Procoagulant platelets promote immune evasion in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive tumor entity, in which immune checkpoint (IC) molecules are primarily synthesized in the tumor environment. Here, we report that procoagulant platelets bear large amounts of such immunomodulatory factors and that the presence of these cellular blood components in TNBC relates to pro-tumorigenic immune cell activity and impaired survival. Mechanistically, tumor-released nucleic acids attract platelets into the aberrant tumor microvasculature where they undergo procoagulant activation, thus delivering specific stimulatory and inhibitory IC molecules. This concomitantly promotes pro-tumorigenic myeloid leukocyte responses and compromises anti-tumorigenic lymphocyte activity, ultimately supporting tumor growth. Interference with platelet-leukocyte interactions prevented immune cell misguidance and suppressed tumor progression, nearly as effective as systemic IC inhibition. Hence, our data uncover a self-sustaining mechanism of TNBC in utilizing platelets to misdirect immune cell responses. Targeting this irregular multicellular interplay might represent a novel immunotherapeutic strategy in TNBC without side effects of systemic IC inhibition.

Impact of 18F-FDG PET Intensity Normalization on Radiomic Features of Oropharyngeal Squamous Cell Carcinomas and Machine Learning-Generated Biomarkers.

We aimed to investigate the effects of 18F-FDG PET voxel intensity normalization on radiomic features of oropharyngeal squamous cell carcinoma (OPSCC) and machine learning-generated radiomic biomarkers. Methods: We extracted 1,037 18F-FDG PET radiomic features quantifying the shape, intensity, and texture of 430 OPSCC primary tumors. The reproducibility of individual features across 3 intensity-normalized images (body-weight SUV, reference tissue activity ratio to lentiform nucleus of brain and cerebellum) and the raw PET data was assessed using an intraclass correlation coefficient (ICC). We investigated the effects of intensity normalization on the features' utility in predicting the human papillomavirus (HPV) status of OPSCCs in univariate logistic regression, receiver-operating-characteristic analysis, and extreme-gradient-boosting (XGBoost) machine-learning classifiers. Results: Of 1,037 features, a high (ICC ≥ 0.90), medium (0.90 > ICC ≥ 0.75), and low (ICC < 0.75) degree of reproducibility across normalization methods was attained in 356 (34.3%), 608 (58.6%), and 73 (7%) features, respectively. In univariate analysis, features from the PET normalized to the lentiform nucleus had the strongest association with HPV status, with 865 of 1,037 (83.4%) significant features after multiple testing corrections and a median area under the receiver-operating-characteristic curve (AUC) of 0.65 (interquartile range, 0.62-0.68). Similar tendencies were observed in XGBoost models, with the lentiform nucleus-normalized model achieving the numerically highest average AUC of 0.72 (SD, 0.07) in the cross validation within the training cohort. The model generalized well to the validation cohorts, attaining an AUC of 0.73 (95% CI, 0.60-0.85) in independent validation and 0.76 (95% CI, 0.58-0.95) in external validation. The AUCs of the XGBoost models were not significantly different. Conclusion: Only one third of the features demonstrated a high degree of reproducibility across intensity-normalization techniques, making uniform normalization a prerequisite for interindividual comparability of radiomic markers. The choice of normalization technique may affect the radiomic features' predictive value with respect to HPV. Our results show trends that normalization to the lentiform nucleus may improve model performance, although more evidence is needed to draw a firm conclusion.

Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.

Introduction: Head and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches. Methods: In the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. Results: In vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor. Discussion: Hence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.

Comparison of Two Measurement Paradigms to Determine Electrically Evoked Cochlear Nerve Responses and Their Correlation to Cochlear Nerve Cross-section in Infants and Young Children With Cochlear Implant.

