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Abstract Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with a poor prognosis for survival. Risk factors include alcohol and tobacco abuse and infection with human papilloma virus (HPV). To enhance anti-tumor immune responses immunotherapeutic approaches are approved for recurrent metastatic disease but only approx. 20% of patients respond to checkpoint blockade of the PD-1/PD-L1 axis. Therefore, preclinical research is needed to better understand molecular and cellular processes and to identify new therapeutic targets. Immunocompetent mouse models can serve these purposes but only few are currently available for HPV-positive HNSCC. Here, we established a mouse cell line overexpressing the oncogenes E6/E7 of the HPV16 genome as well as a constitutively active form of H-Ras and studied the anti-tumor immune response upon orthotopic tumor growth at the floor of the mouth. Moreover, we analyzed the same immunoregulatory pathways in samples of HPV-positive cancer patients. T cells in the tumor of mice and humans exhibited high expression of CD39 and CD73, two ectoenzymes involved in the production of immunosuppressive adenosine from ATP, along with increased expression of PD-1, LAG-3 and GITR. Additionally, B cell responses were elevated in tumor-bearing mice, seen as an increase of germinal center, immunoregulatory marginal zone and follicular B cell subtypes. Taken together, this study suggests that the generated mouse model shares characteristics with human disease and can thus be used as a platform to study anti-tumor responses in HPV-positive HNSCC which will help to identify novel therapeutic targets.

Abstract Background Exosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood. Methods Exosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements. Results Our data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes. Conclusions The study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation.

Failure of immunotherapy in head and neck squamous cell carcinoma (HNSCC) patients represents an unmet need to augment leverage of adaptive immunity. Immunogenic cancer-testis antigen (CTA) expression as well as lymphocyte differentiation and function are regulated by DNA methylation. Therefore, epigenetic therapy via inhibition of DNA-Methyltransferases by 5-Aza-2′-deoxycytidine (DAC) serves a promising adjuvant in immunotherapy.We investigated the effects of DAC on CTA expression and proliferative capacity in HNSCC cell lines and on the expression of 12 immune checkpoint molecules (ICM) on lymphocytes of oropharyngeal squamous cell carcinoma (OPSCC) patients and healthy donors.In all cell lines CTA were upregulated accompanied by decreased proliferation. In lymphocytes pronounced alterations of the ICM repertoire were observed, influenced by donor type and subpopulation. On CD39+ CD4 and CD8 T cells, the expression of co-stimulatory ICM GITR and OX40 increased dose dependently, whereas expression decreased on CD39- CD4 T cells. PD1 expression increased primarily on CD39+ CD8 T cells and decreased on CD39- CD4 T cells. CD27 expression decreased primarily in CD8 T cells, but increased in CD39- CD4 T cells, whereas ICOS expression was lowered in both CD39+ and CD39- subsets of CD4 as well as CD8 T cells.DAC treatment increased immunogenicity and decreased proliferation in HNSCC cells while enhancing expression of co-stimulatory ICM GITR and OX40. We propose low dose DAC treatment as a adjuvant to immunotherapy.

Hereditary angioedema (HAE) is an inherited disorder causing attacks of subcutaneous tissue or mucosa swelling. The disease burden and attack frequencies vary significantly among patients. This is the first pilot study investigating small extracellular vesicles (sEV) as potential disease modulators in HAE.Plasma-derived sEVs from HAE patients and healthy donors (HD) were thoroughly characterized by Western blot, transmission electron microscopy, nanoparticle tracking and bead-based flow cytometry. The miRNA content of sEVs was examined by nCounter technology and used to predict sEV-based pathomechanisms in silico. All sEV readouts were analyzed regarding HAE-related changes and associations with clinical parameters and attack frequency.Total sEV protein levels were elevated in HAE patients compared to HD. In HAE patients, lower levels of sEVs carrying CD8, CD209, CD81, CD24 and CD44 were measured. sEV miRNA profiling revealed 84 HAE-exclusive and 30 significantly HAE-upregulated candidates. Core hubs of their predicted interaction networks were AGO2, VEGF, RGS5, MTA1, IFG1 and BAX. A set of 12 and 36 sEV miRNAs were restricted to patients with absent attacks or patients with present attacks during prophylactic therapy, respectively.sEVs, especially sEV miRNAs, could contribute to disease pathogenesis and differential attack frequencies. They emerged as disease modulators in HAE and require further study to reveal underlying mechanisms.

