Tumour budding-based grading as independent prognostic biomarker in HPV-positive and HPV-negative head and neck cancer.

BACKGROUND: The prognostic significance of tumour budding (TB) and minimal cell nest size (MCNS) was shown in human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC). However, the optimisation of cutpoints, the prognostic impact in HPV-positive HNSCC, and the comparison with other histopathological grading systems are insufficiently investigated. METHODS: TB and MCNS were analysed digitally in 1 and 10 high-power fields (HPF) of 331 HPV-positive and HPV-negative cases from TCGA. Optimising the cutpoints a new cellular dissociation grading (CDG) system was defined and compared to the WHO grading and the Brandwein-Gensler (BG) risk model. RESULTS: The two-tiered CDG system based solely on TB yielded optimal prognostic stratification with shortened overall survival for CDG-high cases. Optimal cut-offs were two buds (1 HPF) and six buds (10 HPF), respectively. Analysing MCNS did not add prognostic significance to quantifying TB. CDG was a significant prognostic marker in HPV-negative and HPV-positive tumours and prognostically superior to the WHO and BG systems. High CDG was associated with clinically occult lymph-node metastases. CONCLUSIONS: The most comprehensive study of TB in HNSCC so far confirmed its prognostic impact in HPV-negative tumours and for the first time in HPV-positive tumours. Further studies are warranted to evaluate its applicability for therapy guidance in HNSCC.

The role of psycho-oncologic screenings in the detection and evaluation of depression in head and neck cancer aftercare patients.

PURPOSE: Cancer and morbidity during a therapeutic regimen can result in somatic and psychiatric impairment. We have evaluated the need of appropriate psychological screening by analyzing a large collective of head and neck cancer (HNC) patients with particularly burdensome symptoms. METHODS: HNC-aftercare patients were asked about somatic and psychological symptoms by means of standardized questionnaires of the European Organization for Research and Treatment of Cancer (EORTC Q30 and QLQ-H&N35). Patients with poor well-being values on the World Health Organization-5-Well-Being Index were screened for depression by using the Mini International Neuropsychiatric Interview, and adequate treatment was initiated, if necessary. RESULTS: Our sample consisted of 453 HNC-aftercare patients (average age 64.5 years; 72.0% male; 28.0% female). 25.1% showed abnormalities based on their WHO-5 questionnaire. A current major depressive episode was observed in 8.5% of the total study group. Patients with lip and oral cavity tumors showed the highest depression prevalence (18.9%). Time since initial HNC diagnosis showed no clear trend with regard to the number of depression cases. 50.0% of patients with a current major depressive episode consented to receiving assistance and/or therapy. Within the total study population, the most burdensome symptoms were found to be "dry mouth" (48.3%), "trouble doing strenuous activities" (46.0%), "trouble taking a long walk" (38.5%), and "worry" (35.5%). Aftercare patients with a depression diagnosis tended to have heavier symptom burdens than people without major depression. CONCLUSIONS: Despite the various cancer-related burdensome factors, prevalence levels of depression among the HNC-aftercare patients and the general population were similar. Nevertheless, since the number of diagnosed depression cases is high, the need for psychological treatment should be considered within the tumor collective. Furthermore, screening for depression should be implemented in clinical routines by using the appropriate standardized questionnaires.

Report on Late Toxicity in Head-and-Neck Tumor Patients with Long Term Survival after Radiochemotherapy.

