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Abstract Background A disintegrin and metalloproteinase (ADAMs) have been associated with multiple malignancies. ADAMs are involved in cell fusion, cell migration, membrane protein shedding and proteolysis. ADAM8 has been found to be overexpressed in squamous cell carcinomas of the lung. A new study showed that ADAM8 is significantly overexpressed in metastasis of squamous cell carcinomas of the head and neck (HNSCC). Methods We determined ADAM8 levels in the serum of 79 HNSCC patients at the time of diagnosis, in 35 patients 3 months after treatment and in 10 patients 1 year after therapy and compared the results to the sera of 31 healthy volunteers. We also constructed tissue microarrays to detect ADAM8 immunohistochemically in 100 patients. The results were correlated with the survival data of the patients to determine the diagnostic and prognostic value. Results The data demonstrated that patients with high ADAM8 expression in the tumor have worse survival rates. We found that high ADAM8 serum levels correlated with high ADAM8 expression in tumor samples. Soluble ADAM8 levels did not show any prognostic or diagnostic properties. Conclusion In summary ADAM8 expression is a prognostic factor for survival of patients with head and neck squamous cell carcinoma.

Objectives: The transforming growth factor-Beta (TGF-ß) pathway may be involved in the radioresistance of head and neck squamous cell carcinoma (HNSCC). This study analyzed TGF-ß receptor 1 (TGFBR1) expression in HNSCC patients and evaluated the antineoplastic and radiosensitizing effects of vactosertib, a novel TGFBR1 inhibitor, in vitro., Materials and Methods: TGFBR1 expression was examined in HNSCC patients at the mRNA level in silico and the protein level by immunohistochemistry, including surgical specimens of primary tumors, matched lymph node metastasis, and recurrent disease. Furthermore, a novel small molecule TGFBR1 inhibitor was evaluated in HNSCC cell lines. Finally, an indirect coculture model using patient-derived cancer-associated fibroblasts was applied to mimic the tumor microenvironment., Results: Patients with high TGFBR1 mRNA levels showed significantly worse overall survival in silico (OS, p = 0.024). At the protein level, an association between TGFBR1 + tumor and OS was observed for the subgroup with TGFBR1-stroma (p = 0.001). Those results prevailed in multivariable analysis. Inhibition of TGFBR1 showed antineoplastic effects in vitro. In combination with radiation, vactosertib showed synergistic effects., Conclusion: Our results indicate a high risk of death in tumor TGFBR1+ |stroma TGFBR1- expressing patients. In vitro data suggest a potential radiosensitizing effect of TGFBR1 inhibition by vactosertib., (© 2023 The Authors. Oral Diseases published by Wiley Periodicals LLC.)

Purpose: Human papilloma virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) displays distinct epidemiological, clinical, and molecular characteristics compared to the negative counterpart. Alterations in autophagy play an important role in cancer, and emerging evidence indicates an interplay of autophagy in HNSCC carcinogenesis and tumor promotion. However, the influence of HPV infection on autophagy in HNSCC has received less attention and has not been previously reviewed. Therefore, we here aimed to systematically review the role of autophagy explicitly in HPV + HNSCC., Methods: Studies accessible in PubMed, Embase, Scopus, and Web of Science investigating HNSCC, highlighting the molecular biological differences between HPV - and HPV + HNSCC and its influences on autophagy in HNSCC were analyzed according to the PRISMA statement. A total of 10 articles were identified, included, and summarized., Results: The HPV16 E7 oncoprotein was reported to be involved in the degradation of AMBRA1 and STING, and to enhance chemotherapy-induced cell death via lethal mitophagy in HNSCC cells. Autophagy-associated gene signatures correlated with HPV-subtype and overall survival. Additionally, immunohistochemical (IHC) analyses indicate that high LC3B expression correlates with poor overall survival in oropharyngeal HNSCC patients., Conclusion: HPV may dampen general bulk autophagic flux via degradation of AMBRA1 but may promote selective autophagic degradation of STING and mitochondria. Interpretations of correlations between autophagy-associated gene expressions or IHC analyses of autophagy-related (ATG) proteins in paraffin embedded tissue with clinicopathological features without biological validation need to be taken with caution., (© 2024. The Author(s).)

Background: Young patients with tongue squamous cell carcinoma (TSCC) mostly lack typical prognostic markers and face a dire prognosis. The aim of this study was to analyze the prognostic relevance of lymphocyte-to-monocyte ratio (LMR) in TSCC patients, with a special emphasis on patients under 45 years., Methods: This retrospective study included all patients primarily treated for TSCC. The prognostic relevance of LMR was investigated in terms of predicting the overallsurvival (OS) and disease-free survival (DFS)., Results: A total of 74 patients were included and the young cohort (<45 years) comprised 27 individuals. The mortality and recurrence rates were 39.2% ( n = 29) and 37.8% ( n = 28), respectively. OS and DFS were significantly shorter in the low LMR group within the whole cohort. Furthermore, low LMR was associated with worse prognosis, particularly inferior OS (median OS 1.7 vs. 14.6 years, p = 0.0156) and worse DFS (median DFS 0.8 years vs. not reached, p = 0.0405) in the young patient cohort., Conclusions: Our results reveal that pretreatment LMR might become a prognostic tool for young TSCC patients, especially due to its availability. However, further studies on larger cohorts are necessary to validate our results.

