Koichi Sakakura

Stromal cells in the tumor microenvironment (TME) closely interact with tumor cells and affect tumor cell behavior in diverse manners.We herein investigated the mechanisms by which cancer-associated fibroblasts (CAFs) affect the functional polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) in vitro and in human cancer samples.The expression of CD68, CD14, CD163, CD200R, CD206, HLA-G, CD80, and CD86 was higher in CD14-positive cells co-cultured with the culture supernatants of CAFs established from OSCC specimens (CAF-educated cells) than in control cells.The gene expression level of ARG1, IL10, and TGFB1 was increased in CAF-educated cells.CAF-educated cells suppressed T cell proliferation more strongly than control cells, and the neutralization of TGF-β IL-10, or arginase I significantly restored T cell proliferation.We then investigated the relationship between the infiltration of CAFs and TAMs using tissue samples obtained from patients with OSCC.The infiltration of CAFs was associated with the numbers of CD68-positive and CD163-positive macrophages.It also correlated with lymphatic invasion, vascular invasion, lymph node involvement, and the TNM stage.The infiltration of CAFs was identified as an independent prognostic factor in OSCC.Our results indicate that CAFs play important roles in shaping the tumor immunosuppressive microenvironment in OSCC by inducing the protumoral phenotype of TAMs.Therapeutic strategies to reverse CAF-mediated immunosuppression need to be considered.

Evidence has accumulated indicating that only a minority of cancer cells with stem cell properties, cancer stem cells (CSCs), are responsible for maintenance and growth of the tumor. CD44 is currently used to identify CSCs as one of the cell surface markers for solid tumors. Here we report the identification, expansion, and characterization of CD44+ cancer stem-like cells from a permanent squamous cell carcinoma of the head and neck (SCCHN) cell line. Under serum-free medium culture conditions, a small population (less than 3%) of CD44+ cells in a permanent cancer cell line was dramatically increased up to around 40%. The CD44+ cell population also showed higher expression of CD133 and ABCG2 as compared with the CD44- cell population. Moreover, CD44+ cells possess not only a marked capacity for forming tumor spheres, proliferation, migration, and invasion in vitro, but also resistance to chemotherapeutic agents. Four genes related to chemoresistance, ABCB1, ABCG2, CYP2C8, and TERT, were up-regulated in a CD44+ cell population. Our findings indicate that a subpopulation of CSCs is maintained in the SCCHN cell line, and the presence of such CSCs has an important clinical implication for head and neck cancer treatment. Further characterization of CSCs may provide new insights for novel therapeutic targets and prognostic markers.

BackgroundThe cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)–based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb.

Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer.Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53.L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis.L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.

Myeloid‐derived suppressor cells ( MDSC ) represent a heterogeneous population and have the potential to suppress immune responses via diverse mechanisms. In recent studies, a new subset of MDSC was identified by the markers CD 14 + and HLA‐DR − in the peripheral blood from cancer patients. In this study, we investigated the proportions and characteristics of CD 14 + HLA‐DR − cells in patients with squamous cell carcinoma of the head and neck ( SCCHN ). As expected, the percentage of CD 14 + HLA‐DR − cells was significantly elevated in patients relative to healthy donors and the sorted CD 14 + HLA‐DR − cells were able to suppress effectively both the proliferation and IFN ‐γ production of anti‐ CD 3/anti‐ CD 28 stimulated T cells, suggesting that CD 14 + HLA‐DR − cells in patients with SCCHN contribute to the immune suppressive status. Furthermore, CD 14 + HLA‐DR − cells revealed a higher level of CD 86 and PD‐L 1 expression and transforming growth factor (TGF )‐β production than CD 14 + HLA‐DR + cells. Addition of anti‐ CD 86 mA b, anti‐PD‐L1 mA b and anti‐ TGF ‐β mA b partially restored T ‐cell proliferation and IFN ‐γ production, respectively, indicating that the suppressive effects of CD 14 + HLA‐DR − cells appear to be mediated by various molecules, including coinhibitory molecules and cytokines. Our data suggest that CD 14 + HLA‐DR − cells act as potent immunosuppressive cells and particularly contribute to tumor escape from the host immune system in patients with SCCHN . ( Cancer Sci 2012; 103: 976–983)

Abstract Background CD44 was found as a surface marker in cancer stem cell (CSC) of squamous cell carcinoma of the head and neck (SCCHN); however, the immunologic properties of such CSCs have not yet been elucidated. Methods The immunologic properties of CD44+ cancer stem‐like cells were compared with those of CD44‐ cells using flow cytometry and enzyme‐linked immunosorbent assay. Results CD44+ cells exhibited weak HLA‐A2 and class II expression. Interestingly, downregulation of transporter antigen processing (TAP)2 was found in CD44+ cells. The CD44+ cell population produced significantly higher levels of interleukin (IL)‐8, granulocyte colony‐stimulating factor (G‐CSF), and transforming growth factor (TGF)‐β than the CD44‐ cell population. Moreover, CD44+ cells have been shown to not only more strongly inhibit T‐cell proliferation, but also to more efficiently inhibit regulatory T cells (Treg cells) and myeloid‐derived suppressor cells (MDSC) as compared with CD44‐ cells. Additionally, CD44+ cells suppressed Th1 responses and enhanced regulatory T cell responses. Conclusion CSCs might have higher malignant potential with numerous escape strategies from immune attack. © 2010 Wiley Periodicals, Inc. Head Neck, 2011

Chondroitin sulfate proteoglycan 4 (CSPG4), also known as High Molecular Weight- Melanoma Associated Antigen, is a cell surface proteoglycan which has been recently shown to be expressed not only by melanoma cells, but also by various types of human carcinoma and sarcoma. Furthermore, at least in squamous cell carcinoma of head and neck and in basal breast carcinoma, CSPG4 is expressed by cancer stem cells. CSPG4 plays an important role in tumor cell growth and survival. These CSPG4-associated functional properties of tumor cells are inhibited by CSPG4-specific monoclonal antibodies (mAb) in vitro. Moreover, CSPG4-specific mAb can also inhibit tumor growth and metastasis in vivo. The anti-tumor effects of CSPG4-specific mAb are likely to reflect the blocking of important migratory, mitogenic and survival signaling pathways in tumor cells. These results indicate that CSPG4 is a promising new target to implement mAb-based immunotherapy of various types of cancer. Keywords: CSPG4, immunotherapy, monoclonal antibody, cancer stem cells

Tumor-associated macrophages (TAM) have been classified into an immunostimulatory M1 subset against microbes and malignancies, and an immunoregulatory M2 subset that secretes immunosuppressive cytokines in order to repair tissues damaged by malignancies. The infiltration of M2 in the tumor microenvironment is known to facilitate immunosuppression and tumor-promoting properties. In the present study, we investigated the phagocytic potential of these macrophage subsets in oral squamous cell carcinoma (OSCC) in relation to the expression of CD47, the 'don't eat me' signal against macrophages. The macrophage subsets M1 (induced by GM-CSF and IFN-γ) and M2 (induced by M-CSF and IL-10) were derived from the CD14(+) cells of healthy donors. Phagocytosis of the CFSE-labeled CD47(+) cell line HSC-3 by M1/M2 was assessed using flow cytometry and suppressed by an anti-CD47 neutralizing antibody or CD47 siRNA. Furthermore, CD68(+) and CD163(+) macrophage subset counts infiltrating tumor tissue and the expression of CD47 on cancer cells were examined immunohistochemically in 74 cases of OSCC, and their relationships with clinicopathological parameters or prognoses were determined. The phagocytic potential of M1 was similar to that of M2 in vitro. Phagocytosis by M1 increased in a CD47-dependent manner by the neutralizing antibody and siRNA, but did not in M2. An immunohistochemical (IHC) analysis revealed that the expression of CD47 did not correlate with macrophage subsets in peritumoral tissue or with any clinicopathological parameters; however, the stronger expression of CD47 by cancer cells and larger number of total macrophages/M2 were independently related to shorter survivals. Our results suggest that the expression of CD47 by cancer cells is related to evasion from phagocytosis, particularly that by M1 in vitro. IHC results indicate that various mechanisms are involved in the engulfing potential of TAM subsets in vivo.

Abstract Background CD44 was identified previously as a surface marker in cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC). Most cancer treatments have been linked to the activation of the apoptosis‐signaling pathway; however, the resistance mechanisms to apoptosis in CSCs have not yet been fully elucidated. Methods The sensitivity of CD44+ cells to diverse apoptosis‐inducing stimuli was compared with that of CD44‐ cells. Furthermore, cell cycle changes and the expression of anti‐apoptosis‐related genes were examined using flow cytometry and real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). Results CD44+ cells were resistant to various apoptosis‐inducing stimuli. Moreover, CD44+ cells showed a higher proportion of cells in G2/M phase of the cell cycle and upregulation of Bcl‐2 and inhibitor of apoptosis (IAP) family genes compared with CD44‐ cells. Conclusion Treatment resistance in CSCs seems to be regulated by various mechanisms, and, therefore, additional treatment strategies to target CSCs are required in patients with HNSCC. © 2011 Wiley Periodicals, Inc. Head Neck, 2012

Abstract Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4+ melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal–regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4. Cancer Res; 71(24); 7410–22. ©2011 AACR.

Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs). Moreover, evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alterations in the patterns of acetylation of histones and are a promising new group of anticancer agents. In this study, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on two CD44+ cancer stem-like cell lines from squamous cell carcinoma of the head and neck (SCCHN) cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. Histone deacetylase inhibitors inhibited the growth of SCCHN cell lines in a dose-dependent manner as measured by MTS assays. Moreover, HDACi induced cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. In addition, HDACi decreased mRNA expression levels of stemness-related genes and suppressed the epithelial-mesencymal transition phenotype of CSCs. As expected, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel, had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter the cancer stem cell phenotype in SCCHN, raising the possibility that HDACi may have therapeutic potential for cancer stem cells of SCCHN.

