Dynamic changes of the EMT spectrum between circulating tumor cells and the tumor microenvironment in human papillomavirus-positive head and neck squamous cell carcinoma.

OBJECTIVES: Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) differs in terms of cellular and molecular biological characteristics from HPV-negative HNSCC. However, differences in circulating tumor cells (CTCs) between HPV-positive and -negative HNSCC remain unclear. MATERIALS AND METHODS: We first analyzed eight epithelial-mesenchymal transition (EMT)-related genes (VIM, CDH1, CDH2, SNAI1, SNAI2, TWIST1, ZEB1, and ZEB2) using The Cancer Genome Atlas (TCGA) database. Next, we isolated CTCs from patients with HNSCC using CD45-negative selection and expression analysis of epithelial-related genes (EPCAM, EGFR, and MET) by RT-qPCR. CTC-positive samples were further analyzed for EMT-related genes. In addition, we investigated the proportion of circulating T cell subsets and CD38+ T cells using flow cytometry and their involvement in CTCs. RESULTS: Compared with HPV-negative HNSCC, expression of CDH1, SNAI1, SNAI2, TWIST1, and ZEB1 was downregulated in HPV-positive HNSCC, as determined by TCGA analysis. CTCs were detected in 19 (52.8 %) of 36 HPV-positive and 26 (68.4 %) of 38 HPV-negative patients with HNSCC. EPCAM-positive and MET-positive CTCs were significantly more frequent in patients with HPV-negative HNSCC. HPV-positive patients with HNSCC exhibited significantly high SNAI1 and ZEB2 expression in CTCs. Interestingly, differences in SNAI1 expression levels differed markedly between CTCs and TCGA based on HPV status. Moreover, HPV-positive patients with HNSCC exhibiting SNAI1-high CTCs showed a superior prognosis and a lower proportion of CD38+ T cells than those with SNAI1-low CTCs. CONCLUSION: Our results provide novel insights into the EMT-MET spectrum of CTCs and may contribute to the development of prognostic biomarkers for HPV-positive HNSCC.

Circulating T Cell Subsets and ILC2s are Altered in Patients With Chronic Rhinosinusitis With Nasal Polyps After Dupilumab Treatment.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is mainly associated with type 2 inflammation and is often unmanageable, regardless of the treatment. Dupilumab, a monoclonal antibody targeting the interleukin (IL)-4 receptor alpha to inhibit IL-4 and IL-13 signaling, has recently been shown to significantly improve the condition of patients with CRSwNP. However, the mechanisms underlying this response to dupilumab are not yet fully understood. OBJECTIVE: We sought to examine whether circulating T cell subset proportions and their functions are altered by dupilumab treatment. METHODS: We first investigated the proportion of circulating T cell subsets and group 2 innate immune cells (ILC2s) in patients with CRSwNP treated with dupilumab using mass and flow cytometry. We then assessed cytokine gene expression and cytokine production in peripheral blood mononuclear cells (PBMCs) using quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The type 2 T helper (Th2) cell proportion significantly decreased after dupilumab treatment, whereas that of type 1 T helper (Th1) cells increased. Moreover, programmed death-1 (PD-1) expression in regulatory T (Treg) cells was significantly reduced. The proportion of ILC2s significantly increased after dupilumab treatment. Unfortunately, neither cytokine gene expression nor cytokine production in PBMCs showed significant changes. CONCLUSIONS: Our findings suggest that in CRSwNP patients treated with dupilumab, Th2, Treg, and ILC2 cells, which regulate type 2 inflammation, are modulated in the peripheral circulation. Further analysis of circulating immune cells could provide novel insights into understanding the pathophysiologic mechanisms of dupilumab and the development of tailored therapeutic strategies for patients with CRSwNP.

Tumor-derived exosomes elicit cancer-associated fibroblasts shaping inflammatory tumor microenvironment in head and neck squamous cell carcinoma.

