Nivolumab as a Promising Treatment Option for Metastatic Salivary Duct Carcinoma.

Salivary duct carcinomas (SDC) of the parotid gland are rarely occurring highly malignant tumors. A 65-year-old man presented with a preauricular mass. After surgical treatment and histologic examination, the findings were interpreted as a squamous cell carcinoma (SCC) metastasis of the parotid gland deriving from a cancer of unknown primary DD primary SCC of the parotid gland. Adjuvant platinum-based radiochemotherapy was administered in domo. However, re-staging revealed multiple size-progressive pulmonary round lesions. After resection and histological examination of a pulmonary mass and in synopsis with the primary tumor, the initial diagnosis of SCC was revised to SDC of the parotid gland. With positive HER-2 status, off-label trastuzumab/docetaxel was initiated in an individual healing attempt, during which the pulmonary metastases showed clear progression. Consequently, the patient received immunotherapy with nivolumab according to his negative PD-L1 status. After 57 cycles of nivolumab, the patient presents with partial remission and in good condition. We report, for the first time, a robust response of metastatic SDC to checkpoint inhibition with nivolumab without additional radiotherapy.

Point Cloud Registration for Measuring Shape Dependence of Soft Tissue Deformation by Digital Twins in Head and Neck Surgery.

Introduction: A 2½ D point cloud registration method was developed to generate digital twins of different tissue shapes and resection cavities by applying a machine learning (ML) approach. This demonstrates the feasibility of quantifying soft tissue shifts. Methods: An ML model was trained using simulated surface scan data obtained from tumor resections in a pig head cadaver model. It hereby uses 438 2½ D scans of the tissue surface. Tissue shift was induced by a temperature change from 7.91 ± 4.1°C to 36.37 ± 1.28°C. Results: Digital twins were generated from various branched and compact resection cavities (RCs) and cut tissues (CT). A temperature increase induced a tissue shift with a significant volume increase of 6 mL and 2 mL in branched and compact RCs, respectively (p = 0.0443; 0.0157). The volumes of branched and compact CT were decreased by 3 and 4 mL (p < 0.001). In the warm state, RC and CT no longer fit together because of the significant tissue deformation. Although not significant, the compact RC showed a greater tissue deformation of 1 µL than the branched RC with 0.5 µL induced by the temperature change (p = 0.7874). The branched and compact CT forms responded almost equally to changes in temperature (p = 0.1461). Conclusions: The simulation experiment of induced soft tissue deformation using digital twins based on 2½ D point cloud models proved that our method helps to quantify shape-dependent tissue shifts.

Risk factors for immune-related adverse effects during CPI therapy in patients with head and neck malignancies - a single center study.

Introduction: Checkpoint inhibitors, such as PD1 inhibitors, represent an important pillar in the therapy of advanced malignancies of the head and neck region. The most relevant complications are immune-related adverse effects (irAEs), which represent an immense burden for patients. Currently, no sufficient stratification measures are available to identify patients at increased risk of irAEs. The aim of this retrospective study was to examine whether demographic, histopathological, clinical, or laboratory values at the start of CPI therapy represent a risk factor for the later occurrence of autoimmune complications. Material and methods: Data from 35 patients between 2018 and 2021 who received therapy with nivolumab or pembrolizumab for head and neck malignancy were analyzed and assessed for any associations with the subsequent occurrence of irAEs. Results: IrAE developed in 37% of patients, with pneumonitis being the most common form (14%). Pneumonitis was found in patients with an average significantly lower T-stage of primary tumors. An increase in basophilic leukocytes was found in patients with dermatitis later in the course. When thyroiditis developed later, the patients had a higher CPS score and lower monocyte levels. Discussion: Even though individual laboratory values at the beginning of therapy might show a statistical association with the later occurrence of irAEs, neither demographic, histopathological, nor laboratory chemistry values seem to be able to generate a sound and reliable risk profile for this type of complication. Therefore, patients need to be educated and sensitized to irAEs, and regular screening for irAEs should be carried out.

Artificial intelligence directed development of a digital twin to measure soft tissue shift during head and neck surgery.