Regular tumor follow-up care provided by ear-nose-throat (ENT) specialists ends when patients reach 5-year survival, but radiotoxicity is a continuous lifelong process. In this study, long-term head-and-neck cancer (HNC) survivors undergoing tumor follow-up (FU) care exceeding five years in a certified HNC center of a German university hospital were analyzed for newly diagnosed late sequelae after radio-(chemo-)therapy. Patients diagnosed with squamous cell carcinoma (SCC) of the oral cavity, larynx or oro-/hypopharynx receiving treatment between 1990 and 2010 with a tumor FU care beyond five years were reviewed retrospectively for signs of late sequelae after radio-(chemo-)therapy (R(C)T) including carotid artery stenosis, stenosis of the cranial esophagus, dysphagia, osteoradionecrosis, and secondary malignancies. Long-term survivors that solely received surgical treatment served as control. Of 1143 analyzed patients we identified 407 patients with an overall survival beyond five years, 311 with R(C)T and 96 patients without R(C)T. Furthermore, 221/1143 patients were lost to FU and the mortality rate within the first 5-years was 45%. Moreover, 27.7% of the long-term survivors were diagnosed with new onset late sequelae within the following five years. RT was significantly associated with a two-fold risk increase for newly diagnosed symptoms, especially after RT of the lymphatic pathways (LP) which showed a hazard ratio of 23.3 to develop alterations on the carotid arteries. Additional chemotherapy had no statistical correlation with any late onset toxicity nor did the mode of R(C)T (adjuvant/definitive). Although the validity of this study might be limited due to its retrospective nature and the dependence on the voluntary participation in a prolonged tumor FU, the results nevertheless provide the need to offer and encourage a tumor FU by ENT specialists exceeding the common 5-year margin. This could prevent secondary morbidities and improve quality of life for long-term cancer survivors.

Radiooncological View on Therapy Outcome after Multidisciplinary Treatment of Sinonasal Tumors.

PURPOSE: We report the outcome of a mono-institutional retrospective study of sinonasal carcinoma with the primary focus on GTV (gross tumor volume) and the effect of radiotherapy. METHODS: 53 patients with sinonasal carcinoma and that of the nasal cavity, paranasal sinus or both except lymphoma were included. All patients were treated between 1999 and 2017. For tumor volume delineation, all pre-therapeutic images were fused to the planning CT (computed tomography). RESULTS: The median follow-up was 17 months [0.3-60], the median age 60 years, 35 males and 18 females were included. Squamous cell carcinoma (SCC) (60.4%) was the predominant histology, followed by adenocarcinoma (15.1%). The mean composite OS (overall survival) time was 33.3 ± 3.5 months. There was no significant difference in the 5 y composite OS between tumor localization or radiotherapy setting. The simultaneous integrated boost concept showed a trend towards improving five-year composite OS compared to the sequential boost concept. The only factor with a significant impact on the 5 y composite OS rate was the pre-therapeutic GTV (cutoff 75 cm3; p = 0.033). The GTV ≥ 100 cm3 has no effect on the 5 y composite OS rate for SCC. CONCLUSIONS: The pre-therapeutic GTV is a prognostic factor for five-year composite OS for the entire group of patients with sinonasal tumors, influencing the outcome after completion of all treatment strategies. The GTV seems to not influence five-year composite OS in SCC. For this rare tumor entity, an intensive, multidisciplinary discussion is essential to finding the best treatment option for the patient.

Fibrin Gel Suspended Autologous Chondrocytes as Cell-based Material for long-term Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Injection laryngoplasty of materials for unilateral vocal-fold paralysis has shown various results regarding the long-term stability of the injected material. We evaluated a fibrin-gel based cell suspension with autologous chondrocytes in-vitro and in-vivo as long-term-stable vocal-fold augmentation material in an animal model. STUDY DESIGN: This study compises an in vitro cell-culture part as well as an in vivo animal study with New Zealand White Rabbits. METHODS: In in-vitro experiments, auricular chondrocytes harvested from 24 New Zealand White Rabbits cadavers were cultivated in pellet cultures to evaluate cartilage formation for 4 weeks using long-term-stable fibrin gel as carrier. Injectability and injection volume for the laryngoplasty was determined in-vitro using harvested cadaveric larynxes. In-vivo 24 Rabbits were biopsied for elastic cartilage of the ear and autologous P1 cells were injected lateral of one vocal cord into the paraglottic space suspended in a long-term-stable fibrin gel. Histologic evaluation was performed after 2, 4, 12, and 24 weeks. RESULTS: During 12-week pellet culture, we found extracellular matrix formation and weight-stable cartilage of mature appearance. In-vivo, mature cartilage was found in two larynxes (n = 6) at 4 weeks, in four (n = 6) at 12 weeks, and in five (n = 6) at 24 weeks mostly located in the paraglottic space and sometimes with spurs into the vocalis muscle. Surrounding tissue was often infiltrated with inflammatory cells. Material tended to dislocate through the cricothyroid space into the extraglottic surrounding tissue. CONCLUSIONS: A cell-based approach with chondrocytes for permanent vocal-fold augmentation has not previously been reported. We have achieved the formation of structurally mature cartilage in the paraglottic space, but this is accompanied by difficulties with dislocated material, deformation of the augmentation, and inflammation. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1624-E1632, 2021.

