Neural Crest Stem Cells in Juvenile Angiofibromas

The etiology of juvenile angiofibroma (JA) has been a controversial topic for more than 160 years. Numerous theories have been proposed to explain this rare benign neoplasm arising predominately in adolescent males, focusing mainly on either the vascular or fibrous component. To assess our hypothesis of JA’s being a malformation arising from neural crest cells/remnants of the first branchial arch plexus, we performed immunohistochemical analyses of neural crest stem cells (NCSC) and epithelial-mesenchymal transition (EMT) candidates. Immunoexpression of the NCSC marker CD271p75 was observed in all investigated JA’s (n = 22), mainly around the pathological vessels. Close to CD271p75-positive cells, high MMP3-staining was also observed. Additionally, from one JA with sufficient material, RT-qPCR identified differences in the expression pattern of PDGFRβ, MMP2 and MMP3 in MACS®-separated CD271p75positive vs. CD271p75 negative cell fractions. Our results, together with the consideration of the literature, provide evidence that JA’s represent a malformation within the first branchial arch artery/plexus remnants deriving from NCSC. This theory would explain the typical site of tumor origin as well as the characteristic tumor blood supply, whereas the process of EMT provides an explanation for the vascular and fibrous tumor component

Prognostic genetic markers in malignant gliomas

Glioblastomas are the most frequent and malignant brain tumors in adults. Surgical cure is virtually impossible and despite of radiation and chemotherapy the clinical course is very poor. Epigenetic silencing of MGMT has been associated with a better response to temozolomide-chemotherapy. We previously showed that temozolomide increases the median survival time of patients with tumors harbouring deletions on 9p within the region for p15(INK4b), p16(INK4a), and 10q (MGMT). The aim of this study was to investigate the methylation status of p15, p16, 14ARF and MGMT in glioblastomas and to correlate the results with the clinical data.Only patients with KPS > 70, radical tumor resection, radiation and temozolomide-chemotherapy after recurrence were included. We observed promoter methylation of MGMT in 56% (15/27) and of p15 in 37% (10/27) of the tumors, whereas methylation of p16 and p14ARF were rare. Interestingly, methylation of p15 emerged as a significant predictor of shorter overall survival (16.9 vs. 23.8 months, p=0.025), whereas MGMT promoter methylation had no significant effect on median overall survival under this treatment regimen (22.5 vs. 22.1 months, p=0.49). In the presence of other clinically relevant factors, p15 methylation remains the only significant predictor (p=0.021; Cox regression).Although these results need to be confirmed in larger series and under different treatment conditions, our retrospective study shows clear evidence that p15 methylation can act as an additional prognostic factor for survival and underlines that this tumor suppressor, involved in cell cycle control, can act as an attractive candidate for therapeutic approaches in glioblastomas

Immunohistochemistry Reveals TRPC Channels in the Human Hearing Organ : A Novel CT-Guided Approach to the Cochlea

TRPC channels are critical players in cochlear hair cells and sensory neurons, as demonstrated in animal experiments. However, evidence for TRPC expression in the human cochlea is still lacking. This reflects the logistic and practical difficulties in obtaining human cochleae. The purpose of this study was to detect TRPC6, TRPC5 and TRPC3 in the human cochlea. Temporal bone pairs were excised from ten body donors, and the inner ear was first assessed based on computed tomography scans. Decalcification was then performed using 20% EDTA solutions. Immunohistochemistry with knockout-tested antibodies followed. The organ of Corti, the stria vascularis, the spiral lamina, spiral ganglion neurons and cochlear nerves were specifically stained. This unique report of TRPC channels in the human cochlea supports the hypothesis of the potentially critical role of TRPC channels in human cochlear health and disease which has been suggested in previous rodent experiments

Antagonizing Sec62 function in intracellular Ca2+ homeostasis represents a novel therapeutic strategy for head and neck cancer