Digital twins derived from 3D scanning data were developed to measure soft tissue deformation in head and neck surgery by an artificial intelligence approach. This framework was applied suggesting feasibility of soft tissue shift detection as a hitherto unsolved problem. In a pig head cadaver model 104 soft tissue resection had been performed. The surface of the removed soft tissue (RTP) and the corresponding resection cavity (RC) was scanned (N = 416) to train an artificial intelligence (AI) with two different 3D object detectors (HoloLens 2; ArtecEva). An artificial tissue shift (TS) was created by changing the tissue temperature from 7,91±4,1°C to 36,37±1,28°C. Digital twins of RTP and RC in cold and warm conditions had been generated and volumes were calculated based on 3D surface meshes. Significant differences in number of vertices created by the different 3D scanners (HoloLens2 51313 vs. ArtecEva 21694, p<0.0001) hence result in differences in volume measurement of the RTC (p = 0.0015). A significant TS could be induced by changing the temperature of the tissue of RC (p = 0.0027) and RTP (p = <0.0001). RC showed more correlation in TS by heating than RTP with a volume increase of 3.1 µl or 9.09% (p = 0.449). Cadaver models are suitable for training a machine learning model for deformable registration through creation of a digital twin. Despite different point cloud densities, HoloLens and ArtecEva provide only slightly different estimates of volume. This means that both devices can be used for the task.TS can be simulated and measured by temperature change, in which RC and RTP react differently. This corresponds to the clinical behaviour of tumour and resection cavity during surgeries, which could be used for frozen section management and a range of other clinical applications.

Modulation of PD­L1 expression by standard therapy in head and neck cancer cell lines and exosomes.

Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand­1 (PD­L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD­L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD­L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM­SCC­11B, UM­SCC­14C and UM­SCC­22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PD­L1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an ex vivo vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un­)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PD­L1 were detected using western blotting and semi­quantitative levels were calculated. The functional impact of exosomes from the (un­)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UM­SCC­11B and UM­SCC­22B showed strong expression of pERK1/2 and PD­L1, respectively. RT upregulated the PD­L1 expression in UM­SCC­11B and UM­SCC­14C and in exosomes from all three cell lines. CT alone induced PD­L1 expression in all cell lines. CRT induced the expression of PD­L1 in all HNSCC cell lines and exosomes from UM­SCC­14C and UM­SCC­22B. The data indicated a potential co­regulation of PD­L1 and activated ERK1/2, most evident in UM­SCC­14C. Exosomes from irradiated UM­SCC­14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell­mediated regulation of PD­L1 upon platinum­based CRT in HNSCC and in exosomes. A co­regulation of PD­L1 and MAPK signaling response was hypothesized.

Plasma-derived small extracellular vesicles unleash the angiogenic potential in head and neck cancer patients.

BACKGROUND: In Head and neck cancer (HNC) angiogenesis is essential for tumor progression and metastasis. Small extracellular vesicles (sEVs) from HNC cell lines alter endothelial cell (EC) functions towards a pro-angiogenic phenotype. However, the role of plasma sEVs retrieved from HNC patients in this process is not clear so far. METHODS: Plasma sEVs were isolated on size exclusion chromatography columns from 32 HNC patients (early-stage UICC I/II: 8, advanced-stage UICC III/IV: 24), 12 patients with no evident disease after therapy (NED) and 16 healthy donors (HD). Briefly, sEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), BCA protein assays and Western blots. Levels of angiogenesis-associated proteins were determined using antibody arrays. The interaction of fluorescently-labeled sEVs with human umbilical vein ECs was visualized by confocal microscopy. The functional effect of sEVs on tubulogenesis, migration, proliferation and apoptosis of ECs was assessed. RESULTS: The internalization of sEVs by ECs was visualized using confocal microscopy. Based on antibody arrays, all plasma sEVs were enriched in anti-angiogenic proteins. HNC sEVs contained more pro-angiogenic MMP-9 and anti-angiogenic proteins (Serpin F1) than HD sEVs. Interestingly, a strong inhibition of EC function was observed for sEVs from early-stage HNC, NED and HD. In contrast, sEVs from advanced-stage HNC showed a significantly increased tubulogenesis, migration and proliferation and induced less apoptosis in ECs than sEVs from HD. CONCLUSIONS: In general, plasma sEVs carry a predominantly anti-angiogenic protein cargo and suppress the angiogenic properties of ECs, while sEVs from (advanced-stage) HNC patients induce angiogenesis compared to HD sEVs. Thus, tumor-derived sEVs within the plasma of HNC patients might shift the angiogenic switch towards angiogenesis.