[GERD and adjuvant radio-chemotherapy predespose to recurrent voice prosthesis leakage]. / GERD und adjuvante Radiochemotherapie prädisponieren für rezidivierende Stimmprotheseninsuffizienz.

After total laryngectomy, regaining ability to speech is a keystone in regards of life quality. Voice prostheses have been shown to be a sufficient tool for satisfying communication, although frequent replacements of prostheses are burdening certain patients. Therefore, a more accurate understanding of mechanisms of prosthetic leakage is urgently needed. METHODS: We performed a retrospective analysis of 58 Patients after laryngectomy. Additionally, we analyzed pre- and post-therapeutic CT-scans of 22 Patients regarding pharyngeal stenosis. RESULTS: In 40 Patients, at least one replacement of voice prosthesis was documented during observation period, median device life was 235 days. Patients treated with adjuvant radiotherapy (RT) showed a significantly longer device life than patients with adjuvant radio-chemotherapy (RCT, p = 0.002). Furthermore, patients suffering of gastroesophageal reflux disease (GERD) showed a significantly shortened device life (p = 0.04).17 patients (42.5 %) suffered of clinically relevant stenosis of the neopharynx, which was treated with dilatation in 14 patients (82 %) and did not affect prosthesis device life. CONCLUSION: GERD is a risk factor for shortened voice prosthesis' device life and therefore should be treated effectively after laryngectomy. Also, adjuvant RCT predisposes a shortened device life.Stenosis is observed frequently after laryngectomy but does not affect device life when effectively treated.

Laryngeal and Pharyngeal Movements During Inner Singing: A Cross-Sectional Study.

INTRODUCTION: Laryngeal and pharyngeal activity during inner singing is discussed in the context of vocal hygiene. Inner singing is defined as imagined singing, reading music silently, and listening to vocal music. When vocal rest is prescribed, doctors, speech therapists, and voice pedagogues recommend avoiding listening to music or reading music silently, since it is suggested that inner singing unconsciously influences the glottis, and thus moves the vocal folds involuntarily. The aim of this study was to compare the degree to which involuntary laryngeal and/or pharyngeal activity occur during inner singing, inner speech, and at rest, and to evaluate if current recommendations concerning vocal hygiene are still reasonable. MATERIAL AND METHOD: Thirty vocally healthy participants were examined transnasally with a flexible videoendoscope. The sample consisted of 10 nonsingers, 10 lay singers, and 10 professional singers. Participants were examined during five tasks including rest, silent reading, imagining a melody, listening to music, and reading music. Two medical doctors specializing in phoniatrics analyzed the videos both qualitatively and quantitatively. RESULTS: During the endoscopic examination, the raters identified movements at the base of the tongue, the posterior and lateral pharynx wall, the arytenoid cartilage, and the vocal folds. The inner singing tasks showed significantly more laryngeal movements as well as significantly more glottal closures than the control tasks (at rest, silent reading). Pharyngeal structures did not show an increase in activity during inner singing. These findings were independent of the level of proficiency in singing. CONCLUSION: When total vocal rest is prescribed, patients should also be advised to avoid music imagination. Still, further research is needed to survey in detail the actual effects of these involuntary movements during inner singing on the regeneration process of vocal fold healing.

The Aurora-Kinase A Phe31-Ile polymorphism as possible predictor of response to treatment in head and neck squamous cell carcinoma.

