ATP synthase modulation leads to an increase of spare respiratory capacity in HPV associated cancers.

Mucosal and skin cancers are associated with infections by human papillomaviruses (HPV). The manner how viral oncoproteins hijack the host cell metabolism to meet their own energy demands and how this may contribute to tumorigenesis is poorly understood. We now show that the HPV oncoprotein E7 of HPV8, HPV11 and HPV16 directly interact with the beta subunit of the mitochondrial ATP-synthase (ATP5B), which may therefore represent a conserved feature across different HPV genera. By measuring both glycolytic and mitochondrial activity we observed that the association of E7 with ATP5B was accompanied by reduction of glycolytic activity. Interestingly, there was a drastic increase in spare mitochondrial respiratory capacity in HPV8-E7 and an even more profound increase in HPV16-E7 expressing cells. In addition, we could show that ATP5B levels were unchanged in betaHPV positive skin cancers. However, comparing HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) we noticed that, while ATP5B expression levels did not correlate with patient overall survival in HPV-negative OPSCC, there was a strong correlation within the HPV16-positive OPSCC patient group. These novel findings provide evidence that HPV targets the host cell energy metabolism important for viral life cycle and HPV-mediated tumorigenesis.

Impact on survival of tobacco smoking for cases with oropharyngeal squamous cell carcinoma and known human papillomavirus and p16-status: a multicenter retrospective study.

BACKGROUND: Human papilloma virus (HPV) and tobacco smoking are important risk factors for development of oropharyngeal squamous cell carcinoma (OPSCC). AIMS/OBJECTIVES: To evaluate the impact of tobacco smoking on survival for cases with OPSCC with known HPV- and p16INK4A(p16)-status. MATERIALS AND METHODS: OPSCC cases at the University Hospital of Copenhagen, Rigshospitalet, Denmark (2000-2014) and at University Hospital of Giessen, Germany (2000-2009) were included. Survival was illustrated with Kaplan-Meier plots. The effect of smoking exposure on survival was evaluated by Cox-regression models. HPV-positivity was defined as positivity for both HPV-DNA and p16. RESULTS: We included 1316 OPSCC cases from 2000-2014 (48% HPV-positive). Smokers had a poorer outcome compared to non-smokers. Considering continuous smoking exposure, adding 10 pack-years of smoking increased hazard ratios irrespective of HPV-status.We observed a tendency to a greater impact on survival for cases with HPV-neg. tumours compared to cases with HPV-pos. tumours at low numbers of pack-years, yet the survival was similar at high numbers of pack-years. There was no significant difference in the impact of HPV-status on survival for non-smokers, however a highly significant difference for smokers. CONCLUSIONS AND SIGNIFICANCE: Smoking-status and number of pack-years at time of diagnosis impact survival for cases with OPSCC independent of HPV-status.

Increasing Incidence rates of Oropharyngeal Squamous Cell Carcinoma in Germany and Significance of Disease Burden Attributed to Human Papillomavirus.

Increasing incidences of head and neck cancers and rising proportions of these associated with human papillomavirus (HPV), especially in the oropharynx, have been reported in international studies. So far, the trends and contribution of HPV to the number of newly diagnosed cases of oropharyngeal squamous cell carcinomas (OPSCC) in Germany are uncertain. We investigated HPV association and incidence rates in a cohort of consecutively included patients with OPSCC in Giessen 2000-2017, and compared our results with regional (Giessen and the federal state of Hesse), national (Germany), and international (United States) databases. Regional data show a significant increase in the overall incidence rates of oropharyngeal cancers and in the incidence of HPV-associated cancers of the subsites tonsils and oropharynx, whereas other oropharyngeal subsites show no significant change. Analysis of national databases shows a significant incidence increase in Germany and in the United States. The rise in incidence is predominantly attributable to male patients in the US population, whereas in Germany rising OPSCC incidence is more associated with females. There is a significant elevation of OPSCC incidence rates in Germany, which corresponds to the recognized incidence increase of HPV-related oropharyngeal cancers based on experimental data from consecutively included patients of our cohort. Our investigation shows different patterns of this increase in Germany and in the United States, which demonstrates spatial heterogeneity and the need for population-based investigations regarding the role of HPV in oropharyngeal cancer.

[HPV - A different view on Head and Neck Cancer]. / HPV ­ Das andere Kopf-Hals-Karzinom.

