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Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with a poor prognosis for survival. Risk factors include alcohol and tobacco abuse and infection with human papilloma virus (HPV). To enhance anti-tumor immune responses immunotherapeutic approaches are approved for recurrent metastatic disease but only approx. 20% of patients respond to checkpoint blockade of the PD-1/PD-L1 axis. Therefore, preclinical research is needed to better understand molecular and cellular processes and to identify new therapeutic targets. Immunocompetent mouse models can serve these purposes but only few are currently available for HPV-positive HNSCC. Here, we established a mouse cell line overexpressing the oncogenes E6/E7 of the HPV16 genome as well as a constitutively active form of H-Ras and studied the anti-tumor immune response upon orthotopic tumor growth at the floor of the mouth. Moreover, we analyzed the same immunoregulatory pathways in samples of HPV-positive cancer patients. T cells in the tumor of mice and humans exhibited high expression of CD39 and CD73, two ectoenzymes involved in the production of immunosuppressive adenosine from ATP, along with increased expression of PD-1, LAG-3 and GITR. Additionally, B cell responses were elevated in tumor-bearing mice, seen as an increase of germinal center, immunoregulatory marginal zone and follicular B cell subtypes. Taken together, this study suggests that the generated mouse model shares characteristics with human disease and can thus be used as a platform to study anti-tumor responses in HPV-positive HNSCC which will help to identify novel therapeutic targets., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Ulm University (Votum #90/15). Consent to participate: All donors of the present study provided informed and written consent. Animal experimentation was approved by the Regierungspräsidium Tübingen (permission number #1607)., (© 2024. The Author(s).)

Due to their high developmental diversity and different regulatory and functional roles, B cell subpopulations can promote or inhibit tumor growth. An orthotopic murine HNSCC model was applied to investigate the B cell composition and function in HNSCCs. Using flow cytometry approaches, cells from the spleen, lymph nodes and tumors were analyzed. Additionally, immunoglobulin (Ig) levels post-tumor induction were tracked via enzyme-linked immunosorbent assays (ELISA). Following tumor induction, GCs, as well as increasing numbers of GL7 + CD95 + GC B cells in the spleen and tumor tissues, were detected. In parallel, we observed CD39 + CD73 + B cells in tumors and spleens of tumor-bearing mice. Notably, CD39 + CD73 + expression was primarily detected on MZ B cells and to a lesser extent on follicular (FO) and non-follicular, newly formed (NF) B cells, supposing an immunosuppressive function of MZ B cells in the TME. Parallel to increased MZ B cell numbers in secondary lymphoid organs (SLOs) as well as in the tumor tissue, IgM antibody (Ab) levels rose continuously. In contrast, IgG1, IgG2, and IgG3 levels increased at later time points. Understanding the complex interactions between B cell subsets and the TME could lead to new strategies for enhancing the treatment and prognosis of HNSCC patients.

Immunotherapeutic approaches are now established in the treatment of various tumor entities, including head and neck squamous cell carcinoma (HNSCC). PD‑1 antibodies are currently approved for HNSCC with palliative intent but are increasingly being investigated in studies with curative objectives, e.g., as neoadjuvant therapy. At ASCO 2024, particular focus was placed on combinations of immunotherapy with therapeutic vaccines for human papillomavirus (HPV)-induced tumors. Moreover, the question of which patients benefit most from immunotherapy remains unresolved. The growing significance of PD-L1 expression, measured by the combined positive score (CPS), is becoming increasingly evident. This article summarizes the latest relevant findings from the largest international cancer congress, the American Society of Clinical Oncology (ASCO) 2024 Annual Meeting., Competing Interests: Einhaltung ethischer Richtlinien Interessenkonflikt J. Doescher: Advisory Board MSD, Merck Serono. T.K. Hoffmann: Advisory Board BMS, MSD, Sanofi. S. Laban: Advisory Board BMS, MSD, Astra Zeneca, Boehringer Ingelheim, Glaxo Smith Kline. A. von Witzleben gibt an, dass kein Interessenkonflikt besteht.Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: Exosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood., Methods: Exosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements., Results: Our data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes., Conclusions: The study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation., (© 2024. The Author(s).)

Background: Head and neck squamous cell carcinoma (HNSCC) represents a common and heterogeneous malignancy of the oral cavity, pharynx and larynx. Surgery and radio(chemo)therapy are the standard treatment options and also have great influence on the composition of the tumor microenvironment and immune cell functions. However, the impact of radio(chemo)therapy on the distribution and characteristics of circulating monocyte subsets in HNSCC are not fully understood., Methods: Expression patterns of adhesion molecules and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor) and checkpoint molecule PD-L1 (programmed cell death ligand-1) were investigated upon radio(chemo)therapeutic treatment using flow cytometry. Furthermore, comprehensive analysis of plasma cytokines was performed before and after treatment using ELISA measurements., Results: Our data reveal a partial recovery of circulating monocytes in HNSCC patients upon radio(chemo)therapeutic treatment, with differential effects of the individual therapy regimen. PD-L1 expression on non-classical monocytes significantly correlates with the individual plasma levels of chemokine CXCL11 (C-X-C motif chemokine 11)., Conclusions: Further comprehensive investigations on larger patient cohorts are required to elucidate the meaningfulness of peripheral blood monocyte subsets and chemokine CXCL11 as potential bioliquid indicators in HNSCC with regard to therapy response and the individual immunological situation., (© 2024. The Author(s).)