Recently the Aurora-Kinases (Aurk) moved into the focus as novel disease related biomarkers and therapeutic targets. Elevated Aurora-Kinase expression has been found in a number of malignancies, amongst them HNSCC. For esophageal cancer, the AurkA Phe31-Ile polymorphism has previously been associated with tumor progression. Here we evaluated the treatment efficiency of HNSCC cell radiation as a function of Aurora-Kinases in HNSCC cell lines. Moreover, we investigated a potential sensitization to radiation by a cell treatment with the inhibitors Alisertib, Barasertib, Docetaxel and VX-680. In parallel the radiation dependent expression and regulation of AurkA/B, p-Akt Ser 473 and Survivin and the AurkA polymorphism were investigated in primary tumor samples. We identified a high-risk collective with elevated AurkA and Survivin or AurkA and p-Akt Ser 473 expression. High AurkA, AurkB, and p-Akt Ser 473 expression was exclusively found in the heterozygous cell line. We found a polymorphism dependent sensitivity to treatments with different Aurk inhibitors: The homozygous cell line UD-SCC-5 could be sensitized to radiation with Docetaxel in combination with any of the Aurora-Kinase inhibitors. In contrast, treatment with Docetaxel or radiation did not enhance the inhibitory effect of Barasertib or VX-680 in the heterozygous SAS cell line. These findings indicate that the Aurora-Kinase A Phe31-Ile-polymorphism is a possibly predictive factor for response to radiation in combination with Docetaxel and Aurora-Kinase inhibitor treatments.

Liposarcoma of the Thyroid: A Case Report with a Review of the Literature.

BACKGROUND: Liposarcomas of the thyroid gland are extremely rare tumors, and, to our knowledge, only 12 cases have been reported in the English literature. An accurate diagnosis is challenging due to the nonspecific clinical presentation of this cancer, frequently defined just by a swelling of the neck. PATIENT FINDINGS: We present an 82-year-old woman with liposarcoma of the thyroid, complaining of a fast-growing neck mass. MRI and neck ultrasound showed a large lipomatous mass, which corresponded to a cold nodule in the thyroid scan. After performing a total thyroidectomy, the diagnosis of a well-differentiated liposarcoma of the thyroid gland was made, showing an MDM2 amplification in fluorescence in situ hybridization. Since neither a metastasis nor a residual tumor was found, no further adjuvant therapy was needed. RESULTS: We searched the literature for previous case reports and identified only 12 cases worldwide to form our database. A demographic as well as clinical and histopathological analysis was made. In most cases, the liposarcoma occurred in patients >60 years of age. All histological subtypes, such as well-differentiated and myxoid liposarcomas, and pleomorphic and dedifferentiated liposarcomas, were found in the literature. In only 38.46% of the cases, an infiltration of the adjacent organs was observed. Surgery was the most common treatment chosen. CONCLUSIONS: Our review provides clinical and histopathological features of a primary liposarcoma of the thyroid to enable the identifi-cation of this rare tumor entity and assist in the decision-making process regarding therapeutic options and tumor follow-up.

Uncultivated stromal vascular fraction is equivalent to adipose-derived stem and stromal cells on porous polyurethrane scaffolds forming adipose tissue in vivo.

OBJECTIVES/HYPOTHESIS: To find an alternative approach to contemporary techniques in tissue augmentation and reconstruction, tissue engineering strategies aim to involve adipose-derived stem and stromal cells (ASCs) harboring a strong differentiation potential into various tissue types such as bone, cartilage, and fat. STUDY DESIGN: Animal research. METHODS: The stromal vascular fraction (SVF) was used directly as a cell source to provide a potential alternative to contemporary ASC-based adipose tissue engineering. Seeded in TissuCol fibrin, we applied ASCs or SVF cells to porous, degradable polyurethane (PU) scaffolds. RESULTS: We successfully demonstrated the in vivo generation of volume-stable, well-vascularized PU-based constructs containing host-derived mature fat pads. Seeded human stem cells served as modulators of host-cell migration rather than differentiating themselves. We further demonstrated that preliminary culture of SVF cells was not necessary. CONCLUSIONS: Our results bring adipose tissue engineering, together with automated processing devices, closer to clinical applicability. The time-consuming and cost-intensive culture and induction of the ASCs is not necessary. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E206-E213, 2018.

