Immunohistochemical Assessment of Microvessel Density in OSCC: Spatial Heterogeneity of Angiogenesis and Its Impact on Survival.

(1) Background Oral squamous cell carcinomas (OSCC) are a common malignancy of the oral cavity and are often diagnosed when they have already spread to the regional lymph nodes. Advanced stages of cancer are characterized by the development of distant metastases. Angiogenesis, a hallmark of cancer, is known to contribute to cancer progression and metastasis. High microvessel density (MVD) has been linked to poor clinical outcomes in various types of cancer. (2) Methods: In this study, we aimed to investigate the spatial heterogeneity of blood vessels by comparing the tumor center and invasion front and to evaluate its prognostic value in OSCC. A total of 71 OSCC patient specimens were collected. The tissue was immunohistochemically stained using CD31 antibody to assess the MVD in the tumor center and the invasion front. Furthermore, the associations between the histopathological parameters, including MVD, disease-free survival (DFS), and overall survival (OS) were computed. (3) Results: In our study, we found a significantly higher presence of blood vessels at the invasion front of OSCCs compared to the tumor center. However, we did not observe any significant differences in MVD between different tumor stages. High intratumoral MVD was shown to be a positive prognostic factor for DFS (p = 0.047). (4) Conclusions: To the best of our knowledge, we were the first to analyze MVD as a prognostic factor by considering its spatial heterogeneity in OSCC. However, further studies are warranted to further elucidate the complexity of microvascular spatial heterogeneity and its influence on prognosis.

Novel Three-Dimensional and Non-Invasive Diagnostic Approach for Distinction between Odontogenic Keratocysts and Ameloblastomas.

Aim of this study was to demonstrate the diagnostic ability to differentiate odontogenic keratocysts (OKCs) from ameloblastomas (AMs) based on computed tomography (CT) or cone beam computed tomography (CBCT) scans. Preoperative CT and CBCT scans from 2004 to 2019 of OKCs and AMs were analyzed in 51 participants. Lesions were three-dimensionally (3D) assessed and Hounsfield units (HU) as well as gray scale values (GSV) were quantified. Calculated HU spectra were compared within the same imaging modalities using unpaired t-tests and correlated with participants characteristics by calculating Pearsons correlation coefficients. Within the CT scans, AMs had highly significantly higher HU values compared to OKCs (43.52 HU and 19.79 HU, respectively; p < 0.0001). Analogous, within the CBCT scans, AMs had significantly higher GSV compared to OKCs (-413.76 HU and -564.76 HU, respectively; p = 0.0376). These findings were independent from participants' gender and age, anatomical site, and lesion size, indicating that the HU- and GSV-based difference reflects an individual configuration of the lesion. HU and GSV spectra calculated from CT and CBCT scans can be used to discriminate between OKCs and AMs. This diagnostic approach represents a faster and non-invasive option for preoperative diagnosis of such entities and has potential to facilitate therapeutic decision making.

TGFß carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Contributions of TGFß to cancer progression are well documented. However, plasma TGFß levels often do not correlate with clinicopathological data. We examine the role of TGFß carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFß expression levels during oral carcinogenesis. In human HNSCC, TGFß and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFß levels were evaluated by ELISA and TGFß bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFß content was quantified using bioassays and bioprinted microarrays. RESULTS: During 4-NQO carcinogenesis, TGFß levels in tumour tissues and in serum increased as the tumour progressed. The TGFß content of circulating exosomes also increased. In HNSCC patients, TGFß, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFß levels. Neither TGFß expression in tumours nor levels of soluble TGFß correlated with clinicopathological data or survival. Only exosome-associated TGFß reflected tumour progression and correlated with tumour size. CONCLUSIONS: Circulating TGFß+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

Elective Tracheotomy in Patients Receiving Mandibular Reconstructions: Reduced Postoperative Ventilation Time and Lower Incidence of Hospital-Acquired Pneumonia.