Patients with oropharyngeal squamous cell carcinoma (OPSCC) caused by human papilloma virus (HPV) exhibit a better prognosis than those with HPV-negative OPSCC. This study investigated the distinct molecular pathways that delineate HPV-negative from HPV-positive OPSCC to identify biologically relevant therapeutic targets. Bulk mRNA from 23 HPV-negative and 39 HPV-positive OPSCC tumors (n = 62) was sequenced to uncover the transcriptomic profiles. Differential expression followed by gene set enrichment analysis was performed to outline the top enriched biological process in the HPV-negative compared with HPV-positive entity. INHBA, the highest overexpressed gene in the HPV-negative tumor, was knocked down. Functional assays (migration, proliferation, cell death, stemness) were conducted to confirm the target's oncogenic role. Correlation analyses to reveal its impact on the tumor microenvironment were performed. We revealed that epithelial-to-mesenchymal transition (EMT) is the most enriched process in HPV-negative compared with HPV-positive OPSCC, with INHBA (inhibin beta A subunit) being the top upregulated gene. INHBA knockdown downregulated the expression of EMT transcription factors and attenuated migration, proliferation, stemness, and cell death resistance of OPSCC cells. We uncovered that INHBA associates with a pro-tumor microenvironment by negatively correlating with antitumor CD8+ T and B cells while positively correlating with pro-tumor M1 macrophages. We identified three miRNAs that are putatively involved in repressing INHBA expression. Our results indicate that the upregulation of INHBA is tumor-promoting. We propose INHBA as an attractive therapeutic target for the treatment of INHBA-enriched tumors in patients with HPV-negative OPSCC to ameliorate prognosis., Significance: Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: High numbers of tumor-infiltrating lymphocytes (TIL) are linked to better survival in patients with cancer. Tissue-resident memory T cells (TRM; CD8+CD103+) are recognized as a key player of anticancer immune response. To assess TRM cells in primary, metastatic, and recurrent head and neck squamous cell carcinoma (HNSCC), we developed a tissue microarray (TMA) and used multiplex IHC (MxIHC)., Experimental Design: Samples from primary tumors of 379 HNSCC cases treated at Southampton Hospitals between 2000 and 2016 were collected and analyzed. Of these, 105 cases had lymph node metastases and 82 recurrences. A TMA was generated with triplicate cores for each sample. MxIHC with a stain-and-strip approach was performed using CD8, CD103, and TIM3. Scanned slides were analyzed (digital image analysis) and quality checked (QC)., Results: After QC, 194 primary tumors, 76 lymph node metastases, and 65 recurrences were evaluable. Alcohol consumption was statistically significantly correlated with a reduction of TRM cells in primary tumors (nondrinker vs. heavy drinker: P = 0.0036). The known survival benefit of TRM cell infiltration in primary tumors was not found for lymph node metastasis. In recurrences, a high TRM cell number led to a favorable outcome after 12 months. The checkpoint molecule TIM3, was expressed significantly higher on TRM and non-TRM cells in the lymph node compared with primary tumors (P < 0.0001), which was also seen in recurrences (P = 0.0134 and P = 0.0007, respectively)., Conclusions: We confirm the prognostic impact of TIL in primary tumors and in recurrences. TRM cell density in lymph node metastases was not linked to outcome. The role of TIM3, as a therapeutic target remains to be defined., (©2023 American Association for Cancer Research.)

Patients with HPV - -localized head and neck cancer (HNC) show inferior outcomes after surgery and radiochemotherapy compared to HPV-associated cancers. The underlying mechanisms remain elusive, but differences in immune status and immune activity may be implicated. In this study, we analyzed immune profiles of CD8 + T cells and myeloid-derived suppressor cells (MDSC) in HPV + versus HPV - disease.The overall frequency of CD8 + T cells was reduced in HNC versus healthy donors but substantially increased after curative therapy (surgery and/or radiochemotherapy). In HPV + patients, this increase was associated with significant induction of peripheral blood CD8 + /CD45RA - /CD62L - effector memory cells. The frequency of HPV-antigen-specific CD8 + cells was low even in patients with virally associated tumors and dropped to background levels after curative therapy. Pre-therapeutic counts of circulating monocytic MDSC, but not PMN-MDSC, were increased in patients with HPV - disease. This increase was accompanied by reduced fractions of terminally differentiated CD8 + effector cells. HPV - tumors showed reduced infiltrates of CD8 + and CD45RO + immune cells compared with HPV + tumors. Importantly, frequencies of tumor tissue-infiltrating PMN-MDSC were increased, while percentages of Granzyme B + and Ki-67 + CD8 T cells were reduced in patients with HPV - disease.We report differences in frequencies and relative ratios of MDSC and effector T cells in HPV - HNC compared with more immunogenic HPV-associated disease. Our data provide new insight into the immunological profiles of these two tumor entities and may be utilized for more tailored immunotherapeutic approaches in the future., (© 2023. The Author(s).)

