Circulating Cell-Free SEPT9 DNA Methylation in Blood Is a Biomarker for Minimal Residual Disease Detection in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND: Tumorous SEPT9 (septin 9, SEPTIN9) circulating cell-free DNA (ccfDNA) methylation in blood plasma is a powerful biomarker for diagnosis, molecular staging, prognosis, and recurrence monitoring in head and neck squamous cell carcinoma (HNSCC) patients. The present study aimed to evaluate the clinical performance of SEPT9 ccfDNA methylation to detect post-surgical minimal residual disease (MRD) in patients with localized or locally advanced HNSCC treated with curative intent. METHODS: We applied quasi-digital methylation-specific real-time PCR to quantify SEPT9 ccfDNA methylation levels 2 to 30 days post-surgically in plasma from n = 219 prospectively enrolled HNSCC patients. We tested the associations of SEPT9 ccfDNA methylation with clinicopathological parameters and used Kaplan-Meier and Cox proportional hazards analyses for univariate, pairwise bivariate, and multivariate analyses of disease-free survival. RESULTS: Of 219 patients, 26.5% (58/219) were post-surgically SEPT9 ccfDNA methylation positive. SEPT9 ccfDNA methylation positivity was significantly associated with tumor site, American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC; 8th edition) tumor stage, nodal category and extracapsular extension, lymphatic and vascular invasion, and surgical margin. Bivariate Cox proportional hazards analysis proved post-surgical SEPT9 ccfDNA methylation positivity to be an independent prognostic factor tested together with AJCC/UICC tumor stage (SEPT9: hazard ratio [HR] = 2.43, 95% CI, 1.37-4.30, P = 0.002; AJCC/UICC stage: HR = 1.48, 95% CI, 1.11-1.98, P = 0.008). CONCLUSIONS: Post-surgical SEPT9 ccfDNA methylation may aid to identify high-risk HNSCC patients who could benefit from an intensified adjuvant treatment and surveillance.

Polyvalent immunoglobulins with vitamin D3 and vitamin B12 in the treatment of Sjogren's syndrome in a vegetarian with stomatitis, glossodynia, xerostomia, and elevated antinuclear antibodies: Case report
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BACKGROUND: Sjogren's syndrome, involving sicca symptoms with xerostomia, stomatitis, and considerable pain is a difficult-to-treat autoimmune disease where the treatment options are limited and, as in the case of methotrexate, have a low therapeutic index. CASE REPORT: This case report concerns a male patient, aged 75 years and vegetarian, with Sjogren's syndrome subsequently confirmed by salivary gland biopsy. Serum antinuclear antibodies (ANA) were elevated (1 : 320). Low serum vitamin B12 and iron levels could be improved after 20 days using vitamin B12 and iron oral supplements. Despite symptomatic treatment, xerostomia, glossitis, and glossodynia were still present, at times marked, after 12 months when the ANA titer was unchanged. Following treatment with an anti-inflammatory polyvalent immunoglobulin formulation (Lactobin®N, 7 g daily), a bovine colostrum concentrate given orally in combination with oral vitamin D3 (2,000 IU daily), sicca symptoms and xerostomia progressively decreased and at day 750 were confined to occasional and minor glossitis of the upper lip. CONCLUSION: This case report demonstrates the satisfactory control of Sjogren's syndrome using oral polyvalent immunoglobulins with vitamin D3. In contrast to treatment options involving antimalarial drugs and methotrexate, there are no safety issues in patients tolerant to milk products.
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Structured Reporting of Head and Neck Sonography Achieves Substantial Interrater Reliability.

Purpose Ultrasound examinations are often criticized for having higher examiner dependency compared to other imaging techniques. Compared to free-text reporting, structured reporting (SR) of head and neck sonography (HNS) achieves superior time efficiency as well as report quality. However, there are no findings concerning the influence of SR on the interrater reliability (IRR) of HNS. Materials and Methods Typical pathologies (n=4) in HNS were documented by video/images by two certified head and neck ultrasound instructors. Consequently, structured reports of these videos/images were created by n=9 senior physicians at departments of otolaryngology or maxillofacial surgery with DEGUM instructors on staff. Reports (n=36) were evaluated regarding overall completeness and IRR. Additionally, user satisfaction was assessed by a visual analog scale (VAS). Results SR yielded very high report completeness (91.8%) in all four cases with a substantial IRR (Fleiss' κ 0.73). Interrater agreement was high at 87.2% with very good user satisfaction (VAS 8.6). Conclusion SR has the potential to ensure high-quality examination reports with substantial comparability and very high user satisfaction. Furthermore, big data collection and analysis are facilitated by SR. Therefore, process quality, workflow, and scientific output are potentially enhanced by SR.

