Efficacy of immune checkpoint inhibitor treatment for head and neck mucosal melanoma recurrence in patients treated with carbon-ion radiotherapy.

BACKGROUND: Carbon-ion radiotherapy (C-ion RT) is effective for head and neck mucosal melanoma (HN-MM), including radioresistant mucosal melanoma. Melanoma also responds effectively to immune checkpoint inhibitors (ICIs). Data on the efficacy and safety of ICIs for HN-MM are insufficient. AIMS: To analyze the efficacy and safety of ICI salvage therapy in patients with HN-MM recurrence after C-ion RT. METHODS AND RESULTS: This retrospective study analyzed the medical records of 52 patients with HN-MM treated with C-ion RT between 2012 and 2020. A dose of 57.6 or 64.0 Gy (relative biological effectiveness) was provided in 16 fractions. The primary endpoint was 3-year overall survival (OS) rate. The median follow-up time was 26.8 months for all patients. A total of 29 patients had local recurrence or distant metastasis, and 16 patients who received ICI therapy. The 3-year OS rate in the ICI group (n = 16) and best supportive care group (n = 13) were 53.8% and 0.0%, respectively (p = 0.837); the difference was not statistically significant. There were no deaths after 1 year among patients who underwent ICI therapy. No adverse events associated with C-ion RT were related to or exacerbated by ICI. CONCLUSION: ICI salvage therapy is effective and safe for patients with HN-MM recurrence after C-ion RT.

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.

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.

The Efficacy of Radiotherapy without Surgery for External Auditory Canal Squamous Cell Carcinoma.

External auditory canal (EAC) cancer is a rare disease for which there are no adequate evidence-based treatment strategies. Radiotherapy is often used as the initial treatment to preserve the organ. This study aimed to elucidate the efficacy of radiotherapy for EAC squamous cell carcinoma (SCC). Patients with T1 disease were treated with radiotherapy alone. Patients with T2-4 disease were treated with chemoradiotherapy. The median follow-up period was 30.4 months. The 3-year local control (LC) rate for all patients was 51%, the disease-free survival (DFS) rate was 44%, and the overall survival (OS) rate was 73%. For T1-3 disease, the 3-year LC rate was 74%, DFS was 62%, and OS was 89%. However, for T4 disease, the 3-year LC rate was 17%, DFS was 17%, and OS was 50%. In a univariate analysis, only the T-category was a significant factor for LC and DFS (p = 0.006 and 0.02, respectively). All local recurrences were within the high-dose irradiated area. The results of this study suggest chemoradiotherapy can be an alternative to a combination of surgery and postoperative radiation for T1-3 SCC of the EAC. However, the efficacy of chemoradiotherapy in T4 cases was inadequate.

Carbon-ion Radiotherapy for Inoperable Head and Neck Bone and Soft-tissue Sarcoma: Prospective Observational Study.

BACKGROUND/AIM: Bone and soft-tissue sarcomas of the head and neck have very poor prognoses. This prospective study aimed to investigate the efficacy and safety of carbon-ion radiotherapy (C-ion RT) for bone and soft-tissue sarcoma of the head and neck. PATIENTS AND METHODS: The present study was a prospective clinical study that included 10 consecutive patients diagnosed with bone and soft-tissue sarcoma of the head and neck who were treated with C-ion RT between 2012 and 2018 at our institution. C-Ion RT consisted of 70.4 Gy (relative biological effectiveness) in 16 fractions. RESULTS: The 3-year local control, overall survival, and progression-free survival rates for patients overall were 72.9%, 77.8%, and 36%, respectively. CONCLUSION: The present study demonstrated the efficacy of C-ion RT for bone and soft-tissue sarcoma of the head and neck; adverse events were within the expected range.

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.

Upregulated glycolysis correlates with tumor progression and immune evasion in head and neck squamous cell carcinoma.

Altered metabolism is an emerging hallmark of cancer. Cancer cells preferentially utilize glycolysis for energy production, termed "aerobic glycolysis." In this study, we performed a comprehensive analysis of the glycolytic activity in head and neck squamous cell carcinoma (HNSCC) using data obtained from The Cancer Genome Atlas database. We first divided 520 patients with HNSCC into four groups based on the mRNA expression of 16 glycolysis-related genes. The upregulated glycolytic activity positively correlated with human papillomavirus-negative tumor type, advanced T factor, and unfavorable prognosis. The gene set enrichment analysis revealed upregulation of several hallmark pathways, including interferon-alpha response, myc targets, unfolded protein response, transforming growth factor-ß signaling, cholesterol homeostasis, and interleukin 6-Janus kinase-signal transducer and activator of transcription 3 signaling, in the glycolysis-upregulated groups. Immune cell enrichment analysis revealed decreased infiltration of T cells, dendritic cells, and B cells in the glycolysis-upregulated groups, suggesting impaired tumor antigen presentation, T cell activation, and antibody production in the TME. Moreover, the expression profile of immune-related genes indicated increased immune evasion in the glycolysis-upregulated tumors. Collectively, these findings suggest that transcriptome analysis of glycolytic activity of tumors has the potential as a biomarker for tumor progression and immunological status in patients with HNSCC.