INTRODUCTION: Electrically evoked compound action potentials (ECAPs) are used for intra-/postoperative monitoring with intracochlear stimulation of cochlear implants. ECAPs are recorded in MED-EL (Innsbruck, Austria) implants using auditory response telemetry (ART), which has been further developed with automatic threshold determination as AutoART. The success of an ECAP measurement also depends on the number of available spiral ganglion cells and the bipolar neurons of the cochlear nerve (CN). It is assumed that a higher population of spiral ganglion cell implies a larger CN cross-sectional area (CSA), which consequently affects ECAP measurements. METHODS: Intraoperative ECAP measurements from 19 implanted ears of children aged 8 to 18 months were retrospectively evaluated. A comparison and correlation of ART/AutoART ECAP thresholds/slopes at electrodes E2 (apical), E6 (medial), E10 (basal), and averaged E1 to E12 with CN CSA on magnetic resonance imaging was performed. RESULTS: A Pearson correlation of the ART/AutoART ECAP thresholds/slopes for E2/E6/E10 and the averaged electrodes E1 to E12 showed a significant correlation. The CN CSA did not correlate significantly with the averaged ART/AutoART ECAP thresholds/slopes across all 12 electrodes. SUMMARY: AutoART provides reliable measurements and is therefore a suitable alternative to ART. No significant influence of CN CSA on ECAP thresholds/slopes was observed. A predictive evaluation of the success of ECAP measurements based on CN CSA for a clinical setting cannot be made according to the present data.

ART and AutoART ECAP measurements and cochlear nerve anatomy as predictors in adult cochlear implant recipients.

PURPOSE: The purpose of this retrospective study is to compare the results of electrically evoked compound action potential (ECAP) measurements using automatic auditory response telemetry (AutoART) with those obtained by ART in adults. The study also aimed to evaluate the predictive value of intraoperative ART and AutoART ECAPs for speech intelligibility (SI) and hearing success (HS), and to determine if cochlear nerve (CN) cross-sectional area (CSA) obtained preoperatively by magnetic resonance imaging (MRI) scans could predict ART and AutoART ECAPs and SI and HS outcome. METHODS: The study analyzed and correlated ART and AutoART ECAP thresholds at electrodes E2, E6, and E10, as well as averaged ECAP thresholds over electrodes E1-E12, using data from 32 implants. Correlations were also examined for ART and AutoART ECAP slopes. In addition, averaged ART and AutoART ECAP thresholds and slopes over all 12 electrodes for each participant were correlated with CN CSA measured from MRI sequences. SI of the monosyllabic Freiburg Speech Test at 65 dB sound pressure level was examined along with averaged ART and AutoART thresholds and slopes over all 12 electrodes. A parallel analysis was performed for HS, derived from the difference between baseline and 6-month SI. Finally, correlations between CN CSA and SI, as well as CN CSA and HS were examined. RESULTS: The results of the study showed a significant positive correlation between ART and AutoART ECAP thresholds and as well as slopes for E2, E6, E10 and averaged thresholds and slopes of E1-E12. However, no significant correlation was observed between ART and AutoART averaged ECAP thresholds and slopes and either SI and HS or CN CSA. Furthermore, no significant correlation was found between CN CSA and SI and HS. CONCLUSION: While AutoART is a reliable and safe program for measuring ECAPs in adults, the study found no preoperative prognostic information on intraoperative ECAP results using parameters extracted from current MRI sequences or pre-/intraoperative information on subsequent hearing outcome using ECAP and CN CSA.

Comparison of speech perception in bimodal cochlear implant patients with respect to the cochlear coverage.

BACKGROUND: The hearing success of patients with bimodal fitting, utilizing both a cochlear implant (CI) and a hearing aid (HA), varies considerably: While some patients benefit from bimodal CI and HA, others do not. OBJECTIVES: This retrospective study aimed to investigate speech perception in bimodally fitted patients and compare it with the cochlear coverage (CC). METHODS: The CC was calculated with the OTOPLAN software, measuring the cochlear duct length on temporal bone CT scans of 39 patients retrospectively. The patients were categorized into two groups: CC ≤ 65% (CC500) and CC > 65% (CC600). Monaural speech intelligibility for monosyllables at a sound pressure level (SPL) of 65 dB in a free-field setting was assessed before and after CI at various time points. The two groups, one with preoperative HA and one with postoperative CI, were compared. Additionally, speech intelligibility was correlated with CC in the entire cohort before CI and at the last available follow-up (last observation time, LOT). RESULTS: Overall, there was no significant difference in speech intelligibility between CC500 and CC600 patients, with both groups demonstrating a consistent improvement after implantation. While CC600 patients tended to exhibit earlier improvement in speech intelligibility, CC500 patients showed a slower initial improvement within the first 3 months but demonstrated a steeper learning curve thereafter. At LOT, the two patient groups converged, with no significant differences in expected speech intelligibility. There was no significant relationship between unimodal/unilateral free-field speech intelligibility and CC. Interestingly, patients with a CC of 70-75% achieved the highest speech intelligibility. CONCLUSION: Despite of the lack of a significant correlation between CC and speech perception, patients appeared to reach their maximum in unimodal/unilateral speech perception primarily at a coverage level of 70-75%. Nevertheless, further investigation is warranted, as CC500 was associated with shorter cochlear duct length, and different types of electrodes were used in both groups.

