Detection and Differentiation of Different Monocyte Subsets: Optimalization of a Multiparameter Flowcytometric Assay

Peripheral blood monocytes are heterogeneous and it was recently reported that three subsets can be identified based on CD14 and CD16 expression: CD14++/CD16- classical monocytes, CD14++/CD16+ intermediate monocytes and CD14+/CD16++ nonclassical monocytes. The mostly used technique to detect and determine the subtypes is multiparameter flowcytometry, however, there are some technical challenges in the determination of the subsets in flowcytometry, including pre-analytic, analytic and postanalytic aspects. This study aims at validation and optimalization the multiparameter flowcytometric determination of monocyte subsets. For this purpose, patients from the outdoor clinic were asked to participate by having an extra EDTA-anti-coagulated blood tube drawn. The stability of the blood sample was investigated by analyzing 20 blood samples directly and 1h, 3h, 4h and 24h after venipuncture. The influence of antibody clone (B73.1 and 3G8; BD Biosciences) against CD16 was investigated making use of another group of 20 blood samples. Reproducibility was tested and three different gating strategies, based on either CD14 back-gating, HLA-DR expression or CD24 and CD56 expression were applied to the flowcytometric results. Samples were analyzed with flowcytometry (FACSCanto II; BD). The relative size of classical monocyte population shows a significant decrease 24h after venipuncture. Using the HLA-DR based gating method, a decrease in the fraction of totoal monocytes was found. No significant differences were found when comparing the B73.1 and 3G8 antibody clone, however, the use of the 3G8 clone made it possible to make a better distinction between monocytes and granulocytes in the HLADR based gating method. all measurements show a good reproducibility (CV < 10%, ICC > 0,8), although the intermediate monocytes show a higher level of variation. In order to make reliable and proper determinations of the different monocyte subsets, sample preparation and acquisition has to take place within 4 hours after venipuncture. the 3G8 clone is preferred for CD16 labeling and the HLA-DR based gating method is assumed to be the most accurate in identifying the monocyte subsets. Using above mentioned recommendations, the test shows a good reproducibility

Switches of SOX17 and SOX2 expression in the development of squamous metaplasia and squamous intraepithelial lesions of the uterine cervix

Aims: The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells. Methods and Results: Normal cervices and squamous intraepithelial lesions (SIL) were studied with immunohistochemistry, methylation of SOX17, human papilloma virus (HPV) genotyping, and in situ hybridization. In the TZ squamous metaplasia originating from these reserve cells can still show SOX17 expression, while also remnants of SOX17-positive immature metaplasia can be recognized in the normal squamous epithelium. SOX17 expression is gradually lost during maturation, resulting in the exclusive expression of SOX2 in the majority of (SIL). This loss of SOX17 expression is independent of methylation of the CpG island in its promotor region. HPV can be detected in SOX17-positive immature metaplastic regions in the immediate vicinity of SOX2-positive SIL, suggesting that switches in SOX17 and 2 expression can occur upon HPV infection. Conclusions: This switch in expression, and the strong association between the distribution of reserve cells and squamous areas within the columnar epithelium in the TZ, suggests that reserve cell proliferations, next to basal cells in the squamous epithelium, are potential targets for the formation of squamous lesions upon viral infection

Ex Vivo Culture Models to Indicate Therapy Response in Head and Neck Squamous Cell Carcinoma

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

Public awareness of the association between human papillomavirus and oropharyngeal cancer

BACKGROUND: Early diagnosis of human papillomavirus (HPV) associated oropharyngeal cancer (OPC) is associated with improved survival. To achieve early diagnosis, it might be beneficial to increase awareness of the link between HPV and OPC. This increase of awareness could also be an important way to increase vaccination rates. The aim of our study was to explore the current public knowledge in the Netherlands regarding the association of HPV with OPC. METHODS: An online cross-sectional survey was used and sent by the company Flycatcher Internet Research to 1539 of their panel members. Data were analyzed statistically by gender, age, educational level and the participants' use of alcohol and tobacco. RESULTS: The response rate was 68% (1044 participants). Our data revealed that 30.6% of the participants had heard of HPV. There was a knowledge gap regarding HPV in males (P < 0.001), people older than 65 years (P < 0.001), people with low education level (P < 0.001) and current smokers (P < 0.001). Of the respondents who had heard of HPV, only 29.2% knew of the association between HPV and OPC. We also found that only 49.7% of the population knew of the existence of an HPV vaccine. CONCLUSIONS: The results of this survey indicate that the public awareness of HPV and the association of HPV with OPC is lacking. Interventions to increase awareness of HPV and its association with non-cervical cancer should be considered. This might help to increase the HPV vaccine uptake both for girls and boys and earlier diagnosis of this disease leading to improved survival

Awareness of HPV-associated oropharyngeal cancers among GPs in The Netherlands:a cross-sectional study