CTLA4 DNA methylation is associated with CTLA-4 expression and predicts response to immunotherapy in head and neck squamous cell carcinoma.

BACKGROUND: The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value. METHODS: We analyzed CTLA4 promoter methylation in tumors of HNSCC patients (N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort (N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine. RESULTS: Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3+, CD4+, CD8+, and CD45+ immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines. CONCLUSIONS: Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.

CTLA4 , PD-1 , PD-L1 , PD-L2 , TIM-3 , TIGIT , and LAG3 DNA Methylation Is Associated With BAP1 -Aberrancy, Transcriptional Activity, and Overall Survival in Uveal Melanoma.

Uveal melanoma (UM) is an aggressive disease with poor response to oncological treatment, including immunotherapy. Loss of the epigenetic modifier BRCA1-associated protein 1 (BAP1) function drives UM oncogenesis and is associated with an immune-suppressive tumor microenvironment, poor prognosis, and a distinct DNA methylation and gene expression profile. Our study aimed to analyze comprehensively the DNA methylation status of the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4, TIM-3 ( HAVCR2 ), TIGIT , and LAG3 and its association with mRNA expression, BAP1 -aberrancy, and patients' survival. We analyzed the DNA methylation landscape of immune checkpoint genes at single CpG resolution in N=80 UM samples provided by The Cancer Genome Atlas. We analyzed CpG methylation levels of the immune checkpoints with regard to their transcriptional signatures and patient outcomes.Methylation of specific CpG sites within the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4 , TIM-3 , TIGIT , and LAG3 correlated strongly with mRNA expression levels, indicating a strong regulation of gene expression through DNA methylation. Moreover, immune checkpoint gene methylation was strongly associated with BAP1 -mutation status and associated with overall survival in UM. Our data indicate an epigenetic regulation of immune checkpoints through DNA methylation in UM. Further, our data highlight the prognostic significance of DNA methylation of immune checkpoint genes in UM thereby providing a rationale for methylation testing as predictive biomarkers for immunotherapy response.

DNA Methylation and mRNA Expression of OX40 (TNFRSF4) and GITR (TNFRSF18, AITR) in Head and Neck Squamous Cell Carcinoma Correlates With HPV Status, Mutational Load, an Interferon-γ Signature, Signatures of Immune Infiltrates, and Survival.

The tumor necrosis factor receptor superfamily members 4 (TNFRSF4, OX40) and 18 (TNFRSF18, GITR, AITR) are under investigation as targets for immunotherapy of various cancers, including head and neck squamous cell carcinomas. Understanding the regulation of OX40 and GITR, particularly on an epigenetic level, might help to develop companion predictive biomarkers. We conducted broad correlation analyses of DNA methylation of 46 CpG sites within the GITR/OX40 gene locus in head and neck squamous cell carcinomas and normal adjacent tissues provided by The Cancer Genome Atlas (TCGA) Research Network. We analyzed methylation levels with regard to transcriptional gene activity (mRNA expression), human papillomavirus (HPV) infection, differential methylation between tumors and normal adjacent tissues, signatures of immune cell infiltrates, an interferon-γ signature, mutational load, and overall survival. Moreover, we investigated methylation levels in HPV-positive and HPV-negative cell lines and in isolated monocytes, granulocytes, CD8+ and CD4+ T cells, and B cells from peripheral blood from healthy donors. Our results revealed a complex and sequence-contextual methylation pattern in accordance with features of epigenetic regulated genes. We detected significant methylation differences between normal adjacent and tumor tissues, between HPV-positive and HPV-negative tumors, between tumor and immune cells, and significant correlations between methylation and mRNA expression. We further found significant correlations of CpG methylation with overall survival, signatures of immune cell infiltrates, an interferon-γ signature, and mutational load. Our study provides a framework to prospectively test specific CpG sites as biomarkers, in particular in the context of immunotherapies.

Simultaneous treatment with pentoxifylline does not adversely affect the neurotrophic effects of brain-derived neurotrophic factor on spiral ganglion neurons.