Abstract Background Homeostasis of circulating T cells is regulated in complex ways that have not yet been well defined. The balance between type 1 and type 2 T‐cell subsets in cancer patients is thought to modulate antitumor immunity. Meanwhile, CD4+CD25+ regulatory T cells (Treg), which are potent inhibitors of antitumor immune responses, also play an invaluable role in maintaining immune homeostasis. Methods Peripheral blood was obtained from 42 patients with squamous cell carcinoma of the head and neck (SCCHN) and 24 healthy age‐selected donors. The percentages of T‐cell subsets and their cytokine profiles expressed in response to ex vivo stimulation were studied by multicolor flow cytometry. Results Although patients with SCCHN had a lower percentage ( p < .05) of circulating CD4+ T cells than healthy donors, CD4+CD25+ regulatory T cells (Treg) were increased in the patients ( p < .01). A significant increase in Th1 and Th2 CD4+ T cells was observed in the patients after ex vivo stimulation with phorbol 12‐myristate 13‐acetate /ionomycin. The percent of Treg inversely correlated with that of total CD8+ T cells ( p < .05), CD8+IFN‐γ+ (Tc1) cells ( p < .05), and CD8+IL‐4+ (Tc2) cells ( p < .01). There was a highly significant correlation between Tc1 and Tc2 CD8+ T cells ( p < .0001) in SCCHN patients but not in controls. Conclusions Treg are increased in proportion in the circulation of patients with SCCHN. These cells appear to downregulate cytokine expression in both Tc1 and Tc2 subsets of CD8+ effector T cells, which may be responsible for antitumor responses. © 2006 Wiley Periodicals, Inc. Head Neck 2007

The immunological significance of autophagy in the tumor microenvironment remains unclear. To explore the relationship between autophagy and anti-tumor immune responses, we investigated the expression of autophagy-related proteins and infiltration of immune cells using immunohistochemistry (IHC). The expression of three representative autophagy components, LC3, Beclin-1 and p62/SQSTM1, as well as the number of dendritic cells (DC), T cells and NK cells were examined by IHC in 74 patients with oral squamous cell carcinoma (OSCC). The relationship between the expression of autophagy-associated molecules and various clinicopathological parameters was also evaluated. The expression of both LC3 and p62/SQSTM1 in the peripheral site significantly correlated with an increase in the infiltration of T cells. Furthermore, the expression of p62/SQSTM1 and Beclin-1 correlated with that of HLA class I and class II in tumor cells, respectively. In addition, several unfavorable clinicopathological parameters correlated with an increase in the expression of LC3 in the peripheral site. The correlation observed between LC3 or p62/SQSTM1 and the infiltration of T cells suggests that autophagy may actively mobilize immune cells toward the cancer bed. Meanwhile, the three autophagy-associated proteins examined were linked to malignant potential and an unfavorable prognosis.

The aim of this study is to evaluate the clinicopathological significance of L-type amino acid transporter 1 (LAT1) expression in patients with advanced laryngeal squamous cell carcinoma (LSCC). A total of 73 patients with advanced LSCC were retrospectively reviewed. Tumor sections were stained by immunohistochemistry for LAT1, 4F2hc, system ASC amino acid transporter-2 (ASCT2), cell proliferation by Ki-67, microvessel density (MVD) determined by CD34 and p53. A positive LAT1, 4F2hc and ASCT2 expression (staining more than a quarter) in the primary sites were recognized in 85, 80 and 45 %, respectively, and a high LAT1, 4F2hc and ASCT2 expression (staining more than a half) yielded 48, 31 and 18 %, respectively. High expression of LAT1 was significantly associated with lymph node metastasis, 4F2hc, ASCT2, Ki-67 and p53. The expression of LAT1 was significantly correlated with ASCT2, 4F2hc, cell proliferation, and MVD. By univariate analysis, there was no statistically significant relationship between LAT1 expression and prognosis in advanced LSCC. LAT1, 4F2hc and ASCT2 were highly expressed in patients with advanced laryngeal cancer. Our study suggests that the expression of LAT1 plays a crucial role in the metastasis and tumor progression in advanced LSCC.

Background/Aim: Programmed death-ligand 1 (PD-L1) expression in tumor cells is regulated by a close interrelation between tumor and stromal cells within the tumor microenvironment. Our aim was to evaluate the clinical and biological significance of PD-L1 expression in oral squamous cell carcinoma (OSCC). Materials and Methods: PD-L1, cluster of differentiation (CD)4, CD8, and forkhead box P3 (FOXP3) expression in tumor tissues obtained from 77 patients with OSCC was evaluated by immunohistochemical staining, and then analyzed for associations with clinical and biological factors. Results: Among the clinicopathological factors tested, only vascular invasion showed a trend toward lower PD-L1 expression (p=0.05). Metabolic tumor volume (MTV), and total lesion glycolysis (TLG) significantly positively correlated with PD-L1 expression (MTV, p=0.04; TLG, p=0.03). In patients with OSCC with high PD-L1 expression, those whose tumors had abundant infiltrating CD4⁺ T-cells showed a longer progression-free survival than those with low CD4⁺ T-cell infiltration (p=0.0452). Conclusion: As regulation of PD-L1 expression is complex, its evaluation combined with other markers may be useful to determine clinical applications of PD-L1 expression.

The main factor that affects the prognosis of patients with oral squamous cell carcinoma (OSCC) is regional lymph node metastases, which usually spreads first to the sentinel lymph nodes (SLNs). Recent studies have demonstrated that tumor cells in several malignancies can induce lymphangiogenesis in SLNs before metastasizing. To elucidate the mechanisms of tumor dissemination of OSCC, we investigated whether primary tumors induce lymphangiogenesis within SLNs in patients with OSCC. The mRNA expression of lymphatic-specific markers, including VEGFR-3, Prox-1, and LYVE-1 in 23 metastasis-negative SLNs obtained from 10 patients with OSCC, was investigated using a quantitative real-time RT-PCR assay, and compared with control lymph nodes from patients with non-cancerous diseases. In addition, VEGF-C and VEGF-D expressions of the primary tumor were examined by immunohistochemistry. In SLNs, there were highly significant correlations between the three lymphatic markers examined. Interestingly, the level of LYVE-1 expression in SLNs, despite the absence of metastasis, was significantly higher than in control lymph nodes. Moreover, SLNs from patients with VEGF-C-positive tumor showed a significantly higher expression of VEGFR-3 than those from patients with VEGF-C-negative tumor. Our findings suggest that in OSCC, the primary tumor actively induces lymphangiogenesis in SLNs prior to the onset of metastases, and where tumor-derived VEGF-C plays an important role.

Both L-type amino acid transporter 1 (LAT1) and CD98 are strongly expressed in primary human cancer and play essential roles in tumor growth. We studied the clinicopathological significance of LAT1 and CD98 expression in hypopharyngeal squamous cell carcinoma (SCC).A total of 70 patients with stage III/IV disease were retrospectively reviewed. Immunohistochemical staining of tumor sections was used to examine LAT1, CD98, Ki-67, CD34, and p53.High LAT1 and CD98 expression were noted in 60.0% and 47.1%, respectively (p = .174). A statistically significant correlation was recognized between LAT1 and CD98 expression and both expressions were closely associated with tumor cell proliferation. Although LAT1 expression was not significantly associated with poor survival, multivariate analysis revealed high CD98 expression to be an independent prognostic factor for predicting a poor outcome.CD98 is a promising prognostic marker for predicting outcomes after surgical treatment in patients with advanced hypopharyngeal SCC.

Abstract 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.

Abstract Background Several inflammatory biomarkers are considered potential prognostic factors in various cancers. This study aimed to investigate the prognostic significance and population dynamics of pretreatment inflammatory biomarker levels in patients with oropharyngeal squamous cell carcinoma (OPSCC). Methods The influence of neutrophil counts, lymphocyte counts, monocyte counts, platelet counts, lymphocyte‐to‐monocyte ratio (LMR), neutrophil‐to‐lymphocyte ratio, and platelet‐to‐lymphocyte ratio on progression‐free survival (PFS), and overall survival (OS) was analyzed. We also analyzed the peripheral blood mononuclear cells collected from patients and healthy donors (HDs). Results Elevated monocyte count was an independent prognostic factor for PFS. Low LMR was an independent prognostic factor for OS. The proportion of intermediate monocytes was lower, and that of classical monocytes was higher in patients than in HDs. Furthermore, PD‐L1 expression on monocytes was higher in patients than in HDs. Conclusions We showed the prognostic significance and population dynamics of peripheral monocytes in patients with OPSCC.

Congenital laryngeal anomalies are less frequent, but their causes are surprisingly variable. In addition, a variety of synchronous airway lesions as well as comorbidities are accompanied. The objective of this study was to review of patients with congenital laryngeal anomalies presenting as chronic stridor in our experiences.Fifty-five patients, 30 male (54.5%) and 25 female (45.4%), were enrolled in this study, and their hospital records were retrospectively reviewed.The most frequent diagnosis was laryngomalacia (36.4%), followed by subglottic stenosis (30.9%) and vocal cord paralysis (29.1%). Twenty-six (47.3%) of the 55 patients had synchronous airway lesions, whereas thirty-one (56.4%) had various comorbidities. Further analysis was performed in patients diagnosed with laryngomalacia, subglottic stenosis, or vocal cord paralysis, which are major causes of congenital laryngeal stridor. The frequency of synchronous airway lesions was not different significantly in these three groups. On the other hand, the frequency of establishment of airway in patients with laryngomalacia was significantly lower compared to those with subglottic stenosis or vocal cord paralysis. Moreover, median duration of the symptoms and the proportion of patients with poor outcome and decease in laryngomalacia were shorter and lower than that in subglottic stenosis or vocal cord paralysis.While a variety of congenital airway anomalies were causes of chronic stridor, laryngomalacia was the most frequent diagnosis. Severe condition and progression of symptoms should increase suspicion of the synchronous airway lesions and/or comorbidities, which may be important factors for outcome as well as indication of surgical intervention.

