface and intracellular localization of PE-149810 but not of PE-1D11 in granulocytes. CONCLUSION: Our results uncover an erroneous and false positive reactivity of PE-labeled (but not of APC-labeled or unconjugated) anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients and raise a note of caution for studies of NKG2D expression on non-lymphoid cells.

Relevance of Human Papillomaviruses in Head and Neck Cancer-What Remains in 2021 from a Clinician's Point of View?

Human papillomaviruses (HPV) cause a subset of head and neck cancers (HNSCC). HPV16 predominantly signs responsible for approximately 10% of all HNSCC and over 50% of tonsillar (T)SCCs. Prevalence rates depend on several factors, such as the geographical region where patients live, possibly due to different social and sexual habits. Smoking plays an important role, with non-smoking patients being mostly HPV-positive and smokers being mostly HPV-negative. This is of unparalleled clinical relevance, as the outcome of (non-smoking) HPV-positive patients is significantly better, albeit with standard and not with de-escalated therapies. The results of the first prospective de-escalation studies have dampened hopes that similar superior survival can be achieved with de-escalated therapy. In this context, it is important to note that the inclusion of p16INK4A (a surrogate marker for HPV-positivity) in the 8th TMN-classification has only prognostic, not therapeutic, intent. To avoid misclassification, highest precision in determining HPV-status is of utmost importance. Whenever possible, PCR-based methods, still referred to as the "gold standard", should be used. New diagnostic antibodies represent some hope, e.g., to detect primaries and recurrences early. Prophylactic HPV vaccination should lead to a decline in HPV-driven HNSCC as well. This review discusses the above aspects in detail.

[The impact of smoking habit alteration on prognosis of head and neck cancer patients]. / Eine Änderung des Rauchverhaltens beeinflusst das Überleben von HNSCC-Patienten.

INTRODUCTION: The influence of smoking on survival in patients with HNSCC is well documented in the literature. There is little data on changes in smoking habits after diagnosis. Here, the effect on survival of the reduction of smoking compared to full smoking cessation is investigated. PATIENTS AND METHODS: Patient records and tumor documentation of 643 consecutive HNSCC cases of the Head and Neck Tumor Center of the University Hospital Kiel are evaluated retrospectively: smoking habits before and after treatment and survival are evaluated. RESULTS: Change in smoking behavior at the initial diagnosis of HNSCC leads to a significant positive effect on the prognosis compared to continued smoking. There is no difference between smoke reduction and weaning. This effect is based solely on those patients who are treated exclusively by surgery. Lifelong non-smokers have a significant survival advantage over active and ex-smokers, with no difference between the latter two groups. CONCLUSIONS: The positive influence of changed smoking habits on the prognosis runs parallel to the negative direct effect of active smoking on therapy, which is attributed to peritumoral hypoxia with a negative effect on the effectiveness of R(C)T. The positive effect of the change in smoking behaviour during surgery alone is most likely due to reduced peri-operative complications. Patients should be encouraged to at least minimize smoking with the cancer diagnosis. In addition, former smokers should be considered active smokers for survival estimates and therapy planning.

[Smoking and co-morbidity - it's impact on dose achievement in radio(chemo)therapy for HNSCC]. / Rauchen und Komorbidität ohne Einfluss auf geplante Zieldosis der Radio(chemo)therapie.

Smoking worsens the prognosis of patients with HNSCC. Furthermore, smoking is associated with the prevalence of co- and multimorbidity, so that it is assumed that not smoking per se, but co-/multimorbidity worsens the prognosis due to lack of compliance to therapy, e. g. by reducing the dose of the planned radio(chemo)therapy (RCT). However, data on this topic are currently sparse and contradictory, especially for HNSCC.Patient records and tumor documentation of 643 consecutive cases of the Head and Neck Tumor Center of the University Hospital Kiel were retrospectively evaluated. Patient characteristics and smoking habits were recorded and correlated with co-/multimorbidity and treatment course.The 643 patient files examined show that 113 (17.6 %) patients did not smoke, 349 (54.3 %) were active and 180 (28 %) patients had previously smoked. 315 (49 %) are treated exclusively by surgery; 121 (18.8 %) by surgery + adjuvant RCT and 72 (11.2 %) by surgery + adjuvant RT. 111 (17.3 %) receive a primary RCT and 24 (3.7 %) a primary RT. 131 (20.4 %) show co-/multimorbidity and 512 (79.6 %) do not. Smoking (> 10 py) is significantly associated with comorbidity (p = 0.002). However, smoking and comorbidity, neither alone nor in combination, are correlated with the achievement of the target dose of RCT (p > 0.05).As expected, smoking is significantly linked to co-/multimorbidity. Dose reduction of R(C)T is just as frequent in active smokers and patients with co-/multimorbidity as in non-smokers and patients without co-/multimorbidity. Thus, smoking and co-/multimorbidity influence the prognosis in other ways than by interfering with planned therapy regimens.