Background Hereditary angioedema with normal C1-inhibitor (HAE-nC1-INH) is a rare genetic disease. The symptoms can resemble other forms of hereditary angioedema (HAE), but the specific laboratory values are inconspicuous. The knowledge about treatment strategies in HAE-nC1-INH remains insufficient; most of the drugs are only licensed and approved for other types of HAE.Methods An analysis of all patients with HAE-nC1-INH was carried out in a certified angioedema treatment center in southern Germany. Only patients with a confirmed HAE-nC1-INH mutation were included. The impact of disease was monitored with validated questionnaires.Results Eighteen patients were included: two families with a factor XII mutation and seven families with a plasminogen mutation. All individuals received icatibant for on-demand therapy—efficient treatment response was reported. Three patients were severely affected, and prophylaxis was initiated with lanadelumab. According to the questionnaires, the clinical course and symptoms improved significantly under this prophylactic regime.Conclusion This is one of the first descriptions of the clinical outcomes as a response to prophylactic treatment with lanadelumab in HAE-nC1-INH patients with a known mutation. The therapeutic management of HAE-1 and HAE-2 should also be the basis of HAE-nC1-INH, including prophylaxis.

Abstract Background Head and neck squamous cell carcinoma (HNSCC) represents a common and heterogeneous malignancy of the oral cavity, pharynx and larynx. Surgery and radio(chemo)therapy are the standard treatment options and also have great influence on the composition of the tumor microenvironment and immune cell functions. However, the impact of radio(chemo)therapy on the distribution and characteristics of circulating monocyte subsets in HNSCC are not fully understood. Methods Expression patterns of adhesion molecules and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor) and checkpoint molecule PD-L1 (programmed cell death ligand-1) were investigated upon radio(chemo)therapeutic treatment using flow cytometry. Furthermore, comprehensive analysis of plasma cytokines was performed before and after treatment using ELISA measurements. Results Our data reveal a partial recovery of circulating monocytes in HNSCC patients upon radio(chemo)therapeutic treatment, with differential effects of the individual therapy regimen. PD-L1 expression on non-classical monocytes significantly correlates with the individual plasma levels of chemokine CXCL11 (C-X-C motif chemokine 11). Conclusions Further comprehensive investigations on larger patient cohorts are required to elucidate the meaningfulness of peripheral blood monocyte subsets and chemokine CXCL11 as potential bioliquid indicators in HNSCC with regard to therapy response and the individual immunological situation.

Due to their high developmental diversity and different regulatory and functional roles, B cell subpopulations can promote or inhibit tumor growth. An orthotopic murine HNSCC model was applied to investigate the B cell composition and function in HNSCCs. Using flow cytometry approaches, cells from the spleen, lymph nodes and tumors were analyzed. Additionally, immunoglobulin (Ig) levels post-tumor induction were tracked via enzyme-linked immunosorbent assays (ELISA). Following tumor induction, GCs, as well as increasing numbers of GL7+CD95+ GC B cells in the spleen and tumor tissues, were detected. In parallel, we observed CD39+CD73+ B cells in tumors and spleens of tumor-bearing mice. Notably, CD39+CD73+ expression was primarily detected on MZ B cells and to a lesser extent on follicular (FO) and non-follicular, newly formed (NF) B cells, supposing an immunosuppressive function of MZ B cells in the TME. Parallel to increased MZ B cell numbers in secondary lymphoid organs (SLOs) as well as in the tumor tissue, IgM antibody (Ab) levels rose continuously. In contrast, IgG1, IgG2, and IgG3 levels increased at later time points. Understanding the complex interactions between B cell subsets and the TME could lead to new strategies for enhancing the treatment and prognosis of HNSCC patients.