Elective tracheotomy (ET) secures the airway and prevents adverse airway-related events as unplanned secondary tracheotomy (UT), prolonged ventilation (PPV) or nosocomial infection. The primary objective of this study was to identify factors predisposing for airway complications after reconstructive lower ja surgery. We reviewed records of patients undergoing mandibulectomy and microvascular bone reconstruction (N = 123). Epidemiological factors, modus of tracheotomy regarding ET and UT, postoperative ventilation time and occurrence of hospital-acquired pneumonia HAP were recorded. Predictors for PPV and HAP, ET and UT were identified. A total of 82 (66.7%) patients underwent tracheotomy of which 12 (14.6%) were performed as UT. A total of 52 (42.3%) patients presented PPV, while 19 (15.4%) developed HAP. Increased operation time (OR 1.004, p = 0.005) and a difficult airway (OR 2.869, p = 0.02) were predictors, while ET reduced incidence of PPV (OR 0.054, p = 0.006). A difficult airway (OR 4.711, p = 0.03) and postoperative delirium (OR 6.761, p = 0.01) increased UT performance. HAP increased with anesthesia induction time (OR 1.268, p = 0.001) and length in ICU (OR 1.039, p = 0.009) while decreasing in ET group (HR 0.32, p = 0.02). OR for ET increased with mounting CCI (OR 1.462, p = 0.002) and preoperative radiotherapy (OR 2.8, p = 0.018). ET should be strongly considered in patients with increased CCI, preoperative radiotherapy and prolonged operation time. ET shortened postoperative ventilation time and reduced HAP.

A20 as a Potential New Tool in Predicting Recurrence and Patient's Survival in Oral Squamous Cell Carcinoma.

A20, known as a potent inhibitor of NF-κB signaling, has been characterized in numerous clinical as well as preclinical studies. Recently, especially in various malignant diseases, the prognostic and therapeutic relevance of A20 was investigated. In oral squamous cell carcinoma (OSCC) however, the characterization of A20 is uncharted territory. We analyzed a tissue microarray (TMA) of 229 surgically-treated OSCC patients (2003-2013). Immunohistochemical (IHC) stainings were performed for A20 and CD3; additionally, standard haematoxylin-eosin staining was applied. IHC findings were correlated with a comprehensive dataset, comprising clinical and pathohistological information. A20 expression was analyzed in tumor cells as well as in tumor infiltrating lymphocytes (TILs) and correlated with the overall survival (OS) and recurrence-free survival (RFS) using uni- and multivariable Cox regression. The median follow-up time was 10.9 years and the A20 expression was significantly decreased in CD3+ TILs compared to mucosa-infiltrating lymphocytes (MILs). In the Kaplan-Meier analyses, higher A20 expression in TILs was correlated with better OS (p = 0.017) and RFS (p = 0.020). In the multivariable survival analysis, A20 overexpression correlated with improved OS (HR: 0.582; 95% CI 0.388-0.873, p = 0.009) and RFS (HR 0.605; 95% CI 0.411-0.889, p = 0.011). Our results indicate a novel prognostic role for A20 in OSCC. Due to its elevated expression in TILs, further research is highly desirable, which therefore could offer new therapeutic opportunities for patients suffering from OSCC.

Statin Use Ameliorates Survival in Oral Squamous Cell Carcinoma-Data from a Population-Based Cohort Study Applying Propensity Score Matching.

The anti-cancer properties of statins have attracted much attention recently, but little is known about the prognostic role of statins in oral squamous cell carcinoma (OSCC). In a retrospective approach, we analyzed a population-based cohort of 602 OSCC patients with primary curative tumor resection to negative margins and concomitant neck dissection between 2005-2017. Long-term medication with statins was correlated with overall survival (OAS) as well as recurrence-free survival (RFS) using uni- and multivariable Cox regression. Additionally, propensity score matching was applied to adjust for confounders. Statin use was present in 96 patients (15.9%) at a median age of 65.7 years. Statin treatment correlated with ameliorated survival in multivariable Cox regression in the complete cohort (OAS: HR 0.664; 95% CI 0.467-0.945, p = 0.023; RFS: HR 0.662; 95% CI 0.476-0.920, p = 0.014) as well as matched-pair cohort of OSCC patients (OAS: HR 0.691; 95% CI 0.479-0.997, p = 0.048; RFS: HR 0.694; 95% CI 0.493-0.976, p = 0.036) when compared to patients not taking statins at time of diagnosis. These findings were even more pronounced by sub-group analysis in the matched-pair cohort (age < 70 years). These data indicate that statin use might ameliorate the oncological outcome in primarily resected OSCC patients, but prospective clinical trials are highly recommended.