Epidermal growth factor receptor variant III in head and neck squamous cell carcinoma is not relevant for targeted therapy and irradiation.

BACKGROUND: The epidermal growth factor receptor (EGFR) is an important regulator of cell growth and survival, and is highly variable in tumor cells. The most prevalent variation of the EGFR extracellular domain is the EGFR variant III (EGFRvIII). Some studies imply that EGFRvIII may be responsible for the poor response to the monoclonal EGFR-antibody Cetuximab, used therapeutically in head and neck squamous cell carcinoma (HNSCC). Due to inconsistent data in the literature regarding EGFRvIII prevalence and clinical relevance in HNSCC, especially its predictive value, we examined EGFRvIII-transfected cell lines and patient tissue samples. RESULTS: In contrast to other recent publications, we were able to demonstrate EGFRvIII expression in HNSCC. However, we noted that the different detection methods yielded inconsistent results. Furthermore, our EGFRvIII transfected and EGFR wild type cell lines exhibited similar characteristics and response rates in the performed in vitro experiments. MATERIALS AND METHODS: We conducted various inhibition and combined irradiation experiments using three EGFRvIII-transfected cell lines. Moreover, a patient cohort of 149 cases consisting of formalin fixed and paraffin embedded (FFPE) and fresh-frozen specimens was assayed via reverse transcriptase PCR (rtPCR) with gel electrophoresis and sequencing for EGFRvIII prevalence. In the rtPCR assays, we used five previously published EGFRvIII primers and EGFRvIII-positive glioblastoma tissue as a positive control. In addition, immunohistochemical staining was conducted. CONCLUSIONS: EGFRvIII can be detected in HNSCC patient samples. Nevertheless, the low prevalence and similar response rates to targeted drugs and irradiation in vitro cast doubt regarding the clinical relevance of EGFRvIII in HNSCC.

Donor-dependent variances of human adipose-derived stem cells in respect to the in-vitro endothelial cell differentiation capability.

Human adipose-derived stem cells (ASC) have been shown to differentiate into mature adipocytes and to play an important role in creating the vasculature, necessary for white adipose tissue to function. To study the stimulatory capacity of ASC on endothelial progenitor cells we used a commercially available co-culture system (V2a - assay). ASC, isolated from lipoaspirates of 18 healthy patients, were co-cultured for 13 d on endothelial progenitor cells. Using anti CD31 immunostaining, cells that had undergone endothelial differentiation were quantified after the defined co-cultivation period. Endothelial cell differentiation was observed and demonstrated by an increase in area covered by CD31+ cells compared with less to no endothelial cell differentiation in negative and media-only controls. Enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) in supernatant medium collected during the co-cultivation period revealed elevated VEGF levels in the co-culture samples as compared with ASC cultures alone, whereas no increase in adiponectin was detected by ELISA. These findings help to provide further insights in the complex interplay of adipose derived cells and endothelial cells and to better understand the diversity of ASCs in respect of their stimulatory capacity to promote angiogenesis in vitro.

HNSCC cells resistant to EGFR pathway inhibitors are hypermutated and sensitive to DNA damaging substances.