IntroductionThe human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets.Materials and methodsSnap-frozen tumor (NLigand/RNA=40), healthy mucosa (NRNA=6), and healthy tonsils (NLigand=5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (NAb=27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins).Results183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR

Abstract Introduction Demographics are important prognostic factors in malignant diseases. A nationwide analysis concerning the prognostic impact of demographics in head and neck cancer (HNC) patients (HNCP) has not been performed previously. Methods A retrospective analysis of data from the Center for Cancer Registry Data (ZfKD) and the Federal Statistical Office (Destatis) between 2002 and 2017 was performed. A total of 212′920 HNCP were included. Incidence, tumor stage, age development, sex distribution, age‐, residence‐, and diagnosis‐time‐specific survival were examined. Results Mean age of HNCP increased more rapidly than in the general population (slope coefficient: 0.29 vs. 0.20; p 70 years was found. Furthermore, an OS disadvantage was observed for East Germany compared to West Germany (median 47 vs. 60 months; p

Abstract Background Demographic development in rural and urban areas differs substantially. Demographics and access to specialized head and neck cancer centers may affect head and neck cancer patients' (HNCP) outcomes. Here, we compare epidemiological indicators and outcomes of HNCP in rural and urban Germany. Patients and Methods In a retrospective analysis of data from the Center for Cancer Registry Data (ZfKD) between 2002 and 2017, 212,920 HNCP were included. Incidence, demographics, travel distance to specialized centers, and ground values were compared between rural and urban areas with a focus on their association with patient outcomes. Results The mean age of HNCP was significantly higher in urban areas (mean difference = 1.4 years; p

Aim: Recently, a tumor cell-platelet interaction was identified in different tumor entities, resulting in a transfer of tumor-derived RNA into platelets, named further “tumor-educated platelets (TEP)”. The present pilot study aims to investigate whether such a tumor-platelet transfer of RNA occurs also in patients suffering from head and neck squamous cell carcinoma (HNSCC). Methods: Sequencing analysis of RNA derived from platelets of tumor patients (TPs) and healthy donors (HDs) were performed. Subsequently, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used for verification of differentially expressed genes in platelets from TPs and HDs in a second cohort of patients and HDs. Data were analyzed by applying bioinformatic tools. Results: Sequencing of RNA derived from the tumor as well as from platelets of TPs and HDs revealed 426 significantly differentially existing RNA, at which 406 RNA were more and 20 RNA less abundant in platelets from TPs in comparison to that of HDs. In TPs’ platelets, abundantly existing RNA coding for 49 genes were detected, characteristically expressed in epithelial cells and RNA, the products of which are involved in tumor progression. Applying bioinformatic tools and verification on a second TP/HD cohort, collagen type I alpha 1 chain (COL1A1) and zinc finger protein 750 (ZNF750) were identified as the strongest potentially platelet-RNA-sequencing (RNA-seq)-based biomarkers for HNSCC. Conclusions: These results indicate a transfer of tumor-derived messenger RNA (mRNA) into platelets of HNSCC patients. Therefore, analyses of a patient’s platelet RNA could be an efficient option for liquid biopsy in order to diagnose HNSCC or to monitor tumorigenesis as well as therapeutic responses at any time and in real time.

BackgroundFor the surgical treatment of early-stage laryngeal cancer, the use of transoral laser microsurgery (TLM) has emerged as the gold standard. However, this procedure requires a straight line of sight to the operating field. Therefore, the patient's neck needs to be brought into a hyperextended position. In a considerable number of patients, this is not possible due to anomalies in the cervical spine anatomy or soft tissue scarring, e.g., after radiation. In these cases, adequate visualization of relevant laryngeal structures cannot be ensured using a conventional rigid operating laryngoscope, which may negatively affect the outcome of these patients.MethodsWe present a system based on a 3D-printed prototype of a curved laryngoscope with three integrated working channels (sMAC). The curved profile of the sMAC-laryngoscope is specifically adapted to the nonlinear anatomy of the upper airway structures. The central working channel provides access for flexible video endoscope imaging of the operating field while the two remaining channels provide access for flexible instrumentation. In a user study (n = 11), visualization and reachability of relevant laryngeal landmarks as well as the feasibility of basic surgical procedures with the proposed system were examined in a patient simulator. In a second setup, the system was evaluated for its applicability in a human body donor.ResultsAll participants of the user study were able to visualize, reach and manipulate the relevant laryngeal landmarks. Reaching those took significantly less time in the second attempt compared to the first one (27.5 s ± 5.2 s vs. 39.7 s ± 16.5 s, p = 0.008) indicating a significant learning curve for handling the system. Instrument changes were performed quickly and reliably by all participants (10.9 s ± 1.7 s). All participants were able to bring the bimanual instruments into position for a vocal fold incision. Relevant laryngeal landmarks could be visualized and reached in the human body donor setup.ConclusionPossibly, the proposed system may develop into an alternative treatment option for patients with early-stage laryngeal cancer and restricted mobility of the cervical spine in the future. Further improvements of the system could include finer end effectors and a flexible instrument with a laser cutting tool.

