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.

[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.

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.

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.

Systemic immunity shapes the oral microbiome and susceptibility to bisphosphonate-associated osteonecrosis of the jaw.

BACKGROUND: Osteonecrosis of the jaw (ONJ) is a rare but serious adverse drug effect linked to long-term and/or high-dose exposure to nitrogen-bisphosphonates (N-BP), the standard of care for the treatment of bone fragility disorders. The mechanism leading to bisphosphonate-associated ONJ (BAONJ) is unclear and optimal treatment strategies are lacking. Recent evidence suggests that BAONJ may be linked to drug-induced immune dysfunction, possibly associated with increased susceptibility to infections in the oral cavity. The objective of this investigation was to comprehensively assess the relationship linking immune function, N-BP exposure, the oral microbiome and ONJ susceptibility. METHODS: Leukocyte gene expression of factors important for immunity, wound healing and barrier function were assessed by real-time quantitative PCR and the oral microbiome was characterized by 454 pyrosequencing of the 16S rRNA gene in 93 subjects stratified by N-BP exposure and a history of ONJ. RESULTS: There were marked differences in the systemic expression of genes regulating immune and barrier functions including RANK (p = 0.007), aryl hydrocarbon receptor (AHR, p < 0.001), and FGF9 (p < 0.001), which were collectively up-regulated in individuals exposed to N-BP without ONJ relative to treatment controls. In contrast, the expression levels of these same genes were significantly down-regulated in those who had experienced BAONJ. Surprisingly, the oral microbiome composition was not directly linked to either BAONJ or N-BP exposure, rather the systemic leukocyte expression levels of RANK, TNFA and AHR each explained 9% (p = 0.04), 12% (p = 0.01), and 7% (p = 0.03) of the oral bacterial beta diversity. CONCLUSIONS: The oral microbiome is unlikely causative of ONJ, rather individuals with BAONJ lacked immune resiliency which impaired their capacity to respond adequately to the immunological stress of N-BP treatment. This may be the common factor linking N-BP and anti-RANK agents to ONJ in at-risk individuals. Preventive and/or therapeutic strategies should target the wound healing deficits present in those with ONJ.

Characterisation of seven newly established head and neck squamous cell carcinoma cell lines.

Seven squamous cell carcinoma cell lines were disintegrated from biopsies of patients with head and neck cancer. Genotyping tests verified the authenticity and the human origin of all seven lines. The cell lines designated as University of Kiel, Head and Neck (UKHN) -1 to -3 and UKHN-6 to -9 were subjected to flow cytometry and indirect immunofluorescence to assess aberrant DNA content. To confirm the squamous epithelial origin of the cells, the cytokeratin profile was immunocytologically analysed. The cell lines showed individual differences in mitotic frequency. UKHN-1, -6, -7 and -9 grew as monolayers, whereas UKHN-2, -3, and -8 tend to multilayer stratification. Overexpression of LOXL4 and Pim-1 proteins as distinctive features of head and neck carcinomas were shown in all seven cell lines. Inoculating SCID mice with these cell lines resulted in tumour formation, hence corroborating the tumourigenicity of all seven cell lines. The cell lines were also tested for high-risk HPV types using different DNA-based assays and found to be negative.

The role of the antileukoprotease SLPI in smoking-induced human papillomavirus-independent head and neck squamous cell carcinomas.