Augmented reality during parotid surgery: real-life evaluation of voice control of a head mounted display.

PURPOSE: Augmented Reality can improve surgical planning and performance in parotid surgery. For easier application we implemented a voice control manual for our augmented reality system. The aim of the study was to evaluate the feasibility of the voice control in real-life situations. METHODS: We used the HoloLens 1® (Microsoft Corporation) with a special speech recognition software for parotid surgery. The evaluation took place in a audiometry cubicle and during real surgical procedures. Voice commands were used to display various 3D structures of the patient with the HoloLens 1®. Commands had different variations (male/female, 65 dB SPL)/louder, various structures). RESULTS: In silence, 100% of commands were recognized. If the volume of the operation room (OR) background noise exceeds 42 dB, the recognition rate decreases significantly, and it drops below 40% at > 60 dB SPL. With constant speech volume at 65 dB SPL male speakers had a significant better recognition rate than female speakers (p = 0.046). Higher speech volumes can compensate this effect. The recognition rate depends on the type of background noise. Mixed OR noise (52 dB(A)) reduced the detection rate significantly compared to single suction noise at 52 dB(A) (p ≤ 0.00001). The recognition rate was significantly better in the OR than in the audio cubicle (p = 0.00013 both genders, 0.0086 female, and 0.0036 male). CONCLUSIONS: The recognition rate of voice commands can be enhanced by increasing the speech volume and by singularizing ambient noises. The detection rate depends on the loudness of the OR noise. Male voices are understood significantly better than female voices.

Optimisation of Conditions for the Formation of Spheroids of Head and Neck Squamous Cell Carcinoma Cell Lines for Use as Animal Alternatives.

The use of in vitro 3-D cell culture models in cancer research has yielded substantial gains in knowledge on various aspects of tumour biology. Such cell culture models could be useful in the study of head and neck squamous cell carcinoma (HNSCC), where mimicking intratumoral and intertumoral heterogeneity is especially challenging. Our research aims to establish 3-D spheroid models for HNSCC that reproduce in vitro the connections between tumour cells and the surrounding microenvironment. The aims of this study were to determine the optimal conditions for the culture and use of spheroids from HNSCC cell lines and optimal timepoint for using the spheroids obtained, to evaluate the effects of coculture with tumour-specific fibroblasts on spheroid formation, and to investigate spheroid responses to cisplatin treatment. Four HNSCC cell lines (UMSCC-11A, UMSCC-11B, UMSCC-22B and UD-SCC-01) were seeded in flat or round bottom well ultra-low attachment spheroid plates, and spheroid formation was evaluated. The HNSCC cell lines were then cocultured with stromal cells of the tumour microenvironment, producing an accelerated formation of dense spheroids. The viability of cells within the spheroids was assessed during cell culture by using a fluorescent dye. Our results suggest that: three out of the four cell lines tested could form usable spheroids with acceptable viability; the addition of stromal cells did not improve the number of viable cells; and the use of round bottom well plates supported the formation of a single spheroid, whereas flat bottom well plates led to the formation of multiple spheroids of different sizes.

Conjoint analysis of OPRPN and SMR3A protein expression as potential predictive biomarkers for head and neck squamous cell carcinoma after radiotherapy.

