A constantly increasing incidence in high-risk Human Papillomaviruses (HPV)s driven head and neck squamous cell carcinomas (HNSCC)s, especially of oropharyngeal origin, is being observed. During persistent infections, viral DNA integration into the host genome may occur. Studies are examining if the physical status of the virus (episomal vs. integration) affects carcinogenesis and eventually has further-reaching consequences on disease progression and outcome. Here, we review the literature of the most recent five years focusing on the impact of HPV integration in HNSCCs, covering aspects of detection techniques used (from PCR up to NGS approaches), integration loci identified, and associations with genomic and clinical data. The consequences of HPV integration in the human genome, including the methylation status and deregulation of genes involved in cell signaling pathways, immune evasion, and response to therapy, are also summarized.

Head and neck squamous cell carcinoma (HNSCC) is characterized by a poor 5 year survival and varying response rates to both standard-of-care and new treatments. Despite advances in medicine and treatment methods, mortality rates have hardly decreased in recent decades. Reliable patient-derived tumor models offer the chance to predict therapy response in a personalized setting, thereby improving treatment efficacy by identifying the most appropriate treatment regimen for each patient. Furthermore, ex vivo tumor models enable testing of novel therapies before introduction in clinical practice. A literature search was performed to identify relevant literature describing three-dimensional ex vivo culture models of HNSCC to examine sensitivity to chemotherapy, radiotherapy, immunotherapy and targeted therapy. We provide a comprehensive overview of the currently used three-dimensional ex vivo culture models for HNSCC with their advantages and limitations, including culture success percentage and comparison to the original tumor. Furthermore, we evaluate the potential of these models to predict patient therapy response.

Human Papilloma Virus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) represents an OPSCC subgroup with an overall good prognosis with a rising incidence in Western countries. Multiple lines of evidence suggest that HPV-associated tumors are not a homogeneous tumor entity, underlining the need for accurate prognostic biomarkers. In this retrospective, multi-institutional study involving 906 patients from four centers and one database, we developed a deep learning algorithm (OPSCCnet), to analyze standard H&E stains for the calculation of a patient-level score associated with prognosis, comparing it to combined HPV-DNA and p16-status. When comparing OPSCCnet to HPV-status, the algorithm showed a good overall performance with a mean area under the receiver operator curve (AUROC) = 0.83 (95% CI = 0.77-0.9) for the test cohort (n = 639), which could be increased to AUROC = 0.88 by filtering cases using a fixed threshold on the variance of the probability of the HPV-positive class - a potential surrogate marker of HPV-heterogeneity. OPSCCnet could be used as a screening tool, outperforming gold standard HPV testing (OPSCCnet: five-year survival rate: 96% [95% CI = 90–100%]; HPV testing: five-year survival rate: 80% [95% CI = 71–90%]). This could be confirmed using a multivariate analysis of a three-tier threshold (OPSCCnet: high HR = 0.15 [95% CI = 0.05–0.44], intermediate HR = 0.58 [95% CI = 0.34–0.98] p = 0.043, Cox proportional hazards model, n = 211; HPV testing: HR = 0.29 [95% CI = 0.15–0.54] p < 0.001, Cox proportional hazards model, n = 211). Collectively, our findings indicate that by analyzing standard gigapixel hematoxylin and eosin (H&E) histological whole-slide images, OPSCCnet demonstrated superior performance over p16/HPV-DNA testing in various clinical scenarios, particularly in accurately stratifying these patients.

Cidofovir (CDV) is an antiviral agent with antiproliferative properties. The aim of our study was to investigate the efficacy of CDV in HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) cell lines and whether it is caused by a difference in response to DNA damage. Upon CDV treatment of HNSCC and normal oral keratinocyte cell lines, we carried out MTT analysis (cell viability), flow cytometry (cell cycle analysis), (immuno) fluorescence and western blotting (DNA double strand breaks, DNA damage response, apoptosis and mitotic catastrophe). The growth of the cell lines was inhibited by CDV treatment and resulted in γ-H2AX accumulation and upregulation of DNA repair proteins. CDV did not activate apoptosis but induced S- and G2/M phase arrest. Phospho-Aurora Kinase immunostaining showed a decrease in the amount of mitoses but an increase in aberrant mitoses suggesting mitotic catastrophe. In conclusion, CDV inhibits cell growth in HPV-positive and -negative HNSCC cell lines and was more profound in the HPV-positive cell lines. CDV treated cells show accumulation of DNA DSBs and DNA damage response activation, but apoptosis does not seem to occur. Rather our data indicate the occurrence of mitotic catastrophe.

Human papillomavirus (HPV)‑positive and -negative head and neck squamous cell carcinoma (HNSCC) are often associated with activation of the phosphatidylinositol 3‑kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway due to mutations or amplifications in PI3KCA, loss of PTEN or activation of receptor tyrosine kinases. In HPV‑negative tumors, CDKN2A (encoding p16 protein) inactivation or CCND1 (encoding Cyclin D1 protein) amplification frequently results in sustained cyclin‑dependent kinase (CDK) 4/6 activation. The present study aimed to investigate the efficacy of the CDK4/6 inhibitors (CDKi) palbociclib and ribociclib, and the PI3K/Akt/mTOR pathway inhibitors (PI3Ki) gedatolisib, buparlisib and alpelisib, in suppressing cell viability of HPV‑positive and ‑negative HNSCC cell lines. Inhibitor efficacy was assessed in vitro using MTT assay and western blotting analysis. Cell cycle analysis was performed using flow cytometry and apoptosis was assessed using annexin V staining. Metabolic changes in terms of glycolysis and oxidative metabolism were measured by Seahorse XF96 extracellular Flux analysis. The results of the present study showed that both HPV‑positive and ‑negative HNSCC cell lines were sensitive to PI3Ki. In general, PI3Ki decreased PI3K/Akt/mTOR pathway activity, resulting in apoptosis, and decreased oxidative and glycolytic metabolism. The CDKi were particularly effective in blocking HPV‑negative cell line viability, showing decreased retinoblastoma expression and G1‑phase cell cycle arrest, whereas apoptosis was not induced. Thus, PI3Ki and CDKi efficiently inhibited their respective pathways and HNSCC cell viability in vitro, with the latter occurring only in HPV‑negative cell lines. Whereas PI3Ki induced apoptosis and attenuated cellular metabolism, CDKi led to cell cycle arrest. Further research should be performed to elucidate whether (a combination of) these inhibitors may be effective therapeutic agents for patients with HNSCC.

Hypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1⁺ EV in the metastatic cascade. GABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O₂) or hypoxia (O₂ < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. The number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. GABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1⁺ EV are essential in other processes related to the metastatic cascade.