Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics.

Molecular diagnostics is an increasingly important clinical tool, especially in routine sampling. We evaluated two non-invasive methods (oral swabs and mouthwashes) for sampling nucleic acids from the oral/pharyngeal area. We created a workflow from sample collection (n = 59) to RT-qPCR based analysis. The samples were further characterized in terms of their cellular composition as well as the purity, degradation and microbial content of the derived DNA/RNA. We determined the optimal housekeeping genes applicable for these types of samples. The cellular composition indicated that mouthwashes contained more immune cells and bacteria. Even though the protocol was not specifically optimized to extract bacterial RNA it was possible to derive microbial RNA, from both sampling methods. Optimizing the protocol allowed us to generate stable quantities of DNA/RNA. DNA/RNA purity parameters were not significantly different between the two sampling methods. Even though integrity analysis demonstrated a high level of degradation of RNA, corresponding parameters confirmed their sequencing potential. RT-qPCR analysis determined TATA-Box Binding Protein as the most favorable housekeeping gene. In summary, we have developed a robust method suitable for multiple downstream diagnostic techniques. This protocol can be used as a foundation for further research endeavors focusing on developing molecular diagnostics for the oropharyngeal cavity.

Inflammatory phenotypes may be more important than age and comorbidities in predicting clinical outcomes in hospitalised patients with COVID-19.

Objectives: In critically ill patients with COVID-19, distinct hyperinflammatory and hypoinflammatory phenotypes have been described, with different outcomes and responses to therapy. We investigated if similar phenotypes exist in non-severe illness. Methods: Consecutive patients with polymerase chain reaction (PCR) confirmed SARS-CoV-2 were examined. Baseline demographics and laboratory investigations were tabulated, including serum C-reactive protein. Patients were divided into those who were hyperinflammatory (defined as C-reactive protein >17 mg/l) or hypoinflammatory. Adverse outcomes, defined as requiring oxygenation, intensive care, or death, were recorded during the hospital stay. Clinical characteristics and outcomes were compared. Results: Of the 1781 patients examined, 276 (15.5%) had a hyperinflammatory phenotype. They were older (51.8 ± 17.2 vs 40.3 ± 13.8 years, P <0.001), had a lower PCR cycle threshold (PCR cycle threshold value 19.3 ± 6.3 vs 22.7 ± 15.4, P = 0.025) at presentation, and more medical comorbidities. The hyperinflammatory phenotype was independently associated with adverse clinical outcomes, even after adjusting for age, medical history and viral load on multivariable analyses (adjusted odds ratio 5.78, 95% confidence interval 2.86-11.63). Conclusion: Even in non-severe COVID-19, there are distinct hyper- and hypoinflammatory phenotypes, with the hyperinflammatory phenotype strongly associated with adverse clinical outcomes, that could be distinguished with a simple biomarker.

Endolymphatic Hydrops Magnet Resonance Imaging in Ménière's Disease Patients after Cochlea Implantation.

INTRODUCTION: Cochlear implantation in patients with Ménière's disease (MD) is the treatment of choice in cases of functional deafness. Additional vertigo control is of central importance in this group of patients. Endolymphatic hydrops (ELH) is the pathophysiological correlate of MD and can be evaluated by magnet resonance imaging (MRI). Bilateral MD occurs in 10-33% and can be the reason for a postoperative persisting or newly occurring vertigo in this group. Recent developments in the field of implant magnets and experience in MRI sequences allow the diagnostic performance of MRI in cochlear implantees to be evaluated. The aim of the present study was to evaluate the possibility of MRI as a visual diagnostic tool for endolymphatic hydrops in cochlear implantees. MATERIAL AND METHODS: This was a retrospective study including three cochlear implantees (age: 61-76 years, one female, two male) suffering from MD who, postoperatively, had a recurrence of vertigo with Ménière's-like symptoms. An MRI was performed for the evaluation of ELH (ELH-MRI). MRI observation was performed by a 4 h iv. delayed Gad 3 D Flair sequence. RESULTS: In all cases, the ipsilateral implant magnet artifact covered the vestibulum, the semicircular canals and the cochlea. The contralateral vestibulum, the semicircular canal and the cochlea were fully observable, and a classification of the ELH-MRI could be performed. CONCLUSION: ELH-MRI scanning allows for the detection of contralateral labyrinthine endolymphatic hydrops and is a tool for the postoperative evaluation of vertigo in cochlear implantees.