Increased submaxillary gland androgen­regulated protein 3A (SMR3A) expression was previously shown to serve as an independent risk factor for oropharyngeal squamous cell carcinoma (OPSCC) and as a surrogate biomarker for active estrogen receptor 2 signaling in radioresistant tumor cells. In the present study, it was aimed to unravel the expression and clinical significance of another member of the opiorphin family, opiorphin prepropeptide (OPRPN), in the radiotherapy for head and neck squamous cell carcinoma (HNSCC). Expression of SMR3A and OPRPN were analyzed for the prior and post fractionated irradiation (4x2 Gy) by double immunofluorescence staining in established HNSCC cell lines as well as by immunohistochemical (IHC) staining in ex vivo tumor tissues. Next, in a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n=96), who received definitive surgery and adjuvant radiotherapy were reviewed, and expression levels of OPRPN protein were detected by IHC. Immunoreactivity scores (IRS) were associated with pathological and clinical risk factors by Chi­square analysis. Survival analysis was performed by using the Kaplan­Meier plot, log­rank test and Cox regression analysis. The expression levels of OPRPN and SMR3A protein were both induced by fractionated irradiation in vitro and ex vivo. In primary tumor samples, IRS of OPRPN was significantly higher than scores of SMR3A expression and positively correlated with expression patterns of SMR3A. SMR3A was confirmed to serve as an unfavorable factor, while OPRPN protein had no significant association with the clinical outcome of patients with OPSCC. A combinational analysis revealed that the subgroup with SMR3AhighOPRPNlow staining pattern had the worst clinical outcome among the various subgroups. Multivariate Cox regression analyses indicated that high expression of SMR3A serves as an independent unfavorable biomarker, while increased expression of OPRPN appears to exert protective function. In summary, the present study indicated that SMR3A and OPRPN serve as potential prognostic markers for HNSCC after radiotherapy.

Biomarkers and 3D models predicting response to immune checkpoint blockade in head and neck cancer (Review).

Immunotherapy has evolved into a powerful tool in the fight against a number of types of cancer, including head and neck squamous cell carcinomas (HNSCC). Although checkpoint inhibition (CPI) has definitely enriched the treatment options for advanced stage HNSCC during the past decade, the percentage of patients responding to treatment is widely varying between 14­32% in second­line setting in recurrent or metastatic HNSCC with a sporadic durability. Clinical response and, consecutively, treatment success remain unpredictable in most of the cases. One potential factor is the expression of target molecules of the tumor allowing cancer cells to acquire therapy resistance mechanisms. Accordingly, analyzing and modeling the complexity of the tumor microenvironment (TME) is key to i) stratify subgroups of patients most likely to respond to CPI and ii) to define new combinatorial treatment regimens. Particularly in a heterogeneous disease such as HNSCC, thoroughly studying the interactions and crosstalking between tumor and TME cells is one of the biggest challenges. Sophisticated 3D models are therefore urgently needed to be able to validate such basic science hypotheses and to test novel immuno­oncologic treatment regimens in consideration of the individual biology of each tumor. The present review will first summarize recent findings on immunotherapy, predictive biomarkers, the role of the TME and signaling cascades eliciting during CPI. Second, it will highlight the significance of current promising approaches to establish HNSCC 3D models for new immunotherapies. The results are encouraging and indicate that data obtained from patient­specific tumors in a dish might be finally translated into personalized immuno­oncology.

3D cell culture alters signal transduction and drug response in head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) upregulation is a typical characteristic of head and neck squamous cell carcinoma (HNSCC). However, tyrosine kinase inhibitors have not yet been able to achieve enough therapeutic benefit in clinical trials to justify their use in standard therapy regimens. At present, little is known about the reasons for this treatment failure. In the present study, the HNSCC cell lines UM-SCC-11B and UM-SCC-22B were tested for their response to tyrosine kinase inhibitors (TKI) under 2D and 3D cell culture conditions. Absorption and luciferase-based viability assays were used for this, as well as optical evaluation via fluorescence microscopy. In addition, EGFR and HER3 expression as well as the downstream signalling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK were investigated using western blotting. Cell line UM-SCC-11B revealed a strong resistance to lapatinib under 3D cell culture conditions, while a good response to TKI therapy was observed under 2D cell culture conditions. An associated overexpression of phosphorylated HER3 under 3D cell culture conditions offered a plausible explanation for the altered treatment response. The results of the present study represent an idea of how signalling mechanisms of cancer cells can be changed using different cell culture methods. Overall, 3D cell culture could be an important component in the analysis of resistance mechanisms in cancer therapy.

[Time management in operating rooms-a cross-sectional study to evaluate estimated and objective durations of otorhinolaryngologic surgical procedures]. / Zeitmanagement im OP ­ eine Querschnittstudie zur Bewertung der subjektiven und objektiven Dauer chirurgischer Prozeduren im HNO-Bereich.