BackgroundEpithelial to mesenchymal transition (EMT) is a key process in carcinogenesis of head and neck squamous cell carcinoma (HNSCC), contributing to tumor invasiveness, distant metastasis, and recurrence. Exosomes are known mediators and regulators of EMT. Here, we analyze the impact of exosomes that were primed by conventional therapy on EMT modulation.MethodsPlasmas of n = 22 HNSCC patients were collected before and after standard of care surgery and adjuvant or primary (chemo)radiotherapy. Exosomes were isolated by size exclusion chromatography. Upon co-incubation of exosomes with HNSCC cells, the cellular EMT profile was analyzed by flow cytometry and RT-qPCR. Wound healing assays were performed to evaluate migratory potential of exosome-treated cells.ResultsReduction of total exosome protein after therapy and in vitro exosome induced EMT profiles were dependent on the type of treatment. Exosomal TFG-β and miRNA cargo were partly responsible for observed exosome induced EMT changes. Exosomes from recurrent patients induced higher tumor cell migration after therapy than exosomes from disease-free patients.ConclusionsHNSCC patients’ exosomes from timepoints before and after therapy were able to confer therapy induced EMT modulation in vitro and have the potential to monitor the EMT process. Exosome induced changes in migratory potential emerged as discriminants of therapy outcome.

Background: Head and neck squamous cell carcinomas (HNSCC) lack tumor-specific biomarkers. Exosomes from HNSCC patients carry immunomodulatory molecules, and correlate with clinical parameters. We compared miRNA profiles of plasma- and saliva-derived exosomes to reveal liquid biomarker candidates for HNSCC.Methods: Exosomes were isolated by differential ultracentrifugation from corresponding plasma and saliva samples from 11 HNSCC patients and five healthy donors (HD). Exosomal miRNA profiles, as determined by nCounter® SPRINT technology, were analyzed regarding their diagnostic and prognostic potential, correlated to clinical data and integrated into network analysis.Results: 119 miRNAs overlapped between plasma- and saliva-derived exosomes of HNSCC patients, from which 29 tumor-exclusive miRNAs, associated with TP53, TGFB1, PRDM1, FOX O 1 and CDH1 signaling, were selected. By intra-correlation of tumor-exclusive miRNAs from plasma and saliva, top 10 miRNA candidates with the strongest correlation emerged as diagnostic panels to discriminate cancer and healthy as well as potentially prognostic panels for disease-free survival (DFS). Further, exosomal miRNAs were differentially represented in human papillomavirus (HPV) positive and negative as well as low and high stage disease.Conclusion: A plasma- and a saliva-derived panel of tumor-exclusive exosomal miRNAs hold great potential as liquid biopsy for discrimination between cancer and healthy as well as HPV status and disease stage. Exosomal miRNAs from both biofluids represent a promising tool for future biomarker studies, emphasizing the possibility to substitute plasma by less-invasive saliva collection.

BackgroundExosomes contribute to immunosuppression in head and neck squamous cell carcinoma (HNSCC), a tumor entity which lacks specific tumor biomarkers. Plasma-derived exosomes from HNSCC patients correlate with clinical parameters and have potential as liquid biopsy. Here, we investigate the cargo and functional profile of saliva-derived exosomes from HNSCC patients and their potential as non-invasive biomarkers for disease detection and immunomodulation.MethodsExosomes were isolated from saliva of HNSCC patients (n = 21) and healthy donors (HD, n = 12) by differential ultracentrifugation. Surface values of immune checkpoints and tumor associated antigens on saliva-derived exosomes were analyzed by bead-based flow cytometry using CD63 capture. Upon co-incubation with saliva-derived exosomes, activity and proliferation of T cells were assessed by flow cytometry (CD69 expression, CFSE assay). Adenosine levels were measured by mass spectrometry after incubation of saliva-derived exosomes with exogenous ATP. miRNA profiling of saliva-derived exosomes was performed using the nCounter® SPRINT system.ResultsSaliva-derived, CD63-captured exosomes from HNSCC patients carried high amounts of CD44v3, PDL1 and CD39. Compared to plasma, saliva was rich in tumor-derived, CD44v3+ exosomes and poor in hematopoietic cell-derived, CD45+ exosomes. CD8+ T cell activity was attenuated by saliva-derived exosomes from HNSCC patients, while proliferation of CD4+ T cells was not affected. Further, saliva-derived exosomes produced high levels of immunosuppressive adenosine. 62 HD- and 31 HNSCC-exclusive miRNAs were identified. Samples were grouped in “Healthy” and “Cancer” based on their saliva-derived exosomal miRNA profile, which was further found to be involved in RAS/MAPK, NF-κB complex, Smad2/3, and IFN-α signaling.ConclusionsSaliva-derived exosomes from HNSCC patients were enriched in tumor-derived exosomes whose cargo and functional profile reflected an immunosuppressive TME. Surface values of CD44v3, PDL1 and CD39 on CD63-captured exosomes, adenosine production and the miRNA cargo of saliva-derived exosomes emerged as discriminators of disease and emphasized their potential as liquid biomarkers specific for HNSCC.