OBJECTIVES: Exosome-mediated reciprocal crosstalk between tumor and stromal cells plays a crucial role in tumor development and progression. This study investigated whether exosomes released from head and neck squamous cell carcinoma (HNSCC) tumor cells can convert normal fibroblasts into cancer-associated fibroblasts (CAF)-like cells and further analyzed the functional characterization of fibroblasts educated by tumor-derived exosomes. MATERIALS AND METHODS: Exosomes secreted from HNSCC cell lines were isolated and normal fibroblasts were established from normal oropharyngeal mucosa. The effects of the exosomes on fibroblasts were examined by proliferation and migration assays, and exosome-educated fibroblasts were analyzed for the expression of eight genes (IL1B, IL6, CXCL8, TGFB1, ACTA2, FAP, CD274, and PDCD1LG2) by RT-qPCR. Moreover, T cells or CD14-positive cells were co-cultured with culture supernatants from exosome-educated fibroblasts. T-cell proliferation and macrophage polarization were examined using flow cytometry. Then, RNA sequencing (RNA-seq) of exosome-educated fibroblasts and the corresponding control fibroblasts was performed. RESULTS: Tumor-derived exosomes enhanced fibroblast proliferation and migration. Moreover, gene expression analysis revealed upregulation of the gene expression of proinflammatory cytokines and immunoregulatory genes, and activated fibroblast marker genes. The culture supernatants of tumor-derived exosome-educated fibroblasts suppressed T cell proliferation and the induction of protumoral macrophages compared with those of control fibroblasts. Next, comprehensive RNA-seq analysis data revealed the activation of 11 signaling pathways, including IL-6- and IL-17-related signaling. CONCLUSION: These results indicate that HNSCC tumor cells induce and/or differentiate into CAFs through exosome-based cell-to-cell communication to create an inflammatory tumor microenvironment.

Dynamic alterations of circulating T lymphocytes and the clinical response in patients with head and neck squamous cell carcinoma treated with nivolumab.

Cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been recognized as a novel therapeutic option for head and neck squamous cell carcinoma (HNSCC). However, only approximately 20-30% of patients with recurrent/metastatic (R/M) HNSCC benefit. Moreover, the mechanisms underlying the response to ICIs remain unclear. We investigated the proportion, activation status, and expression level of immune checkpoint molecules in circulating T cell subsets in R/M HNSCC patients treated with nivolumab using flow cytometry and mass cytometry, and then determined whether treatment response was associated with these values. We also assessed the changes in the frequency of tumor-associated antigens, MAGE-A4 and p53, -specific T cells prior to and after nivolumab treatment using the IFN-γ ELISPOT assay. The proportion of activated CD4+ and CD8+ TEMRA cells significantly increased in the disease-controlled patients but not in disease-progressed patients. As expected, the expression of PD-1 in T cells markedly decreased regardless of the therapeutic response. Meanwhile, T cell immunoglobulin mucin-3 expression on CD8+ T cells was significantly higher in patients with disease progression than in disease-controlled patients after treatment. The frequency of the tumor-associated antigens, MAGE-A4- and p53-specific T cells, was not correlated with clinical responses; however, in the disease-controlled patients, the frequency of MAGE-A4-specific T cells was significantly augmented. We concluded that in R/M HNSCC patients treated with nivolumab, circulating T cells show dynamic alterations depending on treatment efficacy. An analysis of the immunokinetics of circulating T cells could thus provide new insights into rational therapeutic strategies in cancer immunotherapy for HNSCC.

Circulating naïve and effector memory T cells correlate with prognosis in head and neck squamous cell carcinoma.