Tumor gene signatures that correlate with release of extracellular vesicles shape the immune landscape in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) evade immune responses through multiple resistance mechanisms. Extracellular vesicles (EVs) released by the tumor and interacting with immune cells induce immune dysfunction and contribute to tumor progression. This study evaluates the clinical relevance and impact on anti-tumor immune responses of gene signatures expressed in HNSCC and associated with EV production/release. Expression levels of two recently described gene sets were determined in The Cancer Genome Atlas Head and Neck Cancer cohort (n = 522) and validated in the GSE65858 dataset (n = 250) as well as a recently published single-cell RNA sequencing dataset (n = 18). Clustering into HPV(+) and HPV(-) patients was performed in all cohorts for further analysis. Potential associations between gene expression levels, immune cell infiltration, and patient overall survival were analyzed using GEPIA2, TISIDB, TIMER, and the UCSC Xena browser. Compared to normal control tissues, vesiculation-related genes were upregulated in HNSCC cells. Elevated gene expression levels positively correlated (P < 0.01) with increased abundance of CD4(+) T cells, macrophages, neutrophils, and dendritic cells infiltrating tumor tissues but were negatively associated (P < 0.01) with the presence of B cells and CD8(+) T cells in the tumor. Expression levels of immunosuppressive factors NT5E and TGFB1 correlated with the vesiculation-related genes and might explain the alterations of the anti-tumor immune response. Enhanced expression levels of vesiculation-related genes in tumor tissues associates with the immunosuppressive tumor milieu and the reduced infiltration of B cells and CD8(+) T cells into the tumor.

Tumor Immune Microenvironment Heterogeneity at the Invasion Front and Tumor Center in Oral Squamous Cell Carcinoma as a Perspective of Managing This Cancer Entity.

BACKGROUND: Evaluating the tumor microenvironment and its influence on clinical management and therapy response is becoming increasingly important. However, only a few studies deal with the spatial distribution of immune cells within the tumor. This study aimed to describe the topology of immune cells in the microenvironment of oral squamous cell carcinoma (OSCC) sectioned by tumor invasion front and tumor center and to test their prognostic relevance regarding patient survival. METHODS: A total of 55 OSCC patient specimens were collected retrospectively. The cancer tissue was immunohistochemically stained using an automated tissue stainer Ventana Benchmark Ultra (Roche) and analyzed using discrete expression marker profiles on immune cells. We investigated CD4+ lymphocytes, CD8+ lymphocytes, CD68+ macrophages, CD163+ macrophages, and M1 macrophages regarding their spatial distribution. RESULTS: The statistical analysis revealed that the quantity and distribution of CD4+ (p = 0.007), CD8+ (p < 0.001), CD68+ (p < 0.001), CD163+ cells (p = 0.004), and M1 (p < 0.001) macrophages were significantly higher at the invasion front compared to the tumor center in all observed cases. However, high and low immune cell counts in the tumor center and invasion front were not associated with overall survival. CONCLUSION: Our results show two distinct immune microenvironments of the tumor center compared to the invasion front. Future studies are needed to explore how these results can be leveraged to improve patient therapy and outcome.

Impact of phosphorylation of heat shock protein 27 on the expression profile of periodontal ligament fibroblasts during mechanical strain.