Significance of endolymphatic sac surgery with and/or without simultaneous cochlea implant surgery in respect of vertigo control and speech perception in patients with Menière's disease.

PURPOSE: The focus on treating patients with Menière's Disease (MD) lies on the reduction of vertigo attacks and the preservation of sensory function. Endolympathic hydrops is considered as an epiphenomenon in MD, which can potentially be altered by endolymphatic sac surgery (ESS). Purpose of the study was to investigate the influences on vertigo control through manipulation of the perilymphatic system with or without ESS. METHODS: Retrospective data analysis of 86 consecutive patients with MD according to current diagnostic criteria after endolymphatic sac surgery alone (ESSalone; n = 45), cochlear implantation (CI) alone (CIalone; n = 12), and ESS with CI (ESS + CI; n = 29), treated at a tertiary referral center. MAIN OUTCOME MEASURES: vertigo control, speech perception pre- and postoperatively. RESULTS: Gender, side, and preoperative treatment were similar in all groups. Age was younger in the ESSalone-group with 56.2 ± 13.0 years (CIalone = 64.2 ± 11.4 years; ESS + CI = 63.1 ± 9.7 years). Definitive MD was present in all the CIalone, in 79.3% of the ESS + CI and in 59.6% of the ESSalone-patients. Likewise, vertigo control rate was 100% in the CIalone, 89.7% in the ESS + CI and 66.0% in the ESSalone-group. CONCLUSIONS: Vertigo control was improved in all three groups, however, superior in groups treated with CI, potentially contributed by the manipulation of both the endo- and perilymphatic systems. A more systematic characterization of the patients with larger case numbers and documentation of follow up data would be needed to evaluate a clinical effect more properly.

5'-Ectonucleotidase CD73/NT5E supports EGFR-mediated invasion of HPV-negative head and neck carcinoma cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) of malignant cells is a driving force of disease progression in human papillomavirus-negative (HPV-negative) head and neck squamous cell carcinomas (HNSCC). Sustained hyper-activation of epidermal growth factor receptor (EGFR) induces an invasion-promoting subtype of EMT (EGFR-EMT) characterized by a gene signature ("'EGFR-EMT_Signature'") comprising 5´-ectonucleotidase CD73. Generally, CD73 promotes immune evasion via adenosine (ADO) formation and associates with EMT and metastases. However, CD73 regulation through EGFR signaling remains under-explored and targeting options are amiss. METHODS: CD73 functions in EGFR-mediated tumor cell dissemination were addressed in 2D and 3D cellular models of migration and invasion. The novel antagonizing antibody 22E6 and therapeutic antibody Cetuximab served as inhibitors of CD73 and EGFR, respectively, in combinatorial treatment. Specificity for CD73 and its role as effector or regulator of EGFR-EMT were assessed upon CD73 knock-down and over-expression. CD73 correlation to tumor budding was studied in an in-house primary HNSCC cohort. Expression correlations, and prognostic and predictive values were analyzed using machine learning-based algorithms and Kaplan-Meier survival curves in single cell and bulk RNA sequencing datasets. RESULTS: CD73/NT5E is induced by the EGF/EGFR-EMT-axis and blocked by Cetuximab and MEK inhibitor. Inhibition of CD73 with the novel antagonizing antibody 22E6 specifically repressed EGFR-dependent migration and invasion of HNSCC cells in 2D. Cetuximab and 22E6 alone reduced local invasion in a 3D-model. Interestingly, combining inefficient low-dose concentrations of Cetuximab and 22E6 revealed highly potent in invasion inhibition, substantially reducing the functional IC50 of Cetuximab regarding local invasion. A role for CD73 as an effector of EGFR-EMT in local invasion was further supported by knock-down and over-expression experiments in vitro and by high expression in malignant cells budding from primary tumors. CD73 expression correlated with EGFR pathway activity, EMT, and partial EMT (p-EMT) in malignant single HNSCC cells and in large patient cohorts. Contrary to published data, CD73 was not a prognostic marker of overall survival (OS) in the TCGA-HNSCC cohort when patients were stratified for HPV-status. However, CD73 prognosticated OS of oral cavity carcinomas. Furthermore, CD73 expression levels correlated with response to Cetuximab in HPV-negative advanced, metastasized HNSCC patients. CONCLUSIONS: In sum, CD73 is an effector of EGF/EGFR-mediated local invasion and a potential therapeutic target and candidate predictive marker for advanced HPV-negative HNSCC.