The hemorheologic drug pentoxifylline is applied for the treatment of sudden sensorineural hearing loss and tinnitus to improve cochlear microcirculation. Recent studies also suggest protective and trophic effects on neuronal cells. Because the preservation of sensorineural structures of the inner ear is fundamental for normal hearing and hearing restoration with auditory prostheses, pentoxifylline and neurotrophic factors such as brain-derived neurotrophic factor (BDNF) are promising candidates to treat degenerative disorders of the inner ear. We used an in-vitro model to determine the neurotrophic effects of these factors on spiral ganglion cells from postnatal rats. Pentoxifylline, alone and in combination with BDNF, was added at various concentrations to the cultured cells. Cells were immunolabeled and analyzed to determine neuronal survival, neurite length, neuronal branching and morphology. Pentoxifylline did not significantly increase or decrease neuronal survival, neurite length and neuronal branching compared to control cultures. Analysis of cellular morphology showed that diverse neuronal subtypes developed in the presence of pentoxifylline. Our data revealed that pentoxifylline did not interfere with the robust neurotrophic effects of BDNF on spiral ganglion neurons when cultured cells were treated with pentoxifylline and BDNF simultaneously. The results of our study do not suggest major neurotrophic effects of pentoxifylline on cultured spiral ganglion neurons. Because pentoxifylline has no detrimental effects on spiral ganglion neurons and does not reduce the effects of BDNF, both agents could be combined to treat diseases of the inner ear provided that future in vivo experiments and clinical studies support these findings.

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors.

BACKGROUND: Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies. METHODS: We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(-) HNSCC cell lines. RESULTS: We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3-selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1. CONCLUSIONS: Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.

Alveolar Echinococcosis of the Parotid Gland-An Ultra Rare Location Reported from Western Europe.

(1) Background: Alveolar echinococcosis (AE) is restricted to the northern hemisphere with high endemic regions in Central Europe, North and Central Asia as well as Western China. The larval stage of Echinococcus multilocularis (E. multilocularis) causes AE with tumor-like growth. Humans are accidental hosts. This report is on the first case of AE becoming clinically manifested in the parotic gland. (2) Case presentation: A 52-year-old male patient presented with progressive and painful swelling of the right parotid gland persisting for one year. We performed a partial parotidectomy. The histological examination and immunohistological staining revealed larval stage of E. multilocularis. (3) Conclusion: E. multilocularis is known to infect animals and humans coincidentally, and leads to AE. It is one of the most life-threatening zoonoses in Europe. It typically manifests in the liver (50-77%), with further spreading to other organs being a rare phenomenon. Echinococcosis should be considered in the differential diagnosis of lesions of the parotid gland in endemic areas, but AE has not been described so far in the parotid gland as the sole manifestation and, therefore, impedes the correct diagnosis. A complete resection should be the aim, however, preservation of the facial nerve and adjuvant albendazole therapy is mandatory.

Hyaluronic Acid-based Filler Injection: Late-onset Thrombosis of the Frontal Vein.

Thrombosis is a serious complication of a hyaluronic acid-based filler injection. Little is known about the late-onset complications of fillers; therefore, an optimal complication management is necessary. In this case report, we describe a rare complication of thrombosis after a filler injection. A 35-year old woman was admitted to the emergency department, with swelling on her forehead in association with recurrent pain and light flashes in her right eye. Sonographic examination showed a thrombosis of the right frontal vein. The patient reported that a hyaluronic acid filler injection had been administered on the forehead 3 months ago. After several weeks of anticoagulation with heparin and apixaban, the symptoms persisted. The vein with thrombosis was ultimately resected under local anesthesia. Histological findings showed a chronic inflammatory reaction of the tissue to hyaluronic acid. Vascular complications may appear as late-onset complications even several months after the filler injection. Subcutaneous application of low molecular weight heparins is the therapy of first choice. If this treatment is not effective, resection of the thrombosis may be performed.