PURPOSE: Orthodontic tooth movement is a complex process involving the remodeling of extracellular matrix and bone as well as inflammatory processes. During orthodontic treatment, sterile inflammation and mechanical loading favor the production of receptor activator of NF-κB ligand (RANKL). Simultaneously, expression of osteoprotegerin (OPG) is inhibited. This stimulates bone resorption on the pressure side. Recently, heat shock protein 27 (HSP27) was shown to be expressed in the periodontal ligament after force application and to interfere with inflammatory processes. METHODS: We investigated the effects of phosphorylated HSP27 on collagen synthesis (COL1A2 mRNA), inflammation (IL1B mRNA, IL6 mRNA, PTGS2 protein) and bone remodeling (RANKL protein, OPG protein) in human periodontal ligament fibroblasts (PDLF) without and with transfection of a plasmid mimicking permanent phosphorylation of HSP27 using real-time quantitative polymerase chain reaction (RT-qPCR), western blot and enzyme-linked immunosorbent assays (ELISAs). Furthermore, we investigated PDLF-induced osteoclastogenesis after compressive strain in a co-culture model with human macrophages. RESULTS: In particular, phosphorylated HSP27 increased gene expression of COL1A2 and protein expression of PTGS2, while IL6 mRNA levels were reduced. Furthermore, we observed an increasing effect on the RANKL/OPG ratio and osteoclastogenesis mediated by PDLF. CONCLUSION: Phosphorylation of HSP27 may therefore be involved in the regulation of orthodontic tooth movement by impairment of the sterile inflammation response and osteoclastogenesis.

Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force

Abstract Objective Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. Methodology Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients' genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). Results The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). Conclusions Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force.

TIGIT Expression on Intratumoral Lymphocytes Correlates with Improved Prognosis in Oral Squamous Cell Carcinoma.

(1) Background: T-cell immunoglobulin and ITIM domain (TIGIT) is a potential immunotherapeutic target in a variety of malignant entities, and antibody-based treatments are currently under investigation in clinical trials. While promising results were observed in patients with lung cancer, the role of TIGIT in oral squamous cell carcinoma (OSCC) as a biomarker as well as a therapeutic target remains elusive. Therefore, we evaluated the role of TIGIT as a prognostic factor in OSCC. (2) Methods: Here, we describe the results of a retrospective tissue microarray (TMA) OSCC cohort. Using immunohistochemistry, TIGIT expression was correlated with overall and recurrence-free survival (OAS and RFS, respectively). Additionally, in silico analysis was performed based on the TCGA Head and Neck Squamous Cell Carcinoma (HNSCC) cohort in order to correlate patients' survival with TIGIT and CD274 (encoding for PD-L1) gene expression levels. (3) Results: Database analysis revealed a beneficial outcome in OAS for tumor patients with high intraepithelial CD3-TIGIT-expression (n = 327). Hereby, OAS was 53.9 months vs. 30.1 months for patients with lower TIGIT gene expression levels (p = 0.033). In our retrospective OSCC-TMA cohort, elevated TIGIT levels on CD3+ cells correlated significantly with improved OAS (p = 0.025) as well as distant RFS (p = 0.026). (4) Conclusions: This study introduces TIGIT as a novel prognostic factor in OSCC, indicating the improved outcome of OSCC patients relative to their increased TIGIT expression. TIGIT might provide therapeutic implications for future immunotherapy in advanced-stage OSCC patients.

TGFß+ small extracellular vesicles from head and neck squamous cell carcinoma cells reprogram macrophages towards a pro-angiogenic phenotype.

Transforming growth factor ß (TGFß) is a major component of tumor-derived small extracellular vesicles (TEX) in cancer patients. Mechanisms utilized by TGFß+ TEX to promote tumor growth and pro-tumor activities in the tumor microenvironment (TME) are largely unknown. TEX produced by head and neck squamous cell carcinoma (HNSCC) cell lines carried TGFß and angiogenesis-promoting proteins. TGFß+ TEX stimulated macrophage chemotaxis without a notable M1/M2 phenotype shift and reprogrammed primary human macrophages to a pro-angiogenic phenotype characterized by the upregulation of pro-angiogenic factors and functions. In a murine basement membrane extract plug model, TGFß+ TEX promoted macrophage infiltration and vascularization (p < 0.001), which was blocked by using the TGFß ligand trap mRER (p < 0.001). TGFß+ TEX injected into mice undergoing the 4-nitroquinoline-1-oxide (4-NQO)-driven oral carcinogenesis promoted tumor angiogenesis (p < 0.05), infiltration of M2-like macrophages in the TME (p < 0.05) and ultimately tumor progression (p < 0.05). Inhibition of TGFß signaling in TEX with mRER ameliorated these pro-tumor activities. Silencing of TGFß emerges as a critical step in suppressing pro-angiogenic functions of TEX in HNSCC.

