HPV-associated head and neck cancer is characterized by distinct profiles of CD8+ T cells and myeloid-derived suppressor cells.

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.

Evaluation of Immunoregulatory Biomarkers on Plasma Small Extracellular Vesicles for Disease Progression and Early Therapeutic Response in Head and Neck Cancer.

Head and Neck Cancers (HNCs) have highly immunosuppressive properties. Small extracellular vesicles (sEVs), including exosomes, nanosized mediators of intercellular communication in the blood, carry immunosuppressive proteins and effectively inhibit anti-tumor immune responses in HNCs. This study evaluates immunosuppressive markers on sEVs from 40 HNC patients at different disease stages and 3- and 6-month follow-up after surgery and/or chemoradiotherapy. As controls, sEVs from normal donors (NDs) are examined. Immunoregulatory surface markers on sEVs were detected as relative fluorescence intensity (RFI) using on-bead flow cytometry, and their expression levels were monitored in the early and late stages of HNC and during follow-up. In parallel, the sEV-mediated apoptosis of CD8+ Jurkat cells was assessed. Together with TGF-ß1 and PD-L1 abundance, total sEV proteins are elevated with disease progression. In contrast, total sEV protein, including TGF-ß1, PD-1 and PD-L1, decrease upon therapy response during follow-up. Overall survival analysis implies that high sEV PD-1/PD-L1 content is an unfavorable prognostic marker in HNC. Consistently, the sEV-mediated induction of apoptosis in CD8+ T cells correlates with the disease activity and therapy response. These findings indicate that a combination of immunoregulatory marker profiles should be preferred over a single marker to monitor disease progression and therapy response in HNC.

B-Helper Neutrophils in Regional Lymph Nodes Correlate with Improved Prognosis in Patients with Head and Neck Cancer.

The role of neutrophils during cancer formation and elimination is diverse. Here, for the first time, we investigate neutrophil helper cells (NBH), their influence on B cell activity in the regional lymph nodes (RLN) of head-and-neck cancer patients and the effect of this neutrophil/B cell interaction on patient prognosis. Circulating and RLN neutrophils of patients with stage I-IV head-and-neck squamous cell carcinoma were investigated with flow cytometry and qPCR. In addition, neutrophil/B cell co-localization in RLNs was evaluated using immunohistochemistry. B cell proliferation was assessed and correlated with the distance to neutrophils. Patient survival was evaluated. Neutrophils with the helper cell phenotype were identified in the RLN of HNC patients. B cells in close proximity to such NBH showed significantly higher proliferation rates, together with elevated activation-induced cytidine deaminase (AID) expression. Notably, patient survival was significantly higher in individuals with high NBH frequencies in the B follicles of RLNs. Neutrophils in RLN can support T cell-independent activation of the adaptive immune system through B cell stimulation, capturing helper cell phenotype character. The presence of such helper neutrophils in the RLNs of HNC patients positively correlates with patient prognosis.

Identification of a Prognostic Clinical Score for Patients With Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck Treated With Systemic Therapy Including Cetuximab.

Cetuximab-based chemoimmunotherapy has been the standard of care for recurrent or metastatic squamous cell carcinoma of the head and neck (r/m SCCHN) for more than a decade. To date, no predictive or prognostic biomarkers have been established to further guide the systemic treatment with cetuximab-based chemoimmunotherapy in r/m SCCHN. Against this background, we retrospectively analyzed clinical and blood-based parameters from 218 r/m SCCHN patients treated with chemoimmunotherapy including cetuximab. Multivariate Cox-regression models were used to assess their prognostic or predictive value. Eastern Co-operative Oncology Group (ECOG) performance status (≥2), older age (≥61.8 years), anemia (hemoglobin <11.80), and increased neutrophil-to-lymphocyte ratio (NLR ≥5.73) were independently and strongly associated with inferior overall survival (OS). To group patients according to risk profiles we established a prognostic clinical score (PCS) that can easily be used in clinical practice. The PCS stratified the cohort into low, intermediate, poor or very poor risk subgroups with median OS times of 23.4, 12.1, 7.5, and 4.0 months, respectively. Patients with low risk PCS had a prolonged progression-free survival (PFS) and increased overall response rate (ORR) under first-line cetuximab-based therapy. Interestingly, only patients with low and intermediate risk benefitted from the more intensive first-line cisplatin/cetuximab combination compared to carboplatin/cetuximab therapy, whereas the intensity of first-line treatment had no impact in the poor and very poor risk subgroups. Following external validation, particularly in the context of newly established first-line options, the PCS may guide clinical decision making and serve for stratification of patients with r/m SCCHN in future clinical trials.