T-cell memory is an important mechanism for long-term protection against diverse pathogens. Generation and persistence of memory T cells are vital components of anti-tumor immunity, given their ability to persist for prolonged durations, as well as activate and migrate rapidly. In the present study, we investigated the clinical and prognostic significance of T-cell subsets in the peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC). Moreover, we calculated the enrichment scores of T-cell subsets in primary tumor tissues and compared their clinical characteristics using a public database. Multivariate survival analyses of circulating T-cell parameters revealed that clinical parameters, except M factor, were not independent prognostic factors, whereas proportions of CD8+ T cells, naïve T cells (TN s), effector memory T cells (TEM s), and CD38+ CD8+ T cells were independent prognostic factors, suggesting the importance of these peripheral T-cell parameters as independent prognostic biomarkers. Consistent with these results, the T-cell enrichment analysis indicated that enrichment of CD8+ TN s in the tumor microenvironment was an independent prognostic factor. Moreover, an ex vivo experiment demonstrated significantly less cytotoxic activity in CD38+ T cells than in CD38- T cells. These findings suggest that T-cell memory-related parameters in both systemic immunity and the tumor microenvironment could be used as prognostic biomarkers regardless of clinical characteristics. Further characterization of circulating T cells would lead to the development of novel biomarkers for patients with HNSCC.

Systemic immune responses are associated with molecular characteristics of circulating tumor cells in head and neck squamous cell carcinoma.

Systemic immunity mediated by circulating immune cells may affect clinical features, as well as the characteristics of circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC). The present study aimed to analyze the influence of circulating immune cells, using their markers, on clinical features to investigate the association between systemic immunity and the molecular characteristics of CTCs. Circulating immune-cell markers were associated with disease progression and clinical outcomes in patients with HNSCC. Meanwhile, there was no significant association between the presence of CTCs and systemic immune-related markers. Moreover, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a expression in CTCs was significantly associated with higher lymphocyte counts (P=0.035) and an increased prognostic nutrition index (P=0.0157). Patients with CTCs expressing CD47 exhibited significantly higher neutrophil (P=0.0031) and monocyte (P=0.0016) counts. Patients with CTCs expressing programmed cell death 1 ligand 2 exhibited lower C-reactive protein (CRP) levels (P=0.0271) and a decreased CRP/albumin ratio (P=0.0207). The current results suggested that the interaction between CTCs and circulating immune cells may provide survival advantages via molecular alterations to CTCs.

The Blood Microenvironment Influences the Molecular Phenotypes of Circulating Tumor Cells in Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Circulating tumor cells (CTCs) may be affected by the environment encountered during blood circulation. We aimed to explore the association between the molecular phenotype of CTCs and systemic inflammatory markers. PATIENTS AND METHODS: CTCs isolated from patients with recurrent/metastatic head and neck squamous cell carcinoma by CD45-negative selection were analyzed for the expression of multiple genes. The correlations between gene expression levels in CTCs and systemic inflammation markers were examined. RESULTS: Thirty-five (83.3%) of the 42 patients were positive for CTCs. No significant differences in systemic inflammatory markers were observed between CTC-positive and CTC-negative patients. Notably, VIM or ZEB2 expression was strongly correlated with that of CD44 or ALDH1. PIK3CA, CD44, ALDH1A1, and PDCD1LG2 expression in CTCs was correlated with lymphocyte- and/or albumin-related systemic inflammatory markers. CONCLUSION: CTCs acquire a survival advantage through phenotypic alterations in the hostile blood environment, and evade circulatory immune surveillance.