Conclusion: A high GRP78/BiP expression was proved to be a significant marker for predicting poor outcome after surgery. GRP78/BiP may be a promising molecular target for treatment of ACC. Background: The glucose-regulated protein GRP78/BiP plays a crucial role in the endoplasmic reticulum (ER) stress. The level of GRP78 is highly elevated in various human cancers, but the clinicopathological significance of GRP78/BiP remains controversial in patients with adenoid cystic carcinoma (ACC). Methods: A total of 26 ACC patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, Ki-67, and microvessel density (MVD) determined by CD34. Results: GRP78/BiP and PERK were highly expressed in 58% (15/26) and 35% (9/26), respectively. The high expression of GRP78/BiP was significantly associated with PERK, cell proliferation and angiogenesis.

The glucose-regulated protein (GRP78/BiP) and PKR-like endoplasmic reticulum kinase (PERK) plays a crucial role in the endoplasmic reticulum (ER) stress response.GRP78/BiP is highly elevated in various human cancers.Our study is to examine the clinicopathological significance of GRP78/BiP and PERK expression in patients with tongue cancer.A total of 85 tongue cancer patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, GLUT1, Ki-67 and microvessel density (MVD) determined by CD34.GRP78/BiP and PERK were highly expressed in 47% and 35% of all patients, respectively.GRP78/BiP disclosed a significant relationship with PERK expression, lymphatic permeation, vascular invasion, glucose metabolism and cell proliferation.The expression of GRP78/BiP was significantly higher in metastatic sites than in primary sites (79% vs. 47%, p=0.003).We found that the high expression of GRP78/BiP was proven to be an independent prognostic factor for predicting poor outcome in patients with tongue cancer.In the analysis of PFS, PERK was identified as an independent predictor.The increased GRP78/BiP expression was clarified as an independent prognostic marker for predicting worse outcome.Our study suggests that the expression of GRP78/BiP as ER stress marker is important in the pathogenesis and development of tongue cancer.

Tumor cells have evolved sophisticated means of escape from the host immune system. To date, several important immunological phenomena have been revealed in peripheral blood as well as within tumors. In the present study, we first investigated the proportion and activation status of peripheral immune regulatory cells and CD8 + T‐cell subsets in patients with head and neck squamous cell carcinoma (HNSCC) using a multicolor flow cytometer, and then evaluated how therapy with docetaxel, cisplatin, and 5‐fluorouracil modulated the immune cell profile in peripheral blood. The proportion of naïve T cells was lower and that of effector memory T cells (T EM ) was higher in HNSCC patients than in healthy donors. Moreover, the proportions of activated T EM cells and effector T cells (T EFF ) were dramatically increased in patients with advanced stage disease. The proportion of regulatory T cells and CD14 + HLA‐DR − myeloid‐derived suppressor cells was elevated in HNSCC patients. Of note, after therapy, in addition to the transient reduction in immune regulatory cells, decreases in central memory T cells and increases in T EFF cells were observed among CD8 + T‐cell subsets, suggesting differentiation from central memory T cells into T EFF cells. Our results suggested that, despite the immunosuppressive status in HNSCC patients, tumor‐specific immune responses mediated by CD8 + T cells might be induced and maintained. Moreover, chemotherapy can trigger not only a transient reduction in immune regulatory cells but also further activation of CD8 + T cells.

Cancer-associated fibroblasts (CAFs) play vital roles in tumor progression by promoting epithelial-to-mesenchymal transition, angiogenesis, and immunosuppression. In the present study, we sought to identify the key regulators of the pro-tumoral functions of CAFs in head and neck squamous cell carcinoma (HNSCC). mRNA expression data obtained from The Cancer Genome Atlas revealed that CAF-specific mRNA expression correlated with genes that relate to an immunosuppressive microenvironment in a HNSCC cohort. RNA sequencing of CAFs and normal fibroblasts isolated from HNSCC specimens identified 1127 differentially expressed genes (DEGs) and several upregulated pathways in CAFs. Among the 1127 DEGs, we identified 13 immune function-related genes and focused on AKT3 as a potential regulator of CAFs. The targeted depletion of AKT3 in CAFs revealed that AKT3 promotes their myofibroblastic phenotype. AKT3-transduced CAFs exhibited downregulated the expression of immunosuppressive cytokine genes, impairing T-cell suppression and pro-tumoral macrophage induction. The immunohistochemistry of 72 HNSCC patients showed that AKT3 expression in CAFs positively correlated with tumor infiltration by CAFs, tumor-associated macrophages, dendritic cells, and T cells. Moreover, AKT3 expression in CAFs was an independent prognostic factor for overall survival. In conclusion, AKT3 is a potential target for cancer therapy that inhibits the pro-tumoral function of CAFs and reverses CAF-mediated immunosuppression.

Circulating monocytes are classified into three subsets according to their CD14 and CD16 expressions. Here we investigated all three subsets in patients with squamous cell carcinoma of the head and neck (SCCHN). Peripheral blood from 54 patients with SCCHN and 24 healthy donors (HDs) was tested for flowcytometry. Immunohistochemical staining of the primary tumor was performed. SCCHN cells were co-cultured with human monocytes in vitro. The level of intermediate monocytes was significantly lower in SCCHN than in HDs. The expression levels of HLA-G, PD-L1, and CD51 on intermediate monocytes was evidently greater in patients with SCCHN. In vitro co-culturing of SCCHN cells with monocytes revealed a significant increase in CD51 expression levels on monocytes. The decrease in expression levels of the maturation markers CX3CR1 and CD68 was significantly correlated to poor clinical outcomes. The level of intermediate monocytes was decreased in cancer patients in favor of immature and expressed immunosuppressive molecules.

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Six HLA-A2 or HLA-A24-restricted wt p53 peptides were evaluated for their ex vivo immunogenicity and their potential for use in cancer vaccines. Peripheral blood mononuclear cells (PBMC) obtained from HLA-A⁎0201+ and/or HLA-A⁎2402+ normal donors and subjects with squamous cell carcinoma of the head and neck (SCCHN) were analyzed for p53 peptide-specific reactivity in ELISPOT IFN-γ assays. CD8+ T cells in 7/10 normal donors (HD) and 11/23 subjects with SCCHN responded to at least one of the wt p53 peptides. CD8+ T cell precursors responsive to wt p53 epitopes were detected in the circulation of most subjects with early disease, and an elevated blood Tc1/Tc2 ratio distinguished wt p53 peptide responders from non-responders. The identification of multiple wt p53 peptides able to induce cytolytic T lymphocytes in most subjects with cancer promotes the development of multi-epitope p53 vaccines.

The expression of L-type amino acid transporter 1 (LAT1) is correlated with tumor cell growth and survival. However, the clinicopathological significance of LAT1 expression in adenoid cystic carcinoma (ACC) remains to be elucidated. The aim of this study is to investigate the clinicopathological significance of LAT1 expression in ACC. A total of 30 patients with ACC were retrospectively reviewed. Tumor sections were stained by immunohistochemistry for LAT1, p53, and CD98, and cell proliferation and microvessel density (MVD) were determined by Ki-67 and CD34, respectively. High LAT1 and CD98 expression were observed in 27 % (8/30) and 23 % (7/30) of samples, respectively (p > 0.999). The high expression of LAT1 was significantly correlated with cell proliferation (Ki-67) and the cell cycle regulator p53. By univariate analysis, solid histological pattern, maxillary tumor site, LAT1, CD98, Ki-67 and p53 were significantly associated with poor prognosis. Multivariate analysis demonstrated that the high expression of LAT1 was an independent prognostic factor for predicting poor prognosis after surgical resection. LAT1 is a promising clinical marker to predict the outcome after surgery in patients with ACC.

The maximum standardized uptake value (SUVmax) of early oral squamous cell carcinoma (OSCC) may have a role as an imaging biomarker for assessment of malignant potential, including cell metabolism and angiogenesis.The usefulness of (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) has been proven in various cancers, including OSCC. Moreover, in several carcinomas, the SUVmax of the tumor has been shown to correlate with the histological type, tumor stage, differentiation, and prognosis. Here, we investigated whether the SUVmax of early OSCC was associated with the biological features.Twenty-seven patients with newly diagnosed early OSCC who underwent preoperative FDG-PET and curative surgical resection were included in this study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), L-type amino acid transporter 1 (LAT1), CD98, microvessels (CD34), cell proliferation marker (Ki-67), and cell cycle regulator (p53). The correlation between SUVmax and clinicopathological findings or the expression level of these molecules was analyzed.SUVmax of primary OSCC was significantly higher in patients with T2 stage. Moreover, patients whose tumors showed vascular invasion had a tendency to show higher SUVmax. A significant correlation was observed between SUVmax and the expression of LAT1 or microvessel density.

The immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein 78 (GRP78) is important in the endoplasmic reticulum stress, and is highly expressed in various human cancers. The clinical and pathological features of GRP78/BiP are unclear in patients with advanced laryngeal squamous cell carcinoma (SCC). The purpose of this study was to investigate the clinicopathological significance of GRP78/BiP as a prognostic marker for laryngeal SCC.A total of 59 patients with advanced laryngeal SCC (stage III/IV) were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP and Ki-67. Microvessel density was determined by immunohistochemical staining for CD34 and p53.Expression of GRP78/BiP was confirmed in 87% of cases. Decreased expression of GRP78/BiP was highly associated with positive expression of p53. Decreased GRP78/BiP expression was identified on multivariate analysis as an independent factor of decreased progression-free survival (PFS).GRP78/BiP was found to be commonly expressed in laryngeal SCC, whereas its downregulation was found to serve a significant prognostic role for predicting poor survival in patients with laryngeal SCC with advanced disease. GRP78/BiP may be a potentially attractive target for the treatment of various human neoplasms. © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1544, 2016.

The sentinel lymph node (SLN) is regarded as the first tumor-draining lymph node, which may be the initial site activated by tumor antigens. To clarify the immunological functions of SLNs, a total of 89 tumor-free regional lymph nodes (41 SLNs and 48 non-SLNs) were obtained from 12 patients with oral cavity cancer, and infiltration of both DCs and NK cells was determined by immunohistochemistry. S-100+ and CD1a+ DCs infiltrated significantly into SLNs compared to non-SLNs. Analysis in each of the pN0 and pN+ patients showed that all the DC markers in pN0 patients and only S-100+ in pN+ patients were significantly more abundant in SLNs. Moreover, infiltration of CD83+ DCs was less in pN+ patients than in pN0 patients. These results suggest that more significant immune responses against cancer occur in SLNs than in non-SLNs. However the progression of disease including nodal disease may cause systemic immunosuppression.

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.

Vestibular evoked myogenic potential (VEMP) has been used to test vestibulocollic reflex. However, VEMP is not stable on elderly patients because of their weak muscular strength. In this study, we tried to record VEMP on median neck extensor muscles with weak muscular contractionWe recorded VEMP from normal subjects and patients by novel and conventional methods.Thirty-one normal subjects and 56 patients with vertigo or hearing loss were tested in a seated or prone position without muscular tension. The different electrodes were placed on the median surface at the palpable bottom of the occipital bone.Our response showed a clear negative peak at 13 ms on normal subjects, with reversed polarity compared with VEMP on the sternocleidomastoid muscle. This potential is defined as VEMP caused by the proper latencies, dependency of the strength on sound stimulation, and independence of hearing ability. In the cases of acoustic neurinoma, onset latencies were prolonged or nonexistent. The responses on neck extensor muscles could not be recorded on some elderly patients.This new method of recording VEMP is less invasive and suitable for elderly patients.

Anti-tumor immunity plays an important role in the development of and protection from malignancy. However, there is a lack of information regarding induction of CD4+ T helper responses in patients with squamous cell carcinoma (SCCHN). To explore anti-tumor immune responses against SCCHN, a permanent cell line, Gun-1 was established from a squamous cell carcinoma of the hypopharynx. In addition to its characterization, we performed mixed lymphocyte-tumor cell cultures (MLTC) using peripheral blood lymphocytes and autologous tumor cells. Furthermore, T cell responses to wild type (wt) p53-derived peptides were assessed. Gun-1 cells overexpressed p53 and were negative for HLA-A2 expression. No tumor-specific or wt p53-specific CD8+ CTL lines could be established from peripheral blood mononuclear cells (PBMCs) of this patient. Autologous tumor-specific HLA-DR-restricted CD4+ T helper clone was obtained by limiting dilutions using bulk populations from MLTC. This clone produced IFN-γ but not IL-5 in response to autologous tumor cells. In addition, CD4+ T cells were generated from the patient’s PBMCs which responded to two HLA-DP5-restricted wt p53-derived peptides. Our results suggest that the immune cells specific for autologous tumor as well as wt p53-derived epitopes are present in the peripheral circulation of this cancer patient. However, helper-type CD4+ T lymphocytes represent the predominant anti-tumor response.

The immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein 78 (GRP78) plays an essential role in the endoplasmic reticulum (ER) stress, and GRP78/BiP is known to be highly expressed in various human neoplasms. The clinicopathological features of GRP78/BiP expression in patients with advanced hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. The aim of this study is to elucidate the prognostic significance of GRP78/BiP for HSCC.A total of 68 patients with advanced HSCC (stage III/IV) were analyzed, and tumor specimens were stained with immunohistochemistry for GRP78/BiP, Ki-67, and microvessel density (MVD), as determined through CD34 and p53 levels. GRP78/BiP was highly expressed in 80.8% (55/68) of all patients. The expression level of GRP78/BiP disclosed no significant relationship with any variables. Multivariate analysis confirmed that low expression of GRP78/BiP was an independent prognostic factor for predicting poor overall survival and progression-free survival in patients with advanced HSCC. The decreasing expression of GRP78/BiP was identified as a significant predictor related to shorter survival duration after surgery for advanced HSCC. Our study suggests that the reduced expression of GRP78/BiP contributes to worse survival for patients with advanced head and neck cancer.

Salivary gland swelling is a commonly encountered clinical symptom, but the establishment of a diagnosis is occasionally difficult. Here, we present two sialodochitis fibrinosa patients with recurring bilateral parotid swelling. In both patients, secretion of mucous plugs containing numerous eosinophils was observed from Stensen's ducts. As expected, the level of interleukin-5 in the saliva was much higher than that in the serum. One patient had no medical history of allergic disease; the other had allergic rhinitis which had never been associated with parotid gland swelling. Microbiological examination was unable to isolate significant bacterial specimens from the mucous plugs. Thus, although allergy and/or bacterial infection are reportedly implicated as causes of sialodochitis fibrinosa, there may exist other possibilities for its pathogenesis. Interleukin-5 seems to play a crucial role in the pathogenesis of sialodochitis fibrinosa.

In squamous cell carcinoma of the head and neck (SCCHN), tumor cells have been shown to secrete detectable amounts of various cytokines, such as interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-β. These tumor-derived factors might be responsible for promoting malignancy. Here, we describe a SCCHN patient with tumor produced G-CSF and characterized by marked leukocytosis. In this 45-year-old man, severe leukocytosis developed in parallel with aggressive tumor growth. G-CSF production by the tumor was confirmed by immunohistochemistry (IHC). Serum G-CSF levels were elevated. The leukocyte counts and the blood G-CSF level decreased following a course of radiotherapy. Tumor cells were also positive for G-CSF receptor, suggesting autocrine growth regulation by G-CSF. Moreover, the tumor cells were also investigated by IHC with anti-p53, anti-P-glycoprotein (P-gp), anti-thymidylate synthase (TS), and anti-dihydropyrimidine dehydrogenase (DPD), which molecules are thought to contribute the acquisition of therapeutic resistance. The tumor cells were positively stained for TS and DPD, but neither p53 nor P-gp. These results suggest that a variety of molecules may be responsible for acquisition of high malignancy.

Vestibular evoked myogenic potential (VEMP) has been used to test the vestibulocollic reflex. This study establishes a stable recording of VEMPs of animals, and presents useful parameters for vestibular ability. In an acute experiment, rats were decerebrated, and myogenic potential from neck extensor muscles was recorded. The myogenic potentials elicited by a tone-burst stimulus showed a biphasic response in the ipsilateral muscle, and the mean latency of the response was 3.56 ms, the positive peak appearing at 4.63 ms. The onset latencies of the response lengthen as the stimulus becomes weaker; this is the most suitable parameter of vestibular ability. The latencies of the monophasic response from the spinal cord were shorter than those of muscle. After injection of a muscle relaxant, myogenic potentials disappeared immediately, but the spinal cord response remained. We succeeded in recording responses not only from acute experimental animals but also from free-moving animals for the first time. These myogenic potentials were similar to VEMPs in humans; because the threshold of the response was higher than the auditory brainstem response threshold by 40-45 dB, the response could only be recorded with very high spontaneous muscle activity and the latency was shorter than the startle reflex.

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Evidence has been accumulating which indicates that CD4+ Th cells have an important role in generating and maintaining antitumor immune responses. To elucidate the nature of CD4+ Th responses to wt p53 epitopes in patients with squamous cell carcinoma of the head and neck (SCCHN), peripheral blood mononuclear cells (PBMCs) from HLA-DP5+ patients were stimulated with HLA-DP5-restricted wt p53 peptides, p53(108-122) or p53(153-166), and tested for the release of IFN-gamma and IL-5 in ELISPOT assays. Immunohistochemistry for p53 accumulation in tumors, and ELISA for serum antibodies to p53 were also performed. Eleven (57.9%) of 19 HLA-DP5+ patients but none of 5 healthy donors had detectable Th1 and/or Th2 responses to wt p53 peptides by ELISPOT assay. Among these 11 responding patients, 9 (81.8%) and all 11 (100%) patients had a tumor burden and p53 accumulation, respectively. On the other hand, two responding patients were in post-operative condition. Interestingly, among nine patients with a tumor burden, four patients with early disease showed either Th1-polarized or mixed Th1/Th2 responses, while five patients with advanced disease showed either Th2-polarized or mixed Th1/Th2 responses. Our results suggest that wt p53(108-122) and p53(153-166) peptides stimulate both Th1- and Th2-type CD4+ T cell responses in patients with SCCHN, and anti-p53 Th responses may persist even after surgical resection of the tumor; however, the presence of a tumor and its progression may affect the nature of immune responses to wt p53 peptides.