The interaction of smoking habit, SLPI and AnxA2 in HPV associated head and neck and other cancers.

Six own studies confirm a correlation between smoking, expression of the secretory leukocyte protease inhibitor (SLPI, an antileukoproteinase) and expression of Annexin A2 (AnxA2), and their influence on human papilloma virus (HPV)-infections. SLPI and HPV are ligands of AnxA2. This correlation was tested on 928 tissue samples from 892 patients in six independent studies [squamous cell carcinoma of the head and neck (HNSCC), n = 522; non-neoplastic tonsils n = 214; clinically normal mucosa, n = 93 (of these n = 57 were obtained from patients treated for non-malignant diseases and n = 36 were obtained from HNSCC-patients) and vulvar squamous cell carcinoma (VSCC) n = 99]. HPV-DNA-status was determined by GP5+/GP6+-PCR, followed in case of HPV-positivity by Sanger sequencing and RT-PCR using HPV-type specific primers. SLPI- and AnxA2-gene-expression was determined by RT-q-PCR; SLPI-protein-expression was additionally determined by immunohistochemistry (IHC); the data were correlated with each other and with patient characteristics. Smoking results in increased SLPI-gene- and protein- and AnxA2-gene-expression with significantly higher SLPI- than AnxA2-gene-expression. SLPI is decreased in non-smokers with a continuous AnxA2-surplus. HPV-status correlates with smoking habit, with smokers being mostly HPV-negative and non-smokers HPV-positive. We hypothesize that smoking leads to SLPI-overexpression with SLPI-binding to AnxA2. Thus, HPV cannot bind to AnxA2 but this seems pivotal for HPV-cell-entry. Smoking favors SLPI-expression resulting in HPV-negative carcinomas, while HPV-positive carcinomas are more common in non-smokers possibly due to a surplus of unbound AnxA2. In addition, the hypothesis may contribute to understand why smokers show increased oral HPV-prevalence in natural history studies but do not necessarily develop HPV-associated lesions.

HPV DNA/RNA detection in various oral and oropharyngeal biomaterials identifies active HPV infections also in non-neoplastic tonsils.

Previous studies describe a correlation between HPV-positivity and non-smoking in TSCC; p16INK4A-expression as surrogate-marker for HPV-DNA/RNA-positivity is discussed controversially. In the present study, these parameters are assessed prospectively. HPV-status of sputum and tonsillar-swabs was analyzed to determine their validity as surrogate-marker for tissue-HPV-status. TSCC- (n = 52) and non-neoplastic tonsillar tissue (n = 163) were analyzed. HPV-DNA- and HPV-RNA-status of total sputum, cellular fraction and supernatants, tonsillar-swabs and -tissue was determined by (RT)-PCR. Immunohistochemistry determined p16INK4A-expression. 23/163 (14.2%) non-neoplastic tonsils were HPV-DNA-positive; five patients (3 HPV16, 2 HPV11) had active HPV-infections (HPV-RNA-positive), in all biomaterials. 140/163 (85.9%) patients were either HPV-DNA-positive or HPV-DNA-negative in all samples. 21/52 (40.4%) TSCC-tonsils were HPV-DNA-positive; 17 patients were HPV-RNA-positive (14 HPV16; 4 HPV18). 40/52 (76.9%) TSCC-patients were congruent in all biomaterials. p16INK4A-expression alone would have misclassified the HPV-status of 14/52 (26.2%) TSCC-patients. This prospective study confirms the discrepancy between HPV-status and p16INK4A-expression and the significant correlation between non-smoking and HPV-DNA-positivity. HPV-sputum- and/or swab-results do not consistently match tissue-results, possibly having (detrimental) consequences if those were used to assess tissue-HPV-status. In the 5 patients with active HPV infection in the non-neoplasitic tonsils, tonsillectomy likely prevented subsequent development of TSCC.