Corrigendum: A new device for the removal of cochlear schwannoma: A temporal bone study.

[This corrects the article DOI: 10.3389/fsurg.2023.1077407.].

Recurrent Respiratory Papillomatosis (RRP)-Meta-analyses on the use of the HPV vaccine as adjuvant therapy.

Recurrent Respiratory Papillomatosis(RRP) is a rare disease with severe morbidity. Treatment is surgical. Prevailing viewpoint is that prophylactic HPV vaccines do not have therapeutic benefit due to their modus operandi. Studies on HPV vaccination alongside surgery were meta-analysed to test effect on burden of disease. Databases were accessed Nov and Dec 2021 [PubMed, Cochrane, Embase and Web of Science]. Main outcome measured was: Mean paired differences in the number of surgeries or recurrences per month. Analyses was performed using: Random effect maximal likelihood estimation model using the Stata module Mataan(StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX:StataCorp LLC.) Our results found n = 38 patients, suitable for syntheses with one previous meta-analyses (4 published, 2 unpublished studies) n = 63, total of n = 101 patients. Analyses rendered an overall reduction of 0.123 recurrences or surgeries per month (95% confidence interval [0.064, 0.183]). Our meta-analyses concludes that HPV vaccine is a beneficial adjunct therapy alongside surgery.

Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Hit and run oncogeneses in head and neck cancers requires greater investigation.

Head and neck cancers are unique in so far that two major oncogenic viruses, Epstein Barr virus (EBV) and Human papillomavirus (HPV) infect adjacent anatomy and cause nasopharyngeal and oropharyngeal cancers, respectively. Dominant recognized carcinogens are alcohol and tobacco but some head and neck cancers have been found to have mixed carcinogens (including betel leaf, areca nuts, slaked lime, viruses, etc.) involved in their oncogenesis and conversely, groups of patients with unknown or less dominant carcinogens involved in their development. These cancers may have had viral involvement in the past but then lost most of their viral nucleic acids (be they DNA and/or RNA) below a detection threshold, thus rendering them virus-negative. Some of these virus-negative tumors appear to have mutagenic signatures associated with virus-positive cancers,  for example, from the APOBEC defense mechanism which is known to mutate viral nucleic acids as well as cause collateral damage to host DNA, with subsequent development of strongly viral prejudiced mutational signatures. These mechanisms are likely to be less efficient at oncogenesis than traditional EBV and HPV oncogenes directly driving mutagenesis, thus accounting for the smaller frequencies of these cancers found. More profound investigations of these unusual tumors are warranted to dissect out these mechanistic pathways.

Prospective Evaluation of 3 T MRI Effect on Residual Hearing Function of Cochlea Implantees.