Various cancer types including head and neck squamous cell carcinomas (HNSCC) show a frequent amplification of chromosomal region 3q26 that encodes, among others, for the SEC62 gene. Located in the ER membrane, this translocation protein is known to play a critical role as a potential driver oncogene in cancer development. High SEC62 expression levels were observed in various cancer entities and were associated with a poor outcome and increased metastatic burden. Because of its intracellular localization the SEC62 protein is poorly accessible for therapeutic antibodies, therefore a functional SEC62 knockdown represents the most promising mechanism of a potential antineoplastic targeted therapy. By stimulating the Ca2+ efflux from the ER lumen and thereby increasing cellular stress levels, a functional inhibition of SEC62 bears the potential to limit tumor growth and metastasis formation. In this study, two potential anti-metastatic and -proliferative agents that counteract SEC62 function were investigated in functional in vitro assays by utilizing an immortalized human hypopharyngeal cancer cell line as well as a newly established orthotopic murine in vivo model. Additionally, a CRISPR/ Cas9 based SEC62 knockout HNSCC cell line was generated and functionally characterized for its relevance in HNSCC cell proliferation and migration as well as sensitivity to SEC62 targeted therapy in vitro

Inhibition of CK2 Reduces NG2 Expression in Juvenile Angiofibroma

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve–glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-20 -deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA

Expression of 3q Oncogene SEC62 Predicts Survival in Head and Neck Squamous Cell Carcinoma Patients Treated with Primary Chemoradiation

Primary chemoradiotherapy (CRT) is an established treatment option for locally advanced head and neck squamous cell carcinomas (HNSCC) usually combining intensity modified radiotherapy with concurrent platinum-based chemotherapy. Though the majority of patients can be cured with this regimen, treatment response is highly heterogeneous and can hardly be predicted. SEC62 represents a metastasis stimulating oncogene that is frequently overexpressed in various cancer entities and is associated with poor outcome. Its role in HNSCC patients undergoing CRT has not been investigated so far. A total of 127 HNSCC patients treated with primary CRT were included in this study. The median follow-up was 5.4 years. Pretherapeutic tissue samples of the primary tumors were used for immunohistochemistry targeting SEC62. SEC62 expression, clinical and histopathological parameters, as well as patient outcome, were correlated in univariate and multivariate survival analyses. High SEC62 expression correlated with a significantly shorter overall survival (p = 0.015) and advanced lymph node metastases (p = 0.024). Further significant predictors of poor overall and progression-free survival included response to therapy (RECIST1.1), nodal status, distant metastases, tobacco consumption, recurrence of disease, and UICC stage. In a multivariate Cox hazard proportional regression analysis, only SEC62 expression (p = 0.046) and response to therapy (p < 0.0001) maintained statistical significance as independent predictors of the patients’ overall survival. This study identified SEC62 as an independent prognostic biomarker in HNSCC patients treated with primary CRT. The role of SEC62 as a potential therapeutic target and its interaction with radiation-induced molecular alterations in head and neck cancer cells should further be investigated

Cytology-based Cancer Surgery of the Head and Neck (CyCaS-HN): a prospective, randomized, controlled clinical trial

Purpose Liquid-based cytology (LBC) is routinely used in gynecology but is rarely applied in head and neck oncology though many suspicious lesions are easily accessible. While several studies have evaluated the potential use of LBC for early detection and molecular characterization of head and neck squamous cell carcinomas (HNSCCs), no study investigated its potential role in surgical management and therapy planning so far. Methods Twenty-fve patients with cT1-2 squamous cell carcinomas of the oral cavity and oropharynx were prospectively enrolled in this study and were randomized to two treatment arms: in the control arm, a diagnostic panendoscopy with incisional biopsy was followed by a second operation with transoral tumor resection±neck dissection and tracheostomy. In the intervention arm, patients underwent LBC diagnostics and in case of a positive result received one single operation with panendoscopy and incisional biopsy for confrmation of LBC result by rapid section histology followed by transoral tumor resection±neck dissection and tracheostomy in the same session. Results Time between clinical diagnosis and defnitive surgical treatment was signifcantly shorter in the intervention group compared with the control group (p<0.0001). Additionally, time of hospitalization (p<0.0001) and cumulative operation time (p=0.062) were shorter in the intervention group. No signifcant diferences in overall, progression-free, and diseasespecifc survival were observed. Conclusion Cytology-based cancer surgery is a promising therapeutic strategy that can potentially be considered for a well-defned group of early-stage HNSCC patients and help to avoid repetitive general anesthesia, shorten the diagnosis-totreatment interval and spare operation as well as hospitalization time

HPV Status as Prognostic Biomarker in Head and Neck Cancer—Which Method Fits the Best for Outcome Prediction?