Alteration of smoking habit at time of first diagnosis influences survival of patients with HNSCC.

The impact of smoking on survival in patients with squamous cell carcinoma of the head and neck is well established, despite some conflicting data in the literature. However, data on alterations of smoking habit following cancer diagnosis is sparse. In the present study, the effect of reduction of smoking compared with cessation on the course of disease was studied. Data from 643 patients with HNSCC from the tumor documentation registry of the Department of Otorhinolaryngology, Head and Neck Surgery of the Christian-Albrechts-University Kiel were collected and statistically analyzed, looking at pre- and post-treatment smoking habit and survival. Alteration of smoking at the first diagnosis of HNSCC led to a significantly beneficial effect on survival outcomes compared with continued smoking, without significant differences between reduction and cessation of smoking. Detailed analysis revealed that this effect was solely dependent on patients treated by surgery only. Lifelong non-smokers exhibited a significant survival advantage compared with active and former smokers, with no difference in survival between these last two groups. The positive influence of altered smoking habit following first time diagnosis on disease-specific survival paralleled the negative direct effect of active smoking on therapy, which is predominantly attributed to peritumoral tissue hypoxia leading to impaired efficacy of radiochemotherapy (RCT). In the present study cohort, the positive effect of smoking habit alterations were primarily observed in patients treated by surgery only instead of RCT, possibly due to fewer perioperative complications. These findings indicated that patients should be encouraged to at least minimize smoking following cancer diagnosis. Furthermore, for survival estimates and therapy planning, former smokers should be considered as active smokers.

No association between HPV-status in tonsillar tissue and sexual behavior of the patients in a northern German population - Critical view of the link between HPV natural history and HPV-driven carcinogenesis.

HPV-infection in patients with HNSCC is reportedly correlated with sexual behavior, age, and tobacco/alcohol-consumption. HPV-infections of the oral cavity are regarded as sexually transmitted. Comparable data of patient populations outside the U.S. are sparse or missing. Questionnaires regarding sexual behavior, education tobacco- and alcohol-consumption, were given to 28 patients with tonsillar hyperplasia (H) and 128 patients with tonsillar carcinomas (CA), all with tissue-typed HPV-DNA-status performing PCR. Answers were correlated among groups and HPV-status. 106 questionnaires were analyzed. Comparisons between H- (n = 25) and CA- (n = 81) patients showed that CA-patients were older (61.1yrs ± 9.3) than H-patients (45.2yrs ± 11.9; p < 0.0001; Student's t-test); had a lower educational level (p = 0.0095); and lower number of sexual partners (p = 0.0222; Fisher's exact test). All groups showed a significant correlation between smoking and lack of HPV-DNA-positivity (p = 0.001). Further Fisher's exact tests and logistic regression analysis revealed in all 106 patients no significant correlations between tissue-HPV-status and the analyzed parameters. Despite the limited sample size, we were able to confirm the established correlation between smoking and tissue-HPV-status. The correlation between sexual behavior and HPV-infection was not confirmed. No consensus exists in the literature about the latter. Our data does not support the strict classification of oral HPV-infections and HPV-driven HNSCCs as STDs.

The Influence of Smoking and Co-morbidity on Dose Achievement in Primary or Adjuvant Radio(Chemo)Therapy in Head and Neck Squamous Cell Carcinoma (HNSCC).

Introduction: Smoking has a negative impact on survival of HNSCC patients. In addition, smoking is associated with the prevalence of co-morbidities and, thus, it may be assumed that not smoking per se but co-morbidities impact the course of therapy in terms of lower compliance and dose-reduction. However, data addressing this issue is sparse and conflicting at present, specifically for HNSCCs. Patients and methods: Patient files and tumor documentation from 643 consecutive cases of the University Head and Neck Cancer Centre Kiel were analyzed retrospectively. Patient characteristics and smoking habits were assessed and correlated with co-morbidities and course of treatment. Results: The examined 643 patient files showed that 113 (17.6%), 349 (54.3%), and 180 (28%) patients were never, active, and former smokers, respectively. Three hundred fifteen (49%) were treated by surgery only; 121 (18.8%) received surgery + adjuvant RCT and 72 (11.2%) surgery + adjuvant RT. 111 (17.3%) received primary RCT and 24 (3.7%) primary RT. 131 (20.4%) and 512 (79.6%) had no or had co-morbidities, respectively. Smoking (>10 py) was significantly associated with co-morbidities (p = 0.002). However, smoking and co-morbidities, neither alone nor in combination, were correlated with failure in reaching target doses of radio(chemo)therapy (p > 0.05). Applying (verified) Carlson-Comorbidity-Index (CCI) did not change the results. Conclusions: As expected, smoking is significantly associated with co-morbidities. Dose-reduction of radio(chemo)therapy is as common among active smokers and patients with co-morbidities as among never smokers and patients without co-morbidities. Thus, smoking and co-morbidity seems to impact survival by other means than impairing planned therapy regimens.