Alterations in the DNA damage response play a crucial role in radio- and chemoresistance of neoplastic cells. Activation of the Ataxia telangiectasia and Rad3-related (ATR) pathway is an important DNA damage response mechanism in head and neck squamous cell carcinoma (HNSCC). Berzosertib, a selective ATR inhibitor, shows promising radio- and chemosensitizing effects in preclinical studies and is well tolerated in clinical studies. The aim of this study was to elucidate the effect of berzosertib treatment in combination with radiation and cisplatin in HNSCC. The HNSCC cell lines Cal-27 and FaDu were treated with berzosertib alone and in combination with radiation or cisplatin. Cell viability and clonogenic survival were evaluated. The effect of combination treatment was evaluated with the SynergyFinder or combination index. Apoptosis was assessed via measurement of caspase 3/7 activation and migration was evaluated using a wound healing assay. Berzosertib treatment decreased cell viability in a dose-dependent manner and increased apoptosis. The IC 50 of berzosertib treatment after 72 h was 0.25-0.29 µM. Combination with irradiation treatment led to a synergistic increase in radiosensitivity and a synergistic or additive decrease in colony formation. The combination of berzosertib and cisplatin decreased cell viability in a synergistic manner. Additionally, berzosertib inhibited migration at high doses. Berzosertib displays a cytotoxic effect in HNSCC at clinically relevant doses. Further evaluation of combination treatment with irradiation and cisplatin is strongly recommended in HNSCC patients as it may hold the potential to overcome treatment resistance, reduce treatment doses and thus mitigate adverse events., (© 2023. The Author(s).)

Introduction  Age and lymph node ratio have been attributed as independent predictors for survival and recurrence in carcinoma of unknown primary (CUP). Objective  The purpose of this study was to analyze the prognostic value of p16 overexpression for CUP in the absence of true primary (TP). Methods  The study involved 43 patients who underwent therapeutic lymph node dissection (LND) from 2000 to 2015 after all the diagnostic work up for CUP. Immunohistochemistry for p16 overexpression was performed. Cox proportional hazard regression analysis was used to analyze the prognostic impact on 5-year overall survival (OS) and recurrence-free survival (RFS). Results  The male-to-female ratio was 5.1:1, with a median age of 62 years. The clinicopathological data, except for p16 overexpression, did not differ significantly in terms of 5-year OS and RFS. The Cox regression analysis proposed p16 positivity to be an independent prognosticator of regional recurrence-free survival (RRFS) (hazard ratio [HR] 6.180, p  = 0.21). The median time to recurrence and death were 10 and 25 months, respectively. Conclusion  Cervical metastasis with p16 overexpression is a significant prognostic factor of improved RFS after surgery in CUP. The prognostic significance of lymph node p16 positivity should be further studied., Competing Interests: Conflict of Interests The authors have no conflict of interests to declare., (Fundação Otorrinolaringologia. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. ( https://creativecommons.org/licenses/by-nc-nd/4.0/ ).)

Introduction: Thyroid function is frequently impaired in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). In patients treated with pembrolizumab, immune-related adverse events (irAEs) of the thyroid are common. However, the prognostic significance of baseline and on-treatment thyroid dysfunction is currently unclear., Methods: This study included 95 patients who received pembrolizumab for R/M HNSCC between 2016 and 2022. Baseline thyroid status, according to serum hormone levels, and irAEs were assessed. Univariable and multivariable Cox regression analyses were performed for overall survival (OS) and progression-free survival (PFS). Furthermore, the best overall response according to the prognostic groups was examined., Results: Low fT3 (HR: 2.52, p = 0.006), immune-related hyperthyroidism (HR: 0.11, p = 0.038), ECOG performance status ≥2 (HR: 3.72, p = 0.002), and platinum-refractory disease (HR: 3.29, p = 0.020) were independently associated with OS. Furthermore, immune-related hyperthyroidism was associated with longer PFS (HR: 0.13, p = 0.007), a higher objective response rate (83% vs. 31%, p = 0.018), and a higher disease control rate (100% vs. 43%, p = 0.008). Thyroid-related autoantibodies were elevated in 40% of thyroid irAEs cases with available measurements. Out of 16 thyroid irAEs, 15 occurred in patients with fT3 above the lower limit of normal., Conclusion: Low fT3 was associated with worse OS. Immune-related hyperthyroidism was correlated with both improved OS and PFS. Baseline fT3 assessment and close on-treatment monitoring of serum thyroid levels may be valuable for risk stratification in R/M HNSCC patients receiving pembrolizumab., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thorsten Fuereder received honoraria from MSD, Merck Darmstadt, Roche, BMS, Accord, Sanofi, Boehringer Ingelheim, Amgen, and Pfizer. The other authors declare no conflict of interest related to the publication of this article., (Copyright © 2023. Published by Elsevier B.V.)

Purpose: First-line immune checkpoint blockade has improved the prognosis of recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC), but response rates remain low. In this study, we aimed to investigate the prognostic value of CRP and its early kinetics to predict response and survival in R/M HNSCC., Methods: A total of 87 patients who received first-line pembrolizumab for R/M HNSCC were analyzed. Three-fold cross-validation was used to estimate cut-off points of CRP at baseline and on-treatment (day 40 ± 10). Treatment response and survival were analyzed according to early CRP kinetics. The neutrophil-to-lymphocyte ratio (NLR) was used as a benchmark for the prognostic performance of CRP., Results: On-treatment CRP below 2 mg/dl, 4x the upper limit of normal (ULN), was associated with increased overall survival (OS), while on-treatment CRP below 3 mg/dl (6x ULN) was correlated with a higher disease control rate (DCR) and increased progression-free survival (PFS). CRP flare-responders and CRP responders showed a higher DCR and longer PFS than CRP non-responders. An NLR above 6 was a negative prognosticator for progression. In multivariable analysis, on-treatment CRP prevailed as the only significant prognosticator for OS (HR: 4.97, CI95%: 2.18-11.32, p < 0.001) and PFS (HR: 2.07, CI95%: 1.07-3.99, p = 0.030)., Conclusion: On-treatment CRP was identified as a prognostic biomarker for objective response and survival in R/M HNSCC patients receiving first-line pembrolizumab and could be easily incorporated into clinical practice as a widely available and cost-effective biomarker., (© 2023. The Author(s).)