[Speech perception as a function of cochlear coverage-comparison in bimodally hearing cochlear implant patients. German version]. / Sprachverstehen in Abhängigkeit von der cochleären Abdeckung ­ Vergleich bei bimodal versorgten Cochleaimplantatpatienten.

BACKGROUND: Hearing success in bimodally hearing patients with a cochlear implant (CI) and a hearing aid (HA) exhibits different results: while some benefit from bimodal CI and HA, others do not. OBJECTIVE: The aim of this study was to investigate hearing success in terms of speech perception in bimodally fitted patients in relation to the cochlear coverage (CC) of the CI electrodes. MATERIALS AND METHODS: Using the OTOPLAN software (CAScination AG, Bern, Switzerland), CC was retrospectively measured from CT scans of the temporal bone of 39 patients, who were then categorized into two groups: CC ≤ 65% (CC500) and CC > 65% (CC600). Monaural speech intelligibility for monosyllables at a sound pressure level (SPL) of 65 dB in open field was assessed at various timepoints, preoperatively with HA and postoperatively with CI, and compared between the groups. In addition, speech intelligibility was correlated with CC in the entire cohort before surgery and during follow-up (FU). RESULTS: Overall, no significant differences in speech intelligibility were found between CC500 and CC600 patients at any of the FU timepoints. However, both CC500 and CC600 patients showed a steady improvement in speech intelligibility after implantation. While CC600 patients tended to show an earlier improvement in speech intelligibility, CC500 patients tended to show a slower improvement during the first 3 months and a steeper learning curve thereafter. The two patient groups converged during FU, with no significant differences in speech intelligibility. There was no significant relationship between unimodal/unilateral free-field speech intelligibility and CC. However, patients with a CC of 70-75% achieved maximum speech intelligibility. CONCLUSION: Despite a nonsignificant correlation between CC and speech discrimination, patients seem to reach their maximum in unimodal/unilateral speech understanding mainly at 70-75% coverage. However, there is room for further investigation, as CC500 was associated with a shorter cochlear duct length (CDL), and long and very long electrodes were used in both groups.

Randomized, placebo-controlled study on efficacy, safety and tolerability of drug-induced defibrinogenation for sudden sensorineural hearing loss: the lessons learned.

PURPOSE: Disturbance of cochlear microcirculation is discussed as final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma viscosity is a possible factor for a critical reduction of cochlear blood flow that might lead to sudden sensorineural hearing loss (SSHL). The aim was to determine the efficacy and safety of drug-induced defibrinogenation by ancrod for SSHL. METHODS: Double-blind, randomized, placebo-controlled, multicenter, parallel group, phase II (proof-of-concept) study (planned enrollment: 99 patients). Patients received an infusion of ancrod or placebo (day 1) followed by subcutaneous administrations (day 2, 4, 6). Primary outcome was the change in pure tone audiogram air conduction average until day 8. RESULTS: The study was terminated early due to slow recruiting (31 enrolled patients: 22 ancrod, 9 placebo). A significant improvement of hearing loss was registered in both groups (ancrod: - 14.3 dB ± 20.4 dB, - 39.9% ± 50.4%; placebo: - 22.3 dB ± 13.7 dB, - 59.1% ± 38.0%). A statistically significant group-difference was not detected (p = 0.374). Placebo response of 33.3% complete and 85.7% at least partial recovery was observed. Plasma fibrinogen levels were reduced significantly by ancrod (baseline: 325.2 mg/dL, day 2: 107.2 mg/dL). Ancrod was tolerated well, no adverse drug reaction was of severe intensity, no serious adverse events occurred. CONCLUSION: Ancrod reduced fibrinogen levels that support its mechanism of action. The safety profile can be rated positively. Since the planned number of patients could not be enrolled, no efficacy conclusion can be drawn. The high rate of placebo response challenges clinical trials for SSHL and needs to be considered in future investigations. Trial registrations This study was registered in the EU Clinical Trials Register, EudraCT-No. 2012-000066-37 at 2012-07-02.