Histone Deacetylase Inhibitor Modulates NKG2D Receptor Expression and Memory Phenotype of Human Gamma/Delta T Cells Upon Interaction With Tumor Cells.

The functional plasticity and anti-tumor potential of human γδ T cells have been widely studied. However, the epigenetic regulation of γδ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γδ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γδ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in Vδ2 T cells. The detailed analysis of H3K9aclow Vδ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γδ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γδ T-cell-based immunotherapy for the treatment of certain types of cancer.

Changes in proinflammatory gene expression in human whole blood after contact with UV-conditioned implant surfaces.

OBJECTIVES: The aim of this in vitro study was to assess changes in the gene expression of proinflammatory cytokines in human whole blood after contact with titanium implant surfaces conditioned by UV light. To this end, expression levels of proinflammatory cytokines were analyzed in vitro in human whole blood. MATERIALS AND METHODS: Dental implants made of grade 4 titanium were conditioned by UV light in a UV device and submerged in human whole blood. Unconditioned implants served as controls, and blood samples without implants served as the negative control group. Sampling was performed at 1, 8, and 24 h. Changes in the expression levels of interleukin-1ß (IL1B) and tumor necrosis factor alpha (TNF) were assessed using RT-qPCR at the mRNA level. RESULTS: The gene expression of IL1B was significantly suppressed in the test group over the observation period compared to the control group during the 1-8 h after having contact between the implant surface and the blood. The gene expression of TNF was not significantly altered by UV conditioning after 1 and 8 h of observation, but both cytokine expression levels were increased significantly after 24 h. CONCLUSIONS: Differences in the gene expression of proinflammatory cytokines after insertion of UV-conditioned titanium implants can be assessed using a human whole blood test. UV-conditioned implant surfaces apparently suppress the release of IL1B in vitro. CLINICAL RELEVANCE: The results of our publication demonstrate that modulation of the early inflammatory response in human whole blood is possible by surface treatment with UV light. In particular, the suppression of IL1B expression, especially after the initial contact of blood cells, may be beneficial in the osseointegration of titanium implants by positively influence the balance between rejection and acceptance of an implant.

Influence of HPV-status on survival of patients with tonsillar carcinomas (TSCC) treated by CO2-laser surgery plus risk adapted therapy - A 10 year retrospective single centre study.

The positive prognostic value of HPV-infections in oropharyngeal squamous cell cancer (OSCC) patients has led to the initiation of prospective clinical trials testing the value of treatment de-escalation. It is unclear how to define patients potentially benefiting from de-escalated treatment, whether a positive smoking history impacts survival data and what kind of de-escalation might be best. Here, we investigate the effect of HPV-status, smoking habit and treatment design on overall survival (OS) and progression free survival (PFS) of 126 patients with tonsillar SCC (TSCC) who underwent CO2-laser-surgery and risk adapted adjuvant treatment. HPV-DNA-, HPV-mRNA-, and p16INK4A-expression were analysed and results were correlated to OS and PFS. Factors tested for prognostic value included HPV-status, p16INK4A-protein expression, therapy and smoking habit. Log rank test and p-values ≤0.05 defined significant differences between groups. The highest accuracy of data with highest significance in this study is given when the HPV-RNA-status is considered. Using p16INK4A-expression alone or in combination with HPV-DNA-status, would have misclassified 23 and 7 patients, respectively. Smoking fully abrogates the positive impact of HPV-infection in TSCC on survival. Non-smoking HPV-positive TSCC patients show 10-year OS of 100% and 90.9% PFS when treated with adjuvant RCT. The presented data show that high-precision HPV-detection methods are needed, specifically when treatment decisions are based on the results. Furthermore, smoking habit should be included in all studies and clinical trials testing HPV-associated survival. Adjuvant RCT especially for HPV-positive non-smokers may help to avoid distant failure.

miRNA-expression in tonsillar squamous cell carcinomas in relation to HPV infection and expression of the antileukoproteinase SLPI.