Expression Patterns of CD44 and AREG Under Treatment With Selective Tyrosine Kinase Inhibitors in HPV+ and HPV- Squamous Cell Carcinoma.

BACKGROUND: We investigated the expression patterns of cluster of differentiation (CD) 44 and amphiregulin (AREG), two signaling molecules essential for cell proliferation and differentiation, under the influence of selective tyrosine kinase inhibitors (TKIs) in human papillomavirus (HPV)+ and HPV- squamous carcinoma cell lines. MATERIALS AND METHODS: The protein expression of CD44 and AREG was determined by sandwich enzyme-linked immunosorbent assay in HPV- cell lines UMSCC-11A and UMSCC-14C, and HPV+ CERV-196 cells after TKI treatment. RESULTS: The expression of AREG and CD44 was dependent on the cell line's HPV status. AREG expression increased after incubation with nilotinib in HPV+ tumor cells. The expression of CD44 was significantly influenced by all drugs; its expression under selective epidermal growth factor receptor inhibition was mostly reduced, whereas nilotinib led to an exceptional increase of CD44 expression. CONCLUSION: The selective drug treatment options significantly influenced the expression of CD44 and AREG in HPV- and HPV+ tumor cells, constituting the need for personalized treatment options.

HPV16 drives cancer immune escape via NLRX1-mediated degradation of STING.

The incidence of human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) has surpassed that of cervical cancer and is projected to increase rapidly until 2060. The coevolution of HPV with transforming epithelial cells leads to the shutdown of host immune detection. Targeting proximal viral nucleic acid-sensing machinery is an evolutionarily conserved strategy among viruses to enable immune evasion. However, E7 from the dominant HPV subtype 16 in HNSCC shares low homology with HPV18 E7, which was shown to inhibit the STING DNA-sensing pathway. The mechanisms by which HPV16 suppresses STING remain unknown. Recently, we characterized the role of the STING/type I interferon (IFN-I) pathway in maintaining immunogenicity of HNSCC in mouse models. Here we extended those findings into the clinical domain using tissue microarrays and machine learning-enhanced profiling of STING signatures with immune subsets. We additionally showed that HPV16 E7 uses mechanisms distinct from those used by HPV18 E7 to antagonize the STING pathway. We identified NLRX1 as a critical intermediary partner to facilitate HPV16 E7-potentiated STING turnover. The depletion of NLRX1 resulted in significantly improved IFN-I-dependent T cell infiltration profiles and tumor control. Overall, we discovered a unique HPV16 viral strategy to thwart host innate immune detection that can be further exploited to restore cancer immunogenicity.

T cell receptor richness in peripheral blood increases after cetuximab therapy and correlates with therapeutic response.