Vitronectin promotes immunothrombotic dysregulation in the venular microvasculature.

Microvascular immunothrombotic dysregulation is a critical process in the pathogenesis of severe systemic inflammatory diseases. The mechanisms controlling immunothrombosis in inflamed microvessels, however, remain poorly understood. Here, we report that under systemic inflammatory conditions the matricellular glycoproteinvitronectin (VN) establishes an intravascular scaffold, supporting interactions of aggregating platelets with immune cells and the venular endothelium. Blockade of the VN receptor glycoprotein (GP)IIb/IIIa interfered with this multicellular interplay and effectively prevented microvascular clot formation. In line with these experimental data, particularly VN was found to be enriched in the pulmonary microvasculature of patients with non-infectious (pancreatitis-associated) or infectious (coronavirus disease 2019 (COVID-19)-associated) severe systemic inflammatory responses. Targeting the VN-GPIIb/IIIa axis hence appears as a promising, already feasible strategy to counteract microvascular immunothrombotic dysregulation in systemic inflammatory pathologies.

Low Dose Betahistine in Combination With Selegiline Increases Cochlear Blood Flow in Guinea Pigs.

OBJECTIVE: Betahistine is frequently used in the pharmacotherapy for Menière's Disease (MD). Little is known about its mode of action and prescribed dosages vary. While betahistine had an increasing effect on cochlear microcirculation in earlier studies, low dose betahistine of 0.01 mg/kg bw or less was not able to effect this. Selegiline inhibits monoaminooxidase B and therefore potentially the breakdown of betahistine. The goal of this study was to examine whether the addition of selegiline to low dose betahistine leads to increased cochlear blood flow. METHODS: Twelve Dunkin-Hartley guinea pigs were anesthetized, the cochlea was exposed and a window opened to the stria vascularis. Blood plasma was visualized by injecting fluoresceinisothiocyanate-dextrane and vessel diameter and erythrocyte velocity were evaluated over 20 minutes. One group received low dose betahistine (0.01 mg/kg bw) and selegiline (1 mg/kg bw) i.v. while the other group received only selegiline (1 mg/kg bw) and saline (0.9% NaCl) as placebo i.v. RESULTS: Cochlear microcirculation increased significantly (P < .001) in guinea pigs treated with low dose betahistine combined with selegiline by up to 58.3 ± 38.7% above baseline over a period of up to 11 minutes. In one guinea pig, the increase was 104.6%. Treatment with Selegiline alone did not affect microcirculation significantly. CONCLUSIONS: Low dose betahistine increased cochlear microcirculation significantly when combined with selegiline. This should be investigated in further studies regarding dose-effect relation in comparison to betahistine alone. Side effects, in particular regarding circulation, should be considered carefully in view of the clinical applicability of a combination therapy in patients with MD.

Vestibular and audiometric results after endolymphatic mastoid shunt surgery in patients with Menière's disease.

PURPOSE: Treatment of Menière's Disease (MD) comprises an array of both non-destructive and destructive treatment options. In patients who are therapy-refractory to non-destructive medical treatment, endolymphatic mastoid shunt surgery (EMSS) is both recommended and debated controversially. The aim of this study was to investigate safety in terms of hearing, vestibular function, complication rate, and efficacy with regards to vertigo control of EMSS in patients with MD according to the current diagnostic criteria of 2015. METHODS: Retrospective analysis of 47 consecutive patients with definite or probable MD with description of demographic parameters, pre- and postoperative MD treatment, pre- and postoperative audiometric (pure tone audiometry) and vestibular (caloric testing) results. The parameters were compared between patients with and without postoperative vertigo control. RESULTS: 31/47 patients (66.0%) had improved vertigo control postoperatively. Postoperative hearing and vestibular preservation were predominantly stable. No significant differences between patients with improved vertigo control and patients with no change or worse vertigo episodes were found. In the treatment refractory group, 4 patients required a revision EMSS and 6 a destructive MD treatment (5 gentamicin intratympanically, 1 labyrinthectomy). No peri- or postsurgical complications were reported. CONCLUSIONS: EMSS was found to be beneficial in two thirds of the patients with definite or probable Morbus Menière and a safe procedure regarding hearing and vestibular preservation with no postoperative complications. Therefore, EMSS should be considered before inducing destructive treatment options, such as intratympanic gentamicin application or labyrinthectomy.