Introduction: Head and neck squamous cell carcinoma (HNSCC) is among the most common cancers in the world with a low survival rate and common diagnosis at late stages. Deubiquitination of proteins is involved in tumor growth, metastasis, apoptosis, and immunosuppressive pathways. The impact of the ubiquitin-specific protease (USP4) on survival was only scarcely investigated so far. The goal of our research was to analyze the association of USP4 expression with prognosis and clinicopathological features in HNSCC., Methods: USP4 mRNA levels were derived from The Cancer Genome Atlas (TCGA) for a cohort of 510 patients. Protein expression of USP4 was analyzed by immunohistochemistry in a second cohort of 113 patients. Associations between USP4 levels and overall survival, disease-free survival and clinicopathological data were analyzed., Results: High levels of USP4 mRNA were associated with prolonged overall survival in univariable analysis. There was no more association with survival after correction for the confounders HPV, stage and smoker status. High USP4 mRNA levels were linked to a lower T-stage, the patient's age at diagnosis, and a positive HPV status. USP4 protein levels were not associated with prognosis or other features., Conclusion: Since high USP4 mRNA was not an independent prognostic marker, we assume that the association is a result of the correlation of high USP4 mRNA with an HPV-positive status. Therefore, further investigation of USP4 mRNA and its association with the HPV status of HNSCC patients is warranted., (© 2023. The Author(s).)

Purpose: The evolutionary-conserved Wnt/β-CATENIN (WBC) pathway has been implicated in the pathogenesis of different solid malignant tumors. We evaluated the prognostic relevance of β-CATENIN, a pivotal mediator of WBC activation, in patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC)., Methods: We analyzed if patients with HPV-positive HNSCC from the "The Cancer Genome Atlas" (TCGA cohort, n = 41) can be stratified based on their CTNNB1 mRNA expression. Moreover, in a tissue microarray (TMA) of primary tumor sections from HPV-positive HNSCC patients treated in a tertiary academic center (in-house cohort, n = 31), we evaluated the prognostic relevance of β-CATENIN expression on protein level., Results: In silico mining of CTNNB1 expression in HPV-positive HNSCC revealed that high CTNNB1 expression was linked to better overall survival (OS, p = 0.062). Moreover, high β-CATENIN expression was significantly associated with a better OS in our in-house cohort (p = 0.035)., Conclusion: Based on these findings, we postulate that β-CATENIN expression could serve (potentially in conjunction with other WBC pathway members) as a marker for better survival outcomes in patients with HPV-positive HNSCC. However, it is evident that future studies on bigger cohorts are warranted., (© 2023. The Author(s).)

Purpose: PSMD14 is an essential protein for proteasomal degradation. Inhibition of this protein disrupts homeostasis and inhibits cancer cell viability. Overexpression of PSMD14 was associated with advanced cancer characteristics and a worse prognosis in various carcinomas. This study aimed to analyze PSMD14 copy number variation, mRNA and protein expression in HNSCC, and its role as an independent prognostic biomarker., Methods: PSMD14 mRNA expression and copy number variations were analyzed in "The Cancer Genome Atlas (TCGA)" in 510 patients. Protein expression was evaluated using immunohistochemistry in a second cohort including 115 patients. PSMD14 levels were analyzed for correlation with clinicopathological data, overall and disease-free survival., Results: PSMD14 mRNA expression and copy number variation were high in 44 and 50% of patients, respectively. Protein expression of PSMD14 was high in 56%. In both cohorts, high PSMD14 levels were associated with advanced staging. High PSMD14 mRNA expression was additionally associated with a worse prognosis in univariable analysis. However, after correction for possible confounders, PSMD14 mRNA was not an independent prognostic marker., Conclusion: PSMD14 is commonly expressed in HNSCC patients and associated with advanced stages. High expression of PSMD14 mRNA was associated with a worse outcome. However, this may be a result of the association of PSMD14 with poor prognosticators. Based on our study, further evaluation of PSMD14 as a prognostic marker and potential therapeutic target is warranted., (© 2022. The Author(s).)

Background: New chemotherapy agents are warranted for head and neck squamous cell carcinoma (HNSCC), particularly for incidence-rising HPV-positive tumors. Based on the evidence of Notch pathway involvement in cancer promotion and progression, we aimed to gain insights into the in vitro antineoplastic effects of gamma-secretase inhibition in HPV-positive and -negative HNSCC models., Methods: All in vitro experiments were conducted in two HPV-negative (Cal27 and FaDu) and one HPV-associated HNSCC cell line (SCC154). The influence of the gamma-secretase inhibitor PF03084014 (PF) on proliferation, migration, colony forming, and apoptosis was assessed., Results: We observed significant anti-proliferative, anti-migratory, anti-clonogenic, and pro-apoptotic effects in all three HNSCC cell lines. Furthermore, synergistic effects with concomitant radiation were observable in the proliferation assay. Interestingly, effects were slightly more potent in the HPV-positive cells., Conclusion: We provided novel insights into the potential therapeutic relevance of gamma-secretase inhibition in HNSCC cell lines in vitro. Therefore, PF may become a viable treatment option for patients with HNSCC, particularly for patients with HPV-induced malignancy. Indeed, further in vitro and in vivo experiments should be conducted to validate our results and decipher the mechanism behind the observed anti-neoplastic effects., (© 2023. The Author(s).)