The role of T cell receptor (TCR) signaling for adaptive immune responses is essential. The ability to respond to a broad spectrum of tumor antigens requires an adaptive selection of various TCR. So far, little is known about the role of TCR richness and clonality in the cellular immune response to head and neck cancer (HNC), though the Endothelial Growth Factor Receptor (EGFR)-specific CD8+ T cell response can be enhanced by cetuximab therapy. Therefore, we investigated differences in TCR sequences between human papillomavirus (HPV)+ and HPV- HNC patients, as well as differences in TCR sequence characteristics between T cells of peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL). Additionally, we were able to investigate the TCR richness and clonality in samples pre- and post- treatment in a prospective clinical trial of neoadjuvant cetuximab. Interestingly, HPV+ and HPV- HNSCC did not significantly differ in the extent of TCR clonality and richness in PBMC or TIL. However, neoadjuvant cetuximab treatment increased the number of unique TCR sequences in PBMC (p = 0.0003), which was more prominent in the clinical responder patients compared to non-responders (p = 0.04). A trend toward TCR gene focusing was observed in TIL (p = 0.1) post-treatment. Thus, an increase in richness of TCR sequences in the periphery with a focusing at the tumor site is associated with an improved treatment response, suggesting an influence of peripheral quantity and intratumoral quality on adaptive immunity in cetuximab treated patients.

PD-L1 Mediates Dysfunction in Activated PD-1+ NK Cells in Head and Neck Cancer Patients.

Inhibitory immune-checkpoint receptors (ICRs), including programmed death 1 (PD-1), have been characterized as exhaustion markers on T cells that infiltrate the tumor microenvironment (TME) of many cancer types, including head and neck cancer (HNC). However, expression and function of ICRs, including PD-1, on natural killer (NK) cells remains less defined. NK cells are innate immune effector cells that lyse epidermal growth factor receptor-overexpressing HNC cells via cetuximab-mediated antibody-dependent cytotoxicity. Cetuximab is clinically effective but only in 10% to 15% of patients. Therefore, it is necessary to investigate how immunomodulation with cetuximab or PD-1 blockade might enhance NK cell responses in the TME and improve monoclonal antibody therapeutic efficacy. We observed that expression of PD-1 on NK cells marks an activated phenotype, which was suppressed only after binding programmed death ligand-1 (PD-L1). HNC patients who exhibit higher circulating PD-1+ NK cells associate with better clinical outcome, and these cells are enriched in the TME. Cetuximab-mediated NK cell activation increased PD-1 expression on NK cells in vitro, which was confirmed in vivo in a prospective neoadjuvant cetuximab trial. In contrast, PD-L1 ligation of PD-1+ NK cells diminished their activation status, whereas PD-1 blockade increased cetuximab-mediated NK cell activation and cytotoxicity, but only against HNC targets with high PD-L1 expression. Therefore, blocking the PD-1-PD-L1 axis may be a useful strategy to reverse immune evasion of HNC tumors with high PD-L1 expression during cetuximab therapy by reversing NK cell dysfunction.

[Immunotherapy - The New Era of Oncology]. / Immuntherapie ­ Die neue Ära in der Onkologie.

In the field of immunotherapy, essential progress was achieved over the past years partially demonstrating long lasting therapeutic responses in different tumor entities. A better understanding of the interactions between the tumor and the immune system as well as the integration of immunotherapeutic approaches into clinical routine were the foundations for this development. The different approaches intervene on multiple levels of the immune response and directly or indirectly mount the patient's own immune defense against tumor cells. Immunotherapeutic approaches are represented by cytokine therapies, vaccinations, the use of oncolytic viruses and monoclonal antibody therapies as well as adoptive cell transfer strategies.

Phase Ib Study of Immune Biomarker Modulation with Neoadjuvant Cetuximab and TLR8 Stimulation in Head and Neck Cancer to Overcome Suppressive Myeloid Signals.