Despite remarkable successes with targeted therapies in the treatment of cancer, resistance can occur which limits the clinical outcome. In this study, we generated and characterized resistant cell clones derived from two different head and neck squamous cell carcinoma (HNSCC) cell lines (Cal27, UD-SCC-5) by long-term exposure to five targeted- and chemotherapeutics (afatinib, MK2206, BEZ235, olaparib and cisplatin). The resistant tumor cell clones showed an increased ERK1/2 expression and an altered expression of the stem-cell markers CD44, ALDH1, Oct4, Sox2, Nanog and Bmi1. None of the single markers alone was predictive for resistance to all five targeted- and chemotherapeutics. Furthermore, long-term exposure of tumor cells to these five drugs resulted in an eightfold increase in the mutational rate compared to untreated cells. Interestingly, targeted- and chemotherapy resistant cell clones remained sensitive to irradiation. Lastly, clones that were resistant to afatinib, MK2206 or BEZ235 showed cross-resistance to further treatment with therapeutics that affect the same signaling pathway, but remained sensitive to those affecting different pathways such as cisplatin and olaparib. In contrast, cell clones which were once resistant to cisplatin or olaparib were found to be multidrug-resistant. These data might indicate that patients with HNSCC benefit more by a first line targeted therapy followed by cisplatin as a second line therapy.

Management and prevention of acute bleedings in the head and neck area with interventional radiology.

BACKGROUND: The Interventional Neuroradiology is becoming more important in the interdisciplinary treatment of acute haemorrhages due to vascular erosion and vascular tumors in the head and neck area. The authors report on acute extracranial haemorrhage in emergency situations but also on preventive embolization of good vascularized tumors preoperatively and their outcome. METHODS: Retrospective analysis of 52 patients, who underwent an interdisciplinary approach of the ORL Department and the Interventional Neuroradiology over 5 ½ years at the Department of Otorhinolaryngology, Klinikum Rechts der Isar, Technical University of Munich, Germany. Their outcome was analysed in terms of success of the embolization, blood loss, survival rate and treatment failures. RESULTS: 39/52 patients were treated for acute haemorrhage. Twenty-five of them attributable to vascular erosion in case of malignant tumors. Affected vessels were the common carotid artery as well as its internal and external parts with branches like the ascending pharyngeal, the facial and the superior thyroid artery. Altogether 27/52 patients were treated for malignant tumors, 25/52 were attributable to acute haemorrhage due to epistaxis, after tonsillectomy, benign tumors and bleeding attributable to inflammations. Treatment of all patients consisted either of an unsuccessful approach via exposure, package of the bleeding, electrocoagulation or surgical ligature followed by embolization or the primary treatment via interventional embolization/stenting. CONCLUSIONS: The common monitoring of patients at the ORL and interventional neuroradiology is an important alternative especially in the treatment of severe acute haemorrhage, following vascular erosion in malignant tumors or benign diseases. But also the preoperative embolization of good vascularized tumors must be taken into account to prevent severe blood loss or acute intraoperative bleeding.

New aspects in the pathomechanism and diagnosis of the laryngopharyngeal reflux-clinical impact of laryngeal proton pumps and pharyngeal pH metry in extraesophageal gastroesophageal reflux disease.

AIM: To determine the laryngeal H+K+-ATPase and pharyngeal pH in patients with laryngopharyngeal reflux (LPR)-symptoms as well as to assess the symptom scores during PPI therapy. METHODS: Endoscopy was performed to exclude neoplasia and to collect biopsies from the posterior cricoid area (immunohistochemistry and PCR analysis). Immunohistochemical staining was performed with monoclonal mouse antibodies against human H+K+-ATPase. Quantitative real-time RT-PCR for each of the H+K+-ATPase subunits was performed. The pH values were assessed in the aerosolized environment of the oropharynx (DxpH Catheter) and compared to a subsequently applied combined pH/MII measurement. RESULTS: Twenty patients with LPR symptoms were included. In only one patient, the laryngeal H+K+-ATPase was verified by immunohistochemical staining. In another patient, real-time RT-PCR for each H+K+-ATPase subunit was positive. Fourteen out of twenty patients had pathological results in DxpH, and 6/20 patients had pathological results in pH/MII. Four patients had pathological results in both functional tests. Nine out of twenty patients responded to PPIs. CONCLUSION: The laryngeal H+K+-ATPase can only be sporadically detected in patients with LPR symptoms and is unlikely to cause the LPR symptoms. Alternative hypotheses for the pathomechanism are needed. The role of pharyngeal pH-metry remains unclear and its use can only be recommended for patients in a research study setting.