The purpose of this study was to estimate the possibility of using thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and p53 as predictive values of clinical outcome in adenoid cystic carcinoma (ACC). The expressions of TS, DPD, and p53 were examined with immunohistochemistry in 27 ACC patients, and the association with clinicopathological factors was determined. Cases with high DPD expression had significantly higher distant metastasis rates compared to those with low DPD expression (p = 0.001), whereas neither TS nor p53 expression showed any significant correlation to clinicopathological factors. Interestingly, six of 14 early-stage patients had distant metastases and all of their tumors showed high DPD expression. Kaplan–Meier analysis revealed that a solid histological pattern and distant metastasis correlated with a poor prognosis. In early-stage patients, whose tumor was completely resected, those with high TS or DPD expression had a worse prognosis compared to those with low expression, but the difference did not reach statistical significance (TS, p = 0.178; DPD, p = 0.251). Our results suggest that assessment of DPD expression in ACC may be a useful tool in determining the mode of treatment as well as evaluating clinical outcome.

Objectives: The management of dysphagia requires a multidisciplinary approach, especially in large-scale hospitals. We introduce a novel protocol using a Wi-Fi–based flexible endoscopic evaluation of swallowing (FEES) system and aim to verify its effectiveness in evaluation and rehabilitation of inpatients with dysphagia. Method: We conducted novel Wi-Fi–based FEES at the bedside using 3 iPads as monitors and recorders. Functional outcomes of swallowing in 2 different hospitals for acute care with conventional wired or wireless FEES were compared retrospectively. Results: Using the wireless system, we could visit more patients in a short period of time. Furthermore, a large multidisciplinary team was able to be present at the bedside, which made it easy to hold discussions and rapidly devise appropriate rehabilitation strategies. Aspiration pneumonia recurred in a few cases following our intervention with wireless FEES. Functional oral intake score was significantly increased following the intervention. Moreover, the number of deaths during hospitalization using wireless FEES evaluation was lower than those observed using the conventional system. Conclusion: Wi-Fi–based wireless FEES system, the first of its kind, allowed our multidisciplinary team to easily and effectively assess inpatients with dysphagia by facilitating simple examinations and intensive transprofessional discussions for patient rehabilitation.

Abstract The phosphatidylinositol 3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway plays a vital role in cell proliferation, apoptosis, metabolism, and angiogenesis in various human cancers, including head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to clarify the role of AKT, which is a major downstream effector of the PI3K‐AKT‐mTOR pathway, in HNSCC. We first investigated the mRNA expression of AKT isoforms using RNA‐sequencing data from The Cancer Genome Atlas database. We observed a specific elevation of AKT3 expression in HNSCC tissues when compared with that in normal tissues. Furthermore, AKT3 expression correlated with genes related to the immunosuppressive microenvironment more than the other AKT isoforms and PIK3CA . Accordingly, we focused on AKT3 and performed a knockdown approach using an HNSCC cell line. AKT3 knockdown cells exhibited impaired proliferation, a shift in the cell cycle from G2/M to G1/G0 phase, an increase in apoptotic cells, and downregulation of gene expression related to immunosuppression, as well as the knockdown of its upstream regulator PIK3CA . We also performed immunohistochemistry for both AKT3 and PIK3CA using surgical specimens from 72 patients with HNSCC. AKT3 expression in tumor cells correlated with immune cell infiltration and unfavorable prognosis when compared with PIK3CA. These findings suggested that AKT3 expression is a potential biomarker for predicting the immunoreactivity and prognosis of HNSCC. Furthermore, the isoform‐specific inhibition of AKT3 could be developed as a novel cancer therapy that efficiently suppresses the PI3K‐AKT‐mTOR pathway.

Conclusions. Our results suggest that assessment of dihydropyrimidine dehydrogenase (DPD) in oral squamous cell carcinoma (OSCC) may be a useful tool in evaluating clinical outcomes. Objective. The purpose of this study was to estimate the possibility of using thymidylate synthase (TS) and DPD as predictive values of clinical outcomes in OSCC. Materials and methods. The expression of TS and DPD was examined by immunohistochemistry (IHC) and the real-time reverse transcription-polymerase chain reaction method in 23 patients with OSCC, and the association with clinicopathological factors was determined. Immunohistochemical expression of p53 and P-glycoprotein (P-gp) was also examined. Results. Neither TS protein nor TS mRNA expression showed any significant correlation to clinicopathological factors. In contrast, the patients with high DPD expression had significantly higher levels of recurrence compared with those with low DPD expression (p = 0.016). Similarly, six of seven patients with relapse had higher DPD mRNA expression values than the median value in the patients examined. On the other hand, no association was observed between TS or DPD and p53 or P-gp expressions.

Recent clinical trials with the aim of developing tumor antigen (TA)-specific cancer vaccines against a number of malignancies have focused on the identification of TAs presented by tumor cells and recognized by T cells. In the present study, the TA melanoma antigen family A4 (MAGE-A4) protein was produced using a transgenic (TG) silkworm system. Using in vitro stimulation, it was subsequently determined whether MAGE-A4 protein induced MAGE-A4-specific T cells from peripheral blood mononuclear cells of healthy donors. TG silkworm lines expressing a MAGE-A4 gene under an upstream activating sequence (UAS) were mated with those expressing a yeast transcription activator protein (GAL4) at the middle silk glands (MSGs) and embryos that harbored both the GAL4 and UAS constructs were selected. Recombinant MAGE-A4 protein was extracted from the MSGs of TG silkworms and evaluated using SDS-PAGE and western blot analysis. It was observed that MAGE-A4 produced by the TG silkworm system successfully induced MAGE-A4-specific CD4+ T cell responses. Furthermore, MAGE-A4-specific CD4+ T cells recognized antigen-presenting cells when pulsed with a MAGE-A4+ tumor cell lysate. The present data suggests that recombinant tumor antigen production using the TG silkworm system may be a novel tool in the preparation of cancer vaccines.

Over two decades have passed since tumor antigen was discovered and the possibility of a tumor-specific vaccine was demonstrated; however, it is difficult to accept that tumor antigen–specific immunotherapies can be successful and guarantee a promising outcome. The immunosuppressive state of patients induced by cancer cells is the most probable reason for the unsatisfactory results of clinical vaccine trials. Here, we depict immunosuppression due to ‘host-side’ factors and ‘tumor-side’ factors in patients with squamous cell carcinoma of the head and neck (SCCHN). As ‘host-side’ factors, we mention the dysfunction and apoptosis of immune cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and T-helper 2 (Th2) skewing. As ‘tumor-side’ factors, we describe the production of immunosuppressive cytokines, downregulation of human leukocyte antigen (HLA) expression, and the characteristics of cancer stem cells (CSCs). We believe that this review will be helpful to understand immunosuppressive mechanisms in patients with SCCHN.

Objective —Although acute otitis media (AOM) is the commonest infectious disease of childhood, the emergence of drug-resistant bacteria has dramatically changed its clinical outcome. Here, we report the trend of AOM due to drug-resistant Staphylococcus pneumoniae (DRSP) and β-lactamase-negative ampicillin-resistant Haemophilus influenzae (BLNAR), and the relation between antibiotics used for the management of AOM and the isolation of bacterial pathogens. Material and Methods —Bacterial isolation and susceptibility tests were performed on specimens from children with AOM. Clinical information, including antibiotic treatment within the previous 30 days, was analyzed. Results —DRSP was detected in 59.3% of Pneumococci isolates and BLNAR in 26.0% of H. influenzae isolates. As expected, the incidence of AOM caused by such drug-resistant bacteria has been increasing year on year, and 32% of cases have been treated with inappropriate antibiotics. In contrast, 32% of cases of AOM caused by DRSP and 50% caused by BLNAR were given antibiotics with high susceptibility to drug-resistant bacteria. Conclusion —In order to ensure the most appropriate use of antibiotics, clinicians should consider performing tympanocentesis or myringotomy, with subsequent submission of the middle ear fluid for susceptibility testing. Furthermore, these results suggest that, as well as the selection of antibiotics, the dosage and period of dosing should also be considered in the management of AOM. In addition, other factors, in particular horizontal transmission from other infants in day care or nursery school, may affect the rapid spread of such drug-resistant bacteria.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by the activated macrophages and acts as a bridge between apoptotic cells and phagocytes. Aside from macrophages, a variety of malignant cells also express MFG-E8. The objective of this study is to elucidate the clinical relevance and significance of MFG-E8 in the tumor microenvironment (TME) of patients with oral squamous cell carcinoma (OSCC). We investigated MFG-E8 expression in 74 patients with OSCC by immunohistochemistry and evaluated the relationship between MFG-E8 expression and various clinicopathological factors including immune cell infiltration. MFG-E8 expression was detected in 34 of 74 (45.9%) patients with OSCC and a significant correlation was observed with levels of infiltrating T cells, macrophages, and immunosuppressive M2 macrophages. Furthermore, MFG-E8 expression was also associated with clinical stage, lymphatic/vascular invasion, and Ki-67+ tumor cells but not with survival. Our results suggest that MFG-E8 may play an important role in shaping the immune suppressive network in TME as well as tumor progression.

Abstract There is increasing evidence in certain malignancies that tumor cells are organized into a hierarchy originating from a small population of cancer stem cells (CSCs) that possesses extensive proliferative and self-renewal potential, and that these CSCs are responsible for maintaining the tumors. In squamous cell carcinoma of the head and neck (SCCHN), CD44+ cells with significant tumorigenic potential have been identified as CSCs; however, the immunological properties of such CSCs have not yet been elucidated. In this study, we investigated the immunological functions of CD44+ cells from a SCCHN cell line. Analysis of flow cytometry demonstrated that the expression of HLA-A2 molecules was deficient in the parental cell line, whereas the CD44+ cell population restored HLA-A2 expression, although at very low levels. Moreover, CD44+ cells exhibited weak HLA class II expression on the cell surface. Interestingly, down-regulation of TAP2 was found in CD44+ cells. The CD44+ cell population produced significantly higher levels of IL-8, G-CSF, and TGF-β than the CD44- cell population. Moreover, CD44+ cells have been shown to not only more strongly inhibit the proliferation of T cells activated with anti-CD3/CD28 mAb, but also to more efficiently induce CD4+CD25+FOXP3+ Treg cells and myeloid-derived suppressor cells as compared with CD44- cells. Additionally, CD44+ cells also suppressed Th1 responses (IFN-γ production by PBMCs) and enhanced regulatory T cell responses (IL-10 production by PBMCs). Thus, CSCs may have higher immunosuppressive ability promoting evasion from immunosurveillance; therefore, the development of novel immunotherapeutic strategies to efficiently overcome CSCs-driven immune suppression is urgently needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5314.