BACKGROUND: Accurate planning of operating times in surgical clinics is essential. Moreover, high-capacity utilization of operating rooms (ORs) is necessary for economic efficiency. OBJECTIVE: Most planning of operating times is performed by surgeons. Herein, surgeons' estimated times and the objective times for performing surgical procedures were compared to detect sources of error. MATERIALS AND METHODS: In a retrospective analysis, the durations of 1809 operations using general anesthesia (22 types of surgery) by 31 surgeons (12 specialists and 19 residents) were compared. Comparisons were analyzed by Mann-Whitney U­tests. RESULTS: The comparison of objective times of surgical action showed significant differences between specialists and residents in 6 of 15 types of surgeries. The post-processing times estimated by specialists deviated from the objective times in 2 out of 22 surgery types, while the post-processing times estimated by residents deviated in 7 of 15 types. Specialists misjudged the incision-to-suture times in 7 of 22 surgery types, and residents misjudged these times in 3 of 15 types. The preparation times estimated by specialists deviated from the objective times in 16 of 22 types of surgeries and in 7 of 15 types estimated by residents. CONCLUSION: A surgeon's routine must be carefully considered in order to estimate operating times. Specialists generally underestimated preparation and post-processing times and overestimated incision-to-suture times, whereas residents underestimated all three. Preparation and post-processing times must be considered in planning and, ideally, determined together with anesthesiologists and surgical assistants.

Precision Medicine Gains Momentum: Novel 3D Models and Stem Cell-Based Approaches in Head and Neck Cancer.

Despite the current progress in the development of new concepts of precision medicine for head and neck squamous cell carcinoma (HNSCC), in particular targeted therapies and immune checkpoint inhibition (CPI), overall survival rates have not improved during the last decades. This is, on the one hand, caused by the fact that a significant number of patients presents with late stage disease at the time of diagnosis, on the other hand HNSCC frequently develop therapeutic resistance. Distinct intratumoral and intertumoral heterogeneity is one of the strongest features in HNSCC and has hindered both the identification of specific biomarkers and the establishment of targeted therapies for this disease so far. To date, there is a paucity of reliable preclinical models, particularly those that can predict responses to immune CPI, as these models require an intact tumor microenvironment (TME). The "ideal" preclinical cancer model is supposed to take both the TME as well as tumor heterogeneity into account. Although HNSCC patients are frequently studied in clinical trials, there is a lack of reliable prognostic biomarkers allowing a better stratification of individuals who might benefit from new concepts of targeted or immunotherapeutic strategies. Emerging evidence indicates that cancer stem cells (CSCs) are highly tumorigenic. Through the process of stemness, epithelial cells acquire an invasive phenotype contributing to metastasis and recurrence. Specific markers for CSC such as CD133 and CD44 expression and ALDH activity help to identify CSC in HNSCC. For the majority of patients, allocation of treatment regimens is simply based on histological diagnosis and on tumor location and disease staging (clinical risk assessments) rather than on specific or individual tumor biology. Hence there is an urgent need for tools to stratify HNSCC patients and pave the way for personalized therapeutic options. This work reviews the current literature on novel approaches in implementing three-dimensional (3D) HNSCC in vitro and in vivo tumor models in the clinical daily routine. Stem-cell based assays will be particularly discussed. Those models are highly anticipated to serve as a preclinical prediction platform for the evaluation of stable biomarkers and for therapeutic efficacy testing.

[The tumor stem cell niche of head and neck - point of intersection with therapeutic potential?] / Die Tumorstammzellnische im Kopf-Hals-Bereich ­ Knotenpunkt mit therapeutischem Potenzial?

An increasing amount of evidence suggests the existence of a stem cell-like population in head and neck squamous cell carcinoma (HNSCC). These cells have been termed cancer stem cells (CSC) due to the shared properties with somatic stem cells, such as the ability to self-renew and differentiate. Furthermore, the CSC are thought to be resistant to antineoplastic treatments and are therefore clinically relevant. As with somatic stem cells, CSC are thought to reside in a specialized supportive microenvironment, called the stem cell niche. One possible strategy to target the CSC could be through affecting functions of the stem cell niche.Stromal cell-derived factor-1 (SDF-1) is a multifunctional cytokine, which is secreted by e. g. stromal cells within the niche. SDF-1 is known to be the major regulator of stem cell trafficking between the niche and the peripheral vascular system. It elicits the chemotactic activity through interaction with a transmembrane receptor CXCR4, expressed by CSC. The SDF-1-CXCR4-axis is thought to play a crucial role in the interaction between CSC and their supportive cells in the tumor niche. A better understanding of these interactions could help in gaining further insight into the pathophysiology of progression/recurrence of malignant diseases and aid in finding new strategies for therapy.Specialized cell culture models are of advantage for deciphering the mechanisms of interaction between CSC and their niche. We anticipate that the recent technological advancements in bioprinting and the development of complex 3D cell culture model systems will contribute to our understanding of these mechanisms and to the establishment of individualized therapies.Here were provide an overview of the current knowledge on the CSC-tumor stem cell niche interactions in HNSCC with a focus on the SDF-1-CXCR4 axis.