Introduction: Demographics are important prognostic factors in malignant diseases. A nationwide analysis concerning the prognostic impact of demographics in head and neck cancer (HNC) patients (HNCP) has not been performed previously., Methods: A retrospective analysis of data from the Center for Cancer Registry Data (ZfKD) and the Federal Statistical Office (Destatis) between 2002 and 2017 was performed. A total of 212'920 HNCP were included. Incidence, tumor stage, age development, sex distribution, age-, residence-, and diagnosis-time-specific survival were examined., Results: Mean age of HNCP increased more rapidly than in the general population (slope coefficient: 0.29 vs. 0.20; p < 0.0001). Higher age and male sex were associated with a worse prognosis. Whereas overall survival (OS) increased from the early to the later observation period for HNCP <70 years, no OS improvement for HNCP >70 years was found. Furthermore, an OS disadvantage was observed for East Germany compared to West Germany (median 47 vs. 60 months; p < 0.0001). This disparity was associated with a disproportionately high ratio of men in East Germany (men/women: 4.4 vs. 3.1; p < 0.0001) and a lower mean age (61 vs. 63 years; p < 0.0001). In addition to stage, age and sex, residence in East Germany were confirmed as an independent factor for OS in a multivariate analysis., Conclusion: Finally, three decades after the German reunion, a survival disadvantage for patients in East Germany still exists. This discrepancy may be a result of socioeconomic disparities., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The combination of positron-emission tomography (PET) with cross-sectional imaging in particular is becoming increasingly important in the diagnosis of head and neck tumors because, in addition to pure anatomy, the metabolic activity of tissue can be visualized and assessed. The combination of PET and computed tomography (CT) is already an established procedure in head and neck tumor patients in some indications, e.g., for primary tumor detection in cancer of unknown primary (CUP) syndrome or also after completed primary radio(chemo)therapy for evaluation of response, especially also with regard to nodal status. In some cases, salvage neck dissection can thus be avoided in the case of PET-negative findings. In the context of primary diagnosis, PET/CT imaging can be used primarily to evaluate distant metastasis. According to current guidelines, PET-based imaging is not (yet) of value in determining the local extent at initial diagnosis. A challenge is the still limited reimbursement by health insurance companies, which currently allow only certain indications, and the still lack of nationwide coverage., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: Demographic development in rural and urban areas differs substantially. Demographics and access to specialized head and neck cancer centers may affect head and neck cancer patients' (HNCP) outcomes. Here, we compare epidemiological indicators and outcomes of HNCP in rural and urban Germany., Patients and Methods: In a retrospective analysis of data from the Center for Cancer Registry Data (ZfKD) between 2002 and 2017, 212,920 HNCP were included. Incidence, demographics, travel distance to specialized centers, and ground values were compared between rural and urban areas with a focus on their association with patient outcomes., Results: The mean age of HNCP was significantly higher in urban areas (mean difference = 1.4 years; p < 0.0001), but increased at a comparable rate (p = 0.26) in rural and urban areas during the observation period. Gender imbalance was higher in rural areas (mean ratio of men/women: 4.1 vs. 3.1; p < 0.0001), but showed a comparable trend toward equilibration in both, rural and urban districts (p = 0.46). The portion of HNCP of the entire HNCP population living in urban areas increased from 55.9% in the year 2002 to 76.4% in the year 2017. There was no significant difference or change in the ratio of advanced to low UICC stage during the observation period (p = 0.26). However, travel distances to medical centers were higher in rural areas, especially (p < 0.0001) in East Germany. Median survival of HNCP in rural areas was significantly lower than in urban areas (42 months [SEM = 0.7; CI: 40.5-43.5] vs. 54 months [SEM = 1.2; CI: 51.7-56.3]; p < 0.0001) in East Germany, whereas in West Germany no significant difference was observed (59 months [SEM = 0.8; CI: 57.4-60.6] vs. 60 months [SEM = 0.5; CI: 59.0-61.0]; p = 0.15)., Conclusions: Place of residence contributes to survival outcome of HNCP. Access to specialized care and socioeconomic factors could be improved in East Germany., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1 + CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.

Immunotherapeutic agents are nowadays established for treatment of a wide variety of tumor entities, including squamous cell carcinoma of the head and neck region. Originally used in the palliative setting, these are increasingly administered with curative intent, e.g., as neoadjuvant treatment. Current research addresses the questions of which patients benefit from the treatment and which combination therapies are successful. The present article summarizes relevant findings of the two international cancer congresses in 2022., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of cancers and patients have limited therapy options if primary treatment fails. Therefore, additional information about the biology of the tumor is essential. Here we performed a feasibility study of concurrently applying two precision diagnostic tools in a consecutive series of HNSCC patients. We analyzed tumor samples of 31 patients using a genomic (oncomine) and a proteomic, immunohistochemical approach (oncopanel) and compared the result, also in the focus on their overlapping therapeutical targets. We found no strong correlation between the two approaches and observed a higher proportion of marker expression for the immunohistochemical panel. However, both panels show in our HNSCC cohort distinct patterns with druggable targets. The data suggest that both approaches complement one another and can be applied side-by-side to identify the best targets for the development of individual treatment options for HNSCC patients.