INTRODUCTION: The approval process for MRI safety of implants includes physical observations and an experimental evaluation in artificial settings to simulate the in vivo effect. This contains the observation of temperature changes and artificial current generation by the magnetic field. From these findings, the safety of an implant and its effect on the patient can be estimated. MRI safety is based on an in vivo evaluation of adverse events after the approval process, but an actual analysis of the effect on different tissues is not followed. The effect of MRI scanning in cochlea implantees on their residual hearing as the correlate of the hair cell function is so far unknown, therefore the aim of the present study was to observe the effect of 3 T MRI on the residual hearing of cochlea implantees. MATERIAL AND METHODS: In this prospective study, we performed a 3 T MRI T2 2D MS Drive sequence in eight cochlea-implanted ears. Before and after the MRI scan, a bone conduction pure tone audiogram (BC PTA) was performed. All cochlea implantees had a pre-scanning threshold of low frequency residual hearing between 20 dB and 65 dB. RESULTS: Low frequency mean residual hearing was not affected by the 3 T T2 2D MS Drive sequence. We observed a pre-scanning threshold at 250 Hz of 42.9 (SD 3.9) dB and for 500 Hz 57.1 (SD 6.4) dB. Post-scanning BC PTA was for 250 Hz 42.1 (SD 3.9) dB and for 500 Hz 57.1 (SD 5.7) dB. CONCLUSION: 3 T MRI scanning has no significant functional effect on the hair cells in cochlea implantees in low frequencies with a T2 2D MS Drive sequence.

Review of potential medical treatments for middle ear cholesteatoma.

Middle ear cholesteatoma (MEC), is a destructive, and locally invasive lesion in the middle ear driven by inflammation with an annual incidence of 10 per 100,000. Surgical extraction/excision remains the only treatment strategy available and recurrence is high (up to 40%), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review was targeted at connecting the dysregulated inflammatory network of MEC to pathogenesis and identification of pharmaceutical targets. We summarized the numerous basic research endeavors undertaken over the last 30+ years to identify the key targets in the dysregulated inflammatory pathways and judged the level of evidence for a given target if it was generated by in vitro, in vivo or clinical experiments. MEC pathogenesis was found to be connected to cytokines characteristic for Th1, Th17 and M1 cells. In addition, we found that the inflammation created damage associated molecular patterns (DAMPs), which further promoted inflammation. Similar positive feedback loops have already been described for other Th1/Th17 driven inflammatory diseases (arthritis, Crohn's disease or multiple sclerosis). A wide-ranging search for molecular targeted therapies (MTT) led to the discovery of over a hundred clinically approved drugs already applied in precision medicine. Based on exclusion criteria designed to enable fast translation as well as efficacy, we condensed the numerous MTTs down to 13 top drugs. The review should serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history. Video Abstract.

Middle ear cholesteatoma (MEC) is a destructive and locally invasive ulcerated lesion in the middle ear driven by inflammation which occurs in 10 out of 100,000 people annually. Surgical extraction/excision is the only treatment strategy available and recurrence is high (up to 40% after ten years), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review is focused on the connections between inflammation and MEC pathogenesis. These connections can be used as attack points for pharmaceuticals. For this we summarized the results of research undertaken over the last 30 + years. MEC pathogenesis can be described by specific inflammatory dysregulation already known from arthritis, Crohn's disease or multiple sclerosis. A hallmark of this dysregulation are positive feedback loops of the inflammation further amplifying itself in a vicious circle-like manner. We have identified over one hundred drugs which are already used in clinic to treat other inflammatory diseases, and could potentially be repurposed to treat MEC. To improve and expedite clinical success rates, we applied certain criteria based on our literature searches and condensed these drugs down to the 13 top drugs. We hope the review will serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history.

p53 Pathway Inactivation Drives SMARCB1-deficient p53-wildtype Epithelioid Sarcoma Onset Indicating Therapeutic Vulnerability Through MDM2 Inhibition.

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy.

Viral and Clinical Oncology of Head and Neck Cancers.