The transcriptional co-activator BOB.1/OBF.1 is expressed in both B and T cells. The main characteristic of conventional BOB.1/OBF.1 deficient mice is the complete absence of germinal centers (GCs). This defect was mainly attributed to the defective B cell compartment. However, it is unknown whether and how BOB.1/OBF.1 expression in T cells contributes to the GC reaction. To finally clarify this question, we studied the in vivo function of BOB.1/OBF.1 in CD4+ T and follicular T helper (TFH) cell subpopulations by conditional mutagenesis, in the presence of immunocompetent B lymphocytes. BOB.1/OBF.1 deletion in CD4+ T as well as TFH cells resulted in impaired GC formation demonstrating that the impaired GC reaction described for conventional BOB.1/OBF.1-deficient mice cannot exclusively be traced back to the B cell compartment. Furthermore, we show a requirement of BOB.1/OBF.1 for T helper (TH) cell subsets, particularly for TFH cell differentiation.

Chemoradiotherapy (CRT) is a standard treatment for advanced head and neck squamous cell carcinoma (HNSCC). Unfortunately, not all patients respond to this therapy and require further treatment, either salvage surgery or palliative therapy. The addition of immunotherapy to CRT is currently being investigated and early results describe a mixed response. Therefore, it is important to understand the impact of CRT on the tumor microenvironment (TME) to be able to interpret the results of the clinical trials. Paired biopsies from 30 HNSCC patients were collected before and three months after completion of primary CRT and interrogated for the expression of 1392 immune- and cancer-related genes. There was a relevant difference in the number of differentially expressed genes between the total cohort and patients with residual disease. Genes involved in T cell activation showed significantly reduced expression in these tumors after therapy. Furthermore, gene enrichment for several T cell subsets confirmed this observation. The analysis of tissue resident memory T cells (TRM) did not show a clear association with impaired response to therapy. CRT seems to lead to a loss of T cells in patients with incomplete response that needs to be reversed. It is not clear whether the addition of anti-PD-1 antibodies alone to CRT can prevent treatment failure, as no upregulation of the targets was measurable in the TME.

Btk and Vav proteins are all components of the signalosome that builds upon B cell receptor (BCR) activation. However, the role of Vav proteins within the signalosome is quite complex and not yet fully understood. Until now, studies of these have focused predominantly on a deficiency of Vav proteins alone or in combination with other Vav protein family members. Since a physical association of Btk with Vav was shown previously, we asked whether these molecules lie in the same or independent signaling pathways. By analyzing Vav1 and Vav3 single knock-out mice and generating double-knock-out animals deficient for either Vav1 or Vav3 and Btk, we observed, in line with previous publications, no severe B cell developmental defects when either Vav1 or Vav3 alone are not expressed. However, a simultaneous deficiency of Btk together with either Vav1 or Vav3 leads to a severe reduction of splenic B cells, which exhibit an immature phenotype. B cell developmental defects of Btk/Vav1-double deficient mice in the periphery were more severe than those observed in Btk-single-deficient animals. Additionally, morphological changes in splenic microarchitecture were observed in double- but also in single-knock-out mutants. These observations were accompanied by reduced BCR-induced Ca2+ mobilization, proliferation, germinal center formation and immunoglobulin secretion. Although deletion of Btk alone impaired Ca2+ mobilization upon BCR activation, the defect was even more severe when Vav1 or Vav3 were also mutated, indicating that Btk and the Vav proteins act in separate pathways that converge on Ca2+ signaling. In vitro ASC differentiation suggests that both B and T cells contribute to the observed phenotype of a Btk/Vav-double deficiency. Our results show that Vav proteins and Btk are both components of the BCR-activated signalosome but control separate signaling pathways important for B cell development.