The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uniand multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives

Prognostic impact of intra- and peritumoral immune cell subpopulations in head and neck squamous cell carcinomas – comprehensive analysis of the TCGA-HNSC cohort and immunohistochemical validation on 101 patients

BackgroundDue to the expanding role of immune checkpoint inhibition in the treatment of head and neck squamous cell carcinoma, understanding immunological processes in the tumor microevironment (TME) has strong translational importance. Though analytical methods for a comprehensive analysis of the immunological TME have constantly improved and expanded over the past years the prognostic relevance of immune cell composition in head and neck cancer TME largely remains ambiguous with most studies focusing on one or a small subset of immune cells.MethodsThe overall survival (OS) of the TCGA-HNSC patient cohort comprising 513 head and neck cancer patients was correlated with a total of 29 different immune metrics including a wide spectrum of immune cell subpopulations as well as immune checkpoint receptors and cytokines using RNAseq based immune deconvolution analyses. The most significant predictors of survival among these 29 immune metrics were validated on a separate HNSCC patient cohort (n=101) using immunohistochemistry: CD3, CD20+CXCR5, CD4+CXCR5, Foxp3 and CD68.ResultsOverall immune infiltration irrespective of immune cell composition showed no significant correlation with the patients’ overall survival in the TCGA-HNSC cohort. However, when focusing on different immune cell subpopulations, naïve B cells (p=0.0006), follicular T-helper cells (p&lt;0.0001), macrophages (p=0.0042), regulatory T cells (p=0.0306), lymphocytes (p=0.0001), and cytotoxic T cells (p=0.0242) were identified as highly significant predictors of improved patient survival. Using immunohistochemical detection of these immune cells in a second independent validation cohort of 101 HNSCC patients, we confirmed the prognostic relevance of follicular T helper cells, cytotoxic T cells and lymphocytes. In multivariable analysis, HPV negativity and advanced UICC stages were identified as additional prognostic biomarkers associated with poor outcome.ConclusionOur study highlights the prognostic relevance of the immunological tumor environment in head and neck cancer and demonstrates that a more detailed analysis of immune cell composition and immune cell subtypes is necessary to accurately prognosticate. We observed the highest prognostic relevance for lymphocytes, cytotoxic T cells, and follicular T helper cells, suggesting further investigations focusing on these specific immune cell subpopulations not only as predictors of patient prognosis but also as promising targets of new immunotherapeutic strategies

Genetic alterations of malignant gliomas predict response to temozolomide-chemotherapy