Demographically, the German population is aging and becoming more morbid. At the same time, urbanization trends, medical overcapacities, and increasing care costs are being observed in association with a tight healthcare budget. Centralization, specialization, and outpatient care are intended to provide a remedy and can be controlled by modifications to remuneration. This upheaval poses new challenges for patients and physicians, which were analyzed exemplarily at the Head and Neck Tumor (HNC) Center of the University Hospital Ulm. This is a retrospective, monocentric cohort study on the development of patient volume, catchment area, treatment modality, and demographics including 2070 HNC patients at the ENT clinic between the years 2011 and 2020. It was observed that the number (new diagnoses 2011: 134 vs. 2020: 204) and the average age (2011: 61.5 years vs. 2020: 65.8 years; p < 0.0001) of HNC patients increased over time. In addition, patients tended to travel longer distances (2011: 54.4 km vs. 2020: 64.4 km; p = 0.05). At the same time, the mean number of consultations and treatments per patient per 5-year follow-up interval grew (at initial diagnosis in 2011: 7.8 vs. 2016: 10.4; p = 0.0003), with the proportion of outpatient contacts increasing from 58.9 % to 62.4 % (p = 0.09) from 2011 to 2020. Accordingly, clinical centers are expected to become more important in the care of HNC patients as the healthcare system becomes more specialized, and centralized with a growing outpatient setting. The following consequences for patient care should be considered in restructuring strategies., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Background: This year's American Society of Clinical Oncology (ASCO) meeting included interesting data on first-line therapy of nasopharyngeal carcinomas with PD‑1 inhibitors and on checkpoint inhibition in various clinical constellations. At the European Society of Medical Oncology (ESMO) meeting, the results of the CheckMate-651 study were presented., Materials and Methods: All abstracts and presentations from the ASCO and ESMO meetings 2021 on immunotherapy in head and neck cancer (HNSCC) were evaluated for their relevance. The most interesting studies are elaborated upon herein., Results: Studies on locally advanced HNSCC showed an improved response after neoadjuvant pembrolizumab administration. A second cycle did not improve the response rate, but the proportion of patients with a good response was almost doubled. The CheckRad CD8 study showed an improvement in progression-free survival by induction chemoimmunotherapy with tremelimumab and durvalumab followed by stratification according to the CD8 immune cell infiltrate. Two studies were presented on first-line treatment of recurrent/metastatic nasopharyngeal carcinomas. Chemoimmunotherapy showed a higher response rate and prolonged progression-free survival with a similar adverse event profile. In recurrent/metastatic HNSCC, the CheckMate 651 study showed an increased duration of response with nivolumab and ipilimumab and higher response rates than pembrolizumab alone. The primary endpoints for overall survival were not achieved., Conclusion: PD‑1 inhibition has great potential to change the therapeutic landscape for nasopharyngeal carcinomas in the future. In HNSCC, CD8 tumor infiltrate presents a promising predictive marker for selecting patients who can benefit from radioimmunotherapy. The combination of nivolumab and ipilimumab did not improve overall survival in palliative first-line therapy; thus, no change in the current standard is expected., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Under common therapeutic regimens, the prognosis of human papillomavirus (HPV)-positive squamous oropharyngeal carcinomas (OPCs) is more favorable than HPV-negative OPCs. However, the prognosis of some tumors is dismal, and validated prognostic factors are missing in clinical practice. The present work aimed to validate the prognostic significance of our published three-cluster model and to compare its prognostic value with those of the 8 th edition of the tumor-node-metastasis staging system (TNM8) and published signatures and clustering models., Methods: Patients with HPV DNA-positive OPCs with locoregionally advanced nonmetastatic disease treated with curative intent (BD2Decide observational study, NCT02832102) were considered as validation cohort. Patients were treated in seven European centers, with expertise in the multidisciplinary management of patients with head and neck cancer. The median follow-up was 46.2 months (95% CI, 41.2 to 50), and data collection was concluded in September 2019. The primary end point of this study was overall survival (OS). Three-clustering models and seven prognostic signatures were compared with our three-cluster model., Results: The study population consisted of 235 patients. The three-cluster model confirmed its prognostic value. Two-year OS in each cluster was 100% in the low-risk cluster, 96.6% in the intermediate-risk cluster, and 86.3% in the high-risk cluster ( P = .00074). For the high-risk cluster, we observed an area under the curve = 0.832 for 2-year OS, significantly outperforming TNM 8th edition (area under the curve = 0.596), and functional and biological differences were identified for each cluster., Conclusion: The rigorous clinical selection of the cases included in this study confirmed the robustness of our three-cluster model in HPV-positive OPCs. The prognostic value was found to be independent and superior compared with TNM8. The next step includes the translation of the three-cluster model in clinical practice. This could open the way to future exploration of already available therapies in HPV-positive OPCs tailoring de-escalation or intensification according to the three-cluster model., Competing Interests: Loris De Cecco Research Funding: Bristol Myers Squibb Foundation No other potential conflicts of interest were reported. Loris De Cecco Research Funding: Bristol Myers Squibb Foundation No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Programmed-death-1 (PD1) antibodies are approved for recurrent and metastatic head and neck squamous cell carcinoma. Multiple drugs targeting costimulatory and coinhibitory immune checkpoint molecules (ICM) have been discovered. However, it remains unknown how these ICM are affected by curative conventional therapy on different immune cell subsets during the course of treatment. In the prospective noninterventional clinical study titled "Immune Response Evaluation to Curative conventional Therapy" (NCT03053661), 22 patients were prospectively enrolled. Blood samples were drawn at defined time points throughout curative conventional treatment and follow-up. Immune cells (IC) from the different time points were assessed by multicolor flow cytometry. The following ICM were measured by flow cytometry: PD1, CTLA4, BTLA, CD137, CD27, GITR, OX40, LAG3 and TIM3. Dynamics of ICM expression were assessed using nonparametric paired samples tests. Significant changes were noted for PD1, BTLA and CD27 on multiple IC types during or after radiotherapy. Nonsignificant trends for increased expression of OX40 and GITR from baseline until the end of RT were observed on CD4 T cells and CD4 + CD39 + T cells. In patients with samples at recurrence of disease, a nonsignificant increase of TIM3 and LAG3 positive CD4 + CD39 + T cells was evident, accompanied by an increase of double positive cells for TIM3/LAG3. Potential future targets to be combined with RT in the conventional treatment and anti-PD1/PD-L could be BTLA agonists, or agonistic antibodies to costimulatory ICM like CD137, OX40 or GITR. The combination of cetuximab with CD27 agonistic antibodies enhancing ADCC or the targeting of TIM3/LAG3 may be another promising strategy., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Primary platinum-based chemoradiotherapy (CRT) remains the treatment of choice for nonresectable squamous cell carcinoma of the head and neck (HNSCC). Immune-checkpoint modulators are used as palliative therapy and studied in combination with definitive CRT. However, the immunological changes by CRT need yet to be understood., Methods: A cohort consisting of 67 paired tissue biopsies (N = 134) of HNSCC patients before and after CRT was created. The expression of PD-1, PD-L1, and CD27 of tumor and immune cells by immunohistochemistry was evaluated., Results: PD-L1 expression on immune cells of non-responders was significantly lower before CRT (P = .008). CD27 was expressed only on immune cells and not on cancer cells. A significant lower CD27-expression score was observed following CRT (P = .019)., Conclusions: Conventional CRT changes the expression of CD27 in the tumor microenvironment. Whether this is due to a loss of expression or a reduction of CD27+ cells must be evaluated in further analyses., (© 2020 The Authors. Head & Neck published by Wiley Periodicals LLC.)

Background: Despite advances in treatments, 30% to 50% of stage III-IV head and neck squamous cell carcinoma (HNSCC) patients relapse within 2 years after treatment. The Big Data to Decide (BD2Decide) project aimed to build a database for prognostic prediction modeling., Methods: Stage III-IV HNSCC patients with locoregionally advanced HNSCC treated with curative intent (1537) were included. Whole transcriptomics and radiomics analyses were performed using pretreatment tumor samples and computed tomography/magnetic resonance imaging scans, respectively., Results: The entire cohort was composed of 71% male (1097)and 29% female (440): oral cavity (429, 28%), oropharynx (624, 41%), larynx (314, 20%), and hypopharynx (170, 11%); median follow-up 50.5 months. Transcriptomics and imaging data were available for 1284 (83%) and 1239 (80%) cases, respectively; 1047 (68%) patients shared both., Conclusions: This annotated database represents the HNSCC largest available repository and will enable to develop/validate a decision support system integrating multiscale data to explore through classical and machine learning models their prognostic role., (© 2020 The Authors. Head & Neck published by Wiley Periodicals LLC.)