Suppurative acute thyroiditis is caused by pyriform sinus fistula (PSF), and PSF frequently elicits deep neck abscess. However, complete fistulectomy is the ideal management of PSF, and studies on surgical findings of PSF are exceedingly rare. This study aimed to reveal the origins of PSF, each route, and clinical presentation.This is a multicenter study. We have conducted 19 complete fistulectomies of PSF in Japan, analyzed routes of the fistulas, estimated the origins, and investigated their histological and clinical findings.No recurrence was observed in all cases. Five of 12 cases showed thymic and/or parathyroid tissues around the fistulas, passing inside the inferior horn of thyroid cartilage, were regarded as having 3rd pouch origin, and tended to have low frequency of severe deep neck abscess. The remaining 7 cases originated from the 4th pouch running outside of the horn and showed frequent severe infection.PSF have 2 different routes depending on their generation and may present different clinical manifestations.

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<div>Abstract<p>Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4<sup>+</sup> melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited <i>in vitro</i> growth and migration of tumor cells and <i>in vivo</i> growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal–regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4. <i>Cancer Res; 71(24); 7410–22. ©2011 AACR</i>.</p></div>

<div>Abstract<p>Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4<sup>+</sup> melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited <i>in vitro</i> growth and migration of tumor cells and <i>in vivo</i> growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal–regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4. <i>Cancer Res; 71(24); 7410–22. ©2011 AACR</i>.</p></div>

Background Evidence has been accumulated indicating that regulatory T (T-reg) cells play a crucial role in the maintenance of peripheral T-cell tolerance to allergens. To explore the role of FOXP3, which is required for the development of T-reg cells, in allergen-specific immune responses, we examined the relationship between the alteration of FOXP3 gene expression and in vitro immune responses against allergens. Methods Peripheral blood mononuclear cells obtained from 19 human histocompatibility leukocyte antigens (HLA)-DPB1 * 0501 donors, including patients with Japanese cedar pollinosis and nonallergic healthy donors, were stimulated with Cry j 1 p61-75 peptide. On day 7, T cells were tested for peptide-specific reactivity in IFN-γ and interleukin (IL)-5 enzyme-linked immunospot (ELISPOT) assays. Real-time quantitative RT-PCR was performed to assess relative change of FOXP3 gene expression before and after in vitro stimulation. Neutralization assays using anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and anti-IL-10 monoclonal antibody were also performed. Results Of 14 patients with allergic pollinosis tested, 10 responders displayed T-helper type 2 (Th2)-polarized reactivity to Cry j 1 p61-75, and 2 donors showed Th0 responses. Notably, the change of FOXP3 gene expression in donors showing peptide-specific T-helper responses was significantly lower than that in nonresponders, regardless of allergic pollinosis. Conclusion Our data indicate that FOXP3 is functional in nonallergic healthy donors as well as allergic patients, and FOXP3-expressing T cells may be responsible for the down-regulation of allergen-specific T-helper responses in individuals. A better understanding of the nature and specificity of FOXP3-expressing T cells in a suppressive mechanism is necessary to develop new immunotherapies against allergic rhinitis.

Abstract Background Cancer-associated fibroblasts (CAFs) are one of main elements in the tumor microenvironment (TME). Among the innate and adaptive immune cells in the TME, tumor-associated macrophages (TAMs) are particularly abundant and play a protumoral role. However, an interaction between CAFs and TAMs in head and neck squamous cell carcinoma (HNSCC) still remains unclear. In the present study, we investigated whether CAFs skewed macrophage function toward the immune suppressive phenotype. Materials and Methods In vitro assay: CAFs were prepared from surgical tissue of HNSCC patients, and culture supernatants of CAFs were collected. Using this supernatants and peripheral blood mononuclear cells collected from healthy donors, immunological interactions between CAFs and TAMs was investigated. In vivo assay: The immunohistochemistry (IHC) of 73 patients with HNSCC was performed. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs, respectively. The relationship between immunohistochemical parameters and clinical parameters was evaluated. Results The expression levels of CD68, CD163, CD14, CD80, CD86, and HLA-G were up-regulated in CD14+ cells co-cultured with the supernatants of CAFs (CAF-educated cells) compared with control cells. Moreover, the gene expressions of ARG1, IL6, IL10 and TGFB1 were also increased in CAF-educated cells. The CAF-educated cells suppressed T-cell proliferation more intensively than control cells, and neutralization of TGF-β and IL-10 led to significant restoration of T-cell proliferation. In IHC, the intensity of CAFs correlated with the number of CD68-positive macrophages, especially with CD163-positive macrophages. It also correlated with several clinical parameters such as lymphatic invasion, vascular invasion, nodal status, tumor stages and recurrence rate. Moreover, the high intensity of CAFs correlated with poor PFS and OS. The high intensity of CAFs and M2 macrophage infiltration were independent prognostic factors of PFS and OS in HNSCC. Conclusion The present study suggests that CAFs can induce an immunosuppressive phenotype of macrophages and contribute to establishing the immunosuppressive networks in TME, resulting poor prognosis in HNSCC patients. Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Cancer-associated fibroblasts induce immunosuppressive macrophages in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3239.

Abstract Background: With a developing tumor, tumor cells shed a variety of substances into the blood stream, such as tumor cells, tumor DNA, or exosomes, and these are considered to be as attractive circulating biomarkers to monitor disease progression in cancer patients. However, impact of circulating tumor cells (CTCs) in squamous cell carcinoma of the head and neck (SCCHN) is still not fully understood. In this study, we show that CTCs are present in the peripheral blood of patients with SCCHN, and then investigate the correlation with clinicopathological factors. Materials and Methods: 7.5mL of peripheral blood was obtained from 32 patients with pathologically and clinically confirmed SCCHN. Samples were run with a CellSieve™ Microfiltration Assay (Creatv MicroTech, Inc., Rockville, MD) using a low-pressure vacuum system. The CTCs captured by the filter were stained with an antibody mixture of FITC-conjugated anti-CK 8, 18, and 19, PE-conjugated anti-EpCAM, and Cyanine 5-conjugated anti-CD45. Cells that stained positive for DAPI and CK but negative for CD45, were identified as CTCs. Results: The CTCs were found in 29 (90.6%) of 32 patients. Although there was no association between the number of CTCs and tumor sites, the number of CTCs in patients with advanced disease (stage III/IV) was significantly increased, as compared with those with early disease (stage I/II) (p<0.05). Furthermore, patients with T3/4 had higher number of CTCs compared to patients with T1/2 (p<0.01). Meanwhile, nodal status was not significantly associated with the number of CTCs. In 12 of 32 patients tested, the number of CTCs after treatments was also evaluated. As expected, the number of CTCs was significantly decreased after treatments, regardless of the treatment modalities. Conclusion: Our results suggest that microfiltration is very efficient in capturing CTCs from SCCHN patients. Moreover, CTCs analysis in SCCHN may provide useful information for assessment of treatment efficacy. Citation Format: Kazuaki Chikamatsu, Hideyuki Takahashi, Koichi Sakakura, Minoru Toyoda. Detection and clinical significance of circulating tumor cells in squamous cell carcinoma of the head and neck. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 479.

Abstract Introduction: Human circulating monocyte is classified as three populations according to CD14 and CD16 expression: CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes and CD14dimCD16+ nonclassical monocytes. Recent studies have unveiled the specific surface molecules and functions of each subset; however their dynamics in patients with cancer still remains unclear. In this study, we evaluated the proportions and the expressions of immunological/angiogenic molecules of the monocyte subsets in peripheral circulation in patients with squamous cell carcinoma of the head and neck (SCCHN). Materials and Methods: Peripheral blood mononuclear cells were isolated from 9 patients with SCCHN and age-matched 4 healthy donors. The 3 monocyte subsets were identified according to CD14/CD16 expressions using multi-color flow cytometry. Mean fluorescence intensities of various HLA molecules and angiogenic markers were analyzed. Results: Percentage of CD14+CD16+ intermediate monocyte among total monocyte tended to decrease in SCCHN patients than that in normal donors (p = 0.079). Higher surface expression of immunosuppressive HLA-G molecule on circulating classical (p = 0.045) and intermediate (p = 0.052) monocyte was observed in patients with SCCHN. An immunosuppressive receptor ILT4 showed tendency to increase on nonclassical monocyte (p = 0.061). Moreover, intracytoplasmic level of milk fat globule-EGF factor 8 (MFG-E8) in association with phagocytosis and angiogenesis tended to increase in intermediate (p = 0.064) and nonclassical (p = 0.052) monocytes of SCCHN patients. Discussion and Conclusion: CD16+ intermediate and nonclassical monocytes have been reported as mature, proinflammatory and angiogenic population in periphery. In the present study, we showed possible alternations of these subsets in patients with SCCHN. Furthermore, increasing surface expressions of immunosuppressive molecules HLA-G and ILT4 on circulating monocytes implied immunological deterioration in cancer patients. However the sample size is still small, our preliminary data strongly suggests proportional and functional changes of peripheral monocyte subsets in SCCHN patients. Further analyses in more patients are currently underway. Citation Format: Koichi Sakakura, Hideyuki Takahashi, Sei-ichiro Motegi, Kazuaki Chikamatsu. Imbalance of circulating monocyte subsets in patients with squamous cell carcinoma of the head and neck. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3238.