Augmented reality with HoloLens in parotid surgery: how to assess and to improve accuracy.

PURPOSE: Augmented reality improves planning and execution of surgical procedures. The aim of this study was to evaluate the feasibility of a 3D augmented reality hologram in live parotic surgery. Another goal was to develop an accuracy measuring instrument and to determine the accuracy of the system. METHODS: We created a software to build and manually align 2D and 3D augmented reality models generated from MRI data onto the patient during surgery using the HoloLens® 1 (Microsoft Corporation, Redmond, USA). To assess the accuracy of the system, we developed a specific measuring tool applying a standard electromagnetic navigation device (Fiagon GmbH, Hennigsdorf, Germany). RESULTS: The accuracy of our system was measured during real surgical procedures. Training of the experimenters and the use of fiducial markers significantly reduced the accuracy of holographic system (p = 0.0166 and p = 0.0132). Precision of the developed measuring system was very high with a mean error of the basic system of 1.3 mm. Feedback evaluation demonstrated 86% of participants agreed or strongly agreed that the HoloLens will play a role in surgical education. Furthermore, 80% of participants agreed or strongly agreed that the HoloLens is feasible to be introduced in clinical routine and will play a role within surgery in the future. CONCLUSION: The use of fiducial markers and repeated training reduces the positional error between the hologram and the real structures. The developed measuring device under the use of the Fiagon navigation system is suitable to measure accuracies of holographic augmented reality images of the HoloLens.

Organotypic Co-Cultures as a Novel 3D Model for Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response.

Adaptive ERK signalling activation in response to therapy and in silico prognostic evaluation of EGFR-MAPK in HNSCC.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) patients frequently develop treatment resistance to cetuximab, a monoclonal antibody against EGFR, as well as radiotherapy. Here we addressed extracellular signal-regulated kinase 1/2 (ERK1/2) regulation by cetuximab or fractionated irradiation (IR) and conducted in silico prognostic evaluation of the EGFR-MAPK axis in HNSCC. METHODS: Expression of ERK1/2 phosphorylation (pERK1/2) was determined in HNSCC cell lines, which were treated with cetuximab or fractionated-IR. Furthermore, the effect of fractionated IR on pERK1/2 was confirmed in an ex vivo HNSCC tissue culture model. Expression and prognostic significance of EGFR-ERK axis was evaluated in a cohort of radiotherapy plus cetuximab-treated HNSCC. Correlations among EGFR-MAPK signalling components and association between transcript and protein expression profiles and patient survival in HNSCC were analysed using publicly available databases. RESULTS: ERK1/2 phosphorylation was rebounded by prolonged cetuximab administration and was induced by fractionated IR, which could be suppressed by a MEK inhibitor as a radiosensitiser. In silico assessments suggested that EGFR-MAPK cascade genes and proteins could predict HNSCC patients' survival as a prognostic signature. CONCLUSIONS: Activation of ERK1/2 signalling contributes to the cellular defence of HNSCC against cetuximab and fractionated IR treatment. EGFR-MAPK axis has a prognostic significance in HNSCC.

Validating the Xerostomia Inventory in a radiation-induced xerostomia population in German language.