BACKGROUND: The incidence of human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) is increasing in high income countries. HPV-associated OPC generally presents as an invasive disease, often with lymph node involvement, in relatively young patients with minimal or no history of smoking and alcohol consumption. Knowledge on HPV-associated OPC among primary care professionals is essential for disease recognition and early start of treatment. AIM: To examine the knowledge on HPV-associated OPC among GPs in the Netherlands. DESIGN & SETTING: A cross-sectional postal survey among GPs in the Netherlands. METHOD: A 12-item questionnaire was sent to 900 randomly selected general practices. Outcome measures included awareness of the link between HPV and OPC, epidemiological trends, and patient characteristics. Data were statistically analysed for sex, years after graduation, and self-rated knowledge of OPC. RESULTS: A total of 207 GPs participated in this study. Seventy-two per cent recognised HPV as a risk factor for OPC and 76.3% were aware of the increasing incidence rate of HPV-associated OPC. In contrast, 35.7% of participants knew that patients with HPV-associated OPC are more often male, and just over half (53.6%) of the participants were aware of the younger age of these patients. CONCLUSION: More than one-quarter of GPs in the Netherlands are unaware of HPV as a causative factor for OPC. Furthermore, there is a gap in knowledge on characteristics of patients with HPV-associated OPC . Further training on these topics could improve disease recognition and, ultimately, patient survival

The Antiviral Agent Cidofovir Induces DNA Damage and Mitotic Catastrophe in HPV-Positive and -Negative Head and Neck Squamous Cell Carcinomas In Vitro

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 gamma-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.</p

Causes and Consequences of HPV Integration in Head and Neck Squamous Cell Carcinomas: State of the Art

Simple Summary In human papillomavirus (HPV) associated head and neck squamous cell carcinomas (HNSCC) s, the HPV genome is commonly found integrated in the human genome. The event of viral-human genome integration may act as a driver of carcinogenesis. Hence, it is vital to assess the viral integration status of a tumor. In this review, current and emerging techniques for integration detection are thoroughly discussed with their advantages and disadvantages. Additionally, the review also discusses the causes of HPV integration into the cellular genome, as well as its ramifications, impacting possible clinical implications. 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

Switches of SOX17 and SOX2 expression in the development of squamous metaplasia and squamous intraepithelial lesions of the uterine cervix

Aims The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells. Methods and Results Normal cervices and squamous intraepithelial lesions (SIL) were studied with immunohistochemistry, methylation of SOX17, human papilloma virus (HPV) genotyping, and in situ hybridization. In the TZ squamous metaplasia originating from these reserve cells can still show SOX17 expression, while also remnants of SOX17-positive immature metaplasia can be recognized in the normal squamous epithelium. SOX17 expression is gradually lost during maturation, resulting in the exclusive expression of SOX2 in the majority of (SIL). This loss of SOX17 expression is independent of methylation of the CpG island in its promotor region. HPV can be detected in SOX17-positive immature metaplastic regions in the immediate vicinity of SOX2-positive SIL, suggesting that switches in SOX17 and 2 expression can occur upon HPV infection. Conclusions This switch in expression, and the strong association between the distribution of reserve cells and squamous areas within the columnar epithelium in the TZ, suggests that reserve cell proliferations, next to basal cells in the squamous epithelium, are potential targets for the formation of squamous lesions upon viral infection

Evaluation of the Sensitivity of Metabolic Profiling by Rapid Evaporative Ionization Mass Spectrometry:Toward More Radical Oral Cavity Cancer Resections

Radical resection for patients with oral cavity cancer remains challenging. Rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical vapors has been reported for real-time classification of normal and tumor tissues for numerous surgical applications. However, the infiltrative pattern of invasion of oral squamous cell carcinomas (OSCC) challenges the ability of REIMS to detect low amounts of tumor cells. We evaluate REIMS sensitivity to determine the minimal amount of detected tumors cells during oral cavity cancer surgery. A total of 11 OSCC patients were included in this study. The tissue classification based on 185 REIMS ex vivo metabolic profiles from five patients was compared to histopathology classification using multivariate analysis and leave-one-patient-out cross-validation. Vapors were analyzed in vivo by REIMS during four glossectomies. Complementary desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was employed to map tissue heterogeneity on six oral cavity sections to support REIMS findings. REIMS sensitivity was assessed with a new cell-based assay consisting of mixtures of cell lines (tumor, myoblasts, keratinocytes). Our results depict REIMS classified tumor and soft tissues with 96.8% accuracy. In vivo REIMS generated intense mass spectrometric signals. REIMS detected 10% of tumor cells mixed with 90% myoblasts with 83% sensitivity and 82% specificity. DESI-MSI underlined distinct metabolic profiles of nerve features and a metabolic shift phosphatidylethanolamine PE(O-16:1/18:2))/cholesterol sulfate common to both mucosal maturation and OSCC differentiation. In conclusion, the assessment of tissue heterogeneity with DESI-MSI and REIMS sensitivity with cell mixtures characterized sensitive metabolic profiles toward in vivo tissue recognition during oral cavity cancer surgeries