Prognostic Relevance of Type 2 Diabetes and Metformin Treatment in Head and Neck Melanoma: Results from a Population-Based Cohort Study.

BACKGROUND: Type 2 Diabetes (DM2) and the consecutively daily use of antidiabetic medication are characterized by a frequent prevalence worldwide and were shown to impact the initiation and progression of malignant diseases. While these effects were observed in a variety of malignancies, comprehensive data about the role of DM2 and antidiabetic drugs in the outcome of head and neck melanoma (HNM) patients are missing. METHODS: This retrospective population-based cohort study included 382 HNM patients from Eastern Bavaria having received tumor resection to negative margins between 2010 and 2017. Recurrence-free survival (RFS) was evaluated with regard to DM2 and routine metformin intake. Statistical analysis was performed by uni- and multivariate analyses. The median follow-up time was 5.6 years. RESULTS: DM2 was diagnosed in 68 patients (17.8%), routine metformin intake was found in 39 cases (10.2%). The univariate survival analysis revealed impaired 5-year RFS in HNM patients with DM2 compared to non-diabetic controls (p = 0.016; 64.0% and 74.5%, respectively). The multivariate Cox regression substantiated this effect (HR = 1.980, 95% CI = 1.108-3.538, p = 0.021). In detail, the cumulative locoregional recurrence rate displayed the most far-reaching negative effect on the RFS of diabetic HNM patients (HR = 4.173, 95% CI = 1.628-10.697, p = 0.003). For metformin intake, a profound positive effect on the RFS in multivariate statistics was observed, both in the complete cohort (HR = 0.396, 95% CI = 0.177-0.884, p = 0.024) as well as in the cohort of diabetic HNM patients (HR = 0.352, 95% CI = 0.135-0.913, p = 0.032). CONCLUSIONS: This study emphasizes that DM2 is a relevant comorbid condition in HNM patients, impairing patient survival. Metformin intake was associated with a favorable outcome in HNM patients, providing possible therapeutic implications for future adjuvant treatment regimes.

Postoperative Delirium after Reconstructive Surgery in the Head and Neck Region.

Postoperative delirium (POD) is an acute and serious complication following extended surgery. The aim of this study was to identify possible risk factors and scores associated with POD in patients undergoing reconstructive head and neck surgery. A collective of 225 patients was retrospectively evaluated after receiving reconstructive surgery in the head and neck region, between 2013 to 2018. The incidence of POD was examined with regards to distinct patient-specific clinical as well as perioperative parameters. Uni- and multivariate statistics were performed for data analysis. POD occurred in 49 patients (21.8%) and was strongly associated with an increased age-adjusted Charlson Comorbidity Index (ACCI) and a prolonged stay in the ICU (p = 0.009 and p = 0.000, respectively). Analogous, binary logistic regression analysis revealed time in the ICU (p < 0.001), an increased ACCI (p = 0.022) and a Nutritional Risk Screening (NRS) score ≠ 0 (p = 0.005) as significant predictors for a diagnosis of POD. In contrast, the extent of reconstructive surgery in terms of parameters such as type of transplant or duration of surgery did not correlate with the occurrence of POD. The extension of reconstructive interventions in the head and neck region is not decisive for the development of postoperative delirium, whereas patient-specific parameters such as age and comorbidities, as well as nutritional parameters, represent predictors of POD occurrence.

Loss of MMP-27 Predicts Mandibular Bone Invasion in Oral Squamous Cell Carcinoma.