Head and neck cancer is the sixth most common cancer with over 500000 annually reported incident cases worldwide. Besides major risk factors tobacco and alcohol, oropharyngeal squamous cell carcinomas (OSCC) show increased association with human papillomavirus (HPV). HPV-associated and HPV-negative OSCC are 2 different entities regarding biological characteristics, therapeutic response, and patient prognosis. In HPV OSCC, viral oncoprotein activity, as well as genetic (mutations and chromosomal aberrations) and epigenetic alterations plays a key role during carcinogenesis. Based on improved treatment response, the introduction of therapy de-intensification and targeted therapy is discussed for patients with HPV OSCC. A promising targeted therapy concept is immunotherapy. The use of checkpoint inhibitors (e.g. anti-PD1) is currently investigated. By means of liquid biopsies, biomarkers such as viral DNA or tumor mutations in the will soon be available for disease monitoring, as well as detection of treatment failure. By now, primary prophylaxis of HPV OSCC can be achieved by vaccination of girls and boys.

Development and external validation of nomograms in oropharyngeal cancer patients with known HPV-DNA status: a European Multicentre Study (OroGrams).

BACKGROUND: The proxy marker for human papillomavirus (HPV), p16, is included in the new AJCC 8th/UICC 8th staging system, but due to incongruence between p16 status and HPV infection, single biomarker evaluation could lead to misallocation of patients. We established nomograms for overall survival (OS) and progression-free survival (PFS) in patients with oropharyngeal squamous cell carcinoma (OPSCC) and known HPV-DNA and p16 status, and validated the models in cohorts from high- and low-prevalent HPV countries. METHODS: Consecutive OPSCC patients treated in Denmark, 2000-2014 formed the development cohort. The validation cohorts were from Sweden, Germany, and the United Kingdom. We developed nomograms by applying a backward-selection procedure for selection of variables, and assessed model performance. RESULTS: In the development cohort, 1313 patients, and in the validation cohorts, 344 German, 503 Swedish and 463 British patients were included. For the OS nomogram, age, gender, combined HPV-DNA and p16 status, smoking, T-, N-, and M-status and UICC-8 staging were selected, and for the PFS nomogram the same variables except UICC-8 staging. The nomograms performed well in discrimination and calibration. CONCLUSIONS: Our nomograms are reliable prognostic methods in patients with OPSCC. Combining HPV DNA and p16 is essential for correct prognostication. The nomograms are available at www.orograms.org .

Hypoxia-inducible factor-1α activation in HPV-positive head and neck squamous cell carcinoma cell lines.

PURPOSE: Human papillomavirus (HPV) is a causative agent for a rising number of head and neck squamous cell carcinomas (HNSCC), which are characterized by distinct tumor biology. Hypoxia inducible-factor (HIF) signaling influences initiation and progression of carcinogenesis and HPV oncoproteins have evolved to highjack cellular pathways for viral reproduction. Therefore, we investigated whether HPV activates HIF-1α expression in HNSCC. EXPERIMENTAL TECHNIQUE: HPV-positive and -negative HNSCC cells were examined for adaptive responses to hypoxia. Expression of HIF-1α, prolyl hydroxylase-domain protein 2 (PHD2) and E-cadherin was analyzed by Western blotting, immunofluorescence (IF) microscopy and migration/wound healing assays. RESULTS: HPV-positive HNSCC cells showed higher HIF-1α and PHD2 protein levels under normoxia and hypoxia. HIF-1α hydroxylation was reduced in HPV-positive HNSCC cell lines under PHD and proteasomal inhibition. In vitro wound healing assays showed impairment of migration and proliferation by HIF-1α pathway activation in HPV-negative cell lines only. In contrast, migration and proliferation in HPV-positive cell lines was impaired by HIF-1α specific siRNA. CONCLUSIONS: HPV-positive HNSCC cells show activation of the HIF pathway and adaptation to HIF-1α upregulation, representing potential therapeutic targets in this emerging tumor entity.

Prognostic Impact of AJCC/UICC 8th Edition New Staging Rules in Oropharyngeal Squamous Cell Carcinoma.