Background: To create nomograms for better prediction of the oncological outcome in advanced laryngeal (LxCAs) or hypopharyngeal (HpxCAs) cancer after laryngopharyngectomy., Materials: 239 patients who underwent total laryngectomy or laryngopharyngectomy due to LxCA (52.7%) or HpxCA (47.3%) were included in this study. Based on clinical risk factors (tumor site, lymph node involvement, salvage setting), we created nomograms for prediction of disease-specific survival (DSS) and disease-free survival (DFS)., Results: HpxCAs showed a higher rate of lymph node involvement (p < 0.001), a 2.47-fold higher risk of a 2nd head and neck cancer (p = 0.009) and significantly worse loco-regional control rates (p = 0.003) compared to LxCAs. Positive neck nodes and salvage procedures were associated with significantly worse outcome. Nomograms demonstrated that hypopharyngeal tumors with positive neck nodes in salvage situations had the worst oncological outcome with a 5-year DSS of 15-20%., Conclusions: The oncological outcome is worse in hypopharyngeal carcinomas and could be easily quantified by our nomograms that are based on tumor site, lymph node involvement and salvage situation., (© 2022. The Author(s).)

Malnutrition is a frequent comorbidity in head and neck cancer patients and has been shown to impair immunotherapy response in other cancer types. The geriatric nutritional risk index (GNRI) assesses malnutrition using the patient's ideal weight, actual weight, and serum albumin. The aim of this study was to evaluate the prognostic relevance of malnutrition as determined by the GNRI for the response to immunotherapy in recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC). A total of 162 patients with R/M HNSCC who received immune checkpoint inhibitors were included. The associations between the GNRI and progression-free survival (PFS), overall survival (OS), and the disease control rate (DCR) were computed. Univariable analysis showed worse PFS for GNRI ≤ 98 ( p < 0.001), ECOG performance status (PS) ≥ 2 ( p = 0.012), and enteral ( p = 0.009) and parenteral ( p = 0.015) nutritional supplementation, and worse OS for GNRI < 92 ( p < 0.001), ECOG PS ≥ 2 ( p < 0.001), and enteral ( p = 0.008) and parenteral ( p = 0.023) nutritional supplementation. In our multivariable model, GNRI ≤ 98 ( p = 0.012) and ECOG PS ≥ 2 ( p = 0.025) were independent prognostic factors for PFS. For OS, GNRI < 92 ( p < 0.001) and ECOG PS ≥ 2 ( p < 0.001) were independent prognostic factors. A GNRI ≤ 98 was significantly associated with a lower DCR compared to a GNRI > 98 ( p = 0.001). In conclusion, our findings suggest that the GNRI may be an effective predictor for response to immunotherapy in R/M HNSCC.

Beta-catenin is known to be a vital component of the canonical Wnt signaling cascade, involved in the carcinogenesis of different solid tumors. We aimed to evaluate the effects of Beta-catenin inhibition in head and neck squamous cell carcinoma (HNSCC) in vitro. The small molecular compound MSAB was used to inhibit Wnt/Beta-catenin signaling in a human papillomavirus (HPV)-positive and HPV-negative cell line and its effects on cell proliferation, migration, colony formation, apoptosis, as well as radiosensitizing properties were assessed. Significant antineoplastic effects were observed in both cell lines. Interestingly, stronger anti-neoplastic and radiosensitizing effects were observed in the HPV-negative cell line, whereas stronger anti-migratory potential was detected in HPV-positive HNSCC cells. In conclusion, our findings suggest MSAB as a potential therapeutic agent for HNSCC. Further studies are warranted to unravel the mechanistic background of our findings., (© 2022. The Author(s).)

Purpose: Head and neck squamous cell carcinomas (HNSCCs) are a molecularly, histologically, and clinically heterogeneous set of tumors originating from the mucosal epithelium of the oral cavity, pharynx, and larynx. This heterogeneous nature of HNSCC is one of the main contributing factors to the lack of prognostic markers for personalized treatment. The aim of this study was to develop and identify multi-omics markers capable of improved risk stratification in this highly heterogeneous patient population., Methods: In this retrospective study, we approached this issue by establishing radiogenomics markers to identify high-risk individuals in a cohort of 127 HNSCC patients. Hybrid in vivo imaging and whole-exome sequencing were employed to identify quantitative imaging markers as well as genetic markers on pathway-level prognostic in HNSCC. We investigated the deductibility of the prognostic genetic markers using anatomical and metabolic imaging using positron emission tomography combined with computed tomography. Moreover, we used statistical and machine learning modeling to investigate whether a multi-omics approach can be used to derive prognostic markers for HNSCC., Results: Radiogenomic analysis revealed a significant influence of genetic pathway alterations on imaging markers. A highly prognostic radiogenomic marker based on cellular senescence was identified. Furthermore, the radiogenomic biomarkers designed in this study vastly outperformed the prognostic value of markers derived from genetics and imaging alone., Conclusion: Using the identified markers, a clinically meaningful stratification of patients is possible, guiding the identification of high-risk patients and potentially aiding in the development of effective targeted therapies., (© 2022. The Author(s).)