Purpose: The response rate of patients with head and neck squamous cell carcinoma (HNSCC) to cetuximab therapy is only 15% to 20%, despite frequent EGFR overexpression. Because immunosuppression is common in HNSCC, we hypothesized that adding a proinflammatory TLR8 agonist to cetuximab therapy might result in enhanced T-lymphocyte stimulation and anti-EGFR-specific priming.Experimental Design: Fourteen patients with previously untreated HNSCC were enrolled in this neoadjuvant trial and treated preoperatively with 3 to 4 weekly doses of motolimod (2.5 mg/m2) and cetuximab. Correlative tumor and peripheral blood specimens were obtained at baseline and at the time of surgical resection and analyzed for immune biomarker changes. Preclinical in vitro studies were also performed to assess the effect of cetuximab plus motolimod on myeloid cells.Results: TLR8 stimulation skewed monocytes toward an M1 phenotype and reversed myeloid-derived suppressor cell (MDSC) suppression of T-cell proliferation in vitro These data were validated in a prospective phase Ib neoadjuvant trial, in which fewer MDSC and increased M1 monocyte infiltration were found in tumor-infiltrating lymphocytes. Motolimod plus cetuximab also decreased induction of Treg and reduced markers of suppression, including CTLA-4, CD73, and membrane-bound TGFß. Significantly increased circulating EGFR-specific T cells were observed, concomitant with enhanced CD8+ T-cell infiltration into tumors. These T cells manifested increased T-cell receptor (TCR) clonality, upregulation of the costimulatory receptor CD27, and downregulation of inhibitory receptor TIGIT.Conclusions: Enhanced inflammatory stimulation in the tumor microenvironment using a TLR agonist overcomes suppressive myeloid and regulatory cells, enhancing the cellular antitumor immune response by therapeutic mAb in HNSCC. Clin Cancer Res; 24(1); 62-72. ©2017 AACR.

PD-1 Status in CD8+ T Cells Associates with Survival and Anti-PD-1 Therapeutic Outcomes in Head and Neck Cancer.

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients, despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact, because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study, PD-1 expression was indeed upregulated on HNC patient TIL, and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006), who nonetheless experienced significantly better clinical outcome. However, PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover, HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001), while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model, anti-PD-1 mAb therapy differentially modulated PD-1high/low populations, and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus, the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. ©2017 AACR.

[Extracranial schwannomas of the Head and Neck: Clinical -approach based on 20 cases]. / Extrakranielle Schwannome im Kopf-Hals-Bereich: Klinische Implikationen anhand von 20 Fällen.

Background Schwannomas are rare benign tumors originating from the perineural cells forming the myelin layer in the peripheral nervous system (PNS). While well established therapeutic concepts exist for intracranial schwannomas, there is a lack of consistent clinical standards for extracranial schwannomas. Method This retrospective study describes the clinical pathway of 20 patients with histologically proven extracranial schwannomas of the head and neck. The diagnostic and therapeutic strategies for schwannomas are discussed with special emphasis on localization and functional outcome. Results Extracranial schwannomas of the head and neck region mostly originated from the facial nerve (n = 4), vagal nerve (n = 4) or sympathetic chain (n = 3). Most common symptoms were swelling (n = 12) and pain (n = 3). Preoperative imaging included MRI (n = 13), ultrasound (n = 12) and CT (n = 3). Surgical intervention was performed in 18 cases (n = 14 complete extirpation, n = 3 partial extirpation, n = 1 unknown). Regarding completely extirpated schwannomas of motor nerves (n = 10) severing the nerve of origin was more often required in patients with a preexisting functional deficit (3 out of 4 = 75 %) than in patients without preexisting deficits (2 out of 6 = 33 %). Conclusion Representing rare tumors of the head and neck region mostly originating from the facial nerve, sympathetic chain or caudal cranial nerves extracranial schwannomas require a systematic diagnostic and therapeutic approach. Postoperative functional deficits after complete extirpation must especially be anticipated in patients with a preexisting functional deficit.

Adenosine metabolism of human mesenchymal stromal cells isolated from patients with head and neck squamous cell carcinoma.