Parameters for estimating the feasibility of implantation of a semi-implantable bone conduction device (SIBCD) in children and adolescents.

PURPOSE: In children and adolescents, preoperative planning for a semi-implantable bone conduction device (SIBCD) is crucial. The geometric changes of the new version of a common SIBCD should enable a higher rate of successful implantation due to its flatter actuator. Thus, this radioanatomic study compared the rate of successful implantation of both device versions at the traditional mastoidal localization and two alternative sites, retrosigmoidal, and parietal, and investigated parameters helping to estimate the feasibility. METHODS: A retrospective analysis of 136 CT scans of 0 to 20-year-old patients, evaluation of demographic parameters, radioanatomy, and assessment of head diameter was conducted. The feasibility was investigated for certain age groups at three implantation sites. Prediction of feasible implantation by means of different parameters was calculated. RESULTS: A significant higher implantation rate was observed with the new device for all three sites and age groups. The age group of 6-8 years (n = 19) had most striking differences with a 58.1% rate of successful implantation with the new device without spacer (80% with spacer) at the mastoidal localization, whereas none with the old implant. Head diameter was identified as the most predictive parameter regarding all implantation sites (mastoidal: p = 0.030; retrosigmoidal: p = 0.006; parietal: p < 0.0001), age for the mastoidal (p < 0.0001) and retrosigmoidal (p < 0.0001), and gender for the parietal site (p = 0.001). CONCLUSION: The geometric changes of the actuator lead to a higher rate of successful implantation in all age-groups and all three localizations with reducing the requirement for spacers. Parameters age and head diameter might aid in estimating the rate of successful implantation in young patients and may be a novel tool to assist in the decision-making process for a SIBCD.

Isolation and characterization of head and neck cancer-derived peritumoral and cancer-associated fibroblasts.

Introduction: Head and neck squamous cell carcinomas (HNSCC) are characterized by strong cellular and molecular heterogeneity and treatment resistance entailing poor survival. Besides cell-intrinsic properties, carcinoma cells receive important cues from non-malignant cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a major component of the TME that impact on the molecular make-up of malignant cells and have a decisive function in tumor progression. However, the potential functionality of fibroblasts within tumor-adjacent, macroscopically normal tissue remains poorly explored. Methods: Here, we isolated primary peritumoral fibroblasts (PtFs) from macroscopically normal tissue in vicinity of primary human papillomavirus-negative and -positive oropharyngeal HNSCC and compared their phenotype and functionality with matched CAFs (n = 5 pairs) and with human oral fibroblasts (hOFs). Results: Expression patterns of CD90, CD73, CD105, smooth muscle actin, Vimentin, and S100A4 were comparable in PtFs, CAFs, and hOFs. Cell proliferation and doubling times of CAFs and PtFs were heterogeneous across patients (n =2 PtF>CAF; n = 1 CAF>PtF; n = 2 CAF=PtF) and reflected inferior growth than hOFs. Furthermore, PtFs displayed an reduced heterogeneity in cell size compared to matched CAFs, which were characterized by the presence of single large cells. Overall, conditioned supernatants from CAFs had more frequently growth-promoting effects on a panel of carcinoma cell lines of the upper aerodigestive tract carcinoma cell lines (Cal27, Cal33, FaDu, and Kyse30), whereas significant differences in migration-inducing effects demonstrated a higher potential of PtFs. Except for Kyse30, CAFs were significantly superior to hOFs in promoting proliferation, while PtFs induced stronger migration than hOFs in all carcinoma lines tested. Analysis of soluble factors demonstrated significantly increased VEGF-A production in CAFs (except in pat.8), and significantly increased PDGF-BB production in PtFs of two patients. Tube formation assays confirmed a significantly enhanced angiogenic potential of conditioned supernatants from CAFs compared to hOFs on human umbilical vascular endothelial cells (HUVECs) in vitro. Discussion: Hence, matched CAFs and PtFs present in HNSCC patients are heterogeneous in their proliferation-, migration-, and angiogenesis-promoting capacity. Despite this heterogeneity, CAFs induced stronger carcinoma cell proliferation and HUVEC tube formation overall, whereas PtFs promoted migration of tumor cells more strongly.