Sinonasal squamous cell carcinoma (SNSCC) is a malignant tumor associated with poor survival, and easily obtainable prognostic markers are of high interest. Therefore, we aimed to assess the prognostic value of a novel survival index (SI) combining prognostic values of clinical (T and N classifications and invasion across Ohngren's line), inflammatory (neutrophil-to-lymphocyte ratio), and nutritional (albumin and body-mass index) markers. All patients with primarily treated SNSCC between 2002 and 2020 ( n = 51) were included. Each of the six SI components was stratified into a low- (0) and high-risk (1) categories. Subsequently, the cohort was stratified into low- (SI of 0-2) and high-risk SI groups (SI of 3-6). Overall survival (OS) and disease-free survival (DFS) were compared between patients with low- and high-risk SI. The log-rank test was used to test for statistical significance. Overall, the mortality rate was 41.2% ( n = 21), and the recurrence rate was 43.1% ( n = 22). We observed significantly better OS in patients with low-risk SI ( n = 24/51, 47.1%, mean OS: 7.9 years, 95% confidence interval (CI): 6.3-9.6 years) than in high-risk SI ( n = 27/51, 52.9%, mean OS: 3.4 years, 95% CI: 2.2-4.5 years; p = 0.013). Moreover, we also showed that patients with low-risk SI had a longer DFS than patients with high-risk SI (mean DFS: 6.4, 95% CI: 4.8-8.0 vs. mean DFS: 2.4 years, 95% CI 1.3-3.5, p = 0.012). The SI combines the prognostic capacity of well-established clinical, radiologic, inflammatory, and nutritional prognosticators and showed prognostic potential in our cohort of SNSCC patients.

Background: Resistance to radiotherapy is a common cause of treatment failure in advanced head and neck squamous cell carcinoma (HNSCC). ß-Thujaplicin, a natural tropolone derivative, acts as an anti-cancer agent and has recently been shown to radiosensitize non-HNSCC cancer cells. However, no data is currently available on its radiosensitizing potential in HNSCC., Methods: To investigate the effect of ß-Thujaplicin and irradiation in HNSCC cell lines CAL27 and FADU, we performed a cell viability assay, colony forming assay, flow cytometry for cell cycle analysis and a wound healing assay. Drug-irradiation interaction was analyzed using a zero-interaction potency model., Results: Treatment with ß-Thujaplicin led to a dose-dependent decrease in cell viability and enhanced the effect of irradiation. Clonogenic survival was inhibited with synergistic drug-irradiation interaction. ß-Thujaplicin further led to S-phase arrest and increased the sub-G1 population. Moreover, combined ß-Thujaplicin and irradiation treatment had a higher anti-migratory effect compared to irradiation alone., Conclusions: ß-Thujaplicin acts as a radiosensitizer in HNSCC cell lines. Further evaluation of its use in HNSCC therapy is warranted., (© 2022. The Author(s).)

Background: Resistance to radiation therapy poses a major clinical problem for patients suffering from head and neck squamous cell carcinoma (HNSCC). Transforming growth factor ß (TGF-ß) has emerged as a potential target. This study aimed to investigate the radiosensitizing effect of galunisertib, a small molecule TGF-ß receptor kinase I inhibitor, on HNSCC cells in vitro., Methods: Three HNSCC cell lines were treated with galunisertib alone, or in combination with radiation. Of those three cell lines, one has a known inactivating mutation of the TGF-ß pathway (Cal27), one has a TGF-ß pathway deficiency (FaDu) and one has no known alteration (SCC-25). The effect on metabolic activity was evaluated by a resazurin-based reduction assay. Cell migration was evaluated by wound-healing assay, clonogenic survival by colony formation assay and cell cycle by FACS analysis., Results: Galunisertib reduced metabolic activity in FaDu, increased in SCC-25 and had no effect on CAL27. Migration was significantly reduced by galunisertib in all three cell lines and showed additive effects in combination with radiation in CAL27 and SCC-25. Colony-forming capabilities were reduced in SCC-25 by galunisertib and also showed an additive effect with adjuvant radiation treatment. Cell cycle analysis showed a reduction of cells in G 1 phase in response to galunisertib treatment., Conclusion: Our results indicate a potential antineoplastic effect of galunisertib in HNSCC with intact TGF-ß signaling in combination with radiation., (© 2022. The Author(s).)

Purpose: Folate receptor alpha (FRα) is overexpressed in various cancer entities while expression in normal tissue is limited. Thus, FRα is an attractive target in cancer therapy. Currently, various therapeutic and diagnostic approaches are under investigation in clinical trials. The aim of this study was to assess the expression and clinical relevance of FRα in adenoid cystic carcinoma of the head and neck., Patients and Methods: In this retrospective cohort study, 43 patients with adenoid cystic carcinoma (ACC) of the head and neck were included. FRα expression was analyzed in tumor tissue and tumor-free margin in a tissue microarray using immunohistochemical staining. Protein levels were correlated with clinical parameters., Results: FRα staining was positive in 47% of ACC patients. The tumor-free margin was positive in 22%. Patients with positive tumor tissue showed positive margin staining in 55%. FRα expression was not associated with the clinical parameters (sex, age, staging, grading, perineural invasion, lymphovascular invasion)., Conclusion: FRα expression is common in ACC of the head and neck. Therefore, FRα should be further evaluated as a therapeutic target in ACC., Competing Interests: The authors declare that they have no conflicts of interest in this work., (© 2022 Schnoell et al.)