Invasion of the mandibular bone is frequent in oral squamous cell carcinoma (OSCC), which often results in extensive ablative and reconstructive procedures for the patient. The purpose of this single-center, retrospective study was to identify and evaluate potential biomarkers and risk factors for bone invasion in OSCC. Initially, in silico gene expression analysis was performed for different HNSCC tumor T-stages to find factors associated with invasive (T4a) tumor growth. Afterwards, the protein expression of bone-metabolizing MMP-27, TNFRSF11B (Osteoprotegerin, OPG), and TNFSF11 (RANKL) was investigated via Tissue Microarrays (TMAs) for their impact on mandibular bone invasion. TMAs were assembled from the bone-tumor interface of primary OSCCs of the floor of the mouth and gingiva from 119 patients. Sixty-four carcinomas with patho-histological jaw invasion (pT4a) were compared to 55 carcinomas growing along the mandible without invasion (pT2, pT3). Tissue samples were additionally evaluated for patterns of invasion using the WPOI grading system. Statistical analysis of in silico data revealed decreased MMP-27 mRNA expression to be strongly associated with the pT4a-stage in OSCC, indicating invasive tumor growth with infiltration of adjacent anatomical structures. Our own clinico-pathological data on OSCCs presented a significant decrease of MMP-27 in tumors invading the nearby mandible (pT4a), compared to pT2 and pT3 tumors without bone invasion. Loss of MMP27 evolved as the strongest predictor of mandibular bone invasion in binary logistic regression analysis. To our knowledge, this is the first study investigating the role of MMP-27 expression in OSCC and demonstrating the importance of the loss of MMP-27 in mandibular bone invasion.

Clinical significance of panendoscopy in initial staging of oral squamous cell carcinoma and detection of synchronous second malignancies of the upper aerodigestive tract - Insights from a retrospective population-based cohort study.

The aim of this study was to examine the prevalence of synchronous upper aerodigestive tract (UAT) tumors within oral squamous cell carcinoma (OSCC) patients, and to specify distinct risk groups who benefit from panendoscopy. Definite clinical and pathohistological characteristics, as well as overall and recurrence-free survival (OAS and RFS, respectively) of OSCC patients with and without synchronous second UAT tumors, carcinomas in situ, or higher-grade dysplasia/metaplasia, were evaluated based on a retrospective population-based cohort study, including alignment with cancer registry data. Out of 727 included OSCC patients, 465 cases (64.0%) received panendoscopy. Among these, 18 UAT tumors were detected, all of which were linked to patients with a positive history of nicotine abuse. Every synchronous UAT tumor was revealed by panendoscopy, which, analyzed as an independent staging procedure, was accompanied by a low complication rate (1.7%). When illuminating the impact of a second UAT tumor in OSCC patients, survival analysis revealed reduced 5-year OAS (63.9% vs 43.5%, p = 0.010) and RFS (57.1% vs 32.4%, p = 0.016) for patients with a second oncology diagnosis of the UAT. Within the limitations of the study, it seems that panendoscopy should be performed in the majority of patients suffering from OSCC, because most of them have a history of smoking and drinking, which correlates with an increased risk of developing synchronous UAT tumors.

Prognostic Value of Perineural Invasion on Survival and Recurrence in Oral Squamous Cell Carcinoma.

A diagnosis of perineural invasion (PNI) is widely accepted as an unfavorable prognostic factor in various solid malignancies. Although PNI has been described as a high-risk parameter in oral squamous cell carcinoma (OSCC), its role in the current staging manuals of the American Joint Committee on Cancer (AJCC) is rather subordinate. We analysed the prognostic value of PNI on survival and recurrence in a large, multicenter OSCC cohort and a population-based approach. A total of 493 OSCC patients with primary tumor resection to negative margins and concomitant neck dissection between 2010 and 2017 were enrolled. PNI was evaluated in relation to overall survival (OAS) and recurrence-free survival (RFS) using uni- and multi-variable Cox regression. The median follow-up time was 5.0 years and PNI was diagnosed in 48 patients (9.7%). A pathohistological verification of PNI correlated significantly with a deteriorated OAS in uni- (HR 2.312; 95% CI 2.312-3.493, p = 0.001) and multivariable Cox regression (HR 1.820; 95% CI 1.164-2.847, p = 0.009). Additionally, a diagnosis of PNI correlated with increased cumulative, as well as distant, metastasis 5-year-recurrence rates (p = 0.027 and p = 0.011, respectively). The application of adjuvant radiotherapy (RT) or radiochemotherapy (RCT) in patients with PNI did not alter OAS or RFS in survival analysis when compared to patients without PNI. The results underline the adverse impact of PNI on the survival and recurrence of surgically treated OSCC patients. Based on our findings, we highly recommend an emphasis on PNI in the TNM staging concept.