Tissue-resident memory T cells correlate with the inflammatory tumor microenvironment and improved prognosis in head and neck squamous cell carcinoma.

OBJECTIVES: Tumor-infiltrating T cell (TIL) is a major cell type involved in tumor eradication in the tumor microenvironment (TME). Among TILs, tissue-resident memory T cells (TRMs) have been recognized as a subset capable of continuous immunosurveillance to afford long-term immunity. In the present study, we comprehensively profiled TRM in patients with head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We analyzed RNA-sequencing (RNA-seq) data obtained from The Cancer Genome Atlas (TCGA) database. Based on the gene expression of CD69 and CD4/CD8A, we identified TRM-enriched patients and evaluated their clinical and biological significance. In addition, we analyzed peripheral blood mononuclear cells (PBMCs) obtained from 60 patients with HNSCC to evaluate the presence of TRM-like cells in the peripheral circulation. RESULTS: TCGA analysis revealed that TRM-enriched tumors correlated with early T factor, human papillomavirus-positive status, the proportion of oropharynx lesion, upregulated inflammatory pathways, upregulation of immunostimulatory and immune checkpoint molecule genes, and favorable overall survival. Moreover, we clarified the presence of CD69 + TRM-like cells that highly express PD-1 and TIM-3 in the peripheral circulation of patients with HNSCC. CONCLUSION: We highlighted the clinical and transcriptomic significance of TRM in patients with HNSCC. Further characterization of TRM could lead to the development of novel biomarkers, especially for immune checkpoint therapies.

Comprehensive analysis of immune cell enrichment in the tumor microenvironment of head and neck squamous cell carcinoma.

Head and neck squamous carcinoma (HNSCC) is highly infiltrated by immune cells, including tumor-infiltrating lymphocytes and myeloid lineage cells. In the tumor microenvironment, tumor cells orchestrate a highly immunosuppressive microenvironment by secreting immunosuppressive mediators, expressing immune checkpoint ligands, and downregulating human leukocyte antigen expression. In the present study, we aimed to comprehensively profile the immune microenvironment of HNSCC using gene expression data obtained from public database. We calculated enrichment scores of 33 immune cell types based on gene expression data of HNSCC tissues and adjacent non-cancer tissues. Based on these scores, we performed non-supervised clustering and identified three immune signatures-cold, lymphocyte, and myeloid/dendritic cell (DC)-based on the clustering results. We then compared the clinical and biological features of the three signatures. Among HNSCC and non-cancer tissues, human papillomavirus (HPV)-positive HNSCCs exhibited the highest scores in various immune cell types, including CD4+ T cells, CD8+ T cells, B cells, plasma cells, basophils, and their subpopulations. Among the three immune signatures, the proportions of HPV-positive tumors, oropharyngeal cancers, early T tumors, and N factor positive cases were significantly higher in the lymphocyte signature than in other signatures. Among the three signatures, the lymphocyte signature showed the longest overall survival (OS), especially in HPV-positive patients, whereas the myeloid/DC signature demonstrated the shortest OS in these patients. Gene set enrichment analysis revealed the upregulation of several pathways related to inflammatory and proinflammatory responses in the lymphocyte signature. The expression of PRF1, IFNG, GZMB, CXCL9, CXCL10, PDCD1, LAG3, CTLA4, HAVCR2, and TIGIT was the highest in the lymphocyte signature. Meanwhile, the expression of PD-1 ligand genes CD274 and PDCD1LG2 was highest in the myeloid/DC signature. Herein, our findings revealed the transcriptomic landscape of the immune microenvironment that closely reflects the clinical and biological significance of HNSCC, indicating that molecular profiling of the immune microenvironment can be employed to develop novel biomarkers and precision immunotherapies for 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.

In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms.