Introduction: Zerumbone is a phytochemical compound of the ginger plant Zingiber zerumbet with cytotoxic effects in various cancer cell lines. To date, zerumbone has shown an antiproliferative effect in oral squamous cell carcinoma cells lines. However, the effect of combination with radiation or cisplatin in head and neck squamous cell carcinoma (HNSCC) is unclear. The aim of this study was to investigate the effect of zerumbone alone, and in combination with irradiation and cisplatin on HNSCC cell lines., Methods: The three HNSCC cell lines SCC25, Cal27 and FaDu were treated with zerumbone, radiation and/or cisplatin. Cell viability and clonogenic assays were performed. The interaction between zerumbone and radiation or cisplatin was evaluated using the combination index. Apoptosis was measured by flow cytometry and cell migration was assessed using a wound healing assay., Results: Treatment with zerumbone resulted in a dose dependent induction of cytotoxicity and apoptosis in all three cell lines. The combination with cisplatin revealed a synergistic to additive effect in Cal27. The clonogenic assay showed a significant radiosensitizing effect in all three cell lines. The wound healing assay showed a reduction of cell migration in Cal27., Conclusion: The natural compound zerumbone shows a cytotoxic and proapoptotic effect on HNSCC cell lines. Furthermore, zerumbone enhances the radiation effect in all three cell lines and thus may be a suitable candidate for combination therapy in HNSCC., (© 2021. The Author(s).)

Purpose: Tonsillectomies are among the most common surgeries in otorhinolaryngology. A novel electrosurgical temperature-controlled instrument (device) promises rapid tonsillectomies and might reduce postoperative pain, but comparative studies to assess performance are warranted., Methods: This randomized self-controlled clinical trial was conducted from October 2019 to October 2020 at the Department of Otorhinolaryngology, Head and Neck Surgery of the Medical University of Vienna. Forty-eight patients underwent a tonsillectomy with the device on one side and using cold-steel with localized bipolar cauterization on the other side (control). Main outcomes were the time for tonsil removal (per side) and the time to stop bleeding (per side). Secondary measurements were postoperative pain, assessed once on day 0 and five times on days 1, 3, 5, 7, and 10. Postoperative bleeding episodes and consequences were recorded., Results: Device tonsillectomies were performed significantly faster than controls; the mean surgical time difference was 209 s (p < 0.001, 95% CI 129; 288). Intraoperative blood loss was significantly lower on the device side (all p < 0.05). Postoperative measurements of pain and bleeding were similar for both sides. Two return-to-theatre secondary bleeding events were recorded for the control side., Conclusion: The novel electrosurgical temperature-controlled divider reduced the tonsillectomy surgical time and intraoperative blood loss, with no apparent negative effects on postoperative pain or bleeding, compared to a cold-steel tonsillectomy with localized bipolar cauterization. In time-restricted settings, the device could be beneficial, particularly after familiarization with device handling., Trial Registration: ClinicalTrials.gov Identifier: NCT03793816., (© 2021. The Author(s).)

Background: Until recently, vascularized lymph-node flaps were based on arterial and venous donor vessels. Now, venous lymph-node flaps form a novel promising concept in the treatment of advanced-stage lymphedema. In preliminary studies, the external jugular vein has shown promising results as a venous lymph-node flap. However, nothing is known about the number of lymph nodes adjacent to the external jugular vein., Methods: Standardized specimens of the external jugular vein and surrounding fatty tissue directly overlying the sternocleidomastoid muscle were obtained during routine neck dissection. Histologic evaluation was performed in order to evaluate for the presence of lymph nodes within the tissue., Results: A total of 20 specimens were evaluated. There was no vein in 4 of the samples. We found lymph nodes in 9 of the remaining 16 samples. In 7 samples, lymph nodes were absent., Conclusion: Our results suggest that the vein directly overlying the sternocleidomastoid muscle may not be the ideal candidate for a venous lymph-node flap.

Wnt/Beta-Catenin signaling is involved in the carcinogenesis of different solid malignant tumors. The interaction of Creb-binding protein (CBP) with Beta-Catenin is a pivotal component of the Wnt/Beta-Catenin signaling pathway. The first aim of this study was to evaluate the association of CBP expression with survival in patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). Second, the in vitro effects of the inhibition of CBP/Beta-Catenin interaction were analyzed. In particular, the effects of ICG-001, an inhibitor of CBP/Beta-Catenin interaction, on proliferation, cell death, modulation of Wnt/Beta-Catenin target expression, and cell migration were examined in vitro. High CBP expression is significantly associated with better survival on mRNA and protein levels. Furthermore, we observed cytotoxic as well as anti-migratory effects of ICG-001. These effects were particularly more potent in the HPV-positive than in the -negative cell line. Mechanistically, ICG-001 treatment induced apoptosis and led to a downregulation of CBP, c-MYC, and Cyclin D1 in HPV-positive cells, indicating inhibition of Wnt/Beta-Catenin signaling. In conclusion, high CBP expression is observed in HPV-positive HNSCC patients with a good prognosis, and ICG-001 showed a promising antineoplastic potential, particularly in HPV-positive HNSCC cells. Therefore, ICG-001 may potentially become an essential component of treatment de-escalation regimens for HPV-positive HNSCC. Further studies are warranted for additional assessment of the mechanistic background of our in vitro findings.

Mammary analogue secretory carcinomas (MASCs) of the parotid gland are considered as low-grade malignancies with good clinical outcome but lacking data regarding prognostic factors. We performed meta-analysis assessing prognostic factors for disease-free survival (DFS) and overall survival (OS) in 256 patients with MASCs of the parotid gland. A total of 73 studies have met the inclusion criteria and 76.3% of patients were seen with T1 and T2 tumors and negative neck nodes. Lymph node metastasis (57.4%) and distant recurrences (46.2%) were particularly found in T4 tumors (p < 0.001). DFS at 5 and 10 years was 77.9% and 47.2% compared to 88.1% and 77.2% for OS at the same time points. Male sex, T3-T4 tumors, and recurrent disease represented independent worse prognosticators for survival outcome. Altogether, parotid gland MASCs show good long-term outcome, but T4 tumors behave significantly more aggressive and require extended treatment strategies along with close follow-ups., (© 2021 Wiley Periodicals LLC.)