Molecular phenotypes of circulating tumor cells and efficacy of nivolumab treatment in patients with head and neck squamous cell carcinoma.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Biomarkers of the therapeutic efficacy of ICIs have been extensively investigated. In this study, we aimed to analyze whether molecular phenotypes of circulating tumor cells (CTCs) are associated with treatment responses and clinical outcomes in patients with R/M HNSCC treated with nivolumab. Peripheral blood samples were collected before treatment initiation and after four infusions of nivolumab. CTCs isolated by depletion of CD45-positive cells were analyzed to determine the expression of EPCAM, MET, KRT19, and EGFR using real-time quantitative polymerase chain reaction. CTC-positive samples were analyzed to determine the expression of PIK3CA, CCND1, SNAI1, VIM, ZEB2, CD44, NANOG, ALDH1A1, CD47, CD274, and PDCD1LG2. Of 30 patients treated with nivolumab, 28 (93.3%) were positive for CTCs. In 20 CTC-positive patients, molecular alterations in CTCs before and after nivolumab treatment were investigated. Patients with MET-positive CTCs had significantly shorter overall survival than those with MET-negative CTCs (p = 0.027). The expression level of CCND1 in CTCs of disease-controlled patients was significantly higher than that of disease-progressed patients (p = 0.034). In disease-controlled patients, the expression level of CCND1 in CTCs significantly decreased after nivolumab treatment (p = 0.043). The NANOG expression in CTCs was significantly increased in disease-controlled patients after nivolumab treatment (p = 0.036). Our findings suggest that the molecular profiling of CTCs is a promising tool to predict the treatment efficacy of nivolumab.

Rapid Effect of Benralizumab for Hypereosinophilia in a Case of Severe Asthma with Eosinophilic Chronic Rhinosinusitis.

A 56-year-old man with severe asthma underwent bronchial thermoplasty (BT). However, his asthma exacerbated and hypereosinophilia developed 2 months later, thus necessitating oral corticosteroid (OCS) therapy. Six months after BT, a diagnosis of severe asthma with eosinophilic chronic rhinosinusitis (ECRS) was made and benralizumab treatment was initiated; the blood eosinophil count subsequently decreased and lung function improved, thereby permitting OCS dose tapering. Surprisingly, benralizumab both reduced nasal polyps and ameliorated ECRS. Thus, benralizumab may be a useful drug for the rapid treatment of severe asthma with ECRS, especially in patients with hypereosinophilia.

[A Case Report of Acute Angioedema that Showed Dramatic Response to Administration of a C1-inactivator].

Angioedema is characterized by rapid and severe swelling of the subcutaneous and submucosal tissues. Angioedema involving the upper airway can lead to life-threatening airway obstruction, and needs prompt diagnosis and treatment. Herein, we report a case of acute angioedema which was suspected as having been caused by estrogen imbalance. A 32-year-old woman who was taking a fertility drug for infertility treatment, presented with sudden swelling of the face and neck region and breathing difficulty. Her symptoms continued to progress despite antibiotic and corticosteroid administration. We suspected hereditary angioedema (HAE), and administered a C1-inactivator, which led to immediate and dramatic resolution of the symptoms. Since the C4 and C1-inhibitor levels were normal, the possibility of HAE type III was considered. However, another possibility was that her complicated hormonal condition, including oral intake of a fertility drug, menstruation, and mental stress may have led to estrogen imbalance causing angioedema. Currently, a variety of hormone therapies is widely used ; therefore, caution is needed against the development of estrogen-dependent angioedema.

A Case of Maxillary Arteriovenous Fistula Rupture Treated by Vascular Interventional Radiology.

A 64-year-old woman with neurofibromatosis type 1 was admitted because of a sudden hematoma in her right neck and throat. To prevent an airway obstruction, tracheostomy was performed, but bleeding into the trachea persisted and was difficult to stop. Bleeding arose from a vessel lesion in the right parapharyngeal space, passed through the paratracheal space, and finally reached the tracheostomy wound. An arteriovenous fistula (AVF) in the maxillary artery was revealed by angiography. We diagnosed rupture of the varix resulting from venous high pressure caused by the AVF. The patient was treated by vascular interventional radiology (IVR). A large number of platinum coils were inserted and N-butyl-2-cyanoacrylate (NBCA) was embolized in the AVF and varix. Although innovative vascular IVR was better than surgery to treat AVF of the head and neck lesion, some problems were encountered. The cost for the provided medical services was high, and NBCA was not approved by the pharmaceutical affairs law in Japan.