Das Glioblastom, das typische Karzinom des Gehirns, gehört zu den bösartigsten Tumoren des Menschen. Es führt, behandelt wie unbehandelt zum Tode. Eine völlige operative Entfernung ist wegen der ausgeprägten Infiltration ins umgebende Hirngewebe unmöglich. In der Regel wird eine Strahlenbehandlung, häufig auch eine Chemotherapie angeschlossen, die den Zeitraum bis zum Auftreten eines Rezidivs und bis zum Tode des Patienten von weniger als einem Jahr bis auf etwa zwei Jahre zu verlängern vermag. Das Glioblastom ist außerdem ein Tumor mit erheblichen genetischen Veränderungen und einer ungewöhnlichen großen intratumoralen wie intertumoralen genetischen Heterogenität, die im histologischen Befund nur bedingt zum Ausdruck kommt, aber offenbar wesentlichen Einfluss auf den - vorübergehenden- Therapieerfolg ausübt. In der vorliegenden Arbeit wurde geprüft, ob der unterschiedliche Behandlungserfolg von Glioblastomen mit dem handelsüblichen Zytostatikum Temozolomid (TMZ; Temodal®), einem DNA-alkylierenden Agens, durch definierte genetische Veränderungen der Tumorzellen beeinflusst wird. Es wurden 53 Gliome mit Hilfe der vergleichenden genomischen Hybridisierung karyotypiert. Bei diesem Verfahren wird die Gesamt-DNA einer kleinen Tumorbiopsie unter standardisierten Bedingungen auf normale Metaphasechromosomen hybridisiert. Dadurch werden Vermehrungen und Verluste bestimmter Chromosomenabschnitte mit einer Auflösung von etwa 15 Mb für Kopiengewinne (1,5 Mb für höhere Amplifikationen) und 20-30 Mb für Verluste erkennbar, die in ca. 40-50% der untersuchten Zellen vorliegen. Veränderungen in sehr kleinen Subklonen werden nicht erkannt. Als charakteristische einheitliche Veränderungen, die in der überwiegenden Mehrzahl der Tumoren vorlagen, wurden Zugewinne von Chromosomen 7 (komplett oder partiell) und Verluste von 9p, 10 (komplett oder partiell) sowie 13q nachgewiesen. Es wurde sichergestellt, dass kein statistischer Unterschied in diesem Spektrum zwischen den Tumoren der Patienten die nur nachbestrahlt worden waren und denjenigen, die zusätzlich TMZ erhalten hatten, bestand. TMZ-behandelte Patienten zeigten eine deutlich höhere Überlebenszeit von 19.2/7,5 Monaten. Bei den TMZ-unbehandelten Patienten bestätigte sich der bekannte Effekt, dass der Verlust von 9p- und 10q die Überlebenszeit signifikant verschlechtert. Es zeigte sich weiter, dass jedoch gerade diese Untergruppe von TMZ besonders profitiert. Eine Multivarianz-Analyse differenzierte dahingehend, dass der Effekt für 9p-Verluste der allgemein stärkere war, für Patienten über 53 Jahren jedoch 10q-Verluste. In Übereinstimmung mit der Literatur wurde die Hypothese aufgestellt, dass als Markergene in erster Linie für 9p die einander benachbarten Tumorsuppressorgene CDKN2A und CDKN2B (p16, p15) und für 10q das DNA-Reparatur-Gen MGMT zu diskutieren sind. Die für einen weitgehenden oder kompletten Expressionsverlust dieser Gene erforderliche Inaktivierung der jeweils zweiten Kopie der Markergene kann durch (submikroskopische) Deletion, Mutation oder epigenetisch durch Promoter-Hypermethylierung erfolgen. In drei von vier darauf untersuchten Tumoren konnte eine Hypermethylierung und in der RT-PCR eine deutlich reduzierte Expression von MGMT gezeigt werden. Die Ergebnisse genetischer Untersuchungen könnten künftig eine gezieltere Chemotherapie von Glioblastompatienten ermöglichen, da histologische Befunde keine diesbezüglichen Informationen liefern.The Glioblastoma, typical karzinoma of the brain, belongs to the most malignant tumors of humans. It leads, treated as untreated to death. A complete surgical removal is impossible because of the pronounced infiltration in the surrounding brain tissue. Usually a radiotherapy, frequently also a chemotherapy, is attached which is able to extend the period up to the reccurrence and up to the death of the patient of less than one year up to approximately two years. In addition, the Glioblastoma is a tumor with substantial genetic changes and an unusual large intratumoral and intertumoral genetic heterogeneity, which is in the histological findings only conditionally expressed, but obviously exercises substantial influence on - passing therapy success. In the available work it was examined, whether the different treatment success of glioblastoma with the commercial cytostatic drug Temozolomid (TMZ; Temodal®), an DNA alkylating agent, is affected by defined genetic changes of the tumor cells. 53 gliomas were karyotyped with the help of comparative genomischen hybridizing (CGH). With this procedure, the entire DNA of a small tumorbiopsie is hybridized on normal metaphase-chromosomes under standardized conditions. Thus gains and losses of intended chromosomal regions become recognizable with a dissolution of approximately 15 Mb for copy gains (1.5 Mb for higher amplifications) and 20-30 Mb for losses, which are present in approx. 40-50% of the examined cells. Changes in very small subclones are not recognized. As characteristic uniform changes, which were present in the predominant majority of the tumors, increases of chromosome 7 (completely or partially) and losses of 9p, 10 (completely or partially) as well as 13q became proven. It was guaranteed that no statistic difference in this spectrum between the tumors of the patients had been only after-illuminated and those, which had received additionally TMZ existed. TMZ-treated patients showed a clearly higher survival period of 19.2/7,5 months. With the TMZ-untreated patients, the well-known effect was confirmed that the loss of 9p and 10q worsened the survival period significantly. However, straight this sub-group particularly profits from TMZ. A multivariate-analysis differentiated in such a way that the effect of 9p losses was the generally stronger, for patients over 53 years however 10q-losses. In agreement with the literature the hypothesis was set up that as marker genes for 9p primarily the each other neighbouring tumorsuppressorgenes CDKN2A and CDKN2B (p16, p15) and for 10q the DNA repair gene MGMT are to be discussed. that necessary for a large or complete expression Inactivating loss of the second copy of the marker genes can take place via (submicroscopic) deletion, mutation or epigenetically via promoter-hypermethylation. In three of four tumors examined for it a Hypermethylierung could be shown and in the RT-PCR a clearly reduced expression of MGMT. In the future, the results of genetic investigations could make a more purposeful chemotherapy possible of glioblastoma patients, since histological findings do not supply relevant information