腫瘍組織には癌細胞だけでなく線維芽細胞，血管内皮細胞，免疫細胞等の多数の間質細胞が存在し，癌の進展に関与している。なかでも癌関連線維芽細胞（Cancer-associated fibroblasts: CAFs）はcell-to-cell contactや様々な液性因子の産生を介して癌細胞の増殖や転移に影響を及ぼしている細胞として，近年注目されている。

Abstract Introduction: Autophagy is an endogenous process that degrades bulk proteins and organelles to maintain energy homeostasis under cellular stress and nutrient starvation. It has been still unclear whether autophagy promotes or suppresses tumor progression in various cancer patients. To date, several studies have shown that autophagy-related molecules such as LC3 and Beclin-1 play important roles in tumor progression; however, few studies regarding immunological significance of these autophagy components in tumor microenvironment exist. The objective of this study is to clarify association between the expression of autophagy-related proteins and immune infiltrates by immunohistochemistry (IHC). Patients and methods: Expression of two representative autophagy-associated molecules, LC3 and Beclin-1, and CD1a (dendritic cell), CD3 (T cell) and CD56 (NK cell) cells were examined by IHC in 74 patients with oral squamous cell carcinoma (OSCC). HLA class I heavy chain, HLA class II, Ki-67 and p53 were also stained and assessed. Two regions, central site and peripheral site in tumor tissues were evaluated, separately. Moreover, the association with various clinicopathological parameters including progression free and overall survival was evaluated. Results: LC3 and Beclin-1 were accumulated in both 27 of 74 cases (36.5%) OCCC examined. LC3 expression in peripheral margin was significantly associated with increasing dendritic cell (DC) and T cell infiltration. Interestingly, peripheral Beclin-1 expression showed statistically significant relation to increasing HLA class II expression on tumor cells. On the other hand, LC3 and Beclin-1 significantly correlates with several unfavorable clinicopathological parameters including short overall survival. In addition, LC3 and Beclin-1 expression in OSCC significantly related to lymphatic invasion by tumor cells. Conclusion: Significant association between LC3 and DC/T cell infiltration suggests that autophagy is likely to actively participate in immunological phenomena within tumor microenvironment. Furthermore, peripheral Beclin-1 may contribute to antigen presentation by tumor cells, while these two autophagy components also relates to unfavorable prognosis. Taken together, autophagy seems to promote immunogenicity of cancer cells, whereas it facilitates tumor progression. Citation Format: Koichi Sakakura, Hideyuki Takahashi, Kyoichi Kaira, Minoru Toyoda, Tetsunari Oyama, Kazuaki Chikamatsu. Expressions of autophagy-related proteins positively correlate with infiltration of immune cells and disease progression in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2014-155

Abstract Introduction: Autophagy is a prosurvival function that facilitates cellular response to stress by cleaning damaged proteins and organelles, and provides energy to the cells during starvation. p62/SQSTM1, which is a multifunctional, multi-domain scaffolding protein that has been implicated in various cell signaling pathways including autophagy-mediated protein degradation. In autophagy, an accumulation of ubiquitinated proteins retains p62/SQSTM1 in the cell, resulting in an increase of selective autophagy, whereas autophagy impairment can accumulate p62/SQSTM1. To date, evidence has accumulated that, in the tumor microenvironment, autophagy is known to play an important role in tumorigenesis, but also influences immune response to tumor cells. However, it still remains unknown how this autophagy-regulated protein, p62/SQSTM1 in tumor cells takes part in antitumor immune responses. To clarify the relation between the p62/SQSTM1 accumulation and antitumor immunity, we investigated the expression of p62/SQSTM1 and immune cell infiltration in tumor tissues obtained from patients with oral squamous cell carcinoma (OSCC). Patients and methods: Seventy-four patients with OSCC who underwent curative surgical resection were included in this study. Tumor sections were stained by immunohistochemistry for p62/SQSTM1; dendritic cells (CD1a); T cells (CD3); NK cells (CD56); HLA class I heavy chain; HLA class II; Ki-67; p53. Each expression level was evaluated, and the association with clinicopathological factors was also determined. Results: p62/SQSTM1 accumulation was observed in 24 of 74 (32.4 %) OSCC examined. The p62/SQSTM1 accumulation in tumor cells showed statistically significant positive correlation to HLA class I expression on tumor cells. Interestingly, in tumor margin regions, expression of p62/SQSTM1 in tumor cells was significantly associated with CD3+T cell infiltration, suggesting that anti-tumor specific T cells may infiltrate and recognize tumor cells as target cells. Moreover, p62/SQSTM1 accumulation in tumor cells was also significant correlated with vessel invasion by tumor cells. Conclusion: In OSCC, p62/SQSTM1 accumulation in tumor cells was likely to associate with increase of tumor antigenicity, resulting in the mobilization of T cells toward cancer nest. On the other hand, p62/SQSTM1 accumulation in tumor cells may also contribute malignant potential in OSCC. Further elucidation of the role of p62/SQSTM1 in antitumor immunity through autophagy is currently underway. Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kyoichi Kaira, Minoru Toyoda, Tetsunari Oyama, Kazuaki Chikamatsu. Immunological significance of p62/SQSTM1 accumulation in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2014-154

Abstract Introduction: In general, TAM is classified into M1 producing Th1 cytokines and M2 secreting IL-10. Numerous reports have demonstrated that M2 infiltration to tumor microenvironment associates with local immunosuppression and proliferation of cancer cells. In this study, we investigated phagocytic potentials of M1 and M2 in SCCHN in relation to the expression of CD47 molecule, which is known as “Don't eat me” signal against macrophage (Mϕ). Material and Methods: CD68 (pan-Mϕ marker) positive CD163 (M2 marker) negative M1 (derived with GM-CSF and IFN-γ) and CD68+CD163+M2 (derived with M-CSF and IL-10) were induced from positively selected CD14+ cells in PBMC from a healthy donor. Phagocytosis of CD47+/CD47-SCCHN cell lines labeled with CFSE by M1/M2 were assessed using multi-color flow cytometry. Suppressions of the phagocytosis by neutralizing anti-CD47 antibody were also evaluated. Furthermore, we examined CD68, CD163 and CD47 expressions immunohistochemically in 74 cases of oral squamous cell carcinoma, and relation between Mϕ counts or CD47 expression on cancer cells and clinicopathological parameters or prognoses. Results: M1 derived from PBMC showed higher phagocytic potential comparing to M2. Phagocytosis of M1 was dose-dependently inhibited by anti-CD47 neutralizing antibody, however M2 phagocytosis was not suppressed. In immunohistochemical analysis, while CD47 was expressed in almost half cases of oral cancer, CD47 expression did not correlate to the numbers of total Mϕ nor M2. Patients with high CD47 expression on cancer cells, increasing number of total Mϕ or M2 showed significantly shorter overall survival. Conclusion: Only M1 showed CD47-dependent phagocytosis of CD47-expressed cancer cells in vitro. While CD47 expression on patient samples did not correlate with number of total Mϕ or M2, each of them independently showed significant association with prognosis. Various mechanisms were presumed between CD47 expression on SCCHN cells and tumor-infiltrating Mϕ subsets. Citation Format: Koichi Sakakura, Hideyuki Takahashi, Kazuaki Chikamatsu. Relation between tumor-associated macrophage (TAM) subsets and CD47 expression on squamous cell carcinoma of the head and neck (SCCHN) in tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1273. doi:10.1158/1538-7445.AM2015-1273

Abstract Introduction: Evidence has accumulated indicating that cancer-associated fibroblasts (CAFs) play an important role in angiogenesis, invasion, and metastasis. Simultaneously, CAFs might actively participate in inducing immunosuppressive ability and promoting evasion from immunosurveillance. In the present study, we investigated whether CAFs within the tumor microenvironment contribute as one of key components of tumor immune evasion in head and neck squamous cell carcinoma (HNSCC). Materials and methods: CAFs and normal fibroblasts (NFs) were prepared from surgical tissue of HNSCC patients. Flow cytometric analysis was performed for the expression of A-smooth muscle actin (A-SMA) and co-stimulatory molecules. Inhibition of T-cell proliferation, T-cell apoptosis and induction of regulatory T cells (Treg) were also examined using flow cytometry. The mRNA expression of various cytokines was investigated by real-time qRT-PCR. Results: Generated CAFs and NFs were negative for CD11b, CD34, and CD45, and were positive for CD90 and FAP. The A-SMA expression of CAFs was higher than that of NFs. CAFs efficiently suppressed T cell proliferation as compared with NFs. Of note, this suppressive activity was found under co-cultivation with not only CAFs, but also supernatant from CAFs. CAFs, but not NFs expressed co-regulatory molecules, B7H1 and B7DC and CAFs showed increased expression levels of cytokine genes including IL-6, IL-8, TNF-A, TGF-β, and VEGF. Moreover, PBMCs co-cultured with supernatant from CAFs preferentially induced T-cell apoptosis and Treg as compared with those from NFs. Conclusion: Our findings suggest that, in the tumor microenvironment, CAFs might play a pivotal role in establishing an immunosuppressive network along with tumor cells, and subsequent facilitation to activate the immunosuppressive pathway. Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Immunosuppressive activity of cancer-associated fibroblasts in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1546. doi:10.1158/1538-7445.AM2015-1546

Myeloid-derived suppressor cells (MDSCs) are key regulators of adaptive immunity by suppressing T-cell functions. However, their potential action on or interaction with B cells remained poorly understood. Here we demonstrate that human polymorphonuclear MDSCs differentially modulate B-cell function by suppressing B-cell proliferation and antibody production. We further demonstrate that this MDSC-mediated effect is cell contact dependent and involves established mediators such as arginase-1, nitric oxide (NO), reactive oxygen species (ROS) as well as B-cell death. Collectively, our studies provide novel evidence that human MDSCs modulate B cells, which could have future implications for immunotherapy approaches.