The proteins sodium iodide symporter (NIS), μ-crystallin (CRYM), and thyroid hormone receptor beta (THRB) have been associated with prognosis in various cancer entities. While NIS and THRB may serve as possible therapeutic targets, the role of CRYM in cancer is still unclear. Protein levels of 44 patients with adenoid cystic carcinoma of the head and neck were analyzed using immunohistochemistry and correlated with clinicopathological data and outcome. NIS was positive in 72%, CRYM was positive in 55%, and THRB was positive in 39% of the patients. CRYM-positive adenoid cystic carcinomas were associated with a better cause-specific survival. Thus, our data indicate that CRYM might be a suitable positive prognostic marker in adenoid cystic carcinoma of the head and neck. Furthermore, expression of NIS was present in most patients and therefore evaluation of the use of radioiodine treatment is recommended.

Thyroid hormone levels may be associated with disease outcome in head and neck squamous cell carcinoma (HNSCC). µ-Crystallin (CRYM), a thyroid hormone binding protein, blocks intracellular binding of the thyroid hormone T3 to its receptors. In this study, we aimed to analyze the association of CRYM levels with disease outcome in HNSCC patients. We retrospectively assessed immunohistochemical CRYM expression in 121 head and neck cancer patients. Preoperative thyrotropin levels could be extracted for 50 patients. Patients with low thyrotropin levels had a worse prognosis compared to euthyroid patients (5-year overall survival TSH low 20% vs. TSH norm 58%). We observed an association of CRYM+ patients with improved overall survival (5-year overall survival for CRYM+ 78.6% vs. CRYM- 56%). Interaction analysis between CRYM and HPV revealed that this effect was limited to HPV- patients (CRYM+|HPV- HR 0.12, 95% CI 0.01-0.87, p = 0.036). These results were replicated in an independent dataset. CRYM expression identified patients with favorable disease progression for HPV- HNSCC patients and could serve as a useful biomarker in this patient population. This study further confirms a correlation of thyroid hormone levels with adverse disease outcome in HNSCC patients, which could be potentially exploited as a therapeutic target.

Many patients with adenoid-cystic carcinoma (ACC) experience an indolent course of disease over many years but face late recurrence, and long-term survivors are rare. Due to its infrequent occurrence, it is hard to predict outcome in these patients. The fibrinogen-to-lymphocyte ratio (FLR) was recently proposed as an outcome prognosticator in different cancer entities. We aimed to investigate its prognostic relevance in patients with head and neck ACC. This retrospective analysis was performed including all patients treated for ACC between 1998 and 2020. The FLR ratio was calculated based on pretreatment values (0-7 days). The study cohort was dichotomized based on optimized threshold value and compared for differences in outcome (overall survival (OS) and disease-free survival (DFS)). In the cohort of 39 included patients, the OS was significantly longer in the low ( n = 28) compared to the high pretreatment FLR group ( n = 11) (median OS 150.5 months, 95% confidence intervals (CI) 85.3-215.7 months vs. 29.4 months, 95% CI not reached; p = 0.0093). Similarly, the DFS was significantly longer in the low FLR group (median DFS 74.5 months, 95% CI 30.6-118.4 months vs. 11.0 months, 95% CI 5.1-16.9 months; p = 0.019). The FLR is an easily obtainable and simple marker and may be a valuable outcome prognosticator in patients with ACC. Further studies are needed for validation of our results.

Background: The aim was to assess the prognostic value of the newly proposed prognostic index (PI) in patients with p16-positive oropharyngeal squamous cell carcinoma., Methods: Patients treated with primary surgery from 2012 to 2019 with available preoperative (0-2 days) values of C‑reactive protein and white blood cell counts needed for calculation of the PI, were included. Main outcome measures were overall survival (OS) and disease-free survival (DFS). The PI was dichotomized into low (PI = 0) and high (PI ≥ 1)., Results: In this study 36 patients were included. Average overall (OS) and disease-free survival (DFS) were 3.3 years (range 0.2-12.3 years) and 2.8 years (0.0-9.8 years), respectively. The overall mortality was 16.7% (n = 6) and a recurrent disease was observed in 30.6% of patients (n = 11). Low PI was associated with better overall survival (mean OS 10.1 ± 1.4 years, 95% confidence interval, CI 7.3-12.9 years vs. 1.9 ± 0.4, 95% CI 1.3-2.6 years, p < 0.01; mean DFS 8.5 ± 0.7 years, 95% CI 7.1-9.6 years vs. 1.0 ± 0.3 years, 95% CI 0.5-1.5 years, p < 0.01)., Conclusion: The PI might be an easily obtainable outcome prognosticator in p16-positive oropharyngeal squamous cell carcinoma patients. Analyzing routinely obtained blood samples can contribute to identifying high-risk patients., (© 2021. The Author(s).)