PURPOSE OF REVIEW: This study assesses the current state of knowledge of head and neck squamous cell carcinomas (HNSCC), which are malignancies arising from the orifices and adjacent mucosae of the aerodigestive tracts. These contiguous anatomical areas are unique in that 2 important human oncoviruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), are causally associated with nasopharyngeal and oropharyngeal cancers, respectively. Mortality rates have remained high over the last 4 decades, and insufficient attention paid to the unique viral and clinical oncology of the different subgroups of HNSCC. RECENT FINDINGS: We have compared and contrasted the 2 double-stranded DNA viruses and the relevant molecular oncogenesis of their respective cancers against other head and neck cancers. Tobacco and alcohol ingestion are also reviewed, as regard the genetic progression/mutation accumulation model of carcinogenesis. The importance of stringent stratification when searching for cancer mutations and biomarkers is discussed. Evidence is presented for a dysplastic/pre-invasive cancerous phase for HPV+ oropharyngeal cancers, and analogous with other HPV+ cancers. This raises the possibility of strategies for cancer screening as early diagnosis will undoubtedly save lives. Staging and prognostication have changed to take into account the distinct biological and prognostic pathways for viral+ and viral- cancers. Diagnosis of pre-cancers and early stage cancers will reduce mortality rates. Multi-modal treatment options for HNSCC are reviewed, especially recent developments with immunotherapies and precision medicine strategies. Knowledge integration of the viral and molecular oncogenic pathways with sound planning, hypothesis generation, and clinical trials will continue to provide therapeutic options in the future.

Sunburn Prevalence is Underestimated in UK-Based People of African Ancestry.

BACKGROUND: Eumelanin, the primary pigment in human epidermis, has a well-established photo-protective role. It can confer a protection factor of up to approximately 13.4 in some individuals. However, the protection eumelanin affords is not absolute and, further, the susceptibility of human skin to the harmful effects of UV radiation is more complex than skin pigmentation alone. OBJECTIVE: Our survey explored the lifetime prevalence of sunburn in people of African Ancestry based in the UK (Black African or Black Caribbean). RESULTS: A significant number of respondents, 52.2% (n=222), reported a history of sunburn. Interestingly, there was a significant increase in frequency of sunburn in those with a lighter skin tone (self-classified from dark, medium and light - 47.3%, 53.5% and 71.4%, respectively). In total 69% reported that the episode of sunburn occurred when they were not using sunscreen, and another 10% could not recall whether sunscreen was used. A large proportion of respondents (59%) indicated that they had been sunburnt while away from the UK in hot/sunny climates, raising the question of whether intermittent sun exposure at high UV indices is a key factor in sunburn risk for those living in temperate climates. CONCLUSION: Our findings do not support the hypothesis of a simplistic relationship between skin colour and sun sensitivity and encourage us to re-examine this relationship and its implications for public health promotion. It also adds to a body of evidence revealing the need for more up-to-date and appropriate systems to assess the risk UV radiation poses to diverse populations.

Comparative Analyses of Tumour Volume Doubling Times for Periocular and Non-periocular Head and Neck Basal Cell Carcinomas.

Basal cell carcinomas are the commonest solid malignancy in humans and thought to grow faster in the periocular region. We measured growth rates between periocular and non-periocular nodular basal cell carcinomas in the head and neck region from high-resolution digital photos and operative notes. The non-periocular basal cell carcinomas (head and neck) showed a mean tumour volume doubling time of 129.8 ± 21.74 (n = 79) days, and the periocular basal cell carcinoma a mean of 177.5 ± 37.21 (n = 47) days. The unpaired t-test with Welch correction showed that this difference was not significant (p = 0.2719). The mean tumour volume doubling time was 147.59 ± 37.75 days for head and neck basal cell carcinomas overall. For the first time, tumour volume doubling times for nodular basal cell carcinomas in the periocular versus non-periocular regions for the head and neck area were analysed, with no significant differences demonstrated. Further, comparison of basal cell carcinoma growth rates with other common solid tumours confirmed that basal cell carcinomas are slow growing malignancies.

An Effective Primary Head and Neck Squamous Cell Carcinoma In Vitro Model.