Impact of melatonin on periodontal ligament fibroblasts during mechanical strain.

BACKGROUND: The endogenous hormone melatonin regulates the circadian rhythm and impacts on bone metabolism. As patient compliance to wear removable orthodontic appliances is generally higher at night, when melatonin release is increased, a boosting effect on tooth movement would be favourable for therapy, whereas an inhibiting effect would indicate daytime wear to be more therapy-effective. We hypothesize that melatonin has either a stimulating or impeding effect on the expression profile of periodontal ligament fibroblasts (PDLF) during simulated orthodontic compressive and tensile strain, which would suggest either an accelerating or inhibiting impact on orthodontic tooth movement in vivo. METHODS: PDLF were preincubated with melatonin for 24 h and then subjected to tensile or compressive strain to mimic tension and pressure sides in PDL. In addition, the selective melatonin MTNR1B-receptor antagonist 4P-PDOT was used. We investigated melatonin effects on collagen synthesis, expression of inflammatory and bone-remodelling genes/proteins by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and total collagen assays. PDLF-induced osteoclastogenesis was analysed in a coculture model by tartrate-resistant acid phosphatise (TRAP) staining. RESULTS: Expression of melatonin receptors in PDLF was not affected by compressive strain. Melatonin increased expression of inflammatory factors and elevated collagen synthesis during mechanical strain. Melatonin showed no effects on OPG or RANKL expression without mechanical strain, but increased RANKL gene expression during compression. CONCLUSIONS: Expression of melatonin receptors by PDLF enable them to detect fluctuating melatonin concentrations in the periodontal ligament. Melatonin increased collagen synthesis and expression of inflammatory mediators, but had no effect on genes involved in bone remodelling. Therefore, we suggest that melatonin has no accelerating effect on PDLF-induced osteoclastogenesis.

An Evaluation of Different 3D Cultivation Models on Expression Profiles of Human Periodontal Ligament Fibroblasts with Compressive Strain.

The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT-qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain.

Surgical and remote site infections after reconstructive surgery of the head and neck: A risk factor analysis.

The aim of this study was to identify risk factors for surgical (SSI) and remote site (RSI) infections, pathogens and antibiotic resistances in patients after pedicled and free flap reconstruction in the head and neck area. SSI criteria implicated infections affecting superficial or deep tissue in the flap area with purulent discharge, fistula, abscess formation and local infections signs. RSI criteria were defined as infections remote from the surgical site presenting with systemic symptoms like fever, leucocytosis, increase in C-reactive protein, purulent tracheobronchial secretion or deterioration of blood gases. Focus adequate specimen sampling and aerobic and anaerobic incubation and cultivation was performed. Epidemiological data, factors directly related to surgery or reconstruction, perioperative antibiotic regimen, length of stay, autologous blood transfusion and microbiological aspects were retrospectively analysed in 157 patients. 10.8% of patients presented SSI, 12.7% RSI. Cultivated bacteria were sampled from flap sites, blood cultures, central catheters and sputum including mainly gram-negative bacteria (70.3%) being frequently resistant against penicillin (85%) and third generation cephalosporine derivates (48%). Autologous blood transfusion (p = 0.018) and perioperative clindamycin use (p = 0.002) were independent risk factors for overall (SSI and RSI combined) infections. Prior radiation (p = 0.05), autologous blood transfusion (p = 0.034) and perioperative clindamycin use (p = 0.004) were predictors for SSIs. ASA >2 (p = 0.05) was a risk factor for remote site infections and prolonged ICU stay (p = 0.002) was associated with overall infections, especially in irradiated patients. Efforts need to be made in improving patient blood management, antibiotic stewardship and accurate postoperative care to avoid postoperative infections after head and neck reconstructive surgery.