The evaluation of antitumor immune responses is essential for immune monitoring to predict clinical outcomes as well as treatment efficacies in cancer patients. In this study, we produced two tumor antigen (TA) proteins, melanoma antigen family A4 and wild type p53, using TG silkworm systems and evaluated anti-TA-specific immune responses by enzyme-linked immunosorbent spot assays in patients with head and neck cancer. Eleven (61.1%) of 18 patients showed significant IFN-γ production in response to at least one TA; however, the presence of TA-specific immune responses did not significantly contribute to better prognosis (overall survival, p = 0.1768; progression-free survival, p = 0.4507). Further studies will need to be performed on a larger scale to better assess the clinical significance of these systems. The production of multiple TA proteins may provide new avenues for the development of immunotherapeutic strategies to stimulate a potent and specific immune response against tumor cells as well as precise assessment of antitumor immune responses in cancer patients.

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.

Clinical features of anti-transcription intermediary factor 1γ (TIF1γ)-positive dermatomyositis with internal malignancy and investigation of the involvement of TIF1γ expression in tumors in the pathogenesis of cancer-associated dermatomyositis.

Anti-transcription intermediary factor 1γ (anti-TIF1γ) antibody (Ab) is significantly associated with internal malignancies in adult patients with dermatomyositis (DM). Although pathogenesis of cancer-associated DM is unknown, TIF1γ overexpression in tumors has been considered to be critical for the development of DM. The objective of this study was to investigate clinical characteristics of patients with anti-TIF1γ Ab-positive DM and elucidate risk factors that are potentially associated with internal malignancy. In addition, we compared the expression of TIF1γ in tumor tissues of patients with anti-TIF1γ Ab-positive DM, anti-TIF1γ Ab-negative DM and without DM in order to investigate the pathogenesis of cancer-associated DM. We analyzed 77 Japanese patients with DM, and found 19 patients to be positive for anti-TIF1γ Ab. Patients with anti-TIF1γ Ab-positive DM were older and presented heliotrope rash and flagellate erythema more frequently than patients without anti-TIF1γ Ab (P < 0.05). Interstitial lung disease (ILD) and rapidly progressive ILD, as well as palmar violaceous erythema, were less frequent in patients with anti-TIF1γ Ab than in patients without. Furthermore, internal malignancy and dysphagia were significantly more frequent in the anti-TIF1γ Ab-positive group (P < 0.01). Male sex and dysphagia were significantly associated with internal malignancy in patients with anti-TIF1γ Ab-positive DM (P < 0.01 and <0.05, respectively). Using immunohistochemistry, we examined the TIF1γ expression in tumors of 11 patients with cancer-associated DM (anti-TIF1γ Ab-positive, nine; anti-TIF1γ Ab-negative, two) and 25 patients without DM. TIF1γ was highly expressed in all tumors, and there was no significant difference in TIF1γ expression between patients with and without DM. Furthermore, TIF1γ expressions in tumors were similar irrespective of the presence of anti-TIF1γ Ab. These results suggest that anti-TIF1γ antibody may not be simply induced by overexpression of TIF1γ in tumors in patients with DM, but that other mechanisms may exist.

Epithelial-Mesenchymal Transition Status of Circulating Tumor Cells Is Associated With Tumor Relapse in Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: We aimed to elucidate the clinical implication of the epithelial-mesenchymal plasticity of circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: CTCs isolated from 44 patients with non-recurrent/metastatic HNSCC and 42 with recurrent/metastatic (R/M) HNSCC were classified into four epithelial-mesenchymal transition (EMT) statuses based on the expression of epithelial (keratin 19) and mesenchymal (vimentin) markers and the relationships between EMT status in CTCs and clinical factors were investigated. RESULTS: E+M- CTC phenotype was more frequent in patients without recurrence/metastasis (p=0.0468) and was also more frequent in those with a complete response (p=0.0346). The E+M+ phenotype constituted the major proportion of the CTCs detected in patients with R/M HNSCC (p=0.0374). CONCLUSION: CTCs may play unique roles at various stages of metastasis through transitioning from epithelial to mesenchymal phenotypes.

Carbon-ion radiotherapy combined with chemotherapy for head and neck mucosal melanoma: Prospective observational study.