INTRODUCTION: The purpose of this study was to test whether the 8th edition of the AJCC/UICC TNM staging system (UICC) precisely differentiates between stages and reflects disease outcome in human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). PATIENTS AND METHODS: OPSCC patients that were diagnosed between 2000 and 2016 were included in this analysis and HPV status was determined by combined DNA and p16 testing. Stratification was done according to 7th and 8th UICC staging rules. Incidence trends of HPV-associated tumorigenesis, 5-year overall survival (OS) according to tumor stages as well as the influence of therapy and prognostic factors toward the outcome were calculated using Kaplan-Meier method and Cox proportional-hazards model. RESULTS: A significant increase [2000; n = 8/39 (21%)-2015; n = 17/32 (53%); p = 0.002] in HPV-associated OPSCC was seen in the observation period. Together, 150/599 (25.0%) of the patients had HPV-driven OPSCC and 64.7% of curative treatments in all OPSCC patients included upfront surgery of the primary and the neck. 7th edition staging rules led to no discrimination in all respective four UICC stages in HPV OPSCC underlining the need for new staging rules. However, only discrimination between stages I vs. II and III vs. IV was significant in our patients with HPV-OPSCC (94.4 vs. 77.5%; p = 0.031 and 63.9 vs. 25.0%; p = 0.013), and stages II vs. III did not differ in OS rates (p = 0.257), when applying the new staging rules. For HPV-negative OPSCC, significant outcome differences were only seen between UICC stages III vs. IV (57.6 vs. 35.2%; p = 0.012). DISCUSSION: While the 7th edition of UICC shows invalid discrimination between stages, the 8th edition is more suitable for HPV-associated carcinoma. Due to lack of differentiation between stages II and III further adaption is essential.

Inhibition of Low-Grade Inflammation by Anthocyanins after Microbial Fermentation in Vitro.

The anti-inflammatory effects of anthocyanins (ACNs) on vascular functions are discussed controversially because of their low bioavailability. This study was performed to determine whether microorganism (MO)-fermented ACNs influence vascular inflammation in vitro. Therefore, MO growth media were supplemented with an ACN-rich grape/berry extract and growth responses of Escherichia coli, E. faecalis and H. alvei, as well as ACN fermentation were observed. MO supernatants were used for measuring the anti-inflammatory effect of MO-fermented ACNs in an epithelial-endothelial co-culture transwell system. After basolateral enrichment (240 min), endothelial cells were stimulated immediately or after 20 h with TNF-α. Afterwards, leukocyte adhesion, expression of adhesion molecules and cytokine release were measured. Results indicate that E. coli, E. faecalis and H. alvei utilized ACNs differentially concomitant with different anti-inflammatory effects. Whereas E. coli utilized ACNs completely, no anti-inflammatory effects of fermented ACNs were observed on activated endothelial cells. In contrast, ACN metabolites generated by E. faecalis and H. alvei significantly attenuated low-grade stimulated leukocyte adhesion, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 and cytokine secretion (IL-8 and IL-6), as well as NF-κB mRNA expression with a more pronounced effect of E. faecalis than H. alvei. Thus, MO-fermented ACNs have the potential to reduce inflammation.

CD56-positive lymphocyte infiltration in relation to human papillomavirus association and prognostic significance in oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx (OSCC) are clinical and biological distinct from their HPV-unrelated counterparts. Patients with HPV-related OSCC display improved prognosis and therefore we investigated possible immune cell infiltrations associated with this tumor phenotype. We retrospectively analyzed a randomly selected cohort of 140 OSCC for presence of immune cells and HPV by immunohistochemistry and PCR followed by bead-based hybridization (Luminex technology). HPV prevalence was 24.3% as determined by positive staining of p16INK4a and detection of high risk HPV-DNA. We found significantly higher numbers of CD56 positive (CD56+) cells in tumor and surrounding microenvironment in HPV-associated compared to HPV-negative OSCC (t-test: p = 0.004 and p = 0.002). For the entire cohort presence of CD56+ cells was associated with increased overall survival independent from HPV (Kaplan-Meier: p = 0.002; Cox regression: p = 0.042). Presence of CD56+ cells also correlated with a better outcome in HPV-negative and especially in HPV-negative OSCC with alcohol consumption ≤ 2 standard drinks per day (Kaplan-Meier: p = 0.05 and p = 0.003). Immunofluorescence localization of granular Granzyme B (GZMB) within CD56+ cells and coexpression of CD16 and CD56 suggests that detected CD56+ cells mainly represent cytotoxic Natural Killer (NK) cells. The fraction of potentially cytotoxic NK cells was significantly higher in HPV-associated compared to HPV-negative OSCC (Mann-Whitney-U-Test: p = 0.011). The elevated abundance and activity of cytotoxic NK cells in OSCC with HPV driven carcinogenesis might contribute to favorable outcome in HPV-related OSCC.