Relevance of Neurotrophin Receptors CD271 and TrkC for Prognosis, Migration, and Proliferation in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and often has a poor prognosis. The present study investigated the role of the low affinity nerve growth factor receptor CD271 as a putative therapy target in HNSCC. Neurotrophins that bind to CD271 also have a high affinity for the tropomyosin receptor kinase family (Trk), consisting of TrkA, TrkB, and TrkC, which must also be considered in addition to CD271. A retrospective study and functional in vitro cell line tests (migration assay and cell sorting) were conducted in order to evaluate the relevance of CD271 expression alone and with regard to Trk expression. CD271 and Trks were heterogeneously expressed in human HNSCC. The vast majority of tumors exhibited CD271 and TrkA, whereas only half of the tumors expressed TrkB and TrkC. High expression of CD271-positive cells predicted a bad clinical outcome of patients with HNSCC and was associated with distant metastases. However, the human carcinomas that also expressed TrkC had a reduced correlation with distant metastases and better survival rates. In vitro, CD271 expression marked a subpopulation with higher proliferation rates, but proliferation was lower in tumor cells that co-expressed CD271 and TrkC. The CD271 inhibitor LM11A 31 suppressed cell motility in vitro. However, neither TrkA nor TrkB expression were linked to prognosis or cell proliferation. We conclude that CD271 is a promising candidate that provides prognostic information for HNSCC and could be a putative target for HNSCC treatment.

Long-term effect of monophosphoryl lipid A adjuvanted specific immunotherapy in patients with grass pollen allergy.

BACKGROUND: Ultra-short course pollen immunotherapy adjuvanted with monophosphoryl lipid A (MPL) is attractive to conventional allergen-specific immunotherapy (AIT). Long term efficacy of MPL-AIT has not been evaluated. METHODS: 68 patients (age 16.75 ± 5.3 years) with allergic rhinitis to grass pollen were investigated. Group 1: 21 controls; Group 2: 19 after complete AIT, and Group 3: 28 with AIT and treatment cessation: 4 years range 3-6 years ago. RESULTS: The clinical symptoms (running nose, sneezing, conjunctivitis and the weekly overall score) were significantly reduced in patients group 2 and 3 compared with controls without AIT p < 0.0001. T-regulatory cells and TH1/TH2 cytokine pattern did not differ between patient groups. CONCLUSION: The patients in our trial with grass pollen allergy exhibited significant and long-lasting improvements after MPL-AIT, however larger trials are needed to support this finding.

Growth hormone promotes neurite growth of spiral ganglion neurons.

Intact spiral ganglion neurons are a specific requirement for hearing rehabilitation in deaf patients by cochlear implantation. Neurotrophic growth factors have been proposed as effective tools to protect and regenerate spiral ganglion neurons that are degenerated in the majority of patients suffering from hearing loss. Here, we show that growth hormone (GH), a pleiotropic growth factor whose neurotrophic role in the inner ear is still unclear, significantly increases neurite extension, as well as neuronal branching, in spiral ganglion cell cultures derived from early postnatal rats. Our data suggest that GH can act as a potent neurotrophic factor for inner ear neurons, which specifically promotes neurite growth. These effects might be elicited in a direct way or, alternatively, by induction of other growth factors that account for the observed neurotrophic effects. Thus, we conlude that GH might represent a novel candidate for the treatment of neurodegeneration in the hearing-impaired inner ear that has the potential to ultimately improve the performance and outcome of modern auditory implants.

Incidence for Tip Foldover During Cochlear Implantation.

OBJECTIVE: Incidence and clinical presentation of tip foldover during cochlear implantation (CI). STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral university hospital. PATIENTS: One thousand three hundred twenty CI recipients who underwent postoperative Stenvers view, digital computed tomography (CT), or digital volume tomography (DVT). MAIN OUTCOME MEASURE: Tip foldover rates were evaluated with regard to perimodiolar (PM), lateral wall (LW), and mid-scalar (MS) electrode carriers; the electrode insertion angle was estimated and postoperative complications and the necessity for revisions were described. RESULTS: One thousand three hundred twenty CI recipients (1,722 ears) had detailed surgery reports and postoperative imaging available for review. Tip foldover occurred in 15 ears (0.87%) with the highest rate (1.67%) for PM electrodes. In three patients tip foldover was accompanied by costimulation of the facial nerve and one patient reported vertigo. Eleven patients underwent revision surgery. In two of them revision was performed in the same surgical intervention due to abnormality in spread of excitation measurement. In four patients no revision was conducted due to uncompromised aided speech perception. CONCLUSION: In a large academic center with experienced surgeons, tip foldover verified by CT/DVT arose at a rate of 0.87%. PM electrode carriers inherited the highest risk for this electrode malposition. Revision surgery was feasible in these cases.