Cytokines and immune mediators play an important role in the communication between immune cells guiding their response to infectious diseases or cancer. In this study, a comprehensive longitudinal analysis of serum cytokines and immune mediators in head and neck squamous cell carcinoma (HNSCC) patients was performed. In a prospective, non-interventional, longitudinal study, blood samples from 22 HNSCC patients were taken at defined time points (TP) before, during, and every 3 months after completion of (chemo)radio)therapy (CRT/RT) until 12 months after treatment. Serum concentrations of 17 cytokines/immune mediators and High-Mobility-Group-Protein B1 (HMGB1) were measured by fluorescent bead array and ELISA. Concentrations of sFas were significantly elevated during and after CRT/RT, whereas perforin levels were significantly decreased after CRT/RT. Levels of MIP-1β and Granzyme B differed significantly during CRT/RT by HPV status. Increased HMGB1 levels were observed at recurrence, accompanied by high levels of IL-4 and IL-10. The sFas increase and simultaneous perforin decrease may indicate an impaired immune cell function during adjuvant radiotherapy. Increased levels of pro-inflammatory cytokines in HPV+ compared to HPV- patients seem to reflect the elevated immunogenicity of HPV-positive tumors. High levels of HMGB1 and anti-inflammatory cytokines at recurrence may be interpreted as a sign of immune evasion.

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients ( n = 23) and compared to healthy donors ( n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

Adenosine is a signaling molecule that exerts dual effects on tumor growth: while it inhibits immune cell function and thereby prevents surveillance by the immune system, it influences tumorigenesis directly via activation of adenosine receptors on tumor cells at the same time. However, the adenosine-mediated mechanisms affecting oncogenic processes particularly in head and neck squamous cell carcinomas (HNSCC) are not fully understood. Here, we investigated the role of adenosine receptor activity on HNSCC-derived cell lines. Targeting the adenosine receptor A2B (ADORA2B) on these cells with the inverse agonist/antagonist PSB-603 leads to inhibition of cell proliferation, transmigration as well as VEGFA secretion in vitro. At the molecular level, these effects were associated with cell cycle arrest as well as the induction of the apoptotic pathway. In addition, shRNA-mediated downmodulation of ADORA2B expression caused decreased proliferation. Moreover, in in vivo xenograft experiments, chemical and genetic abrogation of ADORA2B activity impaired tumor growth associated with decreased tumor vascularization. Together, our findings characterize ADORA2B as a crucial player in the maintenance of HNSCC and, therefore, as a potential therapeutic target for HNSCC treatment., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Multiple mechanisms of immunosuppression have been identified in the tumor microenvironment including regulatory B cells (B reg ). Recently, we have shown that B reg suppress T cell function by production of adenosine (ADO). However, the autocrine effect of ADO on B cells and the role of B reg in head and neck cancer remains unclear., Methods: Blood (n = 42) and tumor tissue (n = 39) of head and neck cancer patients and healthy donors (n = 60) were analyzed by FACS. The effect of ADO on phenotype, intracellular signaling pathways, Ca 2+ influx and ADO production was analyzed in B reg and effector B cells (B eff ) by FACS, luminescence and mass spectrometry. The blockage of the ADO receptor A 2A was analyzed in a murine head and neck cancer model., Results: ADO-producing B reg were found in tumor tissue and peripheral blood. ADO inhibited the intracellular Bruton's tyrosine kinase (BTK) and Ca 2+ influx only in B eff . The inhibition of BTK by ibrutinib mimicked the effect of ADO, and ibrutinib reduced the production of ADO by downregulation of CD39 in vitro. The inhibition of ADO receptor A 2A significantly reduced tumor mass and increased B cell infiltration, in vivo., Conclusion: Our data demonstrate the presence of a novel ADO-producing B reg population within the tumor microenvironment in mice and humans. A new model is proposed on how ADO-producing B reg can influence the function of B eff cells in healthy donors and cancer patients. Thus, the modulation of the ADO pathway in B cells may serve as a therapeutic approach for cancer patients.

Exosomes, the smallest group of extracellular vesicles, carry proteins, miRNA, mRNA, DNA, and lipids, which they efficiently deliver to recipient cells, generating a communication network. Exosomes strongly contribute to the immune suppressive tumor microenvironment of head and neck squamous cell carcinomas (HNSCC). Isolation of exosomes from HNSCC cell culture or patient's plasma allows for analyzing their molecular cargo and functional role in immune suppression and tumor progression. Immune affinity-based separation of different exosome subsets, such as tumor-derived or T cell-derived exosomes, from patient's plasma simultaneously informs about tumor status and immune dysfunction. In this review, we discuss the recent understanding of how exosomes behave in the HNSCC tumor microenvironment and why they are promising liquid biomarkers for diagnosis, prognosis, and therapy in HNSCC.

Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness., Competing Interests: The authors declare no conflict of interest.

The molecular cargo of tumor-cell-derived exosomes (TEX) mimics that of parental tumor cells. Thus, TEX could potentially serve as noninvasive biomarkers of cancer progression. However, separation of TEX from non-TEX in patients' plasma requires tumor antigen-specific detection reagents. CD44v3 has been of interest as a potential biomarker of disease progression in HNSCC, because its overexpression in tumor cells associates with poor outcome. Here, CD44v3+ TEX immunocaptured from plasma of 44 HNSCC patients and 7 healthy donors (HDs) were evaluated as potential biomarkers of disease activity and stage. Exosomes were isolated from plasma of by size exclusion chromatography. Using anti-CD44v3 or anti-CD3 mAbs on beads, CD44v3+ TEX CD3(-)TEX-enriched exosomes were immunocaptured from supernatants of nonmalignant or HNSCC cell lines and from patients' plasma. On-bead flow cytometry was used for the detection of FAS-L, PD-L1, TGFF-β. CSPG4 or EGFR on exosomes. The TEX expression profiles were correlated to clinicopathological parameters. Relative florescence intensity (RFI) values for CD44v3 were higher ( p < .01) on TEX from HNSCC cell lines or on CD44v3+ CD3(-) plasma-derived exosomes. RFI values of CD44v3 on CD3(-) exosomes were higher ( p < .005) in patients than in HDs and correlated ( p < .05) with the UICC stage and lymph node metastasis. In HNSCC patients, CD44v3+ exosomes higher levels of immunosuppressive proteins compared to CD44v3(-) exosomes ( p < .05- p  < .005), and RFI values for these markers correlated with higher disease stages and lymph node metastasis. Isolation of CD44v3+ exosomes by immunocapture allowed for enrichment of TEX which are potentially promising liquid biomarkers of the tumor burden and disease stage in HNSCC., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions., (© 2019 UICC.)