Head and neck squamous cell carcinoma is a highly malignant disease and research is needed to find new therapeutic approaches. Faithful experimental models are required for this purpose. Here, we describe the specific cell culture conditions enabling the efficient establishment of primary cell culture models. Whereas a classical 10% serum-containing medium resulted in the growth of fibroblast-like cells that outcompeted epithelial cells, we found that the use of specific culture conditions enabled the growth of epithelial tumor cells from HPV+ and HPV- head and neck cancer tissue applicable for research. EpCAM and high Thy-1 positivity on the cell surface were mutually exclusive and distinguished epithelial and fibroblast-like subpopulations in all primary cultures examined and thus can be used to monitor stromal contamination and epithelial cell content. Interestingly, cells of an individual patient developed tumor spheroids in suspension without the use of ultra-low attachment plates, whereas all other samples exclusively formed adherent cell layers. Spheroid cells were highly positive for ALDH1A1 and hence displayed a phenotype reminiscent of tumor stem cells. Altogether, we present a system to establish valuable primary cell culture models from head and neck cancer tissue at high efficiency that might be applicable in other tumor entities as well.

Specific Targeting of Oncogenes Using CRISPR Technology.

In recent decades, tools of molecular biology have enabled researchers to genetically modify model organisms, including human cells. RNAi, zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats and CRISPR-associated protein 9), retro- or lentiviral gene transfer, and many other methods can be utilized to remove genes, add genes, or change their expression. Within the same timeframe, survival rates for many highly malignant tumor diseases have not improved substantially. If modern medicine could apply even a subset of research methods in clinical management, which are already well established and controllable in basic research laboratories, this could strongly impact patients' prognosis. CRISPR-Cas9 is a method to precisely target and manipulate genomic loci and recent studies have attempted to use this method as a genetic treatment for Duchenne muscular dystrophy, blood disorders, autosomal-dominant hearing loss, and cancer. Some of these approaches target mutant genomic sequences specifically and try to avoid affecting the respective normal loci. Considering obvious genetic risks opposing the objected benefits, data are needed to show whether CRISPR technology is suitable as a future cancer therapy approach or not. Here, we develop strategies for the specific targeting of viral cancer drivers and oncogenes activated by mutation, using the latest CRISPR technology. Cancer Res; 78(19); 5506-12. ©2018 AACR.

Stem cells in middle ear cholesteatoma contribute to its pathogenesis.

Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the "stemness" markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin + /S100B + -cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion.

Recurrent respiratory papillomatosis (RRP)-time for a reckoning?

OBJECTIVES: Recurrent respiratory papillomatosis (RRP) is a rare disease, but one with severe morbidity and occasional mortality. The aetiological agent is human papillomavirus (HPV), and HPV types 6 and 11 account for over 90% of all cases. In the active phase of the disease, patients require multiple hospital admissions for surgical removal or ablation of these benign tumors, which are likely to obstruct the airways if left unchecked. Long-term sequelae include scarring of the vocal cords, change in voice timbre, or even muteness if a tracheostomy is required. The aim of this study was to determine if adjuvant vaccination with the quadrivalent HPV L1 vaccine (Gardasil™) would decrease numbers of surgical treatments post-vaccination. METHODS: A prospective pilot study following a cohort of 12 RRP patients, all of whom gave fully informed consent to participate. All patients had their papillomas typed and if they were found to have types 6 or 11, were vaccinated at the time of first surgical treatment in the hospital, according to the manufacturer's protocols. Patients were followed up closely with 3 or 6 month follow-up visits. Standard surgical treatments were given and were not affected by whether they participated in the study. RESULTS: We found a >7-fold decrease in the incidence rates of papillomatosis requiring surgical intervention from the pre-vaccination period (47.44/1000 patient-months) compared to the post-vaccination period (6.71/1000 patient-months). DISCUSSION: Surgical treatments for RRP are robust markers for papillomata which require treatment because of the dangers of obstruction of the airway. Despite the small size of this cohort (due to the rarity of this disease), the data suggests that adjuvant use of quadrivalent HPV L1 vaccine imparts significant benefit to this group of patients. A large multi-center randomized placebo controlled trial is required to definitively establish whether this hypothesis is true and can become the new standard of therapy. LEVEL OF EVIDENCE: 3b.