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

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

Predictive factors for dissection-free sentinel node micrometastases in early oral squamous cell carcinoma.

This sentinel node (SN) biopsy trial aimed to assess its effectiveness in identifying predictive factors of micrometastases and to determine whether elective neck dissection is necessary in oral squamous cell carcinoma. This retrospective study included 55 patients from three previous trials, with positive SNs. The relationship between the sizes of the metastatic focus and metastasis in non-sentinel node (NSN) was investigated. Four of the 55 largest metastatic focus were isolated tumor cells, and the remaining 51 were ranged from 0.2 to 15 mm, with a median of 2.6 mm. The difference of prevalence between 46 negative- and 9 positive-NSN was statistically significant with regard to age, long diameter of primary site and number of cases with regional recurrence. In comparing the size of largest metastatic focus dividing the number of positive SN, with metastaic focus range of < 3.0 mm in one-positive SN group, there were 18 (33%) negative-NSN and no positive-NSN. Regarding prognosis, 3-year overall survival rate of this group (n = 18) and other (n = 37) were 94% and 73% (p = 0.04), and 3-year recurrence free survival rate of this group and other were 94% and 51% (p = 0.03), respectively. Absolutely a further prospective clinical trial would be needed, micrometastases may be defined as solitary SN metastasis with < 3.0 mm of metastatic focus, and approximately 33% of neck dissections could be avoided using these criteria.

Macroscopic and multiple metastases in sentinel lymph node biopsy are respectively associated with poor prognosis in early oral cancer.

BACKGROUND: A multicenter, randomized controlled phase III trial was conducted on sentinel lymph node biopsy (SLNB) and elective neck dissection for T1 (depth of invasion ≥ 4 mm)-T2N0M0 oral cavity squamous cell carcinoma. This study identified factors associated with poor prognosis in patients who underwent SLNB based on a subgroup analysis of this trial. METHODS: We analyzed 418 sentinel lymph nodes (SLNs) from 132 patients who underwent SLNB. The metastatic SLNs were classified into three categories based on size-isolated tumor cells: < 0.2 mm, micrometastasis: ≥ 0.2 mm and < 2 mm, and macrometastasis: ≥ 2 mm. Three groups were formed based on the number of metastatic SLNs: no metastasis, 1 metastatic node, and ≥ 2 metastatic nodes. The size and number of metastatic SLNs on survival were evaluated using Cox proportional hazard models. RESULTS: Patients with macrometastasis and ≥ 2 metastatic SLNs had worse overall survival (OS) and disease-free survival (DFS) after adjustment for potential confounders (HR for OS: macrometastasis, 4.85; 95% CI 1.34-17.60; ≥ 2 metastatic SLN, 3.63; 95% CI 1.02-12.89; HR for DFS: macrometastasis, 2.94; 95% CI 1.16-7.44; ≥ 2 metastatic SLN, 2.97; 95% CI 1.18-7.51). CONCLUSIONS: In patients who underwent SLNB, a poorer prognosis was associated with macrometastasis or having ≥ 2 metastatic SLNs.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Relationship between oral mucositis and the oral bacterial count in patients with head and neck cancer undergoing carbon ion radiotherapy: A prospective study.

BACKGROUND AND PURPOSE: Acute adverse events, such as oral mucositis, can affect treatment success in patients undergoing head and neck radiotherapy. In this study, we aimed to identify the relationship between oral mucositis and oral bacterial counts during carbon ion radiotherapy (C-ion RT) in patients with head and neck cancer. MATERIALS AND METHODS: This prospective study included patients with head and neck tumors treated with C-ion RT between 2017 and 2019. C-ion RT consisted of treatment at 57.6, 64.0, or 70.4 Gy (relative biological effectiveness) in 16 fractions. Bacterial counts in the saliva and the back of the tongue were measured using a rapid oral bacteria quantification system. The relationship between the oral bacterial count and oral mucositis was subsequently analyzed. RESULTS: In total, 46 patients were included in the analysis. The bacterial count in the saliva gradually increased from the commencement of C-ion RT and peaked at 16 fractions. Bacterial counts at the back of the tongue were already high at the beginning of C-ion RT; however, they decreased with continued treatment, peaked at 16 fractions, and subsequently decreased again. Patients with bacterial counts exceeding the mean before C-ion RT (high-count group) did not experience more severe mucositis than those with counts below the mean (low-count group). However, patients in the high-count group tended to experience faster-onset mucositis and slower healing than those in the low-count group. CONCLUSION: Bacterial counts may aid in the development of clinical strategies for C-ion RT-induced oral mucositis.

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

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

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

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

Oral findings during follow-up of nasopharyngeal squamous cell carcinoma treatment: A case report.

A 50-year-old woman with a long history of nasopharyngeal cancer (T2N2M0, squamous cell carcinoma) underwent chemoradiotherapy and surgery. In the past, to prevent tumor recurrence or metastasis, she underwent concurrent chemoradiotherapy or neck dissection. However, during a follow-up 10 years after the surgery, intense F-18 fluorodeoxyglucose uptake was detected in the oral area (SUVmax 6.0). A biopsy of the area with F-18 fluorodeoxyglucose uptake revealed pathological inflammation. Radiography showed the presence of a wisdom tooth, located at the F-18 fluorodeoxyglucose accumulation site, and pericoronitis of this tooth was detected. Our findings indicate the importance of considering the effect of inflammatory conditions, such as periodontal disease, in using F-18 fluorodeoxyglucose positron emission tomography/computed tomography during follow-up after head and neck cancer treatment.

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

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

Clinical features and dosimetric evaluation of carbon ion radiation-induced osteoradionecrosis of mandible in head and neck tumors.

BACKGROUND AND PURPOSE: Osteoradionecrosis (ORN) affects the patient's quality of life by making eating and maintaining oral hygiene painful. This study aimed to analyze carbon ion radiotherapy (C-ion RT)-induced ORN of the mandible. MATERIALS AND METHODS: A retrospective study of 199 patients with head and neck tumors treated with C-ion RT was performed from 2010 to 2019. Only 11 patients with tumors located in the oropharynx and floor of the mouth were analyzed. C-ion RT consisted of 57.6 Gy or 64.0 Gy (relative biological effectiveness) in 16 fractions. The mandible was analyzed for magnetic resonance imaging (MRI) changes and bone exposure. The relationship between the radiation dose and ORN of the mandible was analyzed. RESULTS: Five patients (45.5%) had ORN of the mandible. The median follow-up time was 68 months. The median onset times based on MRI changes and bone exposure were 9 and 15 months, respectively. Doses of 30 Gy (relative biological effectiveness) to the mandible and teeth showed the most significant effect, causing ORN at 29.5 ± 6.7 cc and 3.9 ± 1.8 cc, respectively, with cut-off values at 16.5 cc (p = 0.002) and 1.8 cc (p = 0.0059), respectively. CONCLUSION: This is the first study reporting the incidence, onset time, and risk-predictive dosimetry parameters of C-ion RT-induced ORN of the mandible. Our study will be useful for establishing clinical strategies for C-ion RT to the head and neck near the mandible.

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

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

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

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

AKT3 Is a Novel Regulator of Cancer-Associated Fibroblasts in Head and Neck Squamous Cell Carcinoma.

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.