Purpose: The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status., Experimental Design: A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses., Results: Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as AR high risk ) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. AR high risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000)., Conclusions: We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. AR high risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients., (©2019 American Association for Cancer Research.)

Aim: In a prospective clinical initiative, we selected heavily pretreated head and neck carcinoma patients and assessed the clinical utility of a protein-based oncopanel for identification of potential targetable markers. Patients & methods: Tumor samples of 45 patients were evaluated using a 12-marker immunohistochemistry panel. The primary end point was the prevalence of potentially actionable markers. Results: At least one expressed marker in each case could be identified. We noted a high prevalence of EGFR (80%, 39/45) and MET (57.4%, 28/45). Three patients received oncopanel-based therapy with variable results. Conclusion: Despite the limited number of treated subjects, oncopanel analysis in end-stage head and neck cancer is operationally and technically feasible. Combination with targeted next generation sequencing might provide additional therapy options.

Objective: Although current therapeutic options for cutaneous melanoma (CM) are constantly improving survival, mucosal melanoma (MM) remains a rare tumor disease with a poor clinical outcome. While radical surgery is the gold standard, clear margin resections in the head and neck area are particularly critical due to high density of vulnerable structures. Adjuvant therapeutic options increases local control and data on the effect of systemic agents is sparse. The aim of this study was to elucidate surgical challenges in the craniofacial area and to evaluate the effect of local and systemic therapy in Head and Neck Mucosal Melanoma (HNMM)., Methods: In total, 21 patients with nasal mucosal malignant melanoma were included in this study over the course of 20 years in two German tertiary referral centers. Patient characteristics and conducted therapy as well as clinical outcomes were analyzed retrospectively., Results: By performing survival analysis for multimodal therapies, we observed a superiority effect of interferon therapy compared to surgery with radiation and surgery alone in the first therapeutic approach. However, patients treated with surgery alone in a recurrent setting showed the best outcome., Conclusion: Both, Interferon and radiation as adjuvant therapies, demonstrated survival benefits in initial treatment compared to surgery alone. Analysis after recurrence, however, revealed salvage surgery as a reliable and powerful tool to prolong post-recurrence survival without exposing palliative patients to the risk of severe adverse events from systemic therapies., (Copyright © 2019. Published by Elsevier B.V.)

CD8 + cytotoxic T-cell (CTL) specific for non-mutated, wild type (wt) sequence p53 peptides derived from wt or mutant p53 molecules expressed in head and neck squamous cell carcinomas (HNSCC) have been detected in the circulation of patients with this disease. The frequency and differentiation/maturation phenotypes of these anti-tumor specific CTL can reflect the host's immunologic response. Therefore, we investigated the frequency and phenotypes of wt sequence p53 peptide-specific CTL in patients with HNSCC (n = 33) by flow cytometric analysis using HLA-A*0201 tetrameric peptides (tet) complexed with the wt sequence p53 264-272 or p53 149-157 peptide and co-staining with phenotypic markers. One main finding was that increasing frequencies of tet + CD8 + T cells in patients' circulation correlated with increased frequencies of inactive naïve tet + cells, while those with effector memory and terminally differentiated phenotypes, which are associated with positive anti-tumor immune responses, decreased. We also found that the frequency of circulating tet + CD8 + T cells negatively correlated with p53 expression in tumor tissues and tumor stage. Our findings support further clinical-based investigations to define the frequencies and phenotypes of wt sequence p53 peptide-specific CD8 + T cells to predict disease severity, enhance selection of patients for inclusion in vaccination trials and highlight prerequisites to enhance immune susceptibility by activation of inactive naïve tet + T cells and/or enhancing circulating effector T cell activity by checkpoint blockage.

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

Background/aim: Head and neck squamous cell carcinomas (HNSCCs) form a heterogeneous tumor entity located throughout the oral cavity, pharynx and larynx that is caused predominantly by chemically or virally induced carcinogenesis. Heterozygous germline mutations in cancer susceptibility genes might also lead to increased incidence of HNSCCs. As DNA stability is typically impaired in HNSCC cells and genes of the Fanconi anemia/BRCA DNA repair pathway can be mutated or down-regulated in HNSCCs, we investigated here whether germline mutations occur in the X-chromosomal FANCB as candidate gene., Materials and Methods: Germline DNA of 85 consecutive HNSCC patients was sequenced. Missense alterations in FANCB were functionally tested in reference cells., Results and Conclusion: Four single nucleotide polymorphisms were identified, three of which were located in untranslated regions of FANCB (rs2188383, rs2375729, rs2905223) and predicted to be associated with normal function. One missense alteration, c.1004G>A resulting in p.G335E (rs41309679), in exon 4 was detected in five men in homozygous and in five women in heterozygous state. Four in silico prediction programs uniformally predicted p.G335E to be associated with loss-of-function of the protein. To clarify these predictions, we expressed the FANCB p.G335E protein in primary human FANCB deficient fibroblasts. Cell cycle analysis of these fibroblasts established that the FANCB p.G335E was functionally indistinguishable from the wildtype FANCB protein. Thus, functional studies in genetically defined cells showed that the p.G335E germline alteration in FANCB is not associated with impaired function., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purpose: The microenvironment of head and neck squamous cell carcinomas (HNSCC) is highly immunosuppressive. HNSCCs expressing elevated levels of PD-L1 have especially poor outcome. Exosomes that carry PD-L1 and suppress T-cell functions have been isolated from plasma of patients with HNSCC. The potential contributions of PD-L1 + exosomes to immune suppression and disease activity are evaluated. Experimental Design: Exosomes isolated from plasma of 40 HNSCC patients by size exclusion chromatography were captured on beads using anti-CD63 Abs, stained for PD-1 and PD-L1 and analyzed by flow cytometry. The percentages and mean fluorescence intensities (MFI) of PD-L1 + and PD-1 + exosome/bead complexes were correlated with the patients' clinicopathologic data. PD-L1 high or PD-L1 low exosomes were incubated with activated CD69 + human CD8 + T cells ± PD-1 inhibitor. Changes in CD69 expression levels on T cells were measured. Patients' plasma was tested for soluble PD-L1 (sPD-L1) by ELISA. Results: Levels of PD-L1 carried by exosomes correlated with patients' disease activity, the UICC stage and the lymph node status ( P = 0.0008-0.013). In contrast, plasma levels of sPD-L1 or exosome PD-1 levels did not correlate with any clinicopathologic parameters. CD69 expression levels were inhibited ( P < 0.03) by coincubation with PD-L1 high but not by PD-L1 low exosomes. Blocking of PD-L1 + exosome signaling to PD-1 + T cells attenuated immune suppression. Conclusions: PD-L1 levels on exosomes, but not levels of sPD-L1, associated with disease progression in HNSCC patients. Circulating PD-L1 + exosomes emerge as useful metrics of disease and immune activity in HNSCC patients., Significance: Circulating PD-L1 high exosomes in HNC patients' plasma but not soluble PD-L1 levels associate with disease progression. Clin Cancer Res; 24(4); 896-905. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: The aim of this study was to evaluate the value of 18F-FDG PET/CT (PET/CT) and MRI for local and/or whole-body restaging of adenoid cystic carcinoma of the head and neck (ACC)., Methods: Thirty-six patients with ACC underwent conventional MRI of the head and neck and a whole-body PET/CT and were analysed with regards to detection of a local tumor recurrence, lymph node or distant metastases. A consensus interpretation of all available imaging data was used as reference standard. Sensitivity, specificity, diagnostic accuracy, positive and negative predictive values were calculated for MRI and PET/CT., Results: The sensitivity of PET/CT and MRI was 96% (89%), specificity 89% (89%), PPV 96% (96%), NPV 89% (73%) and accuracy 94% (89%) for detection of local tumors. Additionally, PET/CT revealed lymph node metastases in one patient and distant metastases in 9/36 patients. In three patients secondary primaries were found., Conclusions: Whole-body PET/CT in addition to MRI of the head and neck improves detection of local tumour and metastastic spread in ACC.