OBJECTIVES: Though xerostomia is a frequent oral symptom, there is no validated disease-specific questionnaire in German. The purpose of this study was to translate and validate versions of the Xerostomia Inventory and the Summated Xerostomia Inventory in a German-speaking population. PARTICIPANTS AND METHODS: Thirty-nine patients including 18 patients suffering from radiation-induced xerostomia enrolled in this study. Both questionnaires were translated into German language according to international accepted guidelines. For validation, we evaluated reliability, validity, and responsiveness using the COSMIN manual for cross-cultural adaptation. RESULTS: Cronbach's α was 0.92 for XI and 0.91 for SXI, showing both high internal consistency. Patients suffering from xerostomia showed significantly higher average scores demonstrating its discriminant validity. Confirmatory factor analysis showed excellent "goodness-of-fit" values for SXI and good to moderate values for XI, confirming the assumed factor structures. The Xerostomia Inventory and its summated version both showed excellent test-retest reliability in the non-xerostomia group (ICC = 0.85 and 0.84). CONCLUSIONS: The XI and SXI in their cross-cultural adapted versions are the first validated self-report assessments for xerostomia in German language. They are characterized by practical design and can be easily interpreted by the treating physician.

Differential Activation of ERK Signaling in HPV-Related Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) forms a distinct tumor entity with better survival clinical outcome. Numerous underlying molecular mechanisms have been postulated for differences in treatment response, but the impact of MEK/ERK signaling, a main driver of carcinogenesis in various cancers including OPSCC and key player mediating therapy resistance remains elusive. In a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n = 124) were available on tissue microarrays (TMAs) and expression levels of phosphorylated ERK1/2 (pERK1/2) were detected by immunohistochemical staining. Correlations of pERK1/2 expression patterns with clinicopathological features and clinical outcome were evaluated by statistical analysis. A low pERK1/2 expression was strongly associated with HPV-related OPSCC, while primary tumors with high pERK1/2 staining showed a distinctly worse survival outcome and were associated with higher cellular differentiation. Co-activation of both ERK1/2 and AKT was a common event and was associated with unfavorable prognosis in our cohort. However, the combinatorial analysis of pAKT (Ser473) and pERK1/2 did not strengthen the predictive power of pERK1/2, suggesting that pERK1/2 plays a more significant function in OPSCC. In summary, our data provide a compelling experimental and statistical evidence that low levels of tumor cell intrinsic ERK1/2 activation contribute at least in part to the favorable outcome of HPV-related OPSCC. On the other hand, presented findings indicate that non-HPV-related OPSCC with elevated ERK phosphorylation are at high risk for treatment failure and might benefit from targeted therapy of MEK/ERK signaling.

Low SOX2 expression marks a distinct subset of adenoid cystic carcinoma of the head and neck and is associated with an advanced tumor stage.

INTRODUCTION: The transcription factor SOX2 has been identified as a lineage survival oncogene in squamous cell carcinoma and copy number gain is a common event in several human malignancies including head and neck cancer. However, the regulation and function of SOX2 during carcinogenesis as well as its prognostic value appears to be highly context dependent. As an example, high SOX2 expression in lung squamous cell carcinoma (SCC) is related to a favorable prognosis, while it is associated with poor outcome in lung adenocarcinoma. More recently, higher SOX2 levels and improved survival was also reported for head and neck SCC (HNSCC), and silencing of SOX2 expression in HNSCC cell lines revealed a mesenchymal-like phenotype with prominent vimentin expression. So far, SOX2 expression and its clinical relevance for other head and neck cancers, such as adenoid cystic carcinoma (HNACC) have not been sufficiently investigated. MATERIAL AND METHODS: SOX2, vimentin and E-cadherin expression was assessed by immunohistochemical staining on serial sections from formalin fixed and paraffin embedded tissue samples of a patient cohort (n = 45) with primary ACC and correlated with patient and tumor characteristics as well as survival. RESULTS: High SOX2 expression was found in 14 (31%) primary tumor specimens and was significantly correlated with a N0 lymph node status (p = 0.04), while low SOX2 expression was correlated with a solid growth pattern (p = 0.031). Of the 45 patients, 27 tumor samples resembled an EMT-like phenotype, as assessed by high vimentin and low E-cadherin levels. However, in HNACC SOX2 levels were neither correlated with vimentin nor with E-cadherin expression, further supporting a context dependent regulation and function of SOX2 in distinct tumor entities. CONCLUSION: The absence of SOX2 was predominantly found in solid HNACC, which are characterized by a more aggressive phenotype in ACC. However, the underlying molecular mechanisms of SOX2 regulation and function in distinct HNACC subgroups remain to be fully elucidated.