This study aimed to evaluate the efficacy of carbon-ion radiotherapy in combination with chemotherapy using dacarbazine, nimustine, and vincristine (DAV therapy) in mucosal melanoma. Twenty-one patients with clinically localized mucosal melanoma of the head and neck were enrolled. The primary endpoint was 3-year overall survival (OS). Secondary endpoints included local control, progression-free survival (PFS), and adverse event occurrence. Carbon-ion radiotherapy with a dose of 57.6-64.0 Gy (relative biological effectiveness) in 16 fractions was delivered concurrently with DAV therapy, and 2 cycles of adjuvant DAV therapy were administered every 6 weeks. The median follow-up periods were 15.5 months for all patients, and 31.2 months for 12 surviving patients. All patients had locally advanced T4a or T4b disease in the rhino-sinus area. In 16 patients (76.2%), 3 cycles of planned DAV therapy were completed. The 3-year OS and PFS rates were 49.2% and 37.0% respectively. The 3-year local control rate was 92.3%. Eleven patients (52%) developed distant metastasis, which was the most frequent pattern of the first failure. Commonly presenting acute grade 2-3 toxicities associated with radiotherapy and chemotherapy were mucositis (11 patients [53%]) and leukopenia (9 patients [43%]), which improved with conservative therapy. None of the patients developed grade 3 or greater late toxicities. Carbon-ion radiotherapy in combination with DAV therapy led to excellent local control for advanced mucosal melanoma within acceptable toxicities. The efficacy of additional DAV therapy in improving survival was weaker than expected as distant metastases still occurred frequently. Trial registration no. UMIN000007939.

Expression of immune-regulatory molecules in circulating tumor cells derived from patients with head and neck squamous cell carcinoma.

OBJECTIVES: Circulating tumor cells (CTCs) are cells that have shed from tumor tissue into the bloodstream, and the detection and characterization of CTCs in head and neck squamous cell carcinoma (HNSCC) still remain a challenge. MATERIALS AND METHODS: CTCs were isolated from 30 patients with HNSCC with recurrent and/or distant metastasis, via the depletion of CD45-positive cells with magnetic beads and the expression of multiple epithelial markers (CK19, EpCAM, EGFR, and c-Met) was analyzed by RT-qPCR with a low concentration of RNA from the CTC population. We next investigated the expression of the immune-regulatory molecules, PD-L1, PD-L2, and CD47, in CTC-positive patients and the PD-L1 expression in CTCs was compared with that in tumor tissues. RESULTS: Twenty-four (80.0%) of the 30 patients were positive for at least one epithelial-related gene. Among the 24 CTC-positive patients, 19 (79.2%), 20 (83.3%), and 17 (70.8%) patients were positive for CD47, PD-L1, and PD-L2, respectively. Interestingly, the expression of these three immune-regulatory molecules was positively correlated to each other. As expected, PD-L1 expression in the tumor tissue did not correspond completely with that in the CTCs. CONCLUSION: Although clinical application and/or characterization of CTCs are still developing, our findings suggest that the CTCs are rapidly becoming a powerful tool in cancer treatments that involve the use of immune checkpoint inhibitors.

Association Between Laryngopharyngeal Reflux and Radiation-induced Mucositis in Head and Neck Cancer.

BACKGROUND/AIM: We investigated whether laryngopharyngeal reflux (LPR) is a risk factor for radiation-induced mucositis. PATIENTS AND METHODS: This was a retrospective cohort study using our departmental database. The study included patients with stage I or II laryngeal and hypopharyngeal cancers treated with radiation therapy alone between April 2009 and March 2014. Based on endoscopic findings, baseline laryngeal signs were evaluated using the reflux finding score (RFS), and the severity of mucositis was assessed during and after radiation therapy. RESULTS: Fifty-eight patients were enrolled. Thirty-one patients were categorized as high RFS (LPR-likely), while 27 patients were categorized as low RFS (LPR-unlikely). Grade 3 mucositis occurred more frequently in the high RFS group (p<0.042). Furthermore, grade 3 mucositis developed earlier in the high RFS group (p<0.001). CONCLUSION: High RFS (i.e., increased likelihood of LPR) appears to be a potential risk factor for developing severe radiation-induced mucositis.

Circulating tumor cells in patients with head and neck squamous cell carcinoma: Feasibility of detection and quantitation.

BACKGROUND: The purpose of this study was to present our findings that because circulating tumor cells (CTCs) exist in extremely low numbers, their detection and quantification are challenging. METHODS: Peripheral blood samples were collected from 32 patients with head and neck squamous cell carcinoma (HNSCC), and were subjected to the CellSieve Microfiltration Assay using a low-pressure filtration system. The CTCs captured by the filter were stained with an antibody cocktail (anti-cytokeratin (CK) 8, 18, and 19, anti-epithelial cell adhesion molecule (EpCAM), and anti-CD45 antibodies). RESULTS: The CTCs were detected in 29 of 32 patients (90.6%). Although patients with advanced disease had a significantly higher number of CTCs, the clinical N classification was not associated with the CTC count. After treatment, the CTC count showed a significant decrease. CONCLUSION: The CTCs were successfully detected and quantified in patients with HNSCC by using a low-pressure filtration system equipped with precision microfilters. Further studies using a larger number of patient samples and/or molecular analysis of CTCs are warranted.