Mucosal melanomas of the head and neck (MMHN) are aggressive tumors with poor prognosis, different opposed to cutaneous melanoma. In this study, we characterized primary mucosal malignant melanoma for the expression of Kallikrein-related peptidase 6 (KLK6), a member of the KLK family with relevance to the malignant phenotype in various cancer types including cutaneous melanoma. Paraffin-embedded MMHN of 22 patients were stained immunohistochemically for KLK6 and results were correlated with clinical and pathological data. In 77.3% (17/22) of MMHN cases, positive KLK6 staining was found. Staining pattern for tumor cells showed a predominant cytoplasmic staining. However, in six cases we also observed a prominent nuclear staining. MMHN with a high KLK6 expression showed significantly better outcome concerning local recurrence-free survival (p = 0.013) and nuclear KLK6 staining was significantly associated with the survival status (p = 0.027). Overexpression of KLK6 was detected in more than 70% of MMHN and approximately 40% of tumors showed a strong expression pattern. Correlation between clinical outcome of MMHN patients and overexpression of KLK6 has not been addressed so far. Our data demonstrate for the first time increased levels of KLK6 in MMHN and strengthen the hypothesis that there might be a context-specific regulation and function of KLK6 in mucosal melanoma.

Adenoid cystic carcinoma of the head and neck area is a rare malignant tumor with acceptable short-term but mediocre long-term prognosis. Radical tumor excision with clear resection margins, and sometimes resection of the facial nerve due to perineural growth, remains the fundamental therapy. We present 3 distinct clinical cases and discuss the current therapeutic options with special focus on plastic-reconstructive techniques. For reconstruction, the full armament of local and free flaps, as well as prosthetics, may be necessary. Adjuvant radiotherapy increases local control in advanced stages or close resection margins. However, systemic treatment options are limited. Further multicenter clinical trials are necessary due to the rare occurrence of the tumor.

Background: Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines., Methods: E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites., Results: All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration., Conclusion: HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017., (© 2017 Wiley Periodicals, Inc.)

Melanoma associated antigens (MAGE) are potential targets for immunotherapy and have been associated with poor overall survival (OS) in head and neck squamous cell carcinoma (HNSCC). However, little is known about MAGE in lymph node metastases (LNM) and recurrent disease (RD) of HNSCC.To assess whether MAGE expression increases with metastasis or recurrence, a tissue microarray (TMA) of 552 primary tumors (PT), 219 LNM and 75 RD was evaluated by immunohistochemistry for MAGE antigens using three monoclonal antibodies to multiple MAGE family members. Mean expression intensity (MEI) was obtained from triplicates of each tumor specimen.The median MEI compared between PT, LNM and RD was significantly higher in LNM and RD. In paired samples, MEI was comparable in PT to respective LNM, but significantly different from RD. Up to 25% of patients were negative for pan-MAGE or MAGE-A3/A4 in PT, but positive in RD. The prognostic impact of MAGE expression was validated in the TMA cohort and also in TCGA data (mRNA). OS was significantly lower for patients expressing pan-MAGE or MAGE-A3/A4 in both independent cohorts.MAGE expression was confirmed as a prognostic marker in HNSCC and may be important for immunotherapeutic strategies as a shared antigen.

The treatment of head and neck squamous cell carcinoma (HNSCC) has been a medical challenge with limited improvement in overall survival over the past few decades. However, recently, very interesting innovations in the field of immunotherapy have been shown to be beneficial for patients with advanced HNSCC. In this article, the latest medical developments are reviewed with special focus on the clinical achievements and current clinical studies in the field of immunotherapy. The landscape of clinical studies has changed considerably from antibody-based growth factor inhibition towards immune checkpoint modulation, and new indications in the adjuvant and neoadjuvant setting are being tested in large patient cohorts. Even the combination of 2 or more immune modulatory approaches and the correct synchronization with standard cancer therapy is promising and warrants suitable clinical trials., (© 2017 S. Karger GmbH, Freiburg.)