Abstract Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs) from various tumors. In squamous cell carcinoma of the head and neck (SCCHN), various cell surface markers, including CD44, CD133 and ALDH1, have been identified as the potential markers to isolate CSCs. Evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alteration in patterns of acetylation of histones and are promising new group of anticancer agents. In this study, we investigate the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on two CD44+ cancer stem-like cell lines from SCCHN cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. SAHA and TSA inhibited the growth of SCCHN cell lines in a dose dependent manner as measured by MTS assays. Moreover, SAHA and TSA induced G2/M cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2 on SCCHN cell lines were decreased by SAHA or TSA treatment. HDACi also decreased the mRNA expression levels of stemness-related genes (Notch and Nanog) and TGF-β. As expected, HDACi markedly increased the expression of E-cadherin. In addition, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter stem-like properties of the SCCHN cells, raising the possibility that HDACi may have therapeutic potentials for cancer stem cells of SCCHN. Citation Format: Koichi Sakakura, Takaaki Murata, Kazuaki Chikamatsu. Alteration of cancer stem cell-like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4889. doi:10.1158/1538-7445.AM2013-4889

Abstract The main factor that affects the prognosis of patients with squamous cell carcinoma of the head and neck (SCCHN) is regional lymph node metastases, which usually spreads first to the sentinel lymph nodes (SLNs). Recent studies using animal models have demonstrated that tumor cells can induce lymphangiogenesis in SLNs before metastasizing. In this study, we investigated the association of SLN lymphangiogenesis with the status of primary tumors. Moreover, the immunological status of SLNs was also examined. The expression of lymphatic-specific markers, including VEGFR-3, Prox-1, and LYVE-1 in 23 metastasis-negative SLNs obtained from 10 patients with SCCHN was investigated using quantitative real-time RT-PCR. Moreover, primary tumors obtained from patients were evaluated for the expression of VEGF-A, -C, and -D by immunohistochemistry. The level of LYVE-1 expression in SLNs was significantly higher than in control lymph nodes from patients with non-cancerous diseases. Moreover, SLNs from patients with VEGF-C-positive tumor showed a significantly higher expression of VEGFR-3 than those from patients with VEGF-C-negative tumor. VEGFR-3 is expressed mainly in lymphatic endothelium but also in a subset of antigen-presenting cells including immature dendritic cells (DCs), therefore; we furthermore examined the infiltration of DCs into SLN by immunohistochemistry. Interestingly, S-100+ and CD1a+ immature DCs infiltrated significantly into SLNs compared to non-SLNs in patients with SCCHN, suggesting that predominant migration of immature DCs, which suppress T-cell activation and support regulatory T cell development, would contribute to inhibit rather than develop anti-tumor immunity. Our findings suggest that primary tumor actively induces lymphangiogenesis in SLNs prior to the onset of metastases, and where tumor-derived VEGF-C plays an important role. Moreover, active lymphangiogenesis may concomitantly lead to down-regulation of SLN immunity. Thus, both lymphangiogenesis and immune suppression in SLNs are likely the basis of its susceptibility to tumor metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2011-5177

Abstract Evidence has accumulated that chronic inflammation plays a critical role in carcinogenesis, tumor growth, and metastasis. To date, several inflammatory biomarkers are considered as potential prognostic factors in various cancers. The aim of the present study was to investigate the prognostic significance of the pretreatment levels of inflammatory biomarkers in patients with oropharyngeal squamous cell carcinoma (OPSCC). A total of 67 patients newly diagnosed as OPSCC was finally included. The neutrophil, lymphocyte, monocyte and platelet counts recorded before treatment was initiated, then lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were calculated. These inflammatory biomarkers and clinical parameters including age, differentiation, T-stage, N-stage, TNM-stage, smoking, and drinking were compared with progression-free survival (PFS) and overall survival (OS) using cox regression. Elevated monocyte count and advanced T-stage were significantly associated with poor PFS; in addition, these were independent prognostic factors for PFS. Elevated monocyte count and low LMR were significantly associated with poor OS; moreover, low LMR was an independent prognostic factor for OS. Of note, monocyte count and LMR were associated with prognosis more intensively in patients with p16-negative OPSCC. Following these results, we evaluated the peripheral blood mononuclear cells of 14 patients with OPSCC collected before treatments. CD14+ CD86+ HLR-DR+ cells were gated as monocytes, then divided into three subsets according to the expression of CD14 and CD16. The proportion of intermediate monocytes was lower and that of classical monocytes was higher in OPSCC patients than in healthy donors, especially in patients with elevated monocyte count. Furthermore, the expression level of PD-L1 was higher in patients than in healthy donors on all subsets. In conclusion, our data suggests that elevated monocyte count and low LMR seem to be useful biomarkers for predicting prognosis of OPSCC; furthermore, the population shift of monocytes may relate to poor prognosis of OPSCC. Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Prognostic significance and population shift of peripheral monocytes in patients with oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4627. doi:10.1158/1538-7445.AM2017-4627

白血球はその起源によって，リンパ球を主とするリンパ系と，顆粒球・単球・マクロファージ等を含む骨髄系に大分される。本総説では，頭頸部癌における骨髄系細胞研究の動向と免疫療法への応用を，免疫チェックポイント分子と絡めて概説する。

Head & NeckVolume 39, Issue 11 p. 2145-2147 ISSUE INFORMATION - CONTENTSFree Access Issue Information - Contents First published: 17 October 2017 https://doi.org/10.1002/hed.24624AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume39, Issue11November 2017Pages 2145-2147 RelatedInformation

•A case of invasive sinus aspergillosis with no imaging abnormality at onset was reported. •Invasive sinus aspergillosis may occur with no or subtle radiologic changes which can cause a diagnostic delay. •To obtain an earlier diagnosis, repeated imaging studies of thin-slice CT or a gadolinium-enhanced MRI should be considered.

耳下腺癌は多彩な病理組織像を呈し, しばしば治療に難渋する. 治療の第一選択は手術だが, 切除不能例や顔面神経麻痺などの合併症の問題がある. 当科では耳下腺癌症例の一部に重粒子線治療を行っている. 重粒子線は強い殺細胞効果と優れた線量分布を持ち, 重要臓器の隣接する頭頸部領域で従来の放射線抵抗性の高い悪性腫瘍に有用とされている. 今回, 2011年8月から2016年8月までに当科で初期治療として手術治療を施行した22症例 (9例早期癌, 13例進行癌) と重粒子線治療を行った11症例 (2例早期癌, 9例進行癌) の計33症例について比較検討を行った. その結果, 無増悪生存期間および全生存期間は, 手術群と重粒子線治療群で有意差を認めなかった (無増悪生存期間, p=0.147; 全生存期間, p=0.580). 重粒子線治療群では, 1例で永続的な顔面神経麻痺を治療後に認めたが, 治療前に麻痺を認めた4例中3例で麻痺の部分改善を認めた. 一方, 重粒子線治療に特有の有害事象として全例に放射線皮膚炎 (Grade 2以下), また9例に外耳道炎, 5例に滲出性中耳炎, 1例に脳障害を認めた. 進行癌の比率が多いにもかかわらず, 重粒子線治療は手術療法とほぼ同等の有効性が示唆され, 有害事象では手術と異なる特徴が認められた.

Objective: Suppurative acute thyroiditis is caused by pyriform sinus fistula (PSF), and PSF frequently elicits deep neck abscess. However, complete fistulectomy is the ideal management of PSF, and studies on surgical findings of PSF are exceedingly rare. This study aimed to reveal the origins of PSF, each route, and clinical presentation. Methods: This is a multicenter study. We have conducted 19 complete fistulectomies of PSF in Japan, analyzed routes of the fistulas, estimated the origins, and investigated their histological and clinical findings. Results: No recurrence was observed in all cases. Five of 12 cases showed thymic and/or parathyroid tissues around the fistulas, passing inside the inferior horn of thyroid cartilage, were regarded as having 3rd pouch origin, and tended to have low frequency of severe deep neck abscess. The remaining seven cases originated from the 4th pouch running outside of the horn and showed frequent severe infection. Conclusion: PSF have two different routes depending on their generation and may present different clinical manifestations.

Pediatric Acute Otitis Media Caused by Antimicrobial Resistant H. Influenzae Complicated by Respiratory Tract Infection: Community Hospital Study Over 2 Years 10 Months Koichi Sakakura, Tetsuo Takekoshi, Nobuyo Furukawa Mayumi Shibazaki and Nobuhiko Furuya 1) Department of Otolaryngology-Head and Neck Surgery, Gunma University School of Medicine, Maebashi, Gunma, Japan 2) Department of Otolaryngology, Social Insurance Gunma Chuo General Hospital, Maebashi,Gunma, Japan 3) Department of Laboratory Medicine, Social Insurance Gunma Chuo General Hospital, Maebashi, Gunma, Japan Acute otitis media (AOM) caused by beta-lactamase negative ampicillin-resistant Haemophilus influenzae (BLNAR) is an urgent problem. This study analyzes recent trends in infant AOM due to BLNAR in a community hospital. The percentage of BLNAR among all H. influenzae specimens in 2002 was 40%, and the susceptibility to penicillins and cefems remarkably deteriorated. The 25 cases of infant AOM caused by BLNAR tended to have long periods of otorrhea, nd lower, shorter fevers. Of these cases, 50% had been administered antimicrobials showing high susceptibility to BLNAR. An appropriate administration of antibiotic is required.