Background and objectives: Bradykinin-mediated angioedema (AE) induced by antihypertensive drugs primarily affect the head and neck region and may occur even after several years of uneventful treatment. Many facts about the clinical course remain unknown. Diagnosis is not easy, as the clinical appearance resembles allergic AE. No specific diagnostic markers are known and no officially approved treatment is currently available. Methods: All patients who presented to the ORL department between 2010 and 2016 with acute AE were included. Those with a history of renin-angiotensin-aldosterone system (RAAS) blocker intake were defined as RAE and their pathophysiological characteristics and clinical course of the disease were analyzed. Results: A total of 84 patients (median age of 71 years) with RAE was identified. The majority (80%) was on ACE inhibition. The oral cavity was most often affected. Nearly 60% were medicated for more than 1 year before AE occurred. RAE occurred more often during the morning hours. The necessity for emergency intubation and/or tracheostomy was nine times higher in patients with acute RAE compared to patients with AE due to other reasons. Conclusions: Event-free, long-term therapy with an RAAS blocker before the first development of edema does not exclude RAE. RAE is associated with an increased risk for emergency airway management. Abbreviations ACE: Angiotensin Converting Enzyme; ACEi AE: ACE inhibitor-induced angioedema; AE: Angioedema; ARB: Angiotensin II receptor 1 blocker; C1 INH: C1 Inhibitor; CI: Confidence Interval; CRP: C-reactive protein; DPP IV: Dipeptidyl peptidase IV; ENT: Ear, Nose and Throat; HAE: Hereditary Angioedema; ICD 10: International Statistical Classification of Diseases and Related Health Problems, 10th Edition; OR: Odds Ratio; ORL: Otorhinolaryngology; RAAS: Renin-Angiotensin-Aldosterone System; RAE: RAAS-blocker-induced angioedema

Cone beam computed tomography (CBCT) is increasingly used for dental and maxillofacial imaging. The occurrence of incidental findings has been reported, but clinical implications of these findings remain unclear. The study’s aim was to identify the frequency and clinical impact of incidental findings in CBCT. A total of 374 consecutive CBCT examinations of a 3 year period were retrospectively evaluated for the presence, kind, and clinical relevance of incidental findings. In a subgroup of 54 patients, therapeutic consequences of CBCT incidental findings were queried from the referring physicians. A total of 974 incidental findings were detected, involving 78.6% of all CBCT, hence 2.6 incidental findings per CBCT. Of these, 38.6% were classified to require treatment, with an additional 25.2% requiring follow-up. Incidental findings included dental pathologies in 55.3%, pathologies of the paranasal sinuses and airways in 29.2%, osseous pathologies in 14.9% of all CBCT, and findings in the soft tissue or TMJ in few cases. Clinically relevant dental incidental findings were detected significantly more frequently in CBCT for implant planning compared to other indications (60.7% vs. 43.2%, p < 0.01), and in CBCT with an FOV ≥ 100 mm compared to an FOV < 100 mm (54.7% vs. 40.0%, p < 0.01). Similar results were obtained for paranasal incidental findings. In a subgroup analysis, 29 of 54 patients showed incidental findings which were previously unknown, and the findings changed therapeutical management in 19 patients (35%). The results of our study highlighted the importance of a meticulous analysis of the entire FOV of CBCT for incidental findings, which showed clinical relevance in more than one in three patients. Due to a high number of clinically relevant incidental findings especially in CBCT for implant planning, an FOV of 100 × 100 mm covering both the mandible and the maxilla was concluded to be recommendable for this indication.