Background: Photodynamic therapy (PDT) represents a palliative treatment resulting in induction of inflammatory reactions with importance for the development of an antitumor immunity. Cancer/testis antigens (CTAs) have been associated with poor prognosis in different types of cancer, including head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: Tumor tissue samples before and after PDT were evaluated for the expression of four different CTAs by immunohistochemistry. Expression intensity and subcellular expression pattern were assessed., Results: Before PDT, expression of any CTA was detectable in 91%. Comparing the overall expression of CTAs, a decreased expression of all melanoma-associated antigens (MAGEs) post-treatment and a slightly increased expression of New York esophageal squamous cell carcinoma 1 (NY-ESO-1) was visible. The simultaneous cytoplasmic and nuclear expression of pan-MAGE or MAGE-A3/A4 correlated with reduced treatment-failure-free-survival (TFFS)., Conclusion: This study investigated the impact of PDT on CTA expression in HNSCC, detecting modified expression patterns after PDT. These changes may have been caused by immunological pressure or epigenetic regulation of CTA expression., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose/objective(s): Intensification of radiotherapy and chemotherapy for head-and-neck cancer (HNC) may lead to increased rates of long term dysphagia as a severe side effect. Mucositis and consequent swallowing problems require artificial nutrition in many HNC patients undergoing radiotherapy or chemoradiation. It is unknown, which predict factors for prophylactic PEG tube insertion appear useful., Materials/methods: From an institutional database, 101 patients (72 male, 29 female, mean age 59.5years) were identified who underwent radiotherapy or chemoradiation for HNC. Primary end point of the investigation was the need for artificial nutrition for more than 4days during radiotherapy. Dose volume parameters of defined normal tissue structures potentially of relevance for swallowing ability as well as clinical factors were used to develop a predictive model using a binary multiple logistic regression model., Results: Whereas several dosimetric and clinical factors were significant predictors for the need of artificial nutrition on univariate analysis, on multivariate analysis only three factors remained independently significant: mean dose to the oropharynx+1cm circumferential margin, ECOG performance state (0-1 vs. 2-4), and the use of chemotherapy (yes vs. no)., Conclusions: Using a 3 parameter model we could distinguish HNC-patients with different risks for the need of artificial nutrition during radiotherapy. After independent validation, the model could be helpful to decision on prophylactic PEG tube insertion., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Adenoid cystic carcinoma (ACC) of the head and neck is a rare but highly malignant tumor. Cancer-testis antigens (CTAs) represent an immunogenic family of cancer-specific proteins and thus represent an attractive target for immunotherapy., Methods: Eighty-four cases of ACC were identified, the CTAs pan-Melanoma antigen (pan-MAGE; M3H67) and New York esophageal squamous cell carcinoma (NY-ESO-1; E978) were detected immunohistochemically (IHC) and correlated with clinical data., Results: Expression of NY-ESO-1 was found in 48 of 84 patients (57.1%) and of pan-MAGE in 28 of 84 patients (31.2%). Median overall survival (OS) in NY-ESO-1 positive versus negative patients was 130.8 and 282.0 months (p = .223), respectively. OS in pan-MAGE positive versus negative patients was 105.3 and 190.5 months, respectively (p = .096). Patients expressing both NY-ESO-1 and pan-MAGE simultaneously had significantly reduced OS with a median of 90.5 months compared with 282.0 months in negative patients (p = .047)., Conclusion: A significant fraction of patients with ACC show expression of the CTAs NY-ESO-1 and/or pan-MAGE with promising immunotherapeutic implications. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1008-1016, 2016., (© 2016 Wiley Periodicals, Inc.)

Mucosal melanoma of the head and neck is a rare and aggressive tumor entity with a poor prognosis. The standard treatment is radical tumor resection, with or without adjuvant radiation, where conventional chemotherapies in advanced stage or recurrent diseases have shown little benefit. Overexpression of the programmed cell death ligand 1 (PD-L1) is a common feature in human cancer. Although PD-L1 is an acknowledged prognostic biomarker for dismal prognosis in other tumors of the head and neck, expression and clinical relevance of PD-L1 in mucosal melanoma have not been addressed so far. We assessed PD-L1 expression using immunohistochemical staining in 23 tumor samples from patients with primary mucosal melanoma and correlated expression status with clinicopathological and outcome data. Tumors were derived from the nasal cavity (43.5%), nasal sinuses (43.5%), and the conjunctiva (13%). All patients had undergone surgery; 39% of all patients received adjuvant radiation and 13% were administered systemic interferon therapy. The probability of 1- and 5-year overall survival was 87 and 34.8%, respectively. The mean overall survival was 51 months and the mean recurrence-free survival was 23 months. Immunohistochemical staining showed PD-L1 expression in 13% (3/23) of mucosal melanoma. In contrast, prominent PD-L1 staining was detected in 100% of tissue sections from a control group of cutaneous melanoma (n=9). PD-L1 expression in mucosal melanoma was not correlated with age, sex, nor anatomical localization of the tumor. Interestingly, patients with PD-L1-positive mucosal melanoma had a significantly longer recurrence-free survival (P=0.026). In contrast to cutaneous melanoma and some other malignancies, a relevant PD-L1 overexpression in mucosal melanoma could not be confirmed.

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies., (© 2014 UICC.)

Background/aim: Head and neck adenoid cystic carcinoma (HNACC) is a rare malignancy of the salivary glands with a tendency to metastasize in lung or liver without lymph node involvement, whereas squamous cell carcinoma (HNSCC) preferentially metastasizes to locoregional lymph nodes. The expression patterns of microRNA, a class of small non-coding RNA transcripts, involved in gene regulation and various developmental processes, could be of influence during the metastatic process. The aim of the present study was to compare mircoRNA expression patterns of HNACC and HNSCC., Materials and Methods: In a total of 21 tissue samples, a genome-wide screening for microRNAs was performed. A microRNA array platform was used for the identification of target microRNA., Results: Five microRNAs, hsa-MiR-214, hsa-MiR-125a-5p, hsa-MiR-574-3p, hsa-MiR-199a-3p/199b-3p and hsa-miR-199a-5p were identified to be over-expressed in HNACC compared to HNSCC, whereas hsa-MiR-452 showed a lower expression level., Conclusion: Our data showed significantly different expression patterns of mircoRNA in HNACC and HNSCC supporting the theory of tumor-specific expression and giving hints for different clinical behavior., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients., (© 2014 UICC.)