Although mostly associated with good survival outcomes, some patients with HPV-positive oropharyngeal squamous cell carcinoma develop distant metastasis and face dire prognosis. The aim of this study was to analyze distant metastatic patients in regards to survival, clinical staging, therapy approach and p16/HPV status. This retrospective single-centre study assessed patients with HPV-associated oropharyngeal cancer with distant metastasis treated in a tertiary referral center from 2005 to 2019. Overall- (OS) and survival after diagnosis of distant metastasis (OMS), clinical staging and different therapy approaches were assessed. Moreover, the overall mortality was assessed, as well as the association of different therapy approaches and p16/HPV status with the survival outcome. Out of 211 patients with HPV-associated oropharyngeal cancer that were treated in the study period, 15 developed distant metastases (7.1%). Median OS and OMS of the total group were 11 months (range 0.1-32 months) and 3 months (range 0.1-21 months), respectively. The overall mortality rate was 53.3% (n = 8). Significantly better outcome was present in patients treated with primary chemoradiotherapy (median OS 17 months vs. not reached, p = .03, median OMS 8 months vs not reached, p = .05). The OMS was significantly better in patients treated with chemotherapy initially after diagnosis (mean OMS 21 months vs 4 months; P = .001). Surgical resection after initial diagnosis was associated with a significantly shorter OMS (median OMS 3 vs. 21 months, p = .005). Interestingly, postoperative adjuvant therapy was delayed in all of these cases due to surgical site complications. Systemic treatment after initial diagnosis may be beneficial in clinical outcome of HPV associated distant metastases. Furthermore, surgical site complications should be treated with immediate care in order to avoid delay of adjuvant therapy. Further studies are warranted for validation of our results., Competing Interests: Declaration of competing interest There are no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

(1) Objective: To evaluate long-term functional outcome in patients who underwent primary or salvage total laryngectomy (TL), TL with partial (TLPP), or total pharyngectomy (TLTP), and to establish a new scoring system to predict complication rate and long-term functional outcome; (2) Material and Methods: Between 1993 and 2019, 258 patients underwent TL ( n = 85), TLPP ( n = 101), or TLTP ( n = 72). Based on the extent of tumor resection, all patients were stratified to (i) localization I: TL; II: TLPP; III: TLTP and (ii) surgical treatment (A: primary resection; B: salvage surgery). Type and rate of complication and functional outcome, including oral nutrition, G-tube dependence, pharyngeal stenosis, and voice rehabilitation were evaluated in 163 patients with a follow-up ≥ 12 months and absence of recurrent disease; (3) Results: We found 61 IA, 24 IB, 63 IIA, 38 IIB, 37 IIIA, and 35 IIIA patients. Complications and subsequently revision surgeries occurred most frequently in IIIB cases but rarely in IA patients (57.1% vs. 18%; p = 0.001 and 51.4% vs. 14.8%; p = 0.002), respectively. Pharyngocutaneous fistula (PCF) was the most common complication (33%), although it did not significantly differ among cohorts ( p = 0.345). Pharyngeal stenosis was found in 27% of cases, with the highest incidence in IIIA (45.5%) and IIIB (72.7%) patients ( p < 0.001). Most (91.1%) IA patients achieved complete oral nutrition compared to only 41.7% in class IIIB patients ( p < 0.001). Absence of PCF (odds ratio (OR) 3.29; p = 0.003), presence of complications (OR 3.47; p = 0.004), and no need for pharyngeal reconstruction (OR 4.44; p = 0.042) represented independent favorable factors for oral nutrition. Verbal communication was achieved in 69.3% of patients and was accomplished by the insertion of voice prosthesis in 37.4%. Acquisition of esophageal speech was reached in 31.9% of cases. Based on these data, we stratified patients regarding the extent of surgery and previous treatment into subgroups reflecting risk profiles and expectable functional outcome; (4) Conclusions: The extent of resection accompanied by the need for reconstruction and salvage surgery both carry a higher risk of complications and subsequently worse functional outcome. Both factors are reflected in our classification system that can be helpful to better predict patients' functional outcome.

Purpose: The transcription factors YY1 and CP2 have been associated with tumor promotion and suppression in various cancers. Recently, simultaneous expression of both markers was correlated with negative prognosis in cancer. The aim of this study was to explore the expression of YY1 and CP2 in head and neck squamous cell carcinoma (HNSCC) patients and their association with survival., Methods: First, we analyzed mRNA expression and copy number variations (CNVs) of YY1 and CP2 using "The Cancer Genome Atlas" (TCGA) with 510 HNSCC patients. Secondly, protein expression was investigated via immunohistochemistry in 102 patients, who were treated in the Vienna General Hospital, utilizing a tissue microarray., Results: The median follow-up was 2.9 years (1.8-4.6) for the TCGA cohort and 10.3 years (6.5-12.8) for the inhouse tissue micro-array (TMA) cohort. The median overall survival of the TCGA cohort was decreased for patients with a high YY1 mRNA expression (4.0 vs. 5.7 years, p = 0.030, corr. p = 0.180) and high YY1-CNV (3.53 vs. 5.4 years, p = 0.0355, corr. p = 0.213). Furthermore, patients with a combined high expression of YY1 and CP2 mRNA showed a worse survival (3.5 vs. 5.4 years, p = 0.003, corr. p = 0.018). The mortality rate of patients with co-expression of YY1 and CP2 mRNA was twice as high compared to patients with low expression of one or both (HR 1.99, 95% CI 1.11-3.58, p = 0.021). Protein expression of nuclear YY1 and CP2 showed no association with disease outcome in our inhouse cohort., Conclusion: Our data indicate that simultaneous expression of YY1 and CP2 mRNA is associated with shorter overall survival. Thus, combined high mRNA expression might be a suitable prognostic marker for risk stratification in HNSCC patients. However, since we could not validate this finding at genomic or protein level, we hypothesize that unknown underlying mechanisms which regulate mRNA transcription of YY1 and CP2 are the actual culprits leading to a worse survival.