BACKGROUND: Mesenchymal stromal cells (MSC) are a major component of the tumor microenvironment in patients with head and neck squamous cell carcinoma (HNSCC). MSC display innate and regulatory immunologic functions, very similar to many hematopoietic 'classical' immune cells. Conversion of ATP to immunosuppressive adenosine is an immunosuppressive mechanism utilized by other hematopoietic immune cells. The present study explores the adenosine metabolism of tumor derived MSC in comparison to autologous MSC from non-malignant tissue. METHODS: From HNSCC patients (n=10), paired MSC were generated from tumor tissue (tMSC) and autologous healthy control tissue (cMSC). Differentiation properties and phenotype (CD105, CD73, CD39, CD90, CD26, CD29) were compared by flow cytometry. Production of immunosuppressive adenosine (ADO) by functionally active ectonucleotidases, CD39 and CD73, was determined by luminescence and mass spectrometry. Suppressive activity of ADO was tested in CFSE proliferation assays of isolated T-cells. Plasticity of cMSC was explored after incubation with tumor-cell conditioned media. RESULTS: Differentiation into osteogenic, chondrogenic and adipogenic directions was comparable in tMSC and cMSC. Expression of ectonucleotidases, CD39 and CD73, was decreased in tMSC as compared to corresponding cMSC, which correlated with decreased ATP metabolism in mass spectrometry. Proliferation of CD4+ T-cells was significantly suppressed by exogenous ADO. Tumor-conditioned medium was unable to down-regulate ADO production in cMSC. CONCLUSION: We identified MSC of the oropharyngeal mucosa as an important producer of exogenous ADO. In patients with HNSCC, reduced expression of ADO may contribute to excessive inflammation and tumor growth.

The bidirectional tumor--mesenchymal stromal cell interaction promotes the progression of head and neck cancer.

INTRODUCTION: Mesenchymal stromal cells (MSC) are an integral cellular component of the tumor microenvironment. Nevertheless, very little is known about MSC originating from human malignant tissue and modulation of these cells by tumor-derived factors. The aim of this study was to isolate and characterize MSC from head and neck squamous cell carcinoma (HNSCC) and to investigate their interaction with tumor cells. METHODS: MSC were isolated from tumor tissues of HNSCC patients during routine oncological surgery. Immunophenotyping, immunofluorescence and in vitro differentiation were performed to determine whether the isolated cells met the consensus criteria for MSC. The cytokine profile of tumor-derived MSC was determined by enzyme-linked immunosorbent assay (ELISA). Activation of MSC by tumor-conditioned media was assessed by measuring cytokine release and expression of CD54. The impact of MSC on tumor growth in vivo was analyzed in a HNSCC xenograft model. RESULTS: Cells isolated from HNSCC tissue met the consensus criteria for MSC. Tumor-derived MSC constitutively produced high amounts of interleukin (IL)-6, IL-8 and stromal cell-derived factor (SDF)-1α. HNSCC-derived factors activated MSC and enhanced secretion of IL-8 and expression of CD54. Furthermore, MSC provided stromal support for human HNSCC cell lines in vivo and enhanced their growth in a murine xenograft model. CONCLUSIONS: This is the first study to isolate and characterize MSC from malignant tissues of patients with HNSCC. We observed cross-talk of stromal cells and tumor cells resulting in enhanced growth of HNSCC in vivo.

Cervical lymph node diseases in children.

The lymph nodes are an essential part of the body's immune system and as such are affected in many infectious, autoimmune, metabolic and malignant diseases. The cervical lymph nodes are particularly important because they are the first drainage stations for key points of contact with the outside world (mouth/throat/nose/eyes/ears/respiratory system) - a critical aspect especially among children - and can represent an early clinical sign in their exposed position on a child's slim neck. Involvement of the lymph nodes in multiple conditions is accompanied by a correspondingly large number of available diagnostic procedures. In the interests of time, patient wellbeing and cost, a careful choice of these must be made to permit appropriate treatment. The basis of diagnostic decisions is a detailed anamnesis and clinical examination. Sonography also plays an important role in differential diagnosis of lymph node swelling in children and is useful in answering one of the critical diagnostic questions: is there a suspicion of malignancy? If so, full dissection of the most conspicuous lymph node may be necessary to obtain histological confirmation. Diagnosis and treatment of childhood cervical lymph node disorders present the attending pediatric and ENT physicians with some particular challenges. The spectrum of differential diagnoses and the varying degrees of clinical relevance - from banal infections to malignant diseases - demand a clear and considered approach to the child's individual clinical presentation. Such an approach is described in the following paper.