DeepClassPathway: Molecular pathway aware classification using explainable deep learning.

OBJECTIVE: HPV-associated head and neck cancer is correlated with favorable prognosis; however, its underlying biology is not fully understood. We propose an explainable convolutional neural network (CNN) classifier, DeepClassPathway, that predicts HPV-status and allows patient-specific identification of molecular pathways driving classifier decisions. METHODS: The CNN was trained to classify HPV-status on transcriptome data from 264 (13% HPV-positive) and tested on 85 (25% HPV-positive) head and neck squamous carcinoma patients after transformation into 2D-treemaps representing molecular pathways. Grad-CAM saliency was used to quantify pathways contribution to individual CNN decisions. Model stability was assessed by shuffling pathways within 2D-images. RESULTS: The classification performance of the CNN-ensembles achieved ROC-AUC/PR-AUC of 0.96/0.90 for all treemap variants. Quantification of the averaged pathway saliency heatmaps consistently identified KRAS, spermatogenesis, bile acid metabolism, and inflammation signaling pathways as the four most informative for classifying HPV-positive patients and MYC targets, epithelial-mesenchymal transition, and protein secretion pathways for HPV-negative patients. CONCLUSION: We have developed and applied an explainable CNN classification approach to transcriptome data from an oncology cohort with typical sample size that allows classification while accounting for the importance of molecular pathways in individual-level decisions.

[Hearing tests in practice : Audiometric masking in pure-tone and speech audiometry]. / Hörtests in der Praxis : Vertäubung in der Ton- und Sprachaudiometrie.

Correct execution of hearing tests is essential for audiologic diagnostics and selection of treatment by the ENT physician. Especially in the case of pure-tone and speech audiometry, incorrectly performed audiometric masking can lead to false measurement results. Often, hearing that is too good is feigned by overhearing in the contralateral ear. Herein, a masking strategy is described by which errors in the execution of masking can be recognized and corrected. The aim of this paper is to identify and prevent the most common errors made during the masking process in pure-tone and speech audiometry.

A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is both a driver oncogene and a therapeutic target in advanced head and neck squamous cell carcinoma (HNSCC). However, response to EGFR treatment is inconsistent and lacks markers for treatment prediction. This study investigated EGFR-induced epithelial-to-mesenchymal transition (EMT) as a central parameter in tumor progression and identified novel prognostic and therapeutic targets, and a candidate predictive marker for EGFR therapy response. METHODS: Transcriptomic profiles were analyzed by RNA sequencing (RNA-seq) following EGFR-mediated EMT in responsive human HNSCC cell lines. Exclusive genes were extracted via differentially expressed genes (DEGs) and a risk score was determined through forward feature selection and Cox regression models in HNSCC cohorts. Functional characterization of selected prognostic genes was conducted in 2D and 3D cellular models, and findings were validated by immunohistochemistry in primary HNSCC. RESULTS: An EGFR-mediated EMT gene signature composed of n = 171 genes was identified in responsive cell lines and transferred to the TCGA-HNSCC cohort. A 5-gene risk score comprising DDIT4, FADD, ITGB4, NCEH1, and TIMP1 prognosticated overall survival (OS) in TCGA and was confirmed in independent HNSCC cohorts. The EGFR-mediated EMT signature was distinct from EMT hallmark and partial EMT (pEMT) meta-programs with a differing enrichment pattern in single malignant cells. Molecular characterization showed that ITGB4 was upregulated in primary tumors and metastases compared to normal mucosa and correlated with EGFR/MAPK activity in tumor bulk and single malignant cells. Preferential localization of ITGB4 together with its ligand laminin 5 at tumor-stroma interfaces correlated with increased tumor budding in primary HNSCC tissue sections. In vitro, ITGB4 knock-down reduced EGFR-mediated migration and invasion and ITGB4-antagonizing antibody ASC8 impaired 2D and 3D invasion. Furthermore, a logistic regression model defined ITGB4 as a predictive marker of progression-free survival in response to Cetuximab in recurrent metastatic HNSCC patients. CONCLUSIONS: EGFR-mediated EMT conveyed through MAPK activation contributes to HNSCC progression upon induction of migration and invasion. A 5-gene risk score based on a novel EGFR-mediated EMT signature prognosticated survival of HNSCC patients and determined ITGB4 as potential therapeutic and predictive target in patients with strong EGFR-mediated EMT.