Abstract CD8+ cytotoxic T-cell (CTL) specific for non-mutated, wild type (wt) sequence p53 peptides derived from wt or mutant p53 molecules expressed in head and neck squamous cell carcinomas (HNSCC) have been detected in the circulation of patients with this disease. The frequency and differentiation/maturation phenotypes of these anti-tumor specific CTL can reflect the host’s immunologic response. Therefore, we investigated the frequency and phenotypes of wt sequence p53 peptide-specific CTL in patients with HNSCC (n = 33) by flow cytometric analysis using HLA-A*0201 tetrameric peptides (tet) complexed with the wt sequence p53264–272 or p53149–157 peptide and co-staining with phenotypic markers. One main finding was that increasing frequencies of tet+ CD8+ T cells in patients’ circulation correlated with increased frequencies of inactive naïve tet+ cells, while those with effector memory and terminally differentiated phenotypes, which are associated with positive anti-tumor immune responses, decreased. We also found that the frequency of circulating tet+ CD8+ T cells negatively correlated with p53 expression in tumor tissues and tumor stage. Our findings support further clinical-based investigations to define the frequencies and phenotypes of wt sequence p53 peptide-specific CD8+ T cells to predict disease severity, enhance selection of patients for inclusion in vaccination trials and highlight prerequisites to enhance immune susceptibility by activation of inactive naïve tet+ T cells and/or enhancing circulating effector T cell activity by checkpoint blockage.

Abstract Background The aim of this study was to evaluate the value of 18F–FDG PET/CT (PET/CT) and MRI for local and/or whole-body restaging of adenoid cystic carcinoma of the head and neck (ACC). Methods Thirty-six patients with ACC underwent conventional MRI of the head and neck and a whole-body PET/CT and were analysed with regards to detection of a local tumor recurrence, lymph node or distant metastases. A consensus interpretation of all available imaging data was used as reference standard. Sensitivity, specificity, diagnostic accuracy, positive and negative predictive values were calculated for MRI and PET/CT. Results The sensitivity of PET/CT and MRI was 96% (89%), specificity 89% (89%), PPV 96% (96%), NPV 89% (73%) and accuracy 94% (89%) for detection of local tumors. Additionally, PET/CT revealed lymph node metastases in one patient and distant metastases in 9/36 patients. In three patients secondary primaries were found. Conclusions Whole-body PET/CT in addition to MRI of the head and neck improves detection of local tumour and metastastic spread in ACC.

Abstract Background Angioedema is a common presentation in the emergency department (ED). Airway angioedema can be fatal; therefore, prompt diagnosis and correct treatment are vital. Objective of the review Based on the findings of two expert panels attended by international experts in angioedema and emergency medicine, this review aims to provide practical guidance on the diagnosis, differentiation, and management of histamine- and bradykinin-mediated angioedema in the ED. Review The most common pathophysiology underlying angioedema is mediated by histamine; however, ED staff must be alert for the less common bradykinin-mediated forms of angioedema. Crucially, bradykinin-mediated angioedema does not respond to the same treatment as histamine-mediated angioedema. Bradykinin-mediated angioedema can result from many causes, including hereditary defects in C1 esterase inhibitor (C1-INH), side effects of angiotensin-converting enzyme inhibitors (ACEis), or acquired deficiency in C1-INH. The increased use of ACEis in recent decades has resulted in more frequent encounters with ACEi-induced angioedema in the ED; however, surveys have shown that many ED staff may not know how to recognize or manage bradykinin-mediated angioedema, and hospitals may not have specific medications or protocols in place. Conclusion ED physicians must be aware of the different pathophysiologic pathways that lead to angioedema in order to efficiently and effectively manage these potentially fatal conditions.

The molecular cargo of tumor-cell-derived exosomes (TEX) mimics that of parental tumor cells. Thus, TEX could potentially serve as noninvasive biomarkers of cancer progression. However, separation of TEX from non-TEX in patients’ plasma requires tumor antigen-specific detection reagents. CD44v3 has been of interest as a potential biomarker of disease progression in HNSCC, because its overexpression in tumor cells associates with poor outcome. Here, CD44v3+ TEX immunocaptured from plasma of 44 HNSCC patients and 7 healthy donors (HDs) were evaluated as potential biomarkers of disease activity and stage. Exosomes were isolated from plasma of by size exclusion chromatography. Using anti-CD44v3 or anti-CD3 mAbs on beads, CD44v3+ TEX CD3(-)TEX-enriched exosomes were immunocaptured from supernatants of nonmalignant or HNSCC cell lines and from patients’ plasma. On-bead flow cytometry was used for the detection of FAS-L, PD-L1, TGFF-β. CSPG4 or EGFR on exosomes. The TEX expression profiles were correlated to clinicopathological parameters. Relative florescence intensity (RFI) values for CD44v3 were higher (p