The aim of this study was to determine if micro-(mi-)RNAs are involved in the previously reported inverse correlation between the antileukoproteinase SLPI, HPV, and smoking habit of head and neck squamous cells carcinoma (HNSCC) patients. HPV-status and SLPI-protein expression were determined in tonsillar SCC (TSCC; n=126). Differentially expressed miRNAs dependent on HPV-status and SLPI-expression were detected by microarray; possible binding-sites in SLPI- and HPVE6-mRNAs were determined in silico. Survival rates were estimated testing prognostic values of HPV-status, SLPI- and miRNA-expression. miRNA-array identified 24 up-regulated and 10 down-regulated miRNAs in HPV-positive versus HPV-negative TSCC (p<0.01; HPV-positivity: 42.1%). HPV-positivity resulted in two up-regulated miRNAs in SLPI-positive TSCC. Of 16 further miRNAs, eight miRNAs were up- and eight were down-regulated in SLPI-negative TSCC. RT-q-PCR-validation of the four most differentially expressed miRNAs showed that miR-363 is expressed strongest in SLPI-negative/HPV-positive TSSC. In silico-analysis of all differentially expressed miRNAs identified miR-363, miR-210, miR-130a, and miR-181a with possible binding sites in the HPV16-E6-mRNA, but none were predicted in the SLPI-mRNA. HPV-positivity, low SLPI-levels and high miR-363-levels are significantly associated with better survival rates. The data presented here show that miR-363 is associated with HPV-positive/SPLI-negative TSCC. The prognostic value of miR-363 suggests a role in the assumed inverse correlation of smoking and SPLI-expression in the mode of HPV-infections in tonsillar but possibly also other HNSCC.

In-depth immunophenotyping of patients with glioblastoma multiforme: Impact of steroid treatment.

Despite aggressive treatment regimens based on surgery and radiochemotherapy, the prognosis of patients with grade IV glioblastoma multiforme (GBM) remains extremely poor, calling for alternative options such as immunotherapy. Immunological mechanisms including the Natural Killer Group 2 member D (NKG2D) receptor-ligand system play an important role in tumor immune surveillance and targeting the NKG2D system might be beneficial. However, before considering any kind of immunotherapy, a precise characterization of the immune system is important, particularly in GBM patients where conventional therapies with impact on the immune system are frequently co-administered. Here we performed an in-depth immunophenotyping of GBM patients and age-matched healthy controls and analyzed NKG2D ligand expression on primary GBM cells ex vivo. We report that GBM patients have a compromised innate immune system irrespective of steroid (dexamethasone) medication. However, dexamethasone drastically reduced the number of immune cells in the blood of GBM patients. Moreover, higher counts of immune cells influenced by dexamethasone like CD45+ lymphocytes and non-Vδ2 γδ T cells were associated with better overall survival. Higher levels of NKG2D ligands on primary GBM tumor cells were observed in patients who received radiochemotherapy, pointing towards increased immunogenic potential of GBM cells following standard radiochemotherapy. This study sheds light on how steroids and radiochemotherapy affect immune cell parameters of GBM patients, a pre-requisite for the development of new therapeutic strategies targeting the immune system in these patients.

Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies.

Expression of non-secreted IL-4 is associated with HDAC inhibitor-induced cell death, histone acetylation and c-Jun regulation in human gamma/delta T-cells.

Previously, the expression of a non-secreted IL-4 variant (IL-4δ13) has been described in association with apoptosis and age-dependent Th2 T-cell polarization. Signaling pathways involved in this process have so far not been studied. Here we report the induction of IL-4δ13 expression in human γδ T-cells upon treatment with a sublethal dose of histone deacetylase (HDACi) inhibitor valproic acid (VPA). Induction of IL-4δ13 was associated with increased cytoplasmic IL-4Rα and decreased IL-4 expression, while mRNA for mature IL-4 was concomitantly down-regulated. Importantly, only the simultaneous combination of apoptosis and necroptosis inhibitors prevented IL-4δ13 expression and completely abrogated VPA-induced global histone H3K9 acetylation mark. Further, our work reveals a novel involvement of transcription factor c-Jun in the signaling network of IL-4, HDAC1, caspase-3 and mixed lineage kinase domain-like protein (MLKL). This study provides novel insights into the effects of epigenetic modulator VPA on human γδ T-cell differentiation.

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.