Recently, we showed that increased SLPI levels prevent human papillomavirus (HPV) infections and metastasis in smoking-induced, non-HPV-driven head and neck squamous cell carcinoma (HNSCC). Here, we focus on the role of SLPI in non-HPV-driven HNSCC, investigating tumor tissue and non-neoplastic mucosa from the same patients and from non-HNSCC patients. Gene and protein expression of SLPI and gene expression of annexin 2 (a SLPI receptor), nicotine receptor (α7AChR) and arylhydrocarbon receptor (AhR) were analyzed in HNSCC patients (20 smokers; 16 nonsmokers). SLPI-results were correlated with the patients' HPV status. Non-neoplastic mucosa of HNSCC patients and normal mucosa from non-HNSCC individuals (18 smokers; 20 nonsmokers) was analyzed for the same parameters. Tissue of the inferior turbinate (n = 10) was incubated with nicotine for analysis of the same genes. SLPI gene expression in tumor tissue was 109.26 ± 23.08 times higher in smokers versus nonsmokers. Non-neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49 ± 1.89-fold non-HNSCC; 18.02 ± 3.93-fold HNSCC patients). Annexin 2 gene expression was also increased in smokers. SLPI data were corroborated by immunohistochemistry. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r(2) = 0.15, p < 0.001) was shown ex vivo. Nicotine and smoking increased α7AChR and AhR gene expression. Five patients, showing no/low SLPI expression, were HPV16-positive. A significant correlation between smoking and SLPI expression in tumors and to our knowledge for the first time in mucosa of HNSCC and non-HNSCC patients was established. Together with the finding that all patients with HPV infection showed no/low SLPI expression, these data support our intriguing hypothesis that smoking induced upregulated SLPI prevents HPV infections.

Vδ2 T cell deficiency in granulomatosis with polyangiitis (Wegener's granulomatosis).

Previous studies have characterized phenotypic and functional alterations within T-cell receptor αß-expressing T cells in patients with granulomatosis with polyangiitis (GPA). We analyzed the frequency, subset composition and in vitro activation of blood γδ T cells in GPA patients. We observed a significant reduction of γδ T cell numbers, due to the selective depletion of the Vδ2 subset which remained consistent over time upon repeated analysis. The loss of Vδ2 T cells was not due to migration into the inflamed lesions as very few γδ T cells were detected in inflammatory infiltrates. The memory subset distribution did not differ among Vδ2 T cells from healthy donors and GPA patients. Importantly, the remaining Vδ2 T cells were capable of responding to phosphoantigen stimulation in vitro. The marked depletion of blood Vδ2 T cells in GPA suggests cellular exhaustion, possibly due to chronic exposure to and continuous overstimulation by microbial phosphoantigens.

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.

Assessment of lipopolysaccharide microleakage at conical implant-abutment connections.

OBJECTIVE: The aim of this in vitro study was to assess lipopolysaccharide microleakage at conical implant-abutment connections of two-piece dental implants in terms of the expression levels of genes involved in lipopolysaccharide-mediated proinflammatory cytokine production. MATERIALS AND METHODS: Two implant systems with conical implant-abutment connections were inoculated with lipopolysaccharide and submerged in human whole blood. Positive-control blood samples (without implants) were stimulated with 4 µg/ml, 2 µg/ml, 200 ng/ml, and 20 ng/ml lipopolysaccharide. Sampling was performed after 1, 8, and 24 h of incubation. Changes of gene expression levels of Toll-like receptor 9, tumor necrosis factor-α, nuclear factor kappa light chain enhancer of activated B cells, interleukin-1ß, and interferon-γ were assessed by real-time quantitative PCR. In addition, protein expression levels of interleukin-6, tumor necrosis factor-α, interleukin-1ß, and interferon-γ were determined by immunoassay. RESULTS: Changes in cytokine expression at the genomic and proteomic levels indicated lipopolysaccharide leakage at the interfaces of both tested implant systems, although some implants showed no sign of microleakage. Any tested concentration of lipopolysaccharide stimulated similar gene expression. CONCLUSIONS: Conical implant-abutment connections of two-piece dental implants do not prevent microleakage on a molecular level. Changes in lipopolysaccharide-induced proinflammatory cytokine gene expression facilitate the detection of lipopolysaccharide microleakage at implant-abutment interfaces. CLINICAL RELEVANCE: Small amounts of lipopolysaccharide released from intra-implant cavities can stimulate a detectable immunological response in human whole blood and may induce alveolar bone resorption via the osteoclast-